Triple-negative Breast Cancer (TNBC) is characterized by inflammatory myeloid infiltration and a high metastatic potential, however, the mechanisms by which macrophage-derived signals govern tumor cell plasticity remain poorly understood. Here, through patient transcriptomic analyses, in vitro functional studies, and in vivo metastasis models, we identify the Integrated Stress Response (ISR) as a critical tumor cell-intrinsic pathway that translates inflammatory macrophage-derived cues into metastatic competence. In breast cancer clinical cohorts, ISR programs are enriched in TNBC and associate with poor outcome and inflammatory macrophage infiltration. Functionally, we show that the inflammatory macrophage secretome triggers ISR-dependent invasion in TNBC cells. Mechanistically, we identify CXCL10 as a macrophage-derived mediator necessary and sufficient to induce ISR activation and invasion through CXCR3 receptor. Finally, we demonstrate that tumor-intrinsic ISR signaling promotes TNBC metastatic dissemination in vivo. Together, our findings establish a macrophage-CXCL10-CXCR3-ISR signaling axis that fuels metastatic behavior in TNBC and identify this pathway as a potential node for therapeutic intervention. Statement of significance Inflammatory macrophages promote TNBC metastasis by activating the Integrated Stress Response via a CXCL10-CXCR3 paracrine loop. These results bridge the gap between inflammation and tumor-intrinsic stress signaling, uncovering a novel mechanistic axis for therapeutic intervention.
Resistance to immune checkpoint inhibition (ICI) using antibodies against the PD-1/PD-L1 axis remains a significant clinical challenge. Here, we report that combination of anti-PD-1 with a bifunctional fusion molecule, anti-TCRβ-IL-2, comprised of a monovalent Fab that binds and activates the T-cell receptor (TCR) through distinct variable beta (Vβ) chain residues and an IL-2 molecule, mediates robust anti-tumor control in the context of ICI resistance. Syngeneic murine models with varying levels of ICI-resistance were used to assess sensitization to ICI in combination with the anti-TCRβ-IL-2 therapy. Mechanisms of the combination therapy’s benefit were investigated by profiling tumor-infiltrating lymphocytes via flow cytometric, single cell transcriptomic, and histological analyses. Immune response efficacy and diversification was identified by the generation of multifunctional, tumor-associated antigen-specific and neoantigen-specific T-cell responses post-therapy. Unlike anti-PD-1 therapy alone that resulted in expansion of terminally exhausted CD8+ T cells and lack of anti-tumor effect, administration of anti-TCRβ-IL-2 followed by anti-PD-1 greatly enhanced anti-tumor efficacy leading to tumor cures and long-term protection in several murine models resistant to ICI. Analysis of tumor-infiltrating lymphocytes showed increased proliferation and markers of cytotoxicity in CD8+ and CD4+ T cells, reduced CD8+ T-cell exhaustion, and decreased proportion of regulatory CD4+ T cells in tumors of mice receiving TCRβ-IL-2 followed by anti-PD-1. Diversification of the immune response with increased number of multifunctional, neoantigen-specific T cells was observed in mice receiving anti-TCRβ-IL-2 followed by anti-PD-1, compared to each of the monotherapies. A novel T-cell activation mechanism sensitizes refractory tumors to ICI via T-cell function revival and epitope spreading. From these results, a clinical trial is planned to evaluate the combination of an anti-TCRβ-IL-2 agent (invikafusp alfa) and anti-PD-1 in ICI-refractory non-small cell lung and castration-resistant metastatic prostate cancers.
