Modulation of glycosyltransferase ST6Gal-I in gastric cancer-derived organoids disrupts homeostatic epithelial cell turnover

Programed cell death promotes homeostatic cell turnover in the epithelium but is dysregulated in cancer. The glycosyltransferase ST6Gal-I is known to block homeostatic apoptosis through  2,6-linked sialylation of the death receptor TNFR1 in many cell types. However, its role has not been investigated in gastric epithelial cells or gastric tumorigenesis. We determined that human gastric antral epithelium rarely expressed ST6Gal-I, but the number of ST6Gal-I-expressing epithelial cells increased significantly with advancing premalignancy leading to cancer. The mRNA expression level of ST6GAL-I and SOX9 in human gastric epithelial cells correlated positively with one another through the premalignancy cascade, indicating that increased epithelial cell expression of ST6Gal-I is associated with premalignant progression. To determine the functional impact of increased ST6Gal-I, we generated human gastric antral organoids from epithelial stem cells and differentiated epithelial monolayers from gastric organoids. Gastric epithelial stem cells strongly expressed ST6Gal-I, suggesting a novel biomarker of stemness. In contrast, organoid-derived epithelial monolayers expressed markedly reduced ST6Gal-I and underwent TNF-induced, caspase-mediated apoptosis, consistent with homeostasis. Conversely, epithelial monolayers generated from gastric cancer stem cells retained high levels of ST6Gal-I and resisted TNF-induced apoptosis, supporting prolonged survival. Protection from TNF-induced apoptosis was dependent on ST6Gal-I overexpression, since forced ST6Gal-I overexpression in normal gastric stem cell-differentiated monolayers inhibited TNF-induced apoptosis, and cleavage of  2,6-linked sialic acids from gastric cancer organoid-derived


Abstract
Programed cell death promotes homeostatic cell turnover in the epithelium but is dysregulated in cancer. The glycosyltransferase ST6Gal-I is known to block homeostatic apoptosis through 2,6-linked sialylation of the death receptor TNFR1 in many cell types. However, its role has not been investigated in gastric epithelial cells or gastric tumorigenesis. We determined that human gastric antral epithelium rarely expressed ST6Gal-I, but the number of ST6Gal-I-expressing epithelial cells increased significantly with advancing premalignancy leading to cancer. The mRNA expression level of ST6GAL-I and SOX9 in human gastric epithelial cells correlated positively with one another through the premalignancy cascade, indicating that increased epithelial cell expression of ST6Gal-I is associated with premalignant progression. To determine the functional impact of increased ST6Gal-I, we generated human gastric antral organoids from epithelial stem cells and differentiated epithelial monolayers from gastric organoids. Gastric epithelial stem cells strongly expressed ST6Gal-I, suggesting a novel biomarker of stemness. In contrast, organoidderived epithelial monolayers expressed markedly reduced ST6Gal-I and underwent TNFinduced, caspase-mediated apoptosis, consistent with homeostasis. Conversely, epithelial monolayers generated from gastric cancer stem cells retained high levels of ST6Gal-I and resisted TNF-induced apoptosis, supporting prolonged survival. Protection from TNF-induced apoptosis was dependent on ST6Gal-I overexpression, since forced ST6Gal-I overexpression in normal gastric stem cell-differentiated monolayers inhibited TNF-induced apoptosis, and cleavage of 2,6-linked sialic acids from gastric cancer organoid-derived monolayers restored susceptibility to TNF-induced apoptosis. These findings implicate upregulated ST6Gal-I expression in blocking homeostatic epithelial cell apoptosis in gastric cancer pathogenesis, suggesting a mechanism for prolonged epithelioid tumor cell survival.

Introduction
Epithelial cell homeostasis in the gastrointestinal mucosa is maintained by the finely tuned balance between cell proliferation and apoptosis.
This balance is disrupted transiently in mucosal injury and permanently in mucosal cancer cells, which acquire ineffective apoptosis. The latter leads to increased survival and proliferation with the dysregulated apoptosis unable to eliminate cancer cells and restrict progressive tumor cell development (1)(2)(3)(4). In the stomach, impaired epithelial apoptosis has been detected in tissue sections of premalignant gastric lesions (5), suggesting that dysregulated apoptosis may contribute to the histologic changes leading to gastric cancer. Gastric cancer cells and epithelial stem cells have many features in common, including prolonged survival. Thus, elucidation of the cellular pathways involved in prolonging epithelial stem cell survival could inform our understanding of gastric cancer pathogenesis. However, the mechanism of dysregulated apoptosis during the malignant transformation of gastric epithelial cells, especially at the stem cell level, has received insufficient investigative attention.
