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The scrambled story between hyaluronan and glioblastoma

  • Matías Arturo Pibuel
    Correspondence
    For correspondence: Matías Arturo Pibuel; Silvina Laura Lompardía
    Affiliations
    Departamento de Microbiología, Inmunología, Biotecnología y Genética, Facultad de Farmacia y Bioquímica, Instituto de Estudios de la Inmunidad Humoral (IDEHU)-CONICET, Universidad de Buenos Aires, Capital Federal, Argentina
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  • Daniela Poodts
    Affiliations
    Departamento de Microbiología, Inmunología, Biotecnología y Genética, Facultad de Farmacia y Bioquímica, Instituto de Estudios de la Inmunidad Humoral (IDEHU)-CONICET, Universidad de Buenos Aires, Capital Federal, Argentina
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  • Mariángeles Díaz
    Affiliations
    Instituto de Estudios de la Inmunidad Humoral (IDEHU)-CONICET, Universidad de Buenos Aires, Capital Federal, Argentina
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  • Silvia Elvira Hajos
    Affiliations
    Departamento de Microbiología, Inmunología, Biotecnología y Genética, Facultad de Farmacia y Bioquímica, Instituto de Estudios de la Inmunidad Humoral (IDEHU)-CONICET, Universidad de Buenos Aires, Capital Federal, Argentina
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  • Silvina Laura Lompardía
    Correspondence
    For correspondence: Matías Arturo Pibuel; Silvina Laura Lompardía
    Affiliations
    Departamento de Microbiología, Inmunología, Biotecnología y Genética, Facultad de Farmacia y Bioquímica, Instituto de Estudios de la Inmunidad Humoral (IDEHU)-CONICET, Universidad de Buenos Aires, Capital Federal, Argentina
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Open AccessPublished:March 16, 2021DOI:https://doi.org/10.1016/j.jbc.2021.100549
      Advances in cancer biology are revealing the importance of the cancer cell microenvironment on tumorigenesis and cancer progression. Hyaluronan (HA), the main glycosaminoglycan in the extracellular matrix, has been associated with the progression of glioblastoma (GBM), the most frequent and lethal primary tumor in the central nervous system, for several decades. However, the mechanisms by which HA impacts GBM properties and processes have been difficult to elucidate. In this review, we provide a comprehensive assessment of the current knowledge on HA’s effects on GBM biology, introducing its primary receptors CD44 and RHAMM and the plethora of relevant downstream signaling pathways that can scramble efforts to directly link HA activity to biological outcomes. We consider the complexities of studying an extracellular polymer and the different strategies used to try to capture its function, including 2D and 3D in vitro studies, patient samples, and in vivo models. Given that HA affects not only migration and invasion, but also cell proliferation, adherence, and chemoresistance, we highlight the potential role of HA as a therapeutic target. Finally, we review the different existing approaches to diminish its protumor effects, such as the use of 4-methylumbelliferone, HA oligomers, and hyaluronidases and encourage further research along these lines in order to improve the survival and quality of life of GBM patients.

      Keywords

      Abbreviations:

      4MU (4-methylumbelliferone), ADAM10 (disintegrin and metalloproteinase 10), ADAMTS (disintegrin and metalloproteinase with thrombospondin motifs), Akt (protein kinase B (PKB)), AMPK (AMP-activated protein kinase), AP-1 (activator protein-1), BBB (blood–brain barrier), BEHAN (brain-enriched hyaluronan binding), bFGFR (b-fibroblast growth factors receptor), CD133 (cluster of differentiation 133), CD44 (cluster of differentiation 44), CNS (central nervous system), CS (chondroitin sulfate), CSPG4/NG2 (chondroitin sulfate proteoglycan 4), CXCL12 (CXC chemokine ligand 12), CXCR4 (CXC chemokine receptor 4), DCs (dendritic cells), ECM (extracellular matrix), EGFR (epidermal growth factor receptor), EMT (epithelial-to-mesenchymal transition), ERK (extracellular signal-regulated kinase), ERM (ezrin–radixin–moesin), FAK (focal adhesion kinase), FDA (Food and Drug Administration), GAG (glycosaminoglycans), GBM (glioblastoma), GPI (glycosylphosphatidylinositol), HA (hyaluronan), HA-LNPs (HA-coated lipid-based nanoparticles), HA-M (HA-decorated micelle), HAPLN (HA- and proteoglycan-link proteins), HAS (hyaluronan synthases), HAS2-AS1 (antisense for HAS2), HMW-HA (high-molecular-weight HA), HS (heparan sulfate), HYAL (hyaluronidase), IDH (isocitrate dehydrogenase), IFN-γ (interferon-γ), IL-1α (interleukin 1α), iNOS (inducible nitric oxide synthase), LMW-HA (low-molecular-weight HA), MAPK (mitogen-activated protein kinases), MEK (mitogen-activated protein kinase kinase), MGMT (methyl guanine methyl transferase), MMP (matrix metalloproteinase), MT1-MMP (membrane type 1-matrix metalloproteinase), NF-κB (nuclear factor-κB), NSPC (neural stem and progenitor cells), oHA (oligomers of HA), PAI-1 (plasminogen activator inhibitor-1), PBMCs (peripheral blood mononuclear cells), PCL (poly (ε-caprolactone)), PDGFR (platelet-derived growth factor receptor), PI3K (phosphatidylinositol 3-kinase), PLK1 (Polo-like kinase 1), PTEN (tumor suppressor phosphatase and tensin homolog), Ras (oncogenic rat sarcoma), RHAMM (HA-mediated motility receptor), RhoA (Rho GTPase A), ROK (repressor, open reading frame, kinase), RON (receptor originated from nantes), SIRT1 (Sirtuin 1), TGFβR-1 (transforming growth factor β receptor), TIMP-1 (tissue inhibitor of metalloproteinases), TLR4 (Toll-like receptor 4), TMEM (transmembrane Protein 2), TMZ (temozolomide), UDP-GlcNAc (uridine diphosphate N-acetylglucosamine), uPA (urokinase-type plasminogen activator), uPAR (urokinase-type plasminogen activator receptor), VEGF (vascular endothelial-derived growth factor)
      Glioblastoma (GBM), also known as grade IV astrocytoma by the World Health Organization classification (
      • Louis D.N.
      • Perry A.
      • Reifenberger G.
      • von Deimling A.
      • Figarella-Branger D.
      • Cavenee W.K.
      • Ohgaki H.
      • Wiestler O.D.
      • Kleihues P.
      • Ellison D.W.
      The 2016 World Health Organization classification of tumors of the central nervous system: A summary.
      ), is the most frequent primary tumor of the central nervous system (CNS) in adults. It is characterized by fast growth, invasiveness, and high mortality, with a median survival of less than 15 months after diagnosis (
      • Anjum K.
      • Shagufta B.I.
      • Abbas S.Q.
      • Patel S.
      • Khan I.
      • Shah S.A.A.
      • Akhter N.
      • Hassan S.S.U.
      Current status and future therapeutic perspectives of glioblastoma multiforme (GBM) therapy: A review.
      ,
      • Adamson C.
      • Kanu O.O.
      • Mehta A.I.
      • Di C.
      • Lin N.
      • Mattox A.K.
      • Bigner D.D.
      Glioblastoma multiforme: A review of where we have been and where we are going.
      ). Currently there are few therapeutic options (
      • Anjum K.
      • Shagufta B.I.
      • Abbas S.Q.
      • Patel S.
      • Khan I.
      • Shah S.A.A.
      • Akhter N.
      • Hassan S.S.U.
      Current status and future therapeutic perspectives of glioblastoma multiforme (GBM) therapy: A review.
      ,
      • Le Rhun E.
      • Preusser M.
      • Roth P.
      • Reardon D.A.
      • Van Den Bent M.
      • Wen P.
      • Reifenberger G.
      • Weller M.
      Molecular targeted therapy of glioblastoma.
      ,
      • Perus L.J.M.
      • Walsh L.A.
      • Walsh L.A.
      Microenvironmental heterogeneity in brain malignancies.
      ,
      • Wirsching H.G.
      • Galanis E.
      • Weller M.
      Glioblastoma.
      ,
      • Strobel H.
      • Baisch T.
      • Fitzel R.
      • Schilberg K.
      • Siegelin M.D.
      • Karpel-massler G.
      • Debatin K.
      • Westho M.
      Temozolomide and other alkylating agents in glioblastoma therapy.
      ,
      • Daher A.
      • de Groot J.
      Rapid identification and validation of novel targeted approaches for glioblastoma: A combined ex vivo-in vivo pharmaco-omic model.
      ), with the first line therapy being surgical resection and radiotherapy combined with cycles of temozolomide (TMZ). Unfortunately, TMZ therapy causes severe adverse effects, such as myelosuppression and hepatotoxicity, and almost 50% of patients exhibit resistance to the treatment (
      • Anjum K.
      • Shagufta B.I.
      • Abbas S.Q.
      • Patel S.
      • Khan I.
      • Shah S.A.A.
      • Akhter N.
      • Hassan S.S.U.
      Current status and future therapeutic perspectives of glioblastoma multiforme (GBM) therapy: A review.
      ,
      • Le Rhun E.
      • Preusser M.
      • Roth P.
      • Reardon D.A.
      • Van Den Bent M.
      • Wen P.
      • Reifenberger G.
      • Weller M.
      Molecular targeted therapy of glioblastoma.
      ,
      • Wirsching H.G.
      • Galanis E.
      • Weller M.
      Glioblastoma.
      ,
      • Strobel H.
      • Baisch T.
      • Fitzel R.
      • Schilberg K.
      • Siegelin M.D.
      • Karpel-massler G.
      • Debatin K.
      • Westho M.
      Temozolomide and other alkylating agents in glioblastoma therapy.
      ,
      • Daher A.
      • de Groot J.
      Rapid identification and validation of novel targeted approaches for glioblastoma: A combined ex vivo-in vivo pharmaco-omic model.
      ,
      • Philteos J.
      • Karmur B.S.
      • Mansouri A.
      MGMT testing in glioblastomas pitfalls and opportunities.
      ,
      LiverTox: Clinical and Research Information on Drug-Induced Liver Injury.
      ,
      • Houy N.
      • Le Grand F.
      Administration of temozolomide: Comparison of conventional and metronomic chemotherapy regimens.
      ). Furthermore, high inter- and intratumor heterogeneity, individual variability, and different stages of disease at diagnosis time complicate GBM treatment (
      • Rajaratnam V.
      • Islam M.M.
      • Yang M.
      • Slaby R.
      • Ramirez H.M.
      • Mirza S.P.
      Glioblastoma: Pathogenesis and current status of chemotherapy and other novel treatments.
      ).
      Although tumor aggressiveness and resistance are often thought of as being intrinsic to malignant cells, there is a rising appreciation of the critical importance of the tumor microenvironment, including nontumor cells and the extracellular matrix (ECM), in tumorigenesis. In addition, the ECM plays a crucial role in drug penetration as well as in the modulation of the immune system, also impacting on the mechanisms of tumor evasion and invasion (
      • Albini A.
      • Bruno A.
      • Gallo C.
      • Pajardi G.
      • Noonan D.M.
      Cancer stem cells and the tumor microenvironment: Interplay in tumor heterogeneity.
      ,
      • Yeldag G.
      • Rice A.
      • Río Hernández A.
      Chemoresistance and the self-maintaining tumor microenvironment.
      ,
      • Piperigkou Z.
      • Karamanos N.K.
      Dynamic interplay between miRNAs and the extracellular matrix influences the tumor microenvironment.
      ,
      • Soysal S.D.
      • Muenst S.E.
      Role of the tumor microenvironment in breast cancer.
      ,
      • Manou D.
      • Caon I.
      • Bouris P.
      • Triantaphyllidou I.
      • Giaroni C.
      • Passi A.
      • Karamanos N.K.
      • Vigetti D.
      • Theocharis A.D.
      The complex interplay between extracellular matriz and cells in tissues.
      ,
      • Karamanos N.K.
      • Piperigkou Z.
      • Theocharis A.D.
      • Watanabe H.
      • Franchi M.
      • Baud S.
      • Brézillon S.
      • Götte M.
      • Passi A.
      • Vigetti D.
      • Ricard-Blum S.
      • Sanderson R.D.
      • Neill T.
      • Iozzo R.V.
      Proteoglycan chemical diversity drives multifunctional cell regulation and therapeutics.
      ). For these reasons, there is a growing interest in studying the CNS ECM and the mechanisms through which it impacts the development and progression of brain tumors, with obvious implications for the development of new therapeutic alternatives. This is a particularly intriguing area because the ECM in the brain differs notably from that of other organs. Thus, focused investigations into this ECM are likely to broaden our understanding of neurobiology as well as advance cancer treatments.
      One of the primary components of ECM is hyaluronan (HA), which is present in the parenchymal ECM along with proteoglycans (such as aggrecan, versican, neurocan, and brevican without collagen), the tenascins and link proteins (
      • Lau L.W.
      • Cua R.
      • Keough M.B.
      • Haylock-Jacobs S.
      • Yong V.W.
      Pathophysiology of the brain extracellular matrix: A new target for remyelination.
      ,
      • Miyata S.
      • Kitagawa H.
      Formation and remodeling of the brain extracellular matrix in neural plasticity: Roles of chondritin sulfate and hyaluronan.
