Journal of Experimental & Clinical Cancer Research

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and is often associated with dismal outcomes, underscoring the urgent need for new therapeutic strategies. RMS arises from embryonic skeletal muscle precursor cells that fail to complete the myogenic differentiation program. Fusion-positive rhabdomyosarcoma (FP-RMS), defined by the presence of recurrent gene fusions such as PAX3::FOXO1 or PAX7::FOXO1, is associated with the poorest overall survival. The encoded fusion oncoprotein cause epigenetic reprogramming that defines the biology and behavior of FP-RMS. Polycomb repressive complex 1 (PRC1)-mediated chromatin regulation contributes to the control of developmental gene programs. Here, we investigate the dependency of RMS on epigenetic remodeling mediated by the PRC1.1 subunits ubiquitin specific protease 7 (USP7) and really interesting new gene 1B (RING1B). We found that USP7 is overexpressed in RMS samples, and that high expression correlates with poor patient prognosis. USP7 and RING1B bind to H3K27ac-enriched regions and colocalize with PAX3::FOXO1 at active enhancers controlling key tumorigenic genes in FP-RMS. Moreover, both shRNA-mediated depletion and pharmacological inhibition of USP7 downregulate PAX3::FOXO1 enhancer-driven genes, induce skeletal muscle differentiation and significantly inhibit FP-RMS tumor growth in vivo. Altogether, our findings identify USP7 as a critical regulator of PAX3::FOXO1-bound enhancers and highlight a novel therapeutic opportunity in RMS based on epigenetic dependencies.

Background: It is well known that genetic variants contribute to cellular sensitivity to chemotherapeutic agents and ionizing radiation (IR). The aim of this study was to identify single nucleotide polymorphisms (SNPs) and genes associated with the spectrum of normal cellular sensitivity of lymphoblastoid cell lines (LCLs) towards ionizing radiation and mitomycin C (MMC). Methods: In a first step, we determined the viability of LCLs established from male participants of the Berlin Aging Study II (BASE-II) aged >=62 years following treatments with increasing doses of IR (n=137 cell lines) or MMC (n=140 cell lines) using the alamarBlue assay. Results from intra-experimental triplicates and three independent experiments for each cell line and treatment were used to calculate the area under the curves (AUCs) representing the specific sensitivity to IR and MMC of each LCL. The data from these experiments were subsequently used as outcomes in genome-wide association studies (GWASs). In addition, we calculated polygenic risk scores (PGS) from UK Biobank GWAS results for four cancer-related phenotypes and assessed the extent to which the variance in the IR and MMC sensitivity is explained by these PGS. Results: The GWAS analyses revealed one variant, rs74728080, located in CDH13 on chromosome 16, to show genome-wide significant (p < 5 x 10-8, beta = 2.81) association with cellular viability after treatment with IR. In the GWAS on MMC sensitivity the most interesting signal was elicited by SNP rs113978558 in an intron of the PLD5 gene on chromosome 1 (p = 9.232 x 10-8; beta = 1.44). Several other SNPs with statistically suggestive (i.e., p < 1 x 10-5) evidence of association with IR or MMC sensitivity were identified. PGSs calculations from GWAS of four cancer-related traits in UKB explained ~5% and ~3% of phenotypic variance in IR- and MMC-induced cell viability, respectively. Conclusion: The genome-wide significant association of rs74728080 with IR sensitivity and the location of this variant in CDH13 is interesting and functionally highly plausible given its known involvement in oxidative-stress response and function as tumor suppressor. Taken together, our novel data suggest that CDH13 may be genuinely involved in regulating cellular IR sensitivity.

WNT11, a non-canonical WNT ligand, plays well-defined roles in development and tissue architecture; however, its function in cancer remains ambiguous. Here, we characterize WNT11 as a context-dependent suppressor of cancer stemness, invasion, and in vivo tumor formation in human epithelial cancer models. We show that WNT11 upregulation reduces the expression of stemness-promoting genes, suppresses epithelial-to-mesenchymal transition, and inhibits sphere formation and tumor growth. Conversely, downregulation of WNT11 enhances these aggressive malignant properties of cancer cells. Mechanistically, we found that the ability of WNT11 to inhibit RAC1 GTPase activation is essential for its regulation of invasion and self-renewal. In cells unresponsive to WNT11, the connectivity between WNT11 and RAC1 activity is disengaged. Direct manipulation of RAC1 activity in these cells recapitulates the phenotype and molecular signature of WNT11-responsive cells, establishing RAC1 as a critical effector of WNT11-mediated tumor suppression. Taken together, these findings identify the cellular context in which WNT11 suppresses RAC1 activation as a key determinant of its anti-tumor effects and provide a mechanistic framework for understanding the diverse, and sometimes opposing, roles of WNT11 reported in cancer.

