Cervical cancer progresses from high-grade squamous intraepithelial lesions driven by persistent HPV infection. Identifying the gatekeepers restraining malignant transformation could reveal strategies to prevent and treat cervical cancer. Here, we integrated single-cell transcriptomics, CRISPR-Cas9 screening, and organoid modeling to dissect this process. Single-cell analysis of patient samples revealed progressive activation of RAS and proliferation programs along epithelial differentiation paths. A focused CRISPR screen in HPV-positive pre-tumoroids identified NF1 as the top suppressor of malignant transition. NF1 loss accelerated carcinogenesis in organoids, compressing a 5~10-year process into 3~6 months, recapitulating basal cell expansion, dedifferentiation, and tumorigenicity. Mechanistically, NF1 loss enhanced RAS-MAPK and PI3K-AKT signaling, promoted proliferative programs, and remodeled chromatin accessibility at AP-1/E2F motifs. Conversely, NF1 restoration in cancer organoids induced apoptosis and suppressed malignant maintenance. AI-guided modeling was used to design a minimal NF1-mimetic peptide that engaged RAS-GTP to inhibit RAS signaling, reduce tumor burden in HPV16-driven mouse models, and restore local immune permissiveness without systemic activation. Together, these findings establish NF1 as a critical constraint on HPV-associated epithelial evolution and provide a time-compressed organoid model of cervical carcinogenesis, offering proof-of-concept for therapeutic RAS pathway interception via NF1 restoration.
Chemotherapy remains a cornerstone treatment for pancreatic ductal adenocarcinoma (PDAC), yet it paradoxically promotes metastatic recurrence. Elucidation of the underlying mechanisms mediating chemotherapy-induced metastasis could help identify improved combination treatment strategies. Here, we used a dormancy-tracking system to show that chemotherapy awakens dormant disseminated tumor cells (DTCs) in the liver by inducing hepatocyte senescence and subsequent neutrophil extracellular trap (NET) formation. In human and mouse cohorts, adjuvant and neoadjuvant chemotherapy triggered hepatocyte senescence, leading to TGF-β secretion and SMAD-dependent NETosis. NET-derived DNA engaged the transmembrane receptor CCDC25 on dormant DTCs, activating PI3K-AKT signaling and reprogramming DTCs toward a lipid-laden, proliferative phenotype. Genetic or pharmacologic disruption of NET formation, TGF-β signaling, or CCDC25 function effectively suppressed DTC reactivation and hepatic metastasis without compromising chemotherapy efficacy. Clinically, elevated serum NET-DNA levels correlated with hepatocyte senescence, DTC proliferation, and poor survival in PDAC and other solid tumors. Taken together, these findings unveil a senescence-NET-CCDC25 axis that links chemotherapy-induced tissue damage to metastatic relapse, providing a mechanistic basis for adjuvant and neoadjuvant strategies targeting NETosis and CCDC25 to prevent recurrence in PDAC.
Current immunotherapies often fail in immunologically "cold", macrophage-rich tumor microenvironments (TMEs). Multi-targeting approaches that modulate innate-adaptive immune activation represent a promising frontier in cancer immunotherapy. Here, we developed phagocytic synapse enhancers (PSEs), a class of modular immune engagers comprising a high-affinity PD-L1 binder coupled to a macrophage-stimulating peptide, tuftsin. PSEs strengthened effector-target cell interactions and enhanced tumor phagocytosis by bridging tumor PD-L1 to macrophage neuropilin-1 (NRP1), bypassing the classical FcγR-dependent pathways. The PSEs also acted in cis on PD-L1+ macrophages, accelerating the endocytosis and lysosomal compartmentalization of surface PD-L1, therefore stripping the immunosuppressive checkpoint from the local microenvironment. Beyond physical clearance, PSEs reprogrammed macrophage phenotype and triggered a robust pro-inflammatory cytokine and chemokine response. The lead molecule, longPSE, and its half-life-extended variant fused to the albumin binding domain, ABD- longPSE, showcased superior efficacy than the macrophage enhancer magrolimab in a syngeneic tumor model of colorectal cancer and an orthotopic model of pancreatic cancer. PSE treatment remodeled the TME by inducing phenotypic changes in the lymphoid and myeloid compartments, together with a reduction of tumor-associated macrophages and regulatory T cells. These findings establish PSEs as bifunctional molecules that complement innate and adaptive immune modulation. The bifunctional design offers a versatile approach for next-generation immunotherapies and provides a blueprint for a plug-and-play platform of immune engagers targeting diverse cancer-associated pathways.
