Single-Cell Immune Mapping of Melanoma Sentinel Lymph Nodes Reveals an Actionable Immunotolerant Microenvironment.

PURPOSE: Improving our understanding of the immunologic response to cancer cells within the sentinel lymph nodes (SLN) of primary tumors is expected to identify new approaches to stimulate clinically meaningful cancer immunity. EXPERIMENTAL DESIGN: We used mass cytometry by time-of-flight (CyTOF), flow cytometry, and T-cell receptor immunosequencing to conduct simultaneous single-cell analyses of immune cells in the SLNs of patients with melanoma. RESULTS: We found increased effector-memory αß T cells, TCR clonality, and γδ T cells selectively in the melanoma-bearing SLNs relative to non-melanoma-bearing SLNs, consistent with possible activation of an antitumor immune response. However, we also observed a markedly immunotolerant environment in the melanoma-bearing SLNs indicated by reduced and impaired NK cells and increased levels of CD8+CD57+PD-1+ cells, which are known to display low melanoma killing capabilities. Other changes observed in melanoma-bearing SLNs when compared with non-melanoma-bearing SLNs include (i) reduced CD8+CD69+ T cell/T regulatory cell ratio, (ii) high PD-1 expression on CD4+ and CD8+ T cells, and (iii) high CTLA-4 expression on γδ T cells. CONCLUSIONS: Our data suggest that these immunologic changes compromise antimelanoma immunity and contribute to a high relapse rate. We propose the development of clinical trials to test the neo-adjuvant administration of anti-PD-1 antibodies prior to SLN resection in patients with stage III melanoma. See related commentary by Lund, p. 1996.

Primary lung cancer samples cultured under microenvironment-mimetic conditions enrich for mesenchymal stem-like cells that promote metastasis.

The tumor microenvironment (TME) is composed of a heterogeneous biological ecosystem of cellular and non-cellular elements including transformed tumor cells, endothelial cells, immune cells, activated fibroblasts or myofibroblasts, stem and progenitor cells, as well as the cytokines and matrix that they produce. The constituents of the TME stroma are multiple and varied, however cancer associated fibroblasts (CAF) and their contribution to the TME are important in tumor progression. CAF are hypothesized to arise from multiple progenitor cell types, including mesenchymal stem cells. Currently, isolation of TME stroma from patients is complicated by issues such as limited availability of biopsy material and cell stress incurred during lengthy adaptation to atmospheric oxygen (20% O2) in cell culture, limiting pre-clinical studies of patient tumor stromal interactions. Here we describe a microenvironment mimetic in vitro cell culturing system that incorporates elements of the in vivo lung environment, including lung fibroblast derived extracellular matrix and physiological hypoxia (5% O2). Using this system, we easily isolated and rapidly expanded stromal progenitors from patient lung tumor resections without complex sorting methods or growth supplements. These progenitor populations retained expression of pluripotency markers, secreted factors associated with cancer progression, and enhanced tumor cell growth and metastasis. An understanding of the biology of these progenitor cell populations in a TME-like environment may advance our ability to target these cells and limit their effects on promoting cancer metastasis.

Ovarian Cancer Stem Cells: Unraveling a Germline Connection.

Ovarian cancer is most lethal among gynecological cancers with often fatal consequences due to lack of effective biomarkers and relapse, which propels ovarian cancer research into unique directions to establish solid targeted therapeutics. "Ovarian stem cells" expressing germline pluripotent markers serve as novel paradigm with potential to address infertility, menopause, and probably influence tumor initiation. Cancer stem cells (CSCs) pose vital role in tumor recurrence and hence it is extremely important to study them with respect to ovarian stem cells across various cancer stages and normal ovaries. Pluripotent (OCT4, NANOG, SOX2, SSEA1, and SSEA4), germline (IFITM3, VASA/DDX4), and cancer stem (CD44, LGR5) cell specific markers were characterized for protein and mRNA expression in tumor tissues to understand their distribution in the surface epithelium and ovarian cortex in benign, borderline, and high-grade malignant stages. To elucidate whether pluripotent ovarian germline stem cells and CSCs are common subset of stem cells in tumor tissues, VASA was colocalized with known pluripotent stem (OCT4, SSEA1, SSEA4) and CSC (CD44, LGR5) specific markers by confocal microscopy. Single, smaller spherical (≤5 µm), and larger elliptical fibroblast like (≥10 µm) cells (also in clusters or multiples) were detected implying probable functional behavioral significance of cells in tumor initiation and metastasis across various cancer stages. Cells revealed characteristic staining pattern in ovarian surface epithelium (OSE) and cortex regions exclusive for each marker. Co-expression studies revealed specific subpopulations existing simultaneously in OSE and cortex and that a dynamic hierarchy of (cancer) stem cells with germline properties prevails in normal ovaries and cancer stages. Novel insights into CSC biology with respect to ovarian and germline stem cell perspective were obtained. Understanding molecular signatures and distribution within ovarian tissue may enable identification of precise tumor-initiating CSC populations and signaling pathways thus improving their efficient targeting and strategies to prevent their dissemination causing fatal relapse.