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This study investigates the variability among patients with non-small cell lung cancer (NSCLC) in their responses to immune checkpoint inhibitors (ICIs). Recognizing that patients with advanced-stage NSCLC rarely qualify for surgical interventions, it becomes crucial to identify biomarkers that influence responses to ICI therapy. We conducted an analysis of single-cell transcriptomes from 33 lung cancer biopsy samples, with a particular focus on 14 core samples taken before the initiation of palliative ICI treatment. Our objective was to link tumor and immune cell profiles with patient responses to ICI. We discovered that ICI non-responders exhibited a higher presence of CD4+ regulatory T cells, resident memory T cells, and TH17 cells. This contrasts with the diverse activated CD8+ T cells found in responders. Furthermore, tumor cells in non-responders frequently showed heightened transcriptional activity in the NF-kB and STAT3 pathways, suggesting a potential inherent resistance to ICI therapy. Through the integration of immune cell profiles and tumor molecular signatures, we achieved an discriminative power (area under the curve [AUC]) exceeding 95% in identifying patient responses to ICI treatment. These results underscore the crucial importance of the interplay between tumor and immune microenvironment, including within metastatic sites, in affecting the effectiveness of ICIs in NSCLC., Competing Interests: NK, SP, AJ, HE, HK, KL, JC, BK, HJ, JS, SL, JA, JL, JC, DJ, MN, HK, JK, JC, HL, MA No competing interests declared, (© 2024, Kim, Park et al.)

Single-cell RNA sequencing (scRNA-seq) has emerged as a versatile tool in biology, enabling comprehensive genomic-level characterization of individual cells. Currently, most scRNA-seq methods generate barcoded cDNAs by capturing the polyA tails of mRNAs, which exclude many non-coding RNAs (ncRNAs), especially those transcribed by RNA polymerase III (Pol III). Although previously thought to be expressed constitutively, Pol III-transcribed ncRNAs are expressed variably in healthy and disease states and play important roles therein, necessitating their profiling at the single-cell level. In this study, we developed a measurement protocol for nc886 as a model case and initial step for scRNA-seq for Pol III-transcribed ncRNAs. Specifically, we spiked in an oligo-tagged nc886-specific primer during the polyA tail capture process for the 5'scRNA-seq. We then produced sequencing libraries for standard 5' gene expression and oligo-tagged nc886 separately, to accommodate different cDNA sizes and ensure undisturbed transcriptome analysis. We applied this protocol in three cell lines that express high, low, and zero levels of nc886. Our results show that the identification of oligo tags exhibited limited target specificity, and sequencing reads of nc886 enabled the correction of non-specific priming. These findings suggest that gene-specific primers (GSPs) can be employed to capture RNAs lacking a polyA tail, with subsequent sequence verification ensuring accurate gene expression counting. Moreover, we embarked on an analysis of differentially expressed genes in cell line sub-clusters with differential nc886 expression, demonstrating variations in gene expression phenotypes. Collectively, the primer spike-in strategy allows combined analysis of ncRNAs and gene expression phenotype., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Shin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Immune checkpoint inhibitors unleash inhibitory signals on T cells conferred by tumors and surrounding stromal cells. Despite the clinical efficacy of checkpoint inhibitors, the lack of target expression and persistence of immunosuppressive cells limit the pervasive effectiveness of the therapy. These limitations may be overcome by alternative approaches that co-stimulate T cells and the immune microenvironment., Methods: We analyzed single-cell RNA sequencing data from multiple human cancers and a mouse tumor transplant model to discover the pleiotropic expression of the Interleukin 7 (IL-7) receptor on T cells, macrophages, and dendritic cells., Results: Our experiment on the mouse model demonstrated that recombinant IL-7 therapy induces tumor regression, expansion of effector CD8 T cells, and pro-inflammatory activation of macrophages. Moreover, spatial transcriptomic data support immunostimulatory interactions between macrophages and T cells., Conclusion: These results indicate that IL-7 therapy induces anti-tumor immunity by activating T cells and pro-inflammatory myeloid cells, which may have diverse therapeutic applicability., (© 2024. The Author(s).)