The effectiveness of any cancer therapy depends on its dose and its treatment schedule. A potential third dimension of regimen design is whether intentional fluctuations in administered dose might improve treatment outcomes. In this issue, West and colleagues demonstrate that dose fluctuations can alter both tumor drug response and the evolution of drug resistance, in ways related to the shapes of dose-response functions. In mouse xenograft models of ALK-fusion non-small cell lung cancer treated with the ALK inhibitor alectinib, steady dosing produced the best immediate tumor response, but fluctuations in dose were better at delaying the evolution of drug resistance. Treatment schedules that switched between even and uneven dosing navigated this tradeoff, achieving comparable tumor control with steady dosing while better preserving drug sensitivity. By showing how dose-response measurements can guide when doses should remain steady or fluctuate, this work expands how cancer treatment schedules might be optimized. See related article by West et al., p. 4434.
Potent and sustained inhibition of epidermal growth factor receptor (EGFR) signaling is critical for suppressing colorectal cancer (CRC) growth, yet current EGFR therapies are often limited by incomplete EGFR blockade and on-target/off-tumor toxicities, particularly skin rash. Here, we co-targeted EGFR and cadherin-17 (CDH17) with a bispecific antibody (bsAb), overcoming these limitations by reducing skin-related toxicities and achieving superior tumor growth inhibition compared to EGFR- or c-MET/EGFR-targeting antibodies. Mechanistically, the high-affinity CDH17-binding arm anchored the EGFR/CDH17 bsAb to the tumor cell surface, facilitating rapid engagement of unbound or newly synthesized EGFR. This "anchor and capture" mechanism allowed the EGFR/CDH17 bsAb to achieve superior and more sustained suppression of the EGFR pathway than cetuximab and amivantamab. Further incorporation of an anti-CD16A nanobody transformed the EGFR/CDH17 bsAb into a trispecific natural killer (NK)-cell engager. Extensive format screening revealed that NK cell activation is heavily influenced by the spatial distance between the tumor antigen-binding Fab and the anti-CD16A nanobody, with longer distances impairing the bulky CD45 phosphatase exclusion from the immunological synapse and leading to significantly reduced cytotoxicity. Consequently, the widely adopted Morrison-type antibody consistently underperformed compared to architectures with shorter CD16A-TAA-Fab spacing. The final optimized molecule was IBI3019, a CDH17/EGFR/CD16A trispecific antibody that integrates potent CDH17-enhanced EGFR blockade with optimal architecture for efficient NK cell engagement. It demonstrated superior in vivo efficacy and a good safety profile in cynomolgus monkeys, with no observable skin toxicity. These promising pre-clinical findings warrant the clinical development of IBI3019. .
Interferon (IFN) signaling plays a pivotal role in orchestrating antitumor immunity and shaping the response to immune checkpoint blockade (ICB). Although genetic alterations that impair the IFN pathway have been reported, such events are relatively rare, suggesting a potential contribution of epigenetic dysregulation. Here, we identified a RUNX2-mediated epigenetic mechanism that disrupts the type I interferon (IFN-I) signaling pathway in osteosarcoma (OS), thereby limiting the efficacy of ICB. Development of an algorithm to assess the association of 1,425 transcription factors with IFN pathway activation in human OS tumors enabled identification of RUNX2 as a potential negative regulator of IFN signaling. RUNX2 depletion in OS cells activated the IFNB1-driven IFN-I response. Mechanistically, RUNX2 formed a transcriptional repressor complex with NCOR1 and HDAC3 that reduced H3K9 acetylation at the enhancers of key IFN-I genes, leading to their downregulation. Inhibition of the RUNX2-NCOR1-HDAC3 complex enhanced IFN-I signaling, with cGAS, STING, and IFNB1 being required for the induction of interferon-stimulated genes and tumor suppression. Paradoxically, reactivation of IFN-I signaling also upregulated immune checkpoint molecules PD-L1 and PD-L2. Combination treatment with a selective HDAC3 inhibitor and anti-PD-1 antibody led to durable tumor regression in syngeneic OS mouse models, accompanied by increased cytotoxic T cell infiltration. These findings reveal a mechanistic link between RUNX2-driven epigenetic repression and impaired antitumor immunity via the cGAS-STING-IFN-I axis and suggest a rational combinatorial strategy to overcome OS resistance to ICB.
