Muya Zhou

Chimeric antigen receptor T-cell (CAR-T) therapy has achieved remarkable success in the treatment of hematological malignancies, and its application to solid tumors is currently under active investigation. Upon infusion into the bloodstream, CAR-T cells are exposed to fluid shear stress (SS) generated by blood flow. However, the impacts of SS on CAR-T cells function are not well understood. In this study, we exposed anti-mesothelin (MSLN) CAR-T cells to a shear stress of 5 dynes/cm2, and observed reduced cell viability, enhanced activation, and decreased expression of exhaustion markers. Notably, we found that a one-hour SS stimulation applied seven days after T cell isolation and activation significantly increased the proportion of stem cell memory T cells (Tscm), mitigated exhaustion, and enhanced the anti-tumor efficacy of anti-MSLN CAR-T cells. Furthermore, SS-preconditioned CAR-T cells exhibited resistance to SS-induced cell death and demonstrated increased migratory capacity. In vivo experiments, SS-preconditioned CAR-T cells exhibited greater tumor infiltration, improved persistence, superior tumor control, and a favorable safety profile in tumor-bearing mice. Transcriptomic analysis revealed upregulation of genes associated with activation, infiltration, and microtubule dynamics, alongside downregulation of genes linked to exhaustion, apoptosis, and immunosuppression. Collectively, our findings demonstrate for the first time that mechanical preconditioning with fluid shear stress can reprogram CAR-T cells toward a more functional and persistent phenotype, offering a novel strategy to optimize CAR-T therapy for solid tumors.

Background: Chimeric antigen receptor (CAR)-T cell therapy has shown remarkable success in hematologic malignancies but faces significant challenges in solid tumors due to the immunosuppressive tumor microenvironment (TME). Among these, hypoxia plays a vital role, yet the molecular mediators that link hypoxia to CAR-T dysfunction remain incompletely understood. Methods: Anti-mesothelin (MSLN) CAR-T cells were cultured under normoxic (21% O ₂ ) and hypoxic (1% O ₂ ) conditions for six days. We assessed cell expansion, phenotypes, cytotoxicity, and metabolic features. RNA sequencing was conducted to identify key gene expression changes induced by hypoxia. Findings were further validated using anti-CD70 CAR-T cells. Results: Hypoxia reduced CAR-T proliferation, increased apoptosis, lowered memory phenotypes, raised exhaustion, and weakened cytotoxicity in short-term and long-term assays. Transcriptomic and metabolic analyses showed metabolic reprogramming with increased glycolysis and reduced oxidative phosphorylation. Among the dysregulated genes, the serine/threonine-protein kinase PIM3 emerged as a previously underexplored mediator of hypoxia-driven dysfunction. Genetic or pharmacologic inhibition of PIM3 counteracted hypoxia-induced impairment, enhancing memory phenotypes of CAR-T cells, and improving their anti-tumor activity both in vitro and in vivo. Conclusions: This work identifies PIM3 as a previously underexplored target that links hypoxia to CAR-T cell dysfunction and demonstrates that PIM3 inhibition can reverse these effects. These findings provide a mechanistic rationale for incorporating PIM3 inhibition into CAR-T cell manufacturing or engineering to improve their therapeutic potential in hypoxic solid tumors.

Introduction: Chimeric antigen receptor T cell (CAR-T) therapy has shown success in treating hematological malignancies, but its effectiveness against solid tumors is hindered by T cell exhaustion. During in vitro expansion, tonic signaling induced by CAR expression contributes to CAR-T cell exhaustion, which can be mitigated by inhibiting calcium signaling. Given that sodium citrate can chelate calcium ions and inhibit calcium signaling, in this study, we investigated whether sodium citrate could reduce exhaustion and enhance CAR-T cell function.Methods: We constructed anti-CD70 CAR-T cells and cultured them in the presence of sodium citrate. The characteristics and functionality of sodium citrate-pretreated CAR-T cells were assessed through in vitro and in vivo experiments. To further validate our observation, we also treated anti-mesothelin (MSLN) CAR-T cells with sodium citrate and detected the phenotypes and anti-tumor function of CAR-T cells.Results: We found that sodium citrate-pretreated anti-CD70 CAR-T cells exhibited reduced exhaustion, increased memory T cell proportions, and enhanced anti-tumor efficacy both in vitro and in vivo. Notably, sodium citrate treatment improved the in vivo persistence of CAR-T cells and prevented tumor recurrence. These beneficial effects were also observed in anti-MSLN CAR-T cells. Transcriptomic and metabolite analyses revealed that sodium citrate inhibited calcium signaling, mTORC1 activity, and glycolysis pathways, thus modulating T cell exhaustion and differentiation.Discussion: Our findings suggest that sodium citrate supplementation during CAR-T cell expansion could be a promising strategy to improve CAR-T therapy for solid tumors by preventing exhaustion and promoting memory T cell formation.

Introduction: Allogeneic chimeric antigen receptor T (CAR-T) therapy, also known as universal CAR-T (UCAR-T) therapy, offers broad applicability, high production efficiency, and reduced costs, enabling quicker access for patients. However, clinical application remains limited by challenges such as immune rejection, and issues with potency and durability.Methods: We first screened a safe and effective anti-CD70 scFv to construct anti-CD70 CAR-T cells. Anti-CD70 UCAR-T cells were then generated by knocking out TRAC, B2M, and HLA-DRA. To address the limitations of UCAR-T therapy, we developed an 'all-in-one' self-activated and protective (SAP) module, integrated into the CAR scaffold. The SAP module consists of the CD47 extracellular domain, a mutant interleukin 7 receptor alpha (IL7Rα) transmembrane domain, and the IL7Rα intracellular domain, designed to protect UCAR-T cells from host immune attacks and enhance their survival. Results: SAP UCAR-T cells demonstrated significantly reduced immune rejection from the innate immune system, as evidenced by enhanced survival and functionality both in vitro and in vivo. The modified UCAR-T cells exhibited improved persistence, with no observed safety concerns. Furthermore, SAP UCAR-T cells maintained process stability during scale-up production, indicating the potential for large-scale manufacturing.Discussion: Our findings highlight the SAP module as a promising strategy for the preclinical development of anti-CD70 UCAR-T, paving the way for an 'off-the-shelf' cell therapy product.

When circulating tumor cells (CTCs) travel in circulation, they can be killed by detachment‐induced anoikis and fluidic shear stress (SS)‐mediated apoptosis. Circulatory treatment, which can make CTCs detached but also generate SS, can increase metastasis of cancer cells. To identify SS‐specific mechanosensors without detachment impacts, a microfluidic circulatory system is used to generate arteriosus SS and compare transcriptome profiles of circulating lung cancer cells with suspended cells. Half of the cancer cells can survive SS damage and show higher invasion ability. Mesotrypsin (PRSS3), protease‐activated receptor 2 (PAR2), and the subunit of activating protein 1, Fos‐related antigen 1 (FOSL1), are upregulated by SS, and their high expression is responsible for promoting invasion and metastasis. SS triggers PRSS3 to cleave the N‐terminal inhibitory domain of PAR2 within 2 h. As a G protein‐coupled receptor, PAR2 further activates the Gα_i protein to turn on the Src‐ERK/p38/JNK‐FRA1/cJUN axis to promote the expression of epithelial–mesenchymal transition markers, and also PRSS3, which facilitates metastasis. Enriched PRSS3, PAR2, and FOSL1 in human tumor samples and their correlations with worse outcomes reveal their clinical significance. PAR2 may serve as an SS‐specific mechanosensor cleavable by PRSS3 in circulation, which provides new insights for targeting metastasis‐initiating CTCs.