Journal of Translational Medicine

Purpose: Chemoradiotherapy (CRT) for human papillomavirus-related oropharyngeal cancer (HPV+ OPC) causes substantial treatment-related toxicity, with well-known adverse effects on quality of life (QoL), weight loss, and self-reported physical functioning. However, its impact on objectively measured cardiorespiratory fitness is unknown. This study examined changes in cardiorespiratory fitness, body composition, grip strength, and patient-reported outcomes in patients with HPV+ OPC undergoing CRT. Methods: We invited 20 patients with HPV+ OPC scheduled for CRT (age: 61.2 {+/-} 7.1 years, female: n=4) to complete assessments at three timepoints: pre-CRT (baseline), 2-weeks post-CRT, and 8-weeks post-CRT. Cardiorespiratory fitness was assessed using a maximal incremental cardiopulmonary exercise test (CPET). Body composition was estimated using segmental bioelectrical impedance analysis. QoL was assessed using the EORTC QLQ-C30 and QLQ-H&N43, and physical activity was self-reported using the International Physical Activity Questionnaire-Short Form. The primary outcome was change in oxygen consumption at the anaerobic threshold ([V]O2 at AT) measured during CPET; an objective, effort-independent marker of cardiorespiratory fitness. Results: Mean [V]O2 at AT declined from 16.0 {+/-} 3.8 ml/kg/min at baseline to 12.0 {+/-} 3.4 ml/kg/min at 2-weeks post-CRT (adjusted mean change: -4.2, 95% CI: -5.4 to -3.0 ml/kg/min) and remained low at 8-weeks post-CRT. Peak oxygen consumption ([V]O2peak: -7.4, -9.3 to -5.4 ml/kg/min), body mass (-8.5, -10.7 to -6.2 kg), fat-free mass (-6.4, -7.7 to -5.0 kg), grip strength (-4.1, -7.2 to -0.99 kg), global health status (-26.9, -39.2 to -14.6 points), fatigue (49.8, 33.7 to 65.8 points), and several disease-specific symptoms were also adversely affected at 2-weeks post-CRT and remained impaired at 8 weeks. Conclusion: This is the first study to estimate the impact of CRT on cardiopulmonary fitness in patients with HPV+ OPC. Cardiorespiratory fitness declined by ~25% following CRT and remained reduced at 8-weeks. Targeted interventions to mitigate these adverse physiological effects warrants further investigation.

BackgroundPrimary and secondary resistance to immune checkpoint blockade (ICB) remains a critical challenge in advanced melanoma. Oncolytic Viruses (OV) selectively lyse tumor cells while generating systemic anti-tumor immune responses with minimal side effects. Yet their clinical use is limited to refractory melanoma patients and are only given in combination with second-line ICB regimens. ICB can both help and hinder OV efficacy depending on the source of checkpoint interactions across the tumor-immune microenvironment (TiME). However, functional checkpoint interactions are typically inferred from gene or protein expression and rarely contextualized within myeloid- and antigen presenting cell-associated immune niches during OV therapy, despite these populations dominating melanoma TiMEs and serving as key regulators of anti-viral immunity. MethodsAn integrated multi-omics framework combining Nanostring GeoMx digital spatial profiling (DSP), COMET sequential immunofluorescence (seqIF) and functional oncology mapping (FuncOmap) was applied to melanoma patient tissues collected pre- and post-neoadjuvant Talimogene Laherparepvec (T-VEC) to characterize immune remodeling and directly quantify checkpoint interaction dynamics associated with pathologic responses to OV therapy. ResultsT-VEC induced broad lymphocyte- and myeloid-associated immune transcriptional activation across melanoma TiMEs; however, pathologic responses could not be defined by bulk transcriptomics or cellular deconvolution alone. COMET seqIF analysis identified that HSV-associated M1/APC-like tumor-associated macrophages (TAMs) were enriched in complete pathologic response (CR) tissues and were a major source of PD-1/PD-L1 interaction niches. While partial (PR) and non-pathologic response (NR) tissues retained melanoma-centered PD-1/PD-L1 interaction niches and were enriched for B cell and M2-like TAM populations. FuncOmap analysis indicated that post-T-VEC PD-1/PD-L1 interaction states were consistently elevated in tumor bed, but not in lymph node tissues, across all pathologic response groups. Suggesting that immune checkpoint interactions may benefit T-VEC therapeutic responses depending on their spatial and immune context relative to OV infection. ConclusionsThese findings highlight the importance of integrated transcriptomic and functional proteomic analyses for resolving the spatial distribution and functional status of immune niches during OV therapy. Resolving PD-1/PD-L1 interaction states to specific M1/APC-like TAM and B cell niches may define mechanisms of responses and resistance to OV therapy.

