Journal: Frontiers in Immunology

Article Title: Relaxin-2-secreting CAR-T cells exhibit enhanced efficacy in stromal-rich xenograft tumors

doi: 10.3389/fimmu.2025.1506204

Figure Lengend Snippet: Phenotypic and functional characterization of CD44v6 chimeric antigen receptor (CAR)-T cells. (A) Memory/effector status of CAR-T cells before and after puromycin selection. Flow cytometry was performed using antibodies against the Fc gamma region, which binds to the hinge domain of the CAR expressed on both CD4 + and CD8 + T cells. Representative scatter plots are shown for donor 1-derived CAR-T cells. (B) Memory/effector subset analysis of CAR-T cells following co-culture with or without the indicated cell lines, followed by incubation in cytokine-depleted medium for 3 d Cells were stained with CD62L and CD45RO and gated on CD45 + CD3 + CAR-T cells for flow cytometric analysis. Representative data from donor 1-derived CAR-T cells are shown. (C) Immunofluorescence analysis of antigen-dependent binding of CAR-T cells. Donor 1-derived CAR-T cells were co-cultured for 3 h with Panc-1 (CD44v6-positive) or NIH/3T3 (CD44v6-negative) cells, then stained with anti-CD3 antibody. Fluorescent labeling: CD3 (green), CD44v6 (red), and nuclei (DAPI, blue). (D) Antigen-dependent activation and degranulation of CAR-T cells. Cells were co-cultured for 24 h with or without target cell lines in cytokine-depleted medium in the presence of CD107a antibody and the Golgi transport inhibitor Monensin. Activation and degranulation were assessed using CD69 and CD8a staining. Representative flow cytometry plots are shown. (E) Cytokine secretion by CAR-T cells following co-culture with the indicated cell lines. Levels of TNF-α and IFN-γ were quantified by ELISA after 48 h at an E:T ratio of 1:1. Data are presented as mean ± standard deviation (SD) from three independent experiments using CAR-T cells derived from donors 1, 2, and 3. (F) Cytolytic activity of control T and donor 1-derived CAR-T cells against various cancer cell lines. Cells were co-cultured for 3 d and their viability was assessed using crystal violet staining. Absorbance values were normalized to untreated wells to calculate relative cancer cell survival. Data are presented as mean ± SD of three independent experiments performed in triplicate. ns, not significant; ** P < 0.01; *** P < 0.001 (two-way ANOVA).

Article Snippet: The expression of CD44v6 in various cancer cell lines was assessed using a mouse anti-human FITC-conjugated CD44v6 antibody (clone VFF-18, eBioscience).

Techniques: Functional Assay, Selection, Flow Cytometry, Derivative Assay, Co-Culture Assay, Incubation, Staining, Immunofluorescence, Binding Assay, Cell Culture, Labeling, Activation Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Activity Assay, Control

Journal: Frontiers in Immunology

Article Title: Relaxin-2-secreting CAR-T cells exhibit enhanced efficacy in stromal-rich xenograft tumors

doi: 10.3389/fimmu.2025.1506204

Figure Lengend Snippet: Development of CD44v6 CAR-T cells and engineering for RLN2 secretion. (A) Schematic illustration of plasmid constructs used for CAR-T cell generation, including CD44v6 CAR and co-expression of either Luc2 or human RLN2 via transposon-based vectors. (B) RLN2 protein levels secreted by conventional and RLN2-secreting CAR-T cells quantified using ELISA. Data are presented as mean ± SD from three independent experiments performed in triplicate using CAR-T cells derived from donors 1, 2, and 3. (C) Western blot analysis of the RLN2 receptor LGR7/RXFP1 in multiple cancer cell lines. GAPDH was used as a loading control. (D) Expression of MMPs in cancer cell lines cultured for 48 h in serum-free medium (SF), 1% fetal bovine serum (FBS)-containing medium (1% CM), supernatant from CAR-T cells (CAR-T sup), or supernatant from RLN2-secreting CAR-T cells (RLN2 CAR-T sup), collected after 24 h in SF. RT-qPCR was conducted in three independent runs using CAR-T cells from donors 1, 2, and 3, in triplicate (mean ± SD). * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant (one-way ANOVA followed by Bonferroni’s post hoc test and unpaired t -test for comparisons between CAR-T and RLN2-secreting CAR-T groups). (E) Gelatin zymography of conditioned media from SU86.86 cells cultured in SF, complete medium with 10% FBS (CM), or undiluted (×1) or 5-fold diluted (×1/5) supernatants from CAR-T or RLN2 CAR-T cells. (F) Western blot analysis of LGR7/RXFP1 expression in conventional and RLN2-secreting CAR-T cells. MMP expression in imhPSCs treated with the indicated supernatants (evaluated as described in D ). (G) Western blot analysis of α-SMA and collagen type I alpha 1 (COL1A1) in imhPSCs cultured for 48 h in SF, SF + TGF-β (10 ng/mL), CAR-T sup ± TGF-β, or RLN2 CAR-T sup ± TGF-β. GAPDH was used as a loading control. Supernatants were derived from donor 1-generated CAR-T cells. (H) MMP expression in CAR-T cells. Conventional and RLN2-secreting CAR-T cells were cultured in RPMI-1640 with 10% FBS under resting conditions or with stimulation (anti-CD3/CD28 antibodies + IL-15 and IL-21) for 3 d RT-qPCR was performed using cells from donors 1, 2, and 3 (mean ± SD; triplicate experiments). * P < 0.05; *** P < 0.001; ns, not significant (comparisons between CAR-T and RLN2 CAR-T cells analyzed using unpaired t -test).

