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Diverse chemotherapeutics synergize with CR108 to suppress 4T1 and B16-F10 tumors and remodel the immune microenvironment. ( A ) Treatment schedule for 4T1 model. Tumor-bearing BALB/c mice received a single i.v. dose of oxaliplatin (3 <t>mg</t> <t>kg</t> −1 ), temozolomide (10 mg kg −1 ), gemcitabine (20 mg kg −1 ) or pemetrexed (50 mg kg −1 ) on day 7, followed by peritumoral CR108 on days 9, 16, and 23. ( B ) TUNEL/DAPI staining of 4T1 tumors 48 h after chemotherapy. Red, TUNEL; blue, DAPI; merge, bottom row; 40× magnification. All drugs induce extensive apoptosis/necrosis, with gemcitabine and pemetrexed showing the densest signal. ( C ) Endpoint tumor mass for 4T1 (day 30). All combination regimens significantly reduce tumor weight compared with PBS controls. ( D ) Treatment schedule for B16-F10 model. C57BL/6 mice were implanted with 3 × 10 5 B16-F10 cells (day 0), infused intravenously with cisplatin, oxaliplatin, temozolomide, gemcitabine, or pemetrexed on day 7, and injected with CR108 on days 9, 16 and 23. ( E ) TUNEL/DAPI staining of B16-F10 tumors 48 h after chemotherapy. All drug + CR108 combinations yield broad TUNEL positivity, whereas PBS lesions are largely negative. ( F ) Endpoint tumor mass for B16-F10 (day 30). Each combination markedly inhibits tumor growth ( p < 0.05 to p < 0.001, one-way ANOVA with Tukey post-test). ( G ) Immune-cell composition of 4T1 tumors on day 30. Flow cytometry of single-cell suspensions shows increases in B cells, plasma cells, and migratory dendritic cells (migDCs), the latter most pronounced with gemcitabine and temozolomide. IFN-γ + CD8 + T cells rise significantly with cisplatin, gemcitabine, and oxaliplatin, whereas IFN-γ + CD4 + T cells increase chiefly after gemcitabine. Data are mean ± s.d. ( n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001).
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Diverse chemotherapeutics synergize with CR108 to suppress 4T1 and B16-F10 tumors and remodel the immune microenvironment. ( A ) Treatment schedule for 4T1 model. Tumor-bearing BALB/c mice received a single i.v. dose of oxaliplatin (3 <t>mg</t> <t>kg</t> −1 ), temozolomide (10 mg kg −1 ), gemcitabine (20 mg kg −1 ) or pemetrexed (50 mg kg −1 ) on day 7, followed by peritumoral CR108 on days 9, 16, and 23. ( B ) TUNEL/DAPI staining of 4T1 tumors 48 h after chemotherapy. Red, TUNEL; blue, DAPI; merge, bottom row; 40× magnification. All drugs induce extensive apoptosis/necrosis, with gemcitabine and pemetrexed showing the densest signal. ( C ) Endpoint tumor mass for 4T1 (day 30). All combination regimens significantly reduce tumor weight compared with PBS controls. ( D ) Treatment schedule for B16-F10 model. C57BL/6 mice were implanted with 3 × 10 5 B16-F10 cells (day 0), infused intravenously with cisplatin, oxaliplatin, temozolomide, gemcitabine, or pemetrexed on day 7, and injected with CR108 on days 9, 16 and 23. ( E ) TUNEL/DAPI staining of B16-F10 tumors 48 h after chemotherapy. All drug + CR108 combinations yield broad TUNEL positivity, whereas PBS lesions are largely negative. ( F ) Endpoint tumor mass for B16-F10 (day 30). Each combination markedly inhibits tumor growth ( p < 0.05 to p < 0.001, one-way ANOVA with Tukey post-test). ( G ) Immune-cell composition of 4T1 tumors on day 30. Flow cytometry of single-cell suspensions shows increases in B cells, plasma cells, and migratory dendritic cells (migDCs), the latter most pronounced with gemcitabine and temozolomide. IFN-γ + CD8 + T cells rise significantly with cisplatin, gemcitabine, and oxaliplatin, whereas IFN-γ + CD4 + T cells increase chiefly after gemcitabine. Data are mean ± s.d. ( n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001).