Liver sinusoidal endothelial cells (LSECs) are increasingly recognized as a critical yet underexplored cell type in anti-fibrotic drug development. This study presents a computational drug screening platform integrating LSEC-specific transcriptomic analysis across simple steatosis, fibrotic nonalcoholic steatohepatitis (NASH), and cirrhosis, with tiered gene signature selection combining machine learning, large language model-assisted curation, gene safety assessment, and Connectivity Map-based screening using human endothelial perturbational profiles. The platform identifies 6 clinical-stage and 8 preclinical candidates with LSEC-protective potential. Among these, vorinostat (SAHA), a clinically approved histone deacetylase (HDAC) inhibitor, is selected for experimental validation. In hepatocyte-specific Asah1-deficient mice fed a Paigen diet, SAHA attenuates hepatic inflammation, fibrosis, LSEC dysfunction, and portal hemodynamic abnormalities, with effects confirmed in a hepatotoxin (CCl4)-induced fibrosis model. High mobility group box 1 (HMGB1) is identified as a key hepatocyte-derived paracrine mediator of LSEC injury through Transwell co-culture and glycyrrhizin rescue. Vorinostat dose-dependently reverses HMGB1-induced LSEC dysfunction across inflammation, capillarization, fibrogenesis, and vasoconstriction, associated with endothelial transcription factor reprogramming including KLF2 upregulation, validated in primary LSECs and in vivo. SAHA also protected LSECs from TNF--induced inflammation and reduced monocyte adhesion. These findings establish an LSEC-focused drug repurposing framework and identify candidates for LSEC-protective anti-fibrotic therapy. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/727430v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@a71eb3org.highwire.dtl.DTLVardef@1d090acorg.highwire.dtl.DTLVardef@1ca1450org.highwire.dtl.DTLVardef@1fbb3fb_HPS_FORMAT_FIGEXP M_FIG C_FIG

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and long COVID are complex chronic conditions that often follow infectious triggers with overlapping clinical features but poorly defined pathophysiological relationships. This study aimed to identify disease-specific immune signatures through multiparameter immunophenotyping of monocytes, dendritic cells, and T-cell subsets. A total of 207 participants were included (ME/CFS: n = 103; long COVID: n = 63; healthy controls: n = 41). Peripheral blood mononuclear cells were analyzed using multiparameter flow cytometry. Statistical analyses included non-parametric testing, age-adjusted ANCOVA, correlation network analysis, and principal component analysis (PCA). Long COVID was characterized by increased M2-like monocyte polarization, elevated CD80 expression across monocyte subsets, expansion of dendritic cells, and reduced expression of activation markers, indicating persistent immune activation with features of immune exhaustion. In contrast, ME/CFS exhibited reduced costimulatory molecule expression, impaired CCR7-mediated immune cell trafficking, and less coordinated activation patterns, consistent with a state of immune suppression. Correlation network analysis revealed more extensive and integrated immune interactions in long COVID, while PCA identified distinct immunophenotypic components and enabled moderate discrimination between the two conditions. These findings demonstrate that ME/CFS and long COVID are characterized by distinct immune profiles, supporting the concept of divergent immunopathological mechanisms. The identified signatures may contribute to biomarker development and guide targeted therapeutic approaches.

Background: Black patients and individuals with low socioeconomic status (SES) face significant disparities in accessing curative therapies for hepatocellular carcinoma (HCC), including liver transplantation. This study aimed to develop provider-co-created intervention prototypes in response to patient-identified barriers and recommendations. Methods: A human-centered design session with hepatology and transplant providers at a large academic medical center was conducted. Prior to the session, participants were presented with barriers and preliminary solutions identified through an earlier human-centered design session with Black and low-SES patients. Using structured ideation methods, including brainwriting, challenge mapping, and concept voting, providers co-created intervention prototypes. Final concepts were synthesized from patient insights, provider input, and design methods using affinity diagramming and concept modeling. Results: Nine providers participated in the session. They focused on three key areas for intervention: inefficiencies in transplant pre-evaluation, inadequate social support, and information overload. Solutions included: (1) a structured triage pathway to standardize referrals and reduce delays; (2) a peer navigator model to guide patients through the transplant process; and (3) a multimodal transplant education roadmap to improve comprehension and engagement. These prototypes addressed both patient- and system-level barriers. Conclusions: Protypes developed through provider-led design, grounded in patient-identified barriers and co-created ideas, can yield actionable, scalable strategies to advance equity in HCC care. Future work will refine these prototypes through patient feedback and pilot them in clinical settings.

