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

Single-cell genomics is essential to chart tumor ecosystems. Although single-cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumors, single-nucleus RNA-Seq (snRNA-Seq) is needed to profile frozen or hard-to-dissociate tumors. Each requires customization to different tissue and tumor types, posing a barrier to adoption. Here, we have developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We analyzed 216,490 cells and nuclei from 40 samples across 23 specimens spanning eight tumor types of varying tissue and sample characteristics. We evaluated protocols by cell and nucleus quality, recovery rate and cellular composition. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types, but at different proportions. Our work provides guidance for studies in a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases.

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A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors

Head and neck squamous cell carcinoma (HNSCC) is a common, morbid, and frequently lethal malignancy. To uncover its mutational spectrum, we analyzed whole-exome sequencing data from 74 tumor-normal pairs. The majority exhibited a mutational profile consistent with tobacco exposure; human papillomavirus was detectable by sequencing DNA from infected tumors. In addition to identifying previously known HNSCC genes (TP53, CDKN2A, PTEN, PIK3CA, and HRAS), our analysis revealed many genes not previously implicated in this malignancy. At least 30% of cases harbored mutations in genes that regulate squamous differentiation (for example, NOTCH1, IRF6, and TP63), implicating its dysregulation as a major driver of HNSCC carcinogenesis. More generally, the results indicate the ability of large-scale sequencing to reveal fundamental tumorigenic mechanisms.

The Mutational Landscape of Head and Neck Squamous Cell Carcinoma

Malignant transformation and progression of cancer are driven by the co-evolution of cancer cells and their dysregulated tumor microenvironment (TME) Recent studies on immunotherapy demonstrate the efficacy in reverting the anti-tumoral function of T cells, highlighting the therapeutic potential in targeting certain cell types in TME However, the functions of other immune cell types remain largely unexplored We conduct a single-cell RNA-seq analysis of cells isolated from tumor tissue samples of non-small cell lung cancer (NSCLC) patients, and identify subtypes of tumor-infiltrated B cells and their diverse functions in the progression of NSCLC Flow cytometry and immunohistochemistry experiments on two independent cohorts confirm the co-existence of the two major subtypes of B cells, namely the naive-like and plasma-like B cells The naive-like B cells are decreased in advanced NSCLC, and their lower level is associated with poor prognosis Co-culture of isolated naive-like B cells from NSCLC patients with two lung cancer cell lines demonstrate that the naive-like B cells suppress the growth of lung cancer cells by secreting four factors negatively regulating the cell growth We also demonstrate that the plasma-like B cells inhibit cancer cell growth in the early stage of NSCLC, but promote cell growth in the advanced stage of NSCLC The roles of the plasma-like B cell produced immunoglobulins, and their interacting proteins in the progression of NSCLC are further validated by proteomics data Our analysis reveals versatile functions of tumor-infiltrating B cells and their potential clinical implications in NSCLC

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Single-cell transcriptome and antigen-immunoglobin analysis reveals the diversity of B cells in non-small cell lung cancer

Background: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that mainly transfers from human to human via respiratory and gastrointestinal routes. The S-glycoprotein in the virus is the key factor for the entry of SARS-CoV-2 into the cell, which contains two functional domains: S1 is an angiotensin-converting enzyme 2 (ACE2) receptor binding domain, and S2 is necessary for fusion of the coronavirus and cell membranes. Moreover, it has been reported that ACE2 is likely to be the receptor for SARS-CoV-2. In addition, mRNA level expression of Furin enzyme and ACE2 receptor had been reported in airway epithelia, cardiac tissue, and enteric canals. However, the expression patterns of ACE2 and Furin in different cell types of oral tissues are still unclear. Methods: In order to investigate the potential infective channel of the new coronavirus via the oropharyngeal cavity, we analyze the expression of ACE2 and Furin in human oral mucosa using the public single-cell sequence datasets. Furthermore, immunohistochemistry was performed in mucosal tissue from different oral anatomical sites to confirm the expression of ACE2 and Furin at the protein level. Results: The bioinformatics results indicated the differential expression of ACE2 and Furin on epithelial cells from different oral anatomical sites. Immunohistochemistry results revealed that both the ACE2-positive and Furin-positive cells in the target tissues were mainly positioned in the epithelial layers, partly expressed in fibroblasts, further confirming the bioinformatics results. Conclusions: Based on these findings, we speculated that SARS-CoV-2 could invade oral mucosal cells through two possible routes: binding to the ACE2 receptor and fusion with cell membrane activated by Furin protease. Our results indicated that oral mucosa tissues are susceptible to SARS-CoV-2 that could facilitate COVID-19 infection via respiratory and fecal-oral routes.

