CXCR3 enhanced murine chimeric antigen receptor T cells in the treatment of solid tumors

Introduction

Chimeric antigen receptor T cell (CAR-T) therapy has shown remarkable efficacy in the treatment of hematological malignancies (1-3). However, its effectiveness against solid tumors remains limited. This is primarily due to the complex, intertwined vascular network, dense extracellular matrix, and immunosuppressive tumor microenvironment, which collectively impede CAR-T infiltration into tumors (4-8), which reduces CAR-T recognition, engagement, and cytotoxic activity.

Chemokines and their receptors play a crucial role in mediating the directional migration of various cell types in vivo by establishing concentration gradients (9). CXCR3, a transmembrane G protein-coupled receptor, is predominantly expressed on activated T lymphocytes, dendritic cells, and natural killer cells (10). Its primary function, in conjunction with its key ligand CXCL10, is to regulate the chemotaxis of immune inflammatory cells (11). Limagne et al. showed that the combination of mitogen-activated protein kinase inhibitors with pemetrexed and cisplatin chemotherapy enhanced CXCL10 expression in non-small cell lung cancer (12). This upregulation facilitated the recruitment of CXCR3⁺CD8⁺ T cells into the tumor microenvironment, thereby improving patients’ sensitivity to immune checkpoint inhibitors (ICIs). Additionally, a dipeptidyl-peptidase inhibitor has been shown to enhance CXCL9/10 levels in tumors, promoting the recruitment of CXCR3⁺ T cells and improving therapeutic outcomes in pancreatic ductal carcinoma (13). Similarly, Li et al. found that histone deacetylase 3 (HDAC3) binds to the CXCL10 promoter region, suppressing its expression (14). Tumor cells lacking HDAC3 exhibit elevated CXCL10 levels, which effectively inhibit tumor growth in mice by enhancing CXCR3⁺ T cells infiltration.

A meta-analysis of CXCR3 expression in gastric cancer patients revealed a significant correlation between high CXCR3 expression and a lower tumor grade, as well as longer overall survival (15). The overexpression of CXCR3 in gastric cancer patients was found to be positively correlated with reduced M2 macrophage infiltration, longer overall survival, and lower mortality rates (16). Additionally, an analysis of CXCR3 expression in gastric cancer tissues indicated that elevated CXCR3 levels were associated with increased T-cell infiltration into the tumor microenvironment (17,18). These findings suggest a potential role for CXCR3 in the prognosis and treatment of gastric cancer. A phenotypic analysis of tumor-infiltrating immune cells in renal cell carcinoma revealed that higher CXCR3 expression density on T cells was positively correlated with improved patient overall survival (19). In a phase-I trial of GD2 CAR-T therapy for GD2⁺ solid tumors, high CXCR3 expression on patient monocytes was associated with enhanced CAR-T expansion and functional persistence (20).

However, interleukin (IL)-17 has been shown to inhibit CD8⁺ T-cell infiltration in colorectal cancer and promote tumor progression by suppressing CXCL9/10 production (21). Wang et al. reported that CD8⁺ T-cell infiltration was significantly reduced in late-stage colorectal cancer patients compared to early stage patients (22). This reduction was attributed to the T helper 17 cell-mediated downregulation of CXCR3 expression in CD8⁺ T cells via the IL-17A/STAT3 pathway. Further, Gunderson et al. reported that CD8⁺ T cells lacking transforming growth factor beta (TGF-β) receptors exhibited increased CXCR3 expression, leading to enhanced T-cell infiltration in preclinical colorectal cancer models of TGF-β receptor knockout mice (23). Peng et al. showed that miR-762 inhibited T-cell migration and activation in oral squamous cell carcinoma by directly suppressing CXCR3 expression in CD8⁺ T cells, fostering a highly immunosuppressive tumor microenvironment (24). These findings highlight the critical role of CXCR3 in facilitating T-cell infiltration into tumor tissues.

Recent preclinical investigations have engineered CAR-Ts with chemokines and chemokine receptors, which have shown enhanced tumor-targeting efficacy (25-27). We hypothesized that engineering CAR-Ts to overexpress CXCR3 could enhance their chemotaxis and anti-tumor activity in solid tumors. This study sought to generate EGFRvIII-targeting CAR-Ts overexpressing CXCR3, designated as EGFRvIII·mCAR-T-CXCR3 cells. We evaluated their specific cytotoxicity, activation, and CXCL10-mediated chemotaxis in vitro. Additionally, we assessed their tumor infiltration, anti-tumor efficacy, and safety in a subcutaneous tumor model using immunocompetent mice. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1920/rc).

Methods

Cell lines

HEK-293T (a human embryonic kidney cell line), MC38 (a C57BL/6 mouse colon cancer cell line), CT26 (a BALB/c mouse colon cancer cell line), and Panc02 (a C57BL/6 mouse pancreatic cancer cell line) were obtained from Vigen Biotechnology (Zhenjiang, China).

