KLF5 is frequently high-expressed among various cancers and indicates a poor prognosis, related to Figure 1

Generation of Trp53-knockout; Klf5-overexpressing murine endodermal organoids, related to Figure 1

Background The transition from intraductal to invasive Breast cancer (BC) is driven by complex interactions within the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs), a key stromal component, critically influence extracellular matrix remodeling, immune modulation, and metastatic dissemination. However, the spatially resolved cellular mechanisms through which malignant epithelial subpopulations reprogram fibroblasts to promote metastasis remain incompletely understood. Methods We performed multiregional single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics on breast cancer specimens representing the tumor core, tumor border, and adjacent normal tissue. Malignant mammary luminal (ML) subtypes were identified through Large-scale chromosomal copy number variation (CNV) profiling. Fibroblast sub-clustering, pseudotime trajectory reconstruction, and cell–cell communication analysis (Cell Chat) were integrated with in vitro functional assays using primary fibroblasts and breast cancer cell lines to delineate key signaling interactions. Results Five ML clusters were identified, among which ML4—enriched at tumor borders—displayed transcriptional programs linked to ribosome activation, hypoxia, and hyper-inflammatory, and was associated with high recurrence and metastasis in TCGA-BC. Fibroblasts were classified into three spatially distinct subtypes: myofibroblasts, inflammatory fibroblasts (iFIB), and APOD⁺PTGDS⁺ fibroblasts, with iFIB predominantly localized to tumor borders. Cell–cell interaction analysis revealed hyperactivation of the LGALS9 (tumor)–CD44 (fibroblast) signaling axis in the border regions. Functional validation confirmed that LGALS9-expressing BC cells promote migration and activation of primary fibroblasts, whereas activated fibroblasts enhance epithelial–mesenchymal transition and migration of BC cells via Smad2/3 signaling. Conclusion We identify a spatially restricted malignant luminal subtype, ML4, which drives fibroblast activation through LGALS9–CD44 signaling, establishing a reciprocal, pro-metastatic feedback loop within the TME. This niche remodeling holds prognostic relevance and represents a promising therapeutic target for preventing metastasis in aggressive breast cancer.

Cross-species transcriptomics revealed similarity between LUSC and PK-organoids, related to Figure 4

8037 Background: Concurrent chemoradiotherapy followed by consolidation immunotherapy is the standard treatment for unresectable stage III LA-NSCLC; however, many patients in real-world practice are unable to complete concurrent therapy, and locoregional failure remains common. This phase II study evaluated induction chemoimmunotherapy followed by hypofractionated radiotherapy (hypo-RT) and consolidation immunotherapy to improve feasibility and disease control. Methods: Patients with newly diagnosed unresectable stage III NSCLC received 3-4 cycles of immune checkpoint inhibitors combined with platinum-based doublet chemotherapy. Patients without progression or grade ≥2 pneumonitis underwent eligibility assessment for hypo-RT based on anatomical suitability and organ-at-risk constraints. Eligible patients received definitive sequential hypo-RT (48–60 Gy in 12–15 fractions based on tumor regression) and consolidation immunotherapy when appropriate. The primary endpoint was progression-free survival (PFS) with secondary endpoints of safety and overall survival (OS). Results: Between October 2022 and October 2025, 132 patients received induction chemoimmunotherapy, and 55 underdoing hypo-RT. Median age was 70 years (range, 47–85), and 42 patients (76.4%) had squamous cell carcinoma. Programmed death-ligand 1 (PD-L1) expression ≥1% was observed in 17 patients (30.9%), with unknown PD-L1 status in 14 patients (25.5%). With a median follow-up of 20.1 (95%CI, 17.3-22.9) months, median PFS and OS were not reached. The 1- and 2-year PFS rates were 88.1% (95% CI, 78.8%–98.5%) and 58.5% (95% CI, 43.6%–77.7%), respectively; the 1- and 2-year OS rates were 100% and 77% (95% CI, 61.5%–93.7%), respectively. Isolated in-field recurrence occurred in 4 patients (7.3%). Pneumonitis was the most common acute nonhematologic toxicity, with grade 3 in 4 patients (7.3%) and grade 4 in 1 patient (1.8%). Grade 3 atelectasis occurred in 4 patients (7.3%), and grade 3 radiation esophagitis in 2 patients (3.6%). No grade 5 toxicities were observed. Conclusions: Induction chemoimmunotherapy followed by eligibility-adapted hypofractionated radiotherapy showed promising survival and locoregional control with acceptable toxicity in unresectable locally advanced NSCLC, supporting its feasibility as an alternative strategy for selected patients. Clinical trial information: NCT05784142 .

