Journal: Genes & Diseases

Article Title: Blockade of co-inhibitory receptor immune checkpoint protein TIM3/CD366 augments the anti-cancer activity of CAR-T therapy in solid tumors: An ovarian cancer example

doi: 10.1016/j.gendis.2025.101978

Figure Lengend Snippet: Specific IFN-γ and TNF-α release of T lymphocytes transduced with TIM-3-silenced HER2-specific chimeric antigen receptor (CAR) or HER2-specific CAR. (A, B) TIM-3-silenced CAR-T cells and control T cells were co-incubated with Galectin-9 + or Galectin-9 – SKOV3 tumor cells (E:T ratio 5:1 or 10:1). At 20 h after coculture, a specific enzyme-linked immunosorbent assay was used to analyze the supernatant for IFN-γ cytokine-release. Results were presented as mean ± standard deviation. (C, D) The detection of TNF-α in the same culture supernatant. Results were presented as mean ± standard deviation. ∗ P < 0.05 and ∗∗ P < 0.01.

Article Snippet: Human cervical cancer cell line HeLa, lentivirus packaging cell line HEK 293TD, and human ovarian cancer cell line SKOV3 were purchased from American Type Culture Collection (Manassas, Virginia, USA) and cultured in Dulbecco's modified Eagle's medium (Invitrogen, Grand Island, New York) supplemented with 10% heat-inactivated fetal bovine serum.

Techniques: Transduction, Control, Incubation, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Genes & Diseases

Article Title: Blockade of co-inhibitory receptor immune checkpoint protein TIM3/CD366 augments the anti-cancer activity of CAR-T therapy in solid tumors: An ovarian cancer example

doi: 10.1016/j.gendis.2025.101978

Figure Lengend Snippet: TIM-3 silencing augmented the anti-tumor activity of chimeric antigen receptor-T (CAR-T) cells in vivo . 2 × 10 6 SKOV3 tumor cells expressing luciferase were intraperitoneally inoculated in a xenograft mouse model, and 7 days after inoculation, the 2 × 10 6 HER2-specific CAR-T kdTim-3 cells or CAR-T cells, or untreated T cells were intraperitoneally administered. (A, B) Tumor growth was monitored using an in vivo imaging system. (C) Survival curve of 80-day post-treatment. ∗ P < 0.05 and ∗∗ P < 0.01.

Article Snippet: Human cervical cancer cell line HeLa, lentivirus packaging cell line HEK 293TD, and human ovarian cancer cell line SKOV3 were purchased from American Type Culture Collection (Manassas, Virginia, USA) and cultured in Dulbecco's modified Eagle's medium (Invitrogen, Grand Island, New York) supplemented with 10% heat-inactivated fetal bovine serum.

Techniques: Activity Assay, In Vivo, Expressing, Luciferase, In Vivo Imaging

Journal: Journal of Cell Communication and Signaling

Article Title: Gemcitabine activates the Hippo signaling pathway and suppresses tumor growth by stabilizing large tumor suppressor kinase 2 through the hypoxia‐inducible factor 1‐alpha/ubiquitin protein ligase E3 component N‐recognin 5 axis

