Journal: PLOS One

Article Title: An in vitro tumor recurrence model based on platinum-resistant colon cancer cells as a research tool for studying cancer cell dormancy

doi: 10.1371/journal.pone.0333671

Figure Lengend Snippet: (A,B) LysoTracker-positive acidic lysosomal compartments in quiescent cells. Oxaliplatin-resistant HCT116 oxpl-R cells (A) and cisplatin-resistant HCT116 cspl-R cells (B) before (day 0) and after (day 8) platinum treatment (oxaliplatin or cisplatin, respectively), stained with LysoTracker Green™ and MitoTracker Orange™. Scale bars represent 50 µm. (C,D) Dynamics of autophagy marker expression in the in vitro cancer reсurrence model based on cisplatin-resistant colon cancer cells HCT116 cspl-R, analyzed by immunoblotting (C) and qPCR (D) . (C) Immunoblots probed with LC3, Beclin and Survivin antibodies. α-Tubulin was used as a loading control. Quantification values shown beneath each lane represent band intensity normalized to loading control and relative to day 0 control (set as 1.0). (D) Normalized expression of autophagy-related genes ( lc3, beclin ) after cisplatin exposure (days 0-33), relative to day 0. Expression of the GAPDH gene served as the endogenous control. Data represent biological triplicate experiments and are displayed as mean ± SEM.

Article Snippet: The primary antibodies used were against LC3 #4108 RRID: AB_2137703, Beclin #3738 RRID:AB_490837, Survivin #2808 RRID:AB_2063948 (Cell Signaling, USA), Cyclin A sc-751 RRID:AB_631329, and p27 (C-19) sc-528 RRID:AB_632129 (Santa Cruz Biotechnology, USA) with ɑ-Tubulin sc-32293 RRID:AB_628412 (Santa Cruz Biotechnology, USA) serving as a loading control.

Techniques: Staining, Marker, Expressing, In Vitro, Western Blot, Control

Journal: Journal for Immunotherapy of Cancer

Article Title: Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

doi: 10.1136/jitc-2023-006846

Figure Lengend Snippet: Tuning of CD73 blockade is required to improve the response of low CD73-expressing MC38 tumors to IR. (A) MC38 tumor cells were injected subcutaneously in C57BL/6 mice and when tumors reached 80–100 mm 3 , tumor were irradiated at 12 Gy, and mice were treated with anti-CD73 commencing 1 day before IR then 2, 6 and 9 day post-IR. (B) Tumor growth was monitored in treated mice. (C) The Kaplan-Meier survival curves for the treated mice are shown. Data were obtained from two independent experiments and are represented as the mean±SEM. n=11–12, *p<0.05 ****p<0.0001 (two-way ANOVA). (D, right panel) Representative histograms of CD73 expression in MC38 and TS/A cells 24-hour post-IR at 6 and 12 Gy compared with non-irradiated (NIR) cells. (D, left panel) cultured cells were analyzed by flow cytometry for their CD73 expression, which is represented as the mean fluorescence intensity (∆MFI=MFI of the isotype control - MFI of stained cells). Data were obtained from two independent experiments and are represented as the mean±SEM. n=6, **p<0.01, ***p<0.001, ****p<0.0001 (two-way ANOVA). (E) TS/A tumor cells were injected subcutaneously in BALB/c mice and when tumors reached 60–70 mm 3 , tumor were irradiated at 12 Gy, and mice were treated with anti-CD73 commencing 1 day before IR then 2, 6 and 9 days post-IR. (F) Tumor growth was monitored in treated mice. (G) The Kaplan-Meier survival curves for the treated mice are shown. (I) Tumor growth is shown for individual mice in each treatment group. Data were obtained from two independent experiments and are represented as the mean±SEM. n=12–13, **p<0.01 (two-way ANOVA). ANOVA, analysis of variance; IgG, immunoglobulin G; IR, irradiation.