The glycosyltransferase ST6Gal-I has been shown to contribute to multiple cell processes, including apoptosis, differentiation and survival (6,7). ST6Gal-I is located in the Golgi, where it catalyzes the addition of 2,6linked sialic acids to N-glycans on glycoproteins destined via the trans-Golgi network for the plasma membrane. Homeostatic epithelial cell death receptors tumor necrosis receptor 1 (TNFR1) and Fas are key glycoproteins targeted by ST6Gal-I for addition of 2,6 sialic acids (2,6 sialylation). The 2,6 sialylation of TNFR1 does not block ligand binding but prevents TNFR1 internalization, thereby blocking TNF-induced apoptosis and subsequent cell turnover (8,9).
ST6Gal-I is typically expressed at low levels in differentiated epithelium, but its overexpression occurs in several epithelioid cancers, including colonic, pancreatic and ovarian adenocarcinoma (7,8,(10)(11)(12)(13). The role of ST6Gal-I in gastric epithelial cell homeostasis and the pathogenesis of gastric adenocarcinoma is not known, despite gastric cancer being the fifth most common cancer and the third leading cause of cancer-related mortality worldwide (14).
Here we determined the expression of ST6Gal-I in normal human gastric antrum and in the gastric antrum of subjects throughout the premalignant histologic cascade leading to, and including, gastric adenocarcinoma. We used Lgr5+ stem cells (15,16) from human normal gastric antrum and gastric adenocarcinoma to generate epithelial stem cell organoids and establish dynamic in vitro models of normal antral epithelium and gastric tumor epithelium (17). We employed these model systems, together with ST6Gal-I overexpression and knockdown, to elucidate the role of ST6Gal-I in normal epithelial homeostasis and gastric adenocarcinoma.

Epithelial ST6Gal-I expression is increased during gastric premalignancy and adenocarcinoma.
H. pylori infection is the primary risk factor for gastric cancer (18)(19)(20)(21). Usually aquired during early childhood and persisting for decades if untreated (22,23), H. pylori colonizes the stomach in 50% of the world's population (19). Over time, H. pylori-induced histopathology progresses through chronic gastritis, atrophy, complete metaplasia, incomplete metaplasia and dysplasia (the Correa cascade), to adenocarcinoma in 2-3% of infected persons (24,25). Notably, the premalignancy cascade may progress even after loss of H. pylori in later stages of the cascade due to inflammationinduced destruction of the ecological niche of the bacterium (26). Using gastric biopsy tissue from subjects in Chile, where H. pylori is endemic, we show that epithelial cell expression of ST6Gal-I increased progressively with each advancing histological stage (no dysplasia tissues have been available to us to date) of the Correa cascade, leading to and including gastric cancer (Fig. 1A).
Notably, gastric tissue from uninfected children and children with H. pylori chronic gastritis, who rarely develop gastric cancer (27)(28)(29), contained very few ST6Gal-I-expressing gastric epithelial cells, in contrast to the progressively increased expression in the epithelium from adult gastric tissue (Fig. 1A). We next analyzed ST6Gal-I expression in gastric adenocarcinoma tissue sections. The number of ST6Gal-I-expressing cells was strikingly increased in gastric adenocarcinoma epithelium compared with noninvolved regions of gastric tissue from the same donors, confirming ST6Gal-I overexpression in gastric cancer (Fig. 1B,C).
Further, we correlated ST6GAL-I expression with the expression of SOX9, a transcription factor whose expression is associated with the development and progression of malignancies such as colon cancer (30) and induced by H. pylori, as shown in a mouse model of the infection (31). Many of the gastric epithelial cells that expressed ST6Gal-I also expressed Sox9 (Fig. 1D). Furthermore, the mRNA expression level of ST6GAL1 and SOX9 in gastric epithelial cells correlated positively with one another through the premalignancy cascade (Fig. 1E), indicating that increased epithelial cell expression of ST6Gal-I is associated with premalignant progression in the human stomach. These findings extend the epithelioid tumors that express ST6Gal-I to gastric adenocarcinoma.

ST6Gal-I is a novel biomarker of gastric epithelial stem cells.