Background: The T-cell receptor {beta} (TCR{beta}) repertoire reflects antigen-driven adaptive immune responses and provides insight into tumor-immune interaction. In prostate cancer (PCa), the immunosuppressive tumor microenvironment limits effective T-cell activation, and the antigenic drivers shaping intratumoral TCR repertoires remains poorly defined. This study aimed to characterize matched tumor and peripheral TCR{beta} repertoires from treatment-naive PCa patients and to identify shared clonotypes and antigenic specificities associated with disease severity. Methods: Next-generation sequencing was used to profile TCR{beta} repertoires from matched tumor biopsies and peripheral blood mononuclear cells obtained from treatment-naive PCa patients. Repertoires clonality, diversity, and was assessed using established metrics. Antigenic convergence was evaluated using GLIPH2 to identify shared CDR3{beta} motifs and predicted tumor-associated antigen (TAA) recognition, followed by functional validation using IFN-{gamma} ELISpot and T-cell expansion assays. Results: Tumor-derived TCR{beta} repertoires displayed reduced richness and increased clonality compared with peripheral blood mononuclear cells, consistent with local antigen-driven expansion. High-grade tumors demonstrated greater interpatient clonotype sharing and motif-level convergence, indicative of recognition of common TAAs. GLIPH2 analysis associated expanded clonotypes with epitopes derived from prostate-specific G-protein coupled receptor (PSGR), prostate-specific membrane antigen (PSMA), and prostate-specific antigen (PSA). Functional validation confirmed that peptide pools containing PSGR- and PSMA-derived epitopes induced IFN-{gamma} production and antigen-specific T-cell proliferation in vitro. Conclusions: These findings reveal an oligoclonal, antigen-driven intratumoral TCR{beta} landscape and identify PSGR and PSMA as immunogenic, potentially actionable targets. Integration of TCR profiling with antigen discovery pipelines may support the development of TCR-based biomarkers and precision immunotherapeutic strategies in prostate cancer.

Background: Ewing sarcoma (EwS) is a highly aggressive bone and soft tissue cancer mainly affecting children, adolescents, and young adults. The rarity of the disease, relatively small cohort sizes of prior studies, and overall low mutational burden of EwS have limited the ability to establish robust correlations of genomic findings and clinicopathological factors. Methods: To overcome these limitations, we integrated genomic and clinical data from the seven major sequencing studies encompassing 538 EwS patients. Mutational profiles (SNV, indels and CNVs), and their correlation with clinicopathological features in the aggregated cohort were systematically analyzed to provide an integrated view of the EwS genomic landscape. Results: This study compiles the largest EwS genomic dataset reported to date. In the aggregated cohort (n=538) bone tumors were more common (65.4%) than soft-tissue tumors (34.6%), the latter being more frequent in older male patients and associated with poorer outcomes. EWSR1::FLI1 was the most prevalent fusion (87.2%). No major clinicopathological differences were identified between fusion types. The mutational landscape was dominated by STAG2 (15.6%) and TP53 (7.1%) alterations, associated with younger or with older age at diagnosis and poor survival, respectively. Strikingly, the coexistence of STAG2 and TP53 mutations, although rare (n=12), was associated with lethal outcome in all cases. CDKN2A loss (9.1%) was associated with older age, poor survival, and linked to a higher frequency of TP53-mutations in soft tissue EwS. Among frequent CNVs, gain of chr1q (25.2%) and loss of chr16q (21.9%) were per se frequently associated with fatal outcome and their co-occurrence further increased the risk of lethality. Conclusions: We delineate recurrent genomic alterations with important clinicopathological associations, including a uniformly lethal STAG2/TP53 co-mutation and CNV signatures marking aggressive disease. This comprehensive pooled analysis of EwS genomic studies provides a foundation for refined biological risk-stratification.