Exercise induces a variety of changes in the tumor microenvironment with beneficial effects in several tumor types. However, a better understanding of the clinical effects of exercise and mediating mechanisms is needed to maximize the utility of exercise for patients. In this study, we analyzed tumors from pancreatic ductal adenocarcinoma (PDAC) patients in the PancFit trial and identified an exercise induced reduction in cells expressing alpha smooth muscle actin (αSMA). Interrogation of changes in tumor stromal composition with exercise in a murine PDAC model revealed a microbially-influenced reduction in αSMA+ cells and IL6 expressing inflammatory cancer associated fibroblasts (iCAFs). Cholic acid, a microbial bile acid, was increased in both patients and murine models with exercise, as a potential mediator of exercise induced reduction in iCAFs. Consistent with these findings, patients that exercised more also exhibited fewer iCAFs and lower tumor IL6 expression, supporting a stromal remodeling effect of physical activity. In summary, this study demonstrates that the anti-tumor effect of exercise includes stromal remodeling which is impacted by microbial metabolites.
Abstract Serine is a critical nutrient for cancer cell proliferation due to its numerous downstream biosynthetic functions. Given the ongoing clinical efforts to utilize dietary serine starvation as a potential therapy for serine auxotrophic tumors, it is important to understand how cancer cells respond to serine starvation. In this study, we demonstrated that serine starvation induces dramatic changes in mRNA splicing. These effects are due, in part, to reduced translation of serine-rich proteins, including the serine/arginine-rich splicing factor (SRSF) proteins that are known regulators of mRNA splicing. Indeed, translation of SRSF6 was reduced upon serine starvation in serine auxotrophic cancer cells and contributes to the RNA splicing changes seen upon serine deprivation. Furthermore, reduced SRSF6 affected DNA damage response and cell survival in the absence of exogenous serine, and modulators of RNA splicing combined with dietary serine starvation to inhibit tumor growth. Collectively, this work describes a fundamental role for serine in supporting mRNA splicing, which may have implications in the clinical efforts to bring dietary serine starvation to the clinic. Significance: Serine starvation induces changes in mRNA splicing by inhibiting translation of serine-rich proteins like SRSF6, which impacts the DNA damage response and could affect how cancer cells respond to chemotherapy.
Spatial transcriptomic analyses provide spatially resolved gene expression data that can provide insights into complex biological processes. However, current spatial transcriptomics approaches remain financially prohibitive and restricted in resolution, scalability, and gene coverage, limiting broader adoption for large-scale studies. Here, we developed CarHE (contrastive alignment of gene expression for hematoxylin and eosin images), a multimodal pretraining framework that infers high-dimensional spatial transcriptomic profiles from routine H&E-stained slides. By using contrastive learning to align cell type-specific transcriptomic information with histological features, CarHE achieved high prediction accuracy across evaluated datasets and spatial transcriptomics platforms. CarHE approximated spatially organized pathological microenvironment features consistent with tertiary lymphoid structure (TLS)-associated regions in breast cancer, lung cancer, melanoma, and clear cell renal cell carcinoma. Additionally, CarHE inferred approximated 3D spatial transcriptomic context from 2D images, providing more informative neighborhood context than 2D visualization. In a cohort of 880 lung cancer patients, CarHE-derived features were associated with disease-free survival and outperformed current approaches. Overall, CarHE provides a cost-effective and scalable framework for H&E-based spatial inference, supporting further validation toward translational research applications.