The HEK-293T and MC38 cells were cultured in Dulbecco’s modified Eagle’s high-glucose medium (Vigen Biotechnology) supplemented with 10% fetal bovine serum (FBS; Shanghai Liji Biotechnology, Shanghai, China) and incubated at 37 ℃ with 5% CO₂. The CT26 and Panc02 cells were cultured in RPMI-1640 medium (Vigen Biotechnology) supplemented with 10% FBS. The HEK-293T cells were used for the production of all lentiviruses and retroviruses in this study. The tumor cells labeled with firefly luciferase (ffluc) and EGFRvIII were generated via lentiviral transfection, while the mCAR-Ts were prepared via retroviral transfection.

Generation of retroviral vectors encoding CXCR3-CARs

The EGFRvIII-specific murine CAR (EGFRvIII·mCAR) was designed with an EGFRvIII specific scFv, a CD28 transmembrane domain, and a CD3ζ signaling domain. Using In-Fusion cloning (TaKaRa, Shiga, Japan), this CAR gene was inserted into a retroviral vector either alone or in a T2A-linked configuration with green fluorescent protein (GFP) or CXCR3. All constructs were sequence-verified (Genewiz, Suzhou, China). Retroviral particles were produced by co-transfecting HEK-293T cells with the vector and packaging plasmids, followed by concentration of the supernatant.

Isolation, activation, and transduction of mouse T cells

Six-week-old female specific pathogen-free (SPF) grade C57BL/6 mice were obtained from the Experimental Animal Center of Jiangsu University. After a mouse was euthanized, the spleen was harvested, and the cells were passed through a 0.45-µm sieve. The erythrocytes were then lysed, and 500 IU/mL IL-2 and 10 µg/mL concanavalin (C2010, Sigma-Aldrich, St. Louis, MO, USA) were added to the cells, which were cultured in an incubator for 24 hours to facilitate activation. After 24 hours of activation, mouse activated T cells (mATCs) were transduced with retrovirus at a multiplicity of infection of 10 on RetroNectin (T100A, Takara)-pre-coated plate. The plate was centrifuged at 2,000 g for 90 minutes, completing the transduction process.

Flow cytometry

After transduction and culturing for four days, 2×10⁵ CAR-Ts were resuspended in 100 μL staining buffer. Next, 1 μL of allophycocyanin (APC) conjugated antimouse immunoglobulin G (IgG) F(ab’)2 fragment specific antibody (115-136-072, Jackson ImmunoResearch, West Grove, PA, USA) was added to cell suspension, mixed, and incubated on ice for 30 minutes. After the incubation, the cells were washed once, and then centrifuged at 300 g for 5 minutes. The washing process was repeated, and the cells were diluted to 300 μL with phosphate buffered saline (PBS). The flow cytometry analysis was performed using a BeamCyte flow cytometer (VDO biotech, Suzhou, China).

RNA extraction, reverse transcription, and -real-time quantitative PCR (RT-qPCR)

RNA from tumor cells and CAR-Ts was extracted using the MolPure Cell RNA Kit (Yeasen, Shanghai, China). The RNA was then reverse transcribed into complementary DNA (cDNA) using the HiFair II 1^st Strand cDNA Synthesis SuperMix (Yeasen). The primers for the target genes were designed using Oligo 6 software (OLIGO, Colorado Springs, CO, USA). RT-qPCR was performed using the HiEff UNICON qPCR SYBR Green Master Mix (Yeasen) following a two-step amplification protocol. Detection was carried out using an Analytik Jena system m (Analytik Jena, Jena, Germany). Each sample was tested in triplicate, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal reference gene for normalization. Relative gene expression was calculated using the 2^(−ΔΔCt) method.

Western blot

The CAR-Ts were lysed with radio immunoprecipitation assay lysis (RIPA) buffer, sonicated on ice, and centrifuged at 12,000 ×g for 10 minutes at 4 ℃ to collect protein samples. Protein concentrations were determined using a bicinchoninic acid (BCA) quantification kit (Yeasen). After mixing with loading buffer, samples containing 20 µg protein per lane were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following electrophoretic separation, the proteins were transferred onto nitrocellulose membranes, probed with target-specific antibodies, and visualized by chemiluminescent detection. The Tanon 4600 Chemiluminescence System (Tanon, Shanghai, China) was used for exposure. The antibodies used included CD3-ζ antibody (sc-166435, Santa Cruz Biotechnology, Shanghai, China), horseradish peroxidase (HRP)-conjugated Goat Anti-Mouse IgG (D110087, Sangon Biotechnology, Shanghai, China), and β-actin (4D3) monoclonal antibody (BS6007M, BioWorld, Nanjing, China).

Cytotoxicity assay

All the tumor cells in this experiment were labeled with the ffluc signal. To assess CAR-T cytotoxicity in vitro, the tumor cells were seeded at 3×10⁴ cells/well in a 96-well plate and incubated for 24 hours to allow adherence. Effector CAR-Ts were added at effector-to-target (E:T) ratios of 4:1, 2:1, 1:1, and 0:1. After 24 hours of co-culture, the supernatants were collected and stored at −80 ℃. D-luciferin substrate (Yeasen) was added, and luminescence was measured using a Lumistation-1800 luminometer (Flash Spectrum Biotechnology, Shanghai, China). A parallel plate was washed and stained with crystal violet for visualization.

