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Background Patients with esophageal malignancy have a 5-year survival rate of only 14% in Canada. This high mortality rate is due to three factors: late diagnosis, difficulty to surgically remove the tumor due to its localization and treatment resistance. Treatment resistance has been ascribed to the presence of cancer stem cells (CSCs) inside the tumor. However, no treatment specifically directed against CSCs is available to patients. Therefore, targeting CSCs is a promising strategy to improve survival of patients with esophageal squamous cell carcinoma (ESCC), the most common type of esophageal cancer worldwide. Aims Herein, we developed an unbiased approach to identify new players in chemotherapy and radiotherapy resistance in ESCC. Methods We established radioresistant (RR), chemoresistant (CR) and radiochemoresistant (RCR) human ESCC cell lines using weekly radiation and/or continuous treatment with increasing doses of chemotherapeutic agent 5-FU. We validated that the process of resistance acquisition correlates with enrichment in CSCs as revealed by higher ALDH1 expression, and increased proportion of ALDH1high cells and CD24high/CD44high cells in flow cytometry. We then used a proteomic approach to identify new players in treatment resistance. Results Interestingly, pathway analysis demonstrated enrichment in energy metabolism as well as amino acid metabolism. Seahorse assays showed a more quiescent metabolism in all three types of resistant cells compared to the control cell line. More precisely, resistant cell lines have a lower respiration rate than control cell line, while glycolysis remains unchanged. Surprisingly, our results show a metabolic rewiring very different from the well-known Warburg effect. To further characterise these metabolic changes, we performed an unbiased metabolomic pilot study and confirmed a decrease in amino acid levels such as proline, in resistant cell lines. Preliminary data show that when cultured in DMEM with proline addition, CD44high/CD24high cell proportion is decreased in control and RR cell lines suggesting that proline is a key regulator of CSC population in ESCC. Conclusions To conclude, our results suggest an important role of metabolism in ESCC treatment resistance. This study is a first step towards the identification of new targets to fight treatment resistance in ESCC patients. Funding Agencies CAG, CIHRCanada research chair TIER 2

Background Patients with esophageal malignancy have a 5-year survival rate of only 14% in Canada. This high mortality rate is due to three factors: late diagnosis, difficulty in surgically removing the tumor because of its localization, and treatment resistance. Resistance can be developed after prolonged exposure to anti-cancer drugs and/or radiation. Indeed 30% of patients will not respond to treatment or will relapse. Resistance has been mainly ascribed to the presence of cancer stem cells (CSCs) inside the tumor. However, no treatment specifically directed against CSCs is available to patients. Purpose Thus, targeting CSCs is a promising strategy to improve the survival of patients with esophageal squamous cell carcinoma (ESCC), the most common type of esophageal cancer worldwide. Therefore, a better understanding of the molecular mechanisms occurring during long-term exposure to cancer treatments is imperative. Method Herein, we developed an unbiased approach to identify new players in chemotherapy and radiotherapy resistance development in ESCC. We established radio- (R), chemo- (C), and radiochemo-resistant (RC) human ESCC cell lines using prolonged exposure to radiation and/or chemotherapeutic agent 5-FU, respectively. Result(s) The enrichment in CSCs in all treated cell lines was demonstrated by an increase of ALDH1high cells and CD24high/CD44high cells in flow cytometry. We then used a proteomic approach to identify new players in treatment resistance. Interestingly, pathway analysis pointed out to alterations in energy metabolism as well as amino acid metabolism. Seahorse assays showed that resistant cell lines have a lower respiration rate than control cells, while glycolysis remains unchanged. To further characterize these metabolic changes, we performed an unbiased metabolomic study and confirmed a decrease in amino acid levels such as proline, in resistant cell lines. Recently, metabolic regulation has been linked to a new post-translational modification, lactylation. Proteomic data were re-analyzed looking for lactylated protein and found, amongst