Cancer Letters

The anticancer efficacy of radiotherapy (RT) is limited by acquired radioresistance (RR). Here, we aim to characterize prolonged responses of breast carcinoma cells to hypofractionated irradiation (hFI). To this end, murine mammary 4T1 tumor cells (4T1WT) were subjected to a clinically oriented hFI protocol (56 Gy cumulative dose) to select radioresistant cells in vitro (4T1RS). Furthermore, hFI of subcutaneously growing 4T1CTR tumors (hFI; 24 Gy cumulative dose) was performed to radioselected 4T1IR cells in vivo. Following single irradiation in vitro, radioselected 4T1RS cells revealed increased proliferation, attenuated G2/M arrest and reduced apoptosis as compared to parental 4T1WT cells. Moreover, 4T1RS cells showed increased expression of DNA-damage response (DDR)-related proteins (pKAP1, pCHK2, {gamma}H2AX) and improved DSB repair efficiency as demonstrated by nuclear {gamma}H2AX foci analyses. The mRNA expression of factors regulating cell cycle progression, DDR, apoptosis and oxidative stress was substantially different between both cell variants in vitro. Ten days after hFI of in vivo growing tumors, residual DNA damage and apoptosis were increased in the radioselected 4T1IR tumors, whereas proliferation was reduced as compared to non-irradiated 4T1CTR control tumors. Both irradiated and non-irradiated tumors revealed complex differences in the mRNA expression profile of susceptibility- and metastasis related genes, including GADD45a, DUSP1, CDKN1a and NQO1 as well as CD44 and Rho-related factors, respectively. Moreover, hFI stimulated the infiltration of MPO-positive immune cells into tumor tissue while the presence of CD3-positive cells was reduced in the tumor area. In addition, hFI in vivo resulted in a dysregulated mRNA expression of various immune cell markers, Rho-regulatory factors, tissue remodeling molecules and cell adhesion factors. Summarizing, we identified long-lasting adaptive changes following hFI in vitro and in vivo that are associated with DNA replication, DNA repair, senescence and apoptosis as well as immune cell infiltration and tissue remodeling.

Platinum resistance remains a major barrier in Ovarian cancer (OC) treatment[1]. While hyperactivation of DNA damage response (DDR) is a hallmark of chemoresistance[2], the underlying epigenetic mechanisms driving this adaptation remain poorly understood. Here, we identify a novel post-transcriptional regulatory axis involving miR-221-5p that governs two critical DDR effectors: RAD18, which mediates DNA damage tolerance through trans-lesion synthesis (TLS)[3][4], and RAD51, the central recombinase for homologous recombination (HR)[5][6]. Although the miR-221/222 cluster is traditionally categorized as oncogenic[7][8], we demonstrate that the miR-221-5p arm functions as a potent tumor suppressor in OC. Bioinformatic and luciferase reporter assays confirmed that miR-221-5p directly targets the 3'UTRs of both RAD18 and RAD51. In OC clinical specimens and cell lines, miR-221-5p downregulation inversely correlates with RAD18/RAD51 expression. Functionally, miR-221-5p restoration suppressed platinum-induced PCNA mono-ubiquitination and HR, inducing a "functional BRCAness" that sensitized both established and patient-derived primary OC cells to carboplatin and PARP inhibition. Furthermore, in vivo disseminated xenograft models demonstrated that stable miR-221-5p expression significantly reduced tumor burden. Collectively, our results delineate a novel regulatory mechanism where loss of miR-221-5p drives chemoresistance by derepressing the RAD18/RAD51 axis, identifying this axis as a promising therapeutic target.

The poor prognosis of brain tumors, including IDH-wild-type glioblastoma (GB), as well as brain and leptomeningeal metastases, is partly related to the blood-brain barrier (BBB), which limits the delivery of hydrophilic anticancer drugs to the tumor site and surrounding brain parenchyma. Early studies using vital dyes demonstrated that intracranial injection could bypass the BBB in cats. We confirmed that, in guinea pigs, the vital dye Bleu Patente V diffused efficiently into the brain after a bolus intracranial injection, whereas the brain remained unstained after intravenous administration. Similarly, brain concentrations of the hydrophilic anticancer drug gemcitabine were significantly higher following intracranial injection than after intravenous administration. Consistent with these findings, Bleu Patente penetrated deeply into the cerebral cortex of sheep after a 24-hour intraventricular infusion. At the end of a 24-hour intraventricular infusion of 20 mg gemcitabine in sheep, mean gemcitabine concentrations reached 1,415 {micro}g/L in cerebrospinal fluid and 850 {micro}g/kg in brain tissue. These concentrations exceeded the IC90 values of gemcitabine for A172, U87-MG, and U118-MG human glioblastoma cell lines, as determined in vitro after 24 hours of incubation. We hypothesize that Bleu Patente dye and gemcitabine circumvent the blood-brain barrier (BBB) by utilizing the glymphatic system. Tolerance of a single 24-hour intraventricular infusion of gemcitabine at doses of 5, 10, and 20 mg was good. Taken together, these encouraging preclinical results support the resumption of Phase I clinical trials evaluating intraventricular infusion of gemcitabine in patients with refractory primary or secondary brain tumors.

