Autologous T cell responses to primary human colorectal cancer spheroids are enhanced by ectonucleotidase inhibition.

OBJECTIVE: T cells are major effectors of the antitumoural immune response. Their activation by tumour-associated antigens can unleash their proliferation and cytotoxic functions, leading to tumour cell elimination. However, tumour-related immunosuppressive mechanisms including the overexpression of immune checkpoints like programmed cell death protein-1 (PD-1), are also engaged, promoting immune escape. Current immunotherapies targeting these pathways have demonstrated weak efficacy in colorectal cancer (CRC). It is thus crucial to find new targets for immunotherapy in this cancer type. DESIGN: In a prospective cohort of patients with CRC, we investigated the phenotype of tumour-related and non-tumour related intestinal T cells (n=44), particularly the adenosinergic pathway, correlating with clinical phenotype. An autologous coculture model was developed between patient-derived primary tumour spheroids and their autologous tumour-associated lymphocytes. We used this relevant model to assess the effects of CD39 blockade on the antitumour T cell response. RESULTS: We show the increased expression of CD39, and its co-expression with PD-1, on tumour infiltrating T cells compared with mucosal lymphocytes. CD39 expression was higher in the right colon and early-stage tumours, thus defining a subset of patients potentially responsive to CD39 blockade. Finally, we demonstrate in autologous conditions that CD39 blockade triggers T cell infiltration and tumour spheroid destruction in cocultures. CONCLUSION: In CRC, CD39 is strongly expressed on tumour infiltrating lymphocytes and its inhibition represents a promising therapeutic strategy for treating patients.

Secreted Apoe rewires melanoma cell state vulnerability to ferroptosis.

A major therapeutic barrier in melanoma is the coexistence of diverse cellular states marked by distinct metabolic traits. Transitioning from a proliferative to an invasive melanoma phenotype is coupled with increased ferroptosis vulnerability. However, the regulatory circuits controlling ferroptosis susceptibility across melanoma cell states are unknown. In this work, we identified Apolipoprotein E (APOE) as the top lipid-metabolism gene segregating the melanoma MITFhigh/AXLlow proliferative/ferroptosis-resistant from MITFlow/AXLhigh invasive/ferroptosis-sensitive state. Mechanistically, ApoE secreted by the MITFhigh/AXLlow cells protects the invasive phenotype from ferroptosis-inducing agents by reducing the content of peroxidation-prone polyunsaturated fatty acids and boosting GPX4 levels both in vitro and in vivo. Whole-exome sequencing indicates that APOEhigh expression in patients with melanoma is associated with resistance to ferroptosis, regardless of APOE germline status. In aggregate, we found a ferroptosis-resistance mechanism between melanoma cell states relying on secreted ApoE and APOEhigh expression as a potential biomarker for poor ferroptosis response in melanoma.

Assessment of the reliability of MSI status and dMMR proteins deficiency screening on endoscopic biopsy material in esophagus and gastric adenocarcinoma.

BACKGROUND: Microsatellite instability (MSI) is a negative predictive factor for neoadjuvant chemotherapy in resectable oesogastric adenocarcinoma and a crucial determinant for immunotherapy. We aimed to evaluate reliability of dMMR/MSI status screening performed on preoperative endoscopic biopsies. METHODS: Paired pathological samples from biopsies and surgical specimen of oesogastric adenocarcinoma were retrospectively collected between 2009 and 2019. We compared dMMR status obtained by immunohistochemistry (IHC) and MSI status by PCR. dMMR/MSI status on surgical specimen was considered as reference. RESULTS: PCR and IHC were conclusive on biopsies respectively for 53 (96.4%) and 47 (85.5%) of the 55 patients enrolled. IHC was not contributive for 1 surgical specimen. A third reading of IHC was carried out for 3 biopsies. MSI status was observed in 7 (12.5%) surgical specimens. When analyses were contributive, sensitivity and specificity of biopsies for dMMR/MSI were respectively 85% and 98% for PCR vs. 86% and 98% for IHC. Concordance rate between biopsies and surgical specimen was 96.2% for PCR and 97.8% for IHC. CONCLUSIONS: Endoscopic biopsies are a suitable source of tissue for dMMR/MSI status determination in oesogastric adenocarcinoma which should be routinely performed at diagnosis to better adapt neoadjuvant treatment. MINIABSTRACT: By comparison of dMMR phenotype obtained by immunohistochemistry and MSI status by PCR between match-paired samples of oesogastric cancer's endoscopic biopsies and surgical specimen, we observed that biopsies are a suitable source of tissue for dMMR/MSI status determination.