To determine the role of ST6Gal-I in gastric epithelial cell biology and gastric adenocarcinoma pathogenesis, we established gastric epithelial stem cell organoids and organoid-derived monolayers to model antral stem cells and epithelium, respectively. Biopsies of normal human gastric antrum were digested and enriched for stem cells through culture in the presence of 50% L-WRN conditioned media (BASIC-CM) (32). Upon passaging, individual epithelial stem cells proliferated to form spheroid organoids lined by a single layer of cells with the epithelial apical surface directed toward the lumen ( Fig. 2A). The stem cell organoids expressed high levels of mRNA for the canonical stem cell biomarker LGR5 and decreased levels of the mucin gene MUC5AC, present in differentiated goblet cells, compared with the biopsy tissue from which the organoids were derived (Fig. 2B). The gastric epithelial stem cell organoids also expressed both transmembrane tight junction protein zonula occludens 1 (ZO-1) and epithelial cell adhesion/signaling molecule E-cadherin (Fig. 2C). Epithelial stem cells generated from normal gastric antrum stained strongly for ST6Gal-I (Fig. 2D) and potently bound Sambucus nigra agglutinin (SNA) (Fig.  2E), which binds specifically to the sialic acid attached to a terminal galactose in an 2,6 linkage, confirming functional ST6Gal-I protein activity. To determine whether ST6Gal-I is associated with stemness, we examined the expression of ST6Gal-I in decreasing concentrations of stem cell factor-rich media. Gastric epithelial organoid expression of LGR5 and ST6GAL1 mRNA progressively decreased in the presence of diminishing levels of stem cell growth factor availability ( Fig. 2F,G), indicating that stem cell expression of LGR5 and ST6GAL1 was dependent, at least in part, on Wnt factor signaling.
To generate differentiated gastric epithelium, gastric organoids were trypsinized and the individual stem cells plated on glass slides thinly coated with Matrigel in the presence of reduced Wnt availability (5% L-WRN CM, monolayer media) (Fig. 3A). The epithelial stem cells formed monolayers that upregulated MUC5AC mRNA compared with the organoids (Fig. 3B), indicating differentiated gastric epithelium. Epithelial stem cell monolayers expressed ZO-1 and E-cadherin ( Fig. 3C) but exhibited very low levels of ST6Gal-I protein (Fig. 3D). Indeed, the differentiation of gastric organoids into epithelial monolayers resulted in sharp decreases in mRNA expression of both LGR5 and ST6GAL1 (Fig. 3E,F). Further, reculturing established monolayers in stem cell growth factor complete media (50% L-WRN CM) did not induce significant upregulation of either LGR5 or ST6GAL1 (Fig. 3G). These data suggest ST6Gal-I is a potential biomarker of human antral gastric epithelial stem cells but not differentiated epithelium and that Wnt factor signaling contributes to ST6Gal-I expression.

Gastric epithelial cell ST6Gal-I overexpression enhances resistance to TNFinduced epithelial cell apoptosis.
We next investigated the function of epithelial ST6Gal-I on homeostatic apoptosis. Epithelial stem cell organoids from healthy antral mucosa were transfected with an shRNA interference vector (7) targeting ST6GAL1 to knock down ST6Gal-I or transduced with an ST6GAL1 lentiviral vector (7) to overexpress ST6Gal-I. ST6GAL1 mRNA knockdown and overexpression were confirmed in the organoids at both the mRNA and protein level (Fig. 4A,B). Epithelial stem cells successfully formed organoids in the presence of ST6Gal-I knockdown (Fig. 4B), indicating ST6Gal-I was not essential for organoid formation. Importantly, forced overexpression of ST6Gal-I in gastric organoids resulted in sustained ST6Gal-I expression in the organoid-derived epithelial monolayers at both the mRNA and protein level ( Fig. 4C,D).
TNFR1 participates in a wide variety downstream signaling pathways, including homeostatic apoptosis, and is ubiquitously expressed in most tissues (33). TNFR1 is packaged in the Golgi and trafficked via the trans-Golgi network to the cell surface, where it engages with its ligand TNF (34,35). TNF ligation of TNFR1 initiates cell surface receptor aggregation that induces TNFR1 internalization, activating downstream caspases 3/7 to initiate apoptosis (9,(36)(37)(38)(39). ST6Gal-I-mediated 2,6linked sialylation of TNFR1 N-glycans blocks internalization of the receptor, thereby protecting against caspase-activated apoptosis (8,9). Interrogating this key function of ST6Gal-I, we next determined the impact of ST6Gal-I overexpression on the ability of epithelial monolayers derived from healthy gastric antrum to undergo apoptosis. Stable transduction of the organoids with a lentiviral vector containing the ST6GAL1 gene caused increased levels of surface TNFR1 (p=0.03) in the epithelial cell monolayers (Fig. 4E), consistent with impaired receptor internalization and continued TNFR1 trafficking to the epithelial cell surface (40,41). Importantly, the levels of TNFRSF1 gene expression in ST6Gal-I-overexpressing and -knockdown monolayers, as well as monolayers derived from normal gastric organoids, were similar (Fig. 4F), indicating the increased surface expression of TNFR1 in the ST6Gal-I-overexpressing monolayers was indeed due to reduced receptor internalization and not increased TNFRSF1 mRNA expression.