SUMMARYChordoma, a rare malignant notochordal tumor of the skull base and spine, is typically resistant to chemotherapy and radiotherapy and exhibits aggressive local recurrence. Here we show that chordoma recurrence correlates with a coordinated upregulation of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs), a low PFA/MUFA ratio and an adaptive, lipid peroxidation-resistant state that protects against DNA damage and cell death. Single-cell metabolic profiling identified a tumor subpopulation marked by a fatty acid biosynthesis-high state coupled to stemness. RT-tolerance was directly linked to elevated FASN and lipid droplet (LD) expansion, and MUFA-loading phenocopied RT-tolerance in chordoma cells. Mechanistically, LDs accumulated in response to RT via generation of ROS, and subsequent activation of ER-stress, SREBP1 and Fatty Acid Synthetase (FASN). DESI-MS showed that low-dose irradiation was sufficient to increase MUFAs early and build peroxidation resistant MUFA-LDs, whereas PUFA induction required a higher radiation dose. In a spatially defined manner in a patient-derived xenograft. Finally, in silico knockout and pharmacologic FASN blockade restored radiosensitivity and apoptosis in vitro and in vivo. Collectively, our result support a unifying model in which RT resistance in chordoma is shaped by an adaptive fatty acid metabolic program that buffers oxidative injury and increases survival of RT-resistant, stem-like tumor subpopulations. These findings further support FASN inhibition as a practical radiosensitization strategy for chordoma particulary where RT dose escalation is constrained by anatomy. KEYPOINTSO_LIRecurrent chordoma exhibits fatty acid-associated metabolic reprogramming. C_LIO_LIMUFA-associated lipid droplet accumulation is linked to radioresistance in chordoma cells. C_LIO_LITargeting FASN restores radiotherapy sensitivity of chordoma in vitro and in vivo. C_LI IMPORTANCE OF STUDYThis study underscores the clinical importance of targeting metabolic vulnerabilities to restore radiosensitivity in chordoma. By integrating transcriptomics, metabolomics, and in vitro and in vivo models, we identified adaptive fatty acid metabolic reprogramming as a central mechanism of RT resistance in chordoma. Recurrent tumors were characterized by coordinated enrichment of unsaturated fatty acids, especially monounsaturated fatty acids (MUFAs), together with a low PUFA/MUFA ratio and a lipid peroxidation-resistant state. Mechanistically, RT-tolerance chordoma cells exhibited a high-FASN state driven by activation of the ROS-ER stress-PERK/SREBP1/FASN axis, leading to intracellular lipid droplet expansion. Importantly, genetic and pharmacologic inhibition of FASN restored radiosensitivity and enhanced apoptosis in both in vitro and in vivo models, suggesting a translatable therapeutic strategy. Together, these findings link adaptive metabolic reprogramming to RT resistance and support new therapeutic approaches for chordoma management.

