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. 2026 May 11;75(7):181. doi: 10.1007/s00262-026-04391-2

Mechanical preconditioning by shear stress enhances memory formation and anti-tumor function of CAR-T cells

Lianfeng Zhao 1,3,#, Jiaqi Liu 1,3,#, Yan Zhang 1,3, Xinhao Yang 1,3, Tianyu Chen 1,3, Xinyang Xu 2,3, Muya Zhou 1,3,✉, Zhigang Hu 1,3,✉, Zhigang Guo 1,3,✉
PMCID: PMC13341982  PMID: 42113250

Abstract

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.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00262-026-04391-2.

Keywords: Shear stress, Chimeric antigen receptor T-cell (CAR-T), Immunotherapy, Memory phenotype, T cell activation and exhaustion

Introduction

Chimeric antigen receptor T-cell (CAR-T) therapy has recently emerged as a highly promising immunotherapeutic strategy [1, 2]. It has demonstrated remarkable clinical outcomes in the treatment of various malignant tumors [3, 4], particularly hematological malignancies such as acute lymphoblastic leukemia (ALL) [5, 6] and certain types of lymphoma [7]. Despite these success, the effectiveness of CAR-T cell therapy is often limited by several challenges, including T cell exhaustion [8, 9], development of resistance [10], and inconsistent therapeutic responses [11, 12]. Consequently, optimizing CAR-T cell function to enhance anti-tumor efficacy while minimizing side effects remains a critical area of ongoing research [13, 14].

Accumulating evidence indicates that mechanical forces in the environment can profoundly influence immune cell behavior [15, 16]. In particular, fluid shear stress (SS)—the frictional force exerted by flowing blood on circulating cells—has attracted increasing attention. SS has been shown to modulate macrophage polarization and function, influencing their role in inflammation and tissue remodeling [17]. Long-term exposure to SS induces the transformation of monocyte-derived macrophages (MDMs) to a pro-inflammatory phenotype [18]. In addition, SS has been shown to enhance the cytotoxicity of natural killer (NK) cells against circulating tumor cells (CTCs), a mechanism driven by mechanical sensing through NKG2D [19]. Studies have also reported that ex vivo application of SS can promote the activation, proliferation, and cytotoxicity of primary T cells [20, 21], the specific impacts of fluid shear stress on CAR-T cell therapy remain poorly understood. Considering that CAR-T cells are exposed to SS after intravenous infusion into patients, we sought to investigate how SS would alter the phenotypes and functions of CAR-T cells.

To address this, we employed a microfluidic circulatory system to simulate physiological SS in vitro [22]. We first investigated the effects of SS on unmodified T cells, and subsequently applied optimized conditions to anti-mesothelin (MSLN) CAR-T cells. Our preliminary results indicated that SS both induced cell death and alleviated T cell exhaustion, we hypothesized that SS preconditioning—brief exposure to SS prior to therapeutic use—could improve CAR-T resistance to mechanical stress while enhancing function. Through systematic optimization, we identified an SS preconditioning strategy that not only reduced cell death under shear but also enhanced CAR-T cell activation, memory phenotype formation, migration capacity, and anti-tumor efficacy.

This study aims to elucidate how fluid shear stress modulates T cell and CAR-T cell phenotypes and functions, with particular focus on activation, memory formation, and anti-tumor activity. Our findings suggest that shear stress preconditioning significantly enhances CAR-T persistence and cytotoxicity, offering a new approach to optimize CAR-T therapy by integrating mechanical cues into ex vivo cell preparation protocols.

Results

Fluid shear stress induced cell death, increased activation and reduced exhaustion of both T cells and anti-MSLN CAR-T cells

To simulate shear stress (SS) conditions, we adapted a peristaltic pump-driven circulatory system based on previously established methods (Fig. 1A) [22]. Human T cells were isolated from human PBMCs and activated with anti-human CD3/CD28 dynabeads. On day 4 post-isolation and activation, T cells were activated with shear forces of 2.5, 5, and 10 dynes/cm2 for 1 h. h No significant differences in cell viability were observed among the different SS treatments (Fig. 1B). However, SS treatment led to increased expression of CD69, signifying indicating enhanced T cell activation (Fig. 1C). Notably, the levels of the exhaustion markers PD-1 and TIM-3 were reduced (Fig. 1D), and the proportion of stem cell with a stem memory phenotype (Tscm) increased (Fig. 1E). Among the tested forces, 5 dynes/cm2 exerted most significant impacts on T cells, thus, this SS level of 5 dynes/cm2 was utilized in subsequent experiments.

Fig. 1.