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with poor prognosis and limited treatment options. Ferroptosis has emerged as a potential therapeutic vulnerability in PDAC, but the upstream mechanisms regulating this process remain unclear. This study aimed to identify compounds with therapeutic potential against PDAC, determine their molecular targets, and elucidate how alpha/beta hydrolase domain-containing protein 10 (ABHD10) regulates tumor growth. A high-throughput compound screen against PDAC cell proliferation was performed, followed by limited proteolysis–mass spectrometry to identify the molecular target of halofuginone (HF). Target engagement was validated by surface plasmon resonance, cellular thermal shift assay, and drug affinity responsive target stability assay. Gain- and loss-of-function studies were conducted in PDAC cell lines and xenograft mouse models. Protein interaction, palmitoylation, and ferroptosis-related changes were evaluated using co-immunoprecipitation, acyl-biotinyl exchange, Click-iT labeling, transmission electron microscopy, and biochemical assays. Statistical analyses included Student’s t tests and one-way or two-way analysis of variance, as appropriate. HF significantly suppressed PDAC growth in vitro and in vivo. ABHD10 was identified as a direct binding target of HF and was upregulated in pancreatic cancer tissues and cell lines. Functional studies showed that ABHD10 promoted cell proliferation, survival, and tumor progression. Mechanistically, ABHD10 interacted with acyl-CoA synthetase long-chain family member 4 (ACSL4) and depalmitoylated it at cysteine 157, thereby suppressing ferroptosis. HF treatment or ABHD10 depletion increased ACSL4 palmitoylation, enhanced lipid peroxidation, disrupted redox homeostasis, and induced ferroptotic changes. Depletion of ACSL4 partially reversed these effects. The ABHD10–ACSL4 axis promotes PDAC growth by suppressing ferroptosis. Targeting this pathway may represent a promising therapeutic strategy for PDAC.
The E6 oncoprotein of high-risk human papillomaviruses (HPVs) promotes cervical carcinogenesis by forming a complex with the ubiquitin ligase E6AP to mediate p53 proteasomal degradation. A 15-amino-acid peptide (pep11) was shown to disrupt the HPV16 E6/E6AP complex, to restore p53 stability and to induce apoptosis in HPV16-positive cancer cells. In this study, a short cell-penetrating peptide (CPP) was conjugated to the pep11 N-terminus to improve its solubility and intracellular delivery in HPV-positive cancer cells. CPP-pep11 was synthesized using Boc chemistry. The interactions of CPP-pep11 with HPV16 and HPV18 E6 were modelled using AlphaFold2, while peptide structures were characterised by NMR spectroscopy. HPV16-positive SiHa and HPV18-positive C4-I cervical cancer cells were treated with CPP-pep11 (0.5–20 µM) for 24–72 h. Effects on proliferation and clonogenicity were assessed using xCELLigence and colony formation assays. Peptide uptake and localization were analysed by confocal microscopy and differential cell fractionation, while p53 expression and viability were evaluated by Western blotting and cytotoxicity assays. AlphaFold2 modelling suggested that CPP-pep11 can interact with both HPV16 E6 and HPV18 E6, with a more stable predicted binding to HPV16 E6. CPP-pep11 was soluble in water at 20 µM and, according to NMR analysis, predominantly adopted a disordered conformation. CPP-pep11 efficiently entered the SiHa and C4-I cell membranes, mainly localised to the cytoplasm at lower concentrations (0.5–5 µM) and to the nucleus at higher concentrations (10–20 µM). CPP-pep11 induced dose- and time-dependent inhibition of proliferation, with a significant reduction in colony formation in both cell lines. Treatment also restored p53 levels at 48 and 72 h, consistent with disruption of E6-mediated degradation. In conclusion, CPP-pep11 is a novel peptide-based inhibitor of HPV16 E6 and HPV18 E6 with therapeutic potential in HPV-associated malignancies by restoring p53 levels and inhibiting cancer cell proliferation.
