Journal: Cancer discovery

Article Title: Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids

doi: 10.1158/2159-8290.CD-17-0833

Figure Lengend Snippet: a, Schematic for preparation and analysis of MDOTS/PDOTS (S2 fraction) from murine or patient-derived tumor specimens. b, MC38 immune profiling by flow cytometry comparing bulk tumor (n=5) to S1, S2, S3 (n=6) spheroid fractions (Kruskal-Wallis with Dunn’s multiple comparisons test, α=0.05; ns=not significant). c, B16F10 immune profiling by flow cytometry comparing bulk tumor (n=5) to S1 (n=4), S2 (n=5), and S3 (n=4) evaluated by flow cytometry (Kruskal-Wallis test with Dunn’s multiple comparisons test, α=0.05; p<.05; ns=not significant). d, Phase-contrast imaging (4×) of MC38 MDOTS in 3D microfluidic culture. e–f, Heatmaps of secreted cytokine profiles from cultured (e) MC38 and (f) B16F10 MDOTS expressed as log-2 fold change relative to Day 1. g, Immunofluorescence staining of CD45+ and CD8+ immune cells in MC38 MDOTS.

Article Snippet: MC38 tumor staining was performed as previously described ( 59 ) using anti-CD45 (BD Bioscience, 550539) and anti-CD8 (Synaptic System, 361 003) antibodies employing a citrate buffer pressure cooker for antigen retrieval.

Techniques: Derivative Assay, Flow Cytometry, Imaging, Cell Culture, Immunofluorescence, Staining

Journal: Cancer discovery

Article Title: Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids

doi: 10.1158/2159-8290.CD-17-0833

Figure Lengend Snippet: a, Schematic of MDOTS Live/Dead Imaging workflow. b, MC38 implanted tumor volume following isotype control IgG (n=10) or rat-anti-mouse anti-PD-1 antibody (n=10) treatment (mean ± s.e.m., 2-way ANOVA, Sidak’s multiple comparison test, **p<.01, ****p<0.0001). c, Live (AO = green)/dead (PI = red) quantification of MC38 MDOTS Day 0 (immediately after loading), Day 3, and Day 6 following IgG control or indicated anti-PD-1 antibody doses (n=4, biological replicates, 2-way ANOVA with Dunnett’s with multiple comparisons test, **p<.01, ****p<0.0001). d, Live/dead analysis of MC38 spheroids lacking immune cells ± anti-PD1 (n=4, biological replicates). e, Live/dead analysis of B16F10 MDOTS ± anti-PD1 (n=3, biological replicates). f, Deconvolution fluorescence microscopy of MC38 and B16F10 MDOTS Day 6 ± anti-PD1 (representative images shown). g, Live/dead analysis of CT26 MDOTS ± anti-PD1 (n=3, biological replicates, 2-way ANOVA with Dunnett’s with multiple comparisons test; ****p<0.0001). h, Live/dead analysis of CT26 MDOTS performed on Day 6 following treatment with isotype IgG control (10 μg/mL) or anti-PD-1 (10 μg/mL) ± anti-CD8 (10 μg/mL) (n=6, biological replicates; 2-way ANOVA with Tukey’s multiple comparisons test; ****p<.0001, ns = not significant). i, CT26 tumor volumes for responder (R1+R2) and non-responder (NR1+NR2) Balb/c mice treated with anti-PD-1 (10mg/kg twice weekly × 6 doses, starting at Day 5) with time of tumor harvest for MDOTS preparation indicated (*). j, Live/dead analysis (Day 6) of CT26 MDOTS from responder and non-responder mice following ex vivo treatment with isotype IgG control (10 μg/mL) or anti-PD-1 (10 μg/mL) (n=3, biological replicates; 2-way ANOVA with Tukey’s multiple comparisons test; ***p<.001, ns = not significant).

Article Snippet: MC38 tumor staining was performed as previously described ( 59 ) using anti-CD45 (BD Bioscience, 550539) and anti-CD8 (Synaptic System, 361 003) antibodies employing a citrate buffer pressure cooker for antigen retrieval.