      ), and other glycosaminoglycans (GAGs), including chondroitin sulfate, heparan sulfate, and keratan sulfate (
      • Lau L.W.
      • Cua R.
      • Keough M.B.
      • Haylock-Jacobs S.
      • Yong V.W.
      Pathophysiology of the brain extracellular matrix: A new target for remyelination.
      ,
      • Miyata S.
      • Kitagawa H.
      Formation and remodeling of the brain extracellular matrix in neural plasticity: Roles of chondritin sulfate and hyaluronan.
      ).HA is a linear and nonsulfated GAG made of repetitive units of D-glucuronide acid and N-acetyl-D-glucosamine (
      • Boregowda R.K.
      • Appaiah H.N.
      • Siddaiah M.
      • Kumarswamy S.B.
      • Sunila S.
      • Thimmaiah K.N.
      • Mortha K.
      • Toole B.
      • Banerjee S.D.
      Expression of hyaluronan in human tumor progression.
      ,
      • Auvinen P.
      • Tammi R.
      • Kosma V.M.
      • Sironen R.
      • Soini Y.
      • Mannermaa A.
      • Tumelius R.
      • Uljas E.
      • Tammi M.
      Increased hyaluronan content and stromal cell CD44 associate with HER2 positivity and poor prognosis in human breast cancer.
      ,
      • Provenzano P.P.
      • Hingorani S.R.
      Hyaluronan, fluid pressure, and stromal resistance in pancreas cancer.
      ,
      • Toole B.P.
      Hyaluronan: From extracellular glue to pericellular cue.
      ). It serves as a backbone to which link proteins and proteoglycans can be attached, building a three-dimensional (3D) network (
      • Lau L.W.
      • Cua R.
      • Keough M.B.
      • Haylock-Jacobs S.
      • Yong V.W.
      Pathophysiology of the brain extracellular matrix: A new target for remyelination.
      ,
      • Miyata S.
      • Kitagawa H.
      Formation and remodeling of the brain extracellular matrix in neural plasticity: Roles of chondritin sulfate and hyaluronan.
      ,
      • Ferrer V.P.
      • Moura Neto V.
      • Mentlein R.
      Glioma infiltration and extracellular matrix: Key players and modulators.
      ,
      • Ruoslahti E.
      Brain extracellular matrix.
      ,
      • Ding H.
      • Xie Y.
      • Dong Q.
      • Kimata K.
      • Nishida Y.
      • Ishiguro N.
      • Zhuo L.
      Roles of hyaluronan in cardiovascular and nervous system disorders.
      ,
      • Su W.
      • Matsumoto S.
      • Sorg B.
      • Sherman L.S.
      Distinct roles for hyaluronan in neural stem cell niches and perineuronal nets.
      ), and also serves as a ligand for the cell-surface glycoprotein CD44 and the receptor for HA-mediated cell motility (RHAMM). HA is involved in tissue organization, wound healing, leukocyte traffic, growth and cellular differentiation, and other functions (
      • Termeer C.
      • Sleeman J.P.
      • Simon J.C.
      Hyaluronan - magic glue for the regulation of the immune response?.
      ,
      • Khaldoyanidi S.K.
      • Goncharova V.
      • Mueller B.
      • Schraufstatter I.U.
      Hyaluronan in the healthy and malignant hematopoietic microenvironment.
      ,
      • Joy R.A.
      • Vikkath N.
      • Ariyannur P.S.
      Metabolism and mechanisms of action of hyaluronan in human biology.
      ). In the CNS, HA also participates in the correct generation, proliferation, and maturation of neural stem cell progenitors (NSCP) during brain development and repair (
      • Su W.
      • Matsumoto S.
      • Sorg B.
      • Sherman L.S.
      Distinct roles for hyaluronan in neural stem cell niches and perineuronal nets.
      ,
      • Preston M.
      • Sherman L.S.
      Neural stem cell niches: Roles for the hyaluronan-based extracellular matrix.
      ).
      Reports published in the 1970s and 1980s established that HA has another, undesirable role: these studies demonstrated that production of GAGs—and particularly HA—by malignant glioma cells was higher than their production in normal glial cell lines (
      • Dorfman A.
      • Ho P.L.
      Synthesis of acid mucopolysaccharides by glial tumor cells in tissue culture.
      ,
      • Wasteson Å.
      • Westermark B.
      • Lindahl U.
      • Pontén J.
      Aggregation of feline lymphoma cells by hyaluronic acid.
      ,
      • Glimelius B.
      • Norling B.
      • Westermark B.
      • Wasteson A.
      Composition and distribution of glycosaminoglycans in cultures of human normal and malignant glial cells.
      ,
      • Glimelius B.
      • Norling B.
      • Westermark B.
      • Wasteson
      A comparative study of glycosaminoglycans in cultures of human, normal and malignant glial cells.
      ,
      • Steck P.A.
      • Moser R.P.
      • Bruner J.M.
      • Liang L.
      • Freidman A.N.
      • Hwang T.L.
      • Yung W.K.
      Altered expression and distribution of heparan sulfate proteoglycans in human gliomas.
      ), which was associated with a higher rate of cell proliferation (
      • Glimelius B.
      • Norling B.
      • Nederman T.
      • Carlsson J.
      Extracellular matrices in multicellular spheroids of human glioma origin: Increased incorporation of proteoglycans and fibronectin as compared to monolayer cultures.
      ). Later, studies partially contradicted these reports, showing that the addition of exogenous HA did not modify cell proliferation (
      • Nakagawa T.
      • Kubota T.
      • Kabuto M.
      • Kodera T.
      Hyaluronic acid facilitates glioma cell invasion in vitro.
      ,
      • Radotra B.
      • McCormick D.
      Glioma invasion in vitro is mediated by CD44–hyaluronan interactions.
      ), but several publications did confirm a correlation between the addition of HA and an enhanced rate of invasion across multiple glioma cell lines (
      • Nakagawa T.
      • Kubota T.
      • Kabuto M.
      • Kodera T.
      Hyaluronic acid facilitates glioma cell invasion in vitro.
      ,
      • Radotra B.
      • McCormick D.
      Glioma invasion in vitro is mediated by CD44–hyaluronan interactions.
      ,
      • Radotra B.
      • McCormick D.
      CD44 is involved in migration but not spreading of astrocytoma cells in vitro.
      ,
      • Koochekpour S.
      • Pilkington G.J.
      • Merzak A.
      Hyaluronic acid/CD44H interaction induces cell detachment and stimulates migration and invasion of human glioma cells in vitro.
      ,
      • Giese A.
      • Loo M.A.
      • Rief M.D.
      • Tran N.
      • Berens M.E.
      Substrates for astrocytoma invasion.
      ,
      • Pilkington G.J.
      The role of the extracellular matrix in neoplastic glial invasion of the nervous system.
      ,
      • Chintala S.K.
      • Gokaslan Z.L.
      • Go Y.
      • Sawaya R.
      • Nicolson G.L.
      • Rao J.S.
      Role of extracellular matrix proteins in regulation of human glioma cell invasion in vitro.
      ). Our recent data extended these conclusions, showing that both high-molecular-weight HA (HMW-HA; 1.5–1.8 × 106 Da) and low-molecular-weight HA (LMW-HA; 1–3 × 105 Da) enhance cell migration without modifying cell proliferation on the murine GBM cell line GL26 (
      • Pibuel M.A.
      • Díaz M.
      • Molinari Y.
      • Poodts D.
      • Silvestroff L.
      • Lompardía S.L.
      • Franco P.
      • Hajos S.E.
      4-Methylumbelliferone as a potent and selective anti-tumor drug on a glioblastoma model.
      ).
      We now know that HA strongly impacts tumor development and progression, favoring cell proliferation, angiogenesis, lymphangiogenesis, chemotherapy resistance, evasion of apoptosis, and invasion of surrounding tissues (Fig. 1) (
      • Toole B.P.
      Hyaluronan: From extracellular glue to pericellular cue.
      ,
      • Park J.B.
      • Kwak H.J.
      • Lee S.H.
      Role of hyaluronan in glioma invasion.
      ,
      • Mascaro M.
      • Pibuel M.A.
      • Lompardia S.L.
      • Diaz M.
      • Zotta E.
      • Bianconi M.I.
      • Lago N.
      • Otero S.
      • Jankilevich G.
      • Alvarez E.
      • Hajos S.E.
      Low molecular weight hyaluronan induces migration of human choriocarcinoma JEG-3 cells mediated by RHAMM as well as by PI3K and MAPK pathways.
      ,
      • Lompardía S.L.
      • Papademetrio D.L.
      • Mascaró M.
      • Del Carmen Álvarez E.M.
      • Hajos S.E.
      Human leukemic cell lines synthesize hyaluronan to avoid senescence and resist chemotherapy.
      ,
      • Lompardía S.
      • Díaz M.
      • Pibuel M.
      • Papademetrio D.
      • Poodts D.
      • Mihalez C.
      • Álvarez É.
      • Hajos S.
      Hyaluronan abrogates imatinib-induced senescence in chronic myeloid leukemia cell lines.
      ,
      • Sironen R.K.
      • Tammi M.
      • Tammi R.
      • Auvinen P.K.
      • Anttila M.
      • Kosma V.M.
      Hyaluronan in human malignancies.
      ,
      • Toole B.P.
      Hyaluronan-CD44 interactions in cancer: Paradoxes and possibilities.
      ,
      • Du Y.
      • Liu H.
      • He Y.
      • Liu Y.
      • Yang C.
      • Zhou M.
      • Wang W.
      • Cui L.
      • Hu J.
      • Gao F.
      The interaction between LYVE-1 with hyaluronan on the cell surface may play a role in the diversity of adhesion to cancer cells.
      ). Furthermore, HA accumulation in the tumor milieu is associated with poor prognosis in several types of tumors (
      • Caon I.
      • Bartolini B.
      • Parnigoni A.
      • Caravà E.
      • Moretto P.
      • Viola M.
      • Karousou E.
      • Vigetti D.
      • Passi A.
      Revisiting the hallmarks of cancer: The role of hyaluronan.
      ). Due to HA participation in tumor progression, various strategies to mitigate its effect have been suggested, such as the use of HA oligomers (oHA), treatment with hyaluronidase (HYAL), and the utilization of the HA-synthesis inhibitor 4-Methylumbelliferone (4MU), with promising results (
      • Nagy N.
      • Kuipers H.F.
      • Frymoyer A.R.
      • Ishak H.D.
      • Bollyky J.B.
      • Wight T.N.
      • Bollyky P.L.
      4-Methylumbelliferone treatment and hyaluronan inhibition as a therapeutic strategy in inflammation, autoimmunity, and cancer.
      ,
      • Lompardía S.L.
      • Díaz M.
      • Papademetrio D.L.
      • Pibuel M.
      • Álvarez É.
      • Hajos S.E.
      4-Methylumbelliferone and imatinib combination enhances senescence induction in chronic myeloid leukemia cell lines.
      ,
      • Lompardía S.L.
      • Díaz M.
      • Papademetrio D.L.
      • Mascaró M.
      • Pibuel M.
      • Álvarez E.
      • Hajos S.E.
      Hyaluronan oligomers sensitize chronic myeloid leukemia cell lines to the effect of imatinib.
      ). However, establishing a full therapeutic strategy requires an improved understanding of the role of HA on GBM progression. This is complicated by both the complex biology of this tumor and the variety of HA effects and the redundant pathways in which this GAG is involved.
      Figure thumbnail gr1
      Figure 1Impact of HA on GBM biology. Hyaluronan impacts glioblastoma cells through interaction with its receptors CD44 and RHAMM, which also interact with other receptors, such as EGFR, to increase migration, invasion, proliferation as well as radio and chemotherapy resistance. Furthermore, glioblastoma cells secrete HA, which forms a halo that induces dendritic cell death and hinders the action of immune cells generating an immunoprotective barrier. Therefore, HA enhances GBM progression by amplifying features of malignancy and suppressing immune attack.
      In this review, we examine the main questions facing the field, hoping to shed light on this important disease. For example, which of HA’s many roles impact GBM development? How does the 3D structure of HA influence its effects on GBM cells? Does the effect of HA depend on its concentration or quality? Are interactions with CD44 (
      • Pilkington G.J.
      Hyaluronic acid modulates glioma cell proliferation through its interaction with CD44H in vitro.
      ) and/or RHAMM (
      • Hayen W.
      • Goebeler M.
      • Kumar S.
      • Rießen R.
      • Nehls V.
      Hyaluronan stimulates tumor cell migration by modulating the fibrin fiber architecture.
      ) important, and what other biomolecules might be involved? Given previous observations of increased HA in the tumor context and its successful use as a prognostic marker in other pathologies, can HA be used as a biomarker for this particular neoplasm? Is HA itself really an appropriate therapeutic target, and if so, how can we best influence HA function?
      We first describe existing evidence of HA’s impact on GBM and the receptors and broader signaling pathways that mediate HA functions, including both in vitro and in vivo evidence. We examine how the 3D structure of HA impacts its effects versus soluble HA on GBM cells. We consider the correlations of HA and HA-related molecules with tumor grade and GBM patient survival. Finally, we explore the potential of HA as a therapeutic target, connecting both in vitro and in vivo approaches, and consider therapeutic alternatives to target HA.