Fig. 1

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SS induced cell death, promoted activation and persistence of T cells and anti-MSLN CAR-T cells. (A) Schematicdiagrams showing the microfluidic circulatory system：panel (a) shows the overall device, and panel (b) depicts the flow sequence of the cell suspension. 1 mL of cell suspension was loaded into a pipette tip connected to the system. Setting the desired shear stress and driving the cell suspension to circulate along the flow direction. Upon completion of the circulation process, the cell suspension was transferred to culture flasks. (B-E) T cells cultured for 4 days post-isolation and activation were treated with SS levels of 2.5, 5 and 10 dynes/cm2 (SS-2.5, SS-5 and SS-10) for 1 hour. Cell viability was assessed through trypan blue staining (B). Flow cytometry was used to detect the expression levels of CD69 (C), co-expression of PD-1 and TIM-3 (D), and the proportion of Tscm, determined by CD62L-positive and CD45RO-negative (E). (F) Flowchart illustrating the experimental work flow of identifying suitable SS-conditioning durations. T cells cultured for 4 or 11 dayswere treated with SS-5 for indicated durations. (G and J) Detection of T cell viability after SS treatment. (H and K) The expression of CD69 was detected using flow cytometry. (I and L) Co-expression levels of PD-1 and TIM-3 were measured. (M-O) Anti-MSLN CAR-T cells cultured for 4 days after T cell isolation and activation was treated with SS-5 for 1-4 hours. Cellviability (M), CD69-positive rate (N), and PD-1 and TIM-3 double-positive rate (O) were assessed. Data are present as means ± SEM from three different donors. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; one-way ANOVA

To further investigate the effects of SS exposure durations, T cells were cultured for either 4 or 11 days following isolation and activation, then subjected to SS treatment for varying periods (1–4 h) (Fig. 1F). On day 4, T cell viability declined progressively with prolonged SS exposure, and 3 and 4 h of treatment significantly reduced T cell viability (Fig. 1G). Despite this, longer SS exposure resulted in increased CD69 expression (Fig. 1H). Reduced co-expression of PD-1 and TIM-3 was observed with shorter exposures (Fig. 1I). Similarly, on day 11, extended SS exposure reduced T cell viability (Fig. 1J), and increased CD69 expression (Fig. 1K). For exhaustion markers, however, only the 1-h treatment significantly reduced PD-1/TIM-3 co-expression (Fig. 1L). These results suggest that the immunophenotypic response of T cells to SS is time-dependent: while prolonged exposure induces cell death and enhancing activation, but potentially limiting benefits in exhaustion reduction.

Subsequently, anti-MSLN CAR-T cells were cultured for 4 days post isolation and activation, and treated with a SS of 5 dynes/cm2 for 1–4 h to examine the SS effects on CAR-T cells. Consistent with unmodified T cells, CAR-T cell viability decreased with prolonged SS exposure (Fig. 1M). SS treatment increased also CD69 expression (Fig. 1N), while PD-1/TIM-3 expression generally decreased but showed a rebound at extended durations (Fig. 1O), indicating a similar time-dependent pattern in activation and exhaustion dynamics. Collectively, these results demonstrate that fluid shear stress impairs the viability of both T cells and CAR-T cells, yet promotes cellular activation and alleviates exhaustion. Specifically, exposure to 5 dynes/cm2 for 1 h significantly promotes CAR-T cell activation and reduces exhaustion markers while preserving cell viability, indicating that this condition as an optimal preconditioning strategy.

SS preconditioning enhanced memory phenotype and mitigated exhaustion in T cells and CAR-T cells

Given that SS enhanced CAR-T cell activation and reduced exhaustion, we next investigated whether SS preconditioning could improve the functional characteristics of CAR-T cell products. To address this, we implemented a sequential SS preconditioning strategy. T cells were first treated with shear stress at 5 dynes/cm2 for 1 h on day 4, and these cells were designated as C1. On day 7, C1 cells were subjected to a second round of SS treatment to generate C2 cells. Finally, C2 cells were treated again on day 10 to obtain C3 cells. All groups (C1, C2, and C3) were cultured under static conditions and analyzed on day 12 (Fig. 2A). The approach showed a progressive decline in cell viability with increasing rounds of SS exposure (Fig. 2B). Flow cytometry analysis demonstrated that SS preconditioning promoted the expansion of Tscm cells, with no significant differences observed among C1, C2, and C3 groups (Fig. 2C, D). Co-expression of the exhaustion markers PD-1 and TIM-3 was significantly reduced in all SS-preconditioned cells and decreased further with successive treatments (Fig. 2E).

Fig. 2.

