Clinical drug screening reveals clofazimine potentiates the efficacy while reducing the toxicity of anti-PD-1 and CTLA-4 immunotherapy.

Emerging as the most potent and durable combinational immunotherapy, dual anti-PD-1 and CTLA-4 immune checkpoint blockade (ICB) therapy notoriously increases grade 3-5 immune-related adverse events (irAEs) in patients. Accordingly, attempts to improve the antitumor potency of anti-PD-1+CTLA-4 ICB by including additional therapeutics have been largely discouraged due to concerns of further increasing fatal toxicity. Here, we screened ∼3,000 Food and Drug Administration (FDA)-approved drugs and identified clofazimine as a potential third agent to optimize anti-PD-1+CTLA-4 ICB. Remarkably, clofazimine outperforms ICB dose reduction or steroid treatment in reversing lethality of irAEs, but unlike the detrimental effect of steroids on antitumor efficacy, clofazimine potentiates curative responses in anti-PD-1+CTLA-4 ICB. Mechanistically, clofazimine promotes E2F1 activation in CD8+ T cells to overcome resistance and counteracts pathogenic Th17 cells to abolish irAEs. Collectively, clofazimine potentiates the antitumor efficacy of anti-PD-1+CTLA-4 ICB, curbs intractable irAEs, and may fill a desperate clinical need to improve patient survival.

Induction of tumor cell autosis by myxoma virus-infected CAR-T and TCR-T cells to overcome primary and acquired resistance.

Cytotoxicity of tumor-specific T cells requires tumor cell-to-T cell contact-dependent induction of classic tumor cell apoptosis and pyroptosis. However, this may not trigger sufficient primary responses of solid tumors to adoptive cell therapy or prevent tumor antigen escape-mediated acquired resistance. Here we test myxoma virus (MYXV)-infected tumor-specific T (TMYXV) cells expressing chimeric antigen receptor (CAR) or T cell receptor (TCR), which systemically deliver MYXV into solid tumors to overcome primary resistance. In addition to T cell-induced apoptosis and pyroptosis, tumor eradication by CAR/TCR-TMYXV cells is also attributed to tumor cell autosis induction, a special type of cell death. Mechanistically, T cell-derived interferon γ (IFNγ)-protein kinase B (AKT) signaling synergizes with MYXV-induced M-T5-SKP-1-VPS34 signaling to trigger robust tumor cell autosis. CAR/TCR-TMYXV-elicited autosis functions as a type of potent bystander killing to restrain antigen escape. We uncover an unexpected synergy between T cells and MYXV to bolster solid tumor cell autosis that reinforces tumor clearance.

VOC-alarm: mutation-based prediction of SARS-CoV-2 variants of concern.

SUMMARY: Mutation is the key for a variant of concern (VOC) to overcome selective pressures, but this process is still unclear. Understanding the association of the mutational process with VOCs is an unmet need. Motivation: Here, we developed VOC-alarm, a method to predict VOCs and their caused COVID surges, using mutations of about 5.7 million SARS-CoV-2 complete sequences. We found that VOCs rely on lineage-level entropy value of mutation numbers to compete with other variants, suggestive of the importance of population-level mutations in the virus evolution. Thus, we hypothesized that VOCs are a result of a mutational process across the globe. Results: Analyzing the mutations from January 2020 to December 2021, we simulated the mutational process by estimating the pace of evolution, and thus divided the time period, January 2020-March 2022, into eight stages. We predicted Alpha, Delta, Delta Plus (AY.4.2) and Omicron (B.1.1.529) by their mutational entropy values in the Stages I, III, V and VII with accelerated paces, respectively. In late November 2021, VOC-alarm alerted that Omicron strongly competed with Delta and Delta plus to become a highly transmissible variant. Using simulated data, VOC-alarm also predicted that Omicron could lead to another COVID surge from January 2022 to March 2022. AVAILABILITY AND IMPLEMENTATION: Our software implementation is available at https://github.com/guangxujin/VOC-alarm. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Breast cancer extracellular vesicles-derived miR-1290 activates astrocytes in the brain metastatic microenvironment via the FOXA2→CNTF axis to promote progression of brain metastases.