Techniques: Imaging, Fluorescence, Microscopy, Ex Vivo

Journal: Cancer discovery

Article Title: Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids

doi: 10.1158/2159-8290.CD-17-0833

Figure Lengend Snippet: a, Scheme of impact of TBK1/IKKε inhibition on cytokine production from tumor cells and T cells. b, Compound 1 chemical structure with IC50 towards TBK1/IKKε, and EC50 in HCT116 cells. c, IC50 values for indicated enzymes treated with Compound 1. d, Cytokine heatmaps for CT26 spheroids (lacking immune cells) on Day 1, 3, and 6 ± Compound 1 (n=3, biological replicates) expressed as log2 fold-change (L2FC) relative to vehicle control. e–f, Dose-response curves for Compound 1 on IL-2 (e) and IFNγ (f) in human CD4 (n=3) and CD8 (n=5) T cells.

Article Snippet: MC38 tumor staining was performed as previously described ( 59 ) using anti-CD45 (BD Bioscience, 550539) and anti-CD8 (Synaptic System, 361 003) antibodies employing a citrate buffer pressure cooker for antigen retrieval.

Techniques: Inhibition

Journal: Cancer discovery

Article Title: Ex Vivo Profiling of PD-1 Blockade Using Organotypic Tumor Spheroids

doi: 10.1158/2159-8290.CD-17-0833

Figure Lengend Snippet: a, Immune profiling of PDOTS (S2; n=40) (upper panel = % live cells, lower panel = %CD45+ cells) with indicated patient/tumor characteristics, grouped by tumor type and ranked by %CD8+ T cells. b–c, Immunofluorescence staining identifying (b) CD45+ immune cells and (c) CD8+ T cells with EpCAM+ cancer cells NSCLC PDOTS. d, Immune cell correlation of S2/S3 fractions (CD45, n=14; CD3, n=15; CD4/CD8, n=13; CD4+CD45RO+, n=9; CD8+CD45RO+, n=8; activated = CD38+ and/or CD69+, n=6), R2 significant for all comparisons. e, PD-1, CTLA-4, TIM-3 expression on CD4 and CD8 T cell populations in S2/S3 fractions (n=6), R2 significant for all comparisons.

Article Snippet: MC38 tumor staining was performed as previously described ( 59 ) using anti-CD45 (BD Bioscience, 550539) and anti-CD8 (Synaptic System, 361 003) antibodies employing a citrate buffer pressure cooker for antigen retrieval.

Techniques: Immunofluorescence, Staining, Expressing

Journal: Scientific Reports

Article Title: Enhancing cancer immunotherapy using cordycepin and C ordyceps militaris extract to sensitize cancer cells and modulate immune responses

doi: 10.1038/s41598-024-72833-x

Figure Lengend Snippet: Changes in cytokines, cytotoxic mediators, and subpopulations of effector immune cells following treatment with cordycepin (CD) and Cm-EE. Non-adherent cells were exposed to 100 µM of cordycepin (CD) or 100 µg/mL Cm-EE for 24 h. Culture supernatants were collected for determination of cytokine and cytotoxic mediator production by using cytokine bead array. The fold changes in cytokine ( a ) and cytotoxic mediator ( b ) production from the cordycepin or Cm-EE treatments were compared to the appropriate control. Subpopulations of treated immune cells ( c ), including CD4 (CD3 + and CD4+), CD8 (CD3 + and CD8+), NK cells (CD3- and CD56+), and B cells (CD3- and CD19+) were characterized using specific fluorescence dye-conjugated monoclonal antibodies. Data are presented as the mean ± SEM. Statistically significant values (* p < 0.05) were determined by Student’s t-test ( N = 3).