Tumor-associated macrophages (TAMs) comprise functionally diverse states that can suppress anti-tumor immunity and promote tumor progression, yet the tumor microenvironmental cues and signaling programs that generate these states remain incompletely defined. Here, we systematically stimulate primary human monocyte-derived macrophages with a panel of cytokines and metabolites abundant in the tumor microenvironment (TME), and profile their transcriptomic and phosphoproteomic responses to resolve stimulus-specific molecular programs. We observe that potassium (K+) and adenosine (Ado) stimulation, which accumulate in necrotic tumor cores, downregulate antigen-presentation genes and their master regulator CIITA. K+ stimulation results in the upregulated fibronectin 1 expression, associated with immunosuppressive, metastasis-promoting TAM subsets. Ado induces upregulated expression of tryptophan (Trp) catabolism genes, myeloid checkpoints and metallothioneins (MTs). Although MT-high TAM states have been recurrently observed across tumor single cell RNA sequencing studies, their function remains poorly defined. We show that elevated MT expression in tumor tissue is associated with shorter overall survival. By aligning in vitro transcriptomes with single-cell RNA sequencing (scRNA-seq) signatures from a pan-cancer TAM atlas, we identify significant similarities between several in vitro states and clinically observed TAM populations, with Ado-stimulated macrophages closely resembling a MT-expressing TAM cluster. Overall, this work provides a systematic molecular context linking tumor microenvironmental cues to clinically relevant TAM states and offers a framework for recapitulating their functions in vitro. STATEMENT OF SIGNIFICANCEThis study explores how cytokines and metabolites from the tumor microenvironment shape macrophage molecular phenotypes and lead to the upregulation of clinically relevant marker genes and recapitulation of functional states of interest.

Ewing sarcoma (EwS) is an aggressive malignancy driven by EWSR1::ETS fusions, predominantly EWSR1::FLI1. Previous efforts using both direct and indirect approaches to target these chimeric oncoproteins have yielded limited clinical benefit. Although EWSR1::FLI1 is a well-known source of replication stress and genome instability, targeting DNA damage response (DDR) factors that mitigate these effects remain poorly understood. Here, we identified a marked dependency of EwS cells on exonuclease 1 (EXO1). We demonstrate that EXO1 is essential for EwS cell survival and tumor growth, highlighting its potential as a novel therapeutic target. Intriguingly, we unveil that EXO1 loss impairs mitotic DNA synthesis (MiDAS), promoting EWSR1::FLI1-associated genome instability and cell death. Collectively, our results support the idea that targeting DDR factors, which counteract replication stress and/or DNA damage induced by fusion oncoproteins, represents a promising therapeutic option for EwS.

Immune checkpoint inhibitors targeting the PD-1/PD-L1 axis have shown great promise in treating bladder cancer and are now part of the standard treatment for advanced disease. However, many patients still fail to respond to treatment and at present many biomarkers are assessed but have yet shown only limited results. Therefore, with the advent of combination treatments and the increase of immune related adverse event, the search for reliable predictive biomarkers is paramount. Using a multiplexed enhanced FRET-FLIM based technique (QF-Pro) we quantified the interaction of PD-1/PD-L1, CTLA-4/CD80 and TIGIT/CD155 immune checkpoints in a pre-treatment TMA of 46 patients treated with atezolizumab. The association between higher PD-1/PD-L1 ICP interaction state and treatment efficacy was demonstrated in the male sample cohort, where it identified patients with better PFS. Conversely, patients exhibiting higher CTLA-4/CD80 engagement had a worse response to atezolizumab. Remarkably, the dual assessment of patients with high PD-1/PD-L1 and low CTLA-4/CD80 allowed to identify the best responders. These results indicate that the monitoring of patients immune profile in urothelial carcinoma might be critical in identifying patients who may benefit from combination therapy.

Metastasis is the leading cause of mortality in breast cancer and remains largely untargeted therapeutically. Identifying molecular drivers of metastatic progression is essential for developing effective treatments. This study investigated the role of the transcription factor ELK3 in triple-negative breast cancer (TNBC) metastasis by defining the cellular and molecular processes it regulates. MDA231 cells with ELK3 overexpression (OE) or knockdown (KD) were generated by lentiviral transduction. Transcriptomic alterations induced by ELK3-KD were analyzed by microarray and validated by RT-qPCR. Ingenuity Pathway Analysis and Gene Set Enrichment Analysis identified ELK3-dependent metastasis-associated pathways, which were functionally validated using 3D microfluidic migration assays, mammosphere formation assays, and flow cytometry/ AlamarBlue proliferation assays. High ELK3 expression correlated with a mesenchymal phenotype in BC cell lines and lymph node invasion in patient tumors. ELK3-KD significantly altered 740 genes, many linked to migration and stemness. Functionally, ELK3 enhanced 3D confined migration, likely through regulation of EMT, cell adhesion and protrusion formation. ELK3 also promoted cancer stem cell traits, potentially via hypoxia-related and WNT/{beta}-catenin, JAK/STAT3, TGF-{beta}, Notch1, and NF-{kappa}B signaling pathways. Additionally, ELK3 induced cellular quiescence while suppressing proliferation under adherent conditions. Overall, ELK3 acts as a pro-metastatic regulator in TNBC by promoting migration and stemness.