      How hyaluronan and its receptors alter glioblastoma biology

      As discussed above, HA plays many physiological and pathophysiological roles. Here we capture HA’s functional impacts on GBM cells and introduce the biomolecules that mediate these effects, including its receptors RHAMM and CD44, coreceptors, and HA-binding proteins (
      • Chauzy C.
      • Delpech B.
      • Olivier A.
      • Bastard C.
      • Girard N.
      • Courel M.N.
      • Maingonnat C.
      • Frébourg T.
      • Tayot J.
      • Creissard P.
      Establishment and characterisation of a human glioma cell line.
      ,
      • Asher R.
      • Bignami A.
      Hyaluronate binding and CD44 expression in human glioblastoma cells and astrocytes.
      ,
      • Radotra B.
      • McCormick D.
      • Crockard A.
      CD44 plays a role in adhesive interactions between glioma cells and extracellular matrix components.
      ,
      • Eibl R.H.
      • Pietsch T.
      • Moll J.
      • Skroch-Angel P.
      • Heider K.H.
      • von Ammon K.
      • Wiestler O.D.
      • Ponta H.
      • Kleihues P.
      • Herrlich P.
      Expression of variant CD44 epitopes in human astrocytic brain tumors.
      ,
      • Chovanec M.
      • Smetana K.
      • Purkrábková T.
      • Holíkova Z.
      • Dvoránková B.
      • André S.
      • Pytlík R.
      • Hozák P.
      • Plzák J.
      • Šedo A.
      • Vacík J.
      • Gabius H.
      Detection of cell type and marker specificity of nuclear binding sites for anionic carbohydrate ligands.
      ) (Fig. 2).
      Figure thumbnail gr2
      Figure 2HA and HA-related molecules in samples of GBM patients. The GBM microenvironment is a complex scenario in which GBM cells and GSC are intermixed with tumor-associated cells (TAC). In addition, there are multiple matrix components, such as HA being the main GAG in the CNS. The molecules related to its metabolism are also important in GBM invasion. This fact is reflected in the presence of RHAMM and CD44 on the edge of the tumor. It is noteworthy that although HA failed as a molecular marker, the enzymes responsible for its synthesis (especially HAS2) and its degradation (particularly HYAL-2) are associated with tumor grade and even correlate with patient outcomes.
      CD44 is a multifunctional transmembrane glycoprotein that belongs to the group of link-module proteins and is expressed in numerous cells and tissues (
      • Ponta H.
      • Sherman L.
      • Herrlich P.A.
      CD44: From adhesion molecules to signalling regulators.
      ,
      • Naor D.
      • Wallach-Dayan S.B.
      • Zahalka M.A.
      • Sionov R.V.
      Involvement of CD44, a molecule with a thousand faces, in cancer dissemination.
      ). There are several CD44 protein variants encoded by alternative splicing involved in various biological processes, such as migration and cellular adhesion, lymphocyte homing, as well as cellular differentiation and proliferation (
      • Johnson P.
      • Ruffell B.
      CD44 and its role in inflammation and inflammatory diseases.
      ). In the CNS, CD44 has been implicated in neuronal development and in the response to injury (
      • Mooney K.L.
      • Choy W.
      • Sidhu S.
      • Pelargos P.
      • Bui T.T.
      • Voth B.
      • Barnette N.
      • Yang I.
      The role of CD44 in glioblastoma multiforme.
      ,
      • Dzwonek J.
      • Wilczyński G.M.
      CD44: Molecular interactions, signaling and functions in the nervous system.
      ), while mice lacking this receptor have deficits at neurological levels (
      • Raber J.
      • Olsen R.H.J.
      • Su W.
      • Foster S.
      • Xing R.
      • Acevedo S.F.
      • Sherman L.S.
      CD44 is required for spatial memory retention and sensorimotor functions.
      ). Even though its main ligand is HA, CD44 can interact with other molecules such as collagen, fibronectin, laminin, osteopontin, growth factors, and metalloproteinases (MMPs) (
      • Morath I.
      • Hartmann T.N.
      • Orian-Rousseau V.
      CD44: More than a mere stem cell marker.
      ,
      • Zöller M.
      CD44: Can a cancer-initiating cell profit from an abundantly expressed molecule?.
      ). Several tumor cells overexpress CD44, and it has been related to tumor progression, apoptosis evasion, and multidrug resistance (
      • Lompardía S.L.
      • Papademetrio D.L.
      • Mascaró M.
      • Del Carmen Álvarez E.M.
      • Hajos S.E.
      Human leukemic cell lines synthesize hyaluronan to avoid senescence and resist chemotherapy.
      ,
      • Toole B.P.
      Hyaluronan-CD44 interactions in cancer: Paradoxes and possibilities.
      ,
      • Karousou E.
      • Misra S.
      • Ghatak S.
      • Dobra K.
      • G??tte M.
      • Vigetti D.
      • Passi A.
      • Karamanos N.K.
      • Skandalis S.S.
      Roles and targeting of the HAS/hyaluronan/CD44 molecular system in cancer.
      ,
      • Bourguignon L.Y.W.
      • Peyrollier K.
      • Xia W.
      • Gilad E.
      Hyaluronan-CD44 interaction activates stem cell marker nanog, Stat-3-mediated MDR1 gene expression, and ankyrin-regulated multidrug efflux in breast and ovarian tumor cells.
      ,
      • Jordan A.R.
      • Racine R.R.
      • Hennig M.J.P.
      • Lokeshwar V.B.
      The role of CD44 in disease pathophysiology and targeted.
      ). Indeed, in GBM, CD44 is strongly involved in cell invasion (
      • Mooney K.L.
      • Choy W.
      • Sidhu S.
      • Pelargos P.
      • Bui T.T.
      • Voth B.
      • Barnette N.
      • Yang I.
      The role of CD44 in glioblastoma multiforme.
      ). Moreover, it was demonstrated that optimal levels of CD44 were necessary on GBM cells to generate highly infiltrative tumors in a mouse model (
      • Okada H.
      • Yoshida J.
      • Sokabe M.
      • Wakabayashi T.
      • Hagiwara M.
      Suppression of CD44 expression decreases migration and invasion of human glioma cells.
      ,
      • Klank R.L.
      • Decker Grunke S.A.
      • Bangasser B.L.
      • Forster C.L.
      • Price M.A.
      • Odde T.J.
      • SantaCruz K.S.
      • Rosenfeld S.S.
      • Canoll P.
      • Turley E.A.
      • McCarthy J.B.
      • Ohlfest J.R.
      • Odde D.J.
      Biphasic dependence of glioma survival and cell migration on CD44 expression level.
      ) and that treatment with an anti-CD44 monoclonal antibody inhibited tumor growth of local glioma in a mouse model (
      • Breyer R.
      • Hussein S.
      • Radu D.L.
      • Pütz K.M.
      • Gunia S.
      • Hecker H.
      • Samii M.
      • Walter G.F.
      • Stan A.C.
      Disruption of intracerebral progression of C6 rat glioblastoma by in vivo treatment with anti-CD44 monoclonal antibody.
      ). These reports implicate CD44 in HA-enhanced proliferation and migration.
      Beyond cell migration, HA and CD44 were shown to be involved in the ability of GBM cells to evade immune attack. In the CNS, immunity depends on effective innate immune activity, since most requirements for adaptive immune responses are not available (
      • Friese M.A.
      • Steinle A.
      • Weller M.
      The innate immune response in the central nervous system and its role in glioma immune surveillance.
      ). Thus, microglia and resident macrophages are the key immune effector cell populations in the brain. Physiologically, the CNS parenchyma is secluded from circulation by the blood–brain barrier (BBB), a specialized endothelial barrier formed by endothelial cells, pericytes and astrocytes, which controls the passage of water to the brain among other functions (
      • Friese M.A.
      • Steinle A.
      • Weller M.
      The innate immune response in the central nervous system and its role in glioma immune surveillance.
      ). In the presence of GBM, the BBB is impaired, resulting in an infiltration of monocytes and other immune cells from the periphery (
      • Wolburg H.
      • Noell S.
      • Fallier-Becker P.
      • MacK A.F.
      • Wolburg-Buchholz K.
      The disturbed blood-brain barrier in human glioblastoma.
      ). The microglia and peripheral macrophages recruited to the glioma environment are known as tumor-associated macrophages (TAMs) (
      • Won W.-J.
      • Deshane J.S.
      • Leavenworth J.W.
      • Oliva C.R.
      • Griguer C.E.
      Metabolic and functional reprogramming of myeloid-derived suppressor cells and their therapeutic control in glioblastoma.
      ,
      • Hambardzumyan D.
      • Gutmann D.H.
      • Kettenmann H.
      The role of microglia and macrophages in glioma maintenance and progression.
      ). Yet, cancer cells can subvert the immune functions of these cells, using excreted factors, such as HA, to re-educate TAMs to facilitate tumor proliferation, survival, and migration (
      • Zhang G.
      • Guo L.
      • Yang C.
      • Liu Y.
      • He Y.
      • Du Y.
      • Wang W.
      • Gao F.
      A novel role of breast cancer-derived hyaluronan on inducement of M2-like tumor-associated macrophages formation.
      ,
      • Liu M.
      • Tolg C.
      • Turley E.
      • Turley E.
      Dissecting the dual nature of hyaluronan in the tumor microenvironment.
      ).
      Although dendritic cells (DCs) are less studied than TAMs, DC inoculations are an interesting strategy to attract and stimulate brain-specific T cells (
      • Quail D.F.
      • Joyce J.A.
      The microenvironmental landscape of brain tumors.
      ). However, HA synthesized by 9L glioma cells interacts with CD44 to promote apoptosis of DCs via induction of iNOS (
      • Yang T.
      • Witham T.F.
      • Villa L.
      • Erff M.
      • Attanucci J.
      • Watkins S.
      • Kondziolka D.
      • Okada H.
      • Pollack I.F.
      • Chambers W.H.
      Glioma-associated hyaluronan induces apoptosis in dendritic cells via inducible nitric oxide synthase: Implications for the use of dendritic cells for therapy of gliomas.
      ). Moreover, glioma cells grow HA “halos” when in coculture with peripheral blood mononuclear cells (PBMCs). These halos impede the contact between different cell types and reduced the generation of specific T cells for glioma antigens, which may constitute a suppressor mechanism to evade the cellular immune attack (
      • Gately C.L.
      • Muul L.M.
      • Greenwood M.A.
      • Papazoglou S.
      • Dick S.J.
      • Kornblith P.L.
      • Smith B.H.
      • Gately M.K.
      In vitro studies on the cell-mediated immune response to human brain tumors. II. Leukocyte-induced coats of glycosaminoglycan increase the resistance of glioma cells to cellular immune attack.
      ,
      • Oberc-Greenwood M.A.
      • Muul L.M.
      • Gately M.K.
      • Kornblith P.L.
      • Smith B.H.
      Ultrastructural features of the lymphocyte-stimulated halos produced by human glioma-derived cells in vitro.
      ) (Fig. 1). Interestingly, while treatment with the immune cell-activating interferon-α/β does not modify HA secretion, incubation with anti-inflammatory glucocorticoids decreased HA levels in the glioma culture medium (
      • Mackie A.E.
      • Freshney R.I.
      • Akturk F.
      • Hunt G.
      Glucocorticoids and the cell surface of human glioma cells: Relationship to cytostasis.
      ,
      • Wiranowska M.
      • Naidu A.K.
      Interferon effect on glycosaminoglycans in mouse glioma in vitro.
      ), suggesting that an inflammatory context would be necessary for HA production and GBM progression. Finally, recent data demonstrated that tumor-associated mesenchymal stem-like cells secreted C5a, which activates ERK and triggers expression of the HA synthase HAS2, contributing to HA abundance and enhancing GBM invasiveness in a nude mice model (
      • Lim E.J.
      • Suh Y.
      • Yoo K.C.
      • Lee J.H.
      • Kim I.G.
      • Kim M.J.
      • Chang J.H.
      • Kang S.G.
      • Lee S.J.
      Tumor-associated mesenchymal stem-like cells provide extracellular signaling cue for invasiveness of glioblastoma cells.
      ). These results provide compelling examples of the ways in which tumor cells modulate their microenvironment to favor their own malignant behaviors. Moreover, they show that HA can influence GBM progression by enhancing GBM malignancy, increasing proliferation, migration, and invasion, and by impairing the attack of the immune cells.
      CD44 is also associated with the specific binding of glioma cells to HA, but it is not the only receptor involved (
      • Reichard-Brown J.L.
      • Akeson R.
      Correlation of the cell phenotype of cultured cell lines with their adhesion to components of the extracellular matrix.
      ,
      • Knüpfer M.M.
      • Poppenborg H.
      • Hotfilder M.
      • Domula M.
      • Wolff J.E.A.
      Hyaluronic acid binding capacity of malignant glioma cells.
      ,
      • Knüpfer M.M.
      • Poppenborg H.
      • Hotfilder M.
      • Kühnel K.
      • Wolff J.E.A.
      • Domula M.
      CD44 expression and hyaluronic acid binding of malignant glioma cells.
      ,
      • Knüpfer M.M.
      • Knüpfer H.
      • Van Gool S.
      • Domula M.
      • Wolff J.E.A.
      Interferon gamma inhibits proliferation and hyaluronic acid adhesion of human malignant glioma cells in vitro.
      ,
      • Knüpfer M.M.
      • Knüpfer H.
      • Jendrossek V.
      • Van Gool S.
      • Wolff J.E.A.
      • Keller E.
      Interferon-gamma inhibits growth and migration of A172 human glioblastoma cells.
      ).
      RHAMM has several isoforms obtained by alternative splicing (
      • Cheung W.F.
      • Cruz T.F.
      • Turley E.A.
      Receptor for hyaluronan-mediated motility (RHAMM), a hyaladherin that regulates cell responses to growth factors.
      ) and is found in the cytoplasm, nucleus, on the cellular surface, and even in cell culture supernatant (
      • Wang C.
      • Thor A.D.
      • Moore D.H.
      • Zhao Y.
      • Kerschmann R.
      • Stern R.
      • Watson P.H.
      • Turley E.A.
      The overexpression of RHAMM, a hyaluronan-binding protein that regulates ras signaling, correlates with overexpression of mitogen-activated protein kinase and is a significant parameter in breast cancer progression.
      ,
      • Assmann V.
      • Jenkinson D.
      • Marshall J.F.
      • Hart I.R.
      The intracellular hyaluronan receptor RHAMM/IHABP interacts with microtubules and actin filaments.
      ,
      • Zhang S.
      • Chang M.C.Y.
      • Zylka D.
      • Turley S.
      • Harrison R.
      • Turley E.A.
      The hyaluronan receptor RHAMM regulates extracellular-regulated kinase.
      ,
      • Hardwick C.
      • Hoare K.
      • Owens R.
      • Hohn H.P.
      • Hopok M.
      • Moore D.
      • Cripps V.
      • Austen L.
      • Nance D.M.
      • Turley E.A.
      Molecular cloning of a novel hyaluronan receptor that mediates tumor cell motility.
      ). It has been proposed that the levels of RHAMM in the cytosol must be carefully balanced to enable the correct formation of the mitotic spindle and for genomic stability (