Article Snippet: The non-adherent cells were seeded at a density of 1 × 10 6 cells/well in 12-well plate and treated with either 100 µM cordycepin or 100 µg/mL Cm-EE for 24 h. Following treatment, cells were harvested and stained with monoclonal fluorescent-conjugated antibodies, including anti-CD3 FITC (Clone: UCHT-1), anti-CD4 APC (Clone: OKT-4), anti-CD8 APC (Clone: UCHT-4), anti-CD16 APC (Clone: LNK16), anti-CD19 APC (Clone: LT19) (all sourced from Immunotools, Friesoythe, Germany), and anti-CD56 PE (Clone: HCD56; BioLegend).

Techniques: Control, Fluorescence

Journal: Clinical and Experimental Immunology

Article Title: Lymphocyte‐independent pathways underlie the pathogenesis of murine cytomegalovirus‐associated secondary haemophagocytic lymphohistiocytosis

doi: 10.1111/cei.13084

Figure Lengend Snippet: Early T cell depletion does not reduce disease severity in mouse cytomegalovirus (MCMV)‐infected wild‐type (WT) BALB/c mice. Phosphate‐buffered saline (PBS), anti‐CD4‐antibodies alone or combined with anti‐CD8‐antibodies were injected intraperitoneally on days −1 and 2 post‐infection (p.i.). (a) Percentage change in body weight relative from initial weight at day −1 p.i. Median with interquartile range of four to five mice per group. Weight change differed significantly between WT MCMV aCD4 + 8 and untreated WT MCMV on day 4 p.i. (*) and between WT MCMV aCD4 and untreated WT MCMV on day 5 p.i. (*). (b) Rectal body temperature (temp.) (°C) at day 2 p.i. Dotted line = 38·5°C. (c) Absolute platelet count in whole blood. (d) Plasma concentration of the ferritin heavy chain (ng/ml). Dots represent the average of two dilutions for one mouse. (e) Plasma concentration of sCD25 (pg/ml). (f) Plasma concentration of alanine transaminase (ALT) (IU/l). (g and h) Percentage of singlet, live lymph node‐derived CD4+ T cells positive for CD69 or CD25. (i,j) Percentage of CD4+ or CD8+ T cells in inguinal lymph nodes (LN), gated as CD4+ or CD8+ cells of live singlet cells. (k) Titre of infectious virus in spleen [plaque‐forming units (pfu) per mg tissue]. (c–k) Data obtained on day 5 p.i. (b–c,e–k) Dots represent individual animals. Horizontal bars refer to median values. aCD4 = anti‐CD4‐antibodies, aCD4 + 8 = anti‐CD4‐ and anti‐CD8 antibodies, NI = not infected; ns = not significant (P ≥ 0·05); *P < 0·05; **P < 0·01; ***P < 0·001; Kruskal–Wallis with Dunn's post‐test. Depicted data are from one experiment.

Article Snippet: To deplete CD4 + and/or CD8 + T cells, a monoclonal anti‐CD4‐antibody (clone GK1.5) and/or monoclonal anti‐CD8‐antibody (clone YTS169, both Epirus Biopharmaceuticals, Utrecht, the Netherlands) were injected i.p. as a preventive treatment (300 μg/mouse on day −1 and 200 μg/mouse on day 2 p.i. in 100 μl PBS = early depletion) or as a curative treatment (300 μg/mouse on day 2 p.i., in 100 μl PBS = late depletion).

Techniques: Infection, Injection, Concentration Assay, Derivative Assay

Journal: Clinical and Experimental Immunology

Article Title: Lymphocyte‐independent pathways underlie the pathogenesis of murine cytomegalovirus‐associated secondary haemophagocytic lymphohistiocytosis

doi: 10.1111/cei.13084

Figure Lengend Snippet: Reversal of the interleukin (IL)‐2 consumption hierarchy in mouse cytomegalovirus (MCMV)‐infected wild‐type (WT) and interferon (IFN)‐γ knock‐out (KO) mice. Median fluorescence intensity (MFI) of CD25 on CD25+CD8+, CD4+, regulatory T cells (Treg) and natural killer (NK) cells in inguinal lymph nodes in WT (a) and IFN‐γ KO mice (b). (a,b) Box‐plots represent median ±25% quartile, whiskers from minimum to maximum. White boxes, NI = not infected. Grey boxes, MCMV‐infected; five mice per group. Data obtained on day 5 post‐infection. **P < 0·01; Kruskal–Wallis with Dunn's post‐test. Depicted data are from one experiment and representative of three experiments with at least five mice per group.