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Diverse chemotherapeutics synergize with CR108 to suppress 4T1 and B16-F10 tumors and remodel the immune microenvironment. ( A ) Treatment schedule for 4T1 model. Tumor-bearing BALB/c mice received a single i.v. dose of oxaliplatin (3 <t>mg</t> <t>kg</t> −1 ), temozolomide (10 mg kg −1 ), gemcitabine (20 mg kg −1 ) or pemetrexed (50 mg kg −1 ) on day 7, followed by peritumoral CR108 on days 9, 16, and 23. ( B ) TUNEL/DAPI staining of 4T1 tumors 48 h after chemotherapy. Red, TUNEL; blue, DAPI; merge, bottom row; 40× magnification. All drugs induce extensive apoptosis/necrosis, with gemcitabine and pemetrexed showing the densest signal. ( C ) Endpoint tumor mass for 4T1 (day 30). All combination regimens significantly reduce tumor weight compared with PBS controls. ( D ) Treatment schedule for B16-F10 model. C57BL/6 mice were implanted with 3 × 10 5 B16-F10 cells (day 0), infused intravenously with cisplatin, oxaliplatin, temozolomide, gemcitabine, or pemetrexed on day 7, and injected with CR108 on days 9, 16 and 23. ( E ) TUNEL/DAPI staining of B16-F10 tumors 48 h after chemotherapy. All drug + CR108 combinations yield broad TUNEL positivity, whereas PBS lesions are largely negative. ( F ) Endpoint tumor mass for B16-F10 (day 30). Each combination markedly inhibits tumor growth ( p < 0.05 to p < 0.001, one-way ANOVA with Tukey post-test). ( G ) Immune-cell composition of 4T1 tumors on day 30. Flow cytometry of single-cell suspensions shows increases in B cells, plasma cells, and migratory dendritic cells (migDCs), the latter most pronounced with gemcitabine and temozolomide. IFN-γ + CD8 + T cells rise significantly with cisplatin, gemcitabine, and oxaliplatin, whereas IFN-γ + CD4 + T cells increase chiefly after gemcitabine. Data are mean ± s.d. ( n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001).
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Diverse chemotherapeutics synergize with CR108 to suppress 4T1 and B16-F10 tumors and remodel the immune microenvironment. ( A ) Treatment schedule for 4T1 model. Tumor-bearing BALB/c mice received a single i.v. dose of oxaliplatin (3 <t>mg</t> <t>kg</t> −1 ), temozolomide (10 mg kg −1 ), gemcitabine (20 mg kg −1 ) or pemetrexed (50 mg kg −1 ) on day 7, followed by peritumoral CR108 on days 9, 16, and 23. ( B ) TUNEL/DAPI staining of 4T1 tumors 48 h after chemotherapy. Red, TUNEL; blue, DAPI; merge, bottom row; 40× magnification. All drugs induce extensive apoptosis/necrosis, with gemcitabine and pemetrexed showing the densest signal. ( C ) Endpoint tumor mass for 4T1 (day 30). All combination regimens significantly reduce tumor weight compared with PBS controls. ( D ) Treatment schedule for B16-F10 model. C57BL/6 mice were implanted with 3 × 10 5 B16-F10 cells (day 0), infused intravenously with cisplatin, oxaliplatin, temozolomide, gemcitabine, or pemetrexed on day 7, and injected with CR108 on days 9, 16 and 23. ( E ) TUNEL/DAPI staining of B16-F10 tumors 48 h after chemotherapy. All drug + CR108 combinations yield broad TUNEL positivity, whereas PBS lesions are largely negative. ( F ) Endpoint tumor mass for B16-F10 (day 30). Each combination markedly inhibits tumor growth ( p < 0.05 to p < 0.001, one-way ANOVA with Tukey post-test). ( G ) Immune-cell composition of 4T1 tumors on day 30. Flow cytometry of single-cell suspensions shows increases in B cells, plasma cells, and migratory dendritic cells (migDCs), the latter most pronounced with gemcitabine and temozolomide. IFN-γ + CD8 + T cells rise significantly with cisplatin, gemcitabine, and oxaliplatin, whereas IFN-γ + CD4 + T cells increase chiefly after gemcitabine. Data are mean ± s.d. ( n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001).