Mechanisms underlying breast cancer brain metastasis (BCBM) are still unclear. In this study, we observed that extracellular vesicles (EVs) secreted from breast cancer cells with increased expression of tGLI1, a BCBM-promoting transcription factor, strongly activated astrocytes. EV-derived microRNA/miRNA microarray revealed tGLI1-positive breast cancer cells highly secreted miR-1290 and miR-1246 encapsulated in EVs. Genetic knockin/knockout studies established a direct link between tGLI1 and both miRNAs. Datamining and analysis of patient samples revealed that BCBM patients had more circulating EV-miRs-1290/1246 than those without metastasis. Ectopic expression of miR-1290 or miR-1246 strongly activated astrocytes whereas their inhibitors abrogated the effect. Conditioned media from miR-1290- or miR-1246-overexpressing astrocytes promoted mammospheres. Furthermore, miRs-1290/1246 suppressed expression of FOXA2 transcription repressor, leading to CNTF cytokine secretion and subsequent activation of astrocytes. Finally, we conducted a mouse study to demonstrate that astrocytes overexpressing miR-1290, but not miR-1246, enhanced intracranial colonization and growth of breast cancer cells. Collectively, our findings demonstrate, for the first time, that breast cancer EV-derived miR-1290 and miR-1246 activate astrocytes in the brain metastatic microenvironment and that EV-derived miR-1290 promotes progression of brain metastases through the novel EV-miR-1290→FOXA2→CNTF signaling axis.

NEDD4 degrades TUSC2 to promote glioblastoma progression.

Whether tumor suppressor candidate 2 (TUSC2) plays an important role in glioblastoma (GBM) progression is largely unknown. Whether TUSC2 undergoes polyubiquitination is unknown. Herein, we report that TUSC2 protein expression is reduced/lost in GBM compared to normal brain due to protein destabilization; TUSC2 mRNA is equally expressed in both tissues. NEDD4 E3 ubiquitin ligase polyubiquitinates TUSC2 at residue K71, and the TUSC2-K71R mutant is resistant to NEDD4-mediated proteasomal degradation. Analysis of GBM specimens showed NEDD4 protein is highly expressed in GBM and the level is inversely correlated with TUSC2 protein levels. Furthermore, TUSC2 restoration induces apoptosis and inhibits patient-derived glioma stem cells (PD-GSCs) in vitro and in vivo. Conversely, TUSC2-knockout promotes PD-GSCs in vitro and in vivo. RNA-Seq analysis and subsequent validations showed GBM cells with TUSC2-knockout expressed increased Bcl-xL and were more resistant to apoptosis induced by a Bcl-xL-specific BH3 mimetic. A TUSC2-knockout gene signature created from the RNA-seq data predicts poor patient survival. Together, these findings establish that NEDD4-mediated polyubiquitination is a novel mechanism for TUSC2 degradation in GBM and that TUSC2 loss promotes GBM progression in part through Bcl-xL upregulation.

hDirect-MAP: projection-free single-cell modeling of response to checkpoint immunotherapy.

There is a lack of robust generalizable predictive biomarkers of response to immune checkpoint blockade in multiple types of cancer. We develop hDirect-MAP, an algorithm that maps T cells into a shared high-dimensional (HD) expression space of diverse T cell functional signatures in which cells group by the common T cell phenotypes rather than dimensional reduced features or a distorted view of these features. Using projection-free single-cell modeling, hDirect-MAP first removed a large group of cells that did not contribute to response and then clearly distinguished T cells into response-specific subpopulations that were defined by critical T cell functional markers of strong differential expression patterns. We found that these grouped cells cannot be distinguished by dimensional-reduction algorithms but are blended by diluted expression patterns. Moreover, these identified response-specific T cell subpopulations enabled a generalizable prediction by their HD metrics. Tested using five single-cell RNA-seq or mass cytometry datasets from basal cell carcinoma, squamous cell carcinoma and melanoma, hDirect-MAP demonstrated common response-specific T cell phenotypes that defined a generalizable and accurate predictive biomarker.

