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Chemotherapy, the standard of care treatment for cancer patients with advanced disease, has been increasingly recognized to activate host immune responses to produce durable outcomes. Here, in colorectal adenocarcinoma (CRC) we identify oxaliplatin-induced Thioredoxin-Interacting Protein (TXNIP), a MondoA-dependent tumor suppressor gene, as a negative regulator of Growth/Differentiation Factor 15 (GDF15). GDF15 is a negative prognostic factor in CRC and promotes the differentiation of regulatory T cells (Tregs), which inhibit CD8 T-cell activation. Intriguingly, multiple models including patient-derived tumor organoids demonstrate that the loss of TXNIP and GDF15 responsiveness to oxaliplatin is associated with advanced disease or chemotherapeutic resistance, with transcriptomic or proteomic GDF15/TXNIP ratios showing potential as a prognostic biomarker. These findings illustrate a potentially common pathway where chemotherapy-induced epithelial oxidative stress drives local immune remodeling for patient benefit, with disruption of this pathway seen in refractory or advanced cases., (© 2024. The Author(s).)

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).)

In normal intestines, a fetal/regenerative/revival cell state can be induced upon inflammation. This plasticity in cell fate is also one of the current topics in human colorectal cancer (CRC). To dissect the underlying mechanisms, we generated human CRC organoids with naturally selected genetic mutation profiles and exposed them to two different conditions by modulating the extracellular matrix (ECM). Among tested mutation profiles, a fetal/regenerative/revival state was induced following YAP activation via a collagen type I-enriched microenvironment. Mechanistically, YAP transcription was promoted by activating AP-1 and TEAD-dependent transcription and suppressing intestinal lineage-determining transcription via mechanotransduction. The phenotypic conversion was also involved in chemoresistance, which could be potentially resolved by targeting the underlying YAP regulatory elements, a potential target of CRC treatment., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

CD8 + T cell activation via immune checkpoint blockade (ICB) is successful in microsatellite instable (MSI) colorectal cancer (CRC) patients. By comparison, the success of immunotherapy against microsatellite stable (MSS) CRC is limited. Little is known about the most critical features of CRC CD8 + T cells that together determine the diverse immune landscapes and contrasting ICB responses. Hence, we pursued a deep single cell mapping of CRC CD8 + T cells on transcriptomic and T cell receptor (TCR) repertoire levels in a diverse patient cohort, with additional surface proteome validation. This revealed that CRC CD8 + T cell dynamics are underscored by complex interactions between interferon-γ signaling, tumor reactivity, TCR repertoire, (predicted) TCR antigen-specificities, and environmental cues like gut microbiome or colon tissue-specific 'self-like' features. MSI CRC CD8 + T cells showed tumor-specific activation reminiscent of canonical 'T cell hot' tumors, whereas the MSS CRC CD8 + T cells exhibited tumor unspecific or bystander-like features. This was accompanied by inflammation reminiscent of 'pseudo-T cell hot' tumors. Consequently, MSI and MSS CRC CD8 + T cells showed overlapping phenotypic features that differed dramatically in their TCR antigen-specificities. Given their high discriminating potential for CD8 + T cell features/specificities, we used the single cell tumor-reactive signaling modules in CD8 + T cells to build a bulk tumor transcriptome classification for CRC patients. This "Immune Subtype Classification" (ISC) successfully distinguished various tumoral immune landscapes that showed prognostic value and predicted immunotherapy responses in CRC patients. Thus, we deliver a unique map of CRC CD8 + T cells that drives a novel tumor immune landscape classification, with relevance for immunotherapy decision-making., (© 2023. The Author(s).)

The thyroid gland plays a critical role in the maintenance of whole-body metabolism. However, aging frequently impairs homeostatic maintenance by thyroid hormones due to increased prevalence of subclinical hypothyroidism associated with mitochondrial dysfunction, inflammation, and fibrosis. To understand the specific aging-related changes of endocrine function in thyroid epithelial cells, we performed single-cell RNA sequencing (RNA-seq) of 54 726 cells derived from pathologically normal thyroid tissues from 7 patients who underwent thyroidectomy. Thyroid endocrine epithelial cells were clustered into 5 distinct subpopulations, and a subset of cells was found to be particularly vulnerable with aging, showing functional deterioration associated with the expression of metallothionein (MT) and major histocompatibility complex class II genes. We further validated that increased expression of MT family genes are highly correlated with thyroid gland aging in bulk RNAseq datasets. This study provides evidence that aging induces specific transcriptomic changes across multiple cell populations in the human thyroid gland., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The consensus molecular subtype (CMS) classification of colorectal cancer is based on bulk transcriptomics. The underlying epithelial cell diversity remains unclear. We analyzed 373,058 single-cell transcriptomes from 63 patients, focusing on 49,155 epithelial cells. We identified a pervasive genetic and transcriptomic dichotomy of malignant cells, based on distinct gene expression, DNA copy number and gene regulatory network. We recapitulated these subtypes in bulk transcriptomes from 3,614 patients. The two intrinsic subtypes, iCMS2 and iCMS3, refine CMS. iCMS3 comprises microsatellite unstable (MSI-H) cancers and one-third of microsatellite-stable (MSS) tumors. iCMS3 MSS cancers are transcriptomically more similar to MSI-H cancers than to other MSS cancers. CMS4 cancers had either iCMS2 or iCMS3 epithelium; the latter had the worst prognosis. We defined the intrinsic epithelial axis of colorectal cancer and propose a refined 'IMF' classification with five subtypes, combining intrinsic epithelial subtype (I), microsatellite instability status (M) and fibrosis (F)., (© 2022. The Author(s).)