Purpose: Elective nodal (EN) irradiation (ENI) during radiotherapy for locally advanced head-and-neck squamous cell carcinoma (LA-HNSCC) influences hematotoxicity, anti-tumor immunity, and synergy with immunotherapy. We evaluated whether EN-sparing upfront boosts affect DNA damage, systemic immune signaling in peripheral blood lymphocytes (PBLs), and radiation-induced lymphopenia (RIL). Methods and Materials: Twenty-eight patients with LA-HNSCC were randomized to either adjuvant or definitive chemoradiotherapy with standard ENI or EN-sparing upfront boost (adjuvant: 2x2 Gy; definitive: 5x2 Gy). Blood was collected pre-radiotherapy, 15 min, and 24 h after the first fraction, and before the sixth fraction. DNA damage in PBLs was assessed via {gamma}H2AX and 53BP1 foci and dicentric chromosome (DIC) assay. RNA sequencing was performed in two patients per group (definitive setting) at pre-CRT, before the sixth fraction, and at therapy end. Absolute lymphocyte counts (ALCs) were monitored weekly to assess RIL. Results: DNA damage in PBLs correlated with planning target volume and whole-body dose, both of which were reduced by EN-sparing by 9.9-fold and 4.4-fold, respectively (p < 0.001 each). Correspondingly, EN-sparing significantly reduced radiation-induced foci and DIC levels in PBLs (3-4-fold, p < 0.001) and lowered the fraction of radiation-damaged PBLs per fraction (11% vs. 23% with ENI, p < 0.001). EN-sparing preserved baseline ALCs during week 1 of chemoradiotherapy and delayed RIL, whereas ENI caused an immediate ALC decline and RIL. Lymphocyte counts after week 1 negatively correlated with planning target volume, whole-body dose, and DNA damage in PBLs (p < 0.01). Transcriptomics showed metabolic and interferon signaling associated with EN-sparing, versus sterile inflammatory and damage-associated patterns with ENI. Conclusions: EN-sparing by an upfront boost significantly reduced PBL damage and early RIL with distinct immune responses associated with lymphocyte viability and immune maturation. These findings support upfront EN-sparing strategies to mitigate RIL and improve radiotherapy-immunotherapy synergy in HNSCC.

Lung adenocarcinomas frequently harbour actionable oncogenic mutations that are vulnerable to treatment with targeted therapies. While responses to targeted therapies are often initially dramatic, relapse is almost inevitable and prevents durable responses in advanced-stage patients. Relapse is, in part, caused by drug tolerant persister cells (DTPs) which are able to survive treatment by entering a reversible, dormant state. Although long non-coding RNAs (lncRNAs) regulate processes thought to allow DTPs to survive and become stably resistant, the potential roles of lncRNAs in DTPs are largely unknown. In this study, we sought to investigate the expression of lncRNAs in in vitro DTP models of lung adenocarcinoma. We found that the lncRNAs Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1) and Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) were enriched in DTPs and that knocking down MALAT1 enhanced the effect of targeted therapies in both EGFR- and KRAS-mutant DTP models. To better understand pathways that MALAT1 might regulate in DTPs, bulk RNA-sequencing was performed and several pathways that may contribute to the actions of MALAT1 in DTPs were identified. Overall, our work describes a role for the lncRNA MALAT1 in DTPs in NSCLC and suggests that MALAT1 may be a novel target for the prevention of drug tolerance and subsequent resistance to targeted therapy in NSCLC.

Oligodendroglioma is a primary central nervous system tumor classified by the presence of isocitrate dehydrogenase (IDH) mutations and codeletion of 1p/19q. Here we describe the generation of an IDH-mutant 1p/19q-codeleted oligodendroglioma mouse model using in utero electroporation. We identified IDH1R132H, PIK3CAE545K, CicKO, Fubp1KO and Cdkn2aKO as the optimal combination (termed OligoCdkn2a) to drive fully penetrant tumors that histologically resemble human grade II/III IDH-mutant, 1p/19q-codeleted oligodendroglioma. Replacing Cdkn2a with Trp53 loss in this mouse model shifted tumor histology towards high grade astrocytoma. OligoCdkn2a tumors displayed metabolic and transcriptional changes associated with IDH and CIC mutations, and single cell sequencing identified a bias towards oligodendrocyte differentiation compared to an IDH wild-type glioblastoma mouse model. OligoCdkn2a tumors represent the first mouse model system to recapitulate the genetic, histological and transcriptional features of human IDH-mutant 1p/19q-codeleted oligodendrogliomas, offering a platform to further dissect tumor biology and test new therapeutic strategies.