Article Snippet: The expression of CD44v6 in various cancer cell lines was assessed using a mouse anti-human FITC-conjugated CD44v6 antibody (clone VFF-18, eBioscience).

Techniques: Plasmid Preparation, Construct, Expressing, Enzyme-linked Immunosorbent Assay, Derivative Assay, Western Blot, Control, Cell Culture, Quantitative RT-PCR, Zymography, Generated

Journal: Frontiers in Immunology

Article Title: Relaxin-2-secreting CAR-T cells exhibit enhanced efficacy in stromal-rich xenograft tumors

doi: 10.3389/fimmu.2025.1506204

Figure Lengend Snippet: Histological characterization of subcutaneous xenograft tumor models. (A) Histological analysis of Panc-1, AsPC-1-CD44v6, and SU86.86 xenograft tumors. Tumor sections were analyzed for CD44v6 expression and stromal architecture. Formalin-fixed paraffin-embedded samples were subjected to H&E staining for tissue morphology, Picro-Sirius Red staining for collagen types I and III, and immunohistochemistry for CD44v6 and collagen type IV analysis. Scale bar = 200 µm. (B) Immunofluorescence staining of vascular and stromal markers in Panc-1 and SU86.86 subcutaneous xenograft tumors. Endothelial cells were labeled with anti-CD31 (green), myofibroblasts and pericytes with anti-α-SMA (red), and nuclei with DAPI (blue). Scale bar = 100 µm.

Article Snippet: The expression of CD44v6 in various cancer cell lines was assessed using a mouse anti-human FITC-conjugated CD44v6 antibody (clone VFF-18, eBioscience).

Techniques: Expressing, Formalin-fixed Paraffin-Embedded, Staining, Immunohistochemistry, Immunofluorescence, Labeling

Journal: Frontiers in Immunology

Article Title: Relaxin-2-secreting CAR-T cells exhibit enhanced efficacy in stromal-rich xenograft tumors

doi: 10.3389/fimmu.2025.1506204

Figure Lengend Snippet: Efficacy of conventional CD44v6 CAR-T cell therapy in xenograft models with either scant or abundant stroma. (A–C) In vitro cytotoxicity (left) and in vivo antitumor activity (right) of donor 1-derived CD44v6 CAR-T cells against Panc-1 (A) , AsPC-1-CD44v6 (B) , and SU86.86 (C) tumor models. For in vitro assays, CAR-T cells were co-cultured with the indicated cancer cell lines for 3 d, and surviving cancer cells were quantified using a crystal violet assay. Absorbance at 590 nm (A 590 ) was measured and normalized to untreated controls. Data are presented as mean ± SD from three independent experiments conducted in triplicate. ns, not significant; *** P < 0.001 (two-way ANOVA). For in vivo studies, tumor-bearing mice received a single intravenous injection of either Ctrl-T ( n = 6) or CAR-T cells ( n = 6). Data are presented as mean ± SD. *** P < 0.001; ns, not significant (two-way ANOVA). (D) In vivo bioluminescence imaging of Luc2-expressing CAR-T cell accumulation in subcutaneous xenograft tumors. A total of 1 × 10 7 Luc2-expressing CAR-T cells were injected intravenously via the tail vein. Bioluminescence was measured using the IVIS imaging system on days 7 and 14 post-injection. (E) Immunofluorescence analysis of CAR-T cell infiltration in AsPC-1-CD44v6 and SU86.86 xenograft tumors 7 d after systemic injection of Luc2-expressing CAR-T cells. Tumor sections were stained for CD3 (green), CD44v6 (red), and CD31 (blue). Scale bar = 100 µm.

