Hepatic stellate cells (HSCs) and their derived cancer-associated fibroblasts (CAFs) are integral to the development, progression, and prognosis of hepatocellular carcinoma (HCC). However, the mechanisms underlying the transformation of HSCs into CAFs and their subsequent roles in HCC remain incompletely understood. The expression of METTL3 was analyzed in fibroblasts infiltrating into human HCC specimens compared with adjacent nontumor tissues. The effects of HSC-specific METTL3 deficiency on HCC were analyzed by orthotopically implanting hepatoma cells into the fibrotic mouse livers, as well as through direct and indirect co-culture systems. We found that the expression of METTL3, the methyltransferase responsible for RNA N6-methyladenosine (m6A) modification, was downregulated in fibroblasts infiltrating into HCC compared with that of adjacent nontumor tissues. Orthotopical implantation of HCC cells in the fibrotic liver showed that HSC-specific METTL3 deficiency significantly increased tumor burden. Furthermore, conditioned medium treatment and mixed coculture confirmed that METTL3 deletion in HSCs promoted HCC proliferation and migration in vitro, demonstrating that METTL3 deficiency in HSCs accelerated HCC progression in the fibrotic liver. Mechanistically, METTL3 deficiency reduced m6A modification and expression of bone morphogenetic protein 10 (BMP10), a known tumor suppressor. Similar to the effects of METTL3 deficiency, BMP10 knockdown also promoted hepatoma cell growth, whereas recombinant BMP10 (rBMP10) inhibited it. Notably, BMP10 overexpression or rBMP10 supplementation effectively mitigated the tumor-promoting effects of METTL3-deficient HSCs. BMP10’s effects were mediated through SMAD1/5/8 phosphorylation. Collectively, our study reveals a novel crosstalk between HSCs and hepatoma cells via the METTL3/m6A–BMP10–SMAD1/5/8 axis, highlighting a potential therapeutic target for HCC.Significance:In this study, we found that HSC-specific METTL3 deficiency significantly accelerated HCC progression in the fibrotic liver. Mechanistically, METTL3 deficiency reduced m6A modification and expression of BMP10 and the downstream SMAD1/5/8 phosphorylation. This study reveals a novel crosstalk between HSCs and hepatoma cells, suggesting a potential therapeutic target for HCC.

To identify clinicopathologic factors associated with outcomes in patients with localized synovial sarcoma (SS). We retrospectively reviewed 248 patients with localized SS treated across three US quaternary medical centers between 2000-2024. Demographics, tumor characteristics, and treatments, including perioperative chemotherapy and radiotherapy, were assessed. Disease-free survival (DFS) and overall survival (OS) were estimated via Kaplan-Meier, and Cox regression was used for univariable and multivariable analysis, including significant univariate factors and confounders such as age and tumor size. Median follow-up was 5.2 years; median age at diagnosis 35 years (IQR 26-46). Median DFS was 110.3 months. Perioperative chemotherapy was given to 150 (60%) patients, while perioperative radiotherapy (RT) was given to 173 (69.8%) patients. Multivariable analysis showed that tumor size correlated with worse DFS (P < 0.001) and OS (P < 0.001), while a location in the chest wall, trunk or extremities correlated with improved DFS (P = 0.006). Percent tumor necrosis was associated with worse OS (P = 0.04), while R0 (P = 0.002) resection margins correlated with improved OS. Perioperative RT or chemotherapy did not correlate with OS or DFS in the whole cohort, though patients with tumor size ≥ 10 cm (n = 47) showed worse OS when receiving perioperative chemotherapy only as opposed to combined perioperative chemotherapy and RT (P = 0.006). Tumor size, location, margin status, and tumor necrosis are independent prognostic factors in localized SS. Combined perioperative chemotherapy and radiotherapy correlated with improved OS in patients with tumors larger than 10 cm.