      • Assmann V.
      • Jenkinson D.
      • Marshall J.F.
      • Hart I.R.
      The intracellular hyaluronan receptor RHAMM/IHABP interacts with microtubules and actin filaments.
      ,
      • He Z.
      • Mei L.
      • Connell M.
      • Maxwell C.A.
      Hyaluronan mediated motility receptor (HMMR) encodes an evolutionarily conserved homeostasis, mitosis, and meiosis regulator rather than a hyaluronan receptor.
      ,
      • Maxwell C.A.
      • McCarthy J.
      • Turley E.
      Cell-surface and mitotic-spindle RHAMM: Moonlighting or dual oncogenic functions?.
      ). Within the CNS, RHAMM has also been associated with glial motility in response to CNS injuries, axon extension, mitochondrial trafficking, and in the folding of the neocortex in fetal human brain (
      • Turley E.A.
      • Hossain M.Z.
      • Sorokan T.
      • Jordan L.M.
      • Nagy J.I.
      Astrocyte and microglial motility in vitro is functionally dependent on the hyaluronan receptor RHAMM.
      ,
      • Lynn B.D.
      • Turley E.A.
      • Nagy J.I.
      Subcellular distribution, calmodulin interaction, and mitochondrial association of the hyaluronan-binding protein RHAMM in rat brain.
      ,
      • Lynn B.D.
      • Li X.
      • Cattini P.A.
      • Turley E.A.
      • Nagy J.I.
      Identification of sequence, protein isoforms, and distribution of the hyaluronan-binding protein RHAMM in adult and developing rat brain.
      ,
      • Nagy J.I.
      • Price M.L.
      • Staines W.A.
      • Lynn B.D.
      • Granholm A.C.
      The hyaluronan receptor RHAMM in noradrenergic fibers contributes to axon growth capacity of locus coeruleus neurons in an intraocular transplant model.
      ,
      • Long K.R.
      • Newland B.
      • Florio M.
      • Kalebic N.
      • Langen B.
      • Kolterer A.
      • Wimberger P.
      • Huttner W.B.
      Extracellular matrix components HAPLN1, lumican, and collagen I cause hyaluronic acid-dependent folding of the developing human neocortex.
      ,
      • Connell M.
      • Chen H.
      • Jiang J.
      • Kuan C.W.
      • Fotovati A.
      • Chu T.L.H.
      • He Z.
      • Lengyell T.C.
      • Li H.
      • Kroll T.
      • Li A.M.
      • Goldowitz D.
      • Frappart L.
      • Ploubidou A.
      • Patel M.S.
      • et al.
      HMMR acts in the PLK1-dependent spindle positioning pathway and supports neural development.
      ,
      • Li H.
      • Kroll T.
      • Moll J.
      • Frappart L.
      • Herrlich P.
      • Heuer H.
      • Ploubidou A.
      Spindle misorientation of cerebral and cerebellar progenitors is a mechanistic cause of megalencephaly.
      ,
      • Long K.R.
      • Huttner W.B.
      How the extracellular matrix shapes neural development.
      ). In contrast to CD44, RHAMM is normally expressed in only a few tissues. However, RHAMM is considered a tumor-associated antigen, expression of which is increased during malignant cell transformation (
      • Wang C.
      • Thor A.D.
      • Moore D.H.
      • Zhao Y.
      • Kerschmann R.
      • Stern R.
      • Watson P.H.
      • Turley E.A.
      The overexpression of RHAMM, a hyaluronan-binding protein that regulates ras signaling, correlates with overexpression of mitogen-activated protein kinase and is a significant parameter in breast cancer progression.
      ,
      • Buttermore S.T.
      • Hoffman M.S.
      • Kumar A.
      • Champeaux A.
      • Nicosia S.V.
      • Kruk P.A.
      Increased RHAMM expression relates to ovarian cancer progression.
      ,
      • Kouvidi K.
      • Nikitovic D.
      • Berdiaki A.
      • Tzanakakis G.N.
      Hyaluronan/RHAMM interactions in mesenchymal tumor pathogenesis: role of growth factors.
      ). Particularly in GBM, RHAMM has been associated with an increase in migration and proliferation, and its levels have been correlated with tumor grade (
      • Lim E.J.
      • Suh Y.
      • Yoo K.C.
      • Lee J.H.
      • Kim I.G.
      • Kim M.J.
      • Chang J.H.
      • Kang S.G.
      • Lee S.J.
      Tumor-associated mesenchymal stem-like cells provide extracellular signaling cue for invasiveness of glioblastoma cells.
      ,
      • Virga J.
      • Bognár L.
      • Hortobágyi T.
      • Zahuczky G.
      • Cs É.
      Tumor grade versus expression of invasion-related molecules in astrocytoma.
      ,
      • Akiyama Y.
      • Jung S.
      • Salhia B.
      • Lee S.
      • Hubbard S.
      • Taylor M.
      • Mainprize T.
      • Akaishi K.
      • Van Furth W.
      • Rutka J.T.
      Hyaluronate receptors mediating glioma cell migration and proliferation.
      ,
      • Li J.
      • Zhou Y.
      • Wang H.
      • Gao Y.
      • Li L.
      • Hee S.
      COX-2/sEH dual inhibitor PTUPB suppresses glioblastoma growth by targeting epidermal growth factor receptor and hyaluronan mediated motility receptor.
      ). For example, in 2001, it was demonstrated that high-grade gliomas expressed higher levels of RHAMM and CD44 than low-grade lesions and that RHAMM inhibition hindered proliferation and migration of glioma cells, both in the presence and in the absence of HA-based ECM (
      • Akiyama Y.
      • Jung S.
      • Salhia B.
      • Lee S.
      • Hubbard S.
      • Taylor M.
      • Mainprize T.
      • Akaishi K.
      • Van Furth W.
      • Rutka J.T.
      Hyaluronate receptors mediating glioma cell migration and proliferation.
      ). It is worth noting that interpreting the effect of RHAMM inhibition in the absence of the HA-based ECM is complicated by the fact that HA can be secreted by GBM cells. Finally, in an in vivo xenograft model, it was demonstrated that RHAMM silencing reduced tumor formation and extended mouse survival time, while its overexpression enhanced tumor growth, compared with control (
      • Tilghman J.
      • Wu H.
      • Sang Y.
      • Shi X.
      • Guerrero-Cazares H.
      • Quinones-Hinojosa A.
      • Eberhart C.G.
      • Laterra J.
      • Ying M.
      HMMR maintains the stemness and tumorigenicity of glioblastoma stem-like cells.
      ). These results highlight the relevance of RHAMM, and presumably its ligand HA, in GBM progression.
      While these data establish the clear importance of CD44 and RHAMM in mediating HA’s functions, they are not the only biomolecules involved. As mentioned above, GBM is a heterogeneous disease. It has been linked to multiple oncogenic alterations such as mutations in tp53 and atrx (α-thalassemia/mental retardation X-linked syndrome) pten (phosphatase and tensin homologue) tert (telomerase reverse transcriptase) and h3f3a (histone H3.3) genes, codeletion of chromosome arms 1p and 19q, monosomy of chromosome 10, gains of chromosome 7, and egfr (epidermal growth factor receptor) and pdgfra (platelet-derived growth factor receptor-α) gene amplifications (
      • Waker C.A.
      • Lober R.M.
      Brain tumors of glial origin.
      ,
      • Korshunov A.
      • Casalini B.
      • Chavez L.
      • Hielscher T.
      • Sill M.
      • Ryzhova M.
      • Sharma T.
      • Schrimpf D.
      • Stichel D.
      • Capper D.
      • Reuss D.E.
      • Sturm D.
      • Absalyamova O.
      • Golanov A.
      • Lambo S.
      • et al.
      Integrated molecular characterization of IDH-mutant glioblastomas.
      ,
      • Weller M.
      • Wick W.
      • Aldape K.
      • Brada M.
      • Berger M.
      • Pfister S.M.
      • Nishikawa R.
      • Rosenthal M.
      • Wen P.Y.
      • Stupp R.
      • Reifenberger G.
      Glioma.
      ). Interestingly, the last two alterations have in turn been linked to changes in ECM composition. For instance, GBM tumors overexpressing PDGFRα showed elevated expression of chondroitin sulfate proteoglycan 4 (CSPG4/NG2), aggrecan, and extracellular sulfatase 2. In contrast, the GBM cases with EGFR amplification were associated with increased CSPG4/NG2 expression, but with diminished aggrecan and extracellular sulfatase 1 (
      • Wade A.
      • Robinson A.E.
      • Engler J.R.
      • Petritsch C.
      • James C.D.
      • Phillips J.J.
      Proteoglycans and their roles in brain cancer.
      ). Although aggrecan is able to bind HA, the direct relationship between these molecular alterations and HA levels in the ECM remains unknown and could be an interesting field to explore for GBM classification and treatment in the future.
      EGFR has been studied in the context of GBM separately (
      • An Z.
      • Aksoy O.
      • Zheng T.
      • Fan Q.W.
      • Weiss W.A.
      Epidermal growth factor receptor and EGFRvIII in glioblastoma: Signaling pathways and targeted therapies.
      ), but its association with the HA receptors could explain some of the effects of HA on GBM progression. For example, it was demonstrated that CD44 binds EGFR, and it was postulated that the complex may provide a mechanism for HA-mediated cell invasion and proliferation (
      • Tsatas D.
      • Kanagasundaram V.
      • Kaye A.
      • Novak U.
      EGF receptor modifies cellular responses to hyaluronan in glioblastoma cell lines.
      ). RHAMM is also known to associate with transmembrane receptors including CD44, CD44/EGFR, PDGFR, TGFβR-1, bFGFR, and RON (
      • Zhang S.
      • Chang M.C.Y.
      • Zylka D.
      • Turley S.
      • Harrison R.
      • Turley E.A.
      The hyaluronan receptor RHAMM regulates extracellular-regulated kinase.
      ,
      • Hamilton S.R.
      • Fard S.F.
      • Paiwand F.F.
      • Tolg C.
      • Veiseh M.
      • Wang C.
      • McCarthy J.B.
      • Bissell M.J.
      • Koropatnick J.
      • Turley E.A.
      The hyaluronan receptors CD44 and rhamm (CD168) form complexes with ERK1,2 that sustain high basal motility in breast cancer cells.
      ,
      • Hatano H.
      • Shigeishi H.
      • Kudo Y.
      • Higashikawa K.
      • Tobiume K.
      • Takata T.
      • Kamata N.
      RHAMM/ERK interaction induces proliferative activities of cementifying fibroma cells through a mechanism based on the CD44-EGFR.
      ,
      • Park D.
      • Kim Y.
      • Kim H.
      • Lee Y.
      • Choe J.
      • Hahn J.
      • Lee H.
      • Jeon J.
      • Choi C.
      • Kim Y.
      • Jeoung D.
      Hyaluronic acid promotes angiogenesis by inducing RHAMM-TGF β receptor interaction via CD44-PKC δ.
      ,
      • Savani R.C.
      • Cao G.
      • Pooler P.M.
      • Zaman A.
      • Zhou Z.
      • Delisser H.M.
      Differential involvement of the hyaluronan ( HA ) receptors CD44 and receptor for HA-mediated motility in endothelial cell function and angiogenesis.
      ,
      • Manzanares D.
      • Monzon M.
      • Savani R.C.
      • Salathe M.
      Apical oxidative hyaluronan degradation stimulates airway ciliary beating via RHAMM and RON.
      ). In this way, RHAMM can modulate the pathway associated with each one of these receptors and control the expression of genes involved in the cell cycle, influencing proliferation and cellular migration (
      • Wang C.
      • Thor A.D.
      • Moore D.H.
      • Zhao Y.
      • Kerschmann R.
      • Stern R.
      • Watson P.H.
      • Turley E.A.
      The overexpression of RHAMM, a hyaluronan-binding protein that regulates ras signaling, correlates with overexpression of mitogen-activated protein kinase and is a significant parameter in breast cancer progression.
      ,
      • Maxwell C.A.
      • McCarthy J.
      • Turley E.
      Cell-surface and mitotic-spindle RHAMM: Moonlighting or dual oncogenic functions?.
      ,
      • Buttermore S.T.
      • Hoffman M.S.
      • Kumar A.
      • Champeaux A.
      • Nicosia S.V.
      • Kruk P.A.
      Increased RHAMM expression relates to ovarian cancer progression.
      ,
      • Hamilton S.R.
      • Fard S.F.
      • Paiwand F.F.
      • Tolg C.
      • Veiseh M.
      • Wang C.
      • McCarthy J.B.
      • Bissell M.J.
      • Koropatnick J.
      • Turley E.A.
      The hyaluronan receptors CD44 and rhamm (CD168) form complexes with ERK1,2 that sustain high basal motility in breast cancer cells.
      ,
      • Twarock S.
      • Tammi M.
      • Savani R.C.
      • Fischer J.W.
      Hyaluronan stabilizes focal adhesions, filopodia, and the proliferative phenotype in esophageal squamous carcinoma cells.
      ). Therefore, it seems reasonable to think of these complexes as signaling transducers. In addition to these membrane interactions, CD44 has been shown to partner with the intracellular moesin, a protein that connects the actin cytoskeleton to the plasma membrane. This interaction occurs after HA treatment, enhancing U87MG and U373MG glioma cell migration and invasion (
      • DeSouza L.V.
      • Matta A.
      • Karim Z.
      • Mukherjee J.
      • Wang X.S.
      • Krakovska O.
      • Zadeh G.
      • Guha A.
      • Siu K.W.M.
      Role of moesin in hyaluronan induced cell migration in glioblastoma multiforme.
      ).
      Several other HA-binding proteins have also been identified. Jaworski et al. (
      • Jaworski D.M.
      • Kelly G.M.
      • Piepmeier J.M.
      • Hockfield S.
      BEHAB (brain enriched hyaluronan binding) is expressed in surgical samples of glioma and in intracranial grafts of invasive glioma cell lines.
      ) reported that the extracellular hyaluronan-binding protein known as brevican or brain-enriched HA binding (BEHAN) is consistently expressed by human gliomas and enhanced HA-mediated glioma invasion, especially when cleaved by disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) (
      • Zhang H.
      • Kelly G.
      • Zeritlo C.
      • Jaworski D.M.
      • Hockfield S.
      Expression of a cleaved brain-specific extracellular matrix protein mediates glioma cell invasion in vivo.
      ,
      • Matthews R.T.
      • Gary S.C.
      • Zerillo C.
      • Pratta M.
      • Solomon K.
      • Arner E.C.
      • Hockfield S.
      Brain-enriched hyaluronan binding (BEHAB)/brevican cleavage in a glioma cell line is mediated by a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family member.
      ). Finally, it was demonstrated that HA- and proteoglycan-link protein 4 (HAPLN4) increased adhesion and migration and even potentiated the motogenic effect of brevican on U251MG and U87MG cells (
      • Sim H.
      • Hu B.
      • Viapiano M.S.
      Reduced expression of the hyaluronan and proteoglycan link proteins in malignant gliomas.
      ).
      These combined results paint a picture of growing complexity, in which assigning specific GBM-promoting functions becomes scrambled by the multiple receptors, coreceptors, complexes, and regulatory factors potentially involved. However, HA emerges from this scrambled scenario as the key player that brings together the variety of effects observed in GBM cells, emphasizing the potential of HA as a target for improving GBM treatment. In addition, CD44 and RHAMM rise as the main players to watch over HA actions in the context of GBM. But how do CD44 and RHAMM perform this role? We next explore the downstream signaling pathways that execute HA-mediated functions.