Article Snippet: The expression of CD44v6 in various cancer cell lines was assessed using a mouse anti-human FITC-conjugated CD44v6 antibody (clone VFF-18, eBioscience).

Techniques: In Vitro, In Vivo, Activity Assay, Derivative Assay, Cell Culture, Crystal Violet Assay, Injection, Imaging, Expressing, Immunofluorescence, Staining

Journal: Frontiers in Immunology

Article Title: Relaxin-2-secreting CAR-T cells exhibit enhanced efficacy in stromal-rich xenograft tumors

doi: 10.3389/fimmu.2025.1506204

Figure Lengend Snippet: Antitumor effects of RLN2-secreting CD44v6 CAR-T cell therapy in stromal-rich SU86.86 xenograft tumors. (A) Tumor growth in SU86.86 subcutaneous xenografts after single intravenous injection of Ctrl-T (1 × 10 7 cells, n = 6), conventional CD44v6 CAR-T (1 × 10 7 cells, n = 6), or RLN2-secreting CD44v6 CAR-T cells (1 × 10 7 cells, n = 6). Tumor size was measured over time, and representative images of resected tumors were captured following euthanasia. Data are presented as mean ± SD. ns, not significant; *** P < 0.001 (two-way ANOVA with Tukey’s multiple comparisons test). (B) Histological analysis of SU86.86 xenograft tumors collected 7 d after treatment with conventional or RLN2-secreting CAR-T cells. Formalin-fixed paraffin-embedded sections were subjected to H&E staining, Picro-Sirius Red staining (collagen types I and III), and immunohistochemistry. Immunofluorescence analysis of tumor-infiltrating CAR-T cells was performed on frozen sections. Tumor sections were stained for CD44v6 (red), CD3 (green), and CD31 (blue). Scale bar = 100 µm. (C) Quantitative analysis of MMP expression in SU86.86 xenograft tumors treated with conventional or RLN2-secreting CAR-T cells and harvested at either 3 or 7 d after single treatment with 1 × 10 7 CAR-T cells. MMP-7 and MMP-9 mRNA levels were assessed using RT-qPCR. CAR-T group ( n = 9, day 3; n = 5, day 7), RLN2 CAR-T group ( n = 7, day 3; n = 7, day 7). Data from two independent experiments are shown as mean ± SD. ns, not significant; * P < 0.05; *** P < 0.001; ns, not significant (unpaired t -test).

Article Snippet: The expression of CD44v6 in various cancer cell lines was assessed using a mouse anti-human FITC-conjugated CD44v6 antibody (clone VFF-18, eBioscience).

Techniques: Injection, Formalin-fixed Paraffin-Embedded, Staining, Immunohistochemistry, Immunofluorescence, Expressing, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Relaxin-2-secreting CAR-T cells exhibit enhanced efficacy in stromal-rich xenograft tumors

doi: 10.3389/fimmu.2025.1506204

Figure Lengend Snippet: Phenotypic and functional characterization of CD44v6 chimeric antigen receptor (CAR)-T cells. (A) Memory/effector status of CAR-T cells before and after puromycin selection. Flow cytometry was performed using antibodies against the Fc gamma region, which binds to the hinge domain of the CAR expressed on both CD4 + and CD8 + T cells. Representative scatter plots are shown for donor 1-derived CAR-T cells. (B) Memory/effector subset analysis of CAR-T cells following co-culture with or without the indicated cell lines, followed by incubation in cytokine-depleted medium for 3 d Cells were stained with CD62L and CD45RO and gated on CD45 + CD3 + CAR-T cells for flow cytometric analysis. Representative data from donor 1-derived CAR-T cells are shown. (C) Immunofluorescence analysis of antigen-dependent binding of CAR-T cells. Donor 1-derived CAR-T cells were co-cultured for 3 h with Panc-1 (CD44v6-positive) or NIH/3T3 (CD44v6-negative) cells, then stained with anti-CD3 antibody. Fluorescent labeling: CD3 (green), CD44v6 (red), and nuclei (DAPI, blue). (D) Antigen-dependent activation and degranulation of CAR-T cells. Cells were co-cultured for 24 h with or without target cell lines in cytokine-depleted medium in the presence of CD107a antibody and the Golgi transport inhibitor Monensin. Activation and degranulation were assessed using CD69 and CD8a staining. Representative flow cytometry plots are shown. (E) Cytokine secretion by CAR-T cells following co-culture with the indicated cell lines. Levels of TNF-α and IFN-γ were quantified by ELISA after 48 h at an E:T ratio of 1:1. Data are presented as mean ± standard deviation (SD) from three independent experiments using CAR-T cells derived from donors 1, 2, and 3. (F) Cytolytic activity of control T and donor 1-derived CAR-T cells against various cancer cell lines. Cells were co-cultured for 3 d and their viability was assessed using crystal violet staining. Absorbance values were normalized to untreated wells to calculate relative cancer cell survival. Data are presented as mean ± SD of three independent experiments performed in triplicate. ns, not significant; ** P < 0.01; *** P < 0.001 (two-way ANOVA).

