Translational Oncology

Abstract Objective Commercially available next-generation sequencing (NGS) platforms in China routinely adopt a lung cancer-derived tumor microenvironment (TME) subtyping signature from a European cohort to classify colorectal cancer (CRC), yet its diagnostic performance in Chinese CRC patients remains unvalidated. This study aimed to evaluate the subtyping efficiency of the lung cancer TME signature in a Chinese CRC cohort, screen CRC-specific immune mRNA biomarkers for TME subtyping, and explore the clinical utility of IRF1, CD8A and LAG3 for distinguishing immune-enriched (IE) and immune-desert plus fibrotic (D+F) subtypes. Methods A total of 87 FFPE CRC specimens with complete NGS and clinicopathological data were retrospectively enrolled, including 15 IE subtype and 72 D+F subtype patients. Thirty-one mRNA transcripts covering 13 immune-metabolic homeostasis genes and 18 immune checkpoint/infiltration-related genes were divided into two functional modules. Spearman correlation analysis was performed to assess co-expression patterns among candidate genes. Receiver operating characteristic (ROC) curves combined with five-fold cross-validation were used to compare the discriminatory efficacy of single-gene markers and the three-gene combined panel. Results Strong positive co-expression was observed between IRF1, CD8A and LAG3 (IRF1-CD8A: r=0.93; IRF1-LAG3: r=0.84; CD8A-LAG3: r=0.73). Nominal P-values indicated elevated expression of IRF1, CD8A and LAG3 in IE subtype, though no intergroup significance remained after Benjamini-Hochberg FDR correction, largely attributed to the limited sample size of IE cases. Single-gene ROC analysis showed AUC values of 0.763 (IRF1), 0.752 (CD8A) and 0.771 (LAG3), with LAG3 exhibiting the best individual discriminatory capacity. The three-gene combined panel yielded a cross-validated AUC of 0.717, inferior to single LAG3, due to severe collinearity that generated redundant predictive information. Conclusions The lung cancer-originated TME subtyping system cannot be directly extrapolated to Chinese CRC patients. LAG3 serves as a promising independent transcriptomic candidate marker for distinguishing CRC TME subtypes. The robust collinearity among IRF1, CD8A and LAG3 eliminates additional predictive benefits of the combined signature. Large independent multi-center Chinese CRC cohorts are required to construct population-specific immune transcriptomic biomarkers for standardized clinical NGS TME stratification.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers and more evidence suggests that glucose metabolism plays a significant role in the development and progression with glycosylation at multiple sites potentially being a key characteristic. However, the underlying mechanisms remain insufficiently studied. Here, we first confirmed the presence of O-GlcNAc glycosylation modifications at Thr50 on PKM, which is highly expressed in PDAC and strongly correlated with poor prognosis. Further, we found that PKM expression was significantly positively correlated with CDC27 levels, and mutation of the Thr50 O-GlcNAc site in PKM abolished the upregulation of CDC27. We confirmed that O-GlcNAc-modified PKM enhances nuclear translocation of ARNT, which binds to the CDC27 promoter to upregulate its expression. Finally, we demonstrated that reduced expression of CDC27, as a key component of the APC/C complex, leads to downregulation of ubiquitination at the K11 site of PPP2CA, resulting in upregulation of PPP2CA expression, in turn, reduces AKT phosphorylation, ultimately driving PDAC regression by inhibiting aerobic glycolysis. Thus, we delineate a novel O-GlcNAcylation-dependent pathway where PKM drives PDAC progression through ARNT-mediated CDC27 transcriptional activation and AKT-mediated glycolysis.

Objective: Tumor protein D52 (TPD52) is aberrantly expressed in various malignancies; however, its systematic expression profile, prognostic significance, tumor microenvironment associations, and functional mechanisms in breast cancer remain poorly defined. Methods: GEO and TCGA breast cancer expression datasets were integrated to identify differentially expressed genes (DEGs). We evaluated the diagnostic performance of TPD52 via protein-protein interaction (PPI) network analysis, GO/KEGG enrichment analysis and eleven machine learning algorithms. Immunohistochemistry verified TPD52 protein expression in clinical specimens, and Kaplan-Meier analysis assessed its prognostic significance. Analysis of single-cell transcriptomic data (GSE176078) revealed the cell-type-specific distribution of TPD52 and its intercellular communication network in the breast cancer microenvironment. Weighted gene co-expression network analysis (WGCNA) explored relationships between TPD52 and tumor microbiome, hypoxia signatures as well as microsatellite instability. Moreover, TPD52 was knocked down by siRNA in MCF7 cells, and its impacts on cell migration, invasion, proliferation and the MAPK/ERK signaling pathway were examined through wound healing, Transwell, CCK-8 and Western blot assays. Results: TPD52 was significantly overexpressed in breast cancer tissues at both the mRNA and protein levels. A random forest-based diagnostic model demonstrated high accuracy across multiple datasets. Kaplan-Meier analysis revealed that elevated TPD52 expression was associated with longer overall survival in specific subgroups, including the basal-like subtype, invasive lobular carcinoma, and N0/N1 stages. Single-cell analysis showed that TPD52 was predominantly expressed in tumor epithelial cells, which occupied a central position within the intercellular communication network. WGCNA further identified a positive correlation between TPD52 and a hypoxia-associated microbial module, as well as a negative correlation with a microsatellite instability module. In vitro functional assays confirmed that TPD52 knockdown significantly suppressed the migration, invasion, and proliferation of MCF7 cells, and led to reduced p-ERK1/2 protein levels. Conclusion: TPD52 promotes the malignant phenotypes of breast cancer cells through activation of the MAPK/ERK signaling pathway, yet its prognostic significance is subtype- and microenvironment-dependent. These findings establish TPD52 as both a diagnostically valuable biomarker and a mechanistically defined potential therapeutic target.