Bladder cancer (BC) is one of the most deadly diseases in the USA, with 84,530 new cases and 17,870 estimated deaths in 2026. The growth factor progranulin is involved in several human pathologies, including frontotemporal dementia (FTD), immune response, and cancer. We showed that in BC, progranulin and its signaling receptor, EphA2, drive tumor cell motility, invasion, and in vivo tumor formation, making it a critical pathway in tumor establishment. However, the molecular mechanisms of progranulin/EphA2 action are still poorly defined. Progranulin-dependent EphA2 interactome was explored by proteomic approaches. FAM120A or EphA2 protein levels in BC tissue microarrays (TMAs) and FAM120A protein expression in BC cell lines were analyzed by immunohistochemistry (IHC) and immunoblots, respectively. Progranulin-dependent activation of AKT and ERK1/2 were examined by western immunoblots. EphA2-FAM120A interaction was detected using Co-immunoprecipitation and proximity ligation assays (PLA). In addition, EphA2 and FAM120A colocalization and F-actin cytoskeleton rearrangements were assessed by immunofluorescence. The biological function of FAM120A was determined using lentiviral shRNA approaches, wound healing, motility and invasion, spheroid, soft agar, clonogenic assays, cell cytotoxicity, and in vivo xenograft models. We conducted proteomic approaches and identified novel progranulin-dependent EphA2 interactors, including FAM120A, which is a scaffold protein with a putative role in oncogenic pathways. We confirmed that the EphA2 and FAM120A interaction was enhanced upon progranulin stimulation, and FAM120A was upregulated in BC tissues. We further demonstrated that FAM120A was critical for progranulin-evoked AKT and ERK1/2 activation, clonogenic capacity, wound healing, motility, invasion, and 3D spheroid formation of BC cell lines. In addition, FAM120A was essential for anchorage-independent growth and in vivo tumorigenesis in xenograft models. In addition, FAM120A depletion sensitized BC cells to cisplatin treatment. Mechanistically, we showed that FAM120A depletion inhibited progranulin-dependent F-actin cytoskeleton rearrangement through ERK1/2 and RhoA-dependent pathways. Furthermore, we demonstrated that progranulin-induced activation of RhoA was inhibited upon FAM120A depletion. The discovery of FAM120A as an oncogenic progranulin-dependent EphA2 interactor provides novel insights into the progranulin/EphA2 signaling axis and uncovers putative novel targets for BC therapy. Furthermore, FAM120A expression may work as a biomarker with diagnostic and possibly prognostic value in BC.
BRAF-mutant (BRAF-MT) colorectal cancer (CRC) represents a clinically aggressive subtype characterized by distinct biological features and significantly worse prognosis compared to BRAF wild-type (BRAF-WT) CRC, with median survival reduced by approximately 40
Circulating tumor material can already be used to help in cancer management, but host–microbiota interactions may add complementary value. This study evaluated a “liquid microbiopsy”, combining cell-free microbial DNA (cf-mbDNA) with host biomarkers of microbial translocation/permeability (LBP, sCD14, I-FABP), to refine staging and prognosis in breast cancer. We analyzed 136 individuals: 58 metastatic breast cancer (MBC), 58 early breast cancer (EBC), and 20 healthy donors. Plasma cf-mbDNA was quantified by 16S rRNA qPCR with extraction/run negative controls; LBP, sCD14, I-FABP by ELISA; and CRP by immunoturbidimetry. Circulating tumor cells (CTCs) and PD-L1 on CTCs were assessed by CellSearch® and immunostaining. Diagnostic performance was assessed by area under the ROC curve. In MBC, progression-free survival (PFS) and overall survival (OS) were analyzed using Kaplan–Meier and Cox models. LBP discriminated EBC from MBC with an AUC 0.92, with a slight improvement in the 3- or 5-marker models (AUC 0.936–0.939). For EBC vs healthy donors, CRP showed modest performance (AUC 0.73), and marker combinations offered no improvement.. In MBC (median follow-up 77.5 months), median PFS and OS were 4.5 and 14.5 months, respectively. In univariate analysis, shorter PFS was associated with tumor subtype, > 3 prior treatments, > 2 metastatic sites, CTCs ≥ 5, and PD-L1–positive CTCs detection. In multivariate analysis, sCD14 and I-FABP independently predicted PFS, while sCD14 was independently associated with OS. A measurable host–microbiota–barrier axis (LBP, sCD14, I-FABP) provided stage-related and prognostic information complementary to CTC/PD-L1 status, whereas cf-mbDNA load alone showed limited discriminative value. These findings support liquid microbiopsy as a complementary extension of liquid biopsy but must be considered as exploratory, and warrant multicenter validation in larger independent cohorts with standardized pre-analytics and low-biomass controls. NCT03449264; NCT02866149.