Article Snippet: The cells were then incubated for 16–18 h at 4°C with mouse anti-human CD44v6 (1:500; eBioscience) and rat anti-human CD3 (1:500; Abcam, Cambridge, UK) antibodies.

Techniques: Functional Assay, Selection, Flow Cytometry, Derivative Assay, Co-Culture Assay, Incubation, Staining, Immunofluorescence, Binding Assay, Cell Culture, Labeling, Activation Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Activity Assay, Control

Journal: Frontiers in Immunology

Article Title: Relaxin-2-secreting CAR-T cells exhibit enhanced efficacy in stromal-rich xenograft tumors

doi: 10.3389/fimmu.2025.1506204

Figure Lengend Snippet: Development of CD44v6 CAR-T cells and engineering for RLN2 secretion. (A) Schematic illustration of plasmid constructs used for CAR-T cell generation, including CD44v6 CAR and co-expression of either Luc2 or human RLN2 via transposon-based vectors. (B) RLN2 protein levels secreted by conventional and RLN2-secreting CAR-T cells quantified using ELISA. Data are presented as mean ± SD from three independent experiments performed in triplicate using CAR-T cells derived from donors 1, 2, and 3. (C) Western blot analysis of the RLN2 receptor LGR7/RXFP1 in multiple cancer cell lines. GAPDH was used as a loading control. (D) Expression of MMPs in cancer cell lines cultured for 48 h in serum-free medium (SF), 1% fetal bovine serum (FBS)-containing medium (1% CM), supernatant from CAR-T cells (CAR-T sup), or supernatant from RLN2-secreting CAR-T cells (RLN2 CAR-T sup), collected after 24 h in SF. RT-qPCR was conducted in three independent runs using CAR-T cells from donors 1, 2, and 3, in triplicate (mean ± SD). * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant (one-way ANOVA followed by Bonferroni’s post hoc test and unpaired t -test for comparisons between CAR-T and RLN2-secreting CAR-T groups). (E) Gelatin zymography of conditioned media from SU86.86 cells cultured in SF, complete medium with 10% FBS (CM), or undiluted (×1) or 5-fold diluted (×1/5) supernatants from CAR-T or RLN2 CAR-T cells. (F) Western blot analysis of LGR7/RXFP1 expression in conventional and RLN2-secreting CAR-T cells. MMP expression in imhPSCs treated with the indicated supernatants (evaluated as described in D ). (G) Western blot analysis of α-SMA and collagen type I alpha 1 (COL1A1) in imhPSCs cultured for 48 h in SF, SF + TGF-β (10 ng/mL), CAR-T sup ± TGF-β, or RLN2 CAR-T sup ± TGF-β. GAPDH was used as a loading control. Supernatants were derived from donor 1-generated CAR-T cells. (H) MMP expression in CAR-T cells. Conventional and RLN2-secreting CAR-T cells were cultured in RPMI-1640 with 10% FBS under resting conditions or with stimulation (anti-CD3/CD28 antibodies + IL-15 and IL-21) for 3 d RT-qPCR was performed using cells from donors 1, 2, and 3 (mean ± SD; triplicate experiments). * P < 0.05; *** P < 0.001; ns, not significant (comparisons between CAR-T and RLN2 CAR-T cells analyzed using unpaired t -test).

Article Snippet: The cells were then incubated for 16–18 h at 4°C with mouse anti-human CD44v6 (1:500; eBioscience) and rat anti-human CD3 (1:500; Abcam, Cambridge, UK) antibodies.

Techniques: Plasmid Preparation, Construct, Expressing, Enzyme-linked Immunosorbent Assay, Derivative Assay, Western Blot, Control, Cell Culture, Quantitative RT-PCR, Zymography, Generated

Journal: Frontiers in Immunology

Article Title: Relaxin-2-secreting CAR-T cells exhibit enhanced efficacy in stromal-rich xenograft tumors

doi: 10.3389/fimmu.2025.1506204

Figure Lengend Snippet: Histological characterization of subcutaneous xenograft tumor models. (A) Histological analysis of Panc-1, AsPC-1-CD44v6, and SU86.86 xenograft tumors. Tumor sections were analyzed for CD44v6 expression and stromal architecture. Formalin-fixed paraffin-embedded samples were subjected to H&E staining for tissue morphology, Picro-Sirius Red staining for collagen types I and III, and immunohistochemistry for CD44v6 and collagen type IV analysis. Scale bar = 200 µm. (B) Immunofluorescence staining of vascular and stromal markers in Panc-1 and SU86.86 subcutaneous xenograft tumors. Endothelial cells were labeled with anti-CD31 (green), myofibroblasts and pericytes with anti-α-SMA (red), and nuclei with DAPI (blue). Scale bar = 100 µm.

