Journal: Nature Communications

Article Title: Modulating the PPARγ pathway upregulates NECTIN4 and enhances chimeric antigen receptor (CAR) T cell therapy in bladder cancer

doi: 10.1038/s41467-025-62710-0

Figure Lengend Snippet: a Schematic of the lentiviral NECTIN4-CAR construct: signal peptide, Myc tag, scFv targeting NECTIN4, CAR T backbone construct containing an IgG4Fc(EQ) spacer, the CD28 transmembrane domain and the CD28 costimulatory domain with CD3ζ intracellular signaling domain, T2A self-cleavage peptide, GFP transduction marker. scFv single-chain variable fragment, VL variable light chain, VH variable heavy chain, tm transmembrane, GFP green fluorescent protein. Created with BioRender. Chang, K. (2025) https://BioRender.com/k3j6fgi . b Western blot analysis of NECTIN4 expression in whole cell lysates of RT112 parental cells and NECTIN4 knockout (KO) cells. This was repeated n = 3 independent times with similar results. c NECTIN4 surface staining in RT112 parental cells and NECTIN4 knockout (KO) cells. d, e Growth curves of ( d ) RT112 and ( e ) NECTIN4 KO target cells with fluorescently labeled nuclei (NucLightRed, NLR) co-cultured with NECTIN4-CAR T cells (left) or non-transduced (NTD) T cells (right) at indicated effector-to-target (E:T) cell ratios. f A kill index [1/area under the curve (AUC)] was calculated as a time-dependent measure of killing efficacy against the indicated tumor target cells at an E:T ratio of 1:2. n = 3 biological replicates. g IFNγ quantification by ELISA from co-cultures of NECTIN4-CAR or NTD T cells with the indicated RT112 cell lines in ( b ) at a 1:2 ratio at 24 h. n = 3 biological replicates. h Western blots demonstrating NECTIN4 protein expression across luminal and basal human urothelial carcinoma cell lines. This was repeated n = 3 independent times with similar results. i Growth curves of indicated cell lines co-cultured with NECTIN4-CAR T cells at an E:T ratio of 1:1. A representative experiment of n = 3 biologically independent experiments performed in technical triplicates is shown, and error bars represent mean ± SEM. j Scatter plot showing the NECTIN4-CAR T kill index against target cell lines in ( h ) versus respective expression of NECTIN4. Pearson’s correlation is shown for NECTIN4 expression versus kill index ( r = 0.96, P = 0.0097, two-tailed). For panels ( d-g ) n = 3 biological replicates per conditions were used and data are presented as mean ± SEM. For panels ( b, h ), GAPDH was used as a protein loading control. For panels ( f ) and ( g ), two-way ANOVA with Sidak’s multiple comparison test was used. Source data are provided as a Source Data file.

Article Snippet: Cells were then stained with a NECTIN4 antibody (Miltenyi, clone REA967, 1:100), sorted a second time by FACS (BD Fusion), and negative cells were collected and expanded.

Techniques: Construct, Transduction, Marker, Western Blot, Expressing, Knock-Out, Staining, Labeling, Cell Culture, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Control, Comparison

Journal: Nature Communications

Article Title: Modulating the PPARγ pathway upregulates NECTIN4 and enhances chimeric antigen receptor (CAR) T cell therapy in bladder cancer