Although immune checkpoint blockade therapy has generated dramatic responses in certain cancer types, breast tumors are largely unresponsive. Epithelial-mesenchymal plasticity leads to the assembly of an immunosuppressive tumor microenvironment and drives resistance of breast tumors to immunotherapies. Importantly, targeting CD73 completely sensitizes quasi-mesenchymal breast tumors to anti-CTLA4 immune checkpoint blockade therapy. However, the mechanism(s) of sensitization remained unknown. We demonstrate that targeting CD73 in quasi-mesenchymal breast tumors sensitizes them to anti-CTLA4 immune checkpoint blockade therapy in a CD4+ T-cell dependent manner. Moreover, epithelial-mesenchymal plasticity results in elevated expression of cancer cell-intrinsic CD73 in human triple negative breast cancers. Given the ability of quasi-mesenchymal cancer cells to metastasize and resist multiple therapies, these findings can instruct the formation of translational strategies for the treatment of human breast cancers. These findings also bring to the forefront the attractive possibility of utilizing the phenotypic plasticity of cancer cells along with CD73 and CD4+ T-cells as a predictive criterion for immunotherapy responsiveness.

Clear-cell kidney cancers display a more indolent or aggressive clinical behavior after surgery and different sensitivities to currently available medical therapies: immune therapy or angiogenesis inhibitors. We developed an easy-to-use molecular classification that divides these tumors into four subgroups predicting outcomes after surgery or upon medical therapy.

Cancer-associated fibroblasts (CAF), a major component of the breast tumor microenvironment, drive immune evasion in various cancers by promoting T-cell exclusion and dampening T-cell activation. Previous studies have implicated CAF-derived soluble factors in mediating these immunosuppressive effects. In this study, we investigated whether exosomes secreted by CAFs could suppress T-cell activity. Inhibition of global exosome secretion in breast tumor–bearing mice significantly reduced tumor growth and increased tumor-infiltrating T cells with lower exhaustion marker expression. Conversely, administration of CAF-derived exosomes into tumors produced the opposite effects. Moreover, CAF exosomes associated with T cells in vivo and impaired T-cell activation and cytotoxic potential in ex vivo assays. Proteomic and biochemical analyses of T cells exposed to CAF exosomes revealed dampened early T-cell receptor signaling. Mass spectrometry identified an extracellular matrix (ECM) signature on CAF exosomes. Depleting type I and type V collagens from CAF exosomes restored T-cell proliferation, whereas overexpression of collagen in cancer cells led to its incorporation into exosomes, which suppressed T-cell activation. These findings suggest that a signaling bridge between CAF exosomes and T cells, mediated by collagen, promotes T-cell dysfunction, contributing to immune evasion in breast cancer.Significance:Our data provide the first evidence that ECM proteins associate with mouse and human breast CAF-derived exosomes and directly impair T-cell activation and cytotoxicity. These findings suggest that signaling between collagen-rich CAF exosomes and T cells contribute to local and systemic T-cell dysfunction.

Lysine-specific demethylase 1 (LSD1), encoded by the gene KDM1A, is overexpressed and correlates with poor patient prognosis in Ewing sarcoma. LSD1 and the pathognomonic fusion oncoprotein, EWSR1::FLI1, colocalize throughout the genome, suggesting that LSD1 is a critical co-regulator driving the progression of Ewing sarcoma. However, therapeutic targeting of LSD1 by competitive and noncompetitive inhibitors has yielded mixed results. Irreversible, enzymatic inhibition seems ineffective, but reversible noncompetitive inhibition has predominant off-target mechanisms, leaving open the question of LSD1 function in Ewing sarcoma. In this study, we take a robust approach through multiple methods of depletion in multiple Ewing sarcoma cell lines to define enzymatic and nonenzymatic contributions of LSD1 to transcriptional regulation. We define a core set of 22 genes that are commonly repressed by LSD1 in all cell lines and that repression of these genes downregulates synapse functioning and e-cadherin target genes. Derepression of these genes with LSD1 loss is an early and sustained genotype in all cell lines tested. We further define distinct gene sets in each cell line that are regulated by enzymatic and nonenzymatic LSD1 activity and find repression of e-cadherin target genes to be nonenzymatically regulated. This finding supports the growing body of evidence that in addition to their canonical catalytic activity, chromatin regulatory enzymes serve essential noncanonical roles as well. Furthermore, we uncovered evidence through use of the irreversible inhibitor OG-L002 that 2D cytotoxicity and proliferation assays may be insufficient to determine Ewing sarcoma response to LSD1 inhibition.Significance:In this study, we address a long-standing question in the field surrounding LSD1 and define the distinct enzymatic and nonenzymatic functions of LSD1 in Ewing sarcoma. In doing so, we have created a robust data set using genetic and pharmacologic techniques in multiple models to thoroughly characterize LSD1 function in Ewing sarcoma cell lines.