Article Snippet: The cells were then incubated for 16–18 h at 4°C with mouse anti-human CD44v6 (1:500; eBioscience) and rat anti-human CD3 (1:500; Abcam, Cambridge, UK) antibodies.

Techniques: Expressing, Formalin-fixed Paraffin-Embedded, Staining, Immunohistochemistry, Immunofluorescence, Labeling

Journal: Frontiers in Immunology

Article Title: Relaxin-2-secreting CAR-T cells exhibit enhanced efficacy in stromal-rich xenograft tumors

doi: 10.3389/fimmu.2025.1506204

Figure Lengend Snippet: Efficacy of conventional CD44v6 CAR-T cell therapy in xenograft models with either scant or abundant stroma. (A–C) In vitro cytotoxicity (left) and in vivo antitumor activity (right) of donor 1-derived CD44v6 CAR-T cells against Panc-1 (A) , AsPC-1-CD44v6 (B) , and SU86.86 (C) tumor models. For in vitro assays, CAR-T cells were co-cultured with the indicated cancer cell lines for 3 d, and surviving cancer cells were quantified using a crystal violet assay. Absorbance at 590 nm (A 590 ) was measured and normalized to untreated controls. Data are presented as mean ± SD from three independent experiments conducted in triplicate. ns, not significant; *** P < 0.001 (two-way ANOVA). For in vivo studies, tumor-bearing mice received a single intravenous injection of either Ctrl-T ( n = 6) or CAR-T cells ( n = 6). Data are presented as mean ± SD. *** P < 0.001; ns, not significant (two-way ANOVA). (D) In vivo bioluminescence imaging of Luc2-expressing CAR-T cell accumulation in subcutaneous xenograft tumors. A total of 1 × 10 7 Luc2-expressing CAR-T cells were injected intravenously via the tail vein. Bioluminescence was measured using the IVIS imaging system on days 7 and 14 post-injection. (E) Immunofluorescence analysis of CAR-T cell infiltration in AsPC-1-CD44v6 and SU86.86 xenograft tumors 7 d after systemic injection of Luc2-expressing CAR-T cells. Tumor sections were stained for CD3 (green), CD44v6 (red), and CD31 (blue). Scale bar = 100 µm.

Article Snippet: The cells were then incubated for 16–18 h at 4°C with mouse anti-human CD44v6 (1:500; eBioscience) and rat anti-human CD3 (1:500; Abcam, Cambridge, UK) antibodies.

Techniques: In Vitro, In Vivo, Activity Assay, Derivative Assay, Cell Culture, Crystal Violet Assay, Injection, Imaging, Expressing, Immunofluorescence, Staining

Journal: Frontiers in Immunology

Article Title: Relaxin-2-secreting CAR-T cells exhibit enhanced efficacy in stromal-rich xenograft tumors

doi: 10.3389/fimmu.2025.1506204

Figure Lengend Snippet: Antitumor effects of RLN2-secreting CD44v6 CAR-T cell therapy in stromal-rich SU86.86 xenograft tumors. (A) Tumor growth in SU86.86 subcutaneous xenografts after single intravenous injection of Ctrl-T (1 × 10 7 cells, n = 6), conventional CD44v6 CAR-T (1 × 10 7 cells, n = 6), or RLN2-secreting CD44v6 CAR-T cells (1 × 10 7 cells, n = 6). Tumor size was measured over time, and representative images of resected tumors were captured following euthanasia. Data are presented as mean ± SD. ns, not significant; *** P < 0.001 (two-way ANOVA with Tukey’s multiple comparisons test). (B) Histological analysis of SU86.86 xenograft tumors collected 7 d after treatment with conventional or RLN2-secreting CAR-T cells. Formalin-fixed paraffin-embedded sections were subjected to H&E staining, Picro-Sirius Red staining (collagen types I and III), and immunohistochemistry. Immunofluorescence analysis of tumor-infiltrating CAR-T cells was performed on frozen sections. Tumor sections were stained for CD44v6 (red), CD3 (green), and CD31 (blue). Scale bar = 100 µm. (C) Quantitative analysis of MMP expression in SU86.86 xenograft tumors treated with conventional or RLN2-secreting CAR-T cells and harvested at either 3 or 7 d after single treatment with 1 × 10 7 CAR-T cells. MMP-7 and MMP-9 mRNA levels were assessed using RT-qPCR. CAR-T group ( n = 9, day 3; n = 5, day 7), RLN2 CAR-T group ( n = 7, day 3; n = 7, day 7). Data from two independent experiments are shown as mean ± SD. ns, not significant; * P < 0.05; *** P < 0.001; ns, not significant (unpaired t -test).

