The immune response influences the clinical course of colorectal cancer (CRC). Analyzing the invasive margin of human CRC liver metastases, we identified a mechanism of immune cell exploitation by tumor cells. While two distinct subsets of myeloid cells induce an influx of T cells into the invasive margin via CXCL9/CXCL10, CCL5 is produced by these T cells and stimulates pro-tumoral effects via CCR5. CCR5 blockade in patient-derived functional in vitro organotypic culture models showed a macrophage repolarization with anti-tumoral effects. These anti-tumoral effects were then confirmed in a phase I trial with a CCR5 antagonist in patients with liver metastases of advanced refractory CRC. Mitigation of tumor-promoting inflammation within the tumor tissue and objective tumor responses in CRC were observed.

On a broader scale, T cell density and localization in colorectal cancer liver metastases have prognostic and predictive implications. As T cell distribution at higher resolutions has not been fully investigated, a detailed resolution analysis of T cell distribution was performed. Patient tissues were divided into 10 µm distance classes between the tumor border and adjacent normal liver. Thereby, distinct density patterns of T cell localization in relation to the malignant tissue could be detected. At a distance of 20 to 30 µm to the tumor, a decrease of CD3 T cells is common. Within this area, cytotoxic Granzyme B and CD8+ T cells were found to be significantly reduced as well as CD163 macrophages were increased and identified to be in close contact with T cells. Our data suggests a physical or functional border within this region. Survival analysis revealed improved overall survival in patients with high T cells numbers at the direct tumor border. Interestingly, the decreased T cells in the 20 to 30 µm region were also found to be significantly associated with improved survival. Consequently, the detailed localization of T cells, despite blockade, could be associated with improved clinical outcome. The high-resolution analysis represents new insights into relevant heterogenous T cell distributions especially related to clinical responses. As the paradoxical observation of localization-dependent prognostic relevance of T cell densities is only detectable by detailed spatial analyses, this investigation of spatial profiles at higher resolutions is suggested as a new biomarker for survival and response to therapies.

Multiple reports have highlighted the importance of the local immunological cellular composition (i.e. the density of effector T cells and macrophage polarization state) in predicting clinical outcome in advanced metastatic stage of colorectal cancer. However, in spite of the general association between a high effector T cell density and improved outcome, our recent work has revealed a specific lymphocyte-driven cancer cell-supporting signal. Indeed, lymphocyte-derived CCL5 supports CCR5-positive tumor cell proliferation and thereby fosters tumor growth in metastatic liver lesions. Upon systematic analysis of CCR5 expression by tumor cells using immunohistochemistry, we observed that the intensity of CCR5 increases with primary tumor size and peaks in T4 tumors. In liver metastases however, though CCR5 expression intensity is globally heightened compared to primary tumors, alterations in the expression patterns appear, leading to "patchiness" of the stain. CCR5 patchiness is, therefore, a signature of liver metastases in our cohort (n = 97 specimens) and relates to globally decreased expression intensity, but does not influence the extent of the response to CCR5 inhibitor Maraviroc in patients. Moreover, CCR5 patchiness relates to a poor immune landscape characterized by a low cytotoxic-to-regulatory T cell ratio at the invasive margin and enriched cellular and molecular markers of macrophage M2 polarization. Finally, because higher numbers of PD-1-and CTLA-4-positive cells surround tumors with patchy CCR5 expression, one can speculate that these tumors potentially respond to immune checkpoint blockade. This hypothesis is corroborated by the prolonged disease-free survival and disease-specific survival observed in patients with low gene expression of CCR5 in metastases from two publically available cohorts. These observations highlight the complex role of the CCL5-CCR5 axis in CRC metastatic progression and warrant further investigations.