Neuroblastoma (NB) remains a major cause of pediatric cancer mortality for which new therapeutic strategies are needed. Exploitation of reprogrammed metabolic pathways offers an opportunity for cell-type specific anticancer therapeutics. To identify pediatric solid tumors with an enhanced susceptibility to targeting the nicotinamide adenine dinucleotide (NAD+) salvage pathway, we performed an unbiased high-throughput screen of over 200 cancer cell line models using inhibitors of nicotinamide phosphoribosyltransferase (NAMPT), the rate limiting enzyme in the NAD+ salvage pathway. Our analysis identified NB as one of the most sensitive solid tumor types, prompting further investigation of NAMPT inhibition as a potential therapeutic strategy in this disease. Using two early phase clinical NAMPT inhibitors (OT-82 and KPT-9274), we validated screen results using assays of proliferation and survival in a panel of molecularly diverse NB cell lines. Effects on proliferation, survival, NAD+ abundance, adenosine triphosphate (ATP) levels, and energy-related metabolites were quantified, and downstream consequences of NAD+-consuming enzymatic pathways were examined. Tolerability, antitumor activity and pharmacodynamic effects of OT-82 were evaluated in three orthotopic NB mouse models. In the drug screen, NB models ranked among the most sensitive pediatric solid tumor cells lines to NAMPT inhibition. OT-82 and KPT-9274 potently suppressed proliferation and viability across multiple molecularly diverse NB models in an on-target manner. Mechanistically, NAMPT inhibition resulted in rapid depletion of NAD+ and ATP, disruption of energy metabolism, accumulation of DNA damage, and induction of irreversible non-apoptotic cellular death. In vivo, OT-82 was well tolerated and produced marked antitumor activity, including tumor regressions in orthotopic NB models, including several with regional liver metastases. Pharmacodynamic analysis confirmed intratumoral NAD+ depletion during treatment, demonstrating on-target pathway inhibition in vivo. These findings identify NB as a highly NAMPT inhibitor-sensitive pediatric solid tumor and establish NAMPT-dependent NAD+ biosynthesis as a targetable metabolic vulnerability in this disease. By integrating an unbiased discovery screen with mechanistic and orthotopic in vivo validation using clinically relevant inhibitors, this study provides a strong translational rationale for clinical investigation of NAMPT inhibitors in NB.
Abstract Background B cells are increasingly recognized regulators of antitumor immunity in melanoma, yet their differentiation states, lineage dynamics, spatial organization and integration within the immune microenvironment, and how these are influenced with immunotherapy remain incompletely defined. Methods We conducted deep profiling of tumor-resident B cell differentiation and evolutionary features in the melanoma tumor microenvironment using bulk, single-cell and spatial transcriptomic analyses, long-read antibody sequencing and mass cytometry (Cytometry by Time-Of-Flight, CyTOF). We characterized B cell differentiation trajectories, class-switching patterns, antibody isotype usage, signaling pathways, spatial niches against clinicopathological features and immune cell communication, before and after checkpoint blockade and in clinically defined responders and non-responders. Results Melanoma-infiltrating B cells were enriched across primary and metastatic lesions, were preferentially associated with immune-rich tumors and correlated with T and natural killer (NK) cell densities. Long-read immunoglobulin sequencing demonstrated recurrent class switch recombination (CSR) trajectories with predominant IgG and IgA isotype usage, confirmed by bulk transcriptomic analyses. Across independent single-cell and bulk transcriptomic datasets we identified conserved class-switching, B cell receptor (BCR) activation, and pseudotime analyses showed differentiation trajectories progressing from naïve and memory states toward interferon (IFN)-driven terminal differentiation trajectories that persisted