Single-cell expression profiles of melanoma Tumors harbor multiple cell types that are thought to play a role in the development of resistance to drug treatments. Tirosh et al. used single-cell sequencing to investigate the distribution of these differing genetic profiles within melanomas. Many cells harbored heterogeneous genetic programs that reflected two different states of genetic expression, one of which was linked to resistance development. Following drug treatment, the resistance-linked expression state was found at a much higher level. Furthermore, the environment of the melanoma cells affected their gene expression programs. Science, this issue p. 189 Melanoma cells show transcriptional heterogeneity. To explore the distinct genotypic and phenotypic states of melanoma tumors, we applied single-cell RNA sequencing (RNA-seq) to 4645 single cells isolated from 19 patients, profiling malignant, immune, stromal, and endothelial cells. Malignant cells within the same tumor displayed transcriptional heterogeneity associated with the cell cycle, spatial context, and a drug-resistance program. In particular, all tumors harbored malignant cells from two distinct transcriptional cell states, such that tumors characterized by high levels of the MITF transcription factor also contained cells with low MITF and elevated levels of the AXL kinase. Single-cell analyses suggested distinct tumor microenvironmental patterns, including cell-to-cell interactions. Analysis of tumor-infiltrating T cells revealed exhaustion programs, their connection to T cell activation and clonal expansion, and their variability across patients. Overall, we begin to unravel the cellular ecosystem of tumors and how single-cell genomics offers insights with implications for both targeted and immune therapies.

Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq

Immunotherapy has revolutionized cancer treatment and rejuvenated the field of tumor immunology Several types of immunotherapy, including adoptive cell transfer (ACT) and immune checkpoint inhibitors (ICIs), have obtained durable clinical responses, but their efficacies vary, and only subsets of cancer patients can benefit from them Immune infiltrates in the tumor microenvironment (TME) have been shown to play a key role in tumor development and will affect the clinical outcomes of cancer patients Comprehensive profiling of tumor-infiltrating immune cells would shed light on the mechanisms of cancer–immune evasion, thus providing opportunities for the development of novel therapeutic strategies However, the highly heterogeneous and dynamic nature of the TME impedes the precise dissection of intratumoral immune cells With recent advances in single-cell technologies such as single-cell RNA sequencing (scRNA-seq) and mass cytometry, systematic interrogation of the TME is feasible and will provide insights into the functional diversities of tumor-infiltrating immune cells In this review, we outline the recent progress in cancer immunotherapy, particularly by focusing on landmark studies and the recent single-cell characterization of tumor-associated immune cells, and we summarize the phenotypic diversities of intratumoral immune cells and their connections with cancer immunotherapy We believe such a review could strengthen our understanding of the progress in cancer immunotherapy, facilitate the elucidation of immune cell modulation in tumor progression, and thus guide the development of novel immunotherapies for cancer treatment

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The history and advances in cancer immunotherapy: understanding the characteristics of tumor-infiltrating immune cells and their therapeutic implications.

INTRODUCTION Resident tissue macrophages (RTMs) are a heterogeneous population of immune cells occupying multiple tissue niches and exhibiting microenvironment-specific phenotypes and functions. In certain tissues such as the brain, lung, and liver, embryonically derived RTMs maintain themselves by self-renewal, whereas others, including those in the gut, dermis, and pancreas, are replaced by monocytes, at levels that are tissue specific. Once they arrive in their tissue of residence, monocytes undergo extensive differentiation according to molecular cues provided by their distinct tissue-specific niches, enabling their development into specialized RTMs that support local tissue function. RATIONALE As a result of this ontogenetic and tissue niche heterogeneity, each tissue contains multiple populations of macrophages. For example, in the murine lung, alveolar macrophages are the major embryonically derived population in the alveolar spaces, whereas a minor population named interstitial macrophages (IMs) resides within the lung parenchyma. Previous results reported several phenotypically distinct IM subpopulations, whose relationship remained unknown. Do they represent independent populations or, rather, different points on the spectrum of maturation and activation states? How do these differences relate to their localization in tissue or roles in tissue function in health and disease? Does such macrophage heterogeneity also exist in other tissues? RESULTS Here, using single-cell mRNA sequencing, we unbiasedly identified two independent populations exhibiting distinct gene expression profiles and phenotypes: Lyve1loMHCIIhiCX3CR1hi (Lyve1loMHCIIhi) and Lyve1hiMHCIIloCX3CR1lo (Lyve1hiMHCIIlo) RTMs. We uncovered evidence of parallel populations in multiple others tissues, including the heart, fat, and dermis, as well as in human lung and omental and subcutaneous fat tissues, suggesting that a similar dichotomy is observed in human tissues. We further demonstrated that both populations are slowly replaced by Ly6Chi monocytes. Importantly, using complementary fate-mapping models, we showed that monocyte-derived RTMs (MRTMs) are two separate lineages, rather than representing points along a developmental or maturation continuum. Notably, these distinct MRTM populations preferentially reside within different, but conserved, subtissular niches, located either adjacent to nerve bundles and fibers (Lyve1loMHCIIhi) or blood vessels (Lyve1hiMHCIIlo) across tissues. Finally, by acutely depleting Lyve1hiMHCIIlo MRTMs using a mouse model of inducible macrophage depletion during the induction of fibrosis, we found that the absence of Lyve1hiMHCIIlo IMs exacerbated experimental lung and heart fibrosis, demonstrating their critical role in tissue inflammation. CONCLUSION Two independent MRTMs populations exist across tissues with specific niche-dependent phenotype and functional programming. Their different roles in homeostasis, immune regulation, and fibrosis renders them attractive and separate cellular targets for the therapeutic exploitation of RTM subsets.

