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: Diabetologia

Article Title: Phenotypically distinct anti-insulin B cells repopulate pancreatic islets after anti-CD20 treatment in NOD mice

doi: 10.1007/s00125-019-04974-y

Figure Lengend Snippet: Anti-insulin B cells repopulate pancreatic islets more rapidly than insulin-negative B cells after anti-CD20 treatment. Groups of VH125.hCD20/NOD mice, aged 6–8 weeks old, were injected with 2H7 anti-CD20 or IgG isotype control. Spleen, PLNs and pancreatic islets were analysed for insulin-positive and insulin-negative B cells at 8 weeks and 12 weeks post depletion by flow cytometry. ( a – f ) No. of cells from IgG control-treated (black circles) and 2H7-treated (grey squares) mice for insulin-negative B cells ( a – c ) and insulin-positive B cells ( d – f ) from spleen ( a , d ) PLNs ( b , e ) and islets ( c , f ). ( g – i ) Percentage of B cells repopulated at 8 and 12 weeks after treatment from spleen ( g ), PLNs ( h ) and islets ( i ) of mice shown in ( a – f ). Percentages were calculated as individual numbers from each 2H7-treated mouse / mean number from all IgG control antibody-treated mice. Horizontal lines represent the median value. Data represent three independent experiments. At 8 weeks, n = 7 (spleen), n = 7 (PLNs) and n = 9 (islets) for control IgG-treated mice and n = 10 (spleen), n = 9 (PLNs) and n = 12 (islets) for 2H7-treated mice. At 12 weeks, n = 8 (spleen), n = 6 (PLNs) and n = 7 (islets) for control IgG and n = 11 (spleen), n = 7 (PLNs) and n = 11 (islets) for 2H7-treated mice. * p < 0.05 (one-way ANOVA)

Article Snippet: Female VH125.hCD20/NOD mice, 6–8 weeks of age were chosen at random to receive either anti-hCD20 antibody (clone 2H7; Bio-XCell, West Lebanon, NH, USA) or control IgG2b antibody (clone MPC-11; Bio-XCell [ , , ]), as described previously [ ].

Techniques: Injection, Control, Flow Cytometry

Journal: Diabetologia

Article Title: Phenotypically distinct anti-insulin B cells repopulate pancreatic islets after anti-CD20 treatment in NOD mice

doi: 10.1007/s00125-019-04974-y

Figure Lengend Snippet: CD138 int anti-insulin B cells are enriched in pancreatic islets after anti-CD20 treatment. Groups of 6- to 8-week-old VH125.hCD20/NOD mice were injected with 2H7 anti-CD20 or IgG isotype control. Groups of mice ( n = 2 or 3 per group) were pooled and insulin + B cells from pancreatic islets were analysed for four different populations based on CD138 expression: CD138 − (blue); CD138 int IgM + (orange); CD138 int IgM lo (grey) and CD138 hi IgM lo (red). ( a , b ) Representative flow plots showing gating on live CD3 − CD11b − CD11c − ( a ) and graph showing the overall percentages of the four different populations ( b ). ( c , d ) Representative flow plots showing insulin − CD19 + , insulin + CD19 + and insulin + CD19 − cells ( c ) and graph showing the overall percentages of these cells ( d ); 2H7 (black circles), IgG (grey circles). ( e ) Representative flow plots showing CD138 and IgM expression in insulin + CD19 + and insulin + CD19 − cells. ( f , g ) Graphs showing CD138 and IgM populations on insulin + CD19 + ( f ) and insulin + CD19 − cells ( g ) ( n = 5 groups for control IgG treatment; n = 4 groups for 2H7 treatment). Horizontal lines represent the median values. Data represent two independent experiments. * p < 0.05 (one-way ANOVA)

Article Snippet: Female VH125.hCD20/NOD mice, 6–8 weeks of age were chosen at random to receive either anti-hCD20 antibody (clone 2H7; Bio-XCell, West Lebanon, NH, USA) or control IgG2b antibody (clone MPC-11; Bio-XCell [ , , ]), as described previously [ ].

Techniques: Injection, Control, Expressing

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