Background: Serum proteomic signatures may reflect tumor- and host-related biology and serve as prognostic biomarkers in patients receiving immune checkpoint inhibitors (ICIs). We evaluated the association of the VeriStrat serum proteomic classification with survival outcomes and immune-related adverse events (irAEs) in patients with non-small cell lung cancer (NSCLC) treated with ICIs. Methods: We retrospectively reviewed patients with NSCLC who received ICI-containing therapy and underwent VeriStrat testing at Northwestern Memorial Hospital from October 2015 through June 2023. Patients were classified as proteomic signature Good (PS-Good) or Poor (PS-Poor). Progression-free survival (PFS) and overall survival (OS) were assessed among patients receiving palliative-intent ICI therapy. First any-grade and grade 3 or higher irAEs were evaluated in all ICI-treated patients using cumulative incidence functions and Fine-Gray competing-risk regression. Results: Among 162 ICI-treated patients included in the toxicity analysis, 129 received palliative-intent therapy and were included in the survival analysis; 91 (71%) were PS-Good and 38 (29%) were PS-Poor. PS-Good status was associated with longer PFS (median, 6 vs 3 months; hazard ratio [HR], 0.50; 95% CI, 0.33-0.77; P<0.01) and OS (median, 20 vs 8 months; HR, 0.59; 95% CI, 0.39-0.91; P=0.02). These associations remained significant after multivariable adjustment for PFS (adjusted HR, 0.46; 95% CI, 0.26-0.82; P<0.01) and OS (adjusted HR, 0.50; 95% CI, 0.28-0.87; P=0.01). Any-grade irAEs showed a nonsignificant trend toward a higher cumulative incidence in PS-Good patients. At 12 months, the cumulative incidence was 32.6% for PS-Good versus 22.5% for PS-Poor (subdistribution HR, 1.53; 95% CI, 0.77-3.02; P=0.23). The cumulative incidence of grade 3 or higher irAEs was similar between groups (16.3% vs 15.0%; subdistribution HR, 1.12; 95% CI, 0.48-2.62; P=0.79). Conclusions: PS-Good classification was independently associated with improved survival in patients with NSCLC receiving ICI-containing therapy. Although any-grade irAEs were numerically more frequent among PS-Good patients, proteomic classification was not significantly associated with any-grade or high-grade irAE risk. Prospective validation is warranted.

Epidermal Growth Factor Receptor Pathway Substrate 8 (EPS8) is believed to function as a tumor driver; however, to understand its molecular characteristics across tumors, a comprehensive pan-cancer analysis of EPS8 is lacking. Objective: This study aimed to investigate the prognostic, molecular, and immunological significance of EPS8 across multiple human cancers. Methods: Comprehensive analyses were conducted using GEPIA2, UALCAN, TIMER2.0, cBioPortal, SMART, GSCA, Enrichr, and the TCGAplot R package to evaluate survival prognosis, gene expression, DNA methylation, immune infiltration, proteomic expression, tumor mutational burden (TMB), microsatellite instability (MSI), genetic alterations, drug sensitivity, and enriched pathways. Results: EPS8 overexpression in LGG (p=7.2x10-5) and PAAD (p=9.4x10-6) was significantly associated with poor overall survival and disease-free survival. Amplification was the most common genetic alteration, with alteration frequencies approaching 6% in TGCT and UCEC. In KIRC and PAAD, EPS8 expression correlated with tumor stage. Immune infiltration analysis revealed significant associations between EPS8 expression and immune cells. EPS8 expression also showed significant correlations with both TMB and MSI in STAD and ESCA. Enrichment analysis indicated an association between EPS8 and regulation of the actin cytoskeleton and similar pathways. Conclusion: These findings suggest that EPS8 has prognostic and immunological significance across multiple cancer types and may serve as a potential biomarker. The observed associations with immune-related features and drug response provide a basis for future experimental studies to evaluate its role in cancer biology and its potential relevance to immunotherapy.