      Signaling pathways involved in HA effects

      As discussed above, HA interacts with several receptors and coreceptors, potentially triggering a multitude of intracellular pathways (Fig. 3). For example, using the U87MG and SMA560 glioma cell lines, Tsatas et al. showed that treatment with HA (70 μg/ml) resulted in an increase in ERK1/2 phosphorylation in an EGFR-dependent manner. Conversely, the disruption of the CD44/EGFR complex reduces ERK1/2 activation in U87MG glioma cells (
      • Wakahara K.
      • Kobayashi H.
      • Yagyu T.
      • Matsuzaki H.
      • Kondo T.
      • Kurita N.
      • Sekino H.
      • Inagaki K.
      • Suzuki M.
      • Kanayama N.
      • Terao T.
      Bikunin down-regulates heterodimerization between CD44 and growth factor receptors and subsequently suppresses agonist-mediated signaling.
      ). The EGFR receptor is involved in the hyaluronan-induced expression of urokinase-type plasminogen activator (uPA), urokinase-type plasminogen activator receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1), tissue inhibitor of metalloproteinases (TIMP-1), and c-myc pathway (
      • Tsatas D.
      • Kanagasundaram V.
      • Kaye A.
      • Novak U.
      EGF receptor modifies cellular responses to hyaluronan in glioblastoma cell lines.
      ). All these molecules and signaling pathways were associated with an enhanced proliferation and migration in GBM.
      Figure thumbnail gr3
      Figure 3The scrambled relationship between GBM and HA. HA is accumulated in the GBM microenvironment. Additionally, GBM cells overexpress its receptors, CD44 and RHAMM, as well as tyrosine kinase receptors such as EGFR. In this context, the interaction of HA with CD44, RHAMM, probably in a complex with EGFR, and even TLR4 leads to an hyperactivation of signaling pathways such as MEK/ERK and PI3K/Akt and the overexpression of transcription factors, mainly NFkB. The final molecules of these signaling pathways along with NFkB impact on a transcriptional regulation enhancing all the malignant features of GBM, mostly proliferation, migration, and invasion but also chemoresistance. Moreover, the activation of these signaling pathways increases the activity of MMPs, which cleave CD44 and increase GBM migration and invasion. The tumor suppressor gene, PTEN, inhibits several of these pathways; however, almost 40% of GBM are PTEN-negative. Finally, the transcriptional regulation and, unfortunately, the radiation therapy enhance HAS activity, increasing the HA amount in the GBM microenvironment, leading to a positive feedback loop and favoring GBM progression. The complexity of this scrambled story hinders the understanding of HA effects on GBM cells, at the same time highlighting the potential of HA as a therapeutic target to improve the outcome of GBM patients.
      PTEN is a tumor-suppressor gene whose loss is extremely frequent in GBM (40%), which is correlated with enhanced proliferation as well as TMZ resistance (
      • Luongo F.
      • Colonna F.
      • Calapà F.
      • Vitale S.
      • Fiori M.E.
      • De Maria R.
      Pten tumor-suppressor: The dam of stemness in cancer.
      ). This correlation was explored in a study that expressed PTEN in U87MG cells lacking functional PTEN (
      • Park M.-J.J.
      • Kim M.-S.S.
      • Park I.-C.
      • Kang H.-S.
      • Yoo H.
      • Park S.H.
      • Rhee C.H.
      • Hong S.-I.I.
      • Lee S.-H.H.
      • Park M.-J.J.
      • Kim M.-S.S.
      • Rhee C.H.
      • Lee S.-H.H.
      PTEN suppresses hyaluronic acid-induced matrix metalloproteinase-9 expression in U87MG glioblastoma cells through focal adhesion kinase dephosphorylation.
      ). Treatment of these cells with 100 μg/ml HA significantly decreased the levels of MMP-9 and MMP-2, inhibiting the activation of ERK1/2 and focal adhesion kinase (FAK) while increasing the levels of tissue inhibitor of MMP-1 and MMP-2, which resulted in the inhibition of cell invasion (
      • Park M.-J.J.
      • Kim M.-S.S.
      • Park I.-C.
      • Kang H.-S.
      • Yoo H.
      • Park S.H.
      • Rhee C.H.
      • Hong S.-I.I.
      • Lee S.-H.H.
      • Park M.-J.J.
      • Kim M.-S.S.
      • Rhee C.H.
      • Lee S.-H.H.
      PTEN suppresses hyaluronic acid-induced matrix metalloproteinase-9 expression in U87MG glioblastoma cells through focal adhesion kinase dephosphorylation.
      ). In a second study, 200 μg/ml HA induced the expression of osteopontin, a protein related to GBM migration and invasion, through activation of the PI3K/Akt pathway in a PTEN-dependent manner (
      • Kim M.-S.
      • Park M.-J.
      • Moon E.-J.
      • Kim S.-J.
      • Lee C.-H.
      • Yoo H.
      • Shin S.-H.
      • Song E.-S.
      • Lee S.-H.
      Hyaluronic acid induces osteopontin via the phosphatidylinositol 3-kinase/Akt pathway to enhance the motility of human glioma cells.
      ). Finally, PTEN suppressed HA-induced miR-21 expression, an interference mRNA that increases the strength and duration of Ras/MAPK signaling, enhancing MMP-9 expression and glioma invasion by downregulation of Spry2 (
      • Kwak H.J.
      • Kim Y.J.
      • Chun K.R.
      • Woo Y.M.
      • Park S.J.
      • Jeong J.A.
      • Jo S.H.
      • Kim T.H.
      • Min H.S.
      • Chae J.S.
      • Choi E.J.
      • Kim G.
      • Shin S.H.
      • Gwak H.S.
      • Kim S.K.
      • et al.
      Downregulation of Spry2 by miR-21 triggers malignancy in human gliomas.
      ). Overall, the loss of PTEN seems to create an opportunity for the HA-stimulated ERK1/2 and PI3K/Akt signaling pathways to promote the levels of MMPs, which in turn promotes invasion and migration.
      Two studies converged on these same pathways from alternative starting points: Kim et al. (
      • Kim M.S.
      • Park M.J.
      • Kim S.J.
      • Lee C.H.
      • Yoo H.
      • Shin S.H.
      • Song E.S.
      • Lee S.H.
      Emodin suppresses hyaluronic acid-induced MMP-9 secretion and invasion of glioma cells.
      ) demonstrated that Emodin, an inhibitor of tyrosine kinase proteins, significantly inhibited HA-induced invasion and the secretion of MMP-2 and MMP-9 in U87MG cells via inhibition of FAK, ERK1/2, and Akt activation, as well as partial inhibition of two transcription factors, activator protein-1 (AP-1) and nuclear factor-κB (NF-κB). The same group showed that the inhibition of Hsp90 reduced HA-induced migration and invasion as well as MMP-9 secretion through FAK inhibition, which impedes NF-κB activation (
      • Kim M.S.
      • Kwak H.J.
      • Lee J.W.
      • Kim H.J.
      • Park M.J.
      • Park J.B.
      • Choi K.H.
      • Yoo H.
      • Shin S.H.
      • Shin W.S.
      • Song E.S.
      • Lee S.H.
      17-Allylamino-17-demethoxygeldanamycin down-regulates hyaluronic acid-induced glioma invasion by blocking matrix metalloproteinase-9 secretion.
      ). These studies thus add the FAK signaling pathway and transcription factor NF-κB as components relevant in the migration, invasion, and proliferation of GBM cells.
      Across all of these reports, MMPs are the common target where the signaling pathways converge. However, MMP-2 and MMP-9 are collagenases, and the brain ECM is mostly composed of HA, making it unclear how their enzymatic activity would effectively create openings in the matrix to facilitate migration/invasion. While this activity may be critical for neoangiogenesis (
      • Chintala S.K.
      • Tonn J.C.
      • Rao J.S.
      Matrix metalloproteinases and their biological function in human gliomas.
      ,
      • Kast R.E.
      • Halatsch M.
      Matrix metalloproteinase-2 and -9 in glioblastoma: A trio of old drugs — captopril, disulfiram and nelfinavir — are inhibitors with potential as adjunctive treatments in glioblastoma.
      ), the link to migration seems to be through an indirect route: Chetty et al. (
      • Chetty C.
      • Vanamala S.K.
      • Gondi C.S.
      • Dinh D.H.
      • Gujrati M.
      • Rao J.S.
      MMP-9 induces CD44 cleavage and CD44 mediated cell migration in glioblastoma xenograft cells.
      ) showed that MMP-9 induced cleavage of the extracellular domain of CD44 in xenograft glioma cell lines, promoting cell migration. Similarly, MT1-MMP, a membrane matrix collagenase, mediated CD44 cell surface cleavage through MEK/ERK and RhoA/ROK pathways, inducing detachment of the cell from the HA substrate and promoting the invasive potential of glioma cells (
      • Annabi B.
      • Thibeault S.
      • Moumdjian R.
      • Béliveau R.
      Hyaluronan cell surface binding is induced by type I collagen and regulated by caveolae in glioma cells.
      ,
      • Annabi B.
      • Bouzeghrane M.
      • Moumdjian R.
      • Moghrabi A.
      • Béliveau R.
      Probing the infiltrating character of brain tumors: Inhibition of RhoA/ROK-mediated CD44 cell surface shedding from glioma cells by the green tea catechin EGCg.
      ).
      Adding more complexity, MMPs are not the only triggers of CD44 cleavage. In fact, CD44 can promote its own cleavage: CD44 activation stimulates Rac-mediated cytoskeletal rearrangements that increase CD44 cleavage by disintegrin and metalloproteinase 10 (ADAM10), potentially contributing to the migration and invasion of U251MG cells (
      • Murai T.
      • Miyazaki Y.
      • Nishinakamura H.
      • Sugahara K.N.
      • Miyauchi T.
      • Sako Y.
      • Yanagida T.
      • Miyasaka M.
      Engagement of CD44 promotes Rac activation and CD44 cleavage during tumor cell migration.
      ). Modifications to membrane composition through short-term cholesterol depletion similarly augmented CD44 shedding mediated by ADAM10 and caused changes in CD44 localization, while long-term cholesterol reduction suppressed the CD44 cleavage-induced cell migration on a hyaluronan-coated substrate (
      • Murai T.
      • Maruyama Y.
      • Mio K.
      • Nishiyama H.
      • Suga M.
      • Sato C.
      Low cholesterol triggers membrane microdomain-dependent CD44 shedding and suppresses tumor cell migration.
      ). Interestingly, Lamontagne and Grandbois demonstrated that unstimulated U373MG cells interact with HA through CD44 receptors across their entire surface, while in protein kinase C-activated cells, the interactions were localized at the leading edge of the cells (
      • Lamontagne C.A.
      • Grandbois M.
      PKC-induced stiffening of hyaluronan/CD44 linkage; local force measurements on glioma cells.
      ). These results suggest that not only changes in CD44 expression levels but also even its localization is important in the process of glioma cell migration.
      These studies collectively demonstrate a role for CD44 in HA signaling. RHAMM has been less studied than CD44, but we suspect that this receptor and downstream signaling pathways are similarly important for HA-mediated GBM progression and would benefit from further investigation. Finally, we must consider the nature of the ligand that binds to these receptors. How does the 3D structure of HA, which remains not fully clarified, determine its function?