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.

To elucidate the epigenetic mechanisms of drug resistance, epigenetically reprogrammed H460 cancer cells (R-H460) were established by the transient introduction of reprogramming factors. Then, the R-H460 cells were induced to differentiate by the withdrawal of stem cell media for various durations, which resulted in differentiated R-H460 cells (dR-H460). Notably, dR-H460 cells differentiated for 13 days (13dR-H460 cells) formed a significantly greater number of colonies showing drug resistance to both cisplatin and paclitaxel, whereas the dR-H460 cells differentiated for 40 days (40dR-H460 cells) lost drug resistance; this suggests that 13dR-cancer cells present short-term resistance (less than a month). Similarly, increased drug resistance to both cisplatin and paclitaxel was observed in another R-cancer cell model prepared from N87 cells. The resistant phenotype of the cisplatin-resistant (CR) colonies obtained through cisplatin treatment was maintained for 2-3 months after drug treatment, suggesting that drug treatment transforms cells with short-term resistance into cells with medium-term resistance. In single-cell analyses, heterogeneity was not found to increase in 13dR-H460 cells, suggesting that cancer cells with short-term resistance, rather than heterogeneous cells, may confer epigenetically driven drug resistance in our reprogrammed cancer model. The epigenetically driven short-term and medium-term drug resistance mechanisms could provide new cancer-fighting strategies involving the control of cancer cells during epigenetic transition.

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.

Purpose: In multiple myeloma, extramedullary progression is associated with treatment resistance and a high mortality rate. To understand the molecular mechanisms controlling the devastating progression of myeloma, we applied single-cell RNA-sequencing (RNA-seq) to myeloma in the bone marrow and myelomatous pleural effusions or ascites., Experimental Design: Bone marrow or extramedullary myeloma samples were collected from 15 patients and subjected to single-cell RNA-seq. The single-cell transcriptome data of malignant plasma cells and the surrounding immune microenvironment were analyzed., Results: Comparisons of single-cell transcriptomes revealed the systematic activation of proliferation, antigen presentation, proteasomes, glycolysis, and oxidative phosphorylation pathways in extramedullary myeloma cells. The myeloma cells expressed multiple combinations of growth factors and receptors, suggesting autonomous and pleiotropic growth potential at the single-cell level. Comparisons of the tumor microenvironment revealed the presence of cytotoxic T lymphocytes and natural killer (NK) cells in both the bone marrow and extramedullary ascites, demonstrating a gene-expression phenotype indicative of functional compromise. In parallel, isolated myeloma cells persistently expressed class I MHC molecules and upregulated inhibitory molecules for cytotoxic T and NK cells., Conclusions: These data suggest that myeloma cells are equipped with specialized immune evasion mechanisms in cytotoxic microenvironments. Taken together, single-cell transcriptome analysis revealed transcriptional programs associated with aggressive myeloma progression that support autonomous cell proliferation and immune evasion., (©2019 American Association for Cancer Research.)

Background: Controlled turnover of proteins as mediated by the ubiquitin proteasome system (UPS) is an important element in plant defense against environmental and pathogen stresses. E3 ligases play a central role in subjecting proteins to hydrolysis by the UPS. Recently, it has been demonstrated that a specific class of E3 ligases termed the U-box ligases are directly associated with the defense mechanisms against abiotic and biotic stresses in several plants. However, no studies on U-box E3 ligases have been performed in one of the important staple crops, barley., Results: In this study, we identified 67 putative U-box E3 ligases from the barley genome and expressed sequence tags (ESTs). Similar to Arabidopsis and rice U-box E3 ligases, most of barley U-box E3 ligases possess evolutionary well-conserved domain organizations. Based on the domain compositions and arrangements, the barley U-box proteins were classified into eight different classes. Along with this new classification, we refined the previously reported classifications of U-box E3 ligase genes in Arabidopsis and rice. Furthermore, we investigated the expression profile of 67 U-box E3 ligase genes in response to drought stress and pathogen infection. We observed that many U-box E3 ligase genes were specifically up-and-down regulated by drought stress or by fungal infection, implying their possible roles of some U-box E3 ligase genes in the stress responses., Conclusion: This study reports the classification of U-box E3 ligases in barley and their expression profiles against drought stress and pathogen infection. Therefore, the classification and expression profiling of barley U-box genes can be used as a platform to functionally define the stress-related E3 ligases in barley.