Background: Transcriptomic FOLFIRINOX-component sensitivity signatures have been clinically evaluated in resected and advanced PDAC, but their relevance and longitudinal stability in the neoadjuvant BR/LA setting are unknown. Patients and methods: We retrospectively studied 77 patients with borderline resectable or locally advanced PDAC treated with neoadjuvant FOLFIRINOX followed by resection. Pretreatment sensitivity to 5-fluorouracil, oxaliplatin and irinotecan was determined using previously developed locked transcriptomic classifiers and integrated into a regimen-level FOLFIRINOX classification. The primary molecular cohort comprised 53 patients whose pretreatment biopsies had [&ge;]10% tumour cellularity. Cox models assessed associations with survival. Longitudinal changes were assessed in 35 paired tumours. Results: Thirty-one pretreatment tumours were classified as FOLFIRINOX-sensitive (FFX-Sens), and 22 were not classified as FOLFIRINOX-sensitive (FFX-Res). FFX-Sens status was associated with longer overall survival (OS; hazard ratio [HR] 0.48, 95% confidence interval [CI] 0.25-0.93; p=0.029). In the complete-case model adjusted for baseline carbohydrate antigen 19-9 (CA19-9) and tumour size (n=50), the association with OS persisted (adjusted HR 0.47, 95% CI 0.23-0.96; p=0.037), whereas the adjusted association with disease-free survival was not statistically significant (adjusted HR 0.55, 95% CI 0.28-1.10; p=0.090). Among paired tumours, 15 changed from FFX-Sens to FFX-Res and four in the opposite direction (exact McNemar p=0.019). The OS association also persisted after adjustment for postoperative pathological factors (adjusted HR 0.40; p=0.015). Conclusions: Pretreatment FOLFIRINOX sensitivity was associated with OS under neoadjuvant FOLFIRINOX, while matched pretreatment and residual-tumour analyses revealed significant directional reclassification after treatment. These findings extend the clinical evaluation of validated drug-specific FOLFIRINOX sensitivity classifiers to the neoadjuvant setting and provide a longitudinal assessment of their stability during treatment. They support prospective evaluation of both pretreatment stratification and molecular reassessment of residual disease.

Purpose Modified FOLFIRINOX (FFX) and gemcitabine plus nab-paclitaxel (GNP) are standard first-line treatments for metastatic pancreatic ductal adenocarcinoma (PDAC), but no validated biomarker guides treatment selection. We developed MULTIPL, a multimodal machine learning system, and established the PASS-01 Challenge to benchmark prognostic and predictive biomarkers. Patients and Methods MULTIPL was trained in the COMPASS study (N=268), integrating clinical, digitized histopathology, whole-genome, and RNA-seq data. MULTIPL, PurIST, hENT1 expression, and HRDetect were evaluated in the PASS-01 trial, a randomized phase II trial of FFX versus GNP (N=160), within the Challenge. The primary endpoint was differential treatment benefit measured by concordance-for-benefit for progression-free survival. Results MULTIPL had the highest concordance index for OS among individually evaluated biomarkers (0.595; 95% confidence interval [CI], 0.55-0.65) and separated high- versus low-risk patients (hazard ratio, 1.62; 95% CI, 1.13-2.33; P=0.009). Patients recommended for GNP by MULTIPL had significantly longer OS with GNP than with FFX (hazard ratio, 0.47; 95% CI, 0.28-0.82; P=0.007), whereas patients recommended for FFX had similar OS between treatments. Interpretability analysis of MULTIPL in COMPASS identified KDM6A alterations and SSTR1 expression as prognostic biomarkers, which were validated in PASS-01. However, none of the tested biomarkers significantly predicted differential treatment benefit in the PASS-01 Challenge. Conclusion MULTIPL demonstrated robust prognostic performance in external validation, identified a subgroup enriched for benefit from GNP, and enabled discovery and validation of prognostic biomarkers in metastatic PDAC. However, no biomarker met the primary endpoint for differential treatment benefit, underscoring the value of the PASS-01 Challenge.

Invasive lobular breast carcinoma (ILC) is the most common special histological subtype of breast cancer, which accounts for 10-15% of all cases. To study the phenotype characteristics, metastatic growth kinetic and immune microenvironment of ILC, we developed an orthotopically transplantable cell line model from the spontaneous mammary fat pad tumor of CDH1-PTEN dual knockout C57BL/6 mouse with Cre-loxP system, designated CPT6. CPT6 recapitulates single-file growth pattern of human ILC, with pleomorphic features and a high mitotic index. RNA sequencing together with whole exome sequencing reveals a luminal A subtype with targetable driver mutations such as Kras G12C. As a novel orthotopically transplantable ILC model in immune competent mice, CPT6 shows robust in vivo growth and metastatic rate, and has moderate immunogenicity which appears to be T-cell independent. We also profiled the immune microenvironment of CPT6, revealing a myeloid-rich environment with dominant M2-macrophage population, which is concordant with human ILC. In summary, this model recapitulates human ILC phenotype and represents a valuable preclinical platform for evaluating immunotherapy and other therapeutic strategies for invasive lobular breast carcinoma.