Bulk and Single-Cell Profiling of Breast Tumors Identifies TREM-1 as a Dominant Immune Suppressive Marker Associated With Poor Outcomes.

BACKGROUND: Triggering receptor expressed on myeloid cells (TREM)-1 is a key mediator of innate immunity previously associated with the severity of inflammatory disorders, and more recently, the inferior survival of lung and liver cancer patients. Here, we investigated the prognostic impact and immunological correlates of TREM1 expression in breast tumors. METHODS: Breast tumor microarray and RNAseq expression profiles (n=4,364 tumors) were analyzed for associations between gene expression, tumor immune subtypes, distant metastasis-free survival (DMFS) and clinical response to neoadjuvant chemotherapy (NAC). Single-cell (sc)RNAseq was performed using the 10X Genomics platform. Statistical associations were assessed by logistic regression, Cox regression, Kaplan-Meier analysis, Spearman correlation, Student's t-test and Chi-square test. RESULTS: In pre-treatment biopsies, TREM1 and known TREM-1 inducible cytokines (IL1B, IL8) were discovered by a statistical ranking procedure as top genes for which high expression was associated with reduced response to NAC, but only in the context of immunologically "hot" tumors otherwise associated with a high NAC response rate. In surgical specimens, TREM1 expression varied among tumor molecular subtypes, with highest expression in the more aggressive subtypes (Basal-like, HER2-E). High TREM1 significantly and reproducibly associated with inferior distant metastasis-free survival (DMFS), independent of conventional prognostic markers. Notably, the association between high TREM1 and inferior DMFS was most prominent in the subset of immunogenic tumors that exhibited the immunologically hot phenotype and otherwise associated with superior DMFS. Further observations from bulk and single-cell RNAseq analyses indicated that TREM1 expression was significantly enriched in polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and M2-like macrophages, and correlated with downstream transcriptional targets of TREM-1 (IL8, IL-1B, IL6, MCP-1, SPP1, IL1RN, INHBA) which have been previously associated with pro-tumorigenic and immunosuppressive functions. CONCLUSIONS: Together, these findings indicate that increased TREM1 expression is prognostic of inferior breast cancer outcomes and may contribute to myeloid-mediated breast cancer progression and immune suppression.

Elimination of acquired resistance to PD-1 blockade via the concurrent depletion of tumour cells and immunosuppressive cells.

Antigen release resulting from the death of tumour cells induced by chemotherapies and targeted therapies can augment the antitumour responses induced by immune checkpoint blockade (ICB). However, tumours responding to ICB therapies often become resistant to them. Here we show that the specific targeting of tumour cells promotes the growth of tumour-cell variants that are resistant to ICB, and that the acquired resistance can be overcome via the concurrent depletion of tumour cells and of major types of immunosuppressive cell via a monoclonal antibody binding the enzyme CD73, which we identified as highly expressed on tumour cells and on regulatory T cells, myeloid-derived suppressor cells and tumour-associated macrophages, but not on cytolytic T lymphocytes, natural killer cells and dendritic cells. In mice with murine tumours, the systemic administration of anti-PD1 antibodies and anti-CD73 antibodies conjugated to a near-infrared dye prevented near-infrared-irradiated tumours from acquiring resistance to ICB and resulted in the eradication of advanced tumours. The elimination of immunosuppressive cells may overcome acquired resistance to ICB across a range of tumour types and combination therapies.

Adoptive cell therapy with tumor-specific Th9 cells induces viral mimicry to eliminate antigen-loss-variant tumor cells.