Enzyme-linked immunosorbent assay (ELISA)

The supernatant from the effector cells co-cultured for 24 hours with the target cells was collected to measure the cytokine secretion levels. ELISA kits for murine IL-2 and interferon (IFN)-γ (555148 and 555138, BD Bioscience, Franklin Lakes, NJ, USA) were used to assess the activation ability of the CAR-Ts. The procedure was performed according to the manufacturer’s instructions.

Transwell assay

After 72 hours of cell culture, the supernatant was collected and filtered through a 0.45-µm membrane. A total of 600 µL of the filtered supernatant was added to the lower chamber of a Transwell plate (Jet BIOFIL, Guangzhou, China), and 100 µL of CAR-T suspension (1×10⁶ cells) was added to the upper chamber. The plate was incubated for 2 hours at 37 ℃ to allow chemotaxis. After incubation, the upper chamber was removed, and the cells in the lower chamber were counted to calculate the relative migration rate.

Allograft transplantation model

All the experimental procedures were conducted in accordance with the institutional guidelines for the care and use of animals established by the Institutional Animal Care and Use Committee of Jiangsu University, and approved by the Institutional Animal Care and Use Committee of Jiangsu University (approval No. UJS-IACUC-2023021507). A protocol was prepared before the study without registration. Following subcutaneous inoculation of twenty mice with 1×10⁶ MC38-ffluc-EGFRvIII cells in the right forelimb, the animals were randomly allocated into the following four experimental groups (n=5) using random numbers: the untreated control group (PBS administration), the mATC control group (1×10⁷ cells), the EGFRvIII·mCAR-T group (1×10⁷ cells), and the EGFRvIII·mCAR-T-CXCR3 group (1×10⁷ cells). Treatment initiation occurred 72 hours post-tumor inoculation. Tumor dimensions were measured every three days using digital calipers, and the volume was calculated using the following formula: volume = (length × width²)/2. Concurrent body weight measurements were recorded to monitor treatment toxicity. Animals reaching the predetermined tumor burden threshold (1,000 mm³) were promptly euthanized to minimize distress. At 24 days post-treatment, euthanasia was performed. Tumors and major organs (i.e., the heart, liver, spleen, lungs, and kidneys) were immediately excised, weighed, and fixed in 4% paraformaldehyde for subsequent histological analysis.

Immunohistochemistry

The tumor and normal tissues were embedded in paraffin blocks and sectioned. The normal tissue sections were processed for hematoxylin and eosin staining. For the tumor tissue analysis, immunohistochemical staining was performed with primary antibodies targeting Ki-67, cleaved caspase-3, and CD3 (GB111499, GB11532, and GB111337; Servicebio, Wuhan, China). Following incubation with primary antibodies, HRP-conjugated secondary antibodies (G1302, Servicebio) were applied, followed by chromogenic visualization using diaminobenzidine. The sections were subsequently counterstained with hematoxylin.

Statistical analysis

The data were analyzed and visualized using Prism 8.0 (GraphPad, San Diego, CA, USA). The results are expressed as the mean ± standard error (SE). Statistical significance was determined using the Student’s t-test or one-way analysis of variance with Tukey’s post hoc test. A P value <0.05 was considered statistically significant (*, P<0.05; **, P<0.01; ***, P<0.001). The schematic diagrams were created using Figdraw (Home for Researchers, Hangzhou, China).

Results

Generation and characterization of EGFRvIII-targeted CAR-Ts co-expressing CXCR3

EGFRvIII, a tumor-specific mutant of EGFR, has been reported to be selectively expressed in various malignancies, making it a promising therapeutic target in cancer immunotherapy (28). In this study, we engineered a second-generation chimeric antigen receptor (CAR) construct targeting EGFRvIII, which simultaneously co-expresses CXCR3 and GFP as a marker gene (Figure 1A). To generate the CAR-Ts, primary T cells were isolated from the spleens of the immunocompetent mice, and transduced with a retroviral vector encoding the EGFRvIII-targeted CAR (Figure 1B).

Figure 1 Generation and validation of EGFRvIII·mCAR-T-CXCR3-GFP cells. (A) Schematic of the retroviral plasmid design for EGFRvIII·mCAR-T-CXCR3-GFP cells. (B) Workflow for the in vitro production of mCAR-Ts. (C) Flow cytometry analysis of CAR-T transduction efficiency. (D) Batch-to-batch consistency of the CAR-T transduction rates. (E) Fluorescence microscopy confirming GFP expression in the transduced mCAR-Ts. (F,G) RT-qPCR analysis of CAR and CXCR3 mRNA expression levels. (H) Western blot verification of CAR protein expression. (I) Proliferation kinetics of CAR-Ts post-transduction. Error bars represent the mean ± SE (n=3) (ns, P>0.05; ***, P<0.001). CAR, chimeric antigen receptor; CAR-T, chimeric antigen receptor T cell; GFP, green fluorescence protein; mATC, mouse activated T cell; mCAR-T, murine chimeric antigen receptor T cell; RT-qPCR, real-time quantitative polymerase chain reaction; RV, retroviral vector; SE, standard error.

