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AbstractBackgroundPeritoneal metastasis (PM) is one of the most challenging clinical problems in gastric cancer (GC), largely due to its high recurrence rate and poor response to current therapies. Increasing evidence indicates that remodeling of the extracellular matrix (ECM) plays an important role in therapeutic failure. However, how specific stromal¨Cimmune interactions contribute to PM heterogeneity and immunotherapy resistance remains unclear. In this study, we investigated how ECM composition¡ªparticularly the accumulation of hyaluronic acid (HA)¡ªinfluences the immune microenvironment and therapeutic responses in GC-associated PM.MethodsWe combined histopathological assessment, analyses of patient-derived specimens, single-cell transcriptomic profiling, and murine models of PM to delineate ECM remodeling patterns and immune cell dynamics in therapy-sensitive and therapy-resistant lesions. In addition, functional assays and pharmacological approaches were used to examine HA¨CCD44 signaling and its impact on CD4+ T cell differentiation and responsiveness to immune checkpoint blockade.ResultsTherapy-sensitive PM lesions were characterized by enrichment of elastic fibers, whereas therapy-resistant lesions showed collagen accumulation. Notably, HA deposition emerged as a key feature distinguishing these ECM states and was closely associated with differential therapeutic outcomes. Elevated HA levels activated CD44-dependent signaling in CD4+ T cells, driving regulatory T cell (Treg) differentiation through a CD44¨CIQGAP1¨CRAC1¨CSMAD3 signaling pathway and thereby establishing an immunosuppressive microenvironment. Importantly, pharmacological inhibition of CD44 reduced Treg expansion and markedly enhanced the antitumor efficacy of anti-PD-1 therapy in murine PM models.ConclusionsOur findings identify HA¨CCD44 signaling as a critical link between ECM remodeling and immune evasion in GC PM. Targeting ECM-driven immunosuppressive mechanisms may represent a promising strategy to overcome therapeutic resistance and improve the efficacy of immunotherapy in this aggressive disease.

AbstractBackgroundAdoptive transfer of chimeric antigen receptor (CAR)-expressing natural killer (NK) cells has demonstrated success against hematological malignancies. Efficacy against solid tumors has been limited by poor NK cell survival and function in the suppressive tumor microenvironment (TME). To enhance efficacy against solid tumors, stimulatory cytokines have been incorporated into CAR-NK cell therapeutic approaches. However, current cytokine strategies have limitations, including systemic toxicities, exogenous dependencies, and unwanted TME bystander effects. Here, we aimed to overcome these limitations by modifying CAR-NK cells to express a constitutively active interleukin (IL)-7 receptor, termed C7R, capable of providing intrinsic CAR-NK cell activation that does not rely on or produce exogenous signals nor activate bystander cells.MethodsWe examined persistence, antitumor function, and transcriptional profiles of CAR-NK cells coexpressing C7R in a novel tumor immune microenvironment (TiME) co-culture system and against hematologic and solid tumor xenografts in vivo.ResultsPeripheral blood NK cells expressing a CAR directed against the solid tumor antigen GD2 and modified with C7R demonstrated enhanced tumor killing and persistence in vitro compared with CAR-NK cells without cytokine support and similar functions to CAR-NK cells supplemented with recombinant IL-15. C7R.CAR-NK cells exhibited enhanced survival and proliferation within neuroblastoma TiME xenografts in vivo but produced poor long-term tumor control compared with CAR-NK cells supplemented with IL-15. Similar results were seen using C7R-expressing CD19.CAR-NK cells against CD19+leukemia xenografts. Gene expression analysis revealed that chronic signaling via C7R induced a transcriptional signature consistent with intratumor stressed NK cells with blunted effector function. We identified gene candidates associated with chronic cytokine-stressed NK cells that could be targeted to reduce CAR-NK cell stress within the solid TME.ConclusionC7R promoted CAR-NK cell survival in hostile TMEs independent of exogenous signals but resulted in poor antitumor function in vivo. Our data reveals the detrimental role of continuous IL-7 signaling in CAR-NK cells and provides insights into proper application of cytokine signals when attempting to enhance CAR-NK cell antitumor activity.

DEAD-box helicase 41 (DDX41) is implicated in germline (GL)-predisposed myeloid neoplasms, where pathogenic GL variants often lead to disease following the acquisition of a somatic variant in trans, most commonly p.R525H. However, the precise molecular mechanisms by which DDX41 variants contribute to the pathogenesis of myeloid neoplasms remain poorly understood, partly due to challenges in establishing cellular and animal models that faithfully recapitulate the human disease phenotype. This limitation highlights the necessity of directly analyzing primary human disease cells. In this case report, conducted to pursue this objective, we implemented single-cell RNA sequencing integrated with genotyping at the p.R525 locus in a myelodysplastic neoplasm (MDS) harboring both germline and somatic DDX41 variants, leveraging highly efficient Terminator-Assisted Solid-phase cDNA amplification and sequencing. We found that acquiring p.R525H induced G2/M cell cycle arrest selectively in colony-forming unit-erythroid cells, accompanied by R-loop accumulation, which impaired erythropoiesis through DNA damage. In hematopoietic stem and myeloid progenitor populations, gene expression profiles were largely similar between p.R525H-positive and -negative cells. However, ligand-receptor interaction and transcriptional regulation analyses suggested a non-cell-autonomous influence from p.R525H-expressing cells on GL variant-only cells. This interaction drove convergence toward a shared expression profile, highlighting an intricate interplay shaping the patient¡¯s MDS phenotype.