Quantitative cellular and molecular imaging of the intact tumor microenvironment.

The causes underlying the extent and character of tumor-associated immune responses in cancer are not well defined and are likely multifactorial including cancer cell heterogeneity, host genotype, and the immune status of individual patients. Tumors are complex and organized tissues that include multiple cell types, which together compose the tumor microenvironment (TME). The myeloid cells play a major role in TME and can be composed of heterogeneous cells with functions that can be grossly summarized as: (1) Antigen capture for presentation (dendritic cells, DCs) or for degradation (macrophages); (2) Tissue repair (macrophages) and (3) effector function (mast cells, monocytes, granulocytes). However, the functional status of myeloid cells in human tumors and variation between tumors and patients is not completely understood. This is an important gap in knowledge that needs to be addressed because myeloid antigen presenting cells (mAPCs) control cancer antigen presentation to T cells thereby launching and regulating anti-cancer immunity. Our studies focused therefore on two key approaches necessary to improve our understanding of myeloid cells in TME: 1. Analysis of myeloid cells in situ in tumors. To this end, we developed and applied a microscopy-based approach for quantitative and qualitative mapping of non-dissociated tumors. Indeed, current methods are based on tissue dissociation into single cell suspension, which is associated with cell loss and activation, possibly impacting observed phenotypes. This was combined with laser capture microdissection to lift the cells and transcriptional profiling of APCs based on their tissue location and antigen content; and 2. Humanized mouse models recapitulating human TME. Indeed, while syngeneic and genetically modified mouse models enable in vivo studies of the TME, substantial differences exist between human and mouse immune systems, possibly impacting translation of pre-clinical studies to the clinic. Therefore, we studied human myeloid cells in novel humanized mouse models that support the tumor progression and metastatic spread of human melanoma. Our results show that location of myeloid cells within the tissue, as well as antigen cargo, have a significant impact on cell’s transcriptomic profile and potentially function. Thus, our approach developed in the course of studies discussed herein might bring a new resolution to unraveling the biology of APCs within the TME. This in turn could have an impact beyond melanoma. Furthermore, the new humanized mouse models that we have studied bring the in vivo proof that human myeloid cells and macrophages contribute to tumor development and metastatic colonization.