AbstractAutophagy and Toll-like receptor (TLR) signaling are both implicated in cancer progression, but whether they act as tumor suppressors or promoters remains unclear. To explore this relationship, we investigated the role of autophagy downstream of therapeutic TLR activation in oral squamous cell carcinoma (OSCC). Using the FDA- approved TLR agonists Bacillus Calmette-Guerin (TLR2/4) and imiquimod (TLR7), we assessed their effects in vitro on SCC-4 cells and in vivo in a chemically induced OSCC hamster model. Both agents, alone or in combination with each other or with Monophosphoryl Lipid-A induced robust autophagy, as measured by LC3B staining in vitro and flow cytometry in vivo. Autophagy induction correlated with reduced tumor volume and prolonged survival, with outcomes comparable to cisplatin, the current standard chemotherapeutic, but with less treatment-associated morbidity and mortality. Autophagy was also associated with cisplatin antitumor effects. Notably, imiquimod produced the most pronounced and sustained autophagic and antitumor effects. To our knowledge, this is the first study to directly link the therapeutic efficacy of TLR agonists in OSCC to autophagy modulation, providing both mechanistic and translational insights into their potential as immunotherapeutic agents.

Introduction: Small cell lung cancer (SCLC) patients have an ~8% 5-year survival; new therapies are urgently needed. Approximately 15% of SCLC patients have naturally-occurring low-titer antibodies against neuronal ELAVL proteins, associated with improved response to therapy and significantly improved survival. We previously determined that the anti-ELAVL4 response is triggered by isoaspartylation in the unstructured ELAVL4 N-terminal region. Methods: We used a Tp53fl/fl;Rb1fl/fl inducible SCLC mouse model to test whether 1) immunization with isoaspartylated Elavl4 (isoAsp-Elavl4) prior to SCLC induction improves survival in the absence of any other treatment, and 2) immunization with isoAsp-Elavl4 following completion of 3 rounds of cisplatin+etoposide therapy improves survival. Immunizations contained incomplete Freund's adjuvant with either a recombinant N-terminal fragment of Elavl4 (amino acids 1-117), incubated under isoaspartyl-inducing conditions, or phosphate-buffered saline (used as the negative control, since Elavl4 spontaneously isoaspartylates). Mice were monitored by blinded assessors until euthanasia was indicated. Results: IsoAsp-Elavl4-immunized animals all became immune responsive, and spontaneous anti-isoAsp-Elavl4 antibodies were observed in 7% of the control animals. Kaplan-Meier analyses revealed that pre-SCLC immunization with isoAsp-ELAVL4 in the absence of other treatments did not affect survival. In contrast, immunization of SCLC mice following chemotherapy significantly improved survival. Conclusions: An anti-isoAsp-ELAVL4 response can be actively induced in mice and significantly increases SCLC survival when given following chemotherapy. This indicates that the anti-isoAsp-ELAVL4 immune response can be leveraged to develop new therapies for SCLC patients.

Objective Based on the bioinformatics method of multi-database integration, this study systematically analyzes the expression characteristics, clinical pathological correlation, prognostic value, potential molecular mechanisms, and immune infiltration patterns of hepatocyte growth factor receptor (MET) in gastric cancer. Methods The UALCAN and GEPIA databases were employed to examine the differential expression of MET between gastric cancer and normal gastric mucosal tissues, as well as its associations with clinicopathological features. Kaplan-Meier Plotter was utilized to evaluate the impact of MET expression on overall survival (OS) and progression-free survival (PFS). Protein-protein interaction (PPI) network was constructed via LinkedOmics, followed by Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of co-expressed genes. Four algorithms, TIMER, CIBERSORT, EPIC, and MCPcounter, were used to cross evaluate the correlation between MET expression and immune cell infiltration; Further validate the cell type specific expression of MET using the gastric cancer single-cell sequencing queues (GSE134520, GSE167297) built into the TISCH database. Results MET expression was significantly elevated in gastric cancer tissues compared with normal gastric mucosa (P < .05), and its expression level was significantly correlated with tumor grade and TNM stage. Patients with high MET expression exhibited significantly poorer OS and PFS than those with low MET expression (P < .05). The PPI network revealed that MET could interact with 20 key proteins, including EGFR, ERBB2, HGF, STAT3, and GRB2 etc. GO enrichment analysis suggests that differentially expressed genes are significantly enriched in functions such as the ERBB signaling pathway, cadherin binding, and DNA repair complexes; KEGG enrichment analysis showed that MET related genes were significantly enriched in pathways such as homologous recombination, nuclear cytoplasmic transport, mismatch repair, and oxidative phosphorylation. Immune infiltration analysis showed that the negative association between MET and B cells infiltration has cross algorithm robustness, while the association with neutrophils, CD8+ T cells, CD4+ T cells, and macrophages exhibits algorithmic heterogeneity or insignificance; There is no significant correlation between MET and common immune checkpoint molecules such as PD-1, PD-L1, CTLA4, etc. Single cell validation further confirmed that MET is mainly enriched in malignant epithelial cells and endothelial cells, and is almost not expressed in immune cells. Conclusions Multidimensional bioinformatic analyses demonstrate that elevated MET expression serves as an independent risk factor for unfavorable prognosis in gastric cancer. MET may mediate dual drug resistance in gastric cancer via crosstalk with multiple signaling molecules (including EGFR, ERBB2, HGF, STAT3 and GRB2) and dysregulation of the homologous recombination repair pathway. Results from multiple-algorithm immune infiltration analysis, single-cell dataset analysis and immune checkpoint correlation analysis indicate that MET exerts only modest direct regulatory effects on the gastric cancer immune microenvironment. This exploratory study offers systematic bioinformatic evidence supporting MET as a candidate prognostic biomarker and potential therapeutic target for gastric cancer. Further functional experiments and prospective cohort studies are required to validate its molecular mechanisms and clinical utility.