Fig. 2

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A single round of SS-5 treatment significantly improved the immunophenotypes of T cells and anti-MSLN CAR-T cells. (A) Schematic diagrams showing the timeline of three rounds of SS preconditioning. (B) Trypan blue staining was conducted to assess the viability of T cells with various rounds of SS preconditioning. (C-D) Flow cytometry was used to detect the memory phenotypes of preconditioned T cells. Tscm (stem cell memory T cells), CD62L+ CD45RO-; Tcm (central memory T cells), CD62L+ CD45RO+ ; Tem (effector memory T cells), CD62L-CD45RO + ; Tte (terminal effector T cells), CD62L-CD45RO-. (E) Double-positive rates of PD-1 and TIM-3 in preconditioned T cells were measured via flow cytometry. (F–I) Anti-MSLN CAR-T cells were preconditioned with SS-5 following the procedures in (A). Cell viability (F), memory phenotypes (G-H) and exhaustion (I). Data are present as means ± SEM from three different donors. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; one-way ANOVA

Anti-MSLN CAR-T cells were subjected to the same sequential SS preconditioning process. Similar to T cells, CAR-T cell viability progressively declined with repeated SS exposure (Fig. 2F). Notably, the proportion of Tscm cells significantly increased after the first treatment (C1), but decreased in the C2 and C3 groups (Fig. 2G, H). With respect to exhaustion, PD-1/TIM-3 expression remained unchanged in C1 cells but increased in C2 and C3 cells, indicating elevated exhaustion with repeated SS treatments (Fig. 2I). These results suggest that a single round of SS preconditioning (C1) may serve as an effective strategy to enhance the memory phenotype of CAR-T cells while maintaining cell viability and avoiding increased exhaustion.

SS preconditioning on day 7 significantly enhanced the persistence and tumor-killing ability of anti-MSLN CAR-T cells

Due to tonic signaling induced by the CAR, CAR-T cells undergo dynamic changes in immunophenotypes depending on their culture duration [23], which may influence their response to SS preconditioning. To determine the optimal time point for SS intervention, CAR-T cells cultured for 4, 7, or 10 days were subjected to a single round of SS treatment, generating D4, D7, and D10 CAR-T cells, respectively. Phenotypic and functional analyses were conducted on day 12 (Fig. 3A). Consistent with prior findings, SS preconditioning at these time points did not compromise CAR-T cell viability (Fig. 3B), while significantly increased the proportion of stem cell memory T cells (Tscm) and decreased the expression of exhaustion markers PD-1 and LAG-3 (Fig. 3C-E). Among all groups, D7 CAR-T cells showed the most pronounced enhancement in memory phenotype and reduction in exhaustion.

Fig. 3.

Fig. 3

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SS preconditioning on CAR-T cells cultured for 7 days exhibited the most profound effects of improving immunophenotypes and tumor-killing ability. (A) Schematic diagrams showing the experimental design of identfying the optimal preconditioning time point. (B–E) Cell viability (B), the proportion of Tscm (C), the expression levels of PD-1 (D) and LAG-3 (E) in preconditioned anti-MSLN CAR-T cells were detected. (F–H) Preconditioend CAR-T cells were co-cultured with CAPAN-2 cells at the E:T ratios of 2:1, 1:1, and 1:2 for 24 h. Cell lysis rates were measured through the luciferase reporter assay. (I–K) Preconditionede CAR-T were continuously stimulated with OVCAR3, 1806 and CAPAN-2 cells at the E:T ratio of 1:2 for 24 h for three rounds. Cell lysis rates after each round were measured through the luciferase reporter assay. Data are present as means ± SEM from three different donors. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; one-way ANOVA (B-H) and two-way ANOVA (I–K)

To assess functional improvements, we tested the cytotoxicity of preconditioned CAR-T cells against luciferase-expressing mesothelin-positive CAPAN-2, OVCAR3, and 1806 tumor cells using a luciferase-based reporter assay. Nearly all SS-preconditioned CAR-T cells exhibited enhanced tumor-killing activity, with D7 CAR-T cells demonstrating the most robust cytotoxic effect (Fig. 3F-H and Fig. S1A–F). Furthermore, long-term killing assays further confirmed the superior persistence and sustained anti-tumor efficacy of D7 CAR-T cells (Fig. 3I-K). Collectively, these results identify day 7 post-isolation and activation as the optimal time point for a single round of SS preconditioning, which significantly improves both the persistence and cytotoxic function of CAR-T cells.

SS preconditioning enhances the functional phenotypes, survival ability under SS and migration capacity of CAR-T cells

To further evaluate the functional impacts of SS preconditioning on CAR-T cells, anti-MSLN CAR-T cells were stimulated with OVCAR3 tumor cells and analyzed for immunophenotypic changes. SS-preconditioned anti-MSLN CAR-T (SS-MSLN-CAR-T) cells exhibited significantly higher levels of activation markers CD25 and CD69 (Fig. 4A), along with higher levels of the memory-associated marker CD62L (Fig. 4B), and decreased exhaustion markers PD-1 and LAG-3 (Fig. 4C). Additionally, SS-MSLN-CAR-T cells secreted higher amounts of IFN-γ and TNF-α compared to non-preconditioned controls, indicating enhanced effector function (Fig. 4D).

Fig. 4.