RATIONALE AND OBJECTIVES:Post-SABR radiologic appearances in the lung are dynamic and may imitate local failure. Although radiologic changes after SABR have been described, the dynamic evolution and predictors of pseudoprogression remain unclear. We aimed to characterize serial computed tomography (CT) evolution after SABR and examine its association with pseudoprogression. MATERIALS AND METHODS:We retrospectively included patients who underwent SABR and had ≥24 months radiologic follow-up without definite local recurrence. The maximum diameter of the evolving post-SABR lesion was referenced to the initial (pre-SABR) tumor. Pseudoprogression was defined as any >20% increase in maximum diameter during follow-up that was followed by radiographic stabilization and lacked pathologic or clinical evidence of recurrence. RESULTS:We evaluated 109 lesions from 104 patients (median follow-up, 53 months). Mass-like consolidation developed in 94/109 (86%) lesions. CT evolution followed three phases with typical onset: initiation-reticular infiltrates adjacent to the index tumor (median 3 months; range 1-9); development-increasing density with patchy/streaky opacities and/or fibrotic consolidation (median 4.5 months; range 1.5-15.5); and mass-like consolidation-organization/absorption or retraction culminating in a final mass-like consolidation (median 13 months; range 5.5-39). A total of 74 (68%) lesions showed a >20% increase vs the initial tumor, and 50% occurred during the third phase. Planning target volume (PTV) was an independent predictor of pseudoprogression (adjusted odds ratio 0.98, 95% CI 0.97-0.99; P = 0.002). CONCLUSION:Dynamic CT evolution after SABR commonly manifests as mass-like consolidation with high rates of pseudoprogression. PTV independently predicts this phenomenon, underscoring the need for cautious interpretation of radiographic enlargement during follow-up.

2056 Background: Glioblastoma (GBM) remains a highly lethal malignancy driven by infiltrative growth, molecular complexity, immune evasion, and therapeutic resistance. The limited efficacy of current treatments reflects antigen heterogeneity, an immunosuppressive tumor microenvironment, and restricted delivery across the blood–brain barrier with targeted therapy. Although earlier CAR-T therapies demonstrated preliminary activity in GBM, durable efficacy remains elusive. We have developed EPC-003, a fully human bispecific B7-H3/IL13Rα2 CAR-T engineered with a multi-mechanism armor to mitigate antigen heterogeneity, modulate the tumor microenvironment, and enhance persistence via central memory T-cell enrichment. Methods: Human anti-IL13Rα2, anti-B7-H3, and anti-PD-L1 scFvs were discovered from proprietary human B-cell libraries using mRNA display platform. A multi-mechanism armor was engineered by fusing the anti-PD-L1 scFv to a fine-tuned, immune cell-type specific IL-2 variant, identified through the unique cytokine engineering platform. These binding and functional modules were systematically optimized to generate EPC-003, a bispecific tandem CAR targeting IL13Rα2 and B7-H3 and incorporating a secreted multi-mechanism armor. CAR mechanisms, anti-tumor activity, and preclinical toxicity were evaluated. A proprietary clinical manufacturing process was established to produce a central memory T cell enriched CAR-T product. Results: The secreted armor of EPC-003 suppressed regulatory T-cell activation while selectively activating central memory T-cells, with the potential to enhance CAR-T cell persistence. In a 7-day G-Rex manufacturing process, EPC-003 demonstrated promising druggability with robust and reproducible CAR expression, and the proprietary cytokine cocktail consistently enriched the product to >65% central memory CAR-T cells. In orthotopic GBM models, intracranial administration of EPC-003 (0.4 × 10⁶ and 1.2 × 10⁶ CAR-T cells) induced tumor regression and showed dose-dependent anti-tumor activity. In preclinical toxicity studies, EPC-003 exhibited a favorable safety profile, with no body weight loss, cytokine release syndrome, or systemic clinical signs. No treatment-related pathological changes were observed across 25 examined organs. Immunohistochemistry revealed no evidence of brain hemorrhage in treated groups, with minimal monocyte infiltration in the pia mater observed only at the high dose. Conclusions: EPC-003, a multi-mechanism armored bispecific CAR-T, demonstrated favorable preclinical safety, developability, robust anti-tumor activity, and potential durability. These data support its advancement into an investigator-initiated clinical trial for the treatment of glioblastoma.

Gboxin and CX-5461 inhibited the growth of subcutaneous tumors formed by PK organoids or KLF5-high LUSC organoids in vivo, related to Figure 7

Activation of Ribi/OXPHOS pathways in KLF5-high LUSC samples and Klf5-overexpressing PK organoids, related to Figure 5

Klf5 activation reprogrammed ribosome biosynthesis and energy metabolism, related to Figure 4

Klf5 activation boosts proliferation of colon organoids but not alveolar organoids, related to Figure 1

