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mouse anti-human fitc-conjugated cd44v6 antibody (clone vff-18)  (Thermo Fisher)


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    Thermo Fisher mouse anti-human fitc-conjugated cd44v6 antibody (clone vff-18)
    Phenotypic and functional characterization of <t>CD44v6</t> 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).
    Mouse Anti Human Fitc Conjugated Cd44v6 Antibody (Clone Vff 18), supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mouse+anti-human+cd44v6/anti+cd44v6/pmc12259638-61-13-20
    Average 90 stars, based on 1 article reviews
    mouse anti-human fitc-conjugated cd44v6 antibody (clone vff-18) - by Bioz Stars, 2026-09
    90/100 stars

    Images

    1) Product Images from "Relaxin-2-secreting CAR-T cells exhibit enhanced efficacy in stromal-rich xenograft tumors"

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

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2025.1506204

    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).
    Figure Legend 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).

    Techniques Used: 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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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).
    Figure Legend 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).

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

    Related Articles

    Incubation:

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

    Functional Assay:

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

    Selection:

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

    Flow Cytometry:

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

    Derivative Assay:

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

    Co-Culture Assay:

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

    Staining:

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

    Immunofluorescence:

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

    Binding Assay:

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

    Cell Culture:

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

    Labeling:

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

    Activation Assay:

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

    Enzyme-linked Immunosorbent Assay:

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

    Standard Deviation:

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

    Activity Assay:

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

    Control:

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

    Plasmid Preparation:

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

    Construct:

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

    Expressing:

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

    Western Blot:

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

    Quantitative RT-PCR:

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

    Zymography:

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

    Generated:

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

    Formalin-fixed Paraffin-Embedded:

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

    Immunohistochemistry:

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

    In Vitro:

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

    In Vivo:

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

    Crystal Violet Assay:

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

    Injection:

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

    Imaging:

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



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    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).

    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

    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).

    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

    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.

    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

    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.

    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

    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).

    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

    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).

    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

    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).

    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

    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.

    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

    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.

    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

    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).

    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

    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).

    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

    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).

    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

    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.

    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

    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.

    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

    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).

    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

    Screening of a phage peptide library and phage cell-binding ELISA to identify CD44v6-binding peptides. (A) Experimental schemes for phage peptide library screening. (B) Immunofluorescence staining of CD44v6 with an anti-CD44v6 antibody (red) in HEK 293T cells transfected or not with a GFP-tagged CD44v6 expression vector (green). DAPI was used for nuclear staining (blue). Scale bars = 40 µm. (C) Western blotting analysis of CD44v6 expression in non-transfected (-) and transfected HEK 293T cells at 24 and 48 h after transfection. (D) Western blotting analysis of CD44v6 expression in tumor cells. (E) Enrichment of phage titers during screening rounds. After each round, the phage titers (plaque-forming units; pfu) were measured by plaque assays. Numbers represent the fold ratios relative to the first round. (F) The phage cell-binding ELISA of individual phage clones was performed using HEK 293T cells transfected or not with a CD44v6 expression vector, MDA-MB231 cells, and MCF7 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s, not significant by one-way ANOVA.

    Journal: Theranostics

    Article Title: Identification of novel CD44v6-binding peptides that block CD44v6 and deliver a pro-apoptotic peptide to tumors to inhibit tumor growth and metastasis in mice

    doi: 10.7150/thno.50564

    Figure Lengend Snippet: Screening of a phage peptide library and phage cell-binding ELISA to identify CD44v6-binding peptides. (A) Experimental schemes for phage peptide library screening. (B) Immunofluorescence staining of CD44v6 with an anti-CD44v6 antibody (red) in HEK 293T cells transfected or not with a GFP-tagged CD44v6 expression vector (green). DAPI was used for nuclear staining (blue). Scale bars = 40 µm. (C) Western blotting analysis of CD44v6 expression in non-transfected (-) and transfected HEK 293T cells at 24 and 48 h after transfection. (D) Western blotting analysis of CD44v6 expression in tumor cells. (E) Enrichment of phage titers during screening rounds. After each round, the phage titers (plaque-forming units; pfu) were measured by plaque assays. Numbers represent the fold ratios relative to the first round. (F) The phage cell-binding ELISA of individual phage clones was performed using HEK 293T cells transfected or not with a CD44v6 expression vector, MDA-MB231 cells, and MCF7 cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s, not significant by one-way ANOVA.