Background: Cell surface proteins have provided useful targets and biomarkers for advanced cancer therapies. The recent clinical success of antibody-drug conjugates (ADCs) highlights the importance of finding selective surface antigens for given cancer subtypes. We thus attempted to develop stand-alone software for the analysis of the cell surface transcriptome of patient cancer samples and to prioritize lineage- and/or mutation-specific over-expression markers in cancer cells., Results: A total of 519 genes were selected as surface proteins, and their expression was profiled in 14 cancer subtypes using patient sample transcriptome data. Lineage/mutation-oriented analysis was used to identify subtype-specific surface markers with statistical confidence. Experimental validation confirmed the unique over-expression of predicted surface markers (MUC4, MSLN, and SLC7A11) in lung cancer cells at the protein level. The differential cell surface gene expression of cell lines may differ from that of tissue samples due to the absence of the tumor microenvironment., Conclusions: In the present study, advanced 3D models of lung cell lines successfully reproduced the predicted patterns, demonstrating the physiological relevance of cell line-based 3D models in validating surface markers from patient tumor data. Also QSurface software is freely available at http://compbio.sookmyung.ac.kr/~qsurface .

Although a large collection of cancer cell lines are useful surrogates for patient samples, the physiological relevance of observed molecular phenotypes in cell lines remains controversial. Because transcriptome data are a representative set of molecular phenotypes in cancers, we systematically analyzed the discrepancy of global gene expression profiles between patient samples and cell lines in breast cancers. While the majority of genes exhibited general consistency between patient samples and cell lines, the expression of genes in the categories of extracellular matrix, collagen trimers, receptor activity, catalytic activity and transporter activity were significantly up-regulated only in tissue samples. Genes in the extracellular matrix, particularly collagen trimers, showed a wide variation of expression in tissue, but minimal expression and variation in cell lines. Further analysis of tissue samples exclusively revealed that collagen genes exhibited a cancer stage-dependent expressional variation based on their supramolecular structure. Prognostic collagen biomarkers associated with survival rate were also readily predicted from tissue-oriented transcriptome analysis. This study presents the limitations of cell lines and the exclusive features of tissue samples in terms of functional categories of the cancer transcriptome., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cancer stem-like cells (CSLCs) contribute to the initiation and recurrence of tumors and to their resistance to conventional therapies. In this study, small interfering RNA (siRNA)-based screening of ∼4800 druggable genes in 3-dimensional CSLC cultures in comparison to 2-dimensional bulk cultures of U87 glioma cells revealed 3 groups of genes essential for the following: survival of the CSLC population only, bulk-cultured population only, or both populations. While diverse biologic processes were associated with siRNAs reducing the bulk-cultured population, CSLC-eliminating siRNAs were enriched in a few functional categories, such as lipid metabolism, protein metabolism, and gene expression. Interestingly, siRNAs that selectively reduced CSLC only were found to target genes for cholesterol and unsaturated fatty acid synthesis. The lipidomic profile of CSLCs revealed increased levels of monounsaturated lipids. Pharmacologic blockage of these target pathways reduced CSLCs, and this effect was eliminated by addition of downstream metabolite products. The present CSLC-sensitive target categories provide a useful resource that can be exploited for the selective elimination of CSLCs.-Song, M., Lee, H., Nam, M.-H., Jeong, E., Kim, S., Hong, Y., Kim, N., Yim, H. Y., Yoo, Y.-J., Kim, J. S., Kim, J.-S., Cho, Y.-Y., Mills, G. B., Kim, W.-Y., Yoon, S. Loss-of-function screens of druggable targetome against cancer stem-like cells., (© The Author(s).)

Somatic mutation is a major cause of cancer progression and varied responses of tumors against anticancer agents. Thus, we must obtain and characterize genome-wide mutational profiles in individual cancer subtypes. The Cancer Genome Atlas database includes large amounts of sequencing and omics data generated from diverse human cancer tissues. In the present study, we integrated and analyzed the exome sequencing data from ~3,000 tissue samples and summarized the major mutant genes in each of the diverse cancer subtypes and stages. Mutations were observed in most human genes (~23,000 genes) with low frequency from an analysis of 11 major cancer subtypes. The majority of tissue samples harbored 20-80 different mutant genes, on average. Lung cancer samples showed a greater number of mutations in diverse genes than other cancer subtypes. Only a few genes were mutated with over 5% frequency in tissue samples. Interestingly, mutation frequency was generally similar between non-metastatic and metastastic samples in most cancer subtypes. Among the 12 major mutations, the TP53, USH2A, TTN, and MUC16 genes were found to be frequent in most cancer types, while BRAF, FRG1B, PBRM1, and VHL showed lineage-specific mutation patterns. The present study provides a useful resource to understand the broad spectrum of mutation frequencies in various cancer types.