Autologous organoid co-culture model reveals T cell-driven epithelial cell death in Crohn's Disease.

Lympho-epithelial interactions between intestinal T resident memory cells (Trm) and the epithelium have been associated with inflammatory bowel disease (IBD) activity. We developed ex vivo autologous organoid-mucosal T cell cocultures to functionally assess lymphoepithelial interactions in Crohn's Disease (CD) patients compared to controls. We demonstrate the direct epithelial cell death induced by autologous mucosal T cells in CD patients but not in controls. These findings were positively correlated with T cell infiltration of the organoids. This potential was inhibited by limiting lympho-epithelial interactions through CD103 and NKG2D blocking antibodies. These data directly demonstrate for the first time the direct deleterious effect of mucosal T cells on the epithelium of CD patients. Such ex-vivo models are promising techniques to unravel the pathophysiology of these diseases and the potential mode of action of current and future therapies.

KLRG1 and CD103 Expressions Define Distinct Intestinal Tissue-Resident Memory CD8 T Cell Subsets Modulated in Crohn's Disease.

Intestinal tissue-resident memory CD8 T cells (Trm) are non-recirculating effector cells ideally positioned to detect and react to microbial infections in the gut mucosa. There is an emerging understanding of Trm cell differentiation and functions, but their implication in inflammatory bowel diseases, such as Crohn's disease (CD), is still unknown. Here, we describe CD8 cells in the human intestine expressing KLRG1 or CD103, two receptors of E-cadherin. While CD103 CD8 T cells are present in high numbers in the mucosa of CD patients and controls, KLRG1 CD8 T cells are increased in inflammatory conditions. Mucosal CD103 CD8 T cells are more responsive to TCR restimulation, but KLRG1 CD8 T cells show increased cytotoxic and proliferative potential. CD103 CD8 T cells originate mostly from KLRG1 negative cells after TCR triggering and TGFß stimulation. Interestingly, mucosal CD103 CD8 T cells from CD patients display major changes in their transcriptomic landscape compared to controls. They express Th17 related genes including CCL20, IL22, and IL26, which could contribute to the pathogenesis of CD. Overall, these findings suggest that CD103 CD8 T cells in CD induce a tissue-wide alert increasing innate immune responses and recruitment of effector cells such as KLRG1 CD8 T cells.

Cocultures of human colorectal tumor spheroids with immune cells reveal the therapeutic potential of MICA/B and NKG2A targeting for cancer treatment.

BACKGROUND: Immunotherapies still fail to benefit colorectal cancer (CRC) patients. Relevant functional assays aimed at studying these failures and the efficacy of cancer immunotherapy in human are scarce. 3D tumor cultures, called tumor organoids or spheroids, represent interesting models to study cancer treatments and could help to challenge these issues. METHODS: We analyzed heterotypic cocultures of human colon tumor-derived spheroids with immune cells to assess the infiltration, activation and function of T and NK cells toward human colorectal tumors in vitro. RESULTS: We showed that allogeneic T and NK cells rapidly infiltrated cell line-derived spheroids, inducing immune-mediated tumor cell apoptosis and spheroid destruction. NKG2D, a key activator of cytotoxic responses, was engaged on infiltrating cells. We thus assessed the therapeutic potential of an antibody targeting the specific ligands of NKG2D, MICA and MICB, in this system. Anti-MICA/B enhanced immune-dependent destruction of tumor spheroid by driving an increased NK cells infiltration and activation. Interestingly, tumor cells reacted to immune infiltration by upregulating HLA-E, ligand of the inhibitory receptor NKG2A expressed by CD8 and NK cells. NKG2A was increased after anti-MICA/B treatment and, accordingly, combination of anti-MICA/B and anti-NKG2A was synergistic. These observations were ultimately confirmed in a clinical relevant model of coculture between CRC patients-derived spheroids and autologous tumor-infiltrating lymphocytes. CONCLUSIONS: Altogether, we show that tumor spheroids represent a relevant tool to study tumor-lymphocyte interactions on human tissues and revealed the antitumor potential of immunomodulatory antibodies targeting MICA/B and NKG2A.