Two distinct interstitial macrophage populations coexist across tissues in specific subtissular niches

A pan-cancer single-cell transcriptional atlas of tumor infiltrating myeloid cells.

Cancer immunotherapy targeting co-inhibitory pathways by checkpoint blockade shows remarkable efficacy in a variety of cancer types. However, only a minority of patients respond to treatment due to the stochastic heterogeneity of tumor microenvironment (TME). Recent advances in single-cell RNA-seq technologies enabled comprehensive characterization of the immune system heterogeneity in tumors but posed computational challenges on integrating and utilizing the massive published datasets to inform immunotherapy. Here, we present Tumor Immune Single Cell Hub (TISCH, http://tisch.comp-genomics.org), a large-scale curated database that integrates single-cell transcriptomic profiles of nearly 2 million cells from 76 high-quality tumor datasets across 27 cancer types. All the data were uniformly processed with a standardized workflow, including quality control, batch effect removal, clustering, cell-type annotation, malignant cell classification, differential expression analysis and functional enrichment analysis. TISCH provides interactive gene expression visualization across multiple datasets at the single-cell level or cluster level, allowing systematic comparison between different cell-types, patients, tissue origins, treatment and response groups, and even different cancer-types. In summary, TISCH provides a user-friendly interface for systematically visualizing, searching and downloading gene expression atlas in the TME from multiple cancer types, enabling fast, flexible and comprehensive exploration of the TME.

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TISCH: a comprehensive web resource enabling interactive single-cell transcriptome visualization of tumor microenvironment.

Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer.

Elicitation of tumor cell killing by CD8+ T cells is an effective therapeutic approach for cancer. In addition to using immune checkpoint blockade to reinvigorate existing but unresponsive tumor-specific T cells, alternative therapeutic approaches have been developed, including stimulation of polyclonal T cell cytolytic activity against tumors using bispecific T cell engager (BiTE) molecules that simultaneously engage the TCR complex and a tumor-associated Ag. BiTE molecules are efficacious against hematologic tumors and are currently being explored as an immunotherapy for solid tumors. To understand mechanisms regulating BiTE molecule­–mediated CD8+ T cell activity against solid tumors, we sought to define human CD8+ T cell populations that efficiently respond to BiTE molecule stimulation and identify factors regulating their cytolytic activity. We find that human CD45RA+CCR7− CD8+ T cells are highly responsive to BiTE molecule stimulation, are enriched in genes associated with cytolytic effector function, and express multiple unique inhibitory receptors, including leukocyte Ig-like receptor B1 (LILRB1). LILRB1 and programmed cell death protein 1 (PD1) were found to be expressed by distinct CD8+ T cell populations, suggesting different roles in regulating the antitumor response. Engaging LILRB1 with its ligand HLA-G on tumor cells significantly inhibited BiTE molecule–induced CD8+ T cell activation. Blockades of LILRB1 and PD1 induced greater CD8+ T cell activation than either treatment alone. Together, our data suggest that LILRB1 functions as a negative regulator of human CD8+ effector T cells and that blocking LILRB1 represents a unique strategy to enhance BiTE molecule therapeutic activity against solid tumors.

LILRB1 Blockade Enhances Bispecific T Cell Engager Antibody-Induced Tumor Cell Killing by Effector CD8+ T Cells.

The present invention relates to a human agonistic CD40 multispecific antibody construct for treatment of solid tumors by engineering a molecule that specifically targets the CD40 pathway on tumor-associated APCs, without systemic CD40 activation.

Aeryon Kim

Papers

Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer.

LILRB1 Blockade Enhances Bispecific T Cell Engager Antibody-Induced Tumor Cell Killing by Effector CD8+ T Cells.

Mesothelin-targeted cd40 agonistic multispecific antibody constructs for the treatment of solid tumors