Article Snippet: To deplete CD4 + and/or CD8 + T cells, a monoclonal anti‐CD4‐antibody (clone GK1.5) and/or monoclonal anti‐CD8‐antibody (clone YTS169, both Epirus Biopharmaceuticals, Utrecht, the Netherlands) were injected i.p. as a preventive treatment (300 μg/mouse on day −1 and 200 μg/mouse on day 2 p.i. in 100 μl PBS = early depletion) or as a curative treatment (300 μg/mouse on day 2 p.i., in 100 μl PBS = late depletion).

Techniques: Infection, Knock-Out, Fluorescence

Journal: Clinical and Experimental Immunology

Article Title: Lymphocyte‐independent pathways underlie the pathogenesis of murine cytomegalovirus‐associated secondary haemophagocytic lymphohistiocytosis

doi: 10.1111/cei.13084

Figure Lengend Snippet: CD25‐targeting therapy does not ameliorate the haemophagocytic lymphohistiocytosis (HLH)‐like syndrome in mouse cytomegalovirus (MCMV)‐infected wild‐type (WT) BALB/c mice. Anti‐CD25‐antibodies (blocking clone 7D4 or depleting clone PC61) or phosphate‐buffered saline (PBS) were injected intraperitoneally on day 2 post‐infection (p.i.). (a) Percentage change in body weight relative to initial weight at day 0 p.i. Median with interquartile range of five mice per group. (b) Percentage CD25‐expressing cells of live singlet inguinal lymph node (LN) cells (left panel) and their median fluorescence intensity (MFI) of CD25. Clone 3C7 was used to detect remaining CD25 expression. (c–e) Absolute lymphocyte, platelet and red blood cell (RBC) count in whole blood. (f) Percentage haemophagocytes detected in cytospins of leucocytes derived from lung. Dots represent the average of triplicate counts of 100 cells from one mouse. (g) Plasma concentration of the ferritin heavy chain (ng/ml). Dots represent the average of two dilutions for one mouse. (h) Plasma concentration of sCD25 (pg/ml). (i) Natural killer (NK) cell percentage in lung, gated as CD122+CD49b+ cells of CD3– ZombieAqua– singlet cells. (j) MFI of CD25 on CD4+ and CD8+ T cells in the inguinal lymph nodes. Clone 3C7 was used to detect remaining CD25 expression. (k) Titre of infectious virus in spleen [plaque‐forming units (pfu) per mg tissue]. (b–k) Data obtained on day 5 p.i. (b–e, h–k). Dots represent individual animals. Horizontal bars refer to median values. NI = not infected; ns = not significant (P ≥ 0·05); *P < 0·05; **P < 0·01; ***P < 0·001; Kruskal–Wallis with Dunn's post‐test. Depicted data are from one experiment.

Article Snippet: To deplete CD4 + and/or CD8 + T cells, a monoclonal anti‐CD4‐antibody (clone GK1.5) and/or monoclonal anti‐CD8‐antibody (clone YTS169, both Epirus Biopharmaceuticals, Utrecht, the Netherlands) were injected i.p. as a preventive treatment (300 μg/mouse on day −1 and 200 μg/mouse on day 2 p.i. in 100 μl PBS = early depletion) or as a curative treatment (300 μg/mouse on day 2 p.i., in 100 μl PBS = late depletion).