Mutant KRAS inhibition has revolutionized the treatment of lung adenocarcinoma. Unfortunately, responses to this form of targeted therapy are often of limited duration because of the development of resistance. Targeted protein degradation, including using PROTACs (PROteolysis-TArgeting Chimeras), presents an alternative approach to targeting oncogenic drivers in cancer. In this issue of Cancer Research, Martín and colleagues developed a dTAG-KRASG12V syngeneic mouse model that allows for the study of the effects of degrading the KRASG12V oncoprotein in vivo. The authors discovered that degrading the KRASG12V oncoprotein leads to regression of the resulting lung adenocarcinoma tumors. Most of the regression was based on cancer cell-intrinsic responses, although the tumor microenvironment also underwent substantial remodeling. Despite the initial efficacy of the treatment, the authors found that prolonged PROTAC KRAS degrader treatment eventually resulted in relapse. Resistance to PROTAC treatment seemed to be driven by dysregulation of the ubiquitin-proteasome system that is required for the activity of the PROTAC degraders. Despite developing resistance to the PROTAC degraders, the resulting tumors were still dependent on the KRAS oncoprotein, meaning that they were still sensitive to conventional KRAS inhibitors. Thus, PROTACs that degrade the KRAS oncoprotein are a promising modality for the treatment of lung adenocarcinomas. Resistance to PROTACs may differ from conventional KRAS inhibitors, suggesting potential strategies for overcoming such resistance. See related article by Martín et al., p. 4115.
KRAS mutations are a major driver of pancreatic ductal adenocarcinoma (PDAC). RASA2, a RAS GTPase-activating protein, modulates KRAS protein levels in wild-type contexts, suggesting it could play a potential role in PDAC. Here, we systematically investigated the biological function and molecular mechanisms of RASA2 in PDAC. Integrative analyses of multiple datasets and clinical samples demonstrated that RASA2 was consistently upregulated in KRAS-mutant PDAC and significantly associated with poor prognosis and metastatic progression. Gain- and loss-of-function studies revealed that RASA2 markedly enhanced PDAC cell migration and invasion in both KRAS-mutant and KRAS-wild-type models, suggesting that its pro-metastatic activity is largely independent of KRAS mutational status. Transcriptomic and mechanistic analyses revealed that RASA2 activated GLI1 through a TGFβ2-dependent, non-canonical Hedgehog pathway. Mechanistically, RASA2 interacted with RTF1 to promote H2BK120 ubiquitination at the TGFB2 promoter, thereby enhancing TGFβ2 transcription and activating downstream GLI1 signaling. Pharmacological inhibition of TGFβ signaling or genetic silencing of GLI1 effectively suppressed RASA2-driven migratory, invasive, and metastatic phenotypes in vitro and in vivo. Collectively, these findings reveal a mechanism by which RASA2-dependent epigenetic and transcriptional reprogramming promotes metastatic progression and nominate the RASA2- TGFβ2-GLI1 axis as a potential therapeutic target in PDAC.
Fasting-mimicking diets (FMD) have been reported to increase the anti-tumor efficacy in preclinical studies in various cancers. FMD can impact the tumor microenvironment (TME), and elucidating the mechanisms and cells mediating the effects of FMD may uncover combination treatment strategies. Here, we performed single-cell transcriptomic sequencing to characterize the TME changes induced by FMD intervention in a mouse model of spontaneous breast cancer. The sequencing data indicated that FMD suppressed tumor cell stemness, promoted apoptosis, and increased the infiltration of innate and adaptive immune cells, especially natural killer (NK) cells and effector CD8+T cells. Importantly, FMD induced phenotypic reprogramming of cancer-associated fibroblasts (CAFs), leading to a decrease in the immunosuppressive inflammatory CAF (iCAF) subset. Mechanistically, FMD decreased platelet-derived growth factor C (PDGFC) secretion in tumor cells by reducing glucose and inhibiting glycolysis to reprogram CAFs. PDGFC increased activation of the JAK/STAT3 pathway, which induced iCAF differentiation. Combining FMD with PDGFR inhibitors increased the efficacy of anti-PD-L1 immunotherapy in vivo. Overall, this study reveals a mechanism of metabolic-immune rewiring through which FMD suppresses tumor progression, providing preliminary evidence for the potential of combination FMD-based strategies in breast cancer treatment.