Circulating tumor cells (CTCs) are the potential seeds of distant metastases; however, little is known about how they survive in the bloodstream. Using a large cohort of colorectal cancer (CRC) patients, we found that the pseudokinase receptor PTK7 is highly expressed in primary tumors and metastatic lesions. Consistent with previous reports, high PTK7 expression is associated with reduced disease-free survival and increased metastatic dissemination. Surprisingly, PTK7 is absent from most CTCs and undergoes a cell-autonomous ONtumor/OFFCTC/ONmetastasis switch that can be recapitulated in a xenografted mouse model, in in vitro systems, and a fluidic platform. PTK7-negative cancer cells exhibit increased expression of YAP1-driven genes, senescence-like features, and enhanced resistance to hemodynamic stress following loss of cell-cell and cell-matrix adhesion. This adaptive phenotype depends on metalloproteases, notably ADAM17, whose cleavage activity remodels the CTCs surfaceome. Functionally, the PTK7 OFFCTC state confers enhanced metastatic potential in vivo, and can be pharmacologically suppressed using metalloprotease inhibitors. Collectively, our findings identify a reversible, cell-autonomous, protease-driven surfaceome remodeling program that enables metastatic adaptation during hematogenous dissemination. Highlights / statement of significanceBy investigating potential markers for circulating colorectal tumor cells with strong metastatic potential, we describe a reversible and cell-autonomous remodeling of the circulating tumor cell surfaceome in patients that confers resistance to anoikis and stress induced by entry into the bloodstream. One Sentence SummaryThe dynamic regulation of PTK7 serves as a surrogate marker for tumor cell plasticity, aggressiveness, survival in the bloodstream, and efficiency in forming metastases. Trial registrationCTC colon Cohort: registered on https://ClinicalTrials.gov identifier NCT03256084; date of registration 2017-07-17 B-Org cohort: registered on https://ClinicalTrials.gov NCT05384184; date of registration 2019-06-06 Ethics statement for animal experimentsStudies on animals were conducted in accordance with the current ethical standards of the European Community (Directive 2010/63/EU), the Ethics Committee for Animal Experimentation (CEEA#14) and the French Ministry of Higher Education and Research, which approved and authorized the entire procedure described in this paper (project number APAFIS #35294).

Although the tumor immune microenvironment has been studied in endometrial cancer, the systemic immune alterations associated with disease progression and their potential prognostic significance remain poorly defined. In this study, peripheral blood immune subsets were characterized by multiparametric flow cytometry in 67 patients with EC and 20 healthy controls, including dendritic cells, MDSCs, T-cell subsets, NK cells, and exhaustion and senescence associated markers. Immune profiles were similar between healthy controls and patients with early-stage disease, whereas advanced tumors showed marked changes, including dendritic cell expansion, reduced CD4+ T-cell proportions, and increased frequencies of CD8+CD27-CD28- and CD8+CD57+ populations. Among clinicopathologic features, MDSC levels were associated with tumor grade and myometrial infiltration, while regulatory T cells were increased in TP53-mutated and microsatellite-stable tumors. In the advanced cohort (n=31), non-responders frequently displayed elevated CD8+, CD8+CD27-CD28-, and CD8+CD57+ levels alongside decreased CD27+CD28+ proportions. In multivariable Cox models, higher baseline CD8+ (HR 1.13), CD8+ CD27-CD28- (HR 1.04), and CD8+CD57+ proportions (HR 1.07; all p<0.05) were independently associated with shorter PFS, whereas higher CD27+CD28+ levels were associated with improved PFS. CD27-CD28- proportions were also linked to worse PFS by Kaplan-Meier analysis (HR 5.1, log-rank p=0.005). Longitudinal analysis (n=28) showed that senescent-like lymphocyte levels remained associated with progression across timepoints (OR 18.80), whereas total CD8+ proportions diverged progressively, reaching significance only from 6 months onward. Together, these findings identify systemic immune remodeling as a characteristic of advanced endometrial cancer and support the potential of circulating immune profiling for patient stratification and prognostic assessment, pending validation in larger prospective cohorts.