ImportanceDigital Phenotyping (DP) utilizes digital technologies to assess observable phenotypic traits, enhancing our understanding of various disease states. It aims to support equitable interventions through diverse digital data, fostering inclusivity by integrating various digital devices. Central to DP is identifying digital biomarkers (DBMs), which offer real-time health monitoring and personalized insights into disease conditions. Applying machine learning (ML) techniques on non-invasive signals sets the ideal platform for precision medicine application, especially in rare diseases (RDs). People living with rare diseases (PLWRD) often face significant challenges in receiving timely and accurate diagnoses, leading to what is known as a diagnostic odyssey. Digital phenotyping (DP) offers a promising solution by leveraging advanced technology, such as 3D facial photography, to capture unique digital signatures associated with various rare diseases. This innovative approach not only aids in the identification of these conditions but also facilitates the detection of digital biomarkers (DBM). These biomarkers enable healthcare providers to monitor the progression of the disease over time, enhancing patient care and potentially shortening the duration of the diagnostic odyssey. By utilizing DP and DBMs, we can improve both the diagnosis and management of RDs, ultimately leading to better health outcomes for affected participants. ObjectiveTo identify whether DBMs can be identified by DP utilizing 3D facial imaging techniques in outpatient settings in participants with RDs. The primary objective of this study was to determine if specific facial measurements in participants with RD who experience transient episodes of facial swelling (oedema) differ from established ethnically matched norms. The secondary objective was to assess peri-orbital and/or facial swelling as a potential biomarker for identifying flare-ups in hereditary angioedema (HAE). Design, setting, and participantsThis multicentre observational study was conducted in 3 hospitals in Singapore. The eligible participants were male and female RD participants of various age groups. The study duration was 4 years and 8 months. Interventions: Twenty participants of Chinese genetic ancestry were photographed using a 3D camera. Additionally, two participants with hereditary angioedema (HAE) were photographed during acute stages of disease flare-ups. Main outcomes and measuresThe obtained facial scans of participants (that included participants with HAE in non-acute phase) were plotted using Artificial Intelligence-powered software - Cliniface. The growth curves and facial landmarks obtained were compared against the growth curves of normal RD-unaffected individuals of Chinese genetic ancestry. The two participants with HAE were photographed qualitatively over a longer period of time, and their scans were plotted, yielding growth curves. ResultsDistinct facial markers such as periorbital swelling were identified in two qualitatively assessed HAE participants during flare-up stages. This provides an opportunity to explore and validate further if these facial signatures in a disease condition can be assigned as DBM for HAE. Conclusions and relevanceThis study explores the utility of 3D facial analysis as a DBM in rare diseases such as HAE. Applying non-invasive signals coupled with AI may open new vistas for precision medicine in real-world settings. The individual measurements that yielded small p-values demonstrate significant relevance and potential utility. These findings offer preliminary objective evidence that supports existing subjective reports of facial features in the literature. Additionally, while DPs diagnostic capabilities may be limited, it successfully identified DBM, which could facilitate disease monitoring in conditions such as HAE. Author SummaryRare diseases pose a significant challenge to all stakeholders, including clinicians, patients families, care providers, and the healthcare system. Diagnostic delays are integral and impose a massive financial and emotional burden on everyone involved in care delivery, beyond the patients themselves. A universally acceptable, scalable, and replicable non-invasive mechanism to detect distinct biomarkers associated with a rare disease can help identify the diseases signs and symptoms far earlier and ease the burden. An easy-to-deploy approach could be 3D facial imaging of patients with rare diseases, which are associated with distinct or subtle facial changes at different stages of disease progression. A rare disease, such as hereditary angioedema, which is known to exhibit facial swelling in patients during the acute disease state, is a prime example. The facial changes can be identified and assigned as specific disease markers, also known as facial biomarkers. These facial biomarkers can be identified and measured using 3D facial imaging when patients present to the clinic. Subsequently, these initial signs can be correlated clinically to establish a firm diagnosis earlier than traditional approaches.

BackgroundKidney transplantation is the preferred treatment strategy for end-stage kidney disease. Deceased donor kidneys usually undergo cold storage until kidney transplantation, leading to cold ischemia injury that may contribute to poor graft outcomes. However, the molecular characterization of potential mechanisms of cold ischemia injury remains incomplete. ResultsTo bridge this knowledge gap, we leveraged the 10x Visium spatial transcriptomic technology to perform full transcriptome profiling of murine kidneys subject to varying durations of cold ischemia typical in a deceased donor kidney transplant setting. We developed a computational workflow to identify and compare spatiotemporal transcriptomic changes that accompany the injury pathophysiology in a tissue compartment-specific manner. We identified proportional enrichment of oxidative phosphorylation (OXPHOS) genes with increasing duration of cold ischemia injury within the oxygen-lean inner medulla region, suggestive of atypical metabolic presentation. This was distinct in cold ischemia injury tissue compared to warm ischemia-reperfusion kidney injury tissue. Spatiotemporal trends were validated by qPCR and immunofluorescence in a larger cohort of mice. We provide an interactive online browser at https://jef.works/CellCarto-ColdIschemia/ to facilitate exploration of our results by the broader scientific and clinical community. ConclusionsAltogether, our spatiotemporal transcriptomic analysis identified coordinated molecular changes within metabolic pathways such as OXPHOS deep within the cold ischemic kidney, highlighting the need for increased attention to the inner medulla and potential opportunities for new insights beyond those available from superficial biopsy-focused tissue examinations.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies due to its dense stroma, which limits drug delivery and therapeutic efficacy. Ultrasound (US) mediated strategies using nanobubbles (NBs) offer a promising approach to enhance treatment, yet the biological effects of NB exposure and the timing of US application remain unclear. Here, we investigated how NB exposure with immediate (0h) or delayed (1h) US affects viability, proliferation, metabolism, and stress signaling in PANC-1 and BxPC-3 cells. Immediate US exposure in the presence of extracellular nanobubbles resulted in a greater reduction in cell viability at 24 h compared to delayed US application. Proliferation analysis showed that Ki67 positivity decreased following USNB treatments in both cell lines. Metabolically, NB treatment alone increased cellular activity, whereas combined USNB treatment reduced metabolic activity over time. Seahorse analysis revealed higher basal respiration in PANC-1 cells compared to BxPC-3 cells, consistent with a more glycolytic phenotype, while USNB treatment enhanced glycolytic responses, particularly in PANC-1. Moreover, stress responses were also more pronounced in PANC-1 cells, with HSP70 expression increasing up to 2-fold in NB incubated group and decreasing in USNB groups compared to untreated, whereas BxPC-3 cells exhibited only modest and opposite changes to PANC-1 in HSP70 expression decreasing with NB incubation. Treatment timing critically influenced outcomes, with immediate US producing stronger antiproliferative and cytotoxic effects, highlighting the importance of sequencing in USNB therapeutic strategies. Moreover, NBs alone stimulated metabolic and stress responses that may promote proliferation, whereas NBs combined with US induced stronger stress responses associated with metabolic reprogramming and reduced proliferation.