Article Snippet: The cells were then incubated for 16–18 h at 4°C with mouse anti-human CD44v6 (1:500; eBioscience) and rat anti-human CD3 (1:500; Abcam, Cambridge, UK) antibodies.

Techniques: Injection, Formalin-fixed Paraffin-Embedded, Staining, Immunohistochemistry, Immunofluorescence, Expressing, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Relaxin-2-secreting CAR-T cells exhibit enhanced efficacy in stromal-rich xenograft tumors

doi: 10.3389/fimmu.2025.1506204

Figure Lengend Snippet: Phenotypic and functional characterization of CD44v6 chimeric antigen receptor (CAR)-T cells. (A) Memory/effector status of CAR-T cells before and after puromycin selection. Flow cytometry was performed using antibodies against the Fc gamma region, which binds to the hinge domain of the CAR expressed on both CD4 + and CD8 + T cells. Representative scatter plots are shown for donor 1-derived CAR-T cells. (B) Memory/effector subset analysis of CAR-T cells following co-culture with or without the indicated cell lines, followed by incubation in cytokine-depleted medium for 3 d Cells were stained with CD62L and CD45RO and gated on CD45 + CD3 + CAR-T cells for flow cytometric analysis. Representative data from donor 1-derived CAR-T cells are shown. (C) Immunofluorescence analysis of antigen-dependent binding of CAR-T cells. Donor 1-derived CAR-T cells were co-cultured for 3 h with Panc-1 (CD44v6-positive) or NIH/3T3 (CD44v6-negative) cells, then stained with anti-CD3 antibody. Fluorescent labeling: CD3 (green), CD44v6 (red), and nuclei (DAPI, blue). (D) Antigen-dependent activation and degranulation of CAR-T cells. Cells were co-cultured for 24 h with or without target cell lines in cytokine-depleted medium in the presence of CD107a antibody and the Golgi transport inhibitor Monensin. Activation and degranulation were assessed using CD69 and CD8a staining. Representative flow cytometry plots are shown. (E) Cytokine secretion by CAR-T cells following co-culture with the indicated cell lines. Levels of TNF-α and IFN-γ were quantified by ELISA after 48 h at an E:T ratio of 1:1. Data are presented as mean ± standard deviation (SD) from three independent experiments using CAR-T cells derived from donors 1, 2, and 3. (F) Cytolytic activity of control T and donor 1-derived CAR-T cells against various cancer cell lines. Cells were co-cultured for 3 d and their viability was assessed using crystal violet staining. Absorbance values were normalized to untreated wells to calculate relative cancer cell survival. Data are presented as mean ± SD of three independent experiments performed in triplicate. ns, not significant; ** P < 0.01; *** P < 0.001 (two-way ANOVA).

Article Snippet: The sections were washed three times with PBS-T (0.1% Triton X-100 in PBS), blocked with normal horse serum for 20 min at 25°C and incubated overnight at 4°C with rabbit anti-human CD3 antibody (1:100; ab11089, Abcam), mouse anti-human CD44v6 (1:200; BMS125, eBioscience), and rabbit anti-mouse CD31 antibody (1:200; AF3628, R&D).

Techniques: Functional Assay, Selection, Flow Cytometry, Derivative Assay, Co-Culture Assay, Incubation, Staining, Immunofluorescence, Binding Assay, Cell Culture, Labeling, Activation Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Activity Assay, Control

Journal: Frontiers in Immunology

Article Title: Relaxin-2-secreting CAR-T cells exhibit enhanced efficacy in stromal-rich xenograft tumors

