Objective: Programmed cell death protein 1 (PD-1) checkpoint inhibition and adoptive cellular therapy have had limited success in patients with microsatellite stable colorectal cancer liver metastases (CRLM). We sought to evaluate the effect of interleukin 10 (IL-10) blockade on endogenous T cell and chimeric antigen receptor T (CAR-T) cell antitumour function in CRLM slice cultures. Design: We created organotypic slice cultures from human CRLM (n=38 patients’ tumours) and tested the antitumour effects of a neutralising antibody against IL-10 (αIL-10) both alone as treatment and in combination with exogenously administered carcinoembryonic antigen (CEA)-specific CAR-T cells. We evaluated slice cultures with single and multiplex immunohistochemistry, in situ hybridisation, single-cell RNA sequencing, reverse-phase protein arrays and time-lapse fluorescent microscopy. Results: αIL-10 generated a 1.8-fold increase in T cell-mediated carcinoma cell death in human CRLM slice cultures. αIL-10 significantly increased proportions of CD8 ⁺ T cells without exhaustion transcription changes, and increased human leukocyte antigen - DR isotype (HLA-DR) expression of macrophages. The antitumour effects of αIL-10 were reversed by major histocompatibility complex class I or II (MHC-I or MHC-II) blockade, confirming the essential role of antigen presenting cells. Interrupting IL-10 signalling also rescued murine CAR-T cell proliferation and cytotoxicity from myeloid cell-mediated immunosuppression. In human CRLM slices, αIL-10 increased CEA-specific CAR-T cell activation and CAR-T cell-mediated cytotoxicity, with nearly 70% carcinoma cell apoptosis across multiple human tumours. Pretreatment with an IL-10 receptor blocking antibody also potentiated CAR-T function. Conclusion: Neutralising the effects of IL-10 in human CRLM has therapeutic potential as a stand-alone treatment and to augment the function of adoptively transferred CAR-T cells.

Organotypic tumor slices represent a physiologically-relevant culture system for studying the tumor microenvironment. Systematic characterization of the tumor slice culture system will enable its effective application for translational research. Here, using flow cytometry-based immunophenotyping, we performed a comprehensive characterization of the immune cell composition in organotypic tumor slices prepared from four syngeneic mouse tumor models and a human liver tumor. We found that the immune cell compositions of organotypic tumor slices prepared on the same day as the tumor cores were harvested are similar. Differences were primarily observed in the lymphocyte population of a clinical hepatocellular carcinoma case. Viable populations of immune cells persisted in the tumor slices for 7 days. Despite some changes in the immune cell populations, we showed the utility of mouse tumor slices for assessing responses to immune-modulatory agents. Further, we demonstrated the ability to use patient-derived xenograft tumor slices for assessing responses to targeted and cytotoxic drugs. Overall, tumor slices provide a broadly useful platform for studying the tumor microenvironment and evaluating the preclinical efficacy of cancer therapeutics.

Emerging approaches to study cell–cell interactions in tumor microenvironment Emerging approaches to study cell–cell interactions in tumor microenvironment

Measuring the states of cell signaling pathways in tumor samples promises to advance the understanding of oncogenesis and identify response biomarkers. Here, we describe the use of Reverse Phase Protein Arrays (RPPAs or RPLAs) to profile signaling proteins in 56 breast cancers and matched normal tissue. In RPPAs, hundreds to thousands of lysates are arrayed in dense regular grids and each grid is probed with a different antibody (100 in the current work, of which 71 yielded strong signals with breast tissue). Although RPPA technology is quite widely used, measuring changes in phosphorylation reflective of protein activation remains challenging. Using repeat deposition and well-validated antibodies, we show that diverse patterns of phosphorylation can be monitored in tumor samples and changes mapped onto signaling networks in a coherent fashion. The patterns are consistent with biomarker-based classification of breast cancers and known mechanisms of oncogenesis. We explore in detail one tumor-associated pattern that involves changes in the abundance of the Axl receptor tyrosine kinase (RTK) and phosphorylation of the cMet RTK. Both cMet and Axl have been implicated in breast cancer, or in resistance to anticancer drugs, but the two RTKs are not known to be linked functionally. Protein depletion and overexpression studies in a ‘triple-negative’ breast cell line reveal cross talk between Axl and cMet involving Axl-mediated modification of cMet, a requirement for cMet in efficient and timely signal transduction by the Axl ligand Gas6 and the potential for the two receptors to interact physically. These findings have potential therapeutic implications, as they imply that bi-specific receptor inhibitors (for example, ATP-competitive small-kinase inhibitors such as GSK1363089, BMS-777607 or MP470) may be more efficacious than the mono-specific therapeutic antibodies currently in development (for example, Onartuzumab).

YAP1 is a transcriptional coactivator regulated by the Hippo signaling pathway, including NF2. Meningiomas are the most common primary brain tumors; a large percentage exhibit heterozygous loss of chromosome 22 (harboring the NF2 gene) and functional inactivation of the remaining NF2 copy, implicating oncogenic YAP activity in these tumors. Recently, fusions between YAP1 and MAML2 have been identified in a subset of pediatric NF2 wild-type meningiomas. Here, we show that human YAP1-MAML2-positive meningiomas resemble NF2 mutant meningiomas by global and YAP-related gene expression signatures. We then show that expression of YAP1-MAML2 in mice induces tumors that resemble human YAP1 fusion-positive and NF2 mutant meningiomas by gene expression. We demonstrate that YAP1-MAML2 primarily functions by exerting TEAD-dependent YAP activity that is resistant to Hippo signaling. Treatment with YAP-TEAD inhibitors is sufficient to inhibit the viability of YAP1-MAML2-driven mouse tumors ex vivo. Finally, we show that expression of constitutively active YAP1 (S127/397A-YAP1) is sufficient to induce similar tumors, suggesting that the YAP component of the gene fusion is the critical driver of these tumors. In summary, our results implicate YAP1-MAML2 as a causal oncogenic driver and highlight TEAD-dependent YAP activity as an oncogenic driver in YAP1-MAML2 fusion meningioma as well as NF2 mutant meningioma in general.