The flow cytometry analysis on day 4 post-transduction revealed that the transduction efficiencies of EGFRvIII·mCAR-T-GFP and EGFRvIII·mCAR-T-CXCR3-GFP were 77.17% and 78.40%, respectively, indicating a robust and reproducible transduction process (Figure 1C). Additionally, multi-batch validation showed the stability of CAR expression across independently prepared CAR-T batches (Figure 1D). The GFP signal observed via fluorescence microscopy corroborated the flow cytometry findings, further confirming successful transgene expression (Figure 1E).

The RT-qPCR analysis demonstrated high levels of CAR messenger RNA (mRNA) expression in the EGFRvIII·mCAR-T and EGFRvIII·mCAR-T-CXCR3 cells, while CXCR3 overexpression was specifically detected in the EGFRvIII·mCAR-T-CXCR3 group as expected (Figure 1F,1G). Further, the western blot analysis confirmed the endogenous expression of CD3ζ (35 kDa) in the non-transduced mATCs, as well as in both the EGFRvIII·mCAR-T and EGFRvIII·mCAR-T-CXCR3 cells. Notably, only the transduced CAR-Ts exhibited an additional band at approximately 60 kDa, corresponding to the CAR protein, further verifying successful CAR expression (Figure 1H).

To assess the potential effects of viral transduction and CXCR3 overexpression on T-cell viability and proliferation, we performed serial cell counting over multiple time points. The results revealed no significant differences in the proliferation rates between the non-transduced and transduced T cells, suggesting that neither CAR nor CXCR3 overexpression adversely affected T-cell expansion (Figure 1I).

Taken together, these findings confirmed the successful generation of EGFRvIII·mCAR-T-CXCR3-GFP cells with stable CAR and CXCR3 expression, providing a solid foundation for subsequent functional and therapeutic investigations.

EGFRvIII·mCAR-T-CXCR3 cells exhibited a strong specific killing ability and cytokine secretion ability

To assess the antigen-specific cytotoxicity of our engineered CAR-Ts in vitro, we co-cultured mATCs, EGFRvIII·mCAR-T, and EGFRvIII·mCAR-T-CXCR3 cells with the following two target cells: MC38-ffluc cells (which do not express EGFRvIII), and MC38-ffluc-EGFRvIII cells. The co-cultures were set up at varying E:T ratios (0:1, 1:1, 2:1, and 4:1). Crystal violet staining showed that both the EGFRvIII·mCAR-T and EGFRvIII·mCAR-T-CXCR3 cells exhibited robust cytotoxicity against the MC38-ffluc-EGFRvIII cells, while also exhibiting minimal killing of the MC38-ffluc cells, thereby confirming antigen-specific lysis (Figure 2A,2B). These observations were further corroborated by luciferase assays, which revealed a significant decrease in luminescence—indicative of target cell death—only in the cultures containing the EGFRvIII-expressing target cells (Figure 2C,2D).

Figure 2 EGFRvIII·mCAR-T-CXCR3 cells exhibit potent antigen-specific cytotoxicity in vitro. (A,B) Crystal violet staining of mCAR-T cells co-cultured with MC38-ffluc (A) and MC38-ffluc-EGFRvIII (B) cells in vitro for 24 hours. (C,D) Detection of cytotoxicity of CAR-Ts co-cultured with MC38-ffluc (C) and MC38-ffluc-EGFRvIII (D) by luciferase assay. (E,F) Luciferase assay assessing the cytotoxicity of CAR-Ts co-cultured with CT26-ffluc-EGFRvIII (E) and Panc02-ffluc-EGFRvIII (F). (G,H) Supernatant collected from the CAR-T cells was co-cultured with MC38-ffluc-EGFRvIII for 24 hours to detect IFN-γ (G) and IL-2 (H) secretion by ELISA. Error bars represent the mean ± SE (n=3) (ns, P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001). CAR-T, chimeric antigen receptor T cells; E:T, effector-to-target; ELISA, enzyme-linked immunosorbent assay; IFN, interferon; IL, interleukin; mATC, mouse activated T cell; mCAR-T, murine chimeric antigen receptor T cell; SE, standard error.

Our results also showed that the cytolytic activity of the engineered CAR-Ts extended beyond a single tumor model. Specifically, the EGFRvIII-targeted CAR-Ts efficiently lysed the CT26-ffluc-EGFRvIII cells, derived from a different murine species, as well as the Panc02-ffluc-EGFRvIII cells which representing a distinct tissue origin (Figure 2E,2F). This antigen-dependent, broad-spectrum cytotoxicity underscores the potential versatility of these cells in targeting diverse EGFRvIII-expressing tumors.

To evaluate the functional activation of the CAR-Ts following target cell engagement, we quantified the secretion of key effector cytokines, IFN-γ and IL-2, in the culture supernatants after co-culture with the MC38-ffluc-EGFRvIII cells by ELISA. Notably, the EGFRvIII·mCAR-T-CXCR3 cells produced significantly higher levels of both mIFN-γ and mIL-2 compared to their EGFRvIII·mCAR-T counterparts (Figure 2G,2H). These findings suggest that the co-expression of CXCR3 promoted a more robust activation profile upon antigen encounter, which might result in improved anti-tumor efficacy in vivo.