Background Complex gastrointestinal (GI) oncologic surgeries carry substantial perioperative risk, and nationwide outcomes in low- and middle-income countries (LMICs) are underreported. This study aimed to evaluate national trends in surgical volume, in-hospital mortality, and intensive care unit (ICU) utilization for major GI cancer surgery in Brazils Unified Health System (SUS) over a 14-year period. Methods A population-based analysis was performed using national administrative databases to identify all adult patients undergoing colectomy, gastrectomy, pancreatic resection or esophagectomy for cancer in the SUS from 2010-2023. Annual rates were age-standardized according to the WHO standard population. Temporal trends were assessed using Poisson regression to estimate average annual percent change (AAPC) with 95% confidence intervals (CIs). Results A total of 179,337 hospital admissions were analyzed (median age 63 years; 48% female). Colectomies accounted for 72% of cases, followed by gastrectomies (19%), pancreatic resections (5%), and esophagectomies (3%). Although crude surgical volume increased, population-adjusted rates declined overall (AAPC -2.09%; 95% CI -2.58 to -1.59), mainly due to reductions in gastrectomies and esophagectomies. Median hospital stay decreased from 9 to 7 days (AAPC -1.93%; 95% CI -2.79 to -1.06). Overall in-hospital mortality declined from 8.1% to 5.7% (AAPC -2.88%; 95% CI -4.15 to -1.59). ICU utilization rose from 37% to 43% of admissions (AAPC +1.31%; 95% CI 0.91 to 1.71). Conclusion Over 14 years, in-hospital mortality and length of stay for major gastrointestinal cancer surgery declined within Brazils universal public health system. These temporal trends occurred alongside expansion of accredited oncology services and increased ICU utilization, although causal relationships cannot be established from administrative data. These findings should be interpreted as hypothesis-generating and highlight the need for more granular hospital-level data in LMIC settings.

To elucidate the early mechanisms underlying the long-term neuroprotective effect of FLASH-RT in the normal brain, spatial transcriptomics (Nanostring) were performed after whole-brain irradiation of C57BL/6J mice with either 1 or 3 fractions of 10 Gy at 5.6x106 Gy/s (1 pulse-FLASH) or at conventional dose-rate 0.1 Gy/s. FLASH -RT induced a distinct transcriptomic signature in the CA3 and DG neurons, with upregulation of genes encoding glutamate receptors, involved in calcium signaling, long-term potentiation and mitochondrial OXPHOS. Early transcriptional upregulation of Gria gene translated into increased AMPAR protein levels at 48h in the DG and CA3 region and sustained higher AMPAR expression at 2 and 4 weeks post-FLASH. These findings support a durable activation of AMPAR. We propose a mechanism to explain FLASH-induced neuroprotection initiated by early calcium influx and subsequent sustained expression of glutamate receptor AMPAR in neurons and/or neural progenitors of the CA3, potentially contributing to long-term cognitive sparing. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/725423v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1ae125forg.highwire.dtl.DTLVardef@138357aorg.highwire.dtl.DTLVardef@13f128dorg.highwire.dtl.DTLVardef@1db1cf6_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFLASH-RT induces a stronger transcriptional response in the hippocampus than the cortex. C_LIO_LIFLASH-RT induces calcium signaling, LTP and mitochondrial OXPHOS genes. C_LIO_LIEarly AMPAR upregulation leads to sustained protein expression. C_LIO_LIFLASH-RT induces a AMPAR-dependent signaling program in CA3 neurons. C_LI

Withdrawal StatementThe authors have withdrawn this manuscript to address issues related to data-use permission and authorship review. Therefore, the authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

Chromophobe renal cell carcinoma (ChRCC) accounts for 5% of all renal cancer cases. Despite its generally indolent behavior and low mutational burden, there is no targeted therapy for metastatic ChRCC. Profilin-1 (Pfn1), a cytoskeletal regulator of actin and tubulin dynamics, has emerged as a potential oncogenic driver in several cancers including RCC, but its role in ChRCC, remains undefined. We observed elevated Pfn1 expression in stage IV ChRCC patients, implicating Pfn1 in advanced disease progression. To investigate this, we manipulated Pfn1 expressions in two ChRCC cell lines UOK276 and RCJ41M. Pfn1 knockdown (KD) significantly reduced proliferation, invasion, and colony formation, whereas Pfn1 overexpression (OE) in UOK276 enhanced ChRCC aggressive phenotypes. Pharmacological inhibition of Pfn1 significantly suppressed proliferation and clonogenic growth in both cell lines. Additionally, Pfn1 KD increased intracellular ROS accumulation, while overexpressed reduced ROS levels, linking cytoskeletal regulation to oxidative stress control. Together, these findings position Pfn1 as a critical mediator of ChRCC progression, linking cytoskeletal remodeling to aggressive tumor behavior. This work highlights Pfn1 as a potential therapeutic target and establishes a framework for cytoskeletal-focused strategies in advanced ChRCC.