doi: 10.1002/ccs3.70085

Figure Lengend Snippet: Effects of GEM on hypoxia‐inducible factor 1‐alpha (HIF1A), UBR5, and ovarian cancer Cells. (A) Western blot (WB) analysis of HIF1A and UBR5 expression in SKOV3 cells under hypoxic conditions treated with different concentrations of GEM; (B) WB analysis of HIF1A and UBR5 expression in OVCAR‐3 cells under hypoxic conditions treated with different concentrations of GEM; (C) CCK‐8 assay of cell viability in SKOV3 cells treated with varying concentrations of GEM at 0, 24, and 48 h; (D) CCK‐8 assay of cell viability in OVCAR‐3 cells treated with varying concentrations of GEM at 0, 24, and 48 h; (E) wound healing assay assessing the migration rate of SKOV3 cells treated with different concentrations of GEM at 0 h, 24 h, and 48 h (scale bar: 100 μm); (F) wound healing assay assessing the migration rate of OVCAR‐3 cells treated with different concentrations of GEM at 0, 24, and 48 h (scale bar: 100 μm); (G) transwell invasion assay evaluating the invasive capacity of SKOV3 cells treated with different concentrations of GEM at 0, 24, and 48 h (scale bar: 50 μm); (H) transwell invasion assay evaluating the invasive capacity of OVCAR‐3 cells treated with different concentrations of GEM at 0, 24, and 48 h (scale bar: 50 μm). Data are presented as mean ± SD; all cellular experiments were performed in triplicate. *** p < 0.001, analyzed using ANOVA followed by Tukey's multiple comparison test.

Article Snippet: Human OC cell lines SKOV3 (HTB‐77, ATCC) and OVCAR‐3 (HTB‐161, ATCC) were obtained from ATCC.

Techniques: Western Blot, Expressing, CCK-8 Assay, Wound Healing Assay, Migration, Transwell Invasion Assay, Comparison

Journal: Journal of Cell Communication and Signaling

Article Title: Gemcitabine activates the Hippo signaling pathway and suppresses tumor growth by stabilizing large tumor suppressor kinase 2 through the hypoxia‐inducible factor 1‐alpha/ubiquitin protein ligase E3 component N‐recognin 5 axis

doi: 10.1002/ccs3.70085

Figure Lengend Snippet: Effects of GEM on ovarian cancer proliferation and invasion via HIF1A‐mediated downregulation of UBR5. (A) Western blot (WB) analysis of hypoxia‐inducible factor 1‐alpha (HIF1A) and UBR5 expression levels in SKOV3 cells treated with 20 μM GEM for 48 h; (B) WB analysis of HIF1A and UBR5 expression levels in OVCAR‐3 cells treated with 20 μM GEM for 48 h; (C) CCK8 assay showing cell viability of SKOV3 cells treated with 20 μM GEM for 48 h; (D) CCK8 assay showing cell viability of OVCAR‐3 cells treated with 20 μM GEM for 48 h; (E) wound healing assay assessing the migration rate of SKOV3 cells treated with 20 μM GEM for 48 h (Scale bar: 100 μm); (F) wound healing assay assessing the migration rate of OVCAR‐3 cells treated with 20 μM GEM for 48 h (Scale bar: 100 μm); (G) transwell invasion assay showing the invasive capacity of SKOV3 cells treated with 20 μM GEM for 48 h (Scale bar: 50 μm); (H) transwell invasion assay showing the invasive capacity of OVCAR‐3 cells treated with 20 μM GEM for 48 h (Scale bar: 50 μm); (I) predicted transcription factor binding sites in the UBR5 promoter region based on JASPAR database analysis; (J) ChIP‐PCR analysis showing HIF1A enrichment at the UBR5 promoter region; (K) dual‐luciferase reporter assay evaluating the regulatory effect of HIF1A on UBR5 promoter activity; (L, M) RT‐qPCR analysis of HIF1A and UBR5 mRNA expression levels after shHIF1A knockdown in SKOV3 and OVCAR‐3 cells; (N, O) RT‐qPCR analysis of UBR5 mRNA expression levels in SKOV3 and OVCAR‐3 cells under normoxia, hypoxia, hypoxia + shNC, and hypoxia + shHIF1A conditions. Data are presented as mean ± SD; all cell experiments were performed in triplicate. *** p < 0.001, analyzed by ANOVA followed by Tukey's multiple comparison test.

Article Snippet: Human OC cell lines SKOV3 (HTB‐77, ATCC) and OVCAR‐3 (HTB‐161, ATCC) were obtained from ATCC.