Identification of optimal target antigens that distinguish cancer cells from normal surrounding tissue cells remains a key challenge in chimeric antigen receptor (CAR) cell therapy for tumors with intratumoral heterogeneity. In this study, we dissected tissue complexity to the level of individual cells through the construction of a single-cell expression atlas that integrates ~1.4 million tumor, tumor-infiltrating normal and reference normal cells from 412 tumors and 12 normal organs. We used a two-step screening method using random forest and convolutional neural networks to select gene pairs that contribute most to discrimination between individual malignant and normal cells. Tumor coverage and specificity are evaluated for the AND, OR and NOT logic gates based on the combinatorial expression pattern of the pairing genes across individual single cells. Single-cell transcriptome-coupled epitope profiling validates the AND, OR and NOT switch targets identified in ovarian cancer and colorectal cancer., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Immune checkpoint inhibitors (ICIs) induce activation and expansion of cytotoxic T cells. To depict a comprehensive immune cell landscape reshaped by the CTLA-4 checkpoint inhibitor, we performed single-cell RNA sequencing in a mouse syngeneic tumor transplant model. After CTLA-4 inhibition, tumor regression was accompanied by massive immune cell expansion, especially in T and B cells. We found that B cells in tumor transplant represented follicular, germinal center and plasma B cells, some of which shared identical B cell receptor clonotypes and possessed tumor reactivity. Furthermore, the posttreatment tumor contained a tertiary lymphoid-like structure with intermingled T and B cells. These data suggest germinal center formation within the tumor mass and in situ differentiation of tumor-specific plasma cells. Taken together, our data provide a panoramic view of the immune microenvironment after CTLA-4 inhibition and suggest a role for tumor-specific B cells in antitumor immunity., (© 2023 UICC.)

Background: Single-cell RNA sequencing (scRNA-seq) enables the systemic assessment of intratumoral heterogeneity within tumor cell populations and in diverse stromal cells of the tumor microenvironment. Gain of treatment resistance during tumor progression or drug treatment are important subjects of tumor-centric scRNA-seq analyses, which are hampered by scarce tumor cell portions. To guarantee the inclusion of tumor cells in the data analysis, we developed a prescreening strategy for lung adenocarcinoma., Methods: We obtained candidate genes that were differentially expressed between normal and tumor cells, excluding stromal cells, from the scRNA-seq data. Tumor cell-specific expression of the candidate genes was assessed via real-time reverse transcription-polymerase chain reaction (RT-PCR) using lung cancer cell lines, normal vs. lung cancer tissues, and lymph node biopsy samples with or without metastasis., Results: We found that CEA cell adhesion molecule 5 (CEACAM5) and high mobility group box 3 (HMGB3) were reliable markers for RT-PCR-based prescreening of tumor cells in lung adenocarcinoma., Conclusions: The prescreening strategy using CEACAM5 and HMGB3 expression facilitates tumor-centric scRNA-seq analyses of lung adenocarcinoma., (© 2022. The Author(s).)