Myelofibrosis (MF) originates from the stepwise acquisition of somatic mutations in Hematopoietic Stem and Progenitor Cells (HSPCs). Alongside driver events triggering JAK-STAT pathway hyperactivation, several additional mutations, usually affecting the epigenetic machinery, contribute defining therapeutic response. Specifically, JAK-inhibition (JAKi) relieves MF symptoms but rarely eradicates the neoplastic clone. To elucidate clonal dynamics associated with JAKi, we conducted a longitudinal single-cell proteogenomic study on 6 responders and 6 non-responders MF patients. Mutational analysis revealed that the mutation acquisition order determines JAKi sensitivity. Indeed, driver-only clones are highly sensitive to JAKi, while co-mutated clones persist after treatment. JAKi response is mainly limited to the differentiated myeloid compartment, while mutant HSPCs are often maintained in JAKi-responders. Co-mutated clones may evade JAKi and outcompete other neoplastic cell populations, thus contributing to disease persistence.

Neutrophils display marked functional plasticity in cancer; however, it remains poorly understood how soluble factors derived from hypoxic and irradiated head and neck squamous cell carcinoma (HNSCC) cells reprogram neutrophil phenotype and function. Here, we employed a well characterized and controlled in vitro model to examine how tumor-conditioned media (TCM) from HNSCC cell lines cultured under ambient (21% O2) or hypoxic (1% O2) conditions, with or without 6 Gy gamma irradiation, modulate human neutrophil phenotype, and functional and transcriptional responses. Initial analyses were performed using TCM from three different HNSCC cell lines, whereas subsequent mechanistic characterization focused on FaDu-derived TCM. We show that TCM prolongs neutrophil survival in a cell line-dependent manner. Among the tested cell lines, hypoxia-conditioned FaDu-derived TCM promoted immunomodulatory neutrophil state characterized by enhanced survival, selective priming of ROS production, and elevated TRAIL-R3/TRAIL-R2 ratio. Induction of classical activation markers (CD11b, CD62L) was not evident. Transcriptomic analysis revealed minimal effects of normoxic TCM. Hypoxia-conditioned TCM induced a pronounced transcriptional program enriched in hypoxia- and stress-associated pathways. In contrast, irradiation of tumor cells had a limited additional impact on neutrophil reprogramming. Together, these findings indicate hypoxia-conditioned tumor secretomes as important drivers of neutrophil functional adaptation in vitro, supporting a model in which soluble factors alone are sufficient to induce a persistent, immunomodulatory neutrophil phenotype. This work provides mechanistic insight into tumor-neutrophil crosstalk, highlighting hypoxia-driven signaling as a potential therapeutic target in radioresistant HNSCC and supporting a role for neutrophil reprogramming in this context.

Abstract Background: The lack of effective biomarkers and therapeutic targets to overcome radioresistance in cervical cancer remains a major clinical challenge. Tumor necrosis factor receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase pivotal in immune and inflammatory signaling, has been implicated in various malignancies. However, its role in radioresistance in cervical cancer remains unclear. Methods: TRAF6 expression was evaluated in cervical cancer tissues from 162 patients who underwent postoperative radiotherapy at our institution and in 304 cases from the TCGA-CESC cohort. The prognostic significance of TRAF6 was assessed using Kaplan-Meier and Cox regression analyses. A nomogram integrating TRAF6 expression with clinicopathological factors was constructed to predict overall survival (OS) and progression-free survival (PFS). The functional role of TRAF6 in malignant phenotypes and radiosensitivity was investigated using shRNA-mediated knockdown in HeLa and C33A cervical cancer cells. Immune cell infiltration patterns associated with TRAF6 expression were analyzed using ssGSEA and xCELL algorithms based on TCGA data. Results: TRAF6 expression was significantly elevated in cervical cancer tissues compared with adjacent normal tissues (70.99% vs. control, P < 0.001) and was higher in radioresistant than in radiosensitive patients (P < 0.001). High TRAF6 expression was associated with shorter OS (HR = 18.73, P = 0.004) and PFS (HR = 8.44, P < 0.001) and was identified as an independent risk factor for radiotherapy resistance (OR = 8.44, P < 0.001). The TRAF6-integrated nomogram demonstrated good predictive accuracy for OS (C-index = 0.7351) and PFS (C-index = 0.7444). TRAF6 knockdown in cervical cancer cells significantly suppressed proliferation, migration, and invasion, while substantially enhancing radiosensitivity of tumor cells. Functional enrichment analysis revealed that TRAF6-related genes were enriched in autophagy, mitophagy, and HPV infection pathways. Immune cell infiltration analysis showed that TRAF6 expression correlated with distinct immune cell profiles, characterized by enrichment of activated dendritic cells, M1 macrophages, and regulatory T cells, alongside depletion of cytotoxic effectors such as CD8+ T cells and {gamma}{delta} T cells. Conclusions: TRAF6 could be a prognostic biomarker associated with poor outcomes and indicator of radiotherapy resistance in cervical cancer, TRAF6 represents a potential therapeutic target for overcoming radioresistance in cervical cancer.