Fig. 4

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SS-preconditoning enhanced the activation status, persistence, cytotoxicity, resistance to SS-induced damage and migration ability of anti-MSLN CAR-T cells. (A-D) SS-preconditioned CAR-T (SS-CAR-T) and untreated CAR-T (NC-CAR-T) cells were stimulated with OVCAR3 cells. The expression levels of activation markers CD25 and CD69 (A), memory marker CD62L (B) and exhaustion markers PD-1 and LAG-3 (C) were detected via flow cytometry. The levels of cytokines in the supernatants were detected using CBA assays (D). (E–H) The cytotoxicity of CAR-T cells against OVCAR3 (E–F) and 1806 (G-H) cells was evaluated by RTCA assays. (I-K) NC-CAR-T and SS-CAR-T cells were subjected to SS-5 treatment for 4 h. Flow cytometry was used to detect the apoptosis rate of CAR-T cells under SS conditons (I-J). Cell viaibility was also determined by trypan blue staining (K). (L) Flow cytometry results showing the expression of the migration marker LFA-1 in CAR-T cells. (M) Transwell migration assay was performed. After 18 h, the images of migrated CAR-T cells were taken with a fluorescence microscope and the migrated cell number was counted. Data are present as means ± SEM from three different donors. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; two-way ANOVA (A, C–D) and t-test (B, J-M)

Real-time cell analysis (RTCA) assays demonstrated the enhanced killing ability of SS-MSLN-CAR-T cells against OVCAR3 and 1806 tumor cells, especially at lower E:T ratios (Fig. 4E-H and Fig. S2A-B). Long-term cytotoxicity assays further confirmed more sustained anti-tumor activity against OVCAR3, 1806, and CAPAN-2 cells (Fig. S2C–H).

Since shear stress negatively affects T cell viability, there is a concern that CAR-T cells might be compromised upon infusion due to to hemodynamic forces. To evaluate whether SS preconditioning confers resistance to shear-induced damage,both untreated and SS-preconditioned CAR-T cells were exposed to 5 dynes/cm2 of SS for 4 h. Apoptosis analysis and viability assays demonstrated that SS-MSLN-CAR-T cells were more resilient under SS conditions (Fig. 4I-K).

Moreover, given prior evidence that SS enhances tumor cell motility [22], we investigated whether SS also augments CAR-T cell migration. Flow cytometry revealed elevated expression of the migration-associated protein LFA-1 in SS-preconditioned cells (Fig. 4L), and transwell assays confirmed enhanced migratory capacity (Fig. 4M).

To assess the generalizability of this approach, we applied the same protocol to anti-CD70 CAR-T cells. Similar enhancements in memory phenotype and cytotoxicity were observed (Fig. S2I–K). Collectively, these findings demonstrate that SS preconditioning improves CAR-T cell persistence, effector function, resistance to SS-induced damage, and migratory potential, supporting its applicability across multiple CAR-T products.

SS-CAR-T cells exhibited superior anti-tumor efficacy in vivo

To assess the in vivo functionality of SS-CAR-T cells, NKG mice were subcutaneously implanted with 1806 tumor cells. Ten days post-implantation, mice were randomly divided into three groups and intravenously injected with PBS, NC-MSLN-CAR-T, or SS-MSLN-CAR-T cells (Fig. 5A). One hour after injection, submandibular blood analysis revealed significantly reduced apoptosis in SS-CAR-T cells compared to NC-CAR-T cells, suggesting enhanced resistance to SS-induced damage in vivo (Fig. 5B).

Fig. 5.

Fig. 5

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SS-CAR-T cells displayed superior anti-tumor activity in vivo. (A) Two million 1806 tumor cells were subcutaneously implanted in NKG mice. Ten days later, mice were divided into three groups randomly and injected with PBS, 8 × 105 NC-MSLN-CAR-T or SS-MSLN-CAR-T cells throughh tail vein. (B) One hour after injection of CAR-T, mice blood was collected and the apoptosis of injected cells (CD3+) was detected. (C–I) On the 8th day after administration, three mice from each group were sacrificed and mice blood was collected to detect the number of CAR-T cells (CD3+ CAR+) (C), activation level (D), exhaustion level (E), memory phenotype (Tscm) (F), and the level of INF-γ in serum (G). Tumor tissues were extracted and stained with anti-CD45 antibody for immunohistochemical analysis (H), and quantification of infitrated CAR-T cells was performed using ImageJ (I). (J) Changes of tumor volumes in each group. (K) At the end of the experiment, all mice were sacrificed and tumor tissues were dissected. (L) Representative images of HE staining results performed on the heart, liver, spleen, lung, kidney and brain of mice on D28. Data are present as means ± SEM from three independent experiments. **, p < 0.01; ***, p < 0.001; t-test

Blood sampling on the 4th, 8th and 12th days after administration revealed a proliferation peak on day 8 (Fig S3A), and the number of SS-CAR-T cells was significantly higher than that of NC-CAR-T cells (Fig. 5C). SS-CAR-T cells also exhibited increased activation (Fig. 5D), lower exhaustion (Fig. 5E), an improved memory phenotype, and elevated levels of INF-γ secretion (Fig. 5G). Immunohistochemical analysis of tumor tissues demonstrated markedly improved infiltration of SS-CAR-T cells compared to NC-CAR-T cells (Fig. 5H, I).