BackgroundMesothelioma (Me) is an aggressive cancer with limited response to conventional therapies. The tumors harsh microenvironment contributes to immune escape and therapy resistance and the effects of ICIs on Me are still unclear. Adenosine, an immunosuppressive molecule produced from AMP by the enzyme CD73, accumulates in hypoxic tumor areas. Elevated CD73 and adenosine receptor A2B (A2Br) levels on Me cells are linked to worse patient outcomes, indicating their important role in disease progression and potential as targets for treatment. AimThis study characterizes the Me-ME (micro environment) and evaluates the efficacy of TT-4 (A2B inibitor) and AB680 (CD73 inibitor), alone or with aPD-1, using 3D models in vitro and in vivo. MethodsCD73 and A2B receptor levels were quantified in tumor and normal samples using qRT-PCR and IHC. Cells lines were treated with CoCl2 to mimic hypoxia, then CD73, A2Br and related markers were analyzed. MSTO-211H and REN cells were silenced for CD73, grown as spheroids and adenosine release was measured. Co-culture spheroids of MSTO-211H and Jurkat cells were treated with AMP and CD73 inhibitor, then analyzed for viability and immune markers. An orthotopic Me model was established by injecting AB1-B/c-LUC cells and monitored by in vivo imaging. Proteomic analysis of spheroids was conducted to identify proteins and pathways involved. ResultsHypoxia boosts CD73 and A2Br expression in Me cells, leading to adenosine production via CD73. In 3D co-cultures, AB680 lowered Me cell viability and enhanced activation of Jurkat T cells. In mice, combining aPD-1 therapy with A2Br or CD73 inhibitors strongly reduced tumor growth. Proteomics identified 93 proteins influenced by adenosine signaling through A2B. ConclusionTargeting the adenosine pathway alongside PD-1 blockade offers a promising new immunotherapy strategy for Me.

Treatment of advanced head and neck squamous cell carcinoma (HNSCC) often involves radiotherapy combined with chemotherapy, targeted therapy, or immunotherapy. However, due to its anatomical and molecular heterogeneity, identifying the most effective treatment for each patient remains a major clinical challenge. To address this need, we developed a high-throughput organoid-based drug screening platform that uses patient-derived organoids to assess candidate treatment regimens. We validated the platform by establishing bioprinted 3D organoids of human HNSCC cell lines and exposing them to X-ray radiation in combination with various small-molecule drugs and biologics. We quantified viability using ATP release assays and assessed extracellular matrix (ECM) invasion with a machine learning-based brightfield image analysis pipeline. Proof-of-concept experiments with HPV-negative HNSCC lines (HN30 and HN31, established from primary and metastatic disease from the same patient) and HPV-positive HNSCC cells (SCC154) revealed different therapy agents that can radiosensitize each cell line. Image analysis showed that copanlisib, afatinib, and ibrutinib could limit ECM invasion of HN31, while the AKT inhibitor ipatasertib promotes invasion of HN30 cells, consistent with previous studies. Application of the platform to patient-derived HPV+ oropharyngeal tumor organoids showed that they shared sensitivity to several agents while also exhibiting differences against certain therapies. Cetuximab, sorafenib, and nedisertib significantly radiosensitized organoids from two clinical samples. This work demonstrates the feasibility of performing sensitivity screening by integrating bioprinting, conventional viability assays, and advanced image analysis techniques. This platform has the potential to enable a personalized therapeutic pipeline for patients with advanced HNSCC, optimizing responses to radiotherapy and targeted agents to improve clinical outcomes while avoiding modulators that may promote tumor invasion.

Glypican-3 (GPC3) is an oncofetal protein widely being explored as a diagnostic and therapeutic target in hepatocellular carcinoma (HCC). Given that radiotherapy in the form of external beam and radioembolization are standard-of-care treatments for HCC, we aimed to determine whether there was any relationship between GPC3 and response to radiotherapy. Here, we demonstrate that GPC3 expression confers radioresistance in liver cancer through integrated in vitro, in vivo, and patient-level clinical analyses. Stable GPC3-knockout in liver cancer cell lines (HepG2, Hep3B, Huh7) and ectopic GPC3 expression in GPC3-negative liver cancer cells (SNU449), as well as in non-hepatic A431 cells, demonstrated that GPC3-mediated radioresistance is not restricted to hepatic lineage. Following irradiation, GPC3-deficient cells exhibited reduced proliferation, impaired clonogenic survival, persistent DNA damage, prolonged G2/M arrest, and increased apoptosis. Transcriptomic profiling demonstrated enrichment of cell-cycle and DNA damage response pathways in irradiated GPC3-deficient cells compared with GPC3-positive cells, and protein analyses confirmed sustained activation of the ATM/CHK2 axis. In vivo, GPC3 deletion markedly enhanced radiation-induced tumor growth delay in both HepG2 and A431 xenograft models. Consistent with these findings, high GPC3 expression was associated with inferior clinical outcomes in patients with HCC undergoing external-beam radiotherapy or radioembolization. Together, these findings identify GPC3 as a determinant of radioresistance in liver cancer and suggest its potential utility as a biomarker to guide radiotherapeutic strategies. Significance statementRadiotherapy is an important treatment option for HCC, but biomarkers that predict tumor response remain limited. GPC3 is highly expressed in most HCCs and is being investigated as an important biomarker for diagnosis and treatment of this disease, yet its relationship, if any, on radiosensitivity has not been previously reported. Here, we identify GPC3 as a modulator of radioresistance. GPC3 loss enhances radiosensitivity and is associated with persistent unresolved DNA damage, prolonged G2/M arrest, and sustained activation of the ATM/CHK2 pathway, resulting in delayed tumor growth after irradiation. In a clinical cohort of patients treated with radiotherapy, high GPC3 expression was associated with poorer overall survival. These findings suggest that GPC3 expressing tumors may necessitate either more dose-intense radiotherapy, radiobioligically ablative and/or combined with other modalities, or alternative therapeutic modalities to adequately treat HCC.