doi: 10.3389/fimmu.2025.1506204

Figure Lengend Snippet: Development of CD44v6 CAR-T cells and engineering for RLN2 secretion. (A) Schematic illustration of plasmid constructs used for CAR-T cell generation, including CD44v6 CAR and co-expression of either Luc2 or human RLN2 via transposon-based vectors. (B) RLN2 protein levels secreted by conventional and RLN2-secreting CAR-T cells quantified using ELISA. Data are presented as mean ± SD from three independent experiments performed in triplicate using CAR-T cells derived from donors 1, 2, and 3. (C) Western blot analysis of the RLN2 receptor LGR7/RXFP1 in multiple cancer cell lines. GAPDH was used as a loading control. (D) Expression of MMPs in cancer cell lines cultured for 48 h in serum-free medium (SF), 1% fetal bovine serum (FBS)-containing medium (1% CM), supernatant from CAR-T cells (CAR-T sup), or supernatant from RLN2-secreting CAR-T cells (RLN2 CAR-T sup), collected after 24 h in SF. RT-qPCR was conducted in three independent runs using CAR-T cells from donors 1, 2, and 3, in triplicate (mean ± SD). * P < 0.05; ** P < 0.01; *** P < 0.001; ns, not significant (one-way ANOVA followed by Bonferroni’s post hoc test and unpaired t -test for comparisons between CAR-T and RLN2-secreting CAR-T groups). (E) Gelatin zymography of conditioned media from SU86.86 cells cultured in SF, complete medium with 10% FBS (CM), or undiluted (×1) or 5-fold diluted (×1/5) supernatants from CAR-T or RLN2 CAR-T cells. (F) Western blot analysis of LGR7/RXFP1 expression in conventional and RLN2-secreting CAR-T cells. MMP expression in imhPSCs treated with the indicated supernatants (evaluated as described in D ). (G) Western blot analysis of α-SMA and collagen type I alpha 1 (COL1A1) in imhPSCs cultured for 48 h in SF, SF + TGF-β (10 ng/mL), CAR-T sup ± TGF-β, or RLN2 CAR-T sup ± TGF-β. GAPDH was used as a loading control. Supernatants were derived from donor 1-generated CAR-T cells. (H) MMP expression in CAR-T cells. Conventional and RLN2-secreting CAR-T cells were cultured in RPMI-1640 with 10% FBS under resting conditions or with stimulation (anti-CD3/CD28 antibodies + IL-15 and IL-21) for 3 d RT-qPCR was performed using cells from donors 1, 2, and 3 (mean ± SD; triplicate experiments). * P < 0.05; *** P < 0.001; ns, not significant (comparisons between CAR-T and RLN2 CAR-T cells analyzed using unpaired t -test).

Article Snippet: The sections were washed three times with PBS-T (0.1% Triton X-100 in PBS), blocked with normal horse serum for 20 min at 25°C and incubated overnight at 4°C with rabbit anti-human CD3 antibody (1:100; ab11089, Abcam), mouse anti-human CD44v6 (1:200; BMS125, eBioscience), and rabbit anti-mouse CD31 antibody (1:200; AF3628, R&D).

Techniques: Plasmid Preparation, Construct, Expressing, Enzyme-linked Immunosorbent Assay, Derivative Assay, Western Blot, Control, Cell Culture, Quantitative RT-PCR, Zymography, Generated

Journal: Frontiers in Immunology

Article Title: Relaxin-2-secreting CAR-T cells exhibit enhanced efficacy in stromal-rich xenograft tumors

doi: 10.3389/fimmu.2025.1506204

Figure Lengend Snippet: Histological characterization of subcutaneous xenograft tumor models. (A) Histological analysis of Panc-1, AsPC-1-CD44v6, and SU86.86 xenograft tumors. Tumor sections were analyzed for CD44v6 expression and stromal architecture. Formalin-fixed paraffin-embedded samples were subjected to H&E staining for tissue morphology, Picro-Sirius Red staining for collagen types I and III, and immunohistochemistry for CD44v6 and collagen type IV analysis. Scale bar = 200 µm. (B) Immunofluorescence staining of vascular and stromal markers in Panc-1 and SU86.86 subcutaneous xenograft tumors. Endothelial cells were labeled with anti-CD31 (green), myofibroblasts and pericytes with anti-α-SMA (red), and nuclei with DAPI (blue). Scale bar = 100 µm.

Article Snippet: The sections were washed three times with PBS-T (0.1% Triton X-100 in PBS), blocked with normal horse serum for 20 min at 25°C and incubated overnight at 4°C with rabbit anti-human CD3 antibody (1:100; ab11089, Abcam), mouse anti-human CD44v6 (1:200; BMS125, eBioscience), and rabbit anti-mouse CD31 antibody (1:200; AF3628, R&D).