CXCL10-induced directed migration of EGFRvIII·mCAR-T-CXCR3 cells

As a critical ligand for CXCR3, CXCL10 establishes chemotactic gradients in vivo that facilitate the directional migration of immune cells (29). To investigate whether CXCR3 overexpression enhances the chemotaxis of CAR-Ts, we engineered a lentiviral vector to overexpress CXCL10 (Figure 3A). A packaged lentivirus was used to transduce the 293T cells, thereby generating a stable CXCL10-secreting cell line. Fluorescence microscopy confirmed robust red fluorescence protein (RFP) expression in these cells (Figure 3B), and the RT-qPCR analysis further verified the significant upregulation of CXCL10 transcripts in the transduced 293T cells (Figure 3C).

Figure 3 CXCL10 induces directed chemotaxis in EGFRvIII·mCAR-T-CXCR3 cells. (A) Schematic of the CXCL10-RFP lentiviral plasmid construct. (B) Fluorescence microscopy confirming RFP expression in the 293T-CXCL10 cells. (C) RT-qPCR validation of CXCL10 mRNA expression in the 293T-CXCL10 cells. (D) Experimental design of the Transwell chemotaxis assay. (E) Chemotactic migration of CAR-Ts toward CXCL10 as quantified by Transwell assays. (F,G) Flow cytometry analysis of the migrated CAR-Ts in the lower chamber. Error bars represent the mean ± SE (n=3) (**, P<0.01; ***, P<0.001; ****, P<0.0001). CAR-T, chimeric antigen receptor T cell; CMV, cytomegalovirus; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; LV, lentiviral vector; mCAR-T, murine chimeric antigen receptor T cells; RT-qPCR, real-time quantitative polymerase chain reaction; RFP, red fluorescence protein; SE, standard error; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; WT, wild type.

For the Transwell migration assays, we collected supernatants from the 293T-WT and 293T-CXCL10 cells after 72 hours of culture (Figure 3D). These supernatants were used as chemo-attractants for both the EGFRvIII·mCAR-T and EGFRvIII·mCAR-T-CXCR3 cells. The migration assays revealed that the 293T-CXCL10 supernatant induced a significantly higher directional migration of EGFRvIII·mCAR-T cells compared to the 293T-WT supernatant, likely reflecting the basal expression of CXCR3 on activated T cells. More notably, the EGFRvIII·mCAR-T-CXCR3 cells exhibited a marked enhancement in CXCL10-induced chemotaxis relative to their non-CXCR3-overexpressing counterparts (Figure 3E), indicating that CXCR3 overexpression substantially augments the chemotactic response.

A flow cytometry analysis of cells from the lower chamber showed that the percentage of chemotactic cells in the EGFRvIII·mCAR-T group remained relatively stable following CXCL10 induction, which can be attributed to the intrinsic CXCR3 expression in both the CAR-negative and CAR-positive activated T cells (Figure 3F). Conversely, the overexpression of CXCR3 in the mCAR-T cells resulted in a significant increase in the positive migration rate from 64.92% to 80.26% (Figure 3G). This highlights CXCL10’s preference for inducing migration in cell populations with high CXCR3 expression.

Collectively, these results indicate that CXCL10 effectively induced the directed chemotaxis of CAR⁺ T cells, and that the overexpression of CXCR3 further potentiated this effect. Thus, our findings provide compelling evidence that the CXCL10/CXCR3 axis can be exploited to enhance the migratory capacity of the EGFRvIII-targeted CAR-Ts.

Colorectal cancer cells induce the directed chemotaxis of EGFRvIII·mCAR-T-CXCR3 cells

To validate the chemotactic response of the EGFRvIII·mCAR-T-CXCR3 cells—engineered to overexpress CXCR3—in response to CXCL10 secreted by colorectal cancer cells, we first quantified CXCL10 expression in the CT26 and MC38 cell lines by RT-qPCR. The analysis revealed that the MC38 cells exhibited a significantly higher level of CXCL10 compared to the CT26 cells (Figure 4A). Subsequently, we collected the culture supernatants from the wild-type MC38 and CT26 cells after 72 hours, and performed Transwell migration assays. The results showed that CXCR3 overexpression markedly enhanced the chemotaxis of the mCAR-T cells in response to the MC38 supernatant, while the CT26 supernatant, due to its relatively low CXCL10 expression, did not induce significant migration (Figure 4B,4C).

Figure 4 EGFRvIII·mCAR-T-CXCR3 cells exhibit CXCL10-dependent chemotaxis toward colorectal cancer cells. (A) RT-qPCR analysis of CXCL10 mRNA expression in the colorectal cancer cell lines. (B,C) Transwell assays evaluating the chemotaxis of the CAR-Ts in response to the colorectal cancer cell-conditioned supernatant. (D) Flow cytometry analysis of RFP overexpression efficiency in the colorectal cancer cells. (E,F) RT-qPCR validation of CXCL10 overexpression in the colorectal cancer cells. (G,H) Chemotaxis of CAR-Ts toward the CXCL10-overexpressing colorectal cancer cells as assessed by Transwell assays. Error bars represent the mean ± SE (n=3) (ns, P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001). CAR-T, chimeric antigen receptor T cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mATC, mouse activated T cell; mRNA, messenger RNA; PE, phycoerythrin; RT-qPCR, real-time quantitative polymerase chain reaction; RFP, red fluorescent protein; SE, standard error; WT, wild type.