doi: 10.1038/s41467-025-62710-0

Figure Lengend Snippet: a NECTIN4 mRNA levels in RT112 cells treated with 1μM rosiglitazone and T0070907 (T007) for 72 h. Data are presented as mean ± SEM, n = 6 for DMSO and rosiglitazone, n = 4 for T0070907, all biological replicates. b Western blot for NECTIN4 and HPGD in RT112 cells treated with T0070907, rosiglitazone, and pioglitazone at indicated concentrations for 72 h. This was repeated n = 3 independent times with similar results. c, d NECTIN4 surface staining ( c ) and quantification of median fluorescence intensify (MFI) ( d ) in RT112 cells treated with 1μM rosiglitazone and T0070907 for 72 h. Data are presented as mean ± SEM, n = 4 for DMSO and T0070907, n = 5 for rosiglitazone, all biological replicates. e Western blot for NECTIN4 and HPGD in RT112 cells treated with rosiglitazone for 72 h across a dose series (starting at 1 μM on the right most lane with serial 2-fold dilutions to the left). This was repeated n = 3 independent times with similar results. f Dose response curves of total NECTIN4 and HPGD protein expression after 72 h rosiglitazone treatment in RT112 cells. Data are presented as mean values. g NECTIN4 surface staining in RT112 cells treated with rosiglitazone for 72 h across a dose series. Dose series starts at 1μM with serial 2-fold dilutions. h Dose response curves of surface NECTIN4 and TROP2 expression for rosiglitazone in RT112 cells after 72 h. Data are presented as mean ± SEM, n = 2 biological replicates. i Western blots for NECTIN4, PPARγ and FABP4 in RT112 cells expressing sgRNAs against GAL4 (control), NECTIN4 , and two unique PPARG guides. This was repeated n = 3 independent times with similar results. j Schematic of select predicted PPARG binding sites (labeled 1 and 2) on the NECTIN4 promoter region (500 to −2k) by FIMO using transcription binding motif matrices JASPAR 2018. Created with BioRender. Chang (2025) https://BioRender.com/k3j6fgi . k Chromatin from RT112 cells was precipitated using antibodies against PPARG or IgG. Primers targeting putative PPARG binding motifs 1 and 2 from (j) were used for PCR and the PCR product was visualized by gel electrophoresis. l Quantitative analysis of ChIP-qPCR experiments. Results are represented as fold-enrichment relative to IgG control. Data are presented as mean ± SEM, n = 3 biological replicates. For panels ( a) and ( d ), ordinary one-way ANOVA with Sidak’s multiple comparison was used. For panel ( l ), two-sided, unpaired Student’s t test was used for each primer set. Vinculin shown as a loading control for panels ( b ) and ( e ) and GAPDH for panel ( i ). Source data are provided as a Source Data file.

Article Snippet: Cells were then stained with a NECTIN4 antibody (Miltenyi, clone REA967, 1:100), sorted a second time by FACS (BD Fusion), and negative cells were collected and expanded.

Techniques: Western Blot, Staining, Fluorescence, Expressing, Control, Binding Assay, Labeling, Nucleic Acid Electrophoresis, ChIP-qPCR, Comparison

Journal: Nature Communications

Article Title: Modulating the PPARγ pathway upregulates NECTIN4 and enhances chimeric antigen receptor (CAR) T cell therapy in bladder cancer

doi: 10.1038/s41467-025-62710-0

Figure Lengend Snippet: Representative growth curves of ( a ) RT112, ( c ) UMUC-1, and ( e ) HT1197 cells pretreated for 72 h with DMSO vehicle (black) or 1uM rosiglitazone (gold) and co-cultured with (left) NECTIN4-CAR T cells or (right) NTD T cells at indicated E:T cell ratios. Kill indices for NECTIN4-CAR and NTD T cells against ( b ) RT112, ( d ) UMUC-1, and ( f ) HT1197 cells pretreated for 72 h with DMSO vehicle (black) or 1uM rosiglitazone (gold) across various E:T ratios are shown. For panels a-f , data are presented as mean ± SEM, n = 3 biological replicates for all groups in ( b , d , f ). For panels ( b , d , f ), two-way ANOVA with Sidak’s multiple comparison was used. g , h FN-γ quantification by ELISA from co-cultures of NECTIN4-CAR or NTD T cells with RT112 ( g ) or UMUC-1 ( h ) cells pretreated for 72 h with DMSO control (black) or 1 μM rosiglitazone (gold) at an E:T ratio of 1:1 at 24 h. Data are presented as mean ± SEM, n = 3 biological replicates per condition. For panels ( g , h ), two-way ANOVA with Sidak’s multiple comparison was used, and data are presented as mean ± SEM, n = 4 biological replicates for all groups. Source data are provided as a Source Data file. Created with BioRender. Chang (2025) https://BioRender.com/k3j6fgi .