Techniques: Infection, Blocking Assay, Injection, Expressing, Fluorescence, Derivative Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Tumor-secreted lactate contributes to an immunosuppressive microenvironment and affects CD8 T-cell infiltration in glioblastoma

doi: 10.3389/fimmu.2023.894853

Figure Lengend Snippet: The role of CD8+ T cells in cluster 1 and cluster 2 samples. Cluster 1 and cluster 2 tumor cell communication with macrophage and CD8+ T cells through TNFSF12-TNFRSF12A (A) and GZMA-F2R (B) , respectively. (C) IHC revealed the CD8+ T-cell infiltration difference between cluster 1 and cluster 2 samples. (D) The cluster model in central nervous system tumor cell lines. (E) Lactate concentrations in U251MG, U87MG, and A172. N = 3. *P < 0.05, **P < 0.01, ***P < 0.001. (F) Percentage of migrated of CD8+ T cells after coculture with U87MG and A172 for 48 h. N = 3. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: In brief, slides were incubated with anti-CD8 antibody (66868-1-Ig, Proteintech) at 4°C overnight.

Techniques:

Journal: Journal for Immunotherapy of Cancer

Article Title: Overcoming immunotherapy resistance in bladder cancer with a novel antibody-drug conjugate RC48

doi: 10.1136/jitc-2025-011881

Figure Lengend Snippet: RC48-ADC therapy reshapes the tumor immune microenvironment in BCa. ( A ) Kaplan-Meier survival curves were generated for the anti-PD-1 cohort, stratifying patients based on ERBB2 expression levels. Kaplan-Meier plotter ( https://kmplot.com/analysis/ ) was used to generate survival curves and assess the efficacy of immunotherapy. (B) Boxplot illustrating the relationship between ERBB2 expression and immunotherapy efficacy in the Xiangya Immunotherapy Cohort. (C) The relationship between ERBB2 expression and immune scores in the TCGA-BLCA cohort. (D) Activity levels of the cancer immunity cycle across high and low ERBB2 expression groups. (E) Correlations between ERBB2 and various immune cell types (CD8+T cells, CD4+T cells, dendritic cells, and natural killer cells) analyzed using five independent algorithms (TIMER, CIBERSORT, CIBERSORT-ABS, MCP-COUNTER, and XCELL). (F) Correlations between ERBB2 and tumor-infiltrating immune cells (TIICs) (right) and the cancer immunity cycle (left). (G) Expression patterns of immune cell-related effector genes (including NK cells, Th1 cells, macrophages, CD8+T cells, and dendritic cells) in ADC-treated (n=7) and ADC-untreated (n=18) groups. (H) Cancer immunity cycle activity in the ADC-treated (n=7) and ADC-untreated (n=18) groups. *p<0.05; **p<0.01; ***p<0.001. (I) Correlations between ERBB2 and various immune cell types. *p<0.05; **p<0.01; ***p<0.001. (J) tSNE plot showing the distribution of immune cells in BCa samples from RC48- untreated and RC48-treated patients, with each dot representing a single cell and colors indicating different cell types. (K) Differences in the ratios of three CD8+T cell subgroups (exhausted CD8+T cells, cytotoxic CD8+T cells, and TRM CD8 cells) estimated using the STARTRAC-dist index in ADC-treated (n=4) and ADC-untreated (n=2) groups. +++, Ro/e>1; ++, 0.8

Article Snippet: The primary antibodies included: Anti-PD-L1 antibody ( EPR19759 ), abcam, ab213524; anti-PD-L1 antibody ( EPR20529 ), abcam, ab213480; anti-CD8 alpha antibody ( EPR21769 ), abcam, ab217344; anti-CD8 alpha antibody (CAL66), abcam, ab237709; Granzyme B Polyclonal antibody, Proteintech, 13588-1-AP; TAZ Rabbit Polyclonal Antibody, Proteintech, 23306-1-AP.