To further substantiate these findings, we engineered CT26-EGFRvIII and MC38-EGFRvIII cells to overexpress CXCL10-RFP. Successful overexpression was confirmed by both flow cytometry and RT-qPCR analyses (Figure 4D-4F). Transwell assays using supernatants from both unmodified and CXCL10-overexpressing colorectal cancer cells revealed that enforced CXCL10 expression significantly enhanced the chemotaxis of the EGFRvIII·mCAR-T-CXCR3 cells. Interestingly, the supernatant from the MC38-EGFRvIII cells induced a chemotactic response in the EGFRvIII·mCAR-T-CXCR3 cells that was comparable to the response observed in the MC38 cells that had been engineered to overexpress CXCL10 (Figure 4G,4H). At high CXCL10 concentrations, T-cell migration is driven by ligand saturation despite low CXCR3 expression. Conversely, at low CXCL10 concentrations, ligand limitation balances migration efficiency despite CXCR3 overexpression, resulting in similar numbers of migrating cells.

In conclusion, our results confirmed that colorectal cancer cell-derived CXCL10 effectively drove the chemotaxis of CAR-Ts. Moreover, the EGFRvIII·mCAR-T-CXCR3 cells exhibited significantly enhanced migration compared to the EGFRvIII·mCAR-T cells when stimulated with supernatant from the MC38-EGFRvIII cells, which is attributable to the high endogenous CXCL10 expression in the MC38 cells. These findings underscore the critical role of the CXCL10/CXCR3 axis in mediating targeted CAR-T migration, which might have important implications for improving CAR-T-based immunotherapies in colorectal cancer.

EGFRvIII·mCAR-T-CXCR3 cells showed excellent anti-tumor ability in vivo

In this study, while most preclinical evaluations of humanized CAR-T therapies have employed xenograft tumor NOD SCID Gamma mouse models, we sought to assess the in vivo anti-tumor efficacy of EGFRvIII·mCAR-T-CXCR3 cells in an immunocompetent setting (30). To this end, we established a subcutaneous colorectal cancer model in C57BL/6 mice using the MC38-EGFRvIII cell line. A study has reported that CXCL10 overexpression in CT26 cells significantly suppresses tumorigenicity in BALB/c mice via natural killer cell recruitment, which is consistent with our preliminary findings that the CXCL10-overexpressing colorectal cancer cells exhibited reduced tumor formation in immunocompetent hosts (31). Thus, to ensure robust tumor establishment, we selected the MC38-EGFRvIII cells, whose supernatant had been previously shown to induce the directed chemotaxis of EGFRvIII·mCAR-T-CXCR3 cells.

For the in vivo study, 1×10⁶ MC38-EGFRvIII cells were subcutaneously injected into the right forelimb of C57BL/6 mice. The mice were then randomly assigned to four groups, and on day 3 post-tumor inoculation, they received an intravenous infusion of 1×10⁷ cells of either mATCs (control), EGFRvIII·mCAR-T cells, or EGFRvIII·mCAR-T-CXCR3 cells, with a PBS group serving as an additional control. Tumor volumes and weights were measured at three-day intervals following CAR-T infusion (Figure 5A). By day 24, the tumor volumes of the PBS group exceeded 1,000 mm³ in several mice, necessitating experimental termination. Tumor inhibition curves indicated that the PBS and mATC groups had comparable tumor burdens (approximately 800 mm³), while the mice treated with the EGFRvIII·mCAR-T cells exhibited significant tumor suppression. Notably, the EGFRvIII·mCAR-T-CXCR3 group showed a further marked reduction in tumor volume compared to the EGFRvIII·mCAR-T group (Figure 5B).

Figure 5 EGFRvIII·mCAR-T-CXCR3 cells demonstrate enhanced anti-tumor efficacy in vivo. (A) Schematic of the subcutaneous tumor model in the immunocompetent mice. (B) Tumor growth curves over 24 days post-treatment. (C) Representative photographs of excised tumor tissues 24 days post-treatment. (D) Tumor weights 24 days post-treatment. (E) Histopathological and immunohistochemical analysis of tumor tissues: hematoxylin and eosin staining, Ki-67 (proliferation), cleaved caspase-3 (apoptosis), and CD3 (T-cell infiltration). Error bars represent the mean ± SE (n=5) (ns, P>0.05; *, P<0.05; **, P<0.01; ***, P<0.001). CAR-T, chimeric antigen receptor T cell; HE, hematoxylin and eosin; mATC, mouse activated T cell; mCAR-T, murine chimeric antigen receptor T cell; PBS, phosphate buffered saline; SE, standard error.