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Diverse chemotherapeutics synergize with CR108 to suppress 4T1 and B16-F10 tumors and remodel the immune microenvironment. ( A ) Treatment schedule for 4T1 model. Tumor-bearing BALB/c mice received a single i.v. dose of oxaliplatin (3 <t>mg</t> <t>kg</t> −1 ), temozolomide (10 mg kg −1 ), gemcitabine (20 mg kg −1 ) or pemetrexed (50 mg kg −1 ) on day 7, followed by peritumoral CR108 on days 9, 16, and 23. ( B ) TUNEL/DAPI staining of 4T1 tumors 48 h after chemotherapy. Red, TUNEL; blue, DAPI; merge, bottom row; 40× magnification. All drugs induce extensive apoptosis/necrosis, with gemcitabine and pemetrexed showing the densest signal. ( C ) Endpoint tumor mass for 4T1 (day 30). All combination regimens significantly reduce tumor weight compared with PBS controls. ( D ) Treatment schedule for B16-F10 model. C57BL/6 mice were implanted with 3 × 10 5 B16-F10 cells (day 0), infused intravenously with cisplatin, oxaliplatin, temozolomide, gemcitabine, or pemetrexed on day 7, and injected with CR108 on days 9, 16 and 23. ( E ) TUNEL/DAPI staining of B16-F10 tumors 48 h after chemotherapy. All drug + CR108 combinations yield broad TUNEL positivity, whereas PBS lesions are largely negative. ( F ) Endpoint tumor mass for B16-F10 (day 30). Each combination markedly inhibits tumor growth ( p < 0.05 to p < 0.001, one-way ANOVA with Tukey post-test). ( G ) Immune-cell composition of 4T1 tumors on day 30. Flow cytometry of single-cell suspensions shows increases in B cells, plasma cells, and migratory dendritic cells (migDCs), the latter most pronounced with gemcitabine and temozolomide. IFN-γ + CD8 + T cells rise significantly with cisplatin, gemcitabine, and oxaliplatin, whereas IFN-γ + CD4 + T cells increase chiefly after gemcitabine. Data are mean ± s.d. ( n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001).
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Diverse chemotherapeutics synergize with CR108 to suppress 4T1 and B16-F10 tumors and remodel the immune microenvironment. ( A ) Treatment schedule for 4T1 model. Tumor-bearing BALB/c mice received a single i.v. dose of oxaliplatin (3 <t>mg</t> <t>kg</t> −1 ), temozolomide (10 mg kg −1 ), gemcitabine (20 mg kg −1 ) or pemetrexed (50 mg kg −1 ) on day 7, followed by peritumoral CR108 on days 9, 16, and 23. ( B ) TUNEL/DAPI staining of 4T1 tumors 48 h after chemotherapy. Red, TUNEL; blue, DAPI; merge, bottom row; 40× magnification. All drugs induce extensive apoptosis/necrosis, with gemcitabine and pemetrexed showing the densest signal. ( C ) Endpoint tumor mass for 4T1 (day 30). All combination regimens significantly reduce tumor weight compared with PBS controls. ( D ) Treatment schedule for B16-F10 model. C57BL/6 mice were implanted with 3 × 10 5 B16-F10 cells (day 0), infused intravenously with cisplatin, oxaliplatin, temozolomide, gemcitabine, or pemetrexed on day 7, and injected with CR108 on days 9, 16 and 23. ( E ) TUNEL/DAPI staining of B16-F10 tumors 48 h after chemotherapy. All drug + CR108 combinations yield broad TUNEL positivity, whereas PBS lesions are largely negative. ( F ) Endpoint tumor mass for B16-F10 (day 30). Each combination markedly inhibits tumor growth ( p < 0.05 to p < 0.001, one-way ANOVA with Tukey post-test). ( G ) Immune-cell composition of 4T1 tumors on day 30. Flow cytometry of single-cell suspensions shows increases in B cells, plasma cells, and migratory dendritic cells (migDCs), the latter most pronounced with gemcitabine and temozolomide. IFN-γ + CD8 + T cells rise significantly with cisplatin, gemcitabine, and oxaliplatin, whereas IFN-γ + CD4 + T cells increase chiefly after gemcitabine. Data are mean ± s.d. ( n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001).