Techniques: Generated, Expressing, Activity Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Overcoming immunotherapy resistance in bladder cancer with a novel antibody-drug conjugate RC48

doi: 10.1136/jitc-2025-011881

Figure Lengend Snippet: RC48-ADC inhibits tumor PD-L1 expression, increases CD8+T cell infiltration and activity. ( A ) h-HER2-MB49 mouse bladder cancer cells overexpressing h-HER2 were injected into mice on day 0, and RC48-ADC was administered at either a high dose (HD, 10 mg/kg) or low dose (LD, 5 mg/kg) based on the indicated schedule. (B) Tumor volumes were measured at various time points, with data shown as mean±SD. (C) Representative tumor images from different RC48-ADC dose groups at the final time point in the h-HER2-MB49 tumor-bearing mouse model. (D) Tumor weights were measured on day 15 following RC48-ADC treatment. (E) Representative flow cytometry profiles showing CD8 (CTL marker) detection in h-HER2-MB49 tumors from different treatment groups. (F) Quantification of CD8+/CD3+ cells in tumors from various treatment groups (n=7 per group). (G) Representative flow cytometry profiles displaying GZMB and IFNγ, markers of T cell activity, in h-HER2-MB49 tumor tissues across different treatment groups. (H, I) Quantification of CD8+GZMB+/IFNγ+ CTL percentages in tumor tissues from different treatment groups (n=7 per group). (J) Representative flow cytometry profiles of PD-L1 detection in h-HER2-MB49 tumor tissues from the various treatment groups. (K) Quantification of PD-L1+CD45 cells in tumors from different treatment groups (n=7 mice per group). (L) Representative images of immunohistochemistry staining for CD8 and PD-L1 in h-HER2-MB49 tumors. (M) Representative immunofluorescence staining images showing CD8 and PD-L1 in h-HER2-MB49 tumors. (N, O) Quantification of CD8+ and PD-L1+cell percentages in tumors from different treatment groups (n=7 per group). (P) Representative images of immunofluorescence staining for CD8, GZMB, and PD-L1 in tumor specimens before and after RC48-ADC treatment. (Q) Quantification of CD8+, GZMB+, and PD-L1+cells in tumors RC48-ADC treated or untreated. (n=13 per group). Data are presented as mean±SD, *p<0.05, **p<0.01, ***p<0.001. ADC, antibody-drug conjugate.

Article Snippet: The primary antibodies included: Anti-PD-L1 antibody ( EPR19759 ), abcam, ab213524; anti-PD-L1 antibody ( EPR20529 ), abcam, ab213480; anti-CD8 alpha antibody ( EPR21769 ), abcam, ab217344; anti-CD8 alpha antibody (CAL66), abcam, ab237709; Granzyme B Polyclonal antibody, Proteintech, 13588-1-AP; TAZ Rabbit Polyclonal Antibody, Proteintech, 23306-1-AP.

Techniques: Expressing, Activity Assay, Injection, Flow Cytometry, Marker, Immunohistochemistry, Staining, Immunofluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Overcoming immunotherapy resistance in bladder cancer with a novel antibody-drug conjugate RC48

doi: 10.1136/jitc-2025-011881

Figure Lengend Snippet: RC48-ADC promotes the recruitment and activation of CD8+T cells by inducing the release of chemokines from tumors. ( A) h-HER2-MB49 cells were injected into mice on day 0, and treatment with RC48-ADC (10 mg/kg) and CD8α (100 µg/mouse) was administered based on the indicated schedule. (B) Tumor volume was measured at various time points. Data are presented as mean±SD. (C) Mice were sacrificed on day 15 following RC48-ADC or CD8α treatment, and tumor weights were measured. (D) Representative images of tumors at the final time point after RC48-ADC or CD8α treatment in the h-HER2-MB49 tumor-bearing mouse model. (E) Peripheral blood T cells were extracted and purified for T cell-mediated cytotoxicity assay and Chemotaxis assay. (F) Flow cytometry analysis showing the purification of CD8+T cells for chemotaxis experiments. (G, H) T24 cells were co-incubated with activated T cells for 48 hours, either with or without RC48-ADC (5 µmol), and then stained using crystal violet. The numbers represent the normalized cancer cell survival rates after T cell killing. The ratio of T cells to tumor cells was maintained at 3:1. (I) Bar graph showing the quantitative analysis of cancer cell survival rates from ( E ). (J, K) Flow cytometry analysis showing differences in CD8+T cell activity among various co-culture groups in the T24 or 5637 cell lines. (L-M) Chemotaxis assay demonstrating the different chemotaxis abilities of CTLs in the control versus RC48-ADC-treated group. (N) Bar graph showing the mRNA levels of four chemokines as determined by qRT-PCR after RC48-ADC treatment. (O) Bar graph showing normalized protein secretion concentrations of the four chemokines after RC48-ADC treatment. (P, Q) Use three different neutralizing antibodies to block the chemokines and assess the number of CD8+T cells in T24 and 5637. Data are presented as mean±SD, *p<0.05, **p<0.01, ***p<0.001. ADC, antibody-drug conjugate. FSC, Forward Scatter; SSC, Side Scatter; PBS, phosphate buffered saline.