Techniques: Western Blot, Expressing, CCK-8 Assay, Wound Healing Assay, Migration, Transwell Invasion Assay, Binding Assay, Luciferase, Reporter Assay, Activity Assay, Quantitative RT-PCR, Knockdown, Comparison

Journal: Journal of Cell Communication and Signaling

Article Title: Gemcitabine activates the Hippo signaling pathway and suppresses tumor growth by stabilizing large tumor suppressor kinase 2 through the hypoxia‐inducible factor 1‐alpha/ubiquitin protein ligase E3 component N‐recognin 5 axis

doi: 10.1002/ccs3.70085

Figure Lengend Snippet: GEM regulates LATS2 ubiquitination and YAP/TAZ phosphorylation via the hypoxia‐inducible factor 1‐alpha/UBR5 axis. (A) Western blot (WB) analysis of Hippo pathway‐related proteins in SKOV3 cells under hypoxic conditions; (B) WB analysis of Hippo pathway‐related proteins in OVCAR‐3 cells under hypoxic conditions; (C) co‐IP analysis showing the interaction between LATS2 and UBR5 in SKOV3 cells; (D) Co‐IP analysis showing the interaction between LATS2 and UBR5 in OVCAR‐3 cells; (E) ubiquitination assay measuring LATS2 ubiquitination levels in SKOV3 cells treated with 20 μM GEM for 48 h; (F) ubiquitination assay measuring LATS2 ubiquitination levels in OVCAR‐3 cells treated with 20 μM GEM for 48 h; (G) WB analysis of in vitro ubiquitination to evaluate UBR5‐mediated LATS2 ubiquitination; (H) WB analysis of Hippo pathway‐related proteins in SKOV3 cells treated with 20 μM GEM for 48 h; (I) WB analysis of Hippo pathway‐related proteins in OVCAR‐3 cells treated with 20 μM GEM for 48 h. Results are presented as mean ± standard deviation. All experiments were repeated three times. *** p < 0.001, using ANOVA and Tukey's multiple comparison test.

Article Snippet: Human OC cell lines SKOV3 (HTB‐77, ATCC) and OVCAR‐3 (HTB‐161, ATCC) were obtained from ATCC.

Techniques: Ubiquitin Proteomics, Phospho-proteomics, Western Blot, Co-Immunoprecipitation Assay, In Vitro, Standard Deviation, Comparison

Journal: Journal of Cell Communication and Signaling

Article Title: Gemcitabine activates the Hippo signaling pathway and suppresses tumor growth by stabilizing large tumor suppressor kinase 2 through the hypoxia‐inducible factor 1‐alpha/ubiquitin protein ligase E3 component N‐recognin 5 axis

doi: 10.1002/ccs3.70085

Figure Lengend Snippet: Investigation of GEM's regulation of the Hippo pathway in ovarian cancer inhibition. (A) Western blot (WB) analysis of YAP1 and FGFR1 expression levels in SKOV3 cells treated with 20 μM GEM for 48 h; (B) WB analysis of YAP1 and FGFR1 expression levels in OVCAR‐3 cells treated with 20 μM GEM for 48 h; (C) CCK8 assay measuring cell viability in SKOV3 cells treated with 20 μM GEM for 48 h; (D) CCK8 assay measuring cell viability in OVCAR‐3 cells treated with 20 μM GEM for 48 h; (E) wound healing assay showing migration rates of SKOV3 cells treated with 20 μM GEM for 48 h (scale bar: 100 μm); (F) wound healing assay showing migration rates of OVCAR‐3 cells treated with 20 μM GEM for 48 h (scale bar: 100 μm); (G) transwell assay showing invasion ability of SKOV3 cells treated with 20 μM GEM for 48 h (scale bar: 50 μm); (H) transwell assay showing invasion ability of OVCAR‐3 cells treated with 20 μM GEM for 48 h (scale bar: 50 μm). Results are presented as mean ± standard deviation. Cell experiments were repeated 3 times; *** p < 0.001, using ANOVA and Tukey's multiple comparison test.

