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-γ signature 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.

In 5% of metastatic colorectal cancer (mCRC) patients, tumours display a deficient mismatch repair (dMMR) system. Immunotherapy is beneficial in dMMR mCRC patients and has recently been approved by the Food and Drug Administration for patients with unresectable or metastatic dMMR CRC. Although dMMR and proficient MMR (pMMR) CRC tumours are biologically distinct, they are commonly treated with the same chemotherapy and monoclonal antibodies. This includes dMMR mCRC patients who did not respond to immunotherapy (20-30%). However, it is unclear if these treatments are equally beneficial in dMMR mCRC. Of note, dMMR mCRC patients have a worse prognosis compared to pMMR, which may in part be caused by a lower response to treatment. To avoid unnecessary exposure to ineffective treatments and their associated toxicity, it is important to identify which systemic treatments are most beneficial in dMMR mCRC patients, thus improving their outcome. Indeed, future treatment strategies are likely to involve combinations of immunotherapy, chemotherapy and monoclonal antibodies. In this evidence-based review, we summarize clinical trials reporting treatment efficacy of different types of chemotherapy and monoclonal antibodies in dMMR mCRC patients. We also review the biological rationale behind a potential differential benefit of chemotherapy with or without monoclonal antibodies in dMMR mCRC patients. A barrier in the interpretation of preclinical results is the choice of model systems. They largely comprise traditional models, including cell lines and xenografts, rather than more representative models, such as patient-derived organoids. We provide concrete recommendations for clinical investigators and fundamental researchers to accelerate research regarding which systemic therapy is most effective in dMMR mCRC patients.

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.

Background: Immune checkpoint inhibition (ICI) significantly improves the survival of metastatic melanoma patients; however, a substantial proportion of patients does not respond to these breakthrough therapies. Methods: To improve our understanding of this response variability, we developed high-plex panels for protein imaging of a discovery cohort and validation with RNAseq analyses to examine myeloid and T-cell subsets in pre-anti-PD-1-treatment samples of 14 metastatic melanoma patients (7 responders and 7 non-responders). Results: We demonstrate that a higher abundance of circulating monocyte-derived macrophages (MDMs) and cytotoxic T-cell subsets in the tumor microenvironment (TME) at baseline distinguishes metastatic melanoma patients with a favorable response to anti-PD1 treatment from non-responders, who featured co-localization of suppressive macrophages (M2) and T-cells. Additionally, MDMs expressed high levels of immune checkpoints, and MDM infiltration into the TME was linked to both ICI response and survival. Conclusion: These findings highlight the potential of MDM infiltration as a predictive biomarker for ICI response in metastatic melanoma.

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.