Techniques: Expressing, Formalin-fixed Paraffin-Embedded, Staining, Immunohistochemistry, Immunofluorescence, Labeling

Journal: Frontiers in Immunology

Article Title: Relaxin-2-secreting CAR-T cells exhibit enhanced efficacy in stromal-rich xenograft tumors

doi: 10.3389/fimmu.2025.1506204

Figure Lengend Snippet: Efficacy of conventional CD44v6 CAR-T cell therapy in xenograft models with either scant or abundant stroma. (A–C) In vitro cytotoxicity (left) and in vivo antitumor activity (right) of donor 1-derived CD44v6 CAR-T cells against Panc-1 (A) , AsPC-1-CD44v6 (B) , and SU86.86 (C) tumor models. For in vitro assays, CAR-T cells were co-cultured with the indicated cancer cell lines for 3 d, and surviving cancer cells were quantified using a crystal violet assay. Absorbance at 590 nm (A 590 ) was measured and normalized to untreated controls. Data are presented as mean ± SD from three independent experiments conducted in triplicate. ns, not significant; *** P < 0.001 (two-way ANOVA). For in vivo studies, tumor-bearing mice received a single intravenous injection of either Ctrl-T ( n = 6) or CAR-T cells ( n = 6). Data are presented as mean ± SD. *** P < 0.001; ns, not significant (two-way ANOVA). (D) In vivo bioluminescence imaging of Luc2-expressing CAR-T cell accumulation in subcutaneous xenograft tumors. A total of 1 × 10 7 Luc2-expressing CAR-T cells were injected intravenously via the tail vein. Bioluminescence was measured using the IVIS imaging system on days 7 and 14 post-injection. (E) Immunofluorescence analysis of CAR-T cell infiltration in AsPC-1-CD44v6 and SU86.86 xenograft tumors 7 d after systemic injection of Luc2-expressing CAR-T cells. Tumor sections were stained for CD3 (green), CD44v6 (red), and CD31 (blue). Scale bar = 100 µm.

Article Snippet: The sections were washed three times with PBS-T (0.1% Triton X-100 in PBS), blocked with normal horse serum for 20 min at 25°C and incubated overnight at 4°C with rabbit anti-human CD3 antibody (1:100; ab11089, Abcam), mouse anti-human CD44v6 (1:200; BMS125, eBioscience), and rabbit anti-mouse CD31 antibody (1:200; AF3628, R&D).

Techniques: In Vitro, In Vivo, Activity Assay, Derivative Assay, Cell Culture, Crystal Violet Assay, Injection, Imaging, Expressing, Immunofluorescence, Staining

Journal: Frontiers in Immunology

Article Title: Relaxin-2-secreting CAR-T cells exhibit enhanced efficacy in stromal-rich xenograft tumors

doi: 10.3389/fimmu.2025.1506204

Figure Lengend Snippet: Antitumor effects of RLN2-secreting CD44v6 CAR-T cell therapy in stromal-rich SU86.86 xenograft tumors. (A) Tumor growth in SU86.86 subcutaneous xenografts after single intravenous injection of Ctrl-T (1 × 10 7 cells, n = 6), conventional CD44v6 CAR-T (1 × 10 7 cells, n = 6), or RLN2-secreting CD44v6 CAR-T cells (1 × 10 7 cells, n = 6). Tumor size was measured over time, and representative images of resected tumors were captured following euthanasia. Data are presented as mean ± SD. ns, not significant; *** P < 0.001 (two-way ANOVA with Tukey’s multiple comparisons test). (B) Histological analysis of SU86.86 xenograft tumors collected 7 d after treatment with conventional or RLN2-secreting CAR-T cells. Formalin-fixed paraffin-embedded sections were subjected to H&E staining, Picro-Sirius Red staining (collagen types I and III), and immunohistochemistry. Immunofluorescence analysis of tumor-infiltrating CAR-T cells was performed on frozen sections. Tumor sections were stained for CD44v6 (red), CD3 (green), and CD31 (blue). Scale bar = 100 µm. (C) Quantitative analysis of MMP expression in SU86.86 xenograft tumors treated with conventional or RLN2-secreting CAR-T cells and harvested at either 3 or 7 d after single treatment with 1 × 10 7 CAR-T cells. MMP-7 and MMP-9 mRNA levels were assessed using RT-qPCR. CAR-T group ( n = 9, day 3; n = 5, day 7), RLN2 CAR-T group ( n = 7, day 3; n = 7, day 7). Data from two independent experiments are shown as mean ± SD. ns, not significant; * P < 0.05; *** P < 0.001; ns, not significant (unpaired t -test).

Article Snippet: The sections were washed three times with PBS-T (0.1% Triton X-100 in PBS), blocked with normal horse serum for 20 min at 25°C and incubated overnight at 4°C with rabbit anti-human CD3 antibody (1:100; ab11089, Abcam), mouse anti-human CD44v6 (1:200; BMS125, eBioscience), and rabbit anti-mouse CD31 antibody (1:200; AF3628, R&D).

Techniques: Injection, Formalin-fixed Paraffin-Embedded, Staining, Immunohistochemistry, Immunofluorescence, Expressing, Quantitative RT-PCR