Article Snippet: Human OC cell lines SKOV3 (HTB‐77, ATCC) and OVCAR‐3 (HTB‐161, ATCC) were obtained from ATCC.

Techniques: Inhibition, Western Blot, Expressing, CCK-8 Assay, Wound Healing Assay, Migration, Transwell Assay, Standard Deviation, Comparison

Journal: bioRxiv

Article Title: Context-dependent tonic signaling shapes the performance and manufacturability of a 4-1BB–based HER2 CAR-T cell therapy

doi: 10.64898/2026.05.11.724226

Figure Lengend Snippet: (A) Schematic of CAR constructs used. (B) Population doublings of CD4⁺ and CD8⁺ CAR-T cells at day 10 of expansion. Data are shown as mean ± SD and each dot represents a healthy donor (n=3-7). (C) Percentage and (D) mean fluorescence intensity (MFI) of CAR expression in CD4⁺ and CD8⁺ CAR-T cells at day 8 of expansion. Data are shown as mean ± SD and each dot represents a healthy donor (n=3-8). (E) Representative histogram of CAR expression. (F) Real-time cytotoxicity assay (xCELLigence) of CAR-T cells co-cultured with HER2⁺ SKOV3 ovarian cancer cells (effector-to-target ratio, E:T=1:1). The timepoint of T cell addition is indicated. Data are plotted as mean ± SD of normalized cell index (n=2 healthy donors). (G) IFN-γ (left panel) or IL-2 (right panel) levels after 24 h of co-culture of control T cells or indicated HER2 CAR-T cells with SKOV3 cells (E:T=3:1). Data are shown as mean ± SD and each dot represents a healthy donor (n=2). (H–J) In vivo evaluation in NSG mice bearing subcutaneous SKOV3 tumors treated with a single intravenous dose of 2×10⁶ CAR⁺ or control T cells. (H) Tumor growth kinetics following treatment. Data are shown as mean tumor over time volume ± SEM (n=10-12 tumors per group). (I) Tumor volumes at day 35 post-treatment. Data are shown as mean ± SD and each dot represents an individual tumor (n=10-12 tumors per group). (J) Persistence of CAR-T cells in peripheral blood at day 25 post-infusion. Data are shown as mean ± SD and each dot represents an individual mouse (n=5-6 mice per group). In B - D and H : *p<0.05, ***p<0.001, ****p<0.0001 by two-way ANOVA with Tukey’s multiple comparison test. In G and I - J : *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA with Tukey’s multiple comparison test. Abbreviations: L, leader peptide; H, hinge; TMD, transmembrane domain; CSD, costimulation domain; ICD, intracellular domain.

Article Snippet: Human tumor cell lines SKOV3 (ovarian cystadenocarcinoma) and HCC1954 (breast ductal carcinoma) were purchased from American Tissue Culture Collection (Manassas, Virginia, USA).

Techniques: Construct, Fluorescence, Expressing, Cytotoxicity Assay, Cell Culture, Co-Culture Assay, Control, In Vivo, Comparison

Journal: bioRxiv

Article Title: Context-dependent tonic signaling shapes the performance and manufacturability of a 4-1BB–based HER2 CAR-T cell therapy