Upon sacrifice at day 24, the tumors were excised, photographed, and weighed. Both the tumor volume and mass in the EGFRvIII·mCAR-T-CXCR3 group were significantly lower than those observed in the other three groups (Figure 5C,5D), underscoring the enhanced anti-tumor efficacy conferred by CXCR3 overexpression.

Histological examination via immunohistochemistry further corroborated these findings. Tumor sections from the EGFRvIII·mCAR-T-CXCR3 treatment group displayed a significant decrease in Ki-67-positive proliferative cells, a concomitant increase in cleaved caspase-3-positive apoptotic cells, and a looser internal tumor architecture, all indicative of effective cytotoxicity and apoptosis induction. Moreover, CD3 immunostaining revealed a substantial increase in CD3⁺ T cell infiltration in the tumors from the EGFRvIII·mCAR-T-CXCR3 group compared to the controls (Figure 5E), suggesting that CXCR3 overexpression facilitates the recruitment of T cells and establishes an inflammatory microenvironment conducive to tumor cell destruction.

Importantly, the continuous monitoring of body weight revealed no significant differences between the mice treated with the EGFRvIII·mCAR-T-CXCR3 cells and those in the control groups (Figure 6A). A histopathological analysis of normal tissues by hematoxylin and eosin staining further confirmed the absence of significant tissue toxicity in the EGFRvIII·mCAR-T-CXCR3 group (Figure 6B). This showed that EGFRvIII·mCAR-T-CXCR3 cells are safe for intravenous administration in immunocompetent mice.

Figure 6 In vivo safety assessment of EGFRvIII·mCAR-T-CXCR3 cells. (A) Body weight changes in the mice over 24 days post-CAR-T treatment. (B) Histopathological evaluation of heart, liver, spleen, lung, and kidney tissues via hematoxylin and eosin staining 24 days post-treatment. Error bars represent the mean ± SE (n=5) (ns, P>0.05). CAR-T, chimeric antigen receptor T cell; mATC, mouse activated T cell; mCAR-T, murine chimeric antigen receptor T cell; PBS, phosphate buffered saline; SE, standard error.

Collectively, these results showed that CXCR3 overexpression significantly enhanced the infiltration of the CD3⁺ T cells into tumor tissues, thereby boosting the anti-tumor efficacy of the EGFRvIII-targeted mCAR-T cells in vivo without eliciting adverse systemic toxicity.

Discussion

In this study, we engineered mCAR-Ts that simultaneously target EGFRvIII and overexpress CXCR3, with the aim of enhancing their anti-gen-specific cytotoxicity, activation potential, and migratory capacity. The in vitro experiments confirmed that these engineered cells specifically eliminated EGFRvIII-positive targets and secreted elevated levels of cytokines, reflecting enhanced functional activation. Further, in an immunocompetent subcutaneous tumor model, the EGFRvIII·mCAR-T-CXCR3 cells exhibited superior tumor infiltration, significant tumor growth inhibition, and a favorable safety profile, underscoring the potential of CXCR3 overexpression to improve CAR-T efficacy in solid tumors.

CAR-T therapy has revolutionized the treatment of hematological malignancies; however, its application in solid tumors has been limited by poor T-cell infiltration due to the physical barrier imposed by the extracellular matrix and an immunosuppressive tumor microenvironment. A study has highlighted the role of chemokines such as CXCL9 and CXCL10 in modulating the tumor milieu by recruiting CXCR3-positive T cells, thereby enhancing anti-tumor immunity (32). Building on these insights, we posited that the forced expression of CXCR3 in CAR-Ts could potentiate their therapeutic effect against solid tumors.

Our in vitro data revealed that the mCAR-T cells targeting EGFRvIII not only exerted specific cytotoxicity against the EGFRvIII-expressing tumor cells but also displayed broader anti-tumor activity across different tumor lineages. Notably, the CXCR3-overexpressing mCAR-T cells exhibited an enhanced cytokine secretion profile post-target engagement, suggesting improved activation that could translate into sustained anti-tumor responses. These findings warrant further investigation through continuous killing assays, which may clarify whether limitations in endogenous CXCL10 concentrations can be mitigated by exogenous supplementation (33). Moreover, our Transwell migration assays confirmed that the EGFRvIII·mCAR-T-CXCR3 cells displayed robust chemotactic responses to CXCL10 gradients, an effect that was markedly augmented when CXCL10 was overexpressed in the tumor cells. This enhanced migratory capability likely contributed to the improved CD3⁺ T-cell infiltration observed in the tumor tissues of the mice treated with the EG-FRvIII·mCAR-T-CXCR3 cells. The corresponding reduction in proliferative markers (e.g., Ki-67) and increase in apoptotic indicators (e.g., cleaved caspase-3) in these tumors further validated the enhanced anti-tumor efficacy achieved by CXCR3 modification. The use of an immunocompetent subcutaneous tumor model allowed for a comprehensive evaluation of both tumor suppression and immune cell dynamics. Notably, as all animals were euthanized at the predefined ethical endpoint of tumor volume, survival analysis was not performed in this study. Future investigations will specifically incorporate survival as a key endpoint to further validate the long-term therapeutic benefit. In addition, future studies employing models with delayed treatment schedules will be essential to rigorously evaluate the therapeutic potential of CXCR3 engineering in confronting the formidable barrier of an established, immunosuppressive tumor microenvironment (TME). These studies could also be extended using peritoneal tumor models, which may better recapitulate the complex microenvironment of colorectal cancer (34).