    Article Snippet: Tissue sections (8-μm thick) were prepared and then stained with an anti-human CD44v6 mouse monoclonal antibody (clone no. VFF-7, 1:50 dilution; Santa Cruz Biotechnology) and subsequently with an Alexa Fluor 594-labeled goat anti-mouse IgG antibody.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Library Screening, Immunofluorescence, Staining, Transfection, Expressing, Plasmid Preparation, Western Blot, Clone Assay

    NLN and NEW bind selectively to CD44v6-overexpressing cells and the CD44v6 protein . (A) Cellular binding of FITC-labeled NLN and NEW (green, 25 µM) and staining of CD44v6 (red) in CD44v6-high MDA-MB231, 4T1, and Panc-1 cells and CD44v6-low MCF7 cells. Nuclei were stained with DAPI (blue). Scale bars = 20 µm. (B) Mean fluorescence intensities (MFIs) of FITC-labeled NLN or NEW (25 µM) bound to MDA-MB231, 4T1, Panc-1 and MCF7 cells. Data represent the mean MFIs ± standard errors (S.E.) of peptide-bound cells from three separate experiments. **, P < 0.01; n.s, not significant compared with the control peptide (Ctrl) by one-way ANOVA. (C) Cellular binding of TAMRA-labeled NLN and NEW (red, 25 µM) and expression of CD44v6 (green) in HEK 293T cells transfected or not with a GFP-tagged CD44v6 expression vector. Nuclei were stained with DAPI (blue). Scale bars = 20 µm. (D-E) SPR analysis of the binding affinity (K D value) of NLN and NEW to recombinant CD44v6-Fc (D) and CD44-Fc (E) proteins. Inset are a binding plot for the peptides. RU, resonance unit.

    Journal: Theranostics

    Article Title: Identification of novel CD44v6-binding peptides that block CD44v6 and deliver a pro-apoptotic peptide to tumors to inhibit tumor growth and metastasis in mice

    doi: 10.7150/thno.50564

    Figure Lengend Snippet: NLN and NEW bind selectively to CD44v6-overexpressing cells and the CD44v6 protein . (A) Cellular binding of FITC-labeled NLN and NEW (green, 25 µM) and staining of CD44v6 (red) in CD44v6-high MDA-MB231, 4T1, and Panc-1 cells and CD44v6-low MCF7 cells. Nuclei were stained with DAPI (blue). Scale bars = 20 µm. (B) Mean fluorescence intensities (MFIs) of FITC-labeled NLN or NEW (25 µM) bound to MDA-MB231, 4T1, Panc-1 and MCF7 cells. Data represent the mean MFIs ± standard errors (S.E.) of peptide-bound cells from three separate experiments. **, P < 0.01; n.s, not significant compared with the control peptide (Ctrl) by one-way ANOVA. (C) Cellular binding of TAMRA-labeled NLN and NEW (red, 25 µM) and expression of CD44v6 (green) in HEK 293T cells transfected or not with a GFP-tagged CD44v6 expression vector. Nuclei were stained with DAPI (blue). Scale bars = 20 µm. (D-E) SPR analysis of the binding affinity (K D value) of NLN and NEW to recombinant CD44v6-Fc (D) and CD44-Fc (E) proteins. Inset are a binding plot for the peptides. RU, resonance unit.

    Article Snippet: Tissue sections (8-μm thick) were prepared and then stained with an anti-human CD44v6 mouse monoclonal antibody (clone no. VFF-7, 1:50 dilution; Santa Cruz Biotechnology) and subsequently with an Alexa Fluor 594-labeled goat anti-mouse IgG antibody.