In patients with metastatic colorectal cancer (CRC), the local immune response influences the clinical course. An in-depth analysis of the invasive margin of human CRC liver metastases revealed a distinct immunological microenvironment. Within this microenvironment, two distinct subsets of myeloid cells induce an influx of T cells into the invasive margin via CXCL9/CXCL10. CCL5 is produced by these T cells and stimulates pro-tumoral effects via CCR5, creating an exploitive loop. CCR5 was found on macrophages, lymphocytes and on the vast majority of tumor cells. Inhibition of CCR5 in patient-derived functional in vitro organotypic culture models showed a promising macrophage repolarization with anti-tumoral effects. These effects are mediated by activation of an antiviral program in macrophages, leading to interferon and reactive oxygen species production and subsequent selective tumor cell death. These anti-tumoral effects were confirmed in a phase I trial with a CCR5 antagonist in 14 patients with liver metastases of advanced refractory CRC. Treatment with the oral CCR5 Inhibitor was very well tolerated and objective responses were seen, especially in combination with previously ineffective chemotherapy. Biopsies revealed mitigation of tumor-promoting inflammation within the tumor tissue, confirming the validity of the explant model and highlighting the feasbility of this approach. It furthermore shows the proof-of-concept for macrophage repolarization in cancer patients. Citation Format: Niels Halama, Inka Zoernig, Anna Berthel, Christoph Kahlert, Fee Klupp, Meggy Suarez-Carmona, Karsten Brand, Juergen Krauss, Felix Lasitschka, Alexis Ulrich, Juergen Weitz, Martin Schneider, Markus Buechler, Laurence Zitvogel, Thomas Herrmann, Axel Benner, Christina Kunz, Stephan Luecke, Christoph Springfeld, Christine S. Falk, Dirk Jaeger. CCR5 inhibition: macrophage repolarization therapy for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3021. doi:10.1158/1538-7445.AM2017-3021

Because of the particular anatomy of ovaries, which are in close contact with the adipocyte-rich omentum, ovarian cancer frequently presents itself with established metastasis-like disseminated tumor islets along the omentum. The tropism of ovarian cancer cells for fat is well described. Indeed, adipocyte-derived free fatty acids have been causatively linked to cancer cell proliferation and invasive properties. Obesity is additionally linked to increased risk of ovarian cancer onset and, in ovarian cancer patients, to worse outcome. However, there is to our knowledge no clear association between omentum-anchored dissemination and the anti-tumor immune response. Here we describe an unsuspected association between the presence peritoneal fat in ovarian tumors and massive tumor infiltration by T cells. Still, this massive T cell influx seems to fail in its anti-tumor activity since (1) tumor-infiltrating T cells seem to be hijacked away from tumor cells and accumulate around fatty areas and (2) tumor-infiltrating T cells display an exhausted phenotype. In this presentation, we also describe a new model of tissue culture of explants from primary human tumors that are treated with various drugs in an attempt to circumvent this fat-derived immune exhaustion and restore an efficient anti-tumor immune response. Citation Format: Meggy Suarez-Carmona, Anita Heinzelmann, Nektarios A. Valous, Mareike Hampel, Anna Berthel, Sarah Schott, Inka Zörnig, Dirk Jäger, Niels Halama. The fat in ovarian cancer: Immune-dependent tumor-promoting effects [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A102.

The influence of the local immune response on the clinical course of colorectal cancer (CRC) has been analyzed extensively. Analyzing the invasive margin of human CRC liver metastases, we identified a protumoral mechanism of T-cell-derived CCL5 that leads to immune cell exploitation by tumor cells. Two distinct subsets of myeloid cells produce CXCL9/CXCL10, which induce an influx of T cells into the invasive margin. CCL5 is produced by these exhausted T cells and stimulates tumor cell proliferation and invasive behavior via CCR5 on tumor cells and macrophages. CCR5 inhibition in patient-derived functional in vitro organotypic culture models induced macrophage repolarization with anti-tumoral effects. These immunomodulatory and anti-tumoral effects of CCR5 blockade then could be confirmed in a phase I trial with a CCR5 antagonist in advanced refractory CRC patients with liver metastases. Amelioration of tumor-promoting inflammation on the tumor tissue level and objective tumor responses in advanced metastatic CRC patients were observed. Citation Format: Niels Halama, Inka Zoernig, Anna Berthel, Christoph Kahlert, Fee Klupp, Meggy Suarez-Carmona, Juergen Krauss, Karsten Brand, Felix Lasitschka, Tina Lerchl, Claudia Luckner-Minden, Alexis Ulrich, Juergen Weitz, Martin Schneider, Markus W. Buechler, Laurence Zitvogel, Thomas Herrmann, Axel Benner, Christina Kunz, Stephan Luecke, Christoph Springfeld, Christine Falk, Dirk Jaeger. Macrophage repolarization therapy in metastatic colorectal cancer: CCR5 inhibition [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B037.