BackgroundIn metastatic colorectal cancer, systemic therapies frequently fail, partly due to underlying phenotypic plasticity rooted in pre-existing multi-type cell populations. Intratumoral lineage hierarchies within colorectal tumors require renovated efforts to decode growth principles, design rational therapeutic approaches, and accurately interpret drug response. Understanding the cell-state dynamics of untreated tumors and the degree of cell responsiveness to exogenous stimuli is therefore crucial to improving currently underwhelming therapeutic outcomes. MethodsHere, we leveraged patient-derived organoids established from hepatic metastases of colorectal cancer patients to deconstruct population hierarchies by combining single-cell transcriptomics with single-molecule RNA fluorescent in situ hybridization. Computational frameworks were used to identify independent gene modules. We then employed flow cytometry analysis to track cytokine-induced population shifts using a cell surface marker, validating our findings through bulk RNA analysis, functional assays, and viability assays in response to oxaliplatin. ResultsOur data substantiate the existence of a dual population configuration within untreated metastatic colorectal cancer organoids with diverse genetic backgrounds. Gene modules detected via single-cell transcriptomics delineate a stem-like (LGR5+) and a differentiated-like (KRT20+) population that fluctuate dynamically over time. Spatially and temporally resolved, single-cell level analysis through single molecule FISH captures the inherent stochasticity in cell fate decisions revealing surprising phenotypic variability even across different organoids derived from the same patient. By using GABRA2 as a surface marker we track the emergence of differentiated cells over the course of time and investigate the respective roles of TGF-{beta}1 and IL-6 in the differentiation of organoids. Our findings indicate that IL-6 exerts no major effect within our cell autonomous setting. In stark contrast, TGF-{beta}1 triggered cell cycle arrest and differentiation, while simultaneously reducing clonogenic capacity and significantly amplifying the cytotoxic potency of oxaliplatin. ConclusionsOur findings provide evidence of the dual Stem and Differentiated population hierarchy in metastatic colorectal cancer organoids, and demonstrate how this axis can be effectively hijacked by TGF-{beta}1 to suppress tumor growth. Our results suggest that further mechanistic exploitation of this cell-autonomous, tumor-suppressive arm of TGF-{beta}1 signalling could open unappreciated therapeutic windows in advanced colorectal cancer.