Dendritic cells (DCs) are key antigen-presenting cells that prime naive T cells and initiate adaptive immunity. Although the genetic deficiency and transgenic overexpression of granulocyte macrophage-colony stimulating factor (GM-CSF) signaling were reported to influence the homeostasis of DCs, the in vivo development of DC subsets following injection of GM-CSF has not been analyzed in detail. Among the treatment of mice with different hematopoietic cytokines, only GM-CSF generates a distinct subset of XCR1 - 33D1 - DCs which make up the majority of DCs in the spleen after three daily injections. These GM-CSF-induced DCs (GMiDCs) are distinguished from classical DCs (cDCs) in the spleen by their expression of CD115 and CD301b and by their superior ability to present blood-borne antigen and thus to stimulate CD4 + T cells. Unlike cDCs in the spleen, GMiDCs are exceptionally effective to polarize and expand T helper type 2 (Th2) cells and able to induce allergic sensitization in response to blood-borne antigen. Single-cell RNA sequencing analysis and adoptive cell transfer assay reveal the sequential differentiation of classical monocytes into pre-GMiDCs and GMiDCs. Interestingly, mixed bone marrow chimeric mice of Csf2rb +/+ and Csf2rb -/- demonstrate that the generation of GMiDCs necessitates the cis expression of GM-CSF receptor. Besides the spleen, GMiDCs are generated in the CCR7-independent resident DCs of the LNs and in some peripheral tissues with GM-CSF treatment. Also, small but significant numbers of GMiDCs are generated in the spleen and other tissues during chronic allergic inflammation. Collectively, our present study identifies a splenic subset of CD115 hi CD301b + GMiDCs that possess a strong capacity to promote Th2 polarization and allergic sensitization against blood-borne antigen., Competing Interests: Author CP is employed by Genuv Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ryu, Shin, Eum, Park, Choi, Na, In, Kim, Park, Hwang, Sohn, Kim, Seo, Lee, Lee, Chu and Park.)

The ability of single-cell genomics to resolve cellular heterogeneity is highly appreciated in cancer and is being exploited for precision medicine. In the recent decade, we have witnessed the incorporation of cancer genomics into the clinical decision-making process for molecular-targeted therapies. Compared with conventional genomics, which primarily focuses on the specific and sensitive detection of the molecular targets, single-cell genomics addresses intratumoral heterogeneity and the microenvironmental components impacting the treatment response and resistance. As an exploratory tool, single-cell genomics provides an unprecedented opportunity to improve the diagnosis, monitoring, and treatment of cancer. The results obtained upon employing bulk cancer genomics indicate that single-cell genomics is at an early stage with respect to exploration of clinical relevance and requires further innovations to become a widely utilized technology in the clinic.

Gastric cancer (GC) patients develop malignant ascites as the disease progresses owing to peritoneal metastasis. GC patients with malignant ascites have a rapidly deteriorating clinical course with short survival following the onset of malignant ascites. Better optimized treatment strategies for this subset of patients are needed. To define the cellular characteristics of malignant ascites of GC, we used single-cell RNA sequencing to characterize tumor cells and tumor-associated macrophages (TAMs) from four samples of malignant ascites and one sample of cerebrospinal fluid. Reference transcriptomes for M1 and M2 macrophages were generated by in vitro differentiation of healthy blood-derived monocytes and applied to assess the inflammatory properties of TAMs. We analyzed 180 cells, including tumor cells, macrophages, and mesothelial cells. Dynamic exchange of tumor-promoting signals, including the CCL3-CCR1 or IL1B-IL1R2 interactions, suggests macrophage recruitment and anti-inflammatory tuning by tumor cells. By comparing these data with reference transcriptomes for M1-type and M2-type macrophages, we found noninflammatory characteristics in macrophages recovered from the malignant ascites of GC. Using public datasets, we demonstrated that the single-cell transcriptome-driven M2-specific signature was associated with poor prognosis in GC. Our data indicate that the anti-inflammatory characteristics of TAMs are controlled by tumor cells and present implications for treatment strategies for GC patients in which combination treatment targeting cancer cells and macrophages may have a reciprocal synergistic effect.

Immunotherapy for metastatic colorectal cancer is effective only for mismatch repair-deficient tumors with high microsatellite instability that demonstrate immune infiltration, suggesting that tumor cells can determine their immune microenvironment. To understand this cross-talk, we analyzed the transcriptome of 91,103 unsorted single cells from 23 Korean and 6 Belgian patients. Cancer cells displayed transcriptional features reminiscent of normal differentiation programs, and genetic alterations that apparently fostered immunosuppressive microenvironments directed by regulatory T cells, myofibroblasts and myeloid cells. Intercellular network reconstruction supported the association between cancer cell signatures and specific stromal or immune cell populations. Our collective view of the cellular landscape and intercellular interactions in colorectal cancer provide mechanistic information for the design of efficient immuno-oncology treatment strategies.