This study confirms that CXCR3 modification significantly enhances the antitumor efficacy of CAR-T cells, primarily by improving their ability to infiltrate tumor tissue, as supported by both in vitro transwell assays and in vivo immunohistochemistry results. However, the precise mechanisms underlying this enhancement require further investigation. An interesting observation is that although EGFRvIII·mCAR-T-CXCR3 cells secreted higher levels of cytokines upon target engagement in vitro, their direct cytotoxic activity was not proportionally enhanced. This suggests that the superior in vivo performance is likely attributable to improved homing to tumor sites rather than an increase in intrinsic killing capacity. Existing literature indicates that the CXCL10/CXCR3 axis plays an important role in promoting Th1-type T cell and IFN-γ^high effector/cytotoxic CD8⁺ T cell differentiation, implying that CXCR3 signaling may directly influence the functional polarization or activation state of CAR-T cells (35). Furthermore, a pertinent study demonstrated that the CXCR3 antagonist AMG487 exerted effects by suppressing the differentiation of Th1 cells and the activation of M1 macrophages. This finding indicates that CXCR3 signaling extends beyond simply mediating the targeted chemotaxis of immune cells, it also plays a potent immunoregulatory role in the differentiation and functional maturation of key cytokine-secreting inflammatory cells (36). Therefore, a key future direction will be to clarify whether and how CXCR3 signaling modulates CAR-T cell and endogenous immune cells differentiation and function. To further validate enhanced trafficking as the central mechanism, subsequent studies could employ CXCL10-knockout tumor models to directly verify the dependency on this pathway in vivo. In addition, techniques such as in vivo imaging or flow cytometric analysis of tumor-infiltrating lymphocytes at early time points would provide dynamic and visual assessment of the homing process of CXCR3-engineered CAR-T cells.

Recent studies have highlighted the critical role of CXCR3 in ICI therapy. For instance, combination therapy with CTLA-4 and IL-6 antibodies was shown to significantly enhance T-cell infiltration in pancreatic tumor-bearing mice, leading to tumor growth inhibition (37). This effect was abolished upon CXCR3 blockade, underscoring its importance (37). Further, CXCL9/CXCL10 not only promotes CXCR3⁺ T-cell infiltration into tumors but also drives T-cell differentiation into T helper 1-like and cytotoxic CD8⁺ T cells. Upon binding to CXCR3, CXCL9/CXCL10 induces IFN-γ production, which further stimulates CXCL10 secretion, creating a self-amplifying positive feedback loop. Blocking programmed cell death 1 (PD-1) and CTLA-4 enhances this loop, and CXCR3 is indispensable for ICIs efficacy (35). Chow et al. showed that CXCR3 is essential for the response to PD-1 blockade therapy, as it enhances the proliferation and effector functions of tumor-infiltrating CD8⁺ T cells (38). These findings underscore the pivotal role of CXCR3 in ICI therapy, and suggest that combining CXCR3-modified CAR-Ts with PD-1 blockade could yield promising therapeutic outcomes.

Furthermore, the combination of CXCR3-engineered CAR-T cells with dipeptidyl peptidase inhibitors represents a highly promising translational strategy. The dipeptidyl peptidase inhibitors, which are clinically widely used for conditions such as type 2 diabetes. Critically, several studies have demonstrated that dipeptidyl peptidase inhibitors can successfully increase the local concentration of CXCL10 within the tumor microenvironment (39,40). Given that the efficacy of CXCR3-engineered CAR-T cells is fundamentally dependent on the CXCL10-CXCR3 axis, elevating local CXCL10 levels through dipeptidyl peptidase inhibition could synergistically enhance the recruitment and antitumor activity of these cells.

Additionally, CXCL10-armed oncolytic adenoviruses have been shown to achieve sustained and high-level CXCL10 expression in tumors, effectively recruiting CXCR3⁺ T cells to infiltrate and eliminate tumor cells (41,42). Combining CXCR3-modified CAR-T therapy with oncolytic viruses may further improve therapeutic outcomes (43,44).

Conclusions

Our results provide compelling evidence that the strategic overexpression of CXCR3 in EGFRvIII-targeted mCAR-T cells significantly enhances T-cell infiltration and tumor cell apoptosis, thereby improving the anti-tumor efficacy against solid tumors. These findings support the further development of CXCR3-mCAR-T modifications as a promising strategy for overcoming current limitations in solid tumor immunotherapy.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1920/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1920/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1920/prf

Funding: This research was supported by the Suqian Sci and Tech Program (No. S202006).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1920/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All the experimental procedures were conducted in accordance with the institutional guidelines for the care and use of animals established by the Institutional Animal Care and Use Committee of Jiangsu University, and approved by the Institutional Animal Care and Use Committee of Jiangsu University (approval No. UJS-IACUC-2023021507).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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