      Mimicking the brain: How a complex environment impacts GBM biology

      As discussed above, extracellular HA forms a functional scaffold for other ECM components, which can be extended via extrusion of additional HA by hyaluronic acid synthases to form protective halos or other structures. The molecular mass of the HA polymer affects the number of cross-links that can be formed and the HA saccharides available for receptor binding and thus is a critical factor in its actions, as shown on a GBM cell line (
      • Tian C.
      • Asghar S.
      • Xu Y.
      • Chen Z.
      • Zhang M.
      • Huang L.
      • Ye J.
      • Ping Q.
      • Xiao Y.
      The effect of the molecular weight of hyaluronic acid on the physicochemical characterization of hyaluronic acid-curcumin conjugates and in vitro evaluation in glioma cells.
      ,
      • Chen J.W.E.
      • Pedron S.
      • Shyu P.
      • Hu Y.
      • Sarkaria J.N.
      • Harley B.A.C.
      Influence of hyaluronic acid transitions in tumor microenvironment on glioblastoma malignancy and invasive behavior.
      ). For example, in a cancer context, HMW-HA accumulation is frequently associated with cancer progression, whereas LMW-HA promotes the development of the tumor and o-HA attenuates its development. These disparities are explained by the different manners of interaction with the HA receptors, CD44 and RHAMM (
      • Tavianatou A.G.
      • Caon I.
      • Franchi M.
      • Piperigkou Z.
      • Galesso D.
      • Karamanos N.K.
      Hyaluronan: Molecular size-dependent signaling and biological functions in inflammation and cancer.
      ). Unfortunately, most HA research usually fails to report such data, which hinders the general conclusions that can be drawn about HA’s effects. Moreover, exploring these different molecular sizes, while of value, does not necessarily provide insights into the true tumor microenvironment. Actually, the fact that HA is organized into a 3D structure in the brain ECM suggests a complex scenario that may be difficult to reproduce in vitro by the exogenous addition of soluble HA limiting our ability to fully interrogate HA-mediated mechanisms. Data using HA hydrogels have provided an important entry into this area. Indeed, 3D culture systems have been used to reproduce cancer cell behavior more faithfully than common 2D cultures of various malignancies including GBM (
      • Munson J.M.
      • Bellamkonda R.V.
      • Swartz M.A.
      Interstitial flow in a 3D microenvironment increases glioma invasion by a CXCR4-dependent mechanism.
      ,
      • Rape A.
      • Ananthanarayanan B.
      • Kumar S.
      Engineering strategies to mimic the glioblastoma microenvironment.
      ,
      • Haring A.P.
      • Thompson E.G.
      • Tong Y.
      • Laheri S.
      • Cesewski E.
      • Sontheimer H.
      • Johnson B.N.
      Process- and bio-inspired hydrogels for 3D bioprinting of soft free-standing neural and glial tissues.
      ). Their use in preclinical assays has been proposed as they show better conservation of real growth and sensitivity to drugs and radiation than 2D cultures (
      • Jiguet Jiglaire C.
      • Baeza-Kallee N.
      • Denicolaï E.
      • Barets D.
      • Metellus P.
      • Padovani L.
      • Chinot O.
      • Figarella-Branger D.
      • Fernandez C.
      Ex vivo cultures of glioblastoma in three-dimensional hydrogel maintain the original tumor growth behavior and are suitable for preclinical drug and radiation sensitivity screening.
      ,
      • Hermida M.A.
      • Kumar J.D.
      • Schwarz D.
      • Laverty K.G.
      • Di Bartolo A.
      • Ardron M.
      • Bogomolnijs M.
      • Clavreul A.
      • Brennan P.M.
      • Wiegand U.K.
      • Melchels F.P.
      • Shu W.
      • Leslie N.R.
      Three dimensional in vitro models of cancer: Bioprinting multilineage glioblastoma models.
      ,
      • Florczyk S.J.
      • Wang K.
      • Jana S.
      • Wood D.L.
      • Sytsma S.K.
      • Sham J.
      • Kievit F.M.
      • Miqin Z.
      Porous chitosan-hyaluronic acid scaffolds as a mimic of glioblastoma microenvironment ECM.
      ,
      • Ngo M.T.
      • Harley B.A.
      The influence of hyaluronic acid and glioblastoma cell coculture on the formation of endothelial cell networks in gelatin hydrogels.
      ).
      The first report using HA hydrogel to mimic the brain ECM was published in 1997 (
      • Tamaki M.
      • McDonald W.
      • Amberger V.R.
      • Moore E.
      • Del Maestro R.F.
      Implantation of C6 astrocytoma spheroid into collagen type I gels: Invasive, proliferative, and enzymatic characterizations.
      ) and was followed by several studies, which centered their attention on the differences in the stiffness and the elastic modulus of the hydrogel rather than on the effect of HA per se (
      • Coquerel B.
      • Poyer F.
      • Torossian F.
      • Dulong V.
      • Bellon G.
      • Dubus I.
      • Reber A.
      • Vannier J.-P.
      Elastin-derived peptides: Matrikines critical for glioblastoma cell aggressiveness in a 3-D system.
      ,
      • Ananthanarayanan B.
      • Kim Y.
      • Kumar S.
      Elucidating the mechanobiology of malignant brain tumors using a brain matrix-mimetic hyaluronic acid hydrogel platform.
      ,
      • Lee K.H.
      • Lee K.H.
      • Lee J.
      • Choi H.
      • Lee D.
      • Park Y.
      • Lee S.H.
      Integration of microfluidic chip with biomimetic hydrogel for 3D controlling and monitoring of cell alignment and migration.
      ,
      • Kingsmore K.M.
      • Logsdon D.K.
      • Floyd D.H.
      • Peirce S.M.
      • Purow B.W.
      • Munson J.M.
      Interstitial flow differentially increases patient-derived glioblastoma stem cell invasion via CXCR4, CXCL12, and CD44-mediated mechanisms.
      ,
      • Heffernan J.M.
      • Overstreet D.J.
      • Le L.D.
      • Vernon B.L.
      • Sirianni R.W.
      Bioengineered scaffolds for 3D analysis of glioblastoma proliferation and invasion.
      ,
      • Wang C.
      • Tong X.
      • Yang F.
      Bioengineered 3D brain tumor model to elucidate the effects of matrix stiffness on glioblastoma cell behavior using peg-based hydrogels.
      ,
      • Erickson A.E.
      • Lan Levengood S.K.
      • Sun J.
      • Chang F.-C.
      • Zhang M.
      Fabrication and characterization of chitosan- hyaluronic acid scaffolds with varying stiffness for glioblastoma cell culture.
      ,
      • Wang K.
      • Kievit F.M.
      • Erickson A.E.
      • Silber J.R.
      • Ellenbogen R.G.
      • Zhang M.
      Culture on three-dimensional chitosan-hyaluronic acid scaffolds enhances stem cell marker expression and drug resistance in human glioblastoma cancer stem cells.
      ,
      • Huang Y.J.
      • Hoffmann G.
      • Wheeler B.
      • Schiapparelli P.
      • Quinones-Hinojosa A.
      • Searson P.
      Cellular microenvironment modulates the galvanotaxis of brain tumor initiating cells.
      ,
      • Rape A.D.
      • Zibinsky M.
      • Murthy N.
      • Kumar S.
      A synthetic hydrogel for the high-throughput study of cell-ECM interactions.
      ,
      • Pogoda K.
      • Bucki R.
      • Byfield F.J.
      • Cruz K.
      • Lee T.
      • Marcinkiewicz C.
      • Janmey P.A.
      Soft substrates containing hyaluronan mimic the effects of increased stiffness on morphology, motility, and proliferation of glioma cells.
      ,
      • Simon T.
      • Coquerel B.
      • Petit A.
      • Kassim Y.
      • Demange E.
      • Le Cerf D.
      • Perrot V.
      • Vannier J.P.
      Direct effect of bevacizumab on glioblastoma cell lines in vitro.
      ). In the first studies in which HA was used to stimulate glioma cell migration in a complex 3D approach, the authors reported that the effect might be indirect and partially due to gel polymerization and network structure (
      • Hayen W.
      • Goebeler M.
      • Kumar S.
      • Rießen R.
      • Nehls V.
      Hyaluronan stimulates tumor cell migration by modulating the fibrin fiber architecture.
      ,
      • Yang Y. li
      • Sun C.
      • Wilhelm M.E.
      • Fox L.J.
      • Zhu J.
      • Kaufman L.J.
      Influence of chondroitin sulfate and hyaluronic acid on structure, mechanical properties, and glioma invasion of collagen I gels.
      ,
      • Jin S.G.
      • Jeong Y. Il
      • Jung S.
      • Ryu H.H.
      • Jin Y.H.
      • Kim I.Y.
      The effect of hyaluronic acid on the invasiveness of malignant glioma cells: Comparison of invasion potential at hyaluronic acid hydrogel and Matrigel.
      ). These combined studies demonstrated the importance of considering the maintenance of the elastic modulus, porosity (
      • Ananthanarayanan B.
      • Kim Y.
      • Kumar S.
      Elucidating the mechanobiology of malignant brain tumors using a brain matrix-mimetic hyaluronic acid hydrogel platform.
      ,
      • Heffernan J.M.
      • Overstreet D.J.
      • Le L.D.
      • Vernon B.L.
      • Sirianni R.W.
      Bioengineered scaffolds for 3D analysis of glioblastoma proliferation and invasion.
      ,
      • Wang C.
      • Tong X.
      • Yang F.
      Bioengineered 3D brain tumor model to elucidate the effects of matrix stiffness on glioblastoma cell behavior using peg-based hydrogels.
      ,
      • Erickson A.E.
      • Lan Levengood S.K.
      • Sun J.
      • Chang F.-C.
      • Zhang M.
      Fabrication and characterization of chitosan- hyaluronic acid scaffolds with varying stiffness for glioblastoma cell culture.
      ,
      • Wang K.
      • Kievit F.M.
      • Erickson A.E.
      • Silber J.R.
      • Ellenbogen R.G.
      • Zhang M.
      Culture on three-dimensional chitosan-hyaluronic acid scaffolds enhances stem cell marker expression and drug resistance in human glioblastoma cancer stem cells.
      ,
      • Huang Y.J.
      • Hoffmann G.
      • Wheeler B.
      • Schiapparelli P.
      • Quinones-Hinojosa A.
      • Searson P.
      Cellular microenvironment modulates the galvanotaxis of brain tumor initiating cells.
      ,
      • Rape A.D.
      • Zibinsky M.
      • Murthy N.
      • Kumar S.
      A synthetic hydrogel for the high-throughput study of cell-ECM interactions.
      ,
      • Yang Y. li
      • Sun C.
      • Wilhelm M.E.
      • Fox L.J.
      • Zhu J.
      • Kaufman L.J.
      Influence of chondroitin sulfate and hyaluronic acid on structure, mechanical properties, and glioma invasion of collagen I gels.
      ), and composition of HA hydrogels as compared with HA-free hydrogels when assessing cell features in complex environments (
      • Koh I.
      • Cha J.
      • Park J.
      • Choi J.
      • Kang S.G.
      • Kim P.
      The mode and dynamics of glioblastoma cell invasion into a decellularized tissue-derived extracellular matrix-based three-dimensional tumor model.
      ). Comparison of several recent studies provides an excellent demonstration of these aspects. First, two studies showed that GBM cell migration would be inversely proportional to HA; however, in one of them, the elastic modulus and porosity of HA hydrogels varied with the amount of HA added to the hydrogel (
      • Rao S.S.
      • DeJesus J.
      • Short A.R.
      • Otero J.J.
      • Sarkar A.
      • Winter J.O.
      Glioblastoma behaviors in three-dimensional collagen- hyaluronan composite hydrogels.
      ), while in the other study, N,N-dimethylformamide was used in the HA hydrogel but not in the other scaffolds, potentially hindering clear conclusions (
      • Rao S.S.
      • Nelson M.T.
      • Xue R.
      • DeJesus J.K.
      • Viapiano M.S.
      • Lannutti J.J.
      • Winter J.O.
      • Lowrie W.G.
      • Sarkar A.
      • Winter J.O.
      Mimicking white matter tract topography using core-shell electrospun nanofibers to examine migration of malignant brain tumors.
      ,
      • Li X.N.
      • Du Z.W.
      • Huang Q.
      • Wu J.Q.
      Growth-inhibitory and differentiation-inducing activity of dimethylformamide in cultured human malignant glioma cells.
      ). Other reports that controlled for stiffness and porosity of the gels with and without HA showed that HMW-HA increased migration and haptotaxis of glioma cells (
      • Serres E.
      • Debarbieux F.
      • Stanchi F.
      • Maggiorella L.
      • Grall D.
      • Turchi L.
      • Burel-Vandenbos F.
      • Figarella-Branger D.
      • Virolle T.
      • Rougon G.
      • Van Obberghen-Schilling E.
      Fibronectin expression in glioblastomas promotes cell cohesion, collective invasion of basement membrane in vitro and orthotopic tumor growth in mice.
      ,
      • Logun M.T.
      • Bisel N.S.
      • Tanasse E.A.
      • Zhao W.
      • Gunasekera B.
      • Mao L.
      • Karumbaiah L.
      Glioma cell invasion is significantly enhanced in composite hydrogel matrices composed of chondroitin 4- and 4,6-sulfated glycosaminoglycans.
      ,
      • Gritsenko P.
      • Leenders W.
      • Friedl P.
      Recapitulating in vivo-like plasticity of glioma cell invasion along blood vessels and in astrocyte-rich stroma.
      ). Reinforcing these findings, it was demonstrated that the addition of HMW-HA to hydrogels increased the proliferation and migration of patient-derived GBM stem cells (GSCs) and the expression of epithelial-to-mesenchymal transition (EMT)-related genes on U87MG GBM cells, enhancing their in vivo tumorigenic ability, with respect to cells cultured in scaffolds without HA (
      • Cha J.
      • Kang S.G.
      • Kim P.
      Strategies of mesenchymal invasion of patient-derived brain tumors: Microenvironmental adaptation.
      ,
      • Wang X.
      • Dai X.
      • Zhang X.
      • Li X.
      • Xu T.
      • Lan Q.
      Enrichment of glioma stem cell-like cells on 3D porous scaffolds composed of different extracellular matrix.
      ). In accordance, another work reported that U87MG cells in the presence of 3D hydrogels containing 1500 to 1800 kDa HA showed characteristics resembling the stem cell phenotype, including an increased expression of the markers RHAMM and CD133, compared with hydrogels without HA (
      • Martínez-Ramos C.
      • Lebourg M.
      Three-dimensional constructs using hyaluronan cell carrier as a tool for the study of cancer stem cells.
      ).
      With regard to the importance of the molecular mass of HA in its effect, it was shown that patient-derived xenograft cells cultured in hydrogels containing 500 kDa HA showed less invasiveness than those in hydrogels containing 10 or 60 kDa HA (
      • Chen J.W.E.
      • Pedron S.
      • Shyu P.
      • Hu Y.
      • Sarkaria J.N.
      • Harley B.A.C.
      Influence of hyaluronic acid transitions in tumor microenvironment on glioblastoma malignancy and invasive behavior.
      ). However, a separate study demonstrated that the addition of HA (60 kDa) to the hydrogel did not affect metabolic activity but reduced invasiveness in U251MG cells (
      • Chen E.J.-W.
      • Pedron S.
      • Harley B.A.C.
      The combined influence of hydrogel stiffness and matrix-bound hyaluronic acid content on glioblastoma InvasionPublic access.
      ). Overall, these data suggest that, when the stiffness and porosity of 3D structures are considered, the effect of HA is similar to that seen in assays using soluble HA, enhancing GBM proliferation and migration. However, the response of GBM cells depends not only on the model but also on HA concentration and molecular mass.
      HA hydrogel stiffness also impacts adhesion and migration speed, implying that CD44 signaling is mechanosensitive (
      • Kim Y.
      • Kumar S.
      CD44-mediated adhesion to hyaluronic acid contributes to mechanosensing and invasive motility.
      ) and pointing to additional biomolecules involved in mediating HA activity: CD44 suppression in U373MG and U87MG human GBM cells reduces cell adhesion to HA at short times (0.5 h) while maximal adhesion at 3 h requires both CD44 and integrins. In another study using U87MG cells, the presence of 1630 kDa HA in gelatin or polyethyleneglycol gels also caused cluster growth and modified the expression of fibronectin, MMP-2, MMP-9, vascular endothelial growth factor (VEGF), and hypoxia inducible factor 1 (HIF-1) depending on EGFR status and HA concentration (
      • Pedron S.
      • Harley B.A.C.
      Impact of the biophysical features of a 3D gelatin microenvironment on glioblastoma malignancy.
      ). A second report echoed these conclusions, finding that matrix-bound HA enhances metabolic activity and proliferation depending on the EGFR status of U87MG cells and favors invasion under hypoxia (
      • Chen J.W.E.
      • Lumibao J.
      • Blazek A.
      • Gaskins H.R.
      • Harley B.
      Hypoxia activates enhanced invasive potential and endogenous hyaluronic acid production by glioblastoma cells.
      ). Though both VEGF and HIF-1 were already known to be involved in the malignancy of GBM, these reports help to clarify a possible mechanism for their role, connecting HA to EGFR, probably in a complex with CD44 or RHAMM, and thus to an increase in HIF-1 and VEGF levels.
      Using 3D conditions has also revealed new details regarding the role of HA in drug resistance. A HA/CD44/PI3K/Akt axis has previously been proposed as a pathway involved in drug resistance in several types of tumors (
      • Misra S.
      • Ghatak S.
      • Toole B.P.
      Regulation of MDR1 expression and drug resistance by a positive feedback loop involving hyaluronan, phosphoinositide 3-kinase, and ErbB2.
      ,
      • Torre C.
      • Wang S.J.
      • Xia W.
      • Bourguignon L.Y.W.
      Reduction of hyaluronan-CD44-mediated growth, migration, and cisplatin resistance in head and neck cancer due to inhibition of Rho kinase and PI-3 kinase signaling.
      ,
      • Kashyap T.
      • Pramanik K.K.
      • Nath N.
      • Mishra P.
      • Singh A.K.
      • Nagini S.
      • Rana A.
      • Mishra R.
      Crosstalk between Raf-MEK-ERK and PI3K-Akt-GSK3β signaling networks promotes chemoresistance, invasion/migration and stemness via expression of CD44 variants (v4 and v6) in oral cancer.
      ,
      • Hao J.
      • Madigan M.C.
      • Khatri A.
      • Power C.A.
      • Hung T.T.
      • Beretov J.
      • Chang L.
      • Xiao W.
      • Cozzi P.J.
      • Graham P.H.
      • Kearsley J.H.
      • Li Y.
      In vitro and in vivo prostate cancer metastasis and chemoresistance can be modulated by expression of either CD44 or CD147.
      ). Recent evidence has extended this mechanism to GBM. Specifically, it was shown that the addition of 1630 kDa HA to hydrogels boosted metabolic activity of patient-derived xenograft cells (
      • Pedron S.
      • Hanselman J.S.
      • Schroeder M.A.
      • Sarkaria J.N.
      • Harley B.A.C.
      Extracellular hyaluronic acid influences the efficacy of EGFR tyrosine kinase inhibitors in a biomaterial model of glioblastoma.
      ). In the same work, it was shown that HA upregulated genes associated with matrix remodeling and tumor growth generating resistance to the EGFR inhibitor erlotinib depending on CD44/PI3K axis. However, in cells expressing EGFRvIII, a constitutively activated mutant of EGFR, these effects remain unaltered despite CD44 blocking, thus suggesting the implication of other pathways activated in EGFRvIII within HA matrices (
      • Pedron S.
      • Hanselman J.S.
      • Schroeder M.A.
      • Sarkaria J.N.
      • Harley B.A.C.
      Extracellular hyaluronic acid influences the efficacy of EGFR tyrosine kinase inhibitors in a biomaterial model of glioblastoma.
      ). Besides, in a HA hydrogel, CD44 knockdown restored the cytostatic and cytotoxic effects of erlotinib on resistant GBM cells. However, GBM cells recovered resistance after 12 days. Under such conditions, the authors reported an RHAMM expression pattern resembling that of CD44, which would be able to compensate the resistance in the absence of CD44 (
      • Xiao W.
      • Zhang R.
      • Sohrabi A.
      • Ehsanipour A.
      • Sun S.
      • Liang J.
      • Walthers C.
      • Ta L.
      • Nathanson D.A.
      • Seidits S.K.
      Brain-mimetic 3D culture platforms allow investigation of cooperative effects of extracellular matrix features on therapeutic resistance in glioblastoma.
      ). In fact, RHAMM has been implicated in drug resistance in other tumors, through activation of signaling pathways such as PI3K/Akt and TGFb/Smad2 (
      • Lompardía S.L.
      • Papademetrio D.L.
      • Mascaró M.
      • Del Carmen Álvarez E.M.
      • Hajos S.E.
      Human leukemic cell lines synthesize hyaluronan to avoid senescence and resist chemotherapy.
      ,
      • Zhang H.
      • Ren L.
      • Ding Y.
      • Li F.
      • Chen X.
      • Ouyang Y.
      • Zhang Y.
      • Zhang D.
      Hyaluronan-mediated motility receptor confers resistance to chemotherapy via TGFβ/Smad2-induced epithelial-mesenchymal transition in gastric cancer.
      ,
      • Korkes F.
      • De Castro M.G.
      • De Cassio Zequi S.
      • Nardi L.
      • Del Giglio A.
      • De Lima Pompeo A.C.
      Hyaluronan-mediated motility receptor (RHAMM) immunohistochemical expression and androgen deprivation in normal peritumoral, hyperplasic and neoplastic prostate tissue.
      ). Thus, its study as well as the pathways that trigger would expand our understanding of HA-mediated drug resistance and would be an interesting starting point for future investigations on GBM therapy.
      Overall, the effects of HA observed in 3D assays are certainly similar to those reported using 2D culture approaches in which soluble HA is added to the culture medium, including the same receptors and signaling pathways. However, invasive features are dependent on the composition, elastic modulus, and porosity of hydrogels, and groups that have not considered these variables have reported controversial HA effects regarding those features. Moreover, 3D cultures enable evaluations of additional aspects of GBM biology, such as the mechanosensitive CD44-driven effects. Future research will benefit from additional reporting of hydrogel preparations, ingredients, and material properties, and for conclusions drawn to carefully consider whether outcomes can be linked to the effect of one component of the hydrogels. Increasing incorporation of advanced models for GBM will bring us closer to understanding the complicated details of tumor development, which should similarly increase our ability to diagnose and treat this disease.

