Abstract 3441: Transcriptional profiles of CD14+ cells in situ in melanoma reveal plasticity, localization dependent function and specific T cell interactions
Authors: Martinek Jan , Kim Kyung In , Lin Jianan , Wu Te-Chia , Borouchov Hannah , Gulati Ananya , Sun Lili , Wang Victor , George Joshy , Henrich Philipp , Marches Florentina , Rongvaux Anthony , Chiorazzi Michael , Chuang Jeffrey H. , Robson Paul , Flavell Richard , Banchereau Jacques , Palucka Karolina 
Published: September 2020

Abstract
Mechanisms contributing to immunotherapy resistance in patients are the object of intense studies. The myeloid cells play a major role in tumors and include cells with different 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 is not completely understood. To study intact tumor microenvironments, we have established a comprehensive approach for cellular and molecular analysis. Polychromatic immunofluorescence and histocytometry showed that CD14+ cells represent the majority of the total tumor immune infiltrate. Furthermore, while majority of the T cells are located outside of cancer nests, most of the T cells present in the melanoma tissue are in direct contact with CD14+ cells rather than melanoma cells. The distribution of CD14+ cells shows two distinct patterns: CD14+ cells within cancer nest (intratumoral) are in close interactions with melanoma cells and are loaded with melanoma protein; CD14+ cells in the tissue surrounding cancer nest (stromal) do not show melanoma protein cargo. Using customized immunofluorescence guided laser capture micro-dissection, we harvested CD3+ T cells based on their tissue location and CD14+ cells based on their tissue location and melanoma protein load for downstream analysis. Both stromal and intratumoral CD14+ cells display a macrophage like identity with RNA expression of so called “M2 like” macrophages markers, yet we detect a significant degree of variability in the expression levels for certain markers. Transcriptional profiling showed that CD14+ cells clustered according to their tissue localization, i.e., intratumoral vs. stromal, interestingly this localization imprint was less pronounced for T cells, where samples clustered preferentially by patient, leading to much lower number of localization specific differentially expressed genes. Computational analysis, revealed distinct gene signatures associated with different inflammatory and metabolic pathways in intratumoral and stromal CD14+ cells. Thus, transcriptome differentiates functional status of CD14+ cells related to their localization within tumor. Further the intra/stromal CD14+ signature clusters patient with significantly better long term survival across multiple cancer types in TCGA, which in metastatic melanoma was linked with dendritic cells signature in the stroma. Finally, combining our CD3+ and CD14+ LCM data we identified localization specific pairs of receptor/ligand interactions between myeloid and T cells.
Citation Format: Jan Martinek, Kyung In Kim, Jianan Lin, Te-Chia Wu, Hannah Borouchov, Ananya Gulati, Lili Sun, Victor Wang, Joshy George, Philipp Henrich, Florentina Marches, Anthony Rongvaux, Michael Chiorazzi, Jeffrey H. Chuang, Paul Robson, Richard Flavell, Jacques Banchereau, Karolina Palucka. Transcriptional profiles of CD14+ cells in situ in melanoma reveal plasticity, localization dependent function and specific T cell interactions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3441.