To determine the impact of increased ST6Gal-I expression on TNF-induced epithelial cell apoptosis, we treated epithelial monolayers generated from normal and ST6Gal-I-knockdown or -overexpressing gastric stem cell organoids with TNF for 24 h and measured caspase activity. Caspase activity was increased in both the normal monolayers and the monolayers derived from ST6Gal-I-knockdown organoids (Fig. 4G), indicating susceptibility to TNF-induced apoptosis. In contrast, caspase activity was not significantly augmented by TNF treatment of ST6Gal-I-overexpressing monolayers (Fig. 4G). Thus, increased epithelial ST6Gal-I expression leads to the suppression of TNF-mediated apoptosis, likely contributing to enhanced epithelial cell longevity in vivo.

ST6Gal-I overexpression dysregulates apoptosis in gastric cancer.
To determine the impact of high-level ST6Gal-I expression on epithelial cell function in gastric cancer, we generated epithelial organoids from gastric adenocarcinoma, derived differentiated epithelial cell monolayers, and assessed monolayer ST6Gal-I expression and function. The gastric cancer monolayers displayed diminished LGR5 expression, similar to monolayers derived from normal antral organoids (Fig. 5A). However, in sharp contrast to normal epithelial monolayers, tumor organoidderived monolayers retained high levels of ST6GAL1 expression throughout the entire culture period (4 days) (Fig. 5B). Moreover, gastric cancer-derived and normal antral-derived organoids both expressed SOX9, but only monolayers generated from tumor organoids maintained this expression (Fig. 5C), consistent with the contribution of Sox9 in tumorigenesis (30,42,43) and unfavorable patient prognosis (44).
The elevated ST6GAL1 expression in tumor organoid-derived monolayers was confirmed at the protein level using immunofluorescence (Fig.  5D). Importantly, monolayers generated from tumor organoids did not display significantly increased caspase activity in the presence of TNF (Fig. 5E), indicating resistance to TNF-inducible apoptosis.

Discussion
To date, epithelial cell glycosylation in the gastric mucosa has not been rigorously explored, particularly at the stem cell level and in malignant transformation. Here, we show that epithelial stem cells derived from normal antrum expressed high levels of the glycosyltransferase ST6Gal-I in a Wnt factor-dependent manner, but presence of the enzyme was sharply reduced during and after stem cell differentiation into epithelial cell monolayers, suggesting ST6Gal-I expression reflects epithelial stemness. This finding agrees with a report of the low ST6Gal-I expression in healthy, terminally differentiated epithelium in the pancreas and ovary (7). Although stemness has not been fully defined (48), our study indicates that ST6Gal-I is a candidate biomarker of epithelial stem cells in the antral mucosa of the stomach.
In sharp contrast to the near absence of ST6Gal-I in normal antral epithelium, we show that the number of epithelial cells that express ST6Gal-I increases progressively in advancing stages of H. pylori-associated gastric premalignancy leading to, and including, gastric adenocarcinoma.
This finding extends the repertoire of mucosal epithelioid tumors in which the enzyme is overexpressed to gastric cancer and suggests ST6Gal-I plays a role in the pathogenesis of malignant transformation of gastric epithelium (7,49,50). Infection with H. pylori initiates the premalignant changes in the stomach and is the primary risk factor for the development of gastric cancer. Although the incidence of H. pylori infection has decreased in North America and Europe recently, infection rates in Asia and Latin America have remained constant since the 1970s (19). Notably, gastric biopsies from pediatric subjects, who very rarely develop gastric cancer, displayed low levels of ST6Gal-I-expressing epithelial cells. The highlevel expression of ST6Gal-I in gastric tumor epithelium from adults and the resistance of tumor organoid-derived epithelium to TNFinduced apoptosis is recapitulated in normal epithelial monolayers with forced ST6Gal-I overexpression.