Introduction: Myalgic Encephalomyelitis (ME)/Chronic Fatigue Syndrome (CFS) is a debilitating condition characterised by severe fatigue and post-exertional malaise (PEM). Reported neuropsychophysiological abnormalities suggest ME/CFS is multifactorial, but current knowledge remains fragmented. This study protocol outlines a multimodal investigation designed to (1) compare neuropsychophysiological mechanisms between ME/CFS patients and healthy participants, (2) test an integrative model of ME/CFS, (3) identify neuropsychophysiological subgroups within the patient population, and (4) identify predictors of symptom response during rehabilitation. Methods and analysis: This study will enroll 115 ME/CFS patients and 55 healthy participants. Groups will be comparable in age, sex, and education level, with a larger patient sample enabling subgroup and longitudinal analyses. A cross-sectional assessment at baseline will be carried out in both groups. Patients will then be evaluated longitudinally throughout a standardized cognitive-behavioral therapy rehabilitation program delivered as routine care. Baseline measures include systemic inflammation and general health biomarkers, measures of autonomic and central nervous system function, neuroinflammation (magnetic resonance spectroscopy, [18F]DPA714 PET in a subsample), serum short-chain fatty acid levels, gut microbiota composition and function, and neuroendocrine and self-reported responses to psychosocial stress. Fatigue severity (physical and cognitive) and PEM will be assessed through validated questionnaires, ecological momentary assessment, and laboratory tasks. These will be re-evaluated during therapy, and all non-neuroimaging measures will be repeated after the rehabilitation program. Statistical analyses will comprise multivariate analysis of variance, general linear models, classification algorithms, structural equation models, least absolute shrinkage selection operator principal component regression (LASSO-PCR), cluster analysis and latent class growth analysis (LCGA).

BackgroundSkin aging is multifactorial, and finished multi-ingredient oral beauty supplements require dedicated clinical evaluation because their effects cannot be inferred from individual ingredient data alone. ObjectiveTo explore, in a 56-day single-arm open-label study, whether daily oral intake of NatureU(R) Mind Care BeautyU Caps is associated with within-participant changes in crows-feet wrinkle count (primary endpoint), stratum corneum hydration (secondary endpoint), and additional exploratory skin-aging parameters in adult women. MethodsA single-center, open-label, single-arm exploratory study enrolled 33 healthy women aged 36-56 years; 31 completed the protocol and were included in the completer efficacy analysis. Participants took one capsule orally once daily for 56 consecutive days. Assessments were performed at D0, D28 and D56 using PRIMOS CR, Corneometer CM 825, Cutometer MPA580, Glossymeter, Colorimeter CL400, Mexameter MX18, VISIA CR, DermaScan and a structured self-assessment. ResultsPRIMOS CR crows-feet wrinkle count fell from 965 {+/-} 334 at D0 to 514 {+/-} 171 at D56 (within-participant change -46.74%; nominal P = 0.001). Corneometer hydration rose from 44.3 {+/-} 7.8 to 70.3 {+/-} 9.9 (+58.69%; nominal P = 0.001). Exploratory parameters (other wrinkle metrics, elasticity, gloss, ITA{degrees}, melanin, spots, dermal thickness/density) generally moved in directions consistent with the primary signal. No adverse reactions were reported. ConclusionIn this open-label, single-arm exploratory study, daily NatureU(R) Mind Care BeautyU Caps was associated with within-participant reductions in crows-feet wrinkle count and increases in stratum corneum hydration over 56 days. Findings are hypothesis-generating; randomized placebo-controlled trials are required.