Objective Epstein-Barr virus (EBV) -associated diseases comprise a spectrum of conditions, including infectious mononucleosis (IM) and chronic active EBV infections (CAEBV), as well as malignant lymphomas. The study aimed to compare genomic variations of EBV across different subtypes of EBV-associated diseases to investigate the impact of EBV mutations on disease progression and clinical outcomes. Methods A total of 36 patients with EBV-associated diseases were enrolled. Peripheral blood DNA samples (400µL) were collected and sequenced using Illumina sequencing technology. Following quality control with FastQC, Fastp, and TriTrimmomatic, 27 samples meeting the critera of minimum effective coverage > 10 and coverage > 80% were retained. Single nucleotide polymorphisms (SNP) annotations were generated into VCF files, and mutation profiles were visualized using heatmaps. Mutational rates were compared using Fisher's exact test, based on disease subtype, presence of hemophagocytic lymphohistiocystosis (HLH), and clinical outcomes. Odds ratios (ORs) were calculated using the Haldane-Anscombe correction. Results Compared with the reference stain B95.8, a total of 2182 mutation sites were identified in 27 samples. The mutations located in coding region included 523 missense variants, 1 insertion mutations, and 2 deletion mutations, all of which only involved single amino acid changes in the encoded proteins. Although a large number of mutations were detected, no statistically significant differences were observed among the IM, CAEBV and lymphoma groups, or between patients with and without HLH. However, three mutation sites in BFLF1 were significantly more frequent in deceased patients compared to survivors, which detailedly refers to Ile35Thr, Asp44Glu and Val246Ile [ P = 0.0267, OR = 15.00, 95%CI= (0.7436,301.5)]. Conclusion Single amino acid changes in EBV proteins had no significant effect on the progression status of EBV-associated diseases, but the mutation in BFLF1 were associated with an increased risk of mortality.

Klf5 overexpression activates squamous cell carcinoma marker genes, related to Figure 1

The regulatory sequences of key genes of RiBi and OXPHOS contain binding sites for KLF5

Suppression of ribosome biogenesis and oxidative phosphorylation hinders the proliferation of LUSC cell lines in vitro, related to Figure 6

Primary antibodies used in this study

This study aims to explore the heterogeneity of anticipatory grief experiences among primary family caregivers of patients with advanced lung cancer in China and to identify caregiver personas as a basis for the development of personalized anticipatory grief management strategies. A qualitative study was conducted between July and September 2024 at a tertiary hospital in mainland China. Primary family caregivers of patients with advanced lung cancer were selected through purposive sampling, combined with a maximum difference sampling strategy. Semi-structured interviews were conducted to capture their experiences of anticipatory grief. Interpretive phenomenology and thematic analysis were used to extract factual labels and construct a dimensional model. Following a systematic five-stage workflow, caregiver personas were identified through a hierarchical decision tree based on five core dimensions. Finally, caregiver personas were developed based on dimensional classification, with their experiences of anticipatory grief visualized through labeled character personas. A total of 19 eligible caregivers participated in the study. Five dimensions—family support, emotional expression, emotional burden, meaning construction, and future coping—were identified from the anticipatory grief and coping strategies of primary family caregivers of patients with advanced lung cancer. By combining caregivers’ personal attributes, five user personas were identified: the optimist behind the smiling mask, the lone warrior trudging forward under heavy burdens, the master of meaning reconstruction, the conflicted decision-maker, and the feature-weaving mixer. Precise interventions based on caregiver personas are essential for enhancing the management of anticipatory grief among primary caregivers of patients with advanced lung cancer. This study constructs five distinct caregiver personas, providing a foundation for implementing precision interventions that address the diverse needs of this group in managing anticipatory grief and reshaping their perceptions of life’s value.

BACKGROUND:Lung adenocarcinoma (LUAD) is the most common form of lung cancer. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is abnormally expressed in various tumor tissues and is associated with malignant phenotypes. However, the clinical significance, function, and mechanism of LUAD invasion and metastasis remain unclear. METHODS:We retrospectively enrolled 100 patients with LUAD in this study. Initially, qRT-PCR was performed to detect PCSK9 levels in clinical tissues. Subsequently, bioinformatics analysis of scRNA-seq and The Cancer Genome Atlas Program (TCGA) datasets was performed to predict the role of PCSK9 in tumor cell malignancy and its potential downstream pathways. These predictions were validated experimentally using the CCK-8 assay, TUNEL staining, wound healing, transwell invasion assay, and an in vivo lung metastasis model. Finally, Western blotting and an AKT inhibitor (MK2206) were used to verify the underlying mechanism. RESULTS:PCSK9 was significantly upregulated in LUAD tissues compared to paracancerous tissues and was associated with poorer OS and DFS. Bioinformatics analysis of scRNA-seq data and TCGA analysis predicted that PCSK9 is highly enriched in tumor cells and is involved in EMT, and that the PI3K/AKT pathway plays a significant role in LUAD development. Experiments confirmed that PCSK9 markedly promoted LUAD cell proliferation, migration, and invasion in vitro and lung metastasis in vivo. PCSK9 overexpression significantly upregulated p-AKT, p-PI3K, and p-mTOR levels. Furthermore, the AKT inhibitor, MK2206, reversed the promoting effects of PCSK9. CONCLUSIONS:PCSK9 expression is associated with the prognosis and diagnosis of LUAD. This molecule activates the PI3K/AKT signaling pathway, thereby driving invasion, metastasis, and proliferation in LUAD.

mRNA profiles of Klf5-activated airway organoids during transformation, related to Figure 4