doi: 10.64898/2026.05.11.724226

Figure Lengend Snippet: (A) Schematic of CAR constructs used. (B) Population doublings of CD4⁺ and CD8⁺ CAR-T cells at day 10 of expansion. Data are shown as mean ± SD and each dot represents a healthy donor (n=10). (C) Percentage and (D) mean fluorescence intensity (MFI) of CAR expression in CD4⁺ and CD8⁺ CAR-T cells at day 8 of expansion. Data are shown as mean ± SD and each dot represents a healthy donor (n=10). (E) Representative histogram of CAR expression. (F-G) Real-time cytotoxicity assay (xCELLigence) of CAR-T cells co-cultured with HER2⁺ (F) SKOV3 ovarian or (G) HCC1954 breast cancer cells (E:T=1:1). The timepoint of T cell addition is indicated. Data are plotted as mean ± SD of normalized cell index (n=2 healthy donors). (H-I) IFN-γ (left panel) or IL-2 (right panel) levels after 24h of co-culture of control T cells or indicated HER2 CAR-T cells with (H) SKOV3 or (I) HCC1954 cells (E:T=3:1). Data are shown as mean ± SD and each dot represents a healthy donor (n=3). (J) T cell proliferation at day 7 after co-culture with HCC1954 tumor cells relative to day 0, as analysed by flow cytometry. Data are shown as mean ± SD and each dot represents a healthy donor (n=3). (K-L) In vivo evaluation in NSG mice bearing subcutaneous SKOV3 tumors treated with a single intravenous dose of 2×10⁶ CAR⁺ or control T cells. (K) Tumor growth kinetics following treatment. Data are shown as mean tumor volume ± SEM over time (n=6-8 tumors per group). (L) Persistence of CAR-T cells in peripheral blood at day 25 post-infusion. Data are shown as mean ± SD and each dot represents an individual mouse (n=3-4 mice per group). In B - D and J : *p<0.05, **p<0.01, ****p<0.0001 by two-way ANOVA with Tukey’s multiple comparison test. In H - I and L : *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by one-way ANOVA with Tukey’s multiple comparison test. In I : *p<0.05, ** p<0.01 by one-way ANOVA with Holm-Sidak’s multiple comparison test. Abbreviations: L, leader peptide; H, hinge; TMD, transmembrane domain; CSD, costimulation domain; ICD, intracellular domain.

Article Snippet: Human tumor cell lines SKOV3 (ovarian cystadenocarcinoma) and HCC1954 (breast ductal carcinoma) were purchased from American Tissue Culture Collection (Manassas, Virginia, USA).

Techniques: Construct, Fluorescence, Expressing, Cytotoxicity Assay, Cell Culture, Co-Culture Assay, Control, Flow Cytometry, In Vivo, Comparison

Journal: bioRxiv

Article Title: Context-dependent tonic signaling shapes the performance and manufacturability of a 4-1BB–based HER2 CAR-T cell therapy

doi: 10.64898/2026.05.11.724226

Figure Lengend Snippet: HER2-BBz CAR-T cells were expanded in the absence or presence of increasing doses of dasatinib added to T cell cultures on days 3 and 4. (A) IFN-γ production after 24h of co-culture with SKOV3 cells (E:T=3:1). Absolute IFN-γ levels (left) and fold change relative to untreated HER2-BBz CAR-T cells (right) are shown. Data represent mean ± SD, and each dot indicates an individual healthy donor (n=3-7). (B) Real-time cytotoxicity assay (xCELLigence) of CAR-T cells co-cultured with SKOV3 ovarian cancer cells (E:T=1:1). The timepoint of T cell addition is indicated. Data are shown as mean ± SD of the normalized cell index (n=2 healthy donors). (C) In vivo evaluation in NSG mice bearing subcutaneous SKOV3 tumors treated with a single intravenous dose of 2×10⁶ CAR⁺ or control T cells. Tumor growth kinetics are shown as mean tumor volume ± SEM over time (n=8 tumors per group). (D - G) HER2-BBz CAR-T cells were expanded in the presence of 50 nM dasatinib added at different time intervals (days 3–4, days 7–9, or days 3–9). (D) Population doublings at day 10 of expansion. Data represent mean ± SD, and each dot represents a healthy donor (n=9). (E) Percentage of CAR expression in T cells at day 8 of expansion. Data represent mean ± SD, and each dot represents a healthy donor (n=9). (F) IFN-γ production after 24h of co-culture with SKOV3 cells (E:T=3:1). Absolute IFN-γ levels (left) and fold change relative to untreated HER2-BBz CAR-T cells (right) are shown. Data represent mean ± SD and each dot represents a healthy donor (n=4). (G) Real-time cytotoxicity assay (xCELLigence) of CAR-T cells co-cultured with SKOV3 ovarian cancer cells (E:T=1:1). The timepoint of T cell addition is indicated. Data are plotted as mean ± SD of normalized cell index (n=2 healthy donors). In A and F : *p<0.05, **p<0.01 by one-sample t-test. In D - E : **p<0.01, ***p<0.001, ****p<0.0001 by one-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: Human tumor cell lines SKOV3 (ovarian cystadenocarcinoma) and HCC1954 (breast ductal carcinoma) were purchased from American Tissue Culture Collection (Manassas, Virginia, USA).

