Modeling resistance of colorectal peritoneal metastases to immune checkpoint blockade in humanized mice

Background The immunogenic nature of metastatic colorectal cancer (CRC) with high microsatellite instability (MSI-H) underlies their responsiveness to immune checkpoint blockade (ICB). However, resistance to ICB is commonly observed, and is associated with the presence of peritoneal-metastases and ascites formation. The mechanisms underlying this site-specific benefit of ICB are unknown. Methods We created a novel model for spontaneous multiorgan metastasis in MSI-H CRC tumors by transplanting patient-derived organoids (PDO) into the cecum of humanized mice. Anti-programmed cell death protein-1 (PD-1) and anti-cytotoxic T-lymphocytes-Associated protein 4 (CTLA-4) ICB treatment effects were analyzed in relation to the immune context of primary tumors, liver metastases, and peritoneal metastases. Immune profiling was performed by immunohistochemistry, flow cytometry and single-cell RNA sequencing. The role of B cells was assessed by antibody-mediated depletion. Immunosuppressive cytokine levels (interleukin (IL)-10, transforming growth factor (TGF)b1, TGFb2, TGFb3) were determined in ascites and serum samples by ELISA. Results PDO-initiated primary tumors spontaneously metastasized to the liver and the peritoneum. Peritoneal-metastasis formation was accompanied by the accumulation of ascites. ICB completely cleared liver metastases and reduced primary tumor mass but had no effect on peritoneal metastases. This mimics clinical observations. After therapy discontinuation, primary tumor masses progressively decreased, but peritoneal metastases displayed unabated growth. Therapy efficacy correlated with the formation of tertiary lymphoid structures (TLS)-containing B cells and juxtaposed T cells-and with expression of an interferon-γsignature together with the B cell chemoattractant CXCL13. B cell depletion prevented liver-metastasis clearance by anti-CTLA-4 treatment. Peritoneal metastases were devoid of B cells and TLS, while the T cells in these lesions displayed a dysfunctional phenotype. Ascites samples from patients with cancer with peritoneal metastases and from the mouse model contained significantly higher levels of IL-10, TGFb1, TGFb2 and TGFb3 than serum samples. Conclusions By combining organoid and humanized mouse technologies, we present a novel model for spontaneous multiorgan metastasis by MSI-H CRC, in which the clinically observed organ site-dependent benefit of ICB is recapitulated. Moreover, we provide empirical evidence for a critical role for B cells in the generation of site-dependent antitumor immunity following anti-CTLA-4 treatment. High levels of immunosuppressive cytokines in ascites may underlie the observed resistance of peritoneal metastases to ICB

Modeling metastatic colorectal cancer subtypes to improve therapeutic strategies

Translational cancer research aims to optimize personalized treatment strategies and improve survival rates. However, developing innovative treatment approaches for colorectal cancer (CRC) faces challenges due to tumor subtype heterogeneity and the absence of suitable preclinical models that align with these subtypes. CRC can be categorized into specific tumor subtypes based on gene expression profiles and the status of the mismatch repair (MMR) system, characterized as either proficient (pMMR) or deficient (dMMR). Establishing preclinical models representing these CRC subtypes could rationalize therapeutic strategies. The objectives of the studies in this thesis were to uncover molecular tumor-intrinsic and microenvironmental factors contributing to aggressive behavior and drug resistance. Additionally, we aimed to develop a novel humanized mouse model for studying determinants of immunotherapy response. Furthermore, we investigated the mechanisms behind metastasis formation and its clonal relationship with primary tumors. We utilized patient-derived tumor organoids, animal models, CRC tissue specimens, and clinical data from patients at various stages of the disease. Our findings revealed that the upregulation of a tyrosine kinase receptor enhances tumor cell regenerative capacity and induces a pro-fibrotic tumor microenvironment. However, in vitro experiments showed that dMMR and pMMR tumor organoids had similar sensitivity to chemotherapy and targeted treatments, suggesting that microenvironmental factors may influence drug response and metastatic potential. We observed that metastasis formation in dMMR tumors is associated with the presence of inactive cytotoxic T cells in the pro-fibrotic microenvironment. Furthermore, we introduced a novel humanized mouse model that faithfully replicates the clinically observed benefits of immune checkpoint blockade therapy depending on the organ site, facilitating the evaluation of new immune-oncology approaches. Moreover, by modeling the formation of metastases, we demonstrated that fundamentally distinct processes drive the development of liver, lung, and peritoneal metastases. In summary, these findings contribute to advancing treatment strategies for CRC patients

KIT promotes tumor stroma formation and counteracts tumor-suppressive TGFβ signaling in colorectal cancer

Abstract Expression profiling has identified four consensus molecular subtypes (CMS1-4) in colorectal cancer (CRC). The receptor tyrosine kinase KIT has been associated with the most aggressive subtype, CMS4. However, it is unclear whether, and how, KIT contributes to the aggressive features of CMS4 CRC. Here, we employed genome-editing technologies in patient-derived organoids (PDOs) to study KIT function in CRC in vitro and in vivo. CRISPR-Cas9-mediated deletion of the KIT gene caused a partial mesenchymal-to-epithelial phenotype switch and a strong reduction of intra-tumor stromal content. Vice versa, overexpression of KIT caused a partial epithelial-to-mesenchymal phenotype switch, a strong increase of intra-tumor stromal content, and high expression of TGFβ1. Surprisingly, the levels of phosphorylated SMAD2 were significantly lower in KIT-expressing versus KIT-deficient tumor cells. In vitro analyses showed that TGFβ signaling in PDOs limits their regenerative capacity. Overexpression of KIT prevented tumor-suppressive TGFβ signaling, while KIT deletion sensitized PDOs to TGFβ-mediated growth inhibition. Mechanistically, we found that KIT expression caused a strong reduction in the expression of SMAD2, a central mediator of canonical TGFβ signaling. We propose that KIT induces a pro-fibrotic tumor microenvironment by stimulating TGFβ expression, and protects the tumor cells from tumor-suppressive TGFβ signaling by inhibiting SMAD2 expression

Mismatch Repair Status in Patient-Derived Colorectal Cancer Organoids Does Not Affect Intrinsic Tumor Cell Sensitivity to Systemic Therapy

DNA mismatch repair deficiency (dMMR) in metastatic colorectal cancer (mCRC) is associated with poor survival and a poor response to systemic treatment. However, it is unclear whether dMMR results in a tumor cell-intrinsic state of treatment resistance, or whether alternative mechanisms play a role. To address this, we generated a cohort of MMR-proficient and -deficient Patient-Derived Organoids (PDOs) and tested their response to commonly used drugs in the treatment of mCRC, including 5-fluorouracil (5-FU), oxaliplatin, SN-38, binimetinib, encorafenib, and cetuximab. MMR status did not correlate with the response of PDOs to any of the drugs tested. In contrast, the presence of activating mutations in the KRAS and BRAF oncogenes was significantly associated with resistance to chemotherapy and sensitivity to drugs targeting oncogene-activated pathways. We conclude that mutant KRAS and BRAF impact the intrinsic sensitivity of tumor cells to chemotherapy and targeted therapy. By contrast, tumor cell-extrinsic mechanisms—for instance signals derived from the microenvironment—must underlie the association of MMR status with therapy response. Future drug screens on rationally chosen cohorts of PDOs have great potential in developing tailored therapies for specific CRC subtypes including, but not restricted to, those defined by BRAF/KRAS and MMR status