    Techniques: Binding Assay, Labeling, Staining, Fluorescence, Expressing, Transfection, Plasmid Preparation, Recombinant

    Cellular binding of NLN and NEW is mediated by CD44v6. (A) Western blotting analysis of CD44v6 and CD44 levels in MDA-MB231 cells after the knockdown (KD) of CD44v6 gene expression for 24, 48, and 72 h in cells treated with CD44v6 siRNA or control siRNA. GAPDH is used as a control protein. (B) The MFI of wild-type (WT) and CD44v6 KD MDA-MB231 cells bound to FITC-labeled NLN and NEW (25 µM). Data are shown as mean MFIs ± S.E. of peptide-bound cells from three separate experiments. *, P < 0.05 by Student's t -test. (C) Confocal microscopic images of WT and KD MDA-MB231 cells bound with FITC-labeled NLN and NEW (green, 25 µM) and stained with an anti-CD44v6 antibody (red) and DAPI (blue). Scale bars = 20 µm. (D) Competitive binding of FITC-labeled NLN and NEW (10 µM) following pre-treatment with anti-CD44v6 and anti-CD44 antibodies and IgG control in MDA-MB231 cells. The mean MFIs ± S.E. of peptide-bound cells from three separate experiments are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s, not significant by one-way ANOVA. (E) Competitive binding of an anti-CD44v6 antibody following pre-treatment with NLN and NEW (50 µM) in MDA-MB231 cells. ***, P < 0.001; n.s, not significant compared with untreated control by one-way ANOVA. (F) Pull-down assay of CD44v6 using biotin-labeled NLN and NEW and streptavidin beads, followed by immunoblotting with anti-CD44v6, anti-c-Met, and anti-CD44 antibodies. (G) Pull-down assays of the recombinant human CD44v6-Fc protein using biotin-labeled NLN and NEW and streptavidin followed by immunoblotting (IB) with anti-CD44v6 antibody. Ctrl, control peptide.

    Journal: Theranostics

    Article Title: Identification of novel CD44v6-binding peptides that block CD44v6 and deliver a pro-apoptotic peptide to tumors to inhibit tumor growth and metastasis in mice

    doi: 10.7150/thno.50564

    Figure Lengend Snippet: Cellular binding of NLN and NEW is mediated by CD44v6. (A) Western blotting analysis of CD44v6 and CD44 levels in MDA-MB231 cells after the knockdown (KD) of CD44v6 gene expression for 24, 48, and 72 h in cells treated with CD44v6 siRNA or control siRNA. GAPDH is used as a control protein. (B) The MFI of wild-type (WT) and CD44v6 KD MDA-MB231 cells bound to FITC-labeled NLN and NEW (25 µM). Data are shown as mean MFIs ± S.E. of peptide-bound cells from three separate experiments. *, P < 0.05 by Student's t -test. (C) Confocal microscopic images of WT and KD MDA-MB231 cells bound with FITC-labeled NLN and NEW (green, 25 µM) and stained with an anti-CD44v6 antibody (red) and DAPI (blue). Scale bars = 20 µm. (D) Competitive binding of FITC-labeled NLN and NEW (10 µM) following pre-treatment with anti-CD44v6 and anti-CD44 antibodies and IgG control in MDA-MB231 cells. The mean MFIs ± S.E. of peptide-bound cells from three separate experiments are shown. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s, not significant by one-way ANOVA. (E) Competitive binding of an anti-CD44v6 antibody following pre-treatment with NLN and NEW (50 µM) in MDA-MB231 cells. ***, P < 0.001; n.s, not significant compared with untreated control by one-way ANOVA. (F) Pull-down assay of CD44v6 using biotin-labeled NLN and NEW and streptavidin beads, followed by immunoblotting with anti-CD44v6, anti-c-Met, and anti-CD44 antibodies. (G) Pull-down assays of the recombinant human CD44v6-Fc protein using biotin-labeled NLN and NEW and streptavidin followed by immunoblotting (IB) with anti-CD44v6 antibody. Ctrl, control peptide.