Tumor growth monitoring showed that SS-CAR-T treatment led to rapid tumor regression, achieving complete tumor clearance by day 28, whereas NC-CAR-T cells failed to fully eliminate tumors (Fig. 5J, K). Importantly, there were no significant differences in body weigh among the three groups throughout the treatment period (Fig. S3B), and H&E staining results revealed no additional damage to major organs in the SS-CAR-T group (Fig. 5L). Collectively, these results demonstrate that SS preconditioning enhances CAR-T cell the survival, persistence, and tumor infiltration of CAR-T cells in vivo, thereby conferring superior anti-tumor efficacy without compromising safety.

Gene expression profiles revealed the molecular basis of functional improvements in SS-CAR-T cells

To elucidate the molecular mechanisms underlying the enhanced function of SS-preconditioned CAR-T cells, we performed RNA sequencing on SS-CAR-T and NC-CAR-T cells. Transcriptomic analysis revealed a substantial number of differentially expressed genes (DEGs) between the two groups (Fig. 6A). KEGG pathway enrichment analysis revealed significant upregulation of immune-related pathways, including those involved in cytokine-cytokine receptor interactions, chemokine signaling, and other immune activation processes (Fig. 6B).

Fig. 6.

Fig. 6

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Changes in transcritpomic profiles of SS-MSLN-CAR-T compared with NC-MSLN-CAR-T cells. (A) The volcano plot depictingg the differentially expressed genes (DEGs) under SS pretreatment and normal conditions. (B) KEGG pathway analysis showed the pathhway enrichment of DEGs. (C–G) Heatmaps showing the differential expression of genes related to activation (C), infiltration (D), exhaustion/apoptosis (E), inhibitory factors (F), and cell cycle (G). (H-I) Gene set enrichment analysis (GSEA) displaying the enrichment of genes related to microtubule and microtubule motor activity pathways in SS- CAR-T cells. (J) Schematic diagram: The SS pre-treatment enhances the anti-tumor efficacy of CAR-T cells by promoting cell activation, facilitating cell infiltration, remodeling the cell cycle, reducing cell exhaustion and cell apoptosis

Consistent with the phenotypic changes observed, genes promoting T cell activation—such as CD80 [24, 25] and TNFSF11 [26]—were significantly upregulated in SS-CAR-T cells (Fig. 6C). Additionally, Genes associated with cell infiltration and migration, including CCR2 and CXCR1 [27], exhibited increased expression (Fig. 6D). In contrast, inhibitory receptor genes such as MEF2C [28] and pro-apoptotic genes like IL24 [29] were downregulated (Fig. 6E). Suppression of FCRL3 expression further supported the enhanced immune potency of SS-CAR-T cells [30, 31] (Fig. 6F).

Both KEGG pathway analysis and DEG patterns indicated that SS preconditioning induced transcriptional reprogramming of the cell cycle. Notably, upregulation of CDKN2A and CDKN2B—known inhibitors of the CDK4/6 pathway—suggested maintenance of CAR-T cells in a resting (G0/G1) state [32], which is associated with memory persistence (Fig. 6G). Furthermore, gene set enrichment analysis (GSEA) demonstrated that genes related to microtubule organization and motor activity were significantly enriched in SS-CAR-T cells [33, 34], indicating a potential role in supporting phenotypic remodeling (Fig. 6H-I). Collectively, These results suggest that The SS pre-treatment enhances the anti-tumor efficacy of CAR-T cells by promoting cell activation, facilitating cell infiltration, remodeling the cell cycle, reducing cell exhaustion and cell apoptosis (Fig. 6J).

In conclusion, Our findings demonstrate that conditioning CAR-T cells with a shear force of 5 dyne/cm2 for 1 h on day 7 of cell culturing significantly improve the survival, persistence and anti-tumor activity, establishing SS preconditioning as a promising and broadly applicable strategy e for enhancing CAR-T cell functionality for solid tumor therapy.

Discussion

Despite the remarkable success of CAR-T cell therapy in hematological malignancies, its efficacy in solid tumors remains limited by poor persistence, exhaustion, and inefficient trafficking. In the present study, we systematically investigates the multi-dimensional regulatory effects of fluid shear stress (SS) on T cells and CAR-T cells. We findings identify SS preconditioning as a novel approach to enhance the functionality and survival of CAR-T cells. We found that exposing CAR-T cells to a physiological level of SS (5 dynes/cm2 for 1 h) significantly improved activation, memory differentiation, and cytotoxic function, while reducing exhaustion both in vitro and in vivo. Notably, SS preconditioning on day 7 post-T cell activation yielded the most pronounced functional enhancements, suggesting an optimal window for mechanosensitization during CAR-T cell manufacturing.