Clear cell renal cell carcinoma (ccRCC) exhibits heterogeneity in immune infiltration and clinical outcomes, but the mechanisms governing recruitment and organization of tumor-reactive CD8 T cells remain incompletely defined. We investigated the role of the CXCL13-CXCR5 axis in shaping CD8 T cell recruitment, differentiation, and immune organization in high-risk, non-metastatic ccRCC. Human tumor, plasma, and matched adjacent kidney specimens were analyzed using ELISA, quantitative PCR, migration assays, multiplex immunofluorescence, single-cell RNA sequencing, spatial transcriptomics, and a syngeneic mouse model. CXCL13 was among the most upregulated chemokines in ccRCC relative to matched normal kidney and was embedded within a CD8 T cell-associated inflammatory transcriptional program. In transwell and microphysiological system (MPS) assays, CXCL13 promoted CD8 T cell migration, enriched CXCR5 cells among migrating CD8 T cells and showed reduced migration after CXCL13 or CXCR5 blockade. Single-cell analyses identified CXCR5 expression within stem-like CD8 T cell states associated with TCF7 and IL7R, whereas CXCL13 associated with later cytotoxic/exhausted states along a continuous differentiation landscape. Spatial transcriptomics demonstrated that stem-like CD8 T cells localized within structured lymphoid aggregates enriched for B cells, coordinated CXCL13/CXCR5 expression, and signaling programs. In vivo, tumor-derived CXCL13 suppressed tumor growth, increased intratumoral CD8 T cell infiltration, and enriched CXCR5TCF1CD8 stem-like T cells. In human tumors, higher CXCL13 expression correlated with increased CXCR5CD8 T cell infiltration and improved recurrence-free survival. These findings identify CXCL13 as a regulator of immune recruitment and niche organization and support the CXCL13-CXCR5 axis as a biomarker and possible therapeutic target in ccRCC.

