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Updated: Aug 16, 2025
Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer
Published on: July 18, 2017
Modeling resistance of colorectal peritoneal metastases to immune checkpoint blockade in humanized mice
Emre Küçükköse1, Balthasar A Heesters2, Julien Villaudy3,4
1Laboratory Translational Oncology, Division of Imaging and Cancer, University Medical Center Utrecht, Utrecht, The Netherlands.
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.
Insights
Immune checkpoint blockade (ICB) effectively treats liver metastases in high microsatellite instability colorectal cancer (MSI-H CRC) but not peritoneal metastases. B cells are crucial for ICB efficacy, and immunosuppressive ascites may drive resistance.
Area of Science:
- Oncology
- Immunology
- Cancer Metastasis
Background:
- Metastatic colorectal cancer (CRC) with high microsatellite instability (MSI-H) often responds to immune checkpoint blockade (ICB).
- Resistance to ICB is common, particularly with peritoneal metastases and ascites.
- Mechanisms of ICB resistance in peritoneal metastases are not well understood.
Purpose of the Study:
- To develop a novel model for spontaneous multiorgan metastasis in MSI-H CRC.
- To investigate the mechanisms of ICB efficacy and resistance in different metastatic sites.
- To elucidate the role of B cells and immunosuppressive cytokines in ICB response.
Main Methods:
- Created a spontaneous metastasis model using patient-derived organoids (PDO) in humanized mice.
- Administered anti-PD-1 and anti-CTLA-4 ICB treatments.
- Performed immune profiling via immunohistochemistry, flow cytometry, and single-cell RNA sequencing.
- Assessed B cell function through depletion studies and measured cytokine levels in ascites and serum.
Main Results:
- The model recapitulated clinical observations of ICB efficacy at different metastatic sites.
- ICB cleared liver metastases but failed to impact peritoneal metastases.
- Therapy efficacy correlated with B cell presence and tertiary lymphoid structures (TLS).
- Peritoneal metastases lacked B cells and TLS, with dysfunctional T cells.
- Ascites contained significantly higher immunosuppressive cytokines (IL-10, TGF-β) than serum.
Conclusions:
- The novel PDO-humanized mouse model effectively mimics site-specific ICB benefits and resistance in MSI-H CRC.
- B cells play a critical role in ICB-mediated antitumor immunity.
- Elevated immunosuppressive cytokines in ascites likely contribute to peritoneal metastasis resistance to ICB.