Purpose Tarlatamab, a bispecific T-cell engager, has generated favorable response and survival outcomes in relapsed small cell lung cancer (SCLC). Factors influencing tarlatamab-associated treatment outcomes and toxicity have yet to be established. We performed a multivariate analysis to identify biomarkers associated with cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS) and integrated these variables into a novel risk stratification tool to identify patients at high risk of clinically significant toxicity. Patients and Methods Logistic and ordinal regression analyses evaluated the predictive value of variables contributing to the presence and severity, respectively, of CRS and ICANS in 115 patients with ES-SCLC receiving tarlatamab. Selected variables were then tuned using 5-fold cross-validation and incorporated into novel elastic net-based prediction models. Results Rates of observed CRS, ICANS, and dysgeusia were 46%, 28%, and 47%, respectively. ORR (overall response rate) and DCR (disease control rate) were 44% and 58.2%, while 6-month PFS (progression free survival) and OS (overall survival) rates were 30.4% and 54.9%, respectively. Excluding patients on tarlatamab <30 days, presence of dysgeusia was associated with a lower rate of disease progression (HR 0.44, p=0.00691) and death (HR 0.34, p=0.0145). The presence of, increased size and number of extracranial metastases as well as elevated baseline LDH were significantly associated with increased likelihood of CRS. Increased ECOG performance status, increased volume of brain metastases, development of CRS, and elevated baseline LDH were all associated with increased risk of developing ICANS. Our cross-validated risk model successfully predicted CRS grade [&ge;]2 and ICANS events with 90.9% and 75% sensitivity, respectively. Conclusions This exploratory analysis identified several predictive biomarkers of toxicity that successfully stratified low and high-risk populations as part of an internally validated novel risk scoring tool.