BackgroundTumor-associated macrophages (TAMs) are key drivers of the immunosuppressive tumor microenvironment (TME), supporting tumor progression through diverse functions. However, mechanistic studies of TAM polarization remain limited by the lack of physiologically relevant human model systems that capture stromal-immune interactions and macrophage heterogeneity. MethodsWe established advanced human co-culture systems that integrate healthy donor-derived macrophages with patient-derived organoids and tumoroids (PDOs and PDTs), as well as matched normal fibroblasts (NFs) and cancer-associated fibroblasts (CAFs). These multicellular models enabled the investigation of interactions among stromal, epithelial, and immune cells within tumor and adjacent normal tissue environments. ResultsThe co-culture systems recapitulated distinct macrophage states associated with tumor and adjacent normal environments and identified fibroblasts as major regulators of macrophage phenotypes. CAFs promoted macrophage metabolic remodeling characterized by altered lipid handling and enrichment of TAM-like signatures. Mechanistically, we identified thrombospondin 1 (TSP1) as a CAF-secreted factor linked to metabolic priming. Recombinant TSP1 induced transient lipid accumulation followed by mitochondrial remodeling. In tumor co-culture conditions, CD36 inhibition reduced lipid accumulation in macrophages, supporting a role for TSP1-linked lipid crosstalk in stromal-immune interactions. ConclusionOur study establishes advanced patient-derived co-culture models as a platform to investigate human TAM biology and stromal-immune interactions in CRC. Using these systems, we identify a fibroblast-associated TSP1-lipid axis linked to macrophage metabolic remodeling and TAM-like polarization, highlighting stromal metabolic communication as a potential targetable feature of the CRC microenvironment. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=176 HEIGHT=200 SRC="FIGDIR/small/728363v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@16d60aborg.highwire.dtl.DTLVardef@1e593e7org.highwire.dtl.DTLVardef@15124b0org.highwire.dtl.DTLVardef@3fca92_HPS_FORMAT_FIGEXP M_FIG C_FIG

PurposeDifferentially expressed genes (DEGs) between colorectal cancer liver metastasis (CRLM) epithelium and primary colorectal cancer (CRC) epithelium (LMR DEGs) identified based on single-cell RNA sequencing (scRNA-seq) data may become new biomarkers for CRC prognosis. MethodsAn scRNA-seq dataset was used to describe the cellular landscape of primary CRC and CRLM and identify LMR DEGs. Prognostic LMR DEGs were identified in the bulk RNA-seq dataset. Based on the prognostic LMR DEGs, multiple machine learning algorithm combinations were compared in terms of their C-index, and the best model was selected for the construction of the LMR score. ResultsAmong the 2070 LMR DEGs, 426 prognostic LMR DEGs were ultimately obtained. The combination of the randomized survival forest (RSF) model and ridge regression had the highest C-index and was therefore used to construct a 15-gene scoring system (LMR score). In the external validation set, the 1- and 5-year AUCs of the LMR score were greater than those of the AJCC stage and other scoring systems constructed with a similar dataset. In addition, the LMR score was closely associated with factors that influence CRC outcomes, such as immune infiltration. ConclusionThe LMR score may be a reliable new biomarker for predicting the prognosis of patients with CRC.

Currently, the genetic architecture of Middle Eastern populations is underrepresented in global genomic databases. This gap increases the rate of Variants of Uncertain Significance (VUSs) and clinical misinterpretations of genomic data especially in Middle Eastern populations. Whole exome sequencing was conducted on 90 healthy individuals from Jordan and the data were analysed using Principal Component Analysis (PCA) and multi-computational filtering. PCA revealed a double ancestry (EUR-AFR) admixture rather than a triple admixture (EUR-AFR-AMR). More than 3,500 populations-specific variants (PSVs) were identified, of which 72% were singletons. Additionally, 19 variants were significantly enriched compared to the maximum allele frequencies in public global databases (Fisher's exact test with Benjamini-Hochberg false discovery rate correction, p-value < 0.05). Consequently, the results suggest the reclassification of variants of Uncertain Significance (VUS) which reside in the ECE2 gene to likely benign and the variants of Conflicting Classification of Pathogenicity in the genes IL1RN and THPO to benign based on the significant allele frequency (AF=0.0389, p-value < 0.05). Furthermore, a pathogenic ClinVar variant was identified in a healthy individual, warranting careful interpretation. The findings underscore the importance of identifying PSVs in order to minimize or even prevent clinical misdiagnosis and highlight the unique genetic signature in Jordan. The study serves as a foundational resource for precision medicine in the region.