Background & AimsHepatocellular carcinoma (HCC) frequently exhibits resistance to immune checkpoint inhibitors (ICIs), particularly in {beta} -catenin-driven tumors characterized by immune exclusion. While the Unfolded Protein Response (UPR) and the Integrated Stress Responses (ISR) enable tumor adaptation to metabolic stress their role in shaping tumor immunogenicity remains incompletely understood. We investigated whether ATF4, a central effector of the integrated stress response, couples metabolic reprogramming to suppression of anti-tumor immunity in HCC. MethodsWe combined transcriptomic analyses across three independent human HCC cohorts with mechanistic studies using an immunotherapy-resistant MYC/{beta}-catenin-driven murine HCC model. We integrated CRISPR/Cas9-mediated deletion of Atf4 with RNA-sequencing and targeted metabolomics. The impact of tumor-derived metabolites on macrophage differentiation and polarization was evaluated using primary bone marrow-derived cells. Therapeutic responses were evaluated in orthotopic and subcutaneous models treated with anti-PD-1 and anti-VEGFA. ResultsATF4 and XBP1 transcriptional signatures are selectively enriched in human HCC and associate with poor prognosis, vascular invasion, and an immunosuppressive myeloid-enriched tumor microenvironment. Genetic ablation of Atf4 markedly suppressed tumor growth in immunocompetent but not immunodeficient hosts, establishing a requirement for immune-mediated tumor control. Mechanistically, Atf4 loss downregulated Aldh18a1 and disrupted proline biosynthesis, resulting in extracellular proline depletion. This proline-deficient environment abrogated monocyte-to-macrophage differentiation and decreased M2 polarization, thereby reshaping the tumor microenvironment toward enhanced T cell infiltration and activation. Functionally, Atf4-deficient tumors exhibited restored sensitivity to anti-PD-1 monotherapy and showed pronounced responses to combined anti-PD-1/anti-VEGFA treatment in aggressive orthotopic models. ConclusionATF4 programs a proline-dependent metabolic axis that sustains macrophage-mediated immunosuppression and immune evasion in {beta}-catenin-driven HCC. Disruption of this pathway converts immune-excluded tumors into T cell-inflamed states and restores responsiveness to immunotherapy. By governing proline homeostasis and macrophage-mediated immunosuppression, ATF4 is a key metabolic checkpoint for immune evasion, linking stress adaptation to immune escape and a candidate therapeutic target in HCC. Impact and implicationsWe identify ATF4 as a crucial metabolic-immune orchestrator that sustains myeloid-driven immune evasion in {beta}-catenin-dependent HCC through proline-dependent circuitry. Disrupting the ATF4-proline axis converts immune-desert tumors into T cell-inflamed lesions by blocking macrophage differentiation, thereby sensitizing tumors to immune checkpoint therapy. This work positions ATF4 as a tractable therapeutic target to overcome immunotherapy resistance in HCC. Graphical abstract Highlights- ATF4 orchestrates an immunosuppressive tumor microenvironment in HCC by coupling metabolic stress adaptation to immune evasion. - Ablation of ATF4 disrupts proline biosynthesis, leading to a marked depletion of extracellular proline. - Cancer cell-derived proline availability contributes to macrophage differentiation and M2 polarization; its loss restores T cell-mediated anti-tumor surveillance and sensitizes beta-catenin-driven HCC to immune checkpoint blockade.

PURPOSE: Cancer outcomes in sub-Saharan Africa are driven by delayed diagnosis and treatment initiation. We evaluated the magnitude and determinants of diagnostic and treatment delays among cancer patients in Kinshasa, Democratic Republic of the Congo (DRC). METHODS: We conducted a hospital-based cross-sectional study of 460 adults with confirmed cancer at Nganda Hospital Center in Kinshasa, DRC. Two outcomes were assessed: delay from symptom onset to diagnosis and delay from diagnosis to treatment initiation. Log-normal regression models were fitted for each outcome to estimate adjusted geometric mean ratios (aGMRs) and 95% confidence intervals (CIs). Covariates included demographic, socioeconomic, clinical, behavioral, and stigma-related factors. RESULTS: The median age was 55 years, and 76.2% of participants were women. Overall, 55.0% of participants experienced symptom-to-diagnosis delays >6 months, and 49.4% experienced diagnosis-to-treatment delays >3 months. Older age was associated with longer diagnostic delay (aGMR 1.55, 95% CI 1.03-2.31) and treatment delay (1.51, 1.07-2.14). Unemployment was strongly associated with both diagnostic delay (1.68, 1.15-2.47) and treatment delay (2.27, 1.54-3.33), as was hepatitis B co-infection (1.88, 1.06-3.34 and 2.42, 1.15-5.11, respectively). Longer diagnostic delay was additionally associated with informal trading (1.99, 1.21-3.28), taxi or motorbike transport (1.92, 1.25-2.94), and smoking history (2.25, 1.03-4.91), while high cancer-stereotype stigma was associated with longer treatment delay (1.56, 1.04-2.34). CONCLUSION: Substantial delays exist across the DRC cancer care continuum, driven by socioeconomic vulnerability, transport barriers, hepatitis B co-infection, and cancer-related stigma. These findings highlight the need for integrated interventions to improve timely diagnosis and treatment initiation, including strengthening financial protection, decentralizing cancer services, and reducing stigma in cancer care.

