Positive surgical margin assessment in computational pathology is a low signal-to-noise task because benign inflammation, atrophic glands, and other histological mimics may resemble high-grade carcinoma in restricted fields of view. We propose TD-VAN, a top-down visual attention framework that treats coherent global tissue architecture as a beneficial signal rather than nuisance variation. An Overview-Net extracts low-power architectural and topographical priors, including glandular crowding, infiltrative arrangement, stromal organization, inflammatory aggregation, spatial continuity of suspicious glands, and their relationship to the inked margin. These priors dynamically guide a Focus-Net for context-aware local recognition, helping suppress false positives caused by deceptive mimic noise. Multi-scale sampling integrates architectural and cytological evidence, while automated Topographical Zonal Profiling converts predictions into spatially resolved metrics, including tumor burden, minimum tumor-to-margin distance, and Gleason-grade composition. On a temporally held-out cohort from 2022–2024, TD-VAN achieved an accuracy of 0.971 and a Macro F1-score of 0.974. These results indicate that global context can stabilize surgical-margin assessment and provide interpretable evidence for postoperative risk stratification.

Abstract Although the heterogeneity and plasticity of fibroblasts are recognized hallmarks of tissue fibrosis, their specific contributions to renal fibrosis progression and the therapeutic validity of targeting key effector subsets remain key unanswered questions. Here, we revealed that diverse, functionally exclusive fibroblast subpopulations actively shape their local microenvironment. Pro-inflammatory fibroblasts (i-Fibs) construct immune-active areas, while pro-fibrotic fibroblasts (ECM-Fibs) generate fibrogenic zones. These two distinct microenvironments form a spatial mosaic, occupying mutually exclusive territories within the kidney tissue. We identified mechanotransduction as a signal governing the switch from i-Fibs to the pathogenic ECM-Fib phenotype. Through unbiased bioinformatics and genetic tools, we pinpointed CD248 as a specific cell-surface protein on this matrix-producing subset. Mechanistically, CD248, via its C-type lectin-like domain, senses the disordered matrix, promoting focal adhesion assembly and YAP nuclear translocation through an IQGAP1/ARF6-GTP-dependent axis, leading to a sustained feedback loop between aberrant matrix and myofibroblasts activation. A monoclonal antibody targeting CD248, IgG78, effectively interrupted this feedback loop, mitigating fibrogenesis both in vitro and in vivo in male mice. Collectively, this study established that fibroblast heterogeneity drives pathological niche specification in the fibrotic kidney and validated CD248 as a promising therapeutic target to counteract tissue fibrosis by disrupting aberrant mechanosignaling.

Abstract Background Hepatocellular carcinoma (HCC) is a prominent cause of cancer-related mortality globally. It is urgently necessary to elucidate the pathogenesis of HCC and develop novel therapeutic strategies. Although certain circular RNAs (circRNAs) act as oncogenic drivers in HCC progression, the underlying mechanisms remain poorly understood. Methods CircRNA-miRNA-mRNA network was established to screen potential circRNAs that were associated with HCC progression. CCK8, colony formation, EdU staining, wound healing, transwell assay and xenograft models were implemented to investigate the role of circRPPH1 in HCC progression. RNA-seq, luciferase reporter assays, Turbo-ID, mass spectrometry, co-immunoprecipitation and RNA-binding protein immunoprecipitation assays and rescue experiments were employed to determine the underlying molecular mechanism. Results CircRPPH1 was significantly upregulated in HCC tissues and it promoted HCC progression both in vitro and in vivo. The elevated circRPPH1 expression in HCC was due to its increased stability, which was mediated by m6A methylation. CircRPPH1 could suppress the level of PPARα to promote HCC progression. Mechanistically, we found that circRPPH1 sponged miR-7845-5p to upregulate the expression of SOX4, which inhibited the transcription of PPARα. CircRPPH1 could also competitively bind with USP1, resulting in increased ubiquitination and degradation of PPARα. We also demonstrated that circRPPH1-ASOs could effectively inhibit HCC growth in vivo. Conclusions CircRPPH1 is a tumor-promoting circRNA, which inhibits PPARα transcription through miR-7845-5p/SOX4 axis and accelerates its ubiquitination and degradation through competitively binding with USP1. These findings suggest that circRPPH1 may serve as a promising therapeutic target for HCC treatment.

