IMMUNE EVASION OF COLORECTAL CANCER STEM CELLS IS DEPENDENT ON SLC25A22-MEDIATED WNT SIGNALING

Background & Aims
Colonoscopy surveillance intervals are based on the predicted risk of metachronous colorectal cancer (CRC) after polyp removal. Due to the presence of co-existent findings at baseline colonoscopy, risk estimation per specific polyp subtype is difficult. To investigate the metachronous CRC risk, we evaluated the risk of patient groups with and without co-occuring findings.
Methods
Using screening colonoscopies performed after a positive fecal immunochemical test between 2014-2020 within the Dutch CRC screening program, we applied Cox regression analysis to evaluate the association between findings at baseline colonoscopy and metachronous CRC. Metachronous cancer cases, defined as CRCs diagnosed after 6 months of baseline colonoscopy, were collected from the Dutch Cancer Registry. Risk of subgroups based on polyp findings were compared to individuals without polyps in two different models. In model 1, individuals were classified into eight different subgroups based on the presence or absence of advanced adenomas and/or advanced serrated polyps. In model 2, we included presence of individual polyp characteristics in a multivariable analysis. Both models were adjusted for individuals’ sex and age. A hazard ratio >1.5 was considered clinically relevant. Advanced serrated polyps (ASP) were defined as serrated polyp ≥10mm, sessile serrated lesion with dysplasia, or traditional serrated adenoma.
Results
253,833 colonoscopies were included. Over a median follow-up of 36 months (IQR, 21-57), we identified 504 metachronous CRC cases. HR for CRC was 1.70 (CI95%, 1.07-2.69) for individuals with ASP without advanced adenomas (AA), 1.22 (0.96-1.55) for individuals with AAs without ASPs, and 2.00 (1.19-3.39) for individuals with ASPs and AA, compared to patients without polyps (Table 1). Individuals with non-advanced adenomas and/or non-advanced serrated polyps did not have an increased CRC risk. Model 2 showed that any villous adenoma (HR 2.732, 1.725-4.329), serrated polyp ≥10mm (HR 1.61; 1.06-2.45), sessile serrated lesion with dysplasia (HR 2.09; 1.12-3.88), traditional serrated adenoma (HR 2.668, 1.434-4.965), adenoma with high-grade dysplasia (HR 3.59; 2.17-5.94) and presence of at least five non-advanced adenomas (HR 2.13; 1.10-4.10) resulted in a clinically relevant increased risk for CRC, while any adenoma ≥10mm did not (HR 0.96; 0.74-1.25) (Table 2).
Conclusion
By evaluating high-quality screening colonoscopies with a median follow-up of 3 years and accounting for co-occuring findings, we observed an increased CRC risk in individuals that had ASPs with the presence of AAs, or individuals with ASPs without AAs. These findings could contribute to establish more restrictive post-polypectomy surveillance guidelines.

Background: Post-colonoscopy colorectal cancer (PCCRC) is defined as cancers diagnosed after a colonoscopy in which no cancer is found. The clinical features, prognosis, and risk factors of PCCRC remain poorly characterized.
Methods: In a large healthcare system (Mass General Brigham), we assembled a longitudinal cohort of patients who had undergone a colonoscopy between 2010-2018 by extracting detailed endoscopic, pathologic, and other clinical data from the electronic health records. We identified patients who were diagnosed with CRC between 6 and 48 months after a CRC-free index colonoscopy (PCCRC<4y) and at least 48 months after a CRC-free colonoscopy (PCCRC≥4y). We conducted chart review to categorize the causes of PCCRC according to the World Endoscopy Organization (WEO) recommendations and compared the anatomic subsites, stages, and survival between PCCRC<4y and PCCRC≥4y. We then matched up to 3 controls for each CRC patient based upon age, sex, race/ethnicity, and time and reason of index colonoscopy; and assessed potential risk factors for PCCRC using conditional logistic regression and accounting for missing data via multiple imputation.
Results: During a median follow-up of 5.6 years among 198,066 patients with a CRC-free index colonoscopy, we documented 121 incident PCCRC<4y and 86 PCCRC≥4y cases (females: 42% vs. 53%; mean age=67.7 vs. 70.1 years; median interval between index colonoscopy and diagnosis=2.2 vs. 6.0 years). According to the WEO categorization, 90% of PCCRC<4y cases likely occurred due to possible missed lesions or inadequate exam. Compared to PCCRC≥4ys, PCCRC<4ys had a more advanced stage at diagnosis (p=0.004) but no statistically significant difference in subsites (p=0.56). Among patients with a polyp finding in the index colonoscopy, PCCRC<4ys were more likely to arise in the same broad location (proximal colon, distal colon, and rectum) as prior polyps than PCCRC≥4ys, particularly in the proximal colon (57% vs. 18%). During a median follow-up of 4.6 and 3.3 years of PCCRC<4y and PCCRC≥4y patients, we documented 28 and 9 deaths, respectively; a lower 5-year survival was observed for PCCRC<4y than PCCRC≥4y (68% vs. 76%). By comparing PCCRC<4ys and PCCRC≥4ys and their matched controls (353 and 252 controls, respectively), we found that the presence of large (≥1 cm) index polyps was more strongly associated with higher risk of PCCRC<4ys than PCCRC≥4ys (p for heterogeneity=0.006), whereas no significant difference was found in other risk factors, including histology and multiplicity of index polyps and family history of CRC.
Conclusions: 90% of PCCRC<4y cases are avoidable via improved colonoscopy quality and lesion detection. PCCRC<4y had more unfavorable clinical profiles than PCCRC≥4y. Patients with large index polyps were at a particularly high risk of developing PCCRC<4y, likely in the same location.