Advanced metastatic cancer poses utmost clinical challenges and may present molecular and cellular features distinct from an early-stage cancer. Herein, we present single-cell transcriptome profiling of metastatic lung adenocarcinoma, the most prevalent histological lung cancer type diagnosed at stage IV in over 40% of all cases. From 208,506 cells populating the normal tissues or early to metastatic stage cancer in 44 patients, we identify a cancer cell subtype deviating from the normal differentiation trajectory and dominating the metastatic stage. In all stages, the stromal and immune cell dynamics reveal ontological and functional changes that create a pro-tumoral and immunosuppressive microenvironment. Normal resident myeloid cell populations are gradually replaced with monocyte-derived macrophages and dendritic cells, along with T-cell exhaustion. This extensive single-cell analysis enhances our understanding of molecular and cellular dynamics in metastatic lung cancer and reveals potential diagnostic and therapeutic targets in cancer-microenvironment interactions.

Alternative polyadenylation (APA) in 3' untranslated regions (3' UTR) plays an important role in regulating transcript abundance, localization, and interaction with microRNAs. Length-variation of 3'UTRs by APA contributes to efficient proliferation of cancer cells. In this study, we investigated APA in single cancer cells and tumor microenvironment cells to understand the physiological implication of APA in different cell types. We analyzed APA patterns and the expression level of genes from the 515 single-cell RNA sequencing (scRNA-seq) dataset from 11 breast cancer patients. Although the overall 3'UTR length of individual genes was distributed equally in tumor and non-tumor cells, we found a differential pattern of polyadenylation in gene sets between tumor and non-tumor cells. In addition, we found a differential pattern of APA across tumor types using scRNA-seq data from 3 glioblastoma patients and 1 renal cell carcinoma patients. In detail, 1,176 gene sets and 53 genes showed the distinct pattern of 3'UTR shortening and over-expression as signatures for five cell types including B lymphocytes, T lymphocytes, myeloid cells, stromal cells, and breast cancer cells. Functional categories of gene sets for cellular proliferation demonstrated concordant regulation of APA and gene expression specific to cell types. The expression of APA genes in breast cancer was significantly correlated with the clinical outcome of earlier stage breast cancer patients. We identified cell type-specific APA in single cells, which allows the identification of cell types based on 3'UTR length variation in combination with gene expression. Specifically, an immune-specific APA signature in breast cancer could be utilized as a prognostic marker of early stage breast cancer., Competing Interests: W.P. is employed by a commercial company, GENINUS Inc. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Simultaneous sequencing of the genome and transcriptome at the single-cell level is a powerful tool for characterizing genomic and transcriptomic variation and revealing correlative relationships. However, it remains technically challenging to analyze both the genome and transcriptome in the same cell. Here, we report a novel method for simultaneous isolation of genomic DNA and total RNA (SIDR) from single cells, achieving high recovery rates with minimal cross-contamination, as is crucial for accurate description and integration of the single-cell genome and transcriptome. For reliable and efficient separation of genomic DNA and total RNA from single cells, the method uses hypotonic lysis to preserve nuclear lamina integrity and subsequently captures the cell lysate using antibody-conjugated magnetic microbeads. Evaluating the performance of this method using real-time PCR demonstrated that it efficiently recovered genomic DNA and total RNA. Thorough data quality assessments showed that DNA and RNA simultaneously fractionated by the SIDR method were suitable for genome and transcriptome sequencing analysis at the single-cell level. The integration of single-cell genome and transcriptome sequencing by SIDR (SIDR-seq) showed that genetic alterations, such as copy-number and single-nucleotide variations, were more accurately captured by single-cell SIDR-seq compared with conventional single-cell RNA-seq, although copy-number variations positively correlated with the corresponding gene expression levels. These results suggest that SIDR-seq is potentially a powerful tool to reveal genetic heterogeneity and phenotypic information inferred from gene expression patterns at the single-cell level., (© 2018 Han et al.; Published by Cold Spring Harbor Laboratory Press.)