Techniques: Co-Culture Assay, Cytotoxicity Assay, Cell Culture, In Vivo, Control, Expressing, Comparison

Journal: bioRxiv

Article Title: Programmable synthetic cytokine receptors polarize macrophages to user-defined functional states

doi: 10.64898/2026.05.12.724672

Figure Lengend Snippet: a, Schematic depicting co-expression of a human epidermal growth factor (HER)2-specific chimeric antigen receptor (CAR) and an SCR in human primary macrophages. Also depicted is the co-expression cassette driven by the phosphoglycerate kinase (PGK) promoter (bottom). HER2, human epidermal growth factor; GFP, green fluorescent protein. b, Schematic depicting the experimental design for repeated challenge of αHER2-CAR macrophages with HER2⁺ cancer cells. HER2, human epidermal growth factor 2. c, Representative images of BT474 cell abundance over 9-days of repeated co-culture with macrophages. Macrophages were transduced with the indicated constructs. UT, un-transduced. d, BT474 cell abundance over nine days of repeated co-culture with macrophages transduced with SCR0 ( n = 4), CAR + SCR0 ( n = 4) or CAR + SCR (S2-S2-S2) ( n = 3). BT474 cell abundance was measured as total BFP fluorescence intensity using live-cell imaging. Faint lines represent raw mean value. Darker lines represent smoothed curve for each challenge period. Data are mean ± s.e.m. e, BT474 cell abundance after nine days of repeated co-culture with macrophages transduced with SCRs containing motifs that drove high phagocytosis in , with (light) or without (dark) co-expression of a CAR. Data are mean ± s.e.m. f, Quantification of pHrodo signal intensity after nine days of repeated co-culture with macrophages transduced with SCRs containing motifs that drove high phagocytosis in , with (light) or without (dark) co-expression of a CAR. Data are mean ± s.e.m. g, Quantification of relative K562 growth at the end of a 3-day co-culture between CAR-SCR-Ms and wild-type K562 cells (light) or HER2 + K562 cells (dark). K562 cell growth is normalized growth of K562s in the absence of macrophages (K562 only). Data are mean ± s.e.m. h, Schematic depicting the in vivo experimental design. 1x10 SKOV3 cells expressing firefly luciferase (ffluc) were injected i.p. into each mouse. Mice were then treated i.p. with PBS (grey, n = 5) or 3x10 macrophages expressing SCR0 (grey, n = 5), αHER2-CAR + SCR0 (light blue, n = 4), or αHER2-CAR + SCR(S2-S2-S2) (dark blue, n = 5). Tumor burden was measured using bioluminescent imaging (BLI). i.p., intraperitoneal. i, Representative images from the experiment depicted in f at day 0, day 15, and day 36 after tumor injection. BLI, bioluminescent imaging. j , Tumor burden measured as total flux (photons s⁻¹) of luciferase signal. k, Tumor burden at day 36 post treatment, normalized by the day 0 luciferase signal.

Article Snippet: K562, BT474, and SKOV3 cell lines were originally commercially obtained from the American Type Culture Collection (ATCC).

Techniques: Expressing, Co-Culture Assay, Transduction, Construct, Fluorescence, Live Cell Imaging, In Vivo, Luciferase, Injection, Imaging