before and after immunotherapy. CyTOF analyses independently confirmed proliferative, germinal center (GC)-like, memory and class-switched B cell subsets, suggesting conserved inflammatory differentiation endpoints. Spatial transcriptomic deconvolution identified IFN-rich B cells within tertiary lymphoid structures (TLS). Following immunotherapy, B cells retained class-switched trajectories, displayed enriched GC-associated profiles, enhanced crosstalk with T cells and Dendritic Cells (DCs) and preserved or induced IFN-rich IgM + /IgD + or IgG4 + phenotypes, suggesting persistence or emergence of non-class switched and atypical class-switched isotype profiles. Transcriptomic analyses of B cells pointed to upregulated antigen-driven stimulation and BCR signaling, whereas in non-responders displayed IFN-rich B cells, IFN-associated genes, and IFN-pathway enrichment. Conclusions Melanoma-infiltrating B cells follow class-switched differentiation trajectories culminating in IFN signaling, atypical antibody expressing states that persist or emerge after immunotherapy, aligning dynamic B cell evolution and atypical lineage traits with immunotherapy.
Despite major advances in radiation delivery, clinical outcomes remain constrained by tumor biology rather than technology. Conventional radiobiology has relied on reductionist two-dimensional (2D) systems that fail to capture the spatial, mechanical, and multicellular organization of tumors. Three-dimensional (3D) tumor models now resolve radiation response as a mechanobiological process coordinated across the extracellular matrix (ECM), adhesion signaling, cytoskeleton, and nucleus. This review focuses on a specific and we argue, underappreciated intersection: how 3D models expose integrin-mediated mechanotransduction as a determinant of the DNA damage response (DDR) and therapy resistance epitomized by cell adhesion-mediated radioresistance (CAM-RR) - an organizing principle we frame as the ECM-integrin-nucleus axis. We first delineate which model classes resolve which layer of this biology, distinguishing effects of three-dimensional organization from those of defined ECM-integrin signaling, and we treat the underlying mechanobiology quantitatively rather than descriptively. We then develop a mechanistic framework linking ECM architecture and stiffness to integrin-RTK crosstalk, cytoskeletal tension, LINC-mediated force transfer, and chromatin-dependent DNA repair, in which radiosensitivity emerges as a property of tissue context. From a translational standpoint, 3D models enable functional, radiation-specific assessment of context-dependent radiosensitivity and of mechanically targeted radiosensitization, while their integration with quantitative imaging and computational approaches further supports biomarker-guided and adaptive treatment strategies. We close with testable predictions that this framework generates, intended to guide the next phase of biology-driven radiation oncology.
Melanoma remains one of the most aggressive malignancies, with limited response to immune checkpoint inhibitors (ICIs) due to primary or acquired resistance. Proteolysis-targeting chimeras (PROTACs) have emerged as a novel therapeutic modality that induces selective degradation of target proteins via the ubiquitin–proteasome system, offering distinct advantages over conventional inhibitors. This review summarizes the design principles and molecular mechanisms of PROTACs, and highlights their emerging role in sensitizing melanoma to immunotherapy. Specifically, PROTACs can restore tumor antigen presentation, reprogram the immunosuppressive tumor microenvironment by targeting regulatory T cells, myeloid-derived suppressor cells, and tumor-associated macrophages, and suppress key oncogenic signaling pathways. We also discuss combination strategies with PD-1/PD-L1 and CTLA-4 blockade, as well as current challenges including poor cell permeability, off-target effects, and acquired resistance. Finally, future perspectives on rational design, tissue-specific delivery systems, and clinical translation are addressed.