Resistance can occur in patients receiving adoptive cell therapy (ACT) due to antigen-loss-variant (ALV) cancer cell outgrowth. Here we demonstrate that murine and human T helper (Th) 9 cells, but not Th1/Tc1 or Th17 cells, expressing tumor-specific T cell receptors (TCRs) or chimeric antigen receptors (CARs), eradicate advanced tumors that contain ALVs. This unprecedented antitumor capacity of Th9 cells is attributed to both enhanced direct tumor cell killing and bystander antitumor effects promoted by intratumor release of interferon (IFN) α/ß. Mechanistically, tumor-specific Th9 cells increase the intratumor accumulation of extracellular ATP (eATP; released from dying tumor cells), because of a unique feature of Th9 cells that lack the expression of ATP degrading ectoenzyme cluster of differentiation (CD) 39. Intratumor enrichment of eATP promotes the monocyte infiltration and stimulates their production of IFNα/ß by inducing eATP-endogenous retrovirus-Toll-like receptor 3 (TLR3)/mitochondrial antiviral signaling (MAVS) pathway activation. These results identify tumor-specific Th9 cells as a unique T cell subset endowed with the unprecedented capacity to eliminate ALVs for curative responses.

Inactivating Mutations of the IK Gene Weaken Ku80/Ku70-Mediated DNA Repair and Sensitize Endometrial Cancer to Chemotherapy.

IK is a mitotic factor that promotes cell cycle progression. Our previous investigation of 271 endometrial cancer (EC) samples from the Cancer Genome Atlas (TCGA) dataset showed IK somatic mutations were enriched in a cluster of patients with high-grade and high-stage cancers, and this group had longer survival. This study provides insight into how IK somatic mutations contribute to EC pathophysiology. We analyzed the somatic mutational landscape of IK gene in 547 EC patients using expanded TCGA dataset. Co-immunoprecipitation and mass spectrometry were used to identify protein interactions. In vitro and in vivo experiments were used to evaluate IK's role in EC. The patients with IK-inactivating mutations had longer survival during 10-year follow-up. Frameshift and stop-gain were common mutations and were associated with decreased IK expression. IK knockdown led to enrichment of G2/M phase cells, inactivation of DNA repair signaling mediated by heterodimerization of Ku80 and Ku70, and sensitization of EC cells to cisplatin treatment. IK/Ku80 mutations were accompanied by higher mutation rates and associated with significantly better overall survival. Inactivating mutations of IK gene and loss of IK protein expression were associated with weakened Ku80/Ku70-mediated DNA repair, increased mutation burden, and better response to chemotherapy in patients with EC.

TrkA Interacts with and Phosphorylates STAT3 to Enhance Gene Transcription and Promote Breast Cancer Stem Cells in Triple-Negative and HER2-Enriched Breast Cancers.

JAK2-STAT3 and TrkA signaling pathways have been separately implicated in aggressive breast cancers; however, whether they are co-activated or undergo functional interaction has not been thoroughly investigated. Herein we report, for the first time that STAT3 and TrkA are significantly co-overexpressed and co-activated in triple-negative breast cancer (TNBC) and HER2-enriched breast cancer, as shown by immunohistochemical staining and data mining. Through immunofluorescence staining-confocal microscopy and immunoprecipitation-Western blotting, we found that TrkA and STAT3 co-localize and physically interact in the cytoplasm, and the interaction is dependent on STAT3-Y705 phosphorylation. TrkA-STAT3 interaction leads to STAT3 phosphorylation at Y705 by TrkA in breast cancer cells and cell-free kinase assays, indicating that STAT3 is a novel substrate of TrkA. ß-NGF-mediated TrkA activation induces TrkA-STAT3 interaction, STAT3 nuclear transport and transcriptional activity, and the expression of STAT3 target genes, SOX2 and MYC. The co-activation of both pathways promotes breast cancer stem cells. Finally, we found that TNBC and HER2-enriched breast cancer with JAK2-STAT3 and TrkA co-activation are positively associated with poor overall metastasis-free and organ-specific metastasis-free survival. Collectively, our study uncovered that TrkA is a novel activating kinase of STAT3, and their co-activation enhances gene transcription and promotes breast cancer stem cells in TNBC and HER2-enriched breast cancer.

Origin-independent analysis links SARS-CoV-2 local genomes with COVID-19 incidence and mortality.