    Article Snippet: Tissue sections (8-μm thick) were prepared and then stained with an anti-human CD44v6 mouse monoclonal antibody (clone no. VFF-7, 1:50 dilution; Santa Cruz Biotechnology) and subsequently with an Alexa Fluor 594-labeled goat anti-mouse IgG antibody.

    Techniques: Binding Assay, Western Blot, Expressing, Labeling, Staining, Pull Down Assay, Recombinant

    NLN and NEW inhibit HGF-induced c-Met internalization, c-Met phosphorylation, and cell migration and invasion in MDA-MB231 breast tumor cells. (A) A confocal microscopic Z-section analysis of the internalization of NLN and NEW, and CD44v6 into MDA-MB231 cells. Cells were incubated with FITC-labeled peptides (green, 10 µM) at 37 °C for 10, 30, and 60 min and stained with an anti-CD44v6 antibody (red). Nuclei were stained with DAPI (blue), and images were merged. Numbers in the merged images indicate the distances from the cell surfaces in µm. Scale bars = 10 µm. (B) Confocal microscopic analysis of c-Met (red) in MDA-MB231 cells after pre-treatment with NLN and NEW (10 µM) at 37 °C for 10 min and subsequent treatment with 25 ng/mL HGF for 30 or 60 min. Arrows indicate cytoplasmic c-Met. Nuclei were stained with DAPI (blue). Scale bars = 10 µm. (C) Western blotting analysis of c-Met and Erk phosphorylation in MDA-MB231 cells pre-treated or not with NLN and NEW (20 µM) for 10 min and subsequently treated with 25 ng/mL HGF for 10 min. (D) Phosphorylated c-Met and Erk protein levels normalized by c-Met and Erk total protein levels in MDA-MB231 cells. (E-F) Transwell migration (E) and invasion (F) assays of MDA-MB231 cells pre-treated or not with NLN and NEW (20 µM) for 10 min and subsequently treated with 25 ng/mL HGF for 10 min, followed by incubation for 24 h. Scale bars = 20 µm. Graphs (right panels) represent the quantification of the cell numbers in ten randomly selected fields. Data are shown as the means ± S.E. of three independent experiment. ***, P < 0.001; n.s, not significant compared with HGF by one-way ANOVA.

    Journal: Theranostics

    Article Title: Identification of novel CD44v6-binding peptides that block CD44v6 and deliver a pro-apoptotic peptide to tumors to inhibit tumor growth and metastasis in mice

    doi: 10.7150/thno.50564

    Figure Lengend Snippet: NLN and NEW inhibit HGF-induced c-Met internalization, c-Met phosphorylation, and cell migration and invasion in MDA-MB231 breast tumor cells. (A) A confocal microscopic Z-section analysis of the internalization of NLN and NEW, and CD44v6 into MDA-MB231 cells. Cells were incubated with FITC-labeled peptides (green, 10 µM) at 37 °C for 10, 30, and 60 min and stained with an anti-CD44v6 antibody (red). Nuclei were stained with DAPI (blue), and images were merged. Numbers in the merged images indicate the distances from the cell surfaces in µm. Scale bars = 10 µm. (B) Confocal microscopic analysis of c-Met (red) in MDA-MB231 cells after pre-treatment with NLN and NEW (10 µM) at 37 °C for 10 min and subsequent treatment with 25 ng/mL HGF for 30 or 60 min. Arrows indicate cytoplasmic c-Met. Nuclei were stained with DAPI (blue). Scale bars = 10 µm. (C) Western blotting analysis of c-Met and Erk phosphorylation in MDA-MB231 cells pre-treated or not with NLN and NEW (20 µM) for 10 min and subsequently treated with 25 ng/mL HGF for 10 min. (D) Phosphorylated c-Met and Erk protein levels normalized by c-Met and Erk total protein levels in MDA-MB231 cells. (E-F) Transwell migration (E) and invasion (F) assays of MDA-MB231 cells pre-treated or not with NLN and NEW (20 µM) for 10 min and subsequently treated with 25 ng/mL HGF for 10 min, followed by incubation for 24 h. Scale bars = 20 µm. Graphs (right panels) represent the quantification of the cell numbers in ten randomly selected fields. Data are shown as the means ± S.E. of three independent experiment. ***, P < 0.001; n.s, not significant compared with HGF by one-way ANOVA.