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ACE2 and Furin Expressions in Oral Epithelial Cells Possibly Facilitate COVID-19 Infection via Respiratory and Fecal-Oral Routes

Single-cell sequencing and its applications in head and neck cancer.

Our understanding of epithelial-to-mesenchymal transition (EMT) has slowly evolved from a simple two state, binary model to a multi-step, dynamic continuum of epithelial-to-mesenchymal plasticity, with metastable intermediate transition states that may drive cancer metastasis. Head and neck cancer is no exception, and in this review, we use head and neck as a case study for how partial-EMT (p-EMT) cell states may play an important role in cancer progression. In particular, we summarize recent in vitro and in vivo studies that uncover these intermediate transition states, which exhibit both epithelial and mesenchymal properties and appear to have distinct advantages in migration, survival in the bloodstream, and seeding and propagation within secondary metastatic sites. We then summarize the common and distinct regulators of p-EMT as well as methodologies for identifying this unique cellular subpopulation, with a specific emphasis on the role of cutting-edge technologies, such as single cell approaches. Finally, we propose strategies to target p-EMT cells, highlighting potential opportunities for therapeutic intervention to specifically target the process of metastasis. Thus, although significant challenges remain, including numerous gaps in current knowledge, a deeper understanding of EMT plasticity and a genuine identification of EMT as spectrum rather than a switch will be critical for improving patient diagnosis and treatment across oncology.

Partial EMT in head and neck cancer biology: a spectrum instead of a switch.

Complexities in cell-type composition have rightfully led to skepticism and caution in the interpretation of bulk transcriptomic analyses. Recent studies have shown that deconvolution algorithms can be utilized to computationally estimate cell-type proportions from the gene expression data of bulk blood samples, but their performance when applied to tumor tissues, including those from head and neck, remains poorly characterized. Here, we use single-cell data (~6000 single cells) collected from 21 head and neck squamous cell carcinoma (HNSCC) samples to generate cell-type-specific gene expression signatures. We leverage bulk RNA-seq data from >500 HNSCC samples profiled by The Cancer Genome Atlas (TCGA), and using single-cell data as a reference, apply two newly developed deconvolution algorithms (CIBERSORTx and MuSiC) to the bulk transcriptome data to quantitatively estimate cell-type proportions for each tumor in TCGA. We show that these two algorithms produce similar estimates of constituent/major cell-type proportions and that a high T-cell fraction correlates with improved survival. By further characterizing T-cell subpopulations, we identify that regulatory T-cells (Tregs) were the major contributor to this improved survival. Lastly, we assessed gene expression, specifically in the Treg population, and found that TNFRSF4 (Tumor Necrosis Factor Receptor Superfamily Member 4) was differentially expressed in the core Treg subpopulation. Moreover, higher TNFRSF4 expression was associated with greater survival, suggesting that TNFRSF4 could play a key role in mechanisms underlying the contribution of Treg in HNSCC outcomes.

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Single-Cell Deconvolution of Head and Neck Squamous Cell Carcinoma

Duration of radiation therapy is associated with worse survival in head and neck cancer.

Epithelial-mesenchymal transition (EMT) is a fundamental process that occurs during embryogenesis and tissue repair. However, EMT can be hijacked by malignant cells, where it may promote immune evasion and metastasis. Classically considered a dichotomous transition, EMT in cancer has recently been considered a plastic process whereby malignant cells display and interconvert among hybrid epithelial/mesenchymal (E/M) states. Epithelial-mesenchymal plasticity (EMP) and associated hybrid E/M states are divergent from classical EMT with unique immunomodulatory effects. Here, we review recent insights into the EMP-immune crosstalk, highlighting possible mechanisms of immune evasion conferred by hybrid E/M states and roles of immune cells in EMP.

Thomas F. Barrett

Papers

Single-cell sequencing and its applications in head and neck cancer.

Partial EMT in head and neck cancer biology: a spectrum instead of a switch.

Single-Cell Deconvolution of Head and Neck Squamous Cell Carcinoma

Duration of radiation therapy is associated with worse survival in head and neck cancer.

Epithelial-mesenchymal plasticity in tumor immune evasion.