There is an urgent public health need to better understand Severe Acute Respiratory Syndrome (SARS)-CoV-2/COVID-19, particularly how sequences of the viruses could lead to diverse incidence and mortality of COVID-19 in different countries. However, because of its unknown ancestors and hosts, elucidating the genetic variations of the novel coronavirus, SARS-CoV-2, has been difficult. Without needing to know ancestors, we identified an uneven distribution of local genome similarities among the viruses categorized by geographic regions, and it was strongly correlated with incidence and mortality. To ensure unbiased and origin-independent analyses, we used a pairwise comparison of local genome sequences of virus genomes by Basic Local Alignment Search Tool (BLAST). We found a strong statistical correlation between dominance of the SARS-CoV-2 in distributions of uneven similarities and the incidence and mortality of illness. Genomic annotation of the BLAST hits also showed that viruses from geographic regions with severe infections tended to have more dynamic genomic regions in the SARS-CoV-2 receptor-binding domain (RBD) and receptor-binding motif (RBM) of the spike protein (S protein). Dynamic domains in the S protein were also confirmed by a canyon region of mismatches coincident with RBM and RBD, without hits of alignments of 100% matching. Thus, our origin-independent analysis suggests that the dynamic and unstable SARS-CoV-2-RBD could be the main reason for diverse incidence and mortality of COVID-19 infection.

Clinical Implications of Genetic Signatures in Appendiceal Cancer Patients with Incomplete Cytoreduction/HIPEC.

INTRODUCTION: Clinical decision-making is challenging in patients who undergo cytoreductive surgery/hyperthermic intraperitoneal chemotherapy (CRS/HIPEC) when complete cytoreduction is not feasible. Nevertheless, some patients still benefit with long-term survival after incomplete CRS/HIPEC. There is currently no robust predictive tool that can assist clinical decision-making in this setting. METHODS: We quantified gene expression of 79 appendiceal mucinous neoplasms (AMN) from patients with incomplete CRS/HIPEC (R2 resection) using a custom NanoString gene panel. Using our previously defined, prognostic subtype classification algorithm based on signed nonnegative matrix factorization, we classified AMN cases into three molecular subtypes termed: immune enriched (IE), mixed (M), and oncogene enriched (OE). Kaplan-Meier and Cox proportional hazards analyses were used to associate subtypes and individual genes with overall survival (OS). RESULTS: Median overall survival (OS) was 7.7 years for IE, 3.6 years for M, and 1.4 years for OE. Compared with IE, OE was associated with significantly lower survival [hazard ratio (HR) 3.64, 95% confidence interval (CI) 1.63-8.13; p = 0.0017]. The differences were observed in both low-grade and high-grade tumors. While only two genes were identified to be associated with OS in low-grade tumors, multiple genes were identified to be associated with OS in high-grade tumors, particularly genes with functions in cell cycle/proliferation, mucin production, immune pathways, and cell adhesion/migration. CONCLUSION: Genetic signatures have prognostic value in patients with incomplete cytoreduction and provide valuable information to assist clinical and operative decision-making. Unraveling genetic alterations and involved pathways can direct efforts to design novel therapeutic modalities.

Transcriptomic Features of T Cell-Barren Tumors Are Conserved Across Diverse Tumor Types.