Consistent with ST6Gal-I-mediated inhibition of apoptosis, gastric tumor organoidderived epithelium was resistant to TNF-induced apoptosis, and cleavage of sialic acids implicate increased expression of ST6Gal-I inhibiting homeostatic apoptosis, thereby contributing to gastric cancer cell longevity. In this connection, the correlation between high level ST6Gal-I expression in ovarian cancer and reduced overall survival(7) and increased resistance to chemotherapeutic agents in ST6Gal-I-expressing pancreatic cancer cell lines (47), suggest the possibility that ST6Gal-I expression in gastric cancer could similarly contribute to the poor survival and chemotherapeutic response in subjects with this malignancy.
The generation and analysis of gastric stem cell organoids as described here provides an emerging opportunity to investigate the premalignant stages of gastric cancer at the stem cell level.
Studies of the human gastric epithelium and its interaction with H. pylori have been limited due to the lack of relevant models. The gastric epithelium is highly dynamic, turning over every 2-6 days(51), constantly replenished by stem cells in response to the regenerative requirements of homeostasis and injury. The progressive expression of markers of stemness, including ST6Gal-I, in epithelial monolayers derived from stage-specific organoids could contribute to more informed surveillance for the prevention of gastric cancer. In addition to enhancing prevention surveillance, gastric tissue-derived organoids might be used therapeutically, as reported in mouse (52,53) and human studies (54)(55)(56), to restore defective or injured epithelium.
Relevant to our study, human colon cancer organoids have been cultured with autologous peripheral blood T cells to generate tumor-reactive T cells capable of killing the organoid cells (54).
Further, neuraminidase cleavage of sialic acids from TNFR1 raises the intriguing possibility of tissue-specific neuraminidase delivery to enhance TNF-induced apoptosis in ST6Gal-I overexpressing gastric cancer cells, thereby expanding the treatment options for gastric adenocarcinoma.
Thus, elucidating key cellular events in gastric adenocarcinoma stem cells will enhance our understanding of gastric cancer pathogenesis and potentially inform novel treatment modalities utilizing stem cell organogenesis. In summary, our findings indicate that ST6Gal-I is a novel biomarker of gastric antral epithelial stem cells, is strongly overexpressed in gastric cancer epithelial cells, and mediates dysregulated homeostatic apoptosis, implicating a role for ST6Gal-I in epithelial cell longevity in gastric adenocarcinoma.

Experimental Procedures
Human tissue samples. Biopsy specimens were obtained with IRB approval from the gastric antrum of healthy adults without mucosal pathology and from gastric cancer lesions from subjects with histologically confirmed gastric adenocarcinoma undergoing clinically indicated endoscopy in Birmingham, AL and Santiago, Chile.
Stem cell organogenesis. Epithelial stem cell organoids were generated from gastric tissue specimens in the UAB Stem Cell Organogenesis Unit using previously described protocols(57,58). To generate epithelial stem cell organoids, biopsies were finely minced and digested for up to 50 min at 37oC in collagenase (2 mg/mL Collagenase Type 1; Corning), pipetting every 10 min to release the crypts. The collagenase then was neutralized with Washing Buffer (9 mL (32)), and the cell suspension filtered through a 70-m screen and centrifuged (200 g for 5 min at room temperature). Pelleted cells were either washed and pelleted again, or immediately suspended in Matrigel (Corning) in an ice-cold 24-well plate (15 L/well; Corning). The Matrigel was allowed to polymerize (15 min, 37oC) with the plate upside-down to prevent the epithelial cells from attaching to the plate surface, to which BASIC culture medium (450 L/well; BASIC-CM(32)) containing a 50% mix of L-WRN CM (32) and primary culture medium (Advanced DMEM/F-12; Sigma) both supplemented with 20% fetal bovine serum (Atlanta Biologicals), L-glutamine 2 mM (Fisher Scientific), penicillin 100 U/mL and streptomycin 0.1 mg/mL (Fisher Scientific). BASIC-CM was further supplemented with TGF- R1 inhibitor (10 M SB-431542, Selleck Chemicals), ROCK inhibitor (10 M Y-27632; R&D Biosystems), gentamycin (50 g/mL; Corning) and fungizone (2.5 g/mL; Fisher Scientific) before addition to the wells. Organoids were maintained at 37C and 5% CO2 and passaged every 4-7 days.