Supplementary Table 1: The clinical information of patients and TMA

Supplementary Table 5: Human primers for qRT-PCR analysis

Liver cancer is the fourth leading cause of global cancer mortality, with hepatocellular carcinoma (HCC) accounting for most primary liver cancers. The nucleolus, as the primary site of ribosome biogenesis, plays a crucial role in HCC development. Here, we report that the nucleolar protein KRR1 interacting protein (KRI1) is aberrantly overexpressed in HCC tissues and interacts with nucleophosmin 1 (NPM1) via phase separation to maintain nucleolar structure and ribosome biogenesis. Downregulation of KRI1 significantly impairs HCC cell proliferation and increases apoptosis, accompanied by disrupted nucleolar structure and ribosome biogenesis. Moreover, we show that KRI1 phase separation is enhanced by the phosphorylation of three serine residues (S94, S95, and S97) in its N-terminal intrinsically disordered region, and pyruvate kinase M2 (PKM2) is identified as the key kinase. PKM2 inhibitor shikonin markedly attenuates KRI1 phase separation and its interaction with NPM1, disrupts nucleolar structure and ribosome biogenesis, thereby inhibiting HCC progression. This study uncovers the crucial role of KRI1 in HCC progression and provides theoretical and experimental evidence for the development of nucleolar protein-targeted therapeutic strategies for HCC treatment.

Background:The rising burden of urolithiasis in Southeast Asia and the lack of efficient risk-screening tools for low-resource primary care, such as those in Laos, motivated the development and external validation of a pragmatic nomogram integrating accessible environmental, behavioral, and clinical factors for in-hospital urolithiasis risk stratification, with long-term goal of application in grassroots screening. Methods:A retrospective study was conducted with 665 consecutive inpatients (≥18 years, local residence ≥6 months) at a Lao Military Hospital (March 2024-February 2025). Data were collected using standardized questionnaires and electronic medical records. Least Absolute Shrinkage and Selection Operator (LASSO) regression selected predictors; nomogram performance was assessed by discrimination (area under the curve (AUC)), calibration (Brier score and Hosmer-Lemeshow test), and clinical utility (decision curve analysis (DCA)). Results:Urolithiasis prevalence was 23.0% (153/665). Independent predictors included age (30-59 years, OR = 1.54; ≥60 years, OR = 1.13), sex (OR = 0.59), rural residence (OR = 0.52), high water intake (>1,500 mL/day, OR = 0.02), high sodium consumption (>10 g/day, OR = 21.90), and elevated blood calcium (OR = 9.12). The nomogram presented stable predictive performance, with an AUC of 0.882-0.907, Brier score of 0.086-0.111 (acceptable calibration, Hosmer-Lemeshow p = 0.198-0.892), and optimal clinical threshold at 0.20. Conclusions:This externally validated nomogram uses six accessible environmental, behavioral, and clinical factors to stratify urolithiasis risk in Laos. At present, it helps guide targeted referral and optimize s resource allocation for hospitalized patients in resource-limited settings, while its application in primary care and among broader Southeast Asian populations remains to be prospectively evaluated.