Background: Small cell lung cancer (SCLC) is an aggressive, high-grade neuroendocrine carcinoma (hgNEC) with poor prognosis. Tarlatamab, a DLL3-targeting T cell engager was approved in 2024 for relapsed SCLC, with almost doubled overall survival benefit compared to chemotherapy. However, over half of patients do not respond to tarlatamab and others develop resistance within months. With multiple DLL3-targeting therapies in clinical trials, there are currently no validated predictive biomarkers to identify patients most likely to benefit from tarlatamab. In this study, we evaluate baseline DLL3 IHC level in patients as a predictive biomarker to tarlatamab clinical outcome. Methods: We assembled a cohort of 138 patients within IRB-approved MD Anderson GEMINI database (PA13-0589) with DLL3 expression by CLIA-validated immunohistochemistry (IHC), and those treated with tarlatamab from 7/1/2024 to 3/30/2026. Only those with DLL3 IHC results and treated with tarlatamab were included in this predictive biomarker study. All DLL3 levels were reported as percentage cells with cytoplasmic or surface DLL3 expression, including 24 with additional intensity score and H-score calculation. demographic, clinical, and outcome data were collected. Systemic and intracranial response data were assessed by RECIST and mRANO BM criteria respectively. Longitudinal liquid biopsies were taken from a subset of patients for circulating tumor DNA (ctDNA) and (Cytometry by Time-of-Flight) CyTOF analyses. Results: In the predictive biomarker cohort of 54 patients with SCLC treated with tarlatamab, Median DLL3% level in tarlatamab treated cohort was 75%. Median follow-up time was 9.9 months, median time on tarlatamab treatment (ToT) was 5.8 months. At the data cut-off, 61% (33 out of 54) patients were alive. Kaplan-Meier curve stratified by DLL3 % of <=75% vs. >75 showed significant longer ToT and a trend toward longer median OS (mOS). In DLL3>75% compared to DLL3 <=75%: mToT was 10.1 months vs. 2.9 months (p=0.036), mOS: not reached vs. 9.5 months (p= 0.066). Among 41 patients who completed one cycle of tarlatamab treatment and were eligible for systemic (extra-cranial) response per RECIST criteria; we observed partial response (PR) in 51% (21) patients, stable disease (SD) in 22% (9) patients; and progressive disease (PD) in 27% (11) patients. The median DLL3 % in those with PR vs. PD were 80% vs. 40% (p=0.006); median DLL3% comparing PR and SD were 80% vs. 60% (p=0.051). Among 35 patients evaluable for intracranial response, best objective response prior to any brain radiation were 5 (14%) CR,11(31%) PR, 8 (23%) SD and 11 (31%) PD. 9 received brain radiation (8 SRS, 1 WBRT) during tarlatamab to achieve better intracranial control while systemic control is maintained. DLL3% does not appear to be correlated with intracranial response. Longitudinal ctDNA and CyTOF analyses showed persistent ctDNA positivity, as well as increasing NEUROD1 subtype cells in circulating tumor cells in post-tarlatamab progression samples. We additionally profiled DLL3 expression by IHC in 138 patients with SCLC and found heterogenous DLL3 expression, with Median DLL3% of 70% across different biopsy sites. Conclusions: Our data suggests DLL3 expression in patients with SCLC is heterogeneous and not ubiquitous, with median DLL3 IHC of 70%. DLL3 IHC predicts objective response and long-term outcome with tarlatamab. In addition, dynamic monitoring of ctDNA level and CTCs by CyTOF are suggestive of development of tarlatamab resistance, however further studies with larger cohort of paired longitudinal samples are needed to validate the novel blood-based biomarkers. To our knowledge, this study is the first to validate DLL3 IHC as a predictive biomarker for tarlatamab in SCLC, which leads the way for optimizing treatment selection and combinatorial therapies for the subset of patients less likely to respond to tarlatamab.

Circulating tumor cells (CTCs) are key drivers of distant metastasis, while platelets facilitate this process by protecting them from immune surveillance. However, the detection of CTCs exhibiting platelet markers and their communication with monocytes has not been thoroughly investigated. This study seeks to identify the intercellular communication of CTCs that have acquired platelet traits by integrating single-cell RNA-seq data of 377 CTCs and 2,634 white blood cells. Trajectory analysis identified a subpopulation of CTCs with enhanced platelet-related functions, including platelet aggregation and resistance to NK cell-mediated cytotoxicity, termed Platelet-Programmed CTCs. The Dampened Weighted Least Squares method was implemented to estimate the platelet-programmed tumor cell proportion in 1,102 bulk RNA-seq samples from primary breast cancer tumors. A strong inverse association was found between platelet-programmed tumor cell proportion and overall survival (Cox-PH p-value =0.00419, HR=6 per 10% increase in proportion, 95% CI:1.76-20.7). However, this association did not differ significantly between early- and late-stage patients. Cell-cell communication analyses and molecular docking revealed the CD40LG-ITGA5:ITGB1 pair as a potential immune checkpoint candidate for monocytes in cancer (Hex score=-656.83; TNF-TNFR reference complex=-515). However, further experimental and clinical validation is required to translate these findings. In conclusion, these findings deepen our understanding of the immune evasion mechanisms of CTCs by acquiring platelet characteristics. This immune checkpoint axis offers a novel avenue for further experimental validation and future intervention.

CD47 is a ubiquitously expressed transmembrane protein that functions as a negative immune checkpoint, marking host cells as "self" by delivering an inhibitory "dont-eat-me" signal to phagocytes. Cancer cells co-opt this mechanism, upregulating CD47 to evade immunosurveillance and phagocytosis by innate immune cells, a pattern observed across solid tumours and haematological malignancies. CD47 overexpression correlates with poor prognosis across most cancer types, including therapy-resistant disease. Despite extensive efforts to develop CD47-targeted therapies, the downstream biological consequences of aberrant CD47 expression within tumour cells remain poorly characterised beyond its established anti-phagocytic role. This study investigated non-immunological, pro-tumorigenic functions of CD47 to define the cellular effects, beyond immune evasion, that CD47-targeted therapy might disrupt. We found that CD47 exerts cancer type-specific effects: in DLBCL, CD47 loss impaired mitochondrial metabolism and sensitised cells to R-CHOP standard-of-care chemoimmunotherapy, whereas in triple-negative breast cancer (TNBC), CD47 knockdown delayed cell cycle progression, enhanced migration, and conferred resistance to specific chemotherapeutic agents. These findings indicate that CD47 has multifaceted, context-dependent roles in tumour biology that extend beyond immune checkpoint signalling. Clinically, this suggests CD47-targeted therapies may produce cancer type-specific off-target effects on tumour metabolism, proliferation, and drug sensitivity, which are considerations that should inform their rational combination with existing targeted therapies.