Shear stress (SS), a biomechanical force generated by fluid flow affect various types of cells. Early SS research focused primarily on endothelial cells, which are highly sensitive to SS and cellular signaling pathways are thereby influenced to regulate vascular physiology [35, 36]. In circulating tumor cells, SS exhibited a dual role of triggering apoptosis and inhibiting proliferation, while enhancing the invasion and metastasis [37–39].

The influence of SS on other components in blood vessels has also been investigated, such as platelets [40] and immune cells [41, 42]. Under low-intensity SS conditions, T cells demonstrate enhanced activation, migration and proliferation capabilities, enabling effective participation in immune responses [20, 21, 43]. Conversely, excessive SS may suppress T cell proliferation and promotes functional exhaustion [44]. These results suggest that the effect of SS on T cells is dose-dependent, where moderate SS can enhance immune function, whereas excessive SS may compromise the immune response [45]. In our study, T cells and CAR-T cells were exposed to a moderate level of SS, and our findings are consistent with previous reports suggesting that mechanical cues influence T cell fate and activation. To our knowledge, this is the first time reveals the memory-promoting and exhaustion-reducing effects of SS on T cells and CAR-T cells. In addition, we demonstrate that a defined, brief SS treatment can be leveraged as a practical and scalable method to improve CAR-T cell products, which could resist SS damage and exhibit enhanced persistence, migration ability and tumor-eliminating capacity.

Considering the physiological shear stress levels observed in human circulation—approximately 10–70 dynes/cm2 in arterial vessels, 1–6 dynes/cm2 in venous vessels, and lower, spatially heterogeneous stresses within tumor microenvironments—which collectively represent the mechanical forces experienced by T cells in vivo, we selected a shear stress range of 2.5–10 dynes/cm2 in this study to model physiologically relevant conditions[46]. This selection ensures that the experimental conditions effectively mimic the mechanical cues encountered by T cells as they transit through vascular and tumoral compartments.

We also found that the timing of SS preconditioning plays a critical role in determining its impact on CAR-T cells. When SS treatment was applied on day 4 or day 7 of culture, CAR-T cells demonstrated significantly enhanced functionality, with D7 CAR-T cells exhibiting the most pronounced improvements. In contrast, CAR-T cells preconditioned on day 10 failed to show similar benefits. This discrepancy may be attributed to differences in the differentiation status of CAR-T cells at various time points. By day 7, CAR-T cells typically attain a relatively stable state characterized by robust proliferation and consistent CAR expression. However, by day 10, prolonged culture may induce higher levels of exhaustion, likely due to tonic CAR signaling [47], thereby limiting the effectiveness of SS preconditioning.

Mechanistically, RNA-seq analysis revealed that SS stimulation reshaped the gene expression landscape of CAR-T cells, leading to the upregulation of genes associated with activation, trafficking (CCR2, CXCR1), and memory formation, while downregulating inhibitory and apoptotic genes. Notably, SS-treated cells showed altered cell cycle regulatory pathways, including upregulation of CDKN2A/B, which may contribute to the maintenance of a stem-like resting state (G0/G1). Furthermore, GSEA results suggested a role for microtubule-related gene networks in orchestrating cytoskeletal remodeling and phenotypic reprogramming, which are consistent with previous studies demonstrating the association of cytoskeleton with T cell activation and migration [48]. These findings indicate that biomechanical signals can engage intracellular transport and polarity pathways to optimize T cell optimization.

Despite the valuable insights and findings, several limitations exist. The precise mechanosensing pathways involved in SS-mediated reprogramming warrant further investigation, including the role of various mechanotransduction proteins, as well as potential regulation of mitochondrial function or cellular metabolic pathways [49]. In addition, the RNA‑sequencing analysis in this study was performed with only two biological replicates per condition. Although the two replicates within each group showed high transcriptomic concordance. we acknowledge that three or more replicates represent the gold standard for robust statistical inference. Future studies with larger sample sizes are warranted to validate the transcriptomic signatures reported here. Furthermore, the translational potential of SS preconditioning should be explored in more sophisticated models to take the complexity of tumor microenvironment into consideration, such as patient-derived organoids, humanized mouse models and early-phase clinical trials, to fully assess its safety, efficacy, and reproducibility. To facilitate the application of our strategy in CAR-T cell manufacturing, further improvements in the stability and scalability of the shear stress device are necessary as well.

In conclusion, this study presents the first comprehensive investigation into the regulatory effects of fluid shear stress on T cells and CAR-T cells. We identified SS preconditioning as a robust and reproducible strategy to enhance CAR-T cell functionality, laying a theoretical foundation for the mechanical modulation of immunocellular therapies. By promoting T cell activation, reducing exhaustion, and enriching a stem-like memory phenotype, SS preconditioning provides a simple yet effective means to improve the therapeutic efficacy of CAR-T cells, particularly for the management of solid tumors.