The Tn antigen, a truncated O-glycan, is frequently elevated in pancreatic ductal adenocarcinoma (PDAC). Multiple therapeutic approaches targeting Tn have been developed, but they have not demonstrated clear efficacy signals in early phase clinical studies. Improving Tn-targeted strategies in PDAC will require both overcoming the immunosuppressive tumor microenvironment and defining pathways by which truncated O-glycans promote growth and immune evasion. Here, we showed that Tn reshapes the tumor immune landscape of PDAC. Expression of Tn antigen on PDAC cells enhanced proliferation in vitro and tumor growth in vivo. Tn expression remodeled the immune microenvironment, skewing tumor-associated macrophages toward M2-like phenotypes, reducing cross-presenting dendritic cells, and expanding myeloid-derived suppressor cells (MDSCs). Single-cell RNA sequencing confirmed expansion of MDSCs and downregulation of antigen processing and presentation in the immune cell infiltrate of Tn+ tumors. Tumor-intrinsic transcriptomic analyses revealed activation of TNF-α/NF-κB signaling and induction of IL-34, a cytokine linked to monocyte survival and differentiation in Tn antigen expressing tumors. Additionally, high Tn expression in both organoids derived from pancreatic cancer patients and in PDAC mouse models was associated with increased IL-34 expression. Genetic deletion of Il34 in PDAC cells attenuated Tn-driven tumorigenesis and reduced MDSC infiltration, while recombinant IL-34 promoted myeloid cell differentiation and proliferation in vitro. Together, these findings establish a glyco-immune-cytokine axis in which truncated O-glycans contribute to IL-34-mediated immunosuppression, providing mechanistic insight and potential therapeutic targets in PDAC.
Prostate cancer (PCa) frequently metastasizes to bone, marking incurable disease. This progression is driven by an immunologically cold bone tumor microenvironment that fosters resistance to therapy. To define the mechanisms underlying this uniquely immunosuppressive niche, we applied spatial single cell analyses across primary tumors and metastatic sites. Bone metastases showed marked suppression of tumor-intrinsic type I interferon (IFN-I) signaling and loss of antigen presentation, features that were strongly associated with reduced bone metastasis-free survival. Tumor-intrinsic IFN-I expression correlated with memory T cell infiltration, whereas the bone myeloid compartment was enriched for protumor macrophages and showed reduced dendritic cell (DC) activation and antigen presentation. Digital spatial profiling of matched tumors revealed a broad loss of IFN-I-regulated immunostimulatory and checkpoint molecules. Notably, B7-H3, a putative negative regulator of IFN-I, was highly expressed in bone metastases and inversely associated with antigen presentation. These findings define bone-specific mechanisms of immune resistance and highlight therapeutic vulnerabilities that could inform precision therapeutic strategies for PCa.
High levels of immune suppression are a common intrinsic mechanism of resistance in metastatic breast cancer that calls for developing immunotherapeutic combinations to broaden treatment responses. Histone deacetylase (HDAC) inhibitors can sensitize tumors to dual checkpoint inhibition in patients. Here, we investigated the tumor microenvironment (TME) of breast metastases by combining experimental and clinical data with theory to elucidate the mechanism of response to treatment with the HDAC inhibitor entinostat combined with anti-PD-1 and anti-CTLA-4. Knowledge-guided subclustering of single-cell RNA-sequencing (scRNA-seq) data and cell circuit analyses from murine breast-to-lung metastases identified 39 cell states and salient interactions, of which myeloid, T cell, and B cell subpopulations were most affected. Analyses of patient biopsies and blood via spatial proteomics and flow cytometry corroborated the preclinical findings, showing increased T cell and B cell activation, mature tertiary lymphoid structures, and increased CD8+ T cell-macrophage distances in responders to entinostat + nivolumab + ipilimumab. Combination treatment increased immunoglobulin production in patients and mice, and murine studies demonstrated increased tumor-targeting IgG and implicated B cells as necessary for treatment response. Inhibition of the ICAM1 and IFNγ pathways in myeloid cells partially recapitulated treatment effects on CD8+ T cells observed via scRNA-seq. Mathematical modeling of tumor-immune dynamics implicated simultaneous modulation of multiple TME interactions as required for response to the combination treatment. Overall, this study identifies lymphoid and myeloid cell contributions to response to treatment with HDAC inhibitors and immune checkpoint blockade, providing a framework for discovering interactions driving responses in complex TMEs.