Immune checkpoint blockade targeting cytotoxic T lymphocyte–associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1)/PD-L1 has transformed cancer therapy. However, limited response rates and frequent resistance highlight additional mechanisms of immune suppression within the tumor microenvironment (TME), particularly those driven by myeloid and innate immune compartments. The leukocyte immunoglobulin-like receptor B (LILRB) family has emerged as a central regulatory axis integrating immune and tumor cell–intrinsic programs. Beyond restraining immune activation, LILRB receptors reprogram myeloid cells toward tolerogenic states and, when expressed by tumor cells, directly sustain proliferation, metabolic adaptation, angiogenesis, and immune evasion. This dual functionality positions the LILRB axis as a system-level checkpoint linking immune suppression with tumor cell fitness. Here, we synthesize current knowledge on LILRB biology and discuss its implications for cancer immunotherapy, highlighting how targeting this pathway may overcome resistance to PD-1/PD-L1 blockade and enable more effective combination strategies aimed at reprogramming the tumor–immune ecosystem.
Treatment resistance and recurrence continue to define the clinical landscape of glioblastoma (GBM), yet their study is limited by traditional in vitro frameworks that fail to capture long-term treatment dynamics and tumor evolution. Here we present GliaMimic, a longitudinal in vitro platform incorporating irradiation and multi-dose temozolomide (TMZ). Using patient-derived organoids (PDOs) and cell line-derived spheroids, tumor progression and treatment response were monitored non-invasively over four weeks. Substantial declines in metabolic activity and viability at clinically relevant TMZ concentrations (≤ 10 µM) emerge only after prolonged exposure, whereas short-term assays captured effects only at supraphysiological doses. Notably, PDOs, patient-derived spheroids, and their cell line-derived counterparts exhibited distinct patterns of treatment response and tumor progression, underscoring the importance of model selection in preclinical studies. Following treatment cessation, the platform captured distinct, patient-specific post-treatment tumor behaviors with persistent viable and metabolically active populations across all models with more pronounced changes in PDOs. By moving beyond static molecular diagnostics, GliaMimic provides a longitudinal in vitro treatment evaluation platform for preclinical testing.
Despite significant advances in early detection and therapeutic strategies, breast cancer (BC) continues to pose a major public health challenge. The treatment of HER2-positive (HER2 +) BC has evolved substantially over recent years with the advent of monoclonal antibodies (mAbs), small-molecule tyrosine kinase inhibitors (TKIs), and antibody–drug conjugates (ADCs). Clinical progress has been significantly accelerated by a deeper understanding of the immune-regulatory properties of this subtype and its interactions with the tumor microenvironment. Despite the availability of effective HER2-targeted therapies, approximately one-third of patients develops resistance, highlighting the need for novel and more durable treatment strategies. Indeed, while current treatments have shown promising efficacy by promoting a “passive immune response,” they are associated with the emergence of resistant clones. Conversely, vaccine-based therapies are designed to elicit a durable “active immune response” by presenting tumor-associated antigens that stimulate the host immune system to recognize and eliminate malignant cells. This approach has the potential to generate long-lasting immunological memory, preventing recurrence, disease progression, and the emergence of immune-evasive tumor variants. This review provides a comprehensive overview of cancer vaccines strategies for HER2 + BC with emphasis on their mechanisms, advantages, limitations, and their current developmental status. We explore diverse delivery platforms, including peptide/protein-based, nucleic acid, and cell-based vaccines. The clinical utility of these strategies is assessed across different disease settings: from prevention and interception in pre-invasive lesions, to the (neo)adjuvant treatment of early-stage BC and the management of advanced metastatic disease. While advanced-stage BC often presents an immunosuppressive tumor microenvironment that hinders vaccine efficacy, early-stage disease offers a more favorable immunological milieu for inducing robust and durable T-cell responses. Furthermore, we discuss emerging innovations such as neoantigen discovery, next-generation adjuvants (e.g., TLR and STING agonists), and novel combinatorial approaches with checkpoint inhibitors to overcome immune evasion. Although most BC vaccines remain under clinical investigation, they represent a promising frontier for achieving disease control and the coming decade is expected to yield pivotal insights into their clinical utility and therapeutic potential.