Single-cell transcriptome profiling of tumour tissue isolates allows the characterization of heterogeneous tumour cells along with neighbouring stromal and immune cells. Here we adopt this powerful approach to breast cancer and analyse 515 cells from 11 patients. Inferred copy number variations from the single-cell RNA-seq data separate carcinoma cells from non-cancer cells. At a single-cell resolution, carcinoma cells display common signatures within the tumour as well as intratumoral heterogeneity regarding breast cancer subtype and crucial cancer-related pathways. Most of the non-cancer cells are immune cells, with three distinct clusters of T lymphocytes, B lymphocytes and macrophages. T lymphocytes and macrophages both display immunosuppressive characteristics: T cells with a regulatory or an exhausted phenotype and macrophages with an M2 phenotype. These results illustrate that the breast cancer transcriptome has a wide range of intratumoral heterogeneity, which is shaped by the tumour cells and immune cells in the surrounding microenvironment.

Background: Identification of the causative genes of retinitis pigmentosa (RP) is important for the clinical care of patients with RP. However, a comprehensive genetic study has not been performed in Korean RP patients. Moreover, the genetic heterogeneity found in sensorineural genetic disorders makes identification of pathogenic mutations challenging. Therefore, high throughput genetic testing using massively parallel sequencing is needed., Results: Sixty-two Korean patients with nonsyndromic RP (46 patients from 18 families and 16 simplex cases) who consented to molecular genetic testing were recruited in this study and targeted exome sequencing was applied on 53 RP-related genes. Causal variants were characterised by selecting exonic and splicing variants, selecting variants with low allele frequency (below 1 %), and discarding the remaining variants with quality below 20. The variants were additionally confirmed by an inheritance pattern and cosegregation test of the families, and the rest of the variants were prioritised using in-silico prediction tools. Finally, causal variants were detected from 10 of 18 familial cases (55.5 %) and 7 of 16 simplex cases (43.7 %) in total. Novel variants were detected in 13 of 20 (65 %) candidate variants. Compound heterozygous variants were found in four of 7 simplex cases., Conclusion: Panel-based targeted re-sequencing can be used as an effective molecular diagnostic tool for RP.

Background: Intra-tumoral genetic and functional heterogeneity correlates with cancer clinical prognoses. However, the mechanisms by which intra-tumoral heterogeneity impacts therapeutic outcome remain poorly understood. RNA sequencing (RNA-seq) of single tumor cells can provide comprehensive information about gene expression and single-nucleotide variations in individual tumor cells, which may allow for the translation of heterogeneous tumor cell functional responses into customized anti-cancer treatments., Results: We isolated 34 patient-derived xenograft (PDX) tumor cells from a lung adenocarcinoma patient tumor xenograft. Individual tumor cells were subjected to single cell RNA-seq for gene expression profiling and expressed mutation profiling. Fifty tumor-specific single-nucleotide variations, including KRAS(G12D), were observed to be heterogeneous in individual PDX cells. Semi-supervised clustering, based on KRAS(G12D) mutant expression and a risk score representing expression of 69 lung adenocarcinoma-prognostic genes, classified PDX cells into four groups. PDX cells that survived in vitro anti-cancer drug treatment displayed transcriptome signatures consistent with the group characterized by KRAS(G12D) and low risk score., Conclusions: Single-cell RNA-seq on viable PDX cells identified a candidate tumor cell subgroup associated with anti-cancer drug resistance. Thus, single-cell RNA-seq is a powerful approach for identifying unique tumor cell-specific gene expression profiles which could facilitate the development of optimized clinical anti-cancer strategies.