Asthenozoospermia (AS), a prevalent contributor to male infertility, remains incompletely characterized at the metabolic level. This study aimed to define the energy metabolic profile of AS spermatozoa and identify key proteins associated with observed dysregulations. Sperm samples from 19 AS patients and 21 normozoospermic (NS) controls were analyzed. Aliquots from each sample were used to quantify energy-metabolic parameters [oxidative phosphorylation (OXPHOS)/glycolytic ATP production rates, electron-transport chain complex activities, reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP)] and perform proteomic sequencing. Differentially expressed proteins (DEPs) underwent functional enrichment analysis. Spearman correlation linked DEPs to energy-metabolic parameters to identify pathogenesis-associated candidates. AS spermatozoa demonstrated significantly reduced OXPHOS-derived ATP production, impaired electron-transport chain complex activities, decreased MMP, and elevated ROS levels compared to NS controls. Proteomic analysis identified 205 DEPs, with aerobic respiration as the top enriched pathway. Correlation analysis revealed significant associations between OXPHOS ATP/MMP parameters and six DEPs [Dickkopf (DKK)3, KAD9, TMCO1, DRC10, NDUAD, DCAF8]. DKK3 exhibited pronounced expression in human reproductive tissues and localized to the sperm mitochondrial sheath. Dkk3 knockout (KO) mice displayed phenotypes mirroring clinical AS, including reduced motility and mitochondrial dysfunction. Collectively, our integrated human and murine data demonstrate significant OXPHOS impairment with elevated ROS levels in asthenozoospermic sperm, phenotypes recapitulated in Dkk3-KO models. These findings support DKK3 deficiency as a contributor to AS pathogenesis through mitochondrial bioenergetic dysregulation. Further interventional studies remain essential to define mechanistic links between DKK3 loss and compromised sperm mitochondrial function and motility.

Supplementary Table 6: The clinical information of scRNA-seq data and the numbers of cells in each sample

1111 Background: Palbociclib has significantly improved outcomes in metastatic breast cancer (MBC); however, a substantial proportion of patients experience early treatment resistance. Reliable biomarkers to identify high-risk patients prior to therapy initiation remain limited. Our previous work has demonstrated that circulating tumor cell (CTC) and ctDNA monitoring is associated with poor prognosis in MBC (ASCO 2025 #1042; AACR 2025 #3613). Here, we report new findings of the association between baseline CTC level and timing of resistance in patients receiving palbociclib. Methods: This study included 178 patients with ER+/HER2− MBC treated with palbociclib at the Robert H. Lurie Cancer Center between 2016 and 2025 (IRB: NU16B06), all of whom developed treatment resistance. Time to resistance was defined as the interval from treatment initiation to first documented resistance; the median follow-up period was 8.0 months. Patients were stratified a priori into early resistance (≤6 months; n = 71) and later resistance ( > 6 months; n = 107). Baseline CTCs were enumerated prior to treatment using FDA-approved CELLTRACKS system (Menarini). Associations between baseline CTC levels and time to resistance were assessed using Spearman rank correlation, treating CTC as a continuous variable and analyzing early and later resistance groups separately. Results: Among patients with early resistance, the median time to resistance was 3.25 months (range, 0.16–6.0), compared with 16.0 months (range, 6.5–57) among those with later resistance. Patients with early resistance exhibited higher baseline CTC levels on average than those with later resistance (mean CTC: 36.7 vs 25.8), although CTC distributions were highly right-skewed with substantial overlap between groups. Within the early resistance group, 29 of 71 patients had baseline CTC ≥5, with markedly higher mean CTC levels compared with patients with CTC < 5 (88.6 vs 0.8). In contrast, baseline CTC ≥5 was not associated with timing of resistance among patients with later resistance. Using Spearman rank correlation, higher baseline CTC levels were significantly associated with earlier resistance among patients with early resistance (ρ = −0.24, P = 0.043), whereas no association was observed in patients with resistance occurring beyond 6 months (ρ = 0.04, P = 0.71). Conclusions: Baseline CTC levels were associated with the timing of treatment resistance in patients with metastatic breast cancer receiving palbociclib, with a significant inverse correlation observed exclusively among patients experiencing early resistance within 6 months. These new findings suggest that elevated baseline CTC burden reflects aggressive disease biology underlying rapid treatment failure. Time-dependent assessment of CTC may provide clinically relevant insights into early resistance risk and inform clinical decision-making.