Background: Immune checkpoint inhibitors have transformed cancer treatment, yet a large number of patients fail to respond. Identifying molecular characteristics that predict response before treatment initiation remains an unmet need. Towards that end, this study presents a large-scale integrative analysis of existing single-cell and bulk tissue datasets, aimed at identifying predictive features while providing insights into their cellular origin and potential function within the tumor microenvironment. Methods: A stepwise analysis was performed using single-cell RNA-sequencing data from 60 melanoma patients at baseline, separated into discovery (n=41) and validation (n=19) sets. An integrated bulk transcriptomics dataset (n=128) from melanoma patients and a bladder cancer dataset (n=298) were used for further validation. Results: Integrative analysis of melanoma single-cell datasets revealed that responders exhibit distinct molecular profiles across multiple cell types compared to non-responders. Notably, these included downregulation of the TNFR superfamily and other immunosuppressive genes (TNFRSF18, TNFRSF9, TNFRSF4, LGALS1, BATF, IL12RB2, LINGO1, DUSP4, SDC4, VCAM1) in T-cells. By investigating the findings from the immune cell populations in the bulk tumor context, 13 transcripts were found to be consistently associated with response across all cohorts. These were differentially expressed in T-cells (SELL, EPB41, CD96, UHFR2, LINGO1, LGALS1), B-cells (ALDH5A1), NK cells (PLEC, PDGFRB) and Monocytes (TLR10, ST6GAL1, IKZF1, MPRIP). A predictive model based on these features effectively discriminated responders from non-responders in melanoma (AUC=0.73). The model maintained significant predictive power in an independent bladder cancer dataset (IMvigor210; AUC=0.64). Of high clinical relevance, it demonstrated enhanced performance in identifying responders among patients with low tumor mutational burden (AUC=0.75). Conclusion: Our study reveals pre-treatment molecular features related to immune-cancer crosstalk that are associated with response to immunotherapy. A 13-gene model demonstrates potential added clinical value in stratifying responders, particularly in patients with low tumor mutational burden, meriting further validation.

Background: Head and neck cancer (HNC) patients experience clinically significant anxiety and depression in 65-85% of cases during active treatment. Current supportive care lacks personalized, real-time non-pharmacological interventions. Skitii is a novel HRV-adaptive music therapy system that uses continuous RMSSD (root mean square of successive differences) monitoring via a Polar H10 chest sensor to select music in real-time, targeting parasympathetic recovery (RMSSD >=30ms). Methods: This is a prospective, open-label, randomized controlled trial (1:1 allocation) at Yenepoya Medical College Hospital, Mangalore, India. Adults aged 18-75 years with confirmed head and neck cancer (any subsite, Stage I-IV) undergoing radiotherapy and/or chemotherapy with baseline distress (HADS >=8 or NCCN Distress Thermometer >=4) will be enrolled. Participants are randomized to Skitii adaptive music therapy (20-minute sessions, 3 times daily, 3 weeks) or static music therapy control. Skitii uses a two-phase algorithm: Phase 1 (0-2.5 minutes) uses heart rate as a stress proxy for immediate music selection; Phase 2 (2.5-20 minutes) uses RMSSD to adapt music every 2.5 minutes when physiological state changes by >=20%. Primary endpoints are HADS-Anxiety score and resting RMSSD at Week 3. Sample size is 70 (35 per arm), powered at 80% to detect a 2.5-point HADS difference (SD=3.8, alpha=0.05, 15% dropout). Analysis is ANCOVA, intent-to-treat. Discussion: This is the first randomized controlled trial evaluating RMSSD-based adaptive music therapy in cancer patients. The active control design isolates the effect of the adaptive algorithm from music exposure alone. If positive, results will support a scalable, cost-effective supportive care intervention with objective physiological monitoring, and provide the clinical evidence base for CDSCO Class B medical device approval for Skitii in India, with future CE Mark and FDA applications planned. Trial Registration: Clinical Trials Registry - India CTRI CTRI/2025/11/116732