Article Snippet: Cells were then stained with a NECTIN4 antibody (Miltenyi, clone REA967, 1:100), sorted a second time by FACS (BD Fusion), and negative cells were collected and expanded.

Techniques: Cell Culture, Comparison, Enzyme-linked Immunosorbent Assay, Control

Journal: Nature Communications

Article Title: Modulating the PPARγ pathway upregulates NECTIN4 and enhances chimeric antigen receptor (CAR) T cell therapy in bladder cancer

doi: 10.1038/s41467-025-62710-0

Figure Lengend Snippet: a Schematic of in vivo studies investigating the combination of rosiglitazone plus NECTIN4-CAR T cell therapy. Created with BioRender. Chang (2025) https://BioRender.com/k3j6fgi . Briefly, xenograft models were established by subcutaneous injection of indicated UC cells into NOD/scid/gamma (NSG) mice. When tumors reached approximately 50–100mm 3 , mice were randomized to receive daily rosiglitazone (20 mg/kg) or vehicle for 5 days, followed by a single injection of 2.5–5 × 10 6 NECTIN4-CAR or NTD T cells via IV injection. Some tumors were collected immediately following rosiglitazone or vehicle administration for analysis. b , c Representative western blot ( b ) and quantification ( c ) for NECTIN4 and HPGD in RT112 tumor xenografts from mice treated with either rosiglitazone (gold) or vehicle (black) for 5 days. Data are presented as mean ± SEM, n = 7 vehicle and n = 10 rosiglitazone, all biological replicates. Vinculin shown as a loading control. This was repeated n = 3 independent times with similar results. d Scatterplot showing NECTIN4 versus HPGD expression from western blot analysis of tumors in ( b ). Pearson’s correlation is shown for NECTIN4 expression versus HPGD expression ( r = 0.58, P = 0.01, two-tailed). Data from n = 2 independent cohorts of n = 4–5 mice each. e–f Tumor growth ( e ) and Kaplan–Meier survival curves ( f ) of RT112 subcutaneous xenografts. Mice were treated for 5 days with either vehicle or rosiglitazone followed by an IV injection of NECTIN4-CAR or NTD T cells. n = 5 mice in each NTD T cell group and n = 7 mice in each NECTIN4-CAR T cell group. For panel ( e ), ** indicates p = 0.0058 at day 21, *** indicates p = 0.002, p = 0.008, p = 0.001 at days 25, 28, 31, respectively, and **** indicates p < 0.0001 at day 34, for Vehicle + NECTIN4-CAR T (red) vs Rosi + NECTIN4-CAR T (blue). Error bars represent SEM. ns not significant, NTD non-transduced. For panel ( f ), the log-rank test was used. g Body weights of each group of mice in ( e ) over time. Data are presented as mean ± SEM. h , i Representative western blot for NECTIN4 and HPGD in HT1197 tumor xenografts from mice treated with either rosiglitazone or vehicle for 5 days. Quantification of NECTIN4 is shown in ( i ). Data are presented as mean ± SEM, n = 9 biological replicates in each group. ( j ) Scatterplot showing NECTIN4 versus HPGD expression from western blot analysis. Pearson’s correlation is shown for NECTIN4 expression versus HPGD expression ( r = 0.50, P = 0.04, two-tailed). Data from n = 2 biologically independent cohorts of n = 4–5 mice each. k, l Tumor growth curves ( k ) and body weights of each group ( l ) of HT1197 tumor xenografts treated with vehicle or rosiglitazone followed by single IV injection of NECTIN4-CAR T cells or NTD T cells. The number of mice in each group is shown in parentheses: n = 5 mice (Vehicle+NTD), n = 7 mice (Rosi+NTD), n = 9 mice (Vehicle+NECTIN4-CAR T and Rosi+NECTIN4-CAR T). *** indicates p = 0.004 at day 82 for Vehicle + NTD vs Rosi + NTD and ** indicates p = 0.0068 at day 82 and * indicates p = 0.0167 at day 75 for Vehicle+NECTIN4-CAR T (red) vs Rosi+NECTIN4-CAR T (blue). Data are presented as mean ± SEM. For panels ( c, i ), two-sided, unpaired Student’s t test was used. For panels ( e , g , k , l ), mixed effects model with Sidak’s multiple comparison test was used. Source data are provided as a Source Data file.