Article Snippet: Anti-iCOS mAb (clone 7E.17G9) and the Rat IgG2b isotype control were purchased from Bio X Cell and i.p injected at 100 ug/mouse starting 2 days post-IR and then 6 and 9 days post-IR for a total of three injections.

Techniques: Expressing, Injection, Irradiation, Cell Culture, Flow Cytometry, Fluorescence, Control, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

doi: 10.1136/jitc-2023-006846

Figure Lengend Snippet: CD73 expression level controls the MC38 tumor response to CD73 blockade treatment. (A) Representative histograms of transfected and non-transfected MC38 cell with CD73 gene analyzed by flow cytometry. (B) Cultured MC38 control (Ctrl) and CD73 high MC38 cells were analyzed by flow cytometry 24-hour post-IR at 12 Gy for their CD73 membrane expression, which is represented as the mean fluorescence intensity (∆MFI=MFI of the isotype control - MFI of stained cells). Data were obtained from two independent experiments and are represented as the mean±SEM. n=6, ***p<0.001, ****p<0.0001 (one-way ANOVA). (C) CD73 high MC38 tumor cells were injected subcutaneously in C57BL/6 mice and when tumors reached 80–100 mm 3 , tumor were irradiated at 12 Gy, and mice were treated with anti-CD73 starting 1 day before IR then 2, 6 and 9 days post-IR. (D, F) Tumor growth was monitored in treated mice. (E, G) The Kaplan-Meier survival curves for the treated mice are shown. Data were obtained from two independent experiments and are represented as the mean±SEM. n=13–14, *p<0.05, ***p<0.001 (two-way ANOVA). ANOVA, analysis of variance; IgG, immunoglobulin G; IR, irradiation; NIR, non-irradiated; ssc-a, side scatter-a.

Article Snippet: Anti-iCOS mAb (clone 7E.17G9) and the Rat IgG2b isotype control were purchased from Bio X Cell and i.p injected at 100 ug/mouse starting 2 days post-IR and then 6 and 9 days post-IR for a total of three injections.

Techniques: Expressing, Transfection, Flow Cytometry, Cell Culture, Control, Membrane, Fluorescence, Staining, Injection, Irradiation

Journal: Journal for Immunotherapy of Cancer

Article Title: Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

doi: 10.1136/jitc-2023-006846

Figure Lengend Snippet: CD73 blockade treatment regimen affects the expression level of iCOS in tumor infiltrating CD4 + T lymphocytes. C57BL/6 mice with the subcutaneous MC38 tumors and BALB/c mice with subcutaneous TS/A tumors were irradiated and treated with either one dose or four doses of anti-CD73 starting 1 day before IR, then 2, 6 and 9 days post-IR. At day 10 post-IR, tumors were harvested and analyzed for immune infiltrating tumor cells by flow cytometry. CD4 + T lymphocytes infiltrating MC38 tumor were analyzed by flow cytometry for iCOS (A, left panel) membrane expression, which is represented as the mean fluorescence intensity (∆MFI=MFI of the isotype control - MFI of stained cells). (A, right panel) Representative histograms of iCOS expression, in CD4 + T lymphocytes infiltrating MC38 tumors. (B, left panel) The percentages of iCOS + CD4 + T lymphocytes infiltrating MC38 tumor are presented for each treatment group. (B, right panel) Representative histograms of iCOS + CD4 + T lymphocytes infiltrating MC38 tumor are presented for each treatment group. Data were obtained from two independent experiments and are represented as the mean±SEM. n=8–10, *p<0.05 (two-way ANOVA). CD4 + T lymphocytes infiltrating MC38 CD73 high tumors were analyzed by flow cytometry for iCOS (C, left panel) membrane expression, which is represented as the mean fluorescence intensity (∆MFI=MFI of the isotype control - MFI of stained cells). (C, right panel) Representative histograms of iCOS expression, in CD4 + T lymphocytes infiltrating MC38 CD73 high tumors. (D) The percentages of iCOS + CD4 + T lymphocytes infiltrating MC38 CD73 high tumors are presented for each treatment group. CD4 + T lymphocytes infiltrating TS/A tumor were analyzed by flow cytometry for their iCOS (E, left panel) membrane expression, which is represented as the mean fluorescence intensity (∆MFI=MFI of the isotype control - MFI of stained cells). (E, right panel) Representative histograms of iCOS expression in CD4 + T lymphocytes infiltrating TS/A tumor. (F, left panel) The percentages of iCOS + CD4 + T lymphocytes infiltrating TS/A tumor are presented for each treatment group. (F, right panel) Representative histograms of iCOS + CD4 + T lymphocytes infiltrating MC38 tumor are presented for each treatment group. Data were obtained from two independent experiments and are represented as the mean±SEM. n=4–8, *p<0.05 (two-way ANOVA). ANOVA, analysis of variance; IgG, immunoglobulin G; IR, irradiation.