Background: Understanding how tumors subvert immune destruction is essential to the development of cancer immunotherapies. New evidence suggests that tumors limit anti-tumor immunity by exploiting transcriptional programs that regulate intratumoral trafficking and accumulation of effector cells. Here, we investigated the gene expression profiles that distinguish immunologically "cold" and "hot" tumors across diverse tumor types. Methods: RNAseq profiles of tumors (n = 8,920) representing 23 solid tumor types were analyzed using immune gene signatures that quantify CD8+ T cell abundance. Genes and pathways associated with a low CD8+ T cell infiltration profile (CD8-Low) were identified by correlation, differential expression, and statistical ranking methods. Gene subsets were evaluated in immunotherapy treatment cohorts and functionally characterized in cell lines and mouse tumor models. Results: Among different cancer types, we observed highly significant overlap of genes enriched in CD8-Low tumors, which included known immunomodulatory genes (e.g., BMP7, CMTM4, KDM5B, RCOR2) and exhibited significant associations with Wnt signaling, neurogenesis, cell-cell junctions, lipid biosynthesis, epidermal development, and cancer-testis antigens. Analysis of mutually exclusive gene clusters demonstrated that different transcriptional programs may converge on the T cell-cold phenotype as well as predict for response and survival of patients to Nivo treatment. Furthermore, we confirmed that a top-ranking candidate belonging to the TGF-ß superfamily, BMP7, negatively regulates CD8+ T cell abundance in immunocompetent murine tumor models, with and without anti-PD-L1 treatment. Conclusions: This study presents the first evidence that solid tumors of diverse anatomical origin acquire conserved transcriptional alterations that may be operative in the T cell-cold state. Our findings demonstrate the potential clinical utility of CD8-Low tumor-associated genes for predicting patient immunotherapy outcomes and point to novel mechanisms with potential for broad therapeutic exploitation.

Prognostic Molecular Classification of Appendiceal Mucinous Neoplasms Treated with Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy.

BACKGROUND: Appendiceal mucinous neoplasm (AMN) with peritoneal metastasis is a rare but deadly disease with few prognostic or therapy-predictive biomarkers to guide treatment decisions. Here, we investigated the prognostic and biological attributes of gene expression-based AMN molecular subtypes. METHODS: AMN specimens (n = 138) derived from a population-based subseries of patients treated at our institution with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS/HIPEC) between 05/2000 and 05/2013 were analyzed for gene expression using a custom-designed NanoString 148-gene panel. Signed non-negative matrix factorization (sNMF) was used to define a gene signature capable of delineating robustly-classified AMN molecular subtypes. The sNMF class assignments were evaluated by topology learning, reverse-graph embedding and cross-cohort performance analysis. RESULTS: Three molecular subtypes of AMN were discerned by the expression patterns of 17 genes with roles in cancer progression or anti-tumor immunity. Tumor subtype assignments were confirmed by topology learning. AMN subtypes were termed immune-enriched (IE), oncogene-enriched (OE) and mixed (M) as evidenced by their gene expression patterns, and exhibited significantly different post-treatment survival outcomes. Genes with specialized immune functions, including markers of T-cells, natural killer cells, B-cells, and cytolytic activity showed increased expression in the low-risk IE subtype, while genes implicated in the promotion of cancer growth and progression were more highly expressed in the high-risk OE subtype. In multivariate analysis, the subtypes demonstrated independent prediction power for post-treatment survival. CONCLUSIONS: Our findings suggest a greater role for the immune system in AMN than previously recognized. AMN subtypes may have clinical utility for predicting CRS/HIPEC treatment outcomes.

Tumour-specific amplitude-modulated radiofrequency electromagnetic fields induce differentiation of hepatocellular carcinoma via targeting Cav3.2 T-type voltage-gated calcium channels and Ca2+ influx.

BACKGROUND: Administration of amplitude modulated 27·12 MHz radiofrequency electromagnetic fields (AM RF EMF) by means of a spoon-shaped applicator placed on the patient's tongue is a newly approved treatment for advanced hepatocellular carcinoma (HCC). The mechanism of action of tumour-specific AM RF EMF is largely unknown. METHODS: Whole body and organ-specific human dosimetry analyses were performed. Mice carrying human HCC xenografts were exposed to AM RF EMF using a small animal AM RF EMF exposure system replicating human dosimetry and exposure time. We performed histological analysis of tumours following exposure to AM RF EMF. Using an agnostic genomic approach, we characterized the mechanism of action of AM RF EMF. FINDINGS: Intrabuccal administration results in systemic delivery of athermal AM RF EMF from head to toe at levels lower than those generated by cell phones held close to the body. Tumour shrinkage results from differentiation of HCC cells into quiescent cells with spindle morphology. AM RF EMF targeted antiproliferative effects and cancer stem cell inhibiting effects are mediated by Ca2+ influx through Cav3·2 T-type voltage-gated calcium channels (CACNA1H) resulting in increased intracellular calcium concentration within HCC cells only. INTERPRETATION: Intrabuccally-administered AM RF EMF is a systemic therapy that selectively block the growth of HCC cells. AM RF EMF pronounced inhibitory effects on cancer stem cells may explain the exceptionally long responses observed in several patients with advanced HCC. FUND: Research reported in this publication was supported by the National Cancer Institute's Cancer Centre Support Grant award number P30CA012197 issued to the Wake Forest Baptist Comprehensive Cancer Centre (BP) and by funds from the Charles L. Spurr Professorship Fund (BP). DWG is supported by R01 AA016852 and P50 AA026117.