e13009 Background: ESR 1 mutations are established drivers of endocrine resistance in ER+ metastatic breast cancer (MBC), yet their relevance to chemotherapy resistance remains unclear. Building on our prior work showing circulating tumor DNA (ctDNA) ESR1 monitoring is prognostic in MBC (ASCO 2025 #1042; AACR 2025 #3613), we investigated whether baseline ctDNA ESR 1 hotspots are associated with agent-specific and broad chemotherapy resistance to inform risk stratification and treatment sequencing. Methods: This study included 158 ER + MBC patients (IRB: NU16B06) treated at Robert H. Lurie Cancer Center. Baseline ctDNA was analyzed by Guardant 360 NGS panel. Two clinically motivated analyses were applied to patients with ESR 1 mutations: (1) ESR 1 hotspot mutations were categorized as classic hotspots (D538G, Y537S/Y537N, E380Q) versus non-classic hotspots (L536H, L536R, L536P, K362N, K520K, V392I), and their association with chemotherapy resistance; and (2) very broad resistance was defined a priori as resistance to ≥5 chemotherapeutic agents. Associations were tested using two-sided Fisher’s exact test with odds ratios reported. Results: ESR1 mutations were identified in 10 hotspots among 40 of 158 patients, including 8 E380Q (mean 6.3%), 12 Y537S (mean 5.6%), 13 D538G (mean 11.9%), 1 V392I (0.1%), 1 Y537N (1.7%), 1 L536P (3.4%), 1 L536R (11.0%), 1 L536H (23.9%), 1 K520K (0.6%) and 1 K362 (15.8%). The cases sensitive vs resistant to chemotherapy drugs are as follows: Doxorubicin (22 vs 18), Epirubicin (33 vs 7), Pegylated Liposomal doxorubicin (37 vs 3), Carboplatin (25 vs 15), Paclitaxel (21 vs 19), Docetaxel (23 vs 17), Vinorelbine (29 vs 11) , Ixabepilone (34 vs 6), Eribulin (32 vs 8), Capecitabine (17 vs 23), Cyclophosphamide (16 vs 24), Gemcitabine (36 vs 4), and Abraxane (35 vs 5). Among 40 patients, vinorelbine resistance was more frequent in those harboring non-classic ESR 1 hotspots compared with classic hotspots (66.7% [4/6] vs 20.6% [7/34]; OR 7.71, P = 0.0385). When chemotherapy resistance burden was evaluated, very broad resistance (≥5 agents) was observed in 8/40 (20%) patients, and was absent in D538G cases (0/13, 0%) compared with non-D538G cases (8/27, 29.6%; P = 0.0373), suggesting a distinct resistance spectrum. These findings suggest ESR 1 hotspot mutation information may help identify patients at risk for specific agent resistance and early multi-line chemotherapy failure. Conclusions: Our findings indicate that baseline ctDNA ESR 1 hotspot heterogeneity is associated with distinct chemotherapy resistance patterns. Non-classic ESR 1 hotspots were enriched for vinorelbine resistance, whereas D538G was inversely associated with an extreme multi-drug resistance phenotype (≥5 agents), suggesting ESR 1 hotspots may serve as a clinically relevant biomarker for chemotherapy resistance spectrum, supporting prospective validation and biomarker-guided treatment strategies.

Prostate cancer (PCa) is the most frequently diagnosed cancer in males. Advanced PCa is invasive and may spread rapidly. Current strategies could not fulfill the requirement for clinical application; thus, novel therapeutic strategies are still urgently needed. Nanoparticles are a promising strategy for targeted drug delivery and cancer treatment; however, the strong exogeneity and weak targeting limit their further application. Here, we developed a novel macrophage membrane-coated nanoparticle that has transmembrane-expressed gy-1, a single-chain antibody fragment (scFv) against prostate-specific membrane antigen (PSMA), to endow the immune evasion and targeting property, which we named P-MMCNPs. The Fe3O4@Au nanoparticles were used as the core of P-MMCNPs, which confer P-MMCNPs with the properties of multimodal imaging and photothermal therapy (PTT). Anti-tumor cytotoxic drug maytansine (DM1) was loaded into the nanoparticles to obtain cytotoxicity. P-MMCNPs were shown to have immune evasion capacity and prolonged circulation time and can be specifically distributed in PSMA-positive tumors, thus enabling targeted imaging and targeted drug delivery. The macrophage membrane-coated nanoparticles combined to inhibit tumor growth in vivo when loaded with DM1 and treated with PTT. Additionally, we found that P-MMCNPs alone could inhibit tumor growth, which may be caused by cytokine neutralization by the macrophage membrane. Our work demonstrates that the innovative P-MMCNPs serve as a versatile platform. This platform improves PCa-targeted diagnostic and therapeutic efficacy while avoiding side effects. Moreover, it holds promise for expanding into the diagnosis and treatment of other diseases, with potential for clinical translation. Additionally, it offers novel insights into nanomedicine-based combination therapy.