In some experiments, the percentage of L-WRN CM in BASIC-CM was reduced from 50% to 25%, 5% or 0% by dilution with additional primary culture medium. Stable organoid lines overexpressing ST6Gal-I were generated by transduction with a lentiviral vector containing the ST6GAL-I gene (Genecopoeia) or with knocked down ST6GAL-I using shRNA (Sigma), as previously described (7). ST6Gal-I expression in the organoids was confirmed using flow cytometry. Briefly, organoids were dissociated and trypsinized, washed and then stained with SNA (1:200 dilution, Vectorlabs, FL1301), a lectin specific for 2,6 sialic acids.
Epithelial cell monolayers. Human epithelial stem cell organoid cultures were dissociated in trypsin, washed (Washing Buffer 10 mL(32)), passed through a 40-m screen (Corning) to remove cell clusters, resuspended in 5% L-WRN BASIC medium, counted and seeded onto an 8-well glass chamber slide (200 L/well) or 48-well cell culture plate (350 L/well; both Corning), which was previously coated with Matrigel (1:30 dilution in PBS), polymerized for 20-30 min at 37oC, and supernatant was removed immediately prior to the addition of epithelial stem cells. The individual stem cells were allowed to differentiate, reaching confluence within 2-3 days to form a gastric epithelial cell monolayer. Monolayers typically survived at 100% confluency for 2-4 days, after which the monolayers lost viability beginning in the peripheral region of the monolayer. Each day of the culture, 50% of the media was replaced with fresh media.
Quantitative PCR: Quantitative polymerase chain reaction (qPCR) was performed using a QuantStudio 3 (Thermo Fisher Scientific) instrument. Briefly, organoids or monolayers were lysed in RLT buffer (Qiagen), and RNA was extracted using the RNeasy kit (Qiagen). Equal amounts of RNA were used to generate cDNA with the High Capacity cDNA Reverse Transcription Kit (Fisher). qPCR experiments used TaqMan gene expression assay primers (Thermo Fisher) and TaqMan Fast Advanced Master Mix (Thermo Fisher) to generate reactions, and the comparative CT method was used to analyze the corresponding data.
Immunofluorescence analysis. Organoids and organoid-derived epithelial cell monolayers were fixed in 2% paraformaldehyde and stained with phalloidin-Alexa 594 (Fisher A12381), DAPI (Fisher D1306) and antibodies to ST6Gal-I (goat polyclonal, R&D Systems AF5924) , ZO-1 (mouse monoclonal, BD Biosciences 610967), and E-cadherin (goat polyclonal, R&D Systems AF648). Images were obtained using x10, x20 or x40 objectives with a Nikon Eclipse TE2000-U fluorescent microscope and analyzed with NIS Elements imaging software. Brightfield images of organoids and epithelial cell monolayers were taken using x4 and x10 objectives with a Nikon Eclipse TE2000-U microscope and analyzed with NIS Elements imaging software.
TNFR1 detection. Epithelial cell monolayers were allowed to reach confluence, dissociated in PBS/EDTA to isolate the individual cells, washed in PBS and stained with TNFR1-FITC (Santa Cruz Biotechnology, catalog# sc-731195) for 15 min at room temperature in the dark. Cells then were washed in PBS and analyzed on an LSRII flow cytometer for fluorescence.
Immunohistochemistry. Tissue sections from healthy donors and subjects through the Correa cascade, including gastric cancer, were confirmed by a pathologist (LNC). Parallel sections of the paraffin-embedded tissues were incubated with ST6Gal-I (1g/mL; R&D Systems) or Sox 9 (1g/mL Abcam) antibody or for 1 h at room temperature, and the number of positive cells per high power field (HPF) in 5-10 randomly selected fields per tissue section per donor, according to our published protocol (7).
Epithelial cell monolayers were plated in opaque 96-well culture plates (Corning), cultured until confluent and treated with 50 ng/mL TNF for 24 h. Monolayers were brought to room temperature and 100 L of Cell Caspase-Glo® 3/7 Reagent (Promega) was added to 100 L of the treated cells and shaken at 500 rpm for 30 sec. The plate was incubated for 30 min at room temperature and then analyzed for luminescence (Gen5 Software, BioTek plate reader). In some cases, confluent epithelial cell monolayers were treated with 0.2 U/mL neuraminidase from Arthrobacter ureafaciens (Millipore Sigma) for 30 min to selectively remove 2,6-linked sialic acids (45)(46)(47). Removal of sialic acids was confirmed by flow cytometry after staining with SNA (1:200 dilution, Vectorlabs, FL1301), a lectin specific for 2,6 sialic acids (47).
Study approval. All human specimens were obtained after written informed consent approved by the UAB IRB and abide by the Declaration of Helsinki Principles.