Article Snippet: Cells were then stained with a NECTIN4 antibody (Miltenyi, clone REA967, 1:100), sorted a second time by FACS (BD Fusion), and negative cells were collected and expanded.

Techniques: In Vivo, Injection, IV Injection, Western Blot, Control, Expressing, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Modulating the PPARγ pathway upregulates NECTIN4 and enhances chimeric antigen receptor (CAR) T cell therapy in bladder cancer

doi: 10.1038/s41467-025-62710-0

Figure Lengend Snippet: a Schematic of parental RT112 cells undergoing cycles of treatment with enfortumab vedotin (EV) at escalating doses to 25 μg/ml, yielding EV resistant cells (“RT112-EV Res.”). Created with BioRender. Chang (2025) https://BioRender.com/k3j6fgi . b EV dose-response curves for RT112-Parental and RT112-EV Res. following 9 days of treatment with EV, showing EV IC50 for RT112-parental (2.4 μg/ml) and RT112-EV Res. (25.1 μg/ml). c Western blot for NECTIN4 in RT112-parental and RT112-EV Res. cell lysates. Vinculin shown as a loading control. This was repeated n = 3 independent times with similar results. d , e Representative flow cytometry ( d ) and quantification of surface NECTIN4 MFI ( e ) in RT112-parental and RT112-EV Res. cells. Data are presented as mean MFI ± SEM, n = 4 biological replicates, and two-sided, unpaired Student’s t test was used. f , g H-scores for surface NECTIN4 IHC on matched samples taken from a cohort of n = 17 UC patients who underwent pre-EV and post-EV biopsies. Data are presented as mean ± SEM in ( f ), and a two-sided, paired Student’s t test was used. h Example of NECTIN4 stain on a paired biopsy taken pre-EV (H-score=160) and post-EV (H-score=300). Scale bar denotes 1 mm. Kill indices of ( i ) EV, ( j ) NECTIN4-CAR-1 T cells and ( k ) enfortumab-derived NECTIN4-CAR-2 T cells against RT112-parental (black) and RT112-EV Res. (pink) cells. Error bars represent SEM, n = 3 biological replicates for each group. ( l ) Tumor growth curves of RT112-parental and RT112-EVRes. tumor xenografts treated with a single dose of EV (4 mg/kg) or vehicle. RT112-parental ( n = 10 mice vehicle, black), RT112-EV Res. ( n = 10 mice vehicle, pink), RT112-parental + EV ( n = 4 mice, purple), RT112-EV Res. + EV ( n = 5 mice, teal). Data are presented as mean ± SEM. p values comparig RT112-parental vehicle (black) vs EV (purple) are shown for indicated time points. m Tumor growth curves of RT112-EVRes. tumor xenografts in NSG mice treated with a single dose of 5 × 10 6 NECTIN4-CAR or NTD T cells. n = 5 mice (NTD T cells, pink), n = 3 mice (NECTIN4-CAR T cells, teal). Data are presented as mean ± SEM. p values at days 24, 27 and 31 are shown. For panel ( b ), a nonlinear regression model was used to compare the dose-response curves by the extra sum-of-squares F test. For panel ( g ), a paired, two-tailed Student’s t test was used. For panels ( i–k ), two-way ANOVA with Sidak’s multiple comparison test was used. For panels ( l, m ), a mixed effects model with Sidak’s multiple comparison test was used. Source data are provided as a Source Data file.

Article Snippet: Cells were then stained with a NECTIN4 antibody (Miltenyi, clone REA967, 1:100), sorted a second time by FACS (BD Fusion), and negative cells were collected and expanded.