Tumor innervation, the infiltration of nerves into the tumor microenvironment (TME), is increasingly recognized as a novel hallmark driving cancer progression and is associated with poor patient prognosis across various solid malignancies. This process is orchestrated by a complex, bidirectional crosstalk. Cancer and stromal cells release neurotrophic factors that induce axonogenesis or neurogenesis. In turn, the infiltrating nerves, particularly sensory nerves, secrete neurotransmitters, neuropeptides or form pseudo-synapse with tumor cells to facilitate cancer hallmarks, including sustained proliferation, invasion, metastasis, modulation of the anti-tumor immune response, and cancer plasticity. However, the specific contributions and underlying mechanisms of sensory nerve innervation in orchestrating malignancy remain incompletely elucidated. This review aims to synthesize the current understanding of the multifaceted roles of sensory neurons within the TME, detailing their intricate interactions with cancer and stromal cells, and highlighting the emerging therapeutic strategies that target the sensory nerve-tumor axis.

Immune checkpoint inhibitors (ICIs) have achieved breakthroughs in various solid tumors; however, their efficacy remains limited in prostate cancer (PCa), which is closely associated with its highly immunosuppressive tumor microenvironment (TME). As the most abundant stromal component in the TME, cancer-associated fibroblasts (CAFs) exhibit remarkable heterogeneity and functional plasticity, playing a central role in orchestrating an immunologically "cold" tumor phenotype and driving resistance to immunotherapy. We have moved beyond simple marker-based classification of cancer-associated fibroblast (CAF) subtypes - a framework still widely used in routine pathological assessment - and now define these populations by their functional states and molecular profiles, enabled by advances in single-cell sequencing, spatial multi-omics, and computational biology. Such functional divergence across stromal subsets allows individual CAF populations to engage in complex, multilayered crosstalk with different immune cell types, and together they build an immunosuppressive network that drives tumor immune evasion. This review systematically catalogs the identification methods and signature gene panels for distinct CAF subtypes in prostate cancer, with particular focus on the multilayered regulatory circuits through which these stromal cells shape the immune microenvironment. We also highlight emerging CAF-targeted strategies designed to reverse immunosuppressive states. This integrated perspective frames CAF heterogeneity in prostate cancer immune evasion, and establishes theoretical underpinnings and translational pathways for next-generation precision combination immunotherapies.

Supplementary Table 2: Genotyping Primers and expected band size

BACKGROUND: Male infertility, impacting 8–12% of couples globally, often lacks clear etiology. G-quadruplexes (G4s), noncanonical DNA structures, are implicated in genomic regulation but remain underexplored in spermatogenesis. This study investigates G4 dynamics and their roles in male fertility. METHODS: We employed antibody-based staining, cleavage under targets and tagmentation (CUT&Tag) sequencing, and a novel nanobody-based proximity labeling system (nanoG4BPL) to map G4 distribution and interacting proteins in mouse testicular cells. In vivo G4 stabilization with pyridostatin and clinical analysis of testicular tissues from patients with nonobstructive azoospermia (NOA) were conducted. RESULTS: G4 structures are enriched in testicular tissues, displaying stage-specific dynamics during spermatogonial differentiation, meiosis, and spermiogenesis. Genome-wide profiling revealed the dual roles of G4s in coordinating gene expression with active epigenetic marks and facilitating genome architecture via CTCF interactions. G4 stabilization disrupted double-strand break repair during meiosis, with nanoG4BPL identifying Nijmegen breakage syndrome 1 (NBS1) as a G4-interacting protein promoting phase separation for homologous recombination. Clinically, patients with NOA exhibited significantly elevated G4 levels in spermatocytes. CONCLUSION: G4 structures are critical regulators of spermatogenesis, orchestrating gene expression, chromatin remodeling, and meiotic fidelity. Their dysregulation, particularly in patients with NOA, suggests a mechanistic link to male infertility, providing novel insights into its pathogenesis and highlighting potential avenues for future diagnostic or therapeutic exploration.