Materials and methods

Cell lines and cell culture

HEK293T (human embryonic kidney), OVCAR3 (ovarian cancer), HCC1806 (breast squamous carcinoma), and CAPAN-2 (pancreatic cancer) cell lines were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM (Gibco, Thermo Fisher Scientific, USA) with 10% fetal bovine serum (FBS) (Hysign, China). Human peripheral blood mononuclear lymphocytes (PBMCs) from healthy donors (TPCS#PB025C, Miles-bio, Shanghai) were isolated using Lymphoprep (Stemcell, Canada), and T cells were isolated with the EasySep™ human T Cell Isolation Kit (Stemcell, Canada). On day 0, T cells were activated with anti-human CD3 and CD28 beads (Miltenyi, Germany) and cultured in X-VIVO medium (Ronsa, Switzerland) supplemented with recombinant IL-2 (300 IU/mL) and 5% FBS.

Generation of CAR-T cells

Anti-MSLN and anti-CD70 CAR-T cells were generated as described previously [23]. In brief, lentivirus for generating CAR-T cells was produced by co-transfecting HEK293T cells (ATCC) with lentiviral and packaging plasmids (pMDLg/pRRE, pRSV-Rev, pMD2.G). Supernatants were collected 48 and 72 h post transfection, concentrated by ultracentrifugation, and stored at -80°C. T cells were infected with CAR-expressing lentivirus 24 h post activation.

Fluid shear stress treatment

A commercial microfluidic circulatory system equipped with a peristaltic pump (LabN1, SHENCHEN, Shanghai, China) and silicone tubes was utilized to generate fluid shear stress (SS). Based on the physiological shear stress values observed in human circulation—approximately 10–70 dynes/cm2 in arteries, 1–6 dynes/cm2 in veins, and lower, heterogeneous levels in tumor microenvironments—which represent the mechanical forces encountered by T cells in vivo, the shear stress range of 2.5–10 dynes/cm2 was selected for this study[22] [46, 50]. The system was sterilized with ultraviolet light and 75% alcohol for 15 min, followed by three washed with PBS. T cells or CAR-T cells were suspended at a density of 1 × 106 cells/mL in complete culture medium and circulated within the system under defined flow rates to achieve shear stresses of 2.5–10 dynes/cm2. The shear stress was calculated according to the equation τ = 4μQ/(πr3), where τ was the calculated SS level in dyne cm−2, Q was the flow rate in cm3s−1, η was the dynamic viscosity of the fluid (0.012 dyne s cm−2), and R was the radius of the tubes (250 μm). During the treatment, the entire system was placed inside a standard cell culture incubator maintained at 37°C and 5% CO2, ensuring physiological conditions for cell viability. After exposure for the indicated durations, the cells were collected by centrifugation and subsequently cultured in fresh medium for downstream analyses.

Flow cytometry

Cells were stained with specific fluorescent antibodies at room temperature for 15 min, and then analyzed using a CytoFLEX S flow cytometer (Beckman Coulter, USA). The detailed information of the antibodies were listed in Table S1.

CAR-T cytotoxicity detection

In the short-term killing assay, CAR-T cells were co-cultured with luciferase-expressing OVCAR3, HCC1806, or CAPAN-2 cells at E:T ratios of 2:1, 1:1, 1:2 for 24 h. After incubation, luciferase substrates were added and luminescence was measured using a plate reader. Cytotoxicity was calculated as [(control luminescence—sample luminescence) / control luminescence] × 100%.

For the long-term killing assay, CAR-T cells were challenged with tumor cells for three rounds at E:T ratios of 2:1, 1:1, 1:2. Cytotoxicity was evaluated after each round, and the phenotypes of CAR-T cells upon stimulation were detected via flow cytometry.

CAR-T cytotoxicity was also measured through the real-time cell analysis (RTCA) assay. Tumor cells were seeded on 96-well electrodes that were pre-coated with cysteine, and CAR-T cells were added at E:T ratios of 1:2, 1:4, 1:8 after tumor cells were attached. Data was collected continuously in a CP96 Real-Time Label-Free Cell Growth Analyzer.

Transwell migration assay

CAR-T cell migration was assessed using a Transwell chamber system (Corning, USA). The membrane was pre-coated with matrigel, and CAR-T cells (1 × 104 cells per well) were seeded in the upper chamber. The lower chamber contained 5% FBS, and cells were incubated for 17 h. Migratory cells were counted.

Cytokine measurement

Tumor cells (OVCAR3, HCC1806, CAPAN-2) were co-cultured with CAR-T cells (effector: target ratio 1:2) for 24 h, and supernatants were collected. In animal experiments, serum were isolated from mice blood through centrifugation and cytokine levels in serum were measured. The cytokine levels in cell supernatants and mice serum were analyzed using BD Biosciences’s CBA Human Soluble Protein Master Buffer Kit according to the manufacturer’s instructions.