Dissecting intratumoral myeloid cell plasticity by single cell RNA-seq.

Tumor-infiltrating myeloid cells are the most abundant leukocyte population within tumors. Molecular cues from the tumor microenvironment promote the differentiation of immature myeloid cells toward an immunosuppressive phenotype. However, the in situ dynamics of the transcriptional reprogramming underlying this process are poorly understood. Therefore, we applied single cell RNA-seq (scRNA-seq) to computationally investigate the cellular composition and transcriptional dynamics of tumor and adjacent normal tissues from 4 early-stage non-small cell lung cancer (NSCLC) patients. Our scRNA-seq analyses identified 11 485 cells that varied in identity and gene expression traits between normal and tumor tissues. Among these, myeloid cell populations exhibited the most diverse changes between tumor and normal tissues, consistent with tumor-mediated reprogramming. Through trajectory analysis, we identified a differentiation path from CD14+ monocytes to M2 macrophages (monocyte-to-M2). This differentiation path was reproducible across patients, accompanied by increased expression of genes (eg, MRC1/CD206, MSR1/CD204, PPARG, TREM2) with significantly enriched functions (Oxidative phosphorylation and P53 pathway) and decreased expression of genes (eg, CXCL2, IL1B) with significantly enriched functions (TNF-α signaling via NF-κB and inflammatory response). Our analysis further identified a co-regulatory network implicating upstream transcription factors (JUN, NFKBIA) in monocyte-to-M2 differentiation, and activated ligand-receptor interactions (eg, SFTPA1-TLR2, ICAM1-ITGAM) suggesting intratumoral mechanisms whereby epithelial cells stimulate monocyte-to-M2 differentiation. Overall, our study identified the prevalent monocyte-to-M2 differentiation in NSCLC, accompanied by an intricate transcriptional reprogramming mediated by specific transcriptional activators and intercellular crosstalk involving ligand-receptor interactions.

IL-4 together with IL-1ß induces antitumor Th9 cell differentiation in the absence of TGF-ß signaling.

IL-9-producing CD4+ (Th9) cells are a subset of CD4+ T-helper cells that are endowed with powerful antitumor capacity. Both IL-4 and TGF-ß have been reported to be indispensable for Th9 cell-priming and differentiation. Here we show, by contrast, that Th9 cell development can occur in the absence of TGF-ß signaling. When TGF-ß was replaced by IL-1ß, the combination of IL-1ß and IL-4 efficiently promoted IL-9-producing T cells (Th9IL-4+IL-1ß). Th9IL-4+ IL-1ß cells are phenotypically distinct T cells compared to classic Th9 cells (Th9IL-4+TGF-ß) and other Th cells, and are enriched for IL-1 and NF-κB gene signatures. Inhibition of NF-κB but not TGF-ß-signaling negates IL-9 production by Th9IL-4+IL-1ß cells. Furthermore, when compared with classic Th9IL-4+TGF-ß cells, Th9IL-4+IL-1ß cells are less exhausted, exhibit cytotoxic T effector gene signature and tumor killing function, and exert a superior antitumor response in a mouse melanoma model. Our study thus describes an alternative pathway for Th9 cell differentiation and provides a potential avenue for antitumor therapies.