Article Snippet: The primary antibodies included: Anti-PD-L1 antibody ( EPR19759 ), abcam, ab213524; anti-PD-L1 antibody ( EPR20529 ), abcam, ab213480; anti-CD8 alpha antibody ( EPR21769 ), abcam, ab217344; anti-CD8 alpha antibody (CAL66), abcam, ab237709; Granzyme B Polyclonal antibody, Proteintech, 13588-1-AP; TAZ Rabbit Polyclonal Antibody, Proteintech, 23306-1-AP.

Techniques: Activation Assay, Injection, Purification, Cytotoxicity Assay, Chemotaxis Assay, Flow Cytometry, Incubation, Staining, Activity Assay, Co-Culture Assay, Control, Quantitative RT-PCR, Blocking Assay, Saline

Journal: Journal for Immunotherapy of Cancer

Article Title: Overcoming immunotherapy resistance in bladder cancer with a novel antibody-drug conjugate RC48

doi: 10.1136/jitc-2025-011881

Figure Lengend Snippet: The combination of RC48-ADC and CTLA-4/PD-1 mAb has a synergistic effect in treating BCa in immunocompetent mice. ( A ) h-HER2-MB49 cells were injected into mice on day 0, and treatment with RC48-ADC (10 mg/kg) and 100 µg/mouse of PD-1 or CTLA-4 mAb was administered as indicated. (B) Tumor volume was measured at various time points. (C) Mice were sacrificed on day 15 after treatment with RC48-ADC, PD-1, or CTLA-4 mAb, and tumor weight was measured. (D) Representative tumor images at the end of the experiment after RC48-ADC, PD-1, or CTLA-4 mAb treatment in the h-HER2-MB49 tumor-bearing mouse model. (E) Immunofluorescence staining of CD8 and PD-L1 in h-HER2-MB49 tumors, showing significant differences between treatment groups. (F) Representative flow cytometry profiles detecting CD8 (CTL marker), GZMB, and IFNγ, markers of T cell activity, in h-HER2-MB49 tumors from different treatment groups. (G, H) Quantification of CD8+GZMB+/IFNγ+ CTLs and CD8+/CD3+cell percentages in tumor masses from the different treatment groups (n=5 mice per group). Data are presented as mean±SD, *p<0.05, **p<0.01, ***p<0.001. ADC, antibody-drug conjugate; BCa, bladder cancer. PBS, phosphate buffered saline.

Article Snippet: The primary antibodies included: Anti-PD-L1 antibody ( EPR19759 ), abcam, ab213524; anti-PD-L1 antibody ( EPR20529 ), abcam, ab213480; anti-CD8 alpha antibody ( EPR21769 ), abcam, ab217344; anti-CD8 alpha antibody (CAL66), abcam, ab237709; Granzyme B Polyclonal antibody, Proteintech, 13588-1-AP; TAZ Rabbit Polyclonal Antibody, Proteintech, 23306-1-AP.

Techniques: Injection, Immunofluorescence, Staining, Flow Cytometry, Marker, Activity Assay, Saline