RNA-seq analysis

CAR-T cells were harvested, and total RNA was extracted using the RNeasy Mini Kit (Qiagen). RNA quality was assessed with an Agilent 2100 Bioanalyzer. Sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit (NEB) and sequenced on an Illumina NovaSeq 6000 platform to generate 150‑bp paired‑end reads. Raw reads were quality‑checked with FastQC, trimmed with Trimmomatic, and aligned to the human reference genome (GRCh38) using STAR. Transcript abundance was quantified with RSEM, and differential expression analysis was performed using DESeq2. The sequencing was performed by Genepioneer (Nanjing, China), the resulting differentially expressed gene (DEG) list is provided in Table S2.

Animal experiments

Animal experiments were conducted at the animal laboratory of Nanjing Normal University in accordance with protocols approved by the Animal Welfare Committee of the university (Approval number: 2020-0047). Female B-NKG mice (6 weeks, Biocytogen, Beijing) were subcutaneously implanted with 2 × 106 HCC1806 cells. Ten days post tumor inoculation, mice were randomly divided into three groups (n = 7 for the PBS group; n = 8 for NC and SS groups) and injected with PBS, 8 × 105 NC-MSLN-CAR-T, or SS-MSLN-CAR-T cells through tail vein. Tumor volumes were measured using a caliper and calculated as volume = (length × width2) × 0.5. Mice weight was monitored every 2–3 days.

Peripheral blood was collected from mice on days 4, 8, and 12 following CAR-T cell infusion for phenotypic analysis and cytokine profiling. On day 8 post-treatment, tumors from three mice per group were harvested for immunohistochemical analysis. At the study endpoint (day 28 post-treatment), all remaining mice were euthanized. Tumors were collected for imaging, and major organs—including the heart, liver, spleen, lungs, kidneys, and brain—were excised and subjected to hematoxylin and eosin (H&E) staining to evaluate potential tissue toxicity. Tumor tissues and major organs were fixed in formalin and Further processing and staining were conducted by Servicebio (Wuhan, China). Anti-human CD45 primary antibody was used in IHC staining and quantification was conducted via ImageJ.

Statistical analysis

All experiments were performed at least three times independently. Data were analyzed using GraphPad Prism 8.0 software and presented as mean ± SEM. Student’s t-test was used for two-group comparisons, and one-way or two-way ANOVA was used for multiple-group analysis. Statistical significance was defined as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 871 KB) (870.1KB, docx)

Acknowledgements

We would like to express our gratitude to the National Natural Science Foundation of China (82373183), the Priority Academic Program Development of Jiangsu Higher Education Institutions, General Program of Postdoctoral Science Foundation of China (2025M772868), Young Scientists Fund of the National Natural Science Foundation of China (82503464) and Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. KYCX24_1852), and Department of Research and Development, Nanjing Calmhome Cell & Gene Engineering Institute Co., Ltd., Nanjing, China for generous support of this research.

Author contributions

Lianfeng Zhao: Conceptualization, data curation, methodology, statistical analysis, writing—original draft and writing—review & editing. Jiaqi Liu: Data curation, methodology, statistical analysis, and writing—review & editing. Yan Zhang: Data curation, methodology and statistical analysis. Xinhao Yang, Tianyu Chen and Xinyang Xu: methodology, statistical analysis visualization and writing—review & editing. Muya Zhou: Conceptualization, statistical analysis, investigation, visualization and writing—review & editing. Zhigang Hu: Conceptualization and project administration. Zhigang Guo: Conceptualization, funding acquisition and project administration.

Funding

This work was supported by the National Natural Science Foundation of China (82,373,183), the Priority Academic Program Development of Jiangsu Higher Education Institutions, General Program of Postdoctoral Science Foundation of China (2025M772868),Young Scientists Fund of the National Natural Science Foundation of China (82,503,464), Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. KYCX24_1852) and Department of Research and Development, Nanjing Calmhome Cell & Gene Engineering Institute Co., Ltd., Nanjing, China.

Data availability

The raw RNA‑seq data have been submitted to the NCBI database(Accession number: PRJNA1431589).

Declarations

Conflict of interest

The authors declare no conflict of interests.

Consent for publication

All authors hereby unanimously agree to publish this article.

Ethics approval

Animal experiments were conducted at the animal laboratory of Nanjing Normal University in accordance with protocols approved by the Animal Welfare Committee of the university (Approval number: IACUC-20230311).

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Lianfeng Zhao and Jiaqi Liu have contributed equally to this work.

Contributor Information

Muya Zhou, Email: muyazhou@njnu.edu.cn.

Zhigang Hu, Email: huzg_2000@126.com.

Zhigang Guo, Email: guo@njnu.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary material 1 (DOCX 871 KB) (870.1KB, docx)

Data Availability Statement

The raw RNA‑seq data have been submitted to the NCBI database(Accession number: PRJNA1431589).

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