PurposeGlasdegib is a Sonic Hedgehog (SHH) pathway inhibitor used for treating newly diagnosed acute myeloid leukemia in elders or patients unfit for intensive chemotherapy. This study sought to demonstrate growth inhibition and increased apoptosis of B-cell acute lymphoblastic leukemia (B-ALL) in vitro under glasdegib, alone and combined with inotuzumab, using a novel co-culture system and validated chemosensitivity testing model to determine whether glasdegib with and without inotuzumab may represent a promising treatment strategy in B-ALL. MethodsSeven blood and marrow samples from B-ALL patients were co-cultured with HS-5 stromal cells in a co-culturing system designed to mimic the tumor microenvironment to maintain B-ALL cell viability for chemosensitivity testing under glasdegib and inotuzumab. ResultsCo-culturing improved B-ALL viability from four to nine days. Dosage-dependent responses to glasdegib were consistent among B-ALL samples on day four based on culture viability, and varied based on expressions of SSH genes GLI1, GLI3, SMO, and PTCH1. Combination with inotuzumab had varied effects on treatment response. ConclusionCo-culturing B-ALL cells with HS-5 stromal cells improves B-ALL growth and viability. Glasdegib with and without inotuzumab treatments impact the viability of co-cultured B-ALL cells by day four. SHH gene expressions suggest different B-ALL patients may be sensitive or resistant to glasdegib and inotuzumab.

Background: Immune-oncology has revolutionized cancer treatment, but some patients fail to benefit due to primary resistance and tumour-immune evasion. Extracellular vesicles (EVs) are secreted by both tumour and immune cells and mediate communication between cancer cells and the immune system. Our study used proteomic profiling of circulating EVs collected from NSCLC patients treated with immune checkpoint inhibitors (ICI) to identify predictive biomarkers of response as well as immune evasion mechanisms related to treatment resistance. Methods: EVs were isolated from plasma collected prior to ICI treatment using peptide-affinity purification and high-throughput proteomics was performed using Proximal Extension Assay. Differentially expressed EV proteins between durable (DR) and non-durable responders (NDR) were identified and evaluated using Cox proportional hazards regression, survival analysis, sex-stratified analysis, as well as pathway and network analysis. Results: Proteomics analysis identified 116 differentially expressed EV proteins between DR and NDR. NDR was characterized by enrichment of inflammatory, angiogenic, and immune-suppressive EV proteins, such as IL1RL1, TFRC, IL6ST, galectins, TNF superfamily death receptors, chemokines, and PCSK9. Pathway analysis revealed enrichment of angiogenesis, chemotaxis, ECM remodeling, and neutrophil degranulation associated with poor progression-free survival (PFS). In contrast, DR to ICI treatment was associated with EV proteins related to T- and B-cell activation and adaptive immunity. Sex-related differences in abundance and association with PFS was observed for certain EV proteins, including IL1RL1 and TFRC. A six protein EV model (IL1RL1, TFRC, ERI1, CCN5, IGFBPL1, and TNFRSF13C) demonstrated good prognostic performance for identifying NDR (AUC = 0.907) and stratified patients into three discrete risk groups. Conclusions: High-plex EV proteomics revealed biologically coherent tumour-immune signaling programs that are associated with ICI treatment resistance. Profiling circulating EVs may improve our understanding of EV-mediated immune evasion mechanisms and identify protein signatures that reflect the tumour immune microenvironment and predict response to immune checkpoint blockade.

Background: Non-invasive diagnosis, reliable recurrence surveillance remain critical unmet needs in gliomas. Glioma induces profound systemic immune alterations despite its anatomical confinement to the central nervous system. Circulating immune cells, particularly monocytes, are key mediators of tumor-host crosstalk and may retain tumor-induced transcriptional imprints. However, their potential clinical utility as blood-based biomarkers for detection and monitoring, remain largely unexplored. Methods and findings: In this study, we performed integrated single-cell RNA sequencing of blood immune cells and demonstrated that circulating CD14+ monocytes are significantly expanded in glioma patients, exhibiting features of differentiation arrest and increased transcriptional plasticity. These cells harbor glioma-specific molecular signatures distinct from those observed in healthy controls and patients with other tumors. Leveraging these findings, we developed an ensemble machine learning diagnostic model based on transcriptomic profiles of circulating CD14+ monocytes (training cohort, n=107), which achieved a mean area under the receiver operating characteristic curve (AUC) of 0.971 during cross-validation. In an independent cohort of 567 participants, the model maintained high diagnostic accuracy, yielding an AUC of 0.877 for distinguishing glioma from controls and other tumors. And it achieved a recurrence detection AUC of 0.969 in 51 postoperative samples. Moreover, in a prospective follow-up study involving 30 glioma patients, lower model-derived scores of postoperation were significantly associated with prolonged progression-free survival (log-rank test, P=0.043), supporting its prognostic utility. Conclusion: We demonstrate circulating CD14+ monocytes undergo glioma-specific transcriptional reprogramming, generating systemic tumor-associated signal captured via transcriptomic profiling. This blood-based diagnostic model provides non-invasive, scalable approach for glioma detection, recurrence surveillance, outcome prediction.