Our study identified and characterized NSCLC cell lines with elevated mtdsRNAs, suggesting that mtdsRNA may be a new signaling molecule in lung cancer for mitochondrial stress. We identified that accumulation of mtdsRNAs did not necessarily activate IFN-l response, indicating that NSCLC cell lines may have adaptive mechanisms to reduce IFN-l response.

The current study shows that downregulation of PTPRR induces EGFR activation and resistance to KRASG12C inhibitors in NSCLC, suggesting dual KRAS-EGFR blockade as a rational therapy. PTPRR may help identify patient subgroups that would benefit from the addition of EGFR inhibitors to KRASG12C-targeted therapies.

Three-dimensional (3D) cell culture systems have emerged as powerful tools for modeling tumor biology in ex vivo settings. However, the diverse array of available 3D culture methods presents challenges in selecting the most appropriate model for specific research questions. This study provides a comparative analysis of breast cancer cells (SUM149, IBC-3, and MDA-MB-468) in the mammosphere culture (SphC) model or an “emboli” culture (EmC) model, which enrich for cancer stem cells and epithelial features, respectively. The EmC model, designed originally for inflammatory breast cancer, is characterized by media viscosity and mechanical rocking of the culture vessel. Notably, cells in EmC showed a distinct and durable reduction in cell proliferation ex vivo while demonstrating high capacity to establish experimental lung metastases in vivo. Ultrastructural quantitative analysis of electron microscopy images suggested that cells in EmC acquire nuclear and mitochondrial features that resemble those of tumor tissue. Proteomics, single-cell transcriptomics, and metabolic flux analyses showed that cells in EmC and SphC favor mitochondrial oxidative metabolism (OXPHOS) and glycolysis, respectively. EmC rendered cells hypersensitive to OXPHOS inhibition but more resistant to oxidative stress. Several genes associated with lung metastasis, including ID1, were specifically enriched in EmC. Given the emerging role of OXPHOS in breast cancer cell survival during dissemination and as established metastases, we propose that the EmC paradigm is a suitable ex vivo model to study signaling pathways relevant for basal epithelial breast cancer, including lung metastases, and to assess drug sensitivities and resistance mechanisms of such breast cancer cells ex vivo.Significance:This study provides an in-depth characterization of a resource-efficient, yet powerful 3D culture paradigm to improve the physiologic relevance of ex vivo approaches. Applicable to epithelial cancers, this model offers a platform to accelerate the discovery of physiologically relevant signaling pathways and specific cancer cell vulnerabilities.

Immune checkpoint inhibitors (ICI) have limited efficacy in microsatellite-stable (MSS) metastatic colorectal cancer (mCRC), potentially because of immunosuppressive mechanisms associated with liver metastases. The impact of liver metastases on survival outcomes was evaluated in a retrospective cohort of patients with MSS mCRC treated with ICI-based therapies at Mass General Brigham between January 2015 and December 2022. Patients were stratified by liver metastasis status at ICI initiation. The primary endpoint was progression-free survival (PFS); the secondary endpoint was overall survival (OS). A total of 132 patients were included, of whom 93 (70.5%) had liver metastases at ICI initiation. Most patients in both groups had received ≥2 prior lines of therapy. No significant differences were observed between groups for RAS/BRAF mutation status or tumor mutational burden. Patients without liver metastases demonstrated higher clinical benefit rates (46.2% vs. 16.1%; P = 0.001), longer median PFS (2.5 vs. 2.1 months; HR, 1.68; P = 0.009), and higher 12-month PFS rates (12.8% vs. 1.1%; P = 0.034). The median OS was also prolonged in patients without liver metastases (11.5 vs. 6.2 months; HR, 2.03; P < 0.001). No history of liver metastases was an independent favorable risk factor for PFS and OS in univariable and multivariable analyses. These findings indicate that liver metastases are associated with inferior survival outcomes in patients with MSS mCRC treated with ICI-based therapies, supporting the immunosuppressive role of liver metastases and underscoring the importance of stratifying patients by liver metastasis status to guide patient selection and optimize therapeutic strategies.Significance:The limited efficacy of ICIs in MSS mCRC remains a major challenge. The association between liver metastases and inferior outcomes supports the liver’s immunosuppressive role and suggests that liver metastasis status may guide patient selection and treatment optimization.