Non-small cell lung cancer (NSCLC) is the most common form of lung cancer accounting for most cancer-related deaths worldwide. Despite substantial recent advances in targeted therapies and immunotherapy, the prognosis for advanced-stage disease remains comparably poor, which is why the identification of novel molecular biomarkers as well as therapeutic targets influencing tumor development, progression, and metastasis remain important. This study focusses on SERPINB13, a serine-protease inhibitor expressed in selected tissues that is dysregulated in several tumor entities. However, its role in NSCLC still remains largely unclear. We analyzed SERPINB13 transcription in a cohort of non-small cell lung cancer (NSCLC) cases including both lung squamous cell carcinoma (LUSC) and lung adenocarcinoma (LUAD) by transcriptome profiling. Epigenetic modifications were assessed via Methylation BeadChips. Additionally, SERPINB13 protein expression was assessed by immunohistochemistry (IHC) in an independent cohort of NSCLC comprising 126 LUSC patients. Correlation analyses were performed to associate SERPINB13 expression with key clinico-pathological parameters, including overall survival and extent of tumor-infiltrating immune cells. To functionally investigate the regulatory influence of peripheral blood mononuclear cells (PBMCs) on SERPINB13 expression in LUSC tumor cells in vitro, we utilized the SERPINB13-expressing LUSC cell line LUDLU-1. Here, gene transcription was analyzed by quantitative real-time PCR (RT-qPCR), confirmed by Western blot on the protein level. Transcriptome analysis revealed a significant upregulation of SERPINB13 in lung squamous cell carcinoma (LUSC) compared to lung adenocarcinoma (LUAD), highlighting a subtype-specific expression pattern. This differential expression was further associated with a distinct epigenetic DNA methylation signature at the SERPINB13 loci in LUSC, suggesting transcriptional regulation via hypomethylation. IHC analysis demonstrated that high SERPINB13 protein expression is significantly associated with prolonged overall survival in LUSC. Notably, SERPINB13 expression was enriched in immune-inflamed ("hot") tumors, characterized by elevated infiltrating lymphocytes and immune activation. Mechanistically, co-culture experiments with PBMCs induced SERPINB13 expression in a LUSC cell line in a dose- and time-dependent manner in the absence of direct cell contact. This suggests that soluble factors secreted by immune cells might play a key role in regulating SERPINB13 expression in the tumor microenvironment. Taken together, SERPINB13 is a novel prognostic indicator in LUSC that is modulated by Immune cells. Further studies are necessary to decipher the crosstalk of Immune cells on the Serpin B13 expressing tumor cells in depth, with regard to a possible interventional strategy. immunomodulatory potential strategies and personalized therapeutic approaches in NSCLC.

Long non-coding RNAs (lncRNAs) have attracted increasing interest because of their roles as modulators of tumor progression, acting either as oncogenic drivers or tumor suppressors, depending on the cellular context. LINC01133 has been implicated in regulation of multiple tumor-related mechanisms; however, its role in breast cancer, particularly in the triple-negative subtype, remains poorly characterized. In this study, we investigated the impact of LINC01133 depletion on malignant phenotypes and on the expression of migration- and invasion-associated genes using the Hs578T triple-negative breast cancer (TNBC) cell line, through comparative analyses of parental, control, and LINC01133-knockout cell lines, namely Hs578T_wt, Hs578T_ctr, and Hs578T_ko. Functional characterization included morphological analysis, growth assays, anchorage-independent colony formation, migration, invasion, and quantitative biomolecular experiments. Depletion of LINC01133 led to reduction of cell diameter, a significant increase in colony-forming capacity, and marked enhancement of migratory and invasive potential. At the molecular level, LINC01133 loss induced the expression of genes associated with extracellular matrix remodeling and cellular plasticity, including fibronectin, vimentin, integrins, FOXC1, and TWIST1, concomitant with reduced expression of ZEB1, TWIST2, and N-cadherin. Collectively, these data indicate that LINC01133 acts as a potential fine regulator of in vitro migration and invasion processes in TNBC, with its expression favoring a more asymptomatic mode of tumor progression, whereas its loss markedly enhances tumor malignancy.

BackgroundOvarian cancer (OvCa) ranks as the most lethal gynecological malignancy in women worldwide. This complex disease, which can develop independently of a womans age, is characterized by late diagnosis, pronounced tumor heterogeneity, and an immunosuppressive tumor microenvironment (TME). Incremental diagnostic tools that could better inform clinicians on potential therapy resistance or subsets of patients that could benefit from new drug modalities represent a critical unmet need to improve patient care and potentially the identification of new biomarkers. ObjectiveThis study aimed to establish a reconfigurable patient-derived OvCa-on-chip platform for longitudinal functional profiling of tumor cell death, immune activation, and patient-specific responses to TIL-mediated killing, PD-L1 blockade, and sequential chemo-immunotherapy. MethodsPatient-derived OvCa microtumors (PDM) were integrated with sequential integration of autologous tumor-infiltrating lymphocytes (TILs) into a perfusable microfluidic chip in the presence of different single and combination treatment regimens of chemotherapy and immune checkpoint inhibitors (ICIs). Treatment responses were assessed by longitudinal quantification of caspase-cleaved cytokeratin 18 (ccCK18) as marker of apoptotic epithelial tumor cell death, as well as cytokine/chemokine release in chip effluents, and multiplex flow cytometry-based characterization of autologous TIL subsets. ResultsThe perfusable OvCa-on-chip platform supported long-term culture of PDM while maintaining key structural and microenvironmental features of the primary tumor. Multidimensional analyses, including time-resolved assessment of tumor cell death, secretome profiling and correlative analysis of autologous TIL subsets revealed patient-specific tumor-immune response patterns and heterogenous sensitivity to TIL-mediated killing, PD-L1 blockade, and sequential chemo-immunotherapy. Correlation analyses identified treatment-dependent associations between specific TIL phenotypes and functional tumor cell killing. PD-1-expressing CD4 TIL subsets correlated with enhanced tumor cell killing, whereas terminally exhausted CD8PD-1Tcf1- TILs negatively correlated with durvalumab responses. In contrast, tumor-reactive CD8CD39 TILs were associated with improved responses under sequential chemo-immunotherapy conditions. ConclusionCollectively, this OvCa-on-chip system represents a complex in vitro model (CIVM) that combines 3D tumor tissue with autologous immune cells in a microfluidic platform. Resembling a physiologically relevant human preclinical platform, it allows for the time-resolved functional assessment of patient-specific responsiveness to OvCa therapies, with direct implications for personalized treatment stratification.

