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

AbstractThe 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.</jats:p

Single-cell transcriptomics of melanoma sentinel lymph nodes identifies immune cell signatures associated with metastasis

The sentinel lymph node (SLN) is the first lymph node encountered by a metastatic cancer cell and serves as a predictor of poor prognosis, as cases with clinically occult SLN metastases are classified as stage III with elevated rates of recurrence and diminished overall survival. However, the dynamics of immune infiltrates in SLNs remain poorly characterized. Here, using an unbiased cellular indexing of transcriptomes and epitopes by sequencing technique, we profiled 97,777 cells from SLN tissues obtained from patients with stages I/II and III cutaneous melanoma. We described the transcriptional programs of a multitude of T, B, and myeloid cell subtypes in SLNs. Based on the proportions of cell types, we determined that SLN subtypes stratified along a naive → activated axis; patients with a “high activated” signature score appeared to be undergoing a robust melanoma antigen–driven adaptive immune response and, thus, could be responsive to immunotherapy. Additionally, we identified transcriptomic signatures of SLN-infiltrating dendritic cell subsets that compromise antitumor immune responses. Our analyses provide valuable insights into tumor-driven immune changes in the SLN tissue, offering a powerful tool for the informed design of immune therapies for patients with high-risk melanoma

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

Abstract 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 αβ 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 </jats:sec

Supplementary Figure from Single‐Cell Immune Mapping of Melanoma Sentinel Lymph Nodes Reveals an Actionable Immunotolerant Microenvironment

Supplementary Figure from Single‐Cell Immune Mapping of Melanoma Sentinel Lymph Nodes Reveals an Actionable Immunotolerant Microenvironmen

Supplementary Table from Single‐Cell Immune Mapping of Melanoma Sentinel Lymph Nodes Reveals an Actionable Immunotolerant Microenvironment

Supplementary Table from Single‐Cell Immune Mapping of Melanoma Sentinel Lymph Nodes Reveals an Actionable Immunotolerant Microenvironmen

Supplementary Figure from Single‐Cell Immune Mapping of Melanoma Sentinel Lymph Nodes Reveals an Actionable Immunotolerant Microenvironment

Supplementary Figure from Single‐Cell Immune Mapping of Melanoma Sentinel Lymph Nodes Reveals an Actionable Immunotolerant Microenvironmen

Supplementary Figure from Single‐Cell Immune Mapping of Melanoma Sentinel Lymph Nodes Reveals an Actionable Immunotolerant Microenvironment

Supplementary Figure from Single‐Cell Immune Mapping of Melanoma Sentinel Lymph Nodes Reveals an Actionable Immunotolerant Microenvironmen

Supplementary Data from Single‐Cell Immune Mapping of Melanoma Sentinel Lymph Nodes Reveals an Actionable Immunotolerant Microenvironment

Supplementary Data from Single‐Cell Immune Mapping of Melanoma Sentinel Lymph Nodes Reveals an Actionable Immunotolerant Microenvironmen