Background and Aims: Colorectal cancer (CRC) is one of the commonest cancers worldwide. However, a majority of CRC demonstrate intrinsic resistance to immune checkpoint blockade (ICB) therapy. Cancer stem cells (CSCs) is a subpopulation of cancer cells considered as key players in chemoresistance, tumor relapse and metastasis. However, it is unclear whether CSCs might modulate antitumor immunity and its therapeutic implications.

Methods: Colorectal CSCs (POP66 and LS174T) were established from primary human CRC. Correlation between cancer stemness characteristics and immune checkpoint was examined by single cell RNA-seq (scRNA-seq) of CRC from azoxymethane/Dextran sodium sulfate (AOM-DSS)-treated mice model. TCGA cohort was used to examine correlation of CSC markers with immune checkpoints and SLC25A22. To test the function of SLC25A22 in immune evasion by colorectal CSCs, a colon stem cell-specific SLC25A22 knockout mice (Slc25a22^fl/flLgr5-Cre^ERT2) was established.

Results: Colorectal CSCs have remarkably high expression of immune checkpoints, including Galectin-9 (LGALS9) and PVR (CD155), as compared to a panel of CRC cells. In AOM/DSS-induced CRC, scRNA-seq revealed that Lgr5⁺ CSCs subpopulation have increased immune checkpoints expression compared to non-CSCs fraction. Corroborating these findings, CSCs markers positively correlated to immune checkpoint in TCGA CRC cohort. In support of this notion, co-culture of CSCs (POP66 and LS174T) with human CD8⁺ T cells impaired T cell proliferation and attenuated markers of T cell activity, including Granzyme B, IFN-γ and TNF-α. We next investigated if the blockade of SLC25A22, a driver of WNT-dependent CSCs, could reverse immunosuppressive phenotype of colorectal CSCs. SLC25A22 knockdown in CSCs inhibited stemness and immune checkpoints Galectin-9, PVR, and PD-L1. This in turn reversed suppressive effect of colorectal CSCs on CD8⁺ T cell proliferation and cytotoxic activation. We next established colon stem cell-specific SLC25A22 knockout mice, and demonstrated that SLC25A22 loss impaired AOM/DSS-induced tumorigenesis (P<0.01). Notably, scRNA-seq showed that SLC25A22 depletion decreased Lgr5⁺ CSCs in CRC, leading reduced intratumoral immune checkpoint expression. Assessment of tumor infiltrating immune cells with cytometry by time-of-flight (Cy-TOF) and flow cytometry validated that SLC25A22 knockout increased infiltration of CD8⁺ T cells, together with elevated expression of granzyme B, IFN-γ and TNF-α. Mechanistically, we revealed that SLC25A22-driven WNT signaling underlies the increased expression of immune checkpoints in colorectal CSCs.

Conclusion: Colorectal CSCs are drivers of immune evasion via the overexpression of immune checkpoints to antagonize intratumoral cytotoxic T cells. Targeting of SLC25A22 represents a potential therapeutic approach to reverse CSCs-mediated immunosuppression.