      Hyaluronan and its metabolism as a molecular marker for glioblastoma

      Given the effects of HA on GBM biology, an obvious question is whether HA could be used as a biomarker for this cancer type. Indeed, it was revealed that the quantity of GAGs in malignant tissue samples, particularly in GBM, was considerably higher than that of nontumor specimens (
      • Bertolotto A.
      • Giordana M.T.
      • Magrassi M.L.
      • Mauro A.
      • Schiffer D.
      Glycosaminoglycans (GASs) in human cerebral tumors - part 1. Biochemical findings.
      ). In addition, Delpech et al. (
      • Delpech B.
      • Maingonnat C.
      • Girard N.
      • Chauzy C.
      • Olivier A.
      • Maunoury R.
      • Tayot J.
      • Creissard P.
      Hyaluronan and hyaluronectin in the extracellular matrix of human brain tumour stroma.
      ) showed that in glial tumors the HA content was much higher than in adult normal brains, mainly due to increased quantities of HMW-HA. HA content was also higher in GBM tissue than in lung carcinoma metastases (
      • Varga I.
      • Hutóczki G.
      • Petrás M.
      • Scholtz B.
      • Mikó E.
      • Kenyeres A.
      • Tóth J.
      • Zahuczky G.
      • Bognár L.
      • Hanzély Z.
      • Klekner A.
      Expression of invasion-related extracellular matrix molecules in human glioblastoma versus intracerebral lung adenocarcinoma metastasis.
      ). However, the proportion of HA in comparison to other GAGs was lower in GBM than in other low-grade gliomas (
      • Bertolotto A.
      • Giordana M.T.
      • Magrassi M.L.
      • Mauro A.
      • Schiffer D.
      Glycosaminoglycans (GASs) in human cerebral tumors - part 1. Biochemical findings.
      ), and it was even shown that the concentration of mucopolysaccharide acid, particularly HA, was inversely associated with glioma grade (
      • Engelhardt A.
      Detection of acid mucopolysaccharides in human brain tumors by histochemical methods.
      ,
      • Sadeghi N.
      • Camby I.
      • Goldman S.
      • Gabius H.J.
      • Balériaux D.
      • Salmon I.
      • Decaesteckere C.
      • Kiss R.
      • Metens T.
      Effect of hydrophilic components of the extracellular matrix on quantifiable diffusion-weighted imaging of human gliomas: Preliminary results of correlating apparent diffusion coefficient values and hyaluronan expression level.
      ). It is worth noting that the molecular mass of HA might be underestimated in these reports, which would be an important consideration for further analysis. For now, however, it seems unlikely that HA concentrations could be used for diagnosis. Could its localization point to clusters of GBM cells?
      HA is found both within and surrounding the tumor in an intracranial mouse model (
      • Xiao W.
      • Zhang R.
      • Sohrabi A.
      • Ehsanipour A.
      • Sun S.
      • Liang J.
      • Walthers C.
      • Ta L.
      • Nathanson D.A.
      • Seidits S.K.
      Brain-mimetic 3D culture platforms allow investigation of cooperative effects of extracellular matrix features on therapeutic resistance in glioblastoma.
      ). Although the GBM parenchyma was slightly stained for GAGs in human samples, it was observed that the infiltrated cortex areas exhibited strong staining for HA and CS, suggesting a heterogeneous distribution (
      • Giordana M.T.
      • Bertolotto A.
      • Mauro A.
      • Migheli A.
      • Pezzotta S.
      • Racagni G.
      • Schiffer D.
      Glycosaminoglycans in human cerebral tumors - part II. Histochemical findings and correlations.
      ). Likewise, it was demonstrated that HA was localized in pericellular and perivascular areas, suggesting that both cancer cells and tumor-associated vascular cells secrete it (
      • Delpech B.
      • Maingonnat C.
      • Girard N.
      • Chauzy C.
      • Olivier A.
      • Maunoury R.
      • Tayot J.
      • Creissard P.
      Hyaluronan and hyaluronectin in the extracellular matrix of human brain tumour stroma.
      ). It would be interesting to determine plasma levels of HA and their relation, if any, with tumor stage. However, the current data suggest that an alternative molecule is needed.
      The observed rise in HA levels in brain tumors was accompanied by variations in the content and distribution of other HA metabolism-associated proteins. For example, both HA and RHAMM were found to be elevated in the tumor parenchyma, particularly in the invasive front, suggesting their crucial role in GBM invasiveness (
      • Akiyama Y.
      • Jung S.
      • Salhia B.
      • Lee S.
      • Hubbard S.
      • Taylor M.
      • Mainprize T.
      • Akaishi K.
      • Van Furth W.
      • Rutka J.T.
      Hyaluronate receptors mediating glioma cell migration and proliferation.
      ). Considering that both RHAMM and CD44 are involved in HA-mediated invasion in GBM, it would be expected these receptors to be highly expressed in the migratory edge. In agreement with this, it was reported that CD44 expression was augmented in the glioma–brain interface of a G26 glioma-bearing mouse model, with single infiltrating CD44-enriched cells escaping into the brain parenchyma (
      • Wiranowska M.
      • Ladd S.
      • Smith S.R.
      • Gottschall P.E.
      CD44 adhesion molecule and neuro-glial proteoglycan NG2 as invasive markers of glioma.
      ) (Fig. 2). Strangely, although both HA and CD44 levels were increased in human and rat GBM samples, they showed an opposite distribution, with CD44-enriched regions overlapping with lower HA content (
      • Wang H.H.
      • Liao C.C.
      • Chow N.H.
      • Huang L.L.H.
      • Chuang J.I.
      • Wei K.C.
      • Shin J.W.
      Whether CD44 is an applicable marker for glioma stem cells.
      ). This discrepancy might make it difficult to interpret diagnostic readings.
      Another obvious set of candidates are the enzymes responsible for the synthesis and degradation of HA, as these are primarily responsible for controlling HA levels (
      • Jiang D.
      • Liang J.
      • Noble P.W.
      Hyaluronan as an immune regulator in human diseases.
      ,
      • Hascall V.C.
      • Wang A.
      • Tammi M.
      • Oikari S.
      • Tammi R.
      • Passi A.
      • Vigetti D.
      • Hanson R.W.
      • Hart G.W.
      The dynamic metabolism of hyaluronan regulates the cytosolic concentration of UDP-GlcNAc.
      ,
      • Csoka A.B.
      • Stern R.
      Hypotheses on the evolution of hyaluronan: A highly ironic acid.
      ). HA is synthesized by hyaluronic acid synthases (HASs), which extrude the polymer to the extracellular environment as is produced. Three isoforms of these proteins have been reported, HAS1–3, each with distinct functions (
      • Karbownik M.S.
      • Nowak J.Z.
      Hyaluronan: Towards novel anti-cancer therapeutics.
      ,
      • Vigetti D.
      • Karousou E.
      • Viola M.
      • Deleonibus S.
      • De Luca G.
      • Passi A.
      Hyaluronan: Biosynthesis and signaling.
      ). HA degradation, where the most stringent regulation occurs, is performed by the hyaluronidases (HYALs). There are six HYAL-like genes in the human genome: hyal-1–4, ph-20, and the pseudogene pHyal1 (
      • Csoka A.B.
      • Frost G.I.
      • Stern R.
      The six hyaluronidase-like genes in the human and mouse genomes.
      ,
      • Stern R.
      • Jedrzejas M.J.
      The hyaluronidases: Their genomics, structures, and mechanisms of action.
      ). Similar to the HASs, each HYAL has differential effects on HA (
      • Karbownik M.S.
      • Nowak J.Z.
      Hyaluronan: Towards novel anti-cancer therapeutics.
      ,
      • Csoka A.B.
      • Frost G.I.
      • Stern R.
      The six hyaluronidase-like genes in the human and mouse genomes.
      ,
      • Stern R.
      • Jedrzejas M.J.
      The hyaluronidases: Their genomics, structures, and mechanisms of action.
      ,
      • Bourguignon V.
      • Flamion B.
      Respective roles of hyaluronidases 1 and 2 in endogenous hyaluronan turnover.
      ,
      • Buhren B.A.
      • Schrumpf H.
      • Hoff N.P.
      • Bölke E.
      • Hilton S.
      • Gerber P.A.
      Hyaluronidase: From clinical applications to molecular and cellular mechanisms.
      ). Several studies have examined the possible correlation of HASs or HYALs with GBM prognosis. For example, a connection has been observed between the expression of HAS2 and GBM (
      • Varga I.
      • Hutóczki G.
      • Petrás M.
      • Scholtz B.
      • Mikó E.
      • Kenyeres A.
      • Tóth J.
      • Zahuczky G.
      • Bognár L.
      • Hanzély Z.
      • Klekner A.
      Expression of invasion-related extracellular matrix molecules in human glioblastoma versus intracerebral lung adenocarcinoma metastasis.
      ). Valkonen et al. recently extended these results, reporting tissue microarray data showing that HA and HAS1-3, the HYALs, and CD44 were highly expressed not only in malignant tissue, but also in adjacent gliotic cerebral tissue of grades II to IV diffusely infiltrating astrocytomas. In this study, although the HA content did not exhibit a prognostic value, an association between tumor grade and HAS1, HAS2, and HYAL-2 was observed, and an increase in HAS2 was also correlated with a poor prognosis of the patients (
      • Valkonen M.
      • Haapasalo H.
      • Rilla K.
      • Tyynelä-Korhonen K.
      • Soini Y.
      • Pasonen-Seppänen S.
      Elevated expression of hyaluronan synthase 2 associates with decreased survival in diffusely infiltrating astrocytomas.
      ). Unexpectedly, HAS2 overexpression in GBM cells abolished tumor formation in the brain in a mouse model; the authors proposed that this was due to a lack of HYAL activity, suggesting that gliomas may be more aggressive when coexpressing HAS and HYALs (
      • Enegd B.
      • King J.A.J.
      • Stylli S.
      • Paradiso L.
      • Kaye A.H.
      • Novak U.
      • Enam S.A.
      • Piepmeier J.M.
      Overexpression of hyaluronan synthase-2 reduces the tumorigenic potential of glioma cells lacking hyaluronidase activity.
      ).
      These data suggest that HA metabolism seems to be crucial in GBM, and HA degradation could be more relevant than HA synthesis. However, it’s worth noting that HA synthesis consumes energy and so is regulated by metabolic sensors such as UDP-GlcNAc, AMP-activated protein kinase (AMPK), and sirtuin 1 (SIRT1) (
      • Caon I.
      • Parnigoni A.
      • Viola M.
      • Karousou E.
      • Passi A.
      • Vigetti D.
      Cell energy metabolism and hyaluronan synthesis.
      ). HA synthesis is also regulated at the epigenetic level by HAS2-AS1, an HAS2 antisense transcript (
      • Vigetti D.
      • Deleonibus S.
      • Moretto P.
      • Bowen T.
      • Fischer J.W.
      • Grandoch M.
      • Oberhuber A.
      • Love D.C.
      • Hanover J.A.
      • Cinquetti R.
      • Karousou E.
      • Viola M.
      • D’Angelo M.L.
      • Hascall V.C.
      • De Luca G.
      • et al.
      Natural antisense transcript for hyaluronan synthase 2 (HAS2-AS1) induces transcription of HAS2 via protein O-GlcNAcylation.
      ,
      • Caon I.
      • Bartolini B.
      • Moretto P.
      • Parnigoni A.
      • Caravà E.
      • Vitale D.L.
      • Alaniz L.
      • Viola M.
      • Karousou E.
      • de Luca G.
      • Hascall V.C.
      • Passi A.
      • Vigetti D.
      Sirtuin 1 reduces hyaluronan synthase 2 expression by inhibiting nuclear translocation of NF-κB and expression of the long-noncoding RNA HAS2–AS1.
      ,
      • Vigetti D.
      • Viola M.
      • Karousou E.
      • Deleonibus S.
      • Karamanou K.
      • De Luca G.
      • Passi A.
      Epigenetics in extracellular matrix remodeling and hyaluronan metabolism.
      ). Whether any of these molecules might have potential as GBM biomarkers has not been explored.

      Hyaluronan as a therapeutic target in glioblastoma

      The increases of HA in tumor tissues with respect to the healthy brain discussed above may not have obvious diagnostic potential, but they do implicate HA in GBM progression and suggest its potential as a therapeutic target. New targets are desperately needed for GBM, as the median survival time after diagnosis is only 14.6 months. Moreover, existing treatments can impact healthy brain tissue in addition to disrupting GBM progression. For example, radiotherapy induces a disruption in the BBB, which would enhance the effect of chemotherapy on GBM cells but also expose normal cells to the same chemicals or other complications due to loss of BBB integrity (
      • Van Vulpen M.
      • Kal H.B.
      • Taphoorn M.J.B.
      • El Sharouni S.Y.
      Changes in blood-brain barrier permeability induced by radiotherapy: Implications for timing of chemotherapy? (Review).
      ). Moreover, treatment with TMZ seems to activate the immune system against the tumor cells (
      • Won W.-J.
      • Deshane J.S.
      • Leavenworth J.W.
      • Oliva C.R.
      • Griguer C.E.
      Metabolic and functional reprogramming of myeloid-derived suppressor cells and their therapeutic control in glioblastoma.
      ,
      • Hambardzumyan D.
      • Gutmann D.H.
      • Kettenmann H.
      The role of microglia and macrophages in glioma maintenance and progression.
      ). However, the drug could also generate DNA damage on normal cells that can have negative consequences on the function of the normal brain, diminishing the quality of life of patients.
      HA degradation has been explored as a therapeutic target in several studies (
      • Mooney K.L.
      • Choy W.
      • Sidhu S.
      • Pelargos P.
      • Bui T.T.
      • Voth B.
      • Barnette N.
      • Yang I.
      The role of CD44 in glioblastoma multiforme.
      ,
      • Okada H.
      • Yoshida J.
      • Sokabe M.
      • Wakabayashi T.
      • Hagiwara M.
      Suppression of CD44 expression decreases migration and invasion of human glioma cells.
      ,
      • Klank R.L.
      • Decker Grunke S.A.
      • Bangasser B.L.
      • Forster C.L.
      • Price M.A.
      • Odde T.J.
      • SantaCruz K.S.
      • Rosenfeld S.S.
      • Canoll P.
      • Turley E.A.
      • McCarthy J.B.
      • Ohlfest J.R.
      • Odde D.J.
      Biphasic dependence of glioma survival and cell migration on CD44 expression level.
      ,
      • Tilghman J.
      • Wu H.
      • Sang Y.
      • Shi X.
      • Guerrero-Cazares H.
      • Quinones-Hinojosa A.
      • Eberhart C.G.
      • Laterra J.
      • Ying M.
      HMMR maintains the stemness and tumorigenicity of glioblastoma stem-like cells.
      ,
      • Zhao Z.
      • Liang T.
      • Feng S.
      Silencing of HAS2-AS1 mediates PI3K/AKT signaling pathway to inhibit cell proliferation, migration, and invasion in glioma.
      ,
      • Wiranowska M.
      • Ladd S.
      • Moscinski L.C.
      • Hill B.
      • Haller E.
      • Mikecz K.
      • Plaas A.
      Modulation of hyaluronan production by CD44 positive glioma cells.
      ). Clinical trials have shown that treatment with HYAL as adjuvant chemotherapy improves drug biodistribution and particularly access to the tumor site, contributing to clinically relevant remissions in high-grade astrocytomas and to the efficacy of chemotherapy in pediatric brain tumors (
      • Baumgartner G.
      • Gomar-Höss C.
      • Sakr L.
      • Ulsperger E.
      • Wogritsch C.
      The impact of extracellular matrix on the chemoresistance of solid tumors - experimental and clinical results of hyaluronidase as additive to cytostatic chemotherapy.
      ,
      • Haselsberger K.
      • Radner H.
      • Pendl G.
      Boron neutron capture therapy for glioblastoma: Improvement of boron biodistribution by hyaluronidase.
      ,
      • Martinez-Quintanilla J.
      • He D.
      • Wakimoto H.
      • Alemany R.
      • Shah K.
      Encapsulated stem cells loaded with hyaluronidase-expressing oncolytic virus for brain tumor therapy.
      ). In addition, it was recently reported that treatment with HYAL, alone or in combination with TMZ, showed a cytotoxic effect on the GSC population. Furthermore, HYAL treatment results in upregulation of CD44 and a decrease in stem cell phenotype (
      • Hartheimer J.S.
      • Park S.
      • Rao S.S.
      • Kim Y.
      Targeting hyaluronan interactions for glioblastoma stem cell therapy.
      ). In agreement with this, it was reported that a reduction of CD44 expression increased GSC features (
      • Wang H.H.
      • Liao C.C.
      • Chow N.H.
      • Huang L.L.H.
      • Chuang J.I.
      • Wei K.C.
      • Shin J.W.
      Whether CD44 is an applicable marker for glioma stem cells.
      ). These findings suggest that targeting HA can have direct effects on CD44 and the GBM stem cell phenotype.
      Interestingly, it was observed that treatment with HYAL increases HA synthesis (
      • Wiranowska M.
      • Ladd S.
      • Moscinski L.C.
      • Hill B.
      • Haller E.
      • Mikecz K.
      • Plaas A.
      Modulation of hyaluronan production by CD44 positive glioma cells.
      ,
      • Philipson L.H.
      • Westley J.
      • Schwartz N.B.
      Effect of hyaluronidase treatment of intact cells on hyaluronate synthetase activity.
      ,
      • Asplund T.
      • Brinck J.
      • Suzuki M.
      • Briskin M.J.
      • Heldin P.
      Characterization of hyaluronan synthase from a human glioma cell line.
      ), which could be due to a compensatory mechanism. Moreover, one report did show an increase in U87MG cell proliferation after HYAL treatment (
      • Daginakatte G.C.
      • Gutmann D.H.
      Neurofibromatosis-1 (Nf1) heterozygous brain microglia elaborate paracrine factors that promote Nf1-deficient astrocyte and glioma growth.
      ). Thus, although most of the consulted bibliography reported antitumor results, it is clear that our understanding of HYAL in GBM treatment is incomplete, and new ideas regarding the role of HYAL are needed.
      The in vitro treatment of HA with HYAL generates oHA. Therefore, we propose that the effect of HYAL treatment on glioma cells could be partially explained by the generation of oHA in the medium. These oHA occupy the HA-binding site of the receptor and, because of their small size, fail to cross-link receptors, preventing signaling transduction and the normal consequences of native HA. This proposal is supported by data showing that the treatment with oHA decreased proliferation, downregulated both Akt activation and expression of breast cancer resistance protein (a transporter associated with drug resistance in cancer progenitor cells), and caused increased apoptosis in C6 cells (
      • Gilg A.G.
      • Tye S.L.
      • Tolliver L.B.
      • Wheeler W.G.
      • Visconti R.P.
      • Duncan J.D.
      • Kostova F.V.
      • Bolds L.N.
      • Toole B.P.
      • Maria B.L.
      Targeting hyaluronan interactions in malignant gliomas and their drug-resistant multipotent progenitors.
      ). Reinforcing the opposite effects between oHA and native HA, it was proposed that the last increases the levels of ceruloplasmin, a protein related to hypoxia, inflammation, and angiogenesis in gliomas, while oHA causes a reduction in its levels (
      • Tye S.L.
      • Gilg A.G.
      • Tolliver L.B.
      • Wheeler W.G.
      • Toole B.B.
      • Maria B.L.
      Hyaluronan regulates ceruloplasmin production by gliomas and their treatment-resistant multipotent progenitors.
      ).
      Prior work has explored the direct application of oHA. It improved the effects of both radiation and methotrexate treatment on U87MG cells and enhanced the effects of TMZ, carmustine, and MTX in C6 rat glioma cells (
      • Gilg A.G.
      • Tye S.L.
      • Tolliver L.B.
      • Wheeler W.G.
      • Visconti R.P.
      • Duncan J.D.
      • Kostova F.V.
      • Bolds L.N.
      • Toole B.P.
      • Maria B.L.
      Targeting hyaluronan interactions in malignant gliomas and their drug-resistant multipotent progenitors.
      ,
      • Karbownik M.
      • Pietras T.
      • Szemraj J.
      • Kowalczyk E.
      • Nowak J.Z.
      The ability of hyaluronan fragments to reverse the resistance of C6 rat glioma cell line to temozolomide and carmustine.
      ,
      • Maria B.L.
      • Gupta N.
      • Gilg A.G.
      • Abdel-Wahab M.
      • Leonard A.P.
      • Slomiany M.
      • Wheeler W.G.
      • Tolliver L.B.
      • Babcock M.A.
      • Lucas J.T.
      • Toole B.P.
      Targeting hyaluronan interactions in spinal cord astrocytomas and diffuse pontine gliomas.
      ). Related work has shown that radiation leads to an aggressive phenotype. For example, Yoo et al. (
      • Yoo K.C.
      • Suh Y.
      • An Y.
      • Lee H.J.
      • Jeong Y.J.
      • Uddin N.
      • Cui Y.H.
      • Roh T.H.
      • Shim J.K.
      • Chang J.H.
      • Park J.B.
      • Kim M.J.
      • Kim I.G.
      • Kang S.G.
      • Lee S.J.
      Proinvasive extracellular matrix remodeling in tumor microenvironment in response to radiation.
      ) showed that radiation treatment enriches the HA content in the tumor microenvironment through NF-κB activation, enhancing the invasive properties of GBM cells and leading to poor prognosis. Similarly, it was shown that subcurative radiation enhanced cell proliferation and increased MMP2 and CD44 expression beyond the tumor periphery (
      • Shankar A.
      • Kumar S.
      • Iskander A.
      • Varma N.R.S.
      • Janic B.
      • deCarvalho A.
      • Mikkelsen T.
      • Frank J.A.
      • Ali M.M.
      • Knight R.A.
      • Brown S.
      • Arbab A.S.
      Subcurative radiation significantly increases cell proliferation, invasion, and migration of primary glioblastoma multiforme in vivo.
      ). Taking into account that HA interacts with Toll-like receptor 4 (TLR4) and activates NF-κB, which leads to enhanced proliferation in GSC (
      • Ferrandez E.
      • Gutierrez O.
      • Segundo D.S.
      • Fernandez-Luna J.L.
      NFκB activation in differentiating glioblastoma stem-like cells is promoted by hyaluronic acid signaling through TLR4.
      ), the finding of Yoo et al. could explain the fact that oHA treatment improves the radiotherapy effect on GBM cells, since it might inhibit the protumor effect of HA induced by radiation.
      An alternative strategy is to block synthesis of HA directly. We recently studied the effects of the inhibitor, 4MU, which is known to inhibit HA synthesis but seems to also act via HA-independent mechanisms that are just beginning to be understood. We demonstrated that 4MU decreases metabolic activity, cell proliferation, migration, and MMP-2 activity while causing high levels of apoptosis in murine GL26 GBM cells. Moreover, we showed that 4MU partially diminishes HA synthesis, although several of its effects would be independent of this inhibition (
      • Pibuel M.A.
      • Díaz M.
      • Molinari Y.
      • Poodts D.
      • Silvestroff L.
      • Lompardía S.L.
      • Franco P.
      • Hajos S.E.
      4-Methylumbelliferone as a potent and selective anti-tumor drug on a glioblastoma model.
      ).
      Another direction being explored is the use of HA to deliver other drugs and reagents to GBM cells, taking advantage of their overexpressed HA receptors (
      • Passi A.
      • Vigetti D.
      Hyaluronan as tunable drug delivery system.
      ). For example, in 2015, Cohen et al. developed HA-coated lipid-based nanoparticles (HA-LNPs) that could deliver PLK1 siRNA. They used BALB/c nude mice inoculated with U87MG cells and observed that only those LNPs treated with HA showed specific binding to glioma cells that increased over time, which was attributed to an HA–CD44 interaction (
      • Cohen Z.R.
      • Ramishetti S.
      • Peshes-Yaloz N.
      • Goldsmith M.
      • Wohl A.
      • Zibly Z.
      • Peer D.
      Localized RNAi therapeutics of chemoresistant grade IV glioma using hyaluronan-grafted lipid-based nanoparticles.
      ). Similarly, Liu et al. developed a novel HA-decorated micelle loaded with gemcitabine and honokiol as chemotherapeutic agents. They showed that this system could effectively cross the BBB and target tumor cells, showing effective suppression of cell proliferation, enhanced apoptosis, and extensive necrosis (
      • Liu X.
      • Li W.
      • Chen T.
      • Yang Q.
      • Huang T.
      • Fu Y.
      • Gong T.
      • Zhang Z.
      Hyaluronic acid-modified micelles encapsulating gem-C 12 and HNK for glioblastoma multiforme chemotherapy.
      ). Another group reported that pairing doxorubicin with HA in a liposomal nanoparticle efficiently targets not only glioma cells, but also cancer stem cells and brain macrophages. In addition, the combination lowered the toxic effects of the drug on the heart and bone marrow and increased mice survival, altogether enhancing its antiglioma efficacy (
      • Yang L.
      • Song X.
      • Gong T.
      • Jiang K.
      • Hou Y.
      • Chen T.
      • Sun X.
      • Zhang Z.
      • Gong T.
      Development a hyaluronic acid ion-pairing liposomal nanoparticle for enhancing anti-glioma efficacy by modulating glioma microenvironment.
      ). In summary, the use of oHA or HYAL, especially as adjuvant chemotherapy, appear to be promising strategies. Furthermore, in the field of nanotechnology, the use of HA in decorated micelles to target GBM cells is in a growing phase. We also recommend further investigations of 4MU, both to explore its antitumor effects and to learn more about the underlying mechanism of action.