Background: Gastric carcinoma remains a major cause of cancer related mortality worldwide, with tumor progression increasingly recognized as a consequence of complex interactions within the tumor microenvironment. Hypoxia induced signaling, cancer associated fibroblast (CAF) heterogeneity, and immune checkpoint activation play critical roles in tumor progression and immune evasion. However, their integrated relationship in gastric carcinoma remains insufficiently characterized. Objectives: To evaluate the expression of Hypoxia inducible factor 1 alpha and its association with cancer-associated fibroblast subtypes and Programmed death-ligand 1 expression in gastric carcinoma. Methods: This cross sectional analytical study included 100 histologically confirmed gastric carcinoma cases from Satkhira Medical College. Immunohistochemistry was performed for HIF 1 alpha, smooth muscle actin (SMA), fibroblast activation protein (FAP), and PD L1. CAFs were subclassified into myofibroblastic CAFs (myCAFs) and inflammatory CAFs (iCAFs). Associations between biomarkers and clinicopathological variables were analyzed using chi square test, Spearman correlation, and multivariate logistic regression. Receiver operating characteristic (ROC) curve analysis was used to assess model performance. Result: High HIF 1 alpha expression was observed in 55% of cases and demonstrated significant association with poor differentiation (p = 0.001), advanced tumor stage (p = 0.002), and lymph node metastasis (p = 0.001). iCAF predominance was significantly associated with poor differentiation (p = 0.003), advanced stage (p = 0.004), and nodal metastasis (p = 0.004). High PD L1 expression was significantly associated with poor differentiation (p = 0.03), advanced stage (p = 0.001), and lymph node metastasis (p = 0.002). Multivariate logistic regression identified high HIF 1 alpha expression (OR = 3.8, p = 0.001), iCAF dominance (OR = 4.5, p < 0.001), and advanced tumor stage (OR = 2.9, p = 0.004) as independent predictors of high PD L1 expression. Combined high HIF 1 alpha expression and CAF activation demonstrated the highest rate of PD L1 positivity (76.7%, p < 0.001). ROC curve analysis demonstrated good predictive performance of the model with an area under the curve of 0.81. Conclusion: The present study demonstrates a significant interaction between hypoxia, stromal remodeling, and immune checkpoint activation in gastric carcinoma. High HIF 1 alpha expression and inflammatory CAF predominance are strongly associated with aggressive clinicopathological features and increased PD L1 expression, supporting the existence of a coordinated hypoxia stroma immune axis in gastric carcinoma progression. These findings may have potential implications for prognostic stratification and combined targeted therapeutic strategies.

Triple-negative breast cancer (TNBC) is a highly aggressive malignancy and options for targeted therapy are limited. Although PARP inhibitors (PARPi) like olaparib (Ola) leverage synthetic lethality, their clinical utility is severely impeded by drug resistance and low frequency of BRCA mutations. In this study, 1,2,3,4,6-penta-O-galloyl-{beta}-D-glucose (PGG), a natural polyphenolic compound, can induce a state of homologous recombination deficiency (HRD) in TNBC cells by inhibiting the PALB2-BRCA2 interaction. PGG potentiated the inhibitory effects of Ola on the in vitro proliferation, clonogenic potential, migration, and invasion of TNBC cells. Mechanistically, the dual therapy triggered severe oxidative stress and mitochondrial collapse, which accelerated the classical apoptosis cascade and promoted the emission of immunogenic cell death (ICD)-associated DAMPs. The accumulated cytosolic double-stranded DNA activated the innate immune cGAS-STING pathway, thereby promoting robust intra-tumoral infiltration of CD8+ T cells. Furthermore, since cGAS-STING hyperactivation drives compensatory PD-L1 upregulation on TNBC cells, incorporating anti-PD-L1 antibody into the combination regimen further reversed the immunosuppressive barrier, achieving near-complete tumor eradication and maximum infiltration of cytotoxic T cells in vivo. Bioinformatics analyses further suggested a potential network of PGG targets associated with extracellular matrix organization, offering a bioinformatics-derived hypothesis for its theoretical involvement in modulating stromal tension and vascular homeostasis. Collectively, this study establishes PGG as a multi-functional therapeutic agent integrating intracellular synthetic lethality and innate immune sensing, while providing a preliminary bioinformatic rationale for prospective microenvironmental investigation. This study also shows PGG as a multi-functional therapeutic agent that integrates intracellular synthetic lethality, innate immune sensing, and microenvironmental remodeling to convert immunologically "cold" TNBC into "hot" tumors.