Article Snippet: Anti-iCOS mAb (clone 7E.17G9) and the Rat IgG2b isotype control were purchased from Bio X Cell and i.p injected at 100 ug/mouse starting 2 days post-IR and then 6 and 9 days post-IR for a total of three injections.

Techniques: Expressing, Irradiation, Flow Cytometry, Membrane, Fluorescence, Control, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

doi: 10.1136/jitc-2023-006846

Figure Lengend Snippet: iCOS signaling is involved in CD73 blockade-mediated antitumor effect in MC38 tumor model. (A) MC38 tumor cells were injected subcutaneously in C57BL/6 mice and when tumors reached 80–100 mm 3 , tumor were irradiated at 12 Gy, and mice were treated with anti-CD73 (starting 1 day before IR then 2, 6 and 9 days post-IR) and anti-iCOS (starting 2 days post IR, then 6 and 9 days post-IR). (B and D) Tumor growth was monitored in treated mice. (C and E) The Kaplan-Meier survival curves for the treated mice are shown. (F) Tumor growth is shown for individual mice in each treatment group. Data were obtained from two independent experiments and are represented as the mean±SEM. n=6–7, *p<0.05, ****p<0.001, ***p<0.0001 (two-way ANOVA). ANOVA, analysis of variance; IgG, immunoglobulin G; IR, irradiation.

Article Snippet: Anti-iCOS mAb (clone 7E.17G9) and the Rat IgG2b isotype control were purchased from Bio X Cell and i.p injected at 100 ug/mouse starting 2 days post-IR and then 6 and 9 days post-IR for a total of three injections.

Techniques: Injection, Irradiation

Journal: Journal for Immunotherapy of Cancer

Article Title: Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

doi: 10.1136/jitc-2023-006846

Figure Lengend Snippet: One dose of aCD73 improves the antitumor effect of aPD-L1 and IR treatment in MC38 tumor model. (A) MC38 tumor cells were injected subcutaneously in C57BL/6 mice and when tumors reached 80–100 mm 3 , tumor were irradiated at 12 Gy, and mice were treated with one dose of anti-CD73 (starting 1 day before IR) and anti-PD-L1 (starting the same day as IR, then 3, 6 and 9 days post-IR). (B) Tumor growth was monitored in treated mice. (C) The Kaplan-Meier survival curves for the treated mice are shown. (D) Tumor growth is shown for individual mice in each treatment group. Data were obtained from two independent experiments and are represented as the mean±SEM. n=11–14, *p<0.05, **p<0.01, ****p<0.0001 (two-way ANOVA). ANOVA, analysis of variance; IgG, immunoglobulin G; IR, irradiation.

Article Snippet: Anti-iCOS mAb (clone 7E.17G9) and the Rat IgG2b isotype control were purchased from Bio X Cell and i.p injected at 100 ug/mouse starting 2 days post-IR and then 6 and 9 days post-IR for a total of three injections.

Techniques: Injection, Irradiation