Background & AimsColorectal cancer liver metastasis is the leading cause of mortality in affected patients, with liver sinusoidal endothelial cells playing a pivotal role in metastatic niche formation. Proprotein convertase subtilisin/kexin type 9 has emerged as a regulator of tumor biology, but its function in the hepatic microenvironment remains poorly defined. This study aimed to characterize the role and subcellular localization of PCSK9 in liver sinusoidal endothelial cells and to evaluate the therapeutic potential of its endothelial-specific inhibition in colorectal liver metastasis. MethodsIn vitro studies were performed using human and murine liver sinusoidal endothelial cells stimulated with conditioned media from metastatic colorectal cancer cells and cancer stem cells. Subcellular localization was assessed by immunofluorescence, immunogold electron microscopy, and biochemical fractionation. Protein interactions were investigated using co-immunoprecipitation and proteomic analyses. For in vivo validation, a murine model of colorectal liver metastasis was generated by intrasplenic injection of tumor cells, followed by systemic administration of chondroitin sulfate-targeted nanoparticles delivering PCSK9 siRNA every 5 days for 18 days. ResultsPCSK9 was consistently expressed in liver sinusoidal endothelial cells and displayed a predominant nuclear localization, which increased upon tumor-induced activation. Proteomic integration identified multiple candidate interacting proteins involved in metabolic and tumor-related pathways. Targeted nanoparticle-mediated delivery achieved efficient PCSK9 silencing in vitro. In vivo, endothelial-specific PCSK9 inhibition significantly reduced liver metastatic tumor burden compared with control groups, whereas free siRNA showed no significant effect. ConclusionsPCSK9 exhibits a novel nuclear localization in liver sinusoidal endothelial cells and potentially interacts with proteins implicated in tumor mediated pathways. Selective inhibition of endothelial PCSK9 using targeted nanoparticles significantly reduces colorectal liver metastasis, highlighting a novel therapeutic strategy focused on the hepatic microenvironment. Impact and ImplicationsThis study provides mechanistic insight into how PCSK9 contributes to colorectal liver metastasis by identifying its novel nuclear localization and potential function in liver sinusoidal endothelial cells. These findings are important for researchers and clinicians seeking to understand microenvironment-driven metastasis and resistance to current therapies. The demonstration that endothelial-specific targeting of PCSK9 reduces metastatic burden suggests a new avenue for therapeutic development beyond systemic inhibition. Such strategies could be translated into precision nanomedicine approaches to improve outcomes in patients with metastatic colorectal cancer while minimizing off-target effects.

BackgroundRetrotransposable elements (RE) comprise approximately 45% of the human genome and are typically repressed by DNA methylation to preserve genomic integrity. In cancer, global DNA hypomethylation can lead to RE derepression, resulting in genomic instability and activation of innate immune pathways through viral mimicry. While individual RE classes have been examined in clear cell renal cell carcinoma (ccRCC), the integrated epigenetic landscape of multiple RE families and their clinical relevance remain incompletely characterized. MethodsWe performed a genome-wide prediction of DNA methylation across three major RE classes (Alu, LINE-1, and LTR elements) using a validated computational framework applied to Illumina methylation array data from two independent ccRCC tumor cohorts. Integrated unsupervised clustering of RE methylation profiles was used to define the epigenetic subtypes. Associations with clinicopathologic variables, tumor immune microenvironment composition (DNA Methylation-derived), hypoxia signaling, innate immune activation, and overall survival were evaluated. Prognostic relevance was assessed using multivariable Cox regression models adjusting for age, sex, AJCC stage or AUA risk group, and immune and angiogenic tumor microenvironment features. Key findings were then externally validated in CPTAC-ccRCC and independently replicated in an institutional Dartmouth Cancer Center (DCC) cohort with matched methylation and RNA-sequencing data. ResultsIntegrated clustering identified three reproducible RE methylation subtypes, Repressed, Transient, and Active. In the discovery cohort, the Active subtype showed significantly worse overall survival than the Repressed subtype, with a graded survival pattern across RE methylation states that persisted after multivariable adjustment. RE hypomethylation was associated with reduced EPAS1 (HIF2A) expression, increased immune infiltration, elevated PD-1 expression, and heightened cGAS-STING and interferon signaling, consistent with an immune-inflamed yet immunosuppressed tumor state. In the external CPTAC validation cohort, RE methylation subtypes recapitulated key molecular features and showed supportive survival trends. In the independent DCC replication cohort, an Active RE state was again associated with poorer survival, lower EPAS1 expression, increased PD-1 expression, greater CD8 T-cell and Treg infiltration, and elevated T-cell exhaustion signatures, supporting the reproducibility of the prognostic and immune-exhausted phenotype across cohorts. ConclusionsWe identified RE methylation subtypes with distinct molecular, immunologic, and prognostic features in ccRCC. External validation in CPTAC and independent replication in DCC support the robustness of this RE methylation framework across large-scale and institutional cohorts. These findings highlight the prognostic potential of RE methylation profiles and support their integration into molecular classification strategies to improve risk stratification in ccRCC.

Background: Alpha-1 antitrypsin deficiency (AATD) caused by the PI*ZZ mutation (Glu342Lys) results in hepatic accumulation of misfolded AAT-Z protein and reduced circulating AAT levels, leading to progressive liver disease and emphysema. Gene correction therapy represents a potentially curative approach by directly correcting the underlying genetic defect. We report the first case of successful hepatic gene correction with early histological and functional assessment. Methods/Case presentation: We report the case of a 66-year-old male patient with PI*ZZ AATD who underwent gene correction therapy within the YOLT-202 phase I/Ia clinical trial (clinical trial.gov ID NCT07193615). Ten weeks post treatment a liver biopsy was performed to re-evaluate pre-existing F2 liver fibrosis as measured by elastography before entering the study. Serum samples allowed functional assessment of the AAT-mediated elastase inhibition. Results: Liver biopsy did not show signs of hepatic inflammation and demonstrated 54% (Sanger) and 57% (Illumina) gene correction rate of the PI*ZZ variant on the DNA level with no bystander edits or off-target effects. Following a transient elevation of transaminases during the early post-treatment period, liver enzymes normalized. Monthly serum AAT measurements demonstrated biologically active and stable therapeutic levels throughout follow-up. Conclusions: This case demonstrates efficient and precise hepatic gene correction without concerning histological alterations and with substantial improvement of functional parameters, supporting the feasibility and safety of gene editing approaches for AATD.