Techniques: Western Blot, Control, Flow Cytometry, Staining, Derivative Assay, Two Tailed Test, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Integrin β4 drives immune evasion and therapeutic resistance to PD-1 blockade in bladder cancer via MEK/ERK signaling

doi: 10.1016/j.jbc.2025.110941

Figure Lengend Snippet: ITGB4 positively correlated with enfortumab vedotin target NECTIN4. A , the RNA-Seq analysis in the GSE268855 dataset after overexpressing ITGB4 ; B , the correlation analysis in the DepMap dataset. p values are shown in the figure. C , Pan-cancer data analysis in the DepMap database revealed the correlation between ITGB4 and NECTIN4 . p values were shown in the figure. D – G , HT-1376 and 5637 cells were transfected with shITGB4 or shControl for 72 h. The harvested cells were used for Western blot analysis ( D ) and RT-qPCR detection ( E – G ). H – K , HT-1376 and 5637 cells were transfected with overexpressed ITGB4 plasmid or empty vector for 72 h, and harvested cells were used for Western blot analysis ( H ) and RT-qPCR detection ( I–K ). Data presents as mean ± SD with three replicates. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. L , the tissue microarray of bladder cancer stained with ITGB4 and NECTIN4, respectively. The typical IHC images stained with ITGB4 and NECTIN4 are shown in the panel . M , the correlation of these two proteins is shown in the panel , and the p -value is indicated in the figure.

Article Snippet: The primary antibodies used were as follows: ITGB4 (#21738-1-AP, Proteintech, 1:500 dilution), p-ITGB4(Y1510) (#YP0755, Immunoway, 1:500 dilution), MEK-1(#YM8273, Immunoway, 1:1000 dilution), p-MEK-1 (T386) (#YP0425, Immunoway, 1:500 dilution), ERK1/2(#YM8336, Immunoway, 1:2000 dilution), p-ERK1/2 (T202/T204) (#YM8452, Immunoway, 1:2000 dilution), c-Jun (#YM8321, Immunoway, 1:2000 dilution), p-c-Jun (S63) (#28907-1-AP, Proteintech, 1:500 dilution), PD-L1(#28076-1-AP, Proteintech, 1:300 dilution), NECTIN4 (#21903-1-AP, Proteintech, 1:2000 dilution), HER2(#18299-1-AP, Proteintech, 1:2000 dilution), TROP2(#27360-1-AP, Proteintech, 1:500 dilution), GADPH (#10494-1-AP, Proteintech, 1:5000 dilution).

Techniques: RNA Sequencing, Transfection, Western Blot, Quantitative RT-PCR, Plasmid Preparation, Microarray, Staining

Journal: The Journal of Biological Chemistry

Article Title: Integrin β4 drives immune evasion and therapeutic resistance to PD-1 blockade in bladder cancer via MEK/ERK signaling

doi: 10.1016/j.jbc.2025.110941

Figure Lengend Snippet: ITGB4 plays a vital role in BLCA sequential therapies. A mechanism diagram depicting that the highly expressed ITGB4 in cisplatin-resistant BLCA activated the MEK/ERK pathway through tyrosine-1510 phosphorylation, upregulated the expression level of PD-L1 in BLCA, and caused anti-PD-1 treatment resistance. Meanwhile, the overexpressed ITGB4 was positively correlated to NECTIN4, promoting the sensitivity of BLCA to enfortumab vedotin treatment.

Article Snippet: The primary antibodies used were as follows: ITGB4 (#21738-1-AP, Proteintech, 1:500 dilution), p-ITGB4(Y1510) (#YP0755, Immunoway, 1:500 dilution), MEK-1(#YM8273, Immunoway, 1:1000 dilution), p-MEK-1 (T386) (#YP0425, Immunoway, 1:500 dilution), ERK1/2(#YM8336, Immunoway, 1:2000 dilution), p-ERK1/2 (T202/T204) (#YM8452, Immunoway, 1:2000 dilution), c-Jun (#YM8321, Immunoway, 1:2000 dilution), p-c-Jun (S63) (#28907-1-AP, Proteintech, 1:500 dilution), PD-L1(#28076-1-AP, Proteintech, 1:300 dilution), NECTIN4 (#21903-1-AP, Proteintech, 1:2000 dilution), HER2(#18299-1-AP, Proteintech, 1:2000 dilution), TROP2(#27360-1-AP, Proteintech, 1:500 dilution), GADPH (#10494-1-AP, Proteintech, 1:5000 dilution).

Techniques: Phospho-proteomics, Expressing