T-cell exclusion by tumors is one of the major obstacles for cancer immunotherapies. In colorectal cancer liver metastasis (CRC-LM) patients, effector T-cells are mainly found in the invasive margin and not in the tumor epithelium. Such T-cell exclusion might hinder an effective antitumor immune response and could account for therapy failures with immune checkpoint inhibitors, which is observed in the majority of CRC-LM patients. As T-cell infiltration and distribution are difficult to detect in patients, little is known about detailed immunotherapy effects on tumor-infiltrating lymphocytes (TIL). Therefore, we established a fully human tissue-based ex vivo cell migration analysis model to monitor T-cell infiltration and positioning in the authentic tumor microenvironment of CRC-LM patients. In brief, we isolated T-cells from fresh resected CRC-LM patient tissue samples, labeled the cells with a fluorescent dye and returned the labeled T-cells to the tissues, which were cultured for a certain time period. Besides autologous T-cells, several tissue samples were treated with non-autologous labeled T-cells isolated from a healthy donor. After tissue processing, immunostaining and cell quantification, we observed that remigration of the labeled autologous and donor T-cells into the tissues was significantly associated with numbers of endogenous TIL. The labeled autologous and donor T-cells showed a similar distribution pattern to endogenous TIL with highest densities in the invasive margin of patient tissues. No relevant changes in tumor cell numbers or apoptosis protein concentrations could be observed by both treatments. Furthermore, quantification of the chemokines CXCL9, CXCL10 and CCL5 showed significant associations with labeled autologous and donor T-cell infiltration. Interestingly, treatment with a PD1 inhibitor did not support infiltration of exogenous or endogenous T-cells into the tumor epithelium. Our findings highlight that infiltrating T-cells in CRC-LM are always positioned into the invasive margin independently of the T-cell´s origin. Especially microenvironmental factors such as CXCL9, CXCL10 and CCL5 seem to be involved in this process, preventing T-cell contact with the tumor epithelium, which could also not be abrogated by immune checkpoint inhibition. The similar infiltration and distribution pattern of T-cells in the ex vivo and in vivo settings highlights the functionality and reliability of the human tissue-based cell migration analysis model, emphasizing its use for studying therapy effects on TIL in CRC-LM patient tissues. Citation Format: Anna Berthel, Meggy Suarez-Carmona, Jakob N. Kather, Rodrigo Rojas-Moraleda, Pornpimol Chaorentong, Nektarios A. Valous, Fee Klupp, Martin Schneider, Alexis Ulrich, Markus Buechler, Inka Zoernig, Dirk Jaeger, Niels Halama. A fully human tissue-based ex vivo cell migration analysis model to study T-cell infiltration and distribution in colorectal cancer liver metastases [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A171.

On a broader scale T cell density and localization in colorectal cancer liver metastases (CRC-LM) have prognostic and predictive implications, while T cell distribution at higher resolutions has not been fully investigated. As the invasive front of CRC-LM represents a clear border between malignant tissue and adjacent normal liver tissue, a detailed resolution analysis of T cell distribution in this region could be performed. Tissue areas of 1 square millimeter size from different patients were analyzed by high-throughput whole slide imaging technology, showing distinct density patterns of T cell localization in relation to the malignant tissue across the invasive margins. Unsupervised clustering revealed specific groups of patients with T cell aggregates limited to a given distance from the tumor. Furthermore, regions with T cells in close contact to the tumor usually had elevated T cell numbers further away from the tumor, whereas the intermediate area showed limited enrichment. There seems to be a distance of around 10 to 30 micrometer from the tumor where a decrease in T cells is common. Interestingly, 68 percent of distant T cells were identified to be in close contact with tumor-associated macrophages. Therefore, the spatial heterogeneity of T cell distribution in the invasive margin could be accounted for interactions with immunosuppressive cells, cell-matrix interactions or chemotactic fields potentially generated by tumor or endothelial cells, which is currently investigated. Whether the presence of the observed patterns has clinical importance and might change under therapy remains to be seen. In summary, this high resolution analysis of T cell distribution within the invasive front of CRC-LM reveals insights into the detailed localization of T cells and possible spatial immunosuppressive hurdles that need to be overcome by immunotherapies. The analysis of reversal of these spatial profiles could serve as a biomarker for successful therapies. Citation Format: Anna Berthel, Inka Zoernig, Meggy Suarez-Carmona, Christoph Kahlert, Fee Klupp, Alexis Ulrich, Juergen Weitz, Dirk Jaeger, Halama Niels. Detailed resolution analysis reveals spatial heterogeneity of T cell distribution in the invasive margin of colorectal cancer liver metastases [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A110.