Background and objectiveChronic comorbidities like diabetes mellitus can influence cancer incidence and progression. However, their impact on pathological and molecular tumor characteristics, and dissemination, especially in prostate cancer (PCa), is not fully understood. Here, we investigated differences in the PCa molecular landscape with coexisting diabetes type 2 and its link to tumor dissemination. MethodsDAmico intermediate- or high-risk PCa patients (n=145), with type 2 diabetes or no diabetes, were analyzed for clinico-pathological features, circulating tumor cell (CTC) presence, yields and phenotypes in tumor-draining vein (TDVB) and peripheral blood (PB), primary tumor characteristics, and selected plasma biomarkers. Key findings and limitationsDiabetes type 2 was found in 20/13.8% patients and associated with more advanced clinical outcomes, i.e. pathological tumor stage (p=0.011) and lymph node involvement (p=0.020). Among patients diagnosed before age 65, diabetes and PCa showed a borderline association with shorter time-to-biochemical recurrence (p=0.032). Diabetic PCa patients had significantly higher total CTC counts in TDVB but not in PB, indicating enhanced tumor cell dissemination from primary tumor (PT). PTs from diabetic PCa patients displayed a borderline association with higher ALDH1 expression (p=0.054) and significantly lower vascular vessel density (p=0.008). Similarly, in patients under 65, PTs from diabetic PCa patients expressed genes linked to decreased angiogenesis. Plasma analyses revealed elevated GDF15 levels in PB (p<0.001), increased TRAP5 concentrations in TDVB (p=0.001), and reduced osteonectin levels in TDVB (p=0.026) in diabetic PCa patients. The studys limitation is the relatively small cohort, especially those with coexisting diabetes type 2. Conclusion and clinical implicationsDiabetes in PCa patients associates with advanced tumor stage, enhanced tumor cell dissemination, impaired vascularization, and distinct circulating biomarker alterations. Vascular alterations from diabetes, with systemic factors, especially in PCa patients under 65, may increase tumor dissemination in PCa. However, exact mechanisms need investigation in larger cohorts of patients. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/720986v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@1f5047forg.highwire.dtl.DTLVardef@fa3f43org.highwire.dtl.DTLVardef@14f7765org.highwire.dtl.DTLVardef@27a12d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Melanoma remains one of the most treatment-refractory malignancies due to immune evasion, high mutational burden, and profound tumor heterogeneity. Although immune checkpoint inhibitors have transformed frontline management, a substantial proportion of patients develop resistance or experience relapse, underscoring the need for alternative and complementary immunotherapeutic strategies. Tumor-infiltrating lymphocyte (TIL) therapy and engineered viral vector-based immunotherapies represent mechanistically distinct yet clinically promising approaches for advanced melanoma. This systematic review and Bayesian meta-analysis evaluated the comparative efficacy of TIL therapy and engineered viral vector immunotherapies in advanced melanoma. A structured search of PubMed, Embase, Scopus, and Web of Science (2015-2025) identified 13 eligible studies, including four randomized controlled trials and nine prospective single-arm studies, reporting objective response rate (ORR), progression-free survival (PFS), overall survival (OS), and treatment-related adverse events. Eight studies met criteria for inclusion in the Bayesian quantitative synthesis of ORR outcomes. Risk of bias and certainty of evidence were assessed using Cochrane and GRADE frameworks. TIL therapy demonstrated substantial standalone efficacy, particularly in PD-1-refractory populations, with reported ORRs reaching 49%, median PFS of 7.2 months, and OS extending to 25.8 months. Viral vector-based therapies, including talimogene laherparepvec (T-VEC) and RP1, showed more modest monotherapy activity but demonstrated improved responses when combined with immune checkpoint inhibitors. Among the studies included in the Bayesian quantitative synthesis, the pooled ORR estimate was 37.8% (95% highest density interval [HDI]: 30.6%-45.3%). Sensitivity analysis excluding the small-sample Cui et al. (2022) study yielded a similar pooled estimate of 38.3% (95% HDI: 30.4%-46.2%). Exploratory meta-regression supported the overall robustness of the findings. Certainty of evidence for ORR was moderate, whereas survival and safety outcomes were downgraded due to heterogeneity, sparse reporting, and inconsistent endpoint definitions. Collectively, these findings support complementary rather than competing roles for TIL and engineered viral vector immunotherapies within evolving melanoma treatment paradigms. The results further highlight the potential importance of biomarker-guided sequencing strategies, including viral immune priming followed by adoptive cellular therapy, as a framework for optimizing personalized immunotherapy in both refractory and earlier-line melanoma settings.