Supplementary Figure S1. ScRNA-seq and RNA-seq revealed the ECM-CAF and Lym-CAF. Supplementary Figure S2. Lym-CAF promotes anti-tumor immunity via NF-κB p65. Supplementary Figure S3. ECM-CAF is associated with ECM genes and YAP1. Supplementary Figure S4. YAP1 silencing promotes the secretion of CXCL9/10/11. Supplementary Figure S5. CD248 is a specific biomarker of ECM-CAF.

Abstract Background: Abemaciclib, a CDK4/6 inhibitor widely used in the treatment of metastatic breast cancer (MBC), has significantly improved progression-free survival. However, despite these therapeutic benefits, a substantial proportion of patients still experience recurrence. This ongoing clinical challenge highlights the urgent need for reliable biomarkers that can identify high-risk individuals before treatment initiation which remains insufficiently explored. Our previous work showed that monitoring circulating tumor cells (CTCs) and ctDNA mutations can serve as a predictive tool for poor prognosis in MBC (ASCO 2025 #1042; AACR 2025 #3613; CCR 2024). Here, we report new findings demonstrating that baseline CTC levels provide important predictive value for identifying recurrence risk in patients receiving abemaciclib. Methods: A total of 66 ER+/HER2− MBC patients were enrolled, and due to treatment discontinuation and re-initiation among some individuals, 76 courses of abemaciclib therapy were administered at Northwestern Memorial Hospital (2016-2024, IRB: NU16B06). The median follow-up period was 36.3 months. Blood samples (7.5 mL) were collected from patients prior to initiating abemaciclib therapy. CTC enumeration (classified as CK+/DAPI+/CD45−) was performed in FDA-approved CELLTRACKS System. Statistical analyses were conducted using causal inference with ensemble learning approaches. Results: Of the 76 treatment courses, 28 patients had CTC counts ≥5, while 48 patients had CTC counts <5. All patients were categorized into two cohorts based on progression after abemaciclib treatment. First analysis (cut-off: 6 months): Cohort 1 (early progression <6 months): 29 patients (range: 0.5-5.5 months; median: 2.75 months); Cohort 2 (late progression ≥6 months): 36 patients (range: 6.5-45 months; median: 17.5 months), plus 11 patients with no progression at the end of the study (follow-up: >12 to >70 months). The mean CTC count in Cohort 1 was 9.2, significantly higher than 6.1 in Cohort 2 (P < 0.01). Second analysis (cut-off: 12 months): Cohort 1 (early progression <12 months): 41 patients (range: 0.5-11 months; median: 3.75 months). Cohort 2 (late progression ≥12 months): 24 patients, plus the same 11 patients with no progress. The mean CTC count in Cohort 1 was 11.2, significantly higher than 2.8 in Cohort 2 (P < 0.01). There was no significant difference in CTC levels between the late-progression group (mean 2.3) and the no-progression group (mean 3.1). Conclusions: These findings demonstrate, for the first time, that pre-treatment CTC levels serve as a strong predictor of progress following abemaciclib therapy. Early identification of elevated CTCs in patients with MBC may therefore be critical for optimizing CDK4/6 inhibitor treatment strategies and identifying patients who may benefit from alternative therapeutic approaches. Citation Format: Qiang Zhang, Justin Zhang, Andrew A. Davis, Natalie Heater, Diana Jaber, Paolo D’Amico, Jianhua Jiao, Weijun Qin, Pan Du, Shading Jia, Akhil Chawla, Janice Lu, Lisa Flaum, William J. Gradishar. Pre-treatment circulating tumor cells are a significant predictor of progression in patients with metastatic breast cancer undergoing abemaciclib therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1074.