Melanoma is an aggressive malignancy that rapidly adapts to therapy. While chemotherapy resistance has traditionally been attributed to tumour-intrinsic mechanisms, growing evidence implicates the tumour microenvironment in shaping drug tolerance. However, few in vitro models capture the stromal complexity needed to study this interaction. We developed two multicellular melanoma spheroid models of increasing stromal complexity: a baseline model of melanoma, endothelial, and macrophage cells (BEM), and a fibroblast-containing counterpart (BEMF), and compared their transcriptional response to doxorubicin. Fibroblast inclusion increased the doxorubicin concentration required to achieve comparable growth inhibition. While untreated BEMF spheroids exhibited only modest baseline transcriptional differences, they showed a profoundly reshaped transcriptional response after doxorubicin exposure, displaying broader and higher-magnitude changes. These responses were characterized by suppression of proliferative and cell-cycle programmes, together with activation of inflammatory, immune-associated, metabolic, and stress-adaptive pathways. Higher-resolution pathway analyses further revealed coordinated attenuation of mitotic progression, checkpoint regulation, homologous recombination repair, and Rho GTPase signalling, consistent with a shift toward stress-adaptive and phenotypically plastic states, rather than classical resistance mechanisms. Transcriptome-derived transcription factor activity inference supported this regulatory rewiring. Integration with curated resistance-associated genes and external transcriptomic datasets demonstrated strong conservation of core transcriptional features across heterogeneous experimental systems, including consistent suppression of proliferation-associated genes and induction of inflammatory signalling programmes. Together, these findings indicate that fibroblasts redirect chemotherapy responses toward a stress-adaptive, persister-like phenotype and establish fibroblast-containing 3D melanoma spheroids as a physiologically relevant platform for studying tumour microenvironment-mediated chemotherapy tolerance and stromal-tumour interactions.

Endometrial cancer (EC), the most common gynecologic malignancy in the USA, has seen limited improvement in patient outcomes over recent decades, underscoring the need for relevant preclinical models. To address EC heterogeneity, we established an integrated platform of patient-derived xenografts (PDXs) and matched patient-derived primary cancer cells (PDCs) for disease modeling and systematic drug sensitivity testing. Fresh tumor specimens (n=103) were collected from EC patients to generate PDXs in immunodeficient mice and corresponding PDCs. Fifty-three PDX models were successfully established (52% engraftment rate), with higher success observed in high-grade, recurrent, metastatic tumors (70%), compared with their low-grade counterparts (56%). Histopathologic and immunohistochemical analyses confirmed that PDX tumors faithfully preserved morphology, hormone receptor status, and intertumoral heterogeneity across multiple passages. Using 13 PDC models, we performed an unbiased screening of 179 FDA-approved oncology drugs, revealing marked intertumoral variability in drug response. Almost all PDC models exhibited limited sensitivity to NCCN-recommended therapies, highlighting the need for alternative treatment strategies. In contrast, multiple FDA-approved agents including epigenetic modulators, dual PI3-kinase/HDAC inhibitors, topoisomerase II inhibitors, and proteasome inhibitors demonstrated potent antitumor activity. Importantly, a low-dose combination of the DNA methyltransferase inhibitor 5-azacytidine and the histone deacetylase inhibitor romidepsin significantly suppressed tumor growth across six independent PDX models. Together, these findings establish a comprehensive PDX and PDC platform as a robust translational resource. By capturing the histopathologic and molecular diversity of EC and identifying clinically actionable therapeutic advantages, including an epigenetic combination regimen, this study offers a translational resource for preclinical drug evaluation.