others, PYCRL, an enzyme implicated in proline biosynthesis, as one of the most differentially lactylated proteins in treated cell lines compared to control. PYCRL lactylation was confirmed using immuno-fluorescence colocalization and immuno-precipitation. Lastly, using AlphaFold, preliminary results point toward the importance of PYCRL lactylation impairing PYCRL homomultimerization. Conclusion(s) To conclude, our results suggest an important role of proline metabolism following long-term treatment in ESCC. This study is a first step toward the identification of new targets to fight treatment resistance in ESCC patients. Please acknowledge all funding agencies by checking the applicable boxes below CAG, CIHR, Other Please indicate your source of funding; FRQ Disclosure of Interest None Declared

Background In Canada, patients with esophageal cancer have the 2nd worst 5-year survival rate (14%). Esophageal squamous cell carcinoma (ESCC) is the most common esophageal cancer type worldwide. Its high mortality rate is partly due to the treatment resistance acquired by patients, which has mostly been attributed to the presence of cancer stem cells (CSCs). Therefore, targeting CSCs is a promising strategy to improve treatment and survival of ESCC patients. CSCs are a subpopulation of tumor cells presenting high self-renewal and multipotent capacities, and therefore largely contribute to tumor heterogeneity in addition to treatment resistance. Aims In this study, we aim to establish human ESCC cell lines resistant to radiotherapy (TE15RR), chemotherapy (TE15CR) and both (TE15RCR), and study the relationship between resistance acquisition and CSC properties; Methods To do so, we exposed human ESCC cell line TE15 to chronic doses of radiotherapy (2 Gy/week for a total dose of 60 Gy) and/or increasing doses of chemotherapy (5-FU (1, 2, 5, 10 and 15mM)). We harvested samples at different time points to fully investigate resistance acquisition process. Then, we further validated resistance and properties of the resistant TE15 cell lines. Results We confirmed that TE15RR cells are less susceptible to radiation than control cells and showed that they are more proliferative at baseline using MTT assays. Moreover, we demonstrated that TE15RR cells comprise a higher proportion of CD24high/CD44high CSC population using flow cytometry. We also found that they express higher levels of ALDH1, another well-known CSC marker, by qPCR and Western Blot. Our preliminary observations also suggest a higher invasive phenotype in TE15RR cells in limited dilution spheroid assay than in control cells supporting that TE15RR cells display CSC-like properties. TE15CR and TE15RCR cell lines were validated using similar approaches. Finally, mass spectrometry was performed to compare the proteomic profile of the resistant cell lines to the control cell line in order to identify key players in treatment resistance acquisition. Conclusions Brief, we developed radioresistant, chemoresistant and radiochemoresistant human ESCC cell lines and demonstrated that resistance acquisition correlates with CSC enrichment. Mass spectrophotometry revealed significant proteomic differences between resistant and control cell lines, which should lead to the identification of novel targets to overcome resistance in ESCC patients. Funding Agencies Canada research chair TIER 2

Background Esophageal cancer is a particularly deadly cancer with a 5-year survival rate of only 14% in Canada. Treatment resistance ascribed for at least 30% of the death. The acquisition of resistance to radio- and chemotherapy is mostly attributed to the presence of cancer stem cells (CSCs) and their persistence following classical treatments. CSCs are a subpopulation of tumor cells with high self-renewal and multipotent capacity which amongst others contribute to tumor heterogeneity. Our previous work identified Krt15+ esophageal cells as a rare and long-lived subpopulation of basal cells with higher self-renewal and multipotent capacities than other basal cells. Furthermore, preliminary observations suggest that Krt15+ cells could act as the cell-of-origin for ESCC, the most prevalent type of esophageal cancer worldwide. Though, we still ignore the role of Krt15+ cells in later stages of esophageal cancer such as treatment resistance and if therefore, they could act as CSC. Aims Determine if Krt15+ cells act as CSCs in ESCC patients and if they could contribute to treatment resistance. Methods To do so, we used Krt15-CrePR1;R26mT/mG mice treated with the carcinogen 4 Nitroquinoline-1-oxide (4NQO) in their drinking water for 16 weeks to induce ESCC. Twelve weeks following the beginning of 4NQO treatment, we induced Cre recombination with RU486, a PR1 agonist, leading to GFP expression specifically in Krt15+ cells. Following 4NQO treatment, mice were put back on normal water for 8 to 12 weeks allowing tumors to grow. At euthanasia, esophageal tumor cells were FACS sorted to isolate Krt15+ (GFP+) and Krt15- (GFP-) cells, which were then grown as tumoroids. Results We first validated that 4NQO successfully induced the formation of esophageal lesions in our model, which comprises Krt15+ and Krt15- tumor cells. Tumoroids were then successfully derived from these FACS-sorted cell populations. We demonstrated the increase of CSC-like cells within Krt15+ tumoroids compared to Krt15- tumoroids by measuring the presence of CD44highCD24high cells, two well-known CSC markers, by flow cytometry. Interestingly, Krt15+ and Krt15- tumoroids are histologically distinct. As observed for normal cells, Krt15+ tumoroids appeared as more multipotent and heterogenous than Krt15- tumoroids. Furthermore, Krt15+ tumoroids display higher hyperplasia than Krt15- tumoroids suggesting that Krt15+ tumor cells are functionally distinct from Krt15- tumor cells. Conclusions Krt15+ tumoroids display higher CSC content and hyperplastic capacity suggesting their potential role in esophageal cancer. With this project, we aim to highlight the role of Krt15+ cells in treatment resistance and put forward new targets to overcome this deadly issue in ESCC patients. Funding Agencies CAGCanada research chair TIER 2

BACKGROUND: In Canada, patients with esophageal cancer have a 5-year survival rate of only 14%, the 2(nd) worst in 2017. Esophageal squamous cell carcinoma (ESCC) is the most common esophageal cancer worldwide. Risk factors include tobacco smoking, alcohol use and esophagitis. Esophagitis is developed upon several insults (e.g. acid/bile reflux, radiation, infection). Epithelial damages typically trigger activation of stem cells which will orchestrate tissue regeneration. The existence of esophageal stem cells remained controversial until our studies. We recently identified that Krt15 promotor marks a subpopulation of esophageal basal cells which display self-renewal and multipotent capacities, characteristics consistent with a stem cell population. Our preliminary results suggest that Krt15 esophageal cells have tumor-initiating capacities in mouse. However, we still ignore the role of these cells at later stages of the disease. Cancer stem cells (CSCs) have been linked to treatment resistance and poor survival rate. Therefore, the characterization of esophageal CSCs is a promising strategy to improve survival and treatment in ESCC patients. AIMS: The aim of this study is to investigate the role of Krt15+ esophageal cancer cells in cancer initiation, progression and relapse. We hypothesize that Krt15+ cells initiate tumor formation and contribute to the establishment of treatment resistance in ESCC patients. METHODS: The carcinogen 4-nitroquinoline 1-ozide (4NQO) is used to induce oral-esophageal lesions in mouse that morphologically resemble human ESCC. Krt15-CrePR1;Rosa26(mTomato/mGFP) mice are treated with 4-NQO in the drinking water and expansion of Krt15+ (GFP-labeled) cells was measured. RESULTS: To determine the role of Krt15+ cells in esophageal tumor initiation, we treated Krt15-CrePR1;Rosa26(mTomato/mGFP) mice with 4-NQO. We observed that Krt15+ cells expand in the esophageal lesions. The clones formed by Krt15+ cells (GFP-labeled) were bigger in mice treated with 4-NQO than in control mice suggesting that Krt15+ cells contribute to ESCC expansion. Interestingly, in human ESCC tumors, we observed that K15 expression is localized at the tumor migration edges suggesting a role for K15 in migration and invasion. Furthermore, we isolated esophageal tumors from WT mice treated with 4-NQO. We observed that K15 expression is also increased in the invasive front of mouse esophageal tumors. Finally, we quantified the expression of Krt15 transcript in mouse ESCC cell lines which harbor variable tumorigenicity and aggressiveness. We observed that Krt15 expression correlated with the aggressiveness of ESCC mouse cell lines. CONCLUSIONS: Our results support that Krt15+ stem cells play a role in ESCC initiation and progression suggesting that targeting these stem cells might be a therapeutic approach to improve survival of ESCC patients. FUNDING AGENCIES: Start-up funding (Université de Sherbrooke and CRCHUS)