      Conclusion

      This review seeks to highlight the relevance of HA in GBM progression (Fig. 4). Considering that HA is the main component of parenchymal ECM in the brain, it is necessary to understand its relationship with GBM cells, since the modulation of this interaction might lead to a better patient outcome. As discussed previously, HA is either associated with or related to several proteins implicated in GBM malignancy through several and different interactions and signaling pathways. At the simplest level, HA interacts with RHAMM and CD44, sometimes in complex with EGFR, to activating PI3K/Akt and ERK1/2 pathways, enhancing two key features of GBM malignancy: cell proliferation and migration. However, the plethora of additional mechanisms described and the activation of genes downstream of HA make it difficult to understand its actions. In addition, the different presentations of this GAG (such as HMW, LMW, or oHA) and the various concentrations used for its evaluation have further scrambled the panorama. However, recent exploration of treatments targeting HA, such as the use of oHA, HYALs or 4MU, has shown effective inhibition of the malignant processes in GBM models, providing exciting new directions for GBM therapy. It is imperative to continue with the investigations to fully understand the interaction of HA with GBM cells and, more importantly, to develop an effective treatment that could mitigate its effects to improve survival and life quality in patients with GBM.
      Figure thumbnail gr4
      Figure 4Overview. In this review we analyze the effect of HA on GBM biology, highlighting the receptors and signaling pathways involved in these effects. Likewise, we describe differences and similarities observed in the behavior of GBM cells when treated with HA in its soluble form and as part of a 3D structure. We analyze the possible use of HA as a molecular marker for GBM disease. Finally, and taking together all these topics, we attempt to answer the questions regarding the potential of HA as a therapeutic target, discussing and proposing treatment alternatives targeting HA for improving the outcome in GBM patients.

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We are grateful to Sofía Noli Truant PhD for her assistance with English editing and proofreading. Furthermore, we thank Daniel Grasso PhD for his critical reading.

      Author contributions

      M. A. P. designed the review structure, analyzed the data, prepared figures, and wrote the article. D. P. contributed to the writing, analysis, and editing of the article. M. D. aided in the editing and supervision of the article. S. E. H. is the director of the group and edited and supervised the work. S. L. L. was involved in preparing figures, editing the manuscript, and supervising the work. All authors contributed to this work and have read and approved the final article.

      Funding and additional information

      This work was supported by Universidad de Buenos Aires grant [ UBACYT 20020170100454BA ] to S. E. H. and S. L. L. and by Agencia Nacional de Promoción Científica y Tecnológica , grant [ PICT-2017-2971 ] to S. L. L.

      References

        • Louis D.N.
        • Perry A.
        • Reifenberger G.
        • von Deimling A.
        • Figarella-Branger D.
        • Cavenee W.K.
        • Ohgaki H.
        • Wiestler O.D.
        • Kleihues P.
        • Ellison D.W.
        The 2016 World Health Organization classification of tumors of the central nervous system: A summary.
        Acta Neuropathol. 2016; 131: 803-820
        • Anjum K.
        • Shagufta B.I.
        • Abbas S.Q.
        • Patel S.
        • Khan I.
        • Shah S.A.A.
        • Akhter N.
        • Hassan S.S.U.
        Current status and future therapeutic perspectives of glioblastoma multiforme (GBM) therapy: A review.
        Biomed. Pharmacother. 2017; 92: 681-689
        • Adamson C.
        • Kanu O.O.
        • Mehta A.I.
        • Di C.
        • Lin N.
        • Mattox A.K.
        • Bigner D.D.
        Glioblastoma multiforme: A review of where we have been and where we are going.
        Expert Opin. Investig. Drugs. 2009; 18: 1061-1083
        • Le Rhun E.
        • Preusser M.
        • Roth P.
        • Reardon D.A.
        • Van Den Bent M.
        • Wen P.
        • Reifenberger G.
        • Weller M.
        Molecular targeted therapy of glioblastoma.
        Cancer Treat. Rev. 2019; 80: 101896
        • Perus L.J.M.
        • Walsh L.A.
        • Walsh L.A.
        Microenvironmental heterogeneity in brain malignancies.
        Front. Immunol. 2019; 10: 2294
        • Wirsching H.G.
        • Galanis E.
        • Weller M.
        Glioblastoma.
        Handbook of Clinical Neurology. Vol 134. Elsevier, Amsterdam, The Netherlands2016: 381-397
        • Strobel H.
        • Baisch T.
        • Fitzel R.
        • Schilberg K.
        • Siegelin M.D.
        • Karpel-massler G.
        • Debatin K.
        • Westho M.
        Temozolomide and other alkylating agents in glioblastoma therapy.
        Biomedicines. 2019; 69: 1-17
        • Daher A.
        • de Groot J.
        Rapid identification and validation of novel targeted approaches for glioblastoma: A combined ex vivo-in vivo pharmaco-omic model.
        Exp. Neurol. 2018; 299: 281-288
        • Philteos J.
        • Karmur B.S.
        • Mansouri A.
        MGMT testing in glioblastomas pitfalls and opportunities.
        Am. J. Clin. Oncol. 2019; 42: 117-122
      1. LiverTox: Clinical and Research Information on Drug-Induced Liver Injury.
        National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda (MD)2012
        • Houy N.
        • Le Grand F.
        Administration of temozolomide: Comparison of conventional and metronomic chemotherapy regimens.
        J. Theor. Biol. 2018; 446: 71-78
        • Rajaratnam V.
        • Islam M.M.
        • Yang M.
        • Slaby R.
        • Ramirez H.M.
        • Mirza S.P.
        Glioblastoma: Pathogenesis and current status of chemotherapy and other novel treatments.
        Cancers (Basel). 2020; 12: 937
        • Albini A.
        • Bruno A.
        • Gallo C.
        • Pajardi G.
        • Noonan D.M.
        Cancer stem cells and the tumor microenvironment: Interplay in tumor heterogeneity.
        Connect. Tissue Res. 2015; 8207: 414-425
        • Yeldag G.
        • Rice A.
        • Río Hernández A.
        Chemoresistance and the self-maintaining tumor microenvironment.
        Cancers (Basel). 2018; 10: 471
        • Piperigkou Z.
        • Karamanos N.K.
        Dynamic interplay between miRNAs and the extracellular matrix influences the tumor microenvironment.
        Trends Biochem. Sci. 2019; 44: 1076-1088
        • Soysal S.D.
        • Muenst S.E.
        Role of the tumor microenvironment in breast cancer.
        Pathobiology. 2015; 82: 142-152
        • Manou D.
        • Caon I.
        • Bouris P.
        • Triantaphyllidou I.
        • Giaroni C.
        • Passi A.
        • Karamanos N.K.
        • Vigetti D.
        • Theocharis A.D.
        The complex interplay between extracellular matriz and cells in tissues.
        Methods. Mol. Biol. 2019; 1952: 1-20
        • Karamanos N.K.
        • Piperigkou Z.
        • Theocharis A.D.
        • Watanabe H.
        • Franchi M.
        • Baud S.
        • Brézillon S.
        • Götte M.
        • Passi A.
        • Vigetti D.
        • Ricard-Blum S.
        • Sanderson R.D.
        • Neill T.
        • Iozzo R.V.
        Proteoglycan chemical diversity drives multifunctional cell regulation and therapeutics.
        Chem. Rev. 2018; 118: 9152-9232
        • Lau L.W.
        • Cua R.
        • Keough M.B.
        • Haylock-Jacobs S.
        • Yong V.W.
        Pathophysiology of the brain extracellular matrix: A new target for remyelination.
        Nat. Rev. Neurosci. 2013; 14: 722-729
        • Miyata S.
        • Kitagawa H.
        Formation and remodeling of the brain extracellular matrix in neural plasticity: Roles of chondritin sulfate and hyaluronan.
        Biochim. Biophys. Acta Gen. Subj. 2017; 10: 2420-2434
        • Boregowda R.K.
        • Appaiah H.N.
        • Siddaiah M.
        • Kumarswamy S.B.
        • Sunila S.
        • Thimmaiah K.N.
        • Mortha K.
        • Toole B.
        • Banerjee S.D.
        Expression of hyaluronan in human tumor progression.
        J. Carcinog. 2006; 5: 1-19
        • Auvinen P.
        • Tammi R.
        • Kosma V.M.
        • Sironen R.
        • Soini Y.
        • Mannermaa A.
        • Tumelius R.
        • Uljas E.
        • Tammi M.
        Increased hyaluronan content and stromal cell CD44 associate with HER2 positivity and poor prognosis in human breast cancer.
        Int. J. Cancer. 2013; 132: 531-539
        • Provenzano P.P.
        • Hingorani S.R.
        Hyaluronan, fluid pressure, and stromal resistance in pancreas cancer.
        Br. J. Cancer. 2013; 108: 1-8
        • Toole B.P.
        Hyaluronan: From extracellular glue to pericellular cue.
        Nat. Rev. Cancer. 2004; 4: 528-539
        • Ferrer V.P.
        • Moura Neto V.
        • Mentlein R.
        Glioma infiltration and extracellular matrix: Key players and modulators.
        Glia. 2018; 66: 1542-1565
        • Ruoslahti E.
        Brain extracellular matrix.
        Glycobiology. 1996; 6: 489-492
        • Ding H.
        • Xie Y.
        • Dong Q.
        • Kimata K.
        • Nishida Y.
        • Ishiguro N.
        • Zhuo L.
        Roles of hyaluronan in cardiovascular and nervous system disorders.
        J. Zhejiang Univ. Sci. B. 2019; 20: 428-436
        • Su W.
        • Matsumoto S.
        • Sorg B.
        • Sherman L.S.
        Distinct roles for hyaluronan in neural stem cell niches and perineuronal nets.
        Matrix Biol. 2019; 78–79: 272-283
        • Termeer C.
        • Sleeman J.P.
        • Simon J.C.
        Hyaluronan - magic glue for the regulation of the immune response?.
        Trends Immunol. 2003; 24: 112-114
        • Khaldoyanidi S.K.
        • Goncharova V.
        • Mueller B.
        • Schraufstatter I.U.
        Hyaluronan in the healthy and malignant hematopoietic microenvironment.
        Adv. Cancer Res. 2014; 123: 149-189
        • Joy R.A.
        • Vikkath N.
        • Ariyannur P.S.
        Metabolism and mechanisms of action of hyaluronan in human biology.
        Drug Metab. Pers. Ther. 2018; 33: 15-32
        • Preston M.
        • Sherman L.S.
        Neural stem cell niches: Roles for the hyaluronan-based extracellular matrix.
        Front. Biosci. (Schol. Ed.). 2011; 3: 1165-1179
        • Dorfman A.
        • Ho P.L.
        Synthesis of acid mucopolysaccharides by glial tumor cells in tissue culture.
        Proc. Natl. Acad. Sci. U. S. A. 1970; 66: 495-499
        • Wasteson Å.
        • Westermark B.
        • Lindahl U.
        • Pontén J.
        Aggregation of feline lymphoma cells by hyaluronic acid.
        Int. J. Cancer. 1973; 12: 169-178
        • Glimelius B.
        • Norling B.
        • Westermark B.
        • Wasteson A.
        Composition and distribution of glycosaminoglycans in cultures of human normal and malignant glial cells.
        Biochem. J. 1978; 172: 443-456
        • Glimelius B.
        • Norling B.
        • Westermark B.
        • Wasteson
        A comparative study of glycosaminoglycans in cultures of human, normal and malignant glial cells.
        J. Cell. Physiol. 1979; 98: 527-537
        • Steck P.A.
        • Moser R.P.
        • Bruner J.M.
        • Liang L.
        • Freidman A.N.
        • Hwang T.L.
        • Yung W.K.
        Altered expression and distribution of heparan sulfate proteoglycans in human gliomas.
        Cancer Res. 1989; 49: 2096-2103
        • Glimelius B.
        • Norling B.
        • Nederman T.
        • Carlsson J.
        Extracellular matrices in multicellular spheroids of human glioma origin: Increased incorporation of proteoglycans and fibronectin as compared to monolayer cultures.
        APMIS. 1988; 96: 433-444
        • Nakagawa T.
        • Kubota T.
        • Kabuto M.
        • Kodera T.
        Hyaluronic acid facilitates glioma cell invasion in vitro.
        Anticancer