    Article Snippet: Tissue sections (8-μm thick) were prepared and then stained with an anti-human CD44v6 mouse monoclonal antibody (clone no. VFF-7, 1:50 dilution; Santa Cruz Biotechnology) and subsequently with an Alexa Fluor 594-labeled goat anti-mouse IgG antibody.

    Techniques: Migration, Incubation, Labeling, Staining, Western Blot

    In vivo whole-body fluorescence imaging of NLN and NEW homing to MDA-MB231 breast tumor in mice. (A) In vivo whole-body fluorescence imaging of the homing of Flamma 675 NIR dye-labeled NLN, NEW, or control peptide to tumors at 1-6 h after injection into BALB/c nude mice. Dotted circles represent the tumor region. The scale bar indicates the normalized fluorescent intensity. (B) Ex vivo imaging of the accumulation of Flamma 675 NIR dye-labeled NLN, NEW, or control peptide in the tumors and other organs isolated from mice 6 h after peptide injection. The scale bar indicates the normalized fluorescence intensity. T, tumor; H, heart; L, lung; Li, liver; S, spleen; K, kidney. (C) Quantification of the ex vivo fluorescence intensities in the tumor and organs. Data are shown as the means ± S.E. ( n = 3/group). **, P < 0.01; ***, P < 0.001 compared with the control peptide by one-way ANOVA. (D) Co-localization of CD44v6 (green) with NLN and NEW (red) in tumor tissue sections. Nuclei were counter-stained with DAPI (blue). Scale bars = 20 µm. Ctrl, control peptide.

    Journal: Theranostics

    Article Title: Identification of novel CD44v6-binding peptides that block CD44v6 and deliver a pro-apoptotic peptide to tumors to inhibit tumor growth and metastasis in mice

    doi: 10.7150/thno.50564

    Figure Lengend Snippet: In vivo whole-body fluorescence imaging of NLN and NEW homing to MDA-MB231 breast tumor in mice. (A) In vivo whole-body fluorescence imaging of the homing of Flamma 675 NIR dye-labeled NLN, NEW, or control peptide to tumors at 1-6 h after injection into BALB/c nude mice. Dotted circles represent the tumor region. The scale bar indicates the normalized fluorescent intensity. (B) Ex vivo imaging of the accumulation of Flamma 675 NIR dye-labeled NLN, NEW, or control peptide in the tumors and other organs isolated from mice 6 h after peptide injection. The scale bar indicates the normalized fluorescence intensity. T, tumor; H, heart; L, lung; Li, liver; S, spleen; K, kidney. (C) Quantification of the ex vivo fluorescence intensities in the tumor and organs. Data are shown as the means ± S.E. ( n = 3/group). **, P < 0.01; ***, P < 0.001 compared with the control peptide by one-way ANOVA. (D) Co-localization of CD44v6 (green) with NLN and NEW (red) in tumor tissue sections. Nuclei were counter-stained with DAPI (blue). Scale bars = 20 µm. Ctrl, control peptide.

    Article Snippet: Tissue sections (8-μm thick) were prepared and then stained with an anti-human CD44v6 mouse monoclonal antibody (clone no. VFF-7, 1:50 dilution; Santa Cruz Biotechnology) and subsequently with an Alexa Fluor 594-labeled goat anti-mouse IgG antibody.

    Techniques: In Vivo, Fluorescence, Imaging, Labeling, Injection, Ex Vivo, Isolation, Staining