Women who carry a pregnancy to term have long been known to have a reduced lifetime risk of breast cancer, yet the mechanisms underlying this protection remain poorly understood. While parity-induced differentiation and hormonal remodeling of mammary epithelium have been considered primary drivers of breast cancer protection, the contribution of immune adaptation has remained largely unexplored. In a recent issue of Nature Immunology, Hussain and colleagues identify pregnancy-induced tissue-resident memory-like (TRM-like) CD8⁺ T cells as key mediators of parity-associated breast cancer protection. The authors show that pregnancy establishes a nurturing niche in the mammary gland that supports the expansion of TRM-like cells through epithelial-derived IL-15 and TGF-β, and that depletion of these cells abolishes the cancer-protective effect of parity. Moreover, using therapeutic agents to enhance IL-2Rβ signaling was sufficient to induce expansion of breast cancer-protective TRM-like cells in nulliparous mice, raising the possibility that pregnancy's protective imprint could be pharmacologically induced. Given the established link between TRM cells and immune checkpoint blockade responsiveness in breast cancer, these findings also raise the possibility that pregnancy-induced TRM-like populations could be leveraged to improve immunotherapy outcomes. By linking physiological developmental tissue remodeling to durable local immunosurveillance, this study expands current concepts of tissue-resident immunity and positions pregnancy as an immune-educating event with lasting consequences for breast cancer susceptibility.
Induction of ferroptosis is a potential strategy for treating cancer and improving the efficacy of immunotherapy. Ferroptosis is driven by excessive peroxidation of polyunsaturated fatty acid-containing phospholipids, suggesting that microenvironmental lipid metabolites may regulate ferroptotic sensitivity. By integrating single-cell and bulk transcriptomics from immunotherapy cohorts, we identified the prostaglandin (PG) pathway as closely associated with tumor ferroptosis and therapeutic efficacy. Further screening revealed PGF2α as a potent endogenous ferroptosis sensitizer. Mechanistically, microenvironmental PGF2α bound to ferroptosis suppressor protein 1 (FSP1) at alanine 295 (A295) and inhibited its enzymatic activity, leading to lipid peroxidation accumulation upon ferroptotic stimuli. Preclinically, PGF2α supplementation or FSP1 ablation enhanced tumoral ferroptosis, potentiated CD8+ T cell-mediated immunity, and suppressed tumor progression in immunocompetent mice. Moreover, PGF2α improved immunotherapy efficacy across multiple mouse models, including subcutaneous allografts, Braf/Pten-driven spontaneous melanoma, and humanized mice. Clinically, a high PGF2α activity-related transcriptomic signature correlated with elevated ferroptosis and improved patient survival. Collectively, these findings establish PGF2α as a pro-ferroptotic metabolite and propose that targeting the PGF2α/FSP1 axis may offer an effective cancer immunotherapeutic strategy.
Antibody-drug conjugates (ADCs) show clinical efficacy against several solid malignancies, inducing cytotoxic effects in malignant cells accompanied by changes in the composition of the tumor stroma. The therapeutic effects of some ADCs have been shown to be potentiated by combination with anti-PD(L)1 checkpoint inhibitors, highlighting the potential of combining ADCs and immunotherapy. Here, we investigated the potential of combining ADCs with agonist monoclonal antibodies targeting CD137 (4-1BB), a costimulatory receptor expressed on antigen-primed T lymphocytes and activated NK cells. The combination of MMAE- and deruxtecan-conjugated ADCs towards NECTIN-4 or HER2 with anti-CD137 agonists elicited synergistic effects in mouse tumor models. Treatment with the ADCs increased CD137 expression on intratumor CD8+ and CD4+ T lymphocytes. Moreover, the synergistic antitumor therapeutic efficacy was dependent on cDC1 dendritic cells and CD8+ T cells. ADCs induced features of immunogenic cell death in malignant cells, resulting in cross-presentation and maturation of cDC1 dendritic cells that cross-prime CD8+ T cells to become CD137-positive. Importantly, the anti-CD137 plus ADC synergistic combination attained efficacy against concomitant tumor lesions that failed to express the ADC target. Together, these findings demonstrate that anti-CD137 agonists hold promise for combination strategies with ADCs.
