High Dimensional Imaging Mass Cytometry Panel to Visualize the Tumor Immune Microenvironment Contexture.

The integrative analysis of tumor immune microenvironment (TiME) components, their interactions and their microanatomical distribution is mandatory to better understand tumor progression. Imaging Mass Cytometry (IMC) is a high dimensional tissue imaging system which allows the comprehensive and multiparametric in situ exploration of tumor microenvironments at a single cell level. We describe here the design of a 39-antibody IMC panel for the staining of formalin-fixed paraffin-embedded human tumor sections. We also provide an optimized staining procedure and details of the experimental workflow. This panel deciphers the nature of immune cells, their functions and their interactions with tumor cells and cancer-associated fibroblasts as well as with other TiME structural components known to be associated with tumor progression like nerve fibers and tumor extracellular matrix proteins. This panel represents a valuable innovative and powerful tool for fundamental and clinical studies that could be used for the identification of prognostic biomarkers and mechanisms of resistance to current immunotherapies.

Tumor-Associated Neutrophils Dampen Adaptive Immunity and Promote Cutaneous Squamous Cell Carcinoma Development.

Cutaneous squamous cell carcinoma (cSCC) development has been linked to immune dysfunctions but the mechanisms are still unclear. Here, we report a progressive infiltration of tumor-associated neutrophils (TANs) in precancerous and established cSCC lesions from chemically induced skin carcinogenesis. Comparative in-depth gene expression analyses identified a predominant protumor gene expression signature of TANs in lesions compared to their respective surrounding skin. In addition, in vivo depletion of neutrophils delayed tumor growth and significantly increased the frequency of proliferating IFN-γ (interferon-γ)-producing CD8+ T cells. Mechanisms that limited antitumor responses involved high arginase activity, production of reactive oxygen species (ROS) and nitrite (NO), and the expression of programmed death-ligand 1 (PD-L1) on TAN, concomitantly with an induction of PD-1 on CD8+ T cells, which correlated with tumor size. Our data highlight the relevance of targeting neutrophils and PD-L1-PD-1 (programmed death-1) interaction in the treatment of cSCC.

NK Cell and Fibroblast-Mediated Regulation of Skin Squamous Cell Carcinoma Invasion by CLEC2A Is Compromised in Xeroderma Pigmentosum.

The ability of cancer cells to invade and disseminate can be affected by components of the surrounding microenvironment. To identify dermal components that regulate the growth of epidermal carcinomas, we studied the genetic disease called xeroderma pigmentosum that bears mutations in genes involved in the nucleotide excision repair of DNA. Patients with xeroderma pigmentosum are more prone to develop cutaneous tumors than the general population and their dermal fibroblasts display the features of dermal cancer-associated fibroblasts, which promote the invasion of keratinocytes. Here, we report that 3-dimensional dermal cultures of fibroblasts from healthy donors but not from patients with xeroderma pigmentosum complementation group C express CLEC2A, which is the ligand of the activating NK cell receptor NKp65. A similar loss of CLEC2A was observed in sporadic dermal cancer-associated fibroblasts and upon the culture of fibroblasts with cutaneous squamous cell carcinoma-conditioned medium. Using an innovative 3-dimensional organotypic skin culture model that contain NK cells in addition to fibroblasts and squamous cell carcinoma cells, we unveiled a key role of CLEC2A that orchestrates a crosstalk between fibroblasts and NK cells, thereby leading to the control of squamous cell carcinoma invasion. These findings indicate that CLEC2A-expressing dermal fibroblasts play a major role in immune surveillance of the skin.

Expression of LLT1 and its receptor CD161 in lung cancer is associated with better clinical outcome.

Co-stimulatory and inhibitory receptors expressed by immune cells in the tumor microenvironment modulate the immune response and cancer progression. Their expression and regulation are still not fully characterized and a better understanding of these mechanisms is needed to improve current immunotherapies. Our previous work has identified a novel ligand/receptor pair, LLT1/CD161, that modulates immune responses. Here, we extensively characterize its expression in non-small cell lung cancer (NSCLC). We show that LLT1 expression is restricted to germinal center (GC) B cells within tertiary lymphoid structures (TLS), representing a new hallmark of the presence of active TLS in the tumor microenvironment. CD161-expressing immune cells are found at the vicinity of these structures, with a global enrichment of NSCLC tumors in CD161+ CD4+ and CD8+ T cells as compared to normal distant lung and peripheral blood. CD161+ CD4+ T cells are more activated and produce Th1-cytokines at a higher frequency than their matched CD161-negative counterparts. Interestingly, CD161+ CD4+ T cells highly express OX40 co-stimulatory receptor, less frequently 4-1BB, and display an activated but not completely exhausted PD-1-positive Tim-3-negative phenotype. Finally, a meta-analysis revealed a positive association of CLEC2D (coding for LLT1) and KLRB1 (coding for CD161) gene expression with favorable outcome in NSCLC, independently of the size of T and B cell infiltrates. These data are consistent with a positive impact of LLT1/CD161 on NSCLC patient survival, and make CD161-expressing CD4+ T cells ideal candidates for efficient anti-tumor recall responses.