Hepatocellular carcinoma (HCC) is particularly aggressive and difficult to treat. Due to the lack of early clinical diagnosis and the unsatisfactory clinical treatment effect, it is particularly important to identify novel markers that can predict tumor behavior in HCC. biogenesis of ribosomes BRX1 (BRIX1) is abundant in various tissues of the human body. However, the regulatory mechanisms and its role in various tissues are not fully understood. Here, we analyzed the expression pattern of BRIX1 in HCC from public gene expression databases and tissue samples from clinical HCC. We confirmed that BRIX1 was upregulated in both HCC cell lines and HCC paraffin section samples. BRIX1 depletion significantly dicreased the capacity of cells to grow and migrate in vitro, and knockdown BRIX1 suppressed tumor growth in xenograft tumor model. Mechanistically, BRIX1 depletion suppressed the MAPK/ERK pathway, as reflected by reduced phosphorylated ERK (p-ERK) levels. In summary, we provide a rational clue for the further investigation of BRIX1 as an invaluable biological marker for diagnosing and predicting prognosis of patients with HCC.

Background: Triple-negative breast cancer (TNBC) tumors lack expression of estrogen receptor (ER), progesterone receptor (PR), and HER2, limiting the availability of targeted therapeutic options. As a result, TNBC is characterized by a high propensity for metastasis, rapid recurrence, and poor overall prognosis. Despite significant research efforts, the molecular mechanisms that drive TNBC progression and metastasis remain incompletely understood. Y box binding protein 1 (YB1) is a multifunctional DNA/RNA-binding protein that regulates transcription, translation, and mRNA stability. Aberrant activation of YB1 has been implicated in multiple oncogenic processes, including proliferation, survival, invasion, metastasis, and therapy resistance in several cancer types, including TNBC. Our investigations have revealed a central role for YB1 in regulating key hallmarks of cancer that drive TNBC tumor progression and metastatic dissemination. Methods: Cell viability and drug sensitivity in TNBC cell lines were assessed using the MTT assay to determine IC50 values for pharmacologic inhibitors. Western blotting and quantitative RT PCR were used to quantify protein and mRNA expression levels in TNBC cell lines and tumor samples. Oncogenic phenotypes were evaluated using 2D colony formation assays, 3D tumorsphere growth assays, limiting dilution, and transwell migration assays to assess proliferative, stem-like, and migratory capacities. Cell cycle progression was analyzed using flow cytometry based assays. In vivo therapeutic efficacy was evaluated using preclinical xenograft and patient-derived xenograft (PDX) mouse models of TNBC to assess tumor growth and metastatic progression following treatment. Results: Our studies demonstrate that the small-molecule inhibitor SU056, which specifically targets YB1 for degradation, significantly suppresses the oncogenic behavior of TNBC cell lines and tumors. Pharmacologic inhibition of YB1 reduced TNBC cell proliferation, clonogenic growth, tumorsphere formation, stemness and migratory capacity. Importantly, combined treatment with SU056 and the CDK4/6 inhibitor Palbociclib enhanced the inhibitory effect on TNBC cell growth and tumor progression compared with either agent alone. Mechanistically, both genetic and pharmacologic targeting of YB1 suppressed TNBC tumor growth and metastatic potential through modulation of the Cyclin D/CDK4/6/Rb signaling axis, leading to inhibition of cell cycle progression and induction of G1 arrest. Conclusion: Collectively, these findings identify SU056/CDK4/6i combination as a potential therapeutic option for the treatment of Rb+ TNBC.

Melittin, a highly active natural antimicrobial peptide derived from honeybee venom, holds immense pharmacological potential against solid tumors. However, its specific anti-tumor efficacy and transcriptomic dynamics in cervical carcinoma remain to be systematically characterized. This study evaluated the anti-cancer properties of melittin on murine U14 cervical cancer cells following by transcriptomic investigation of the underlying mechanism. Phenotypic evaluations revealed that melittin potently inhibited U14 cell viability, while wound healing assays demonstrated a profound, dose-dependent suppression of cellular migration, culminating in near-complete migratory arrest at high concentrations. Furthermore, flow cytometry quantified a dramatic, dose-dependent surge predominantly in late apoptotic cell populations. These apoptotic events were structurally corroborated by scanning electron microscopy (SEM), which revealed severe plasma membrane perforation and morphological exhaustion. Enrichment analyses indicated that the physical membrane disruption inflicted by melittin translated into a severe metabolic crisis, marked by a global suppression of ribosomal biogenesis and mitochondrial oxidative phosphorylation. Furthermore, melittin profoundly repressed the Tie2-mediated angiogenic pathway (Etv2 down-regulation) while triggering the lethal hyperactivation of the AP-1 transcriptional stress complex (Jun, Fos, Fosb). Collectively, these findings elucidate the pharmacological networks underlying melittins cytotoxicity, providing solid molecular evidence for its development as a natural therapeutic agent against cervical cancer.