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Diverse chemotherapeutics synergize with CR108 to suppress 4T1 and B16-F10 tumors and remodel the immune microenvironment. ( A ) Treatment schedule for 4T1 model. Tumor-bearing BALB/c mice received a single i.v. dose of oxaliplatin (3 mg kg −1 ), temozolomide (10 mg kg −1 ), gemcitabine (20 mg kg −1 ) or pemetrexed (50 mg kg −1 ) on day 7, followed by peritumoral CR108 on days 9, 16, and 23. ( B ) TUNEL/DAPI staining of 4T1 tumors 48 h after chemotherapy. Red, TUNEL; blue, DAPI; merge, bottom row; 40× magnification. All drugs induce extensive apoptosis/necrosis, with gemcitabine and pemetrexed showing the densest signal. ( C ) Endpoint tumor mass for 4T1 (day 30). All combination regimens significantly reduce tumor weight compared with PBS controls. ( D ) Treatment schedule for B16-F10 model. C57BL/6 mice were implanted with 3 × 10 5 B16-F10 cells (day 0), infused intravenously with cisplatin, oxaliplatin, temozolomide, gemcitabine, or pemetrexed on day 7, and injected with CR108 on days 9, 16 and 23. ( E ) TUNEL/DAPI staining of B16-F10 tumors 48 h after chemotherapy. All drug + CR108 combinations yield broad TUNEL positivity, whereas PBS lesions are largely negative. ( F ) Endpoint tumor mass for B16-F10 (day 30). Each combination markedly inhibits tumor growth ( p < 0.05 to p < 0.001, one-way ANOVA with Tukey post-test). ( G ) Immune-cell composition of 4T1 tumors on day 30. Flow cytometry of single-cell suspensions shows increases in B cells, plasma cells, and migratory dendritic cells (migDCs), the latter most pronounced with gemcitabine and temozolomide. IFN-γ + CD8 + T cells rise significantly with cisplatin, gemcitabine, and oxaliplatin, whereas IFN-γ + CD4 + T cells increase chiefly after gemcitabine. Data are mean ± s.d. ( n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001).

Journal: Cancers

Article Title: Chemotherapy Liberates a Broadening Repertoire of Tumor Antigens for TLR7/8/9-Mediated Potent Antitumor Immunity

doi: 10.3390/cancers17193277

Figure Lengend Snippet: Diverse chemotherapeutics synergize with CR108 to suppress 4T1 and B16-F10 tumors and remodel the immune microenvironment. ( A ) Treatment schedule for 4T1 model. Tumor-bearing BALB/c mice received a single i.v. dose of oxaliplatin (3 mg kg −1 ), temozolomide (10 mg kg −1 ), gemcitabine (20 mg kg −1 ) or pemetrexed (50 mg kg −1 ) on day 7, followed by peritumoral CR108 on days 9, 16, and 23. ( B ) TUNEL/DAPI staining of 4T1 tumors 48 h after chemotherapy. Red, TUNEL; blue, DAPI; merge, bottom row; 40× magnification. All drugs induce extensive apoptosis/necrosis, with gemcitabine and pemetrexed showing the densest signal. ( C ) Endpoint tumor mass for 4T1 (day 30). All combination regimens significantly reduce tumor weight compared with PBS controls. ( D ) Treatment schedule for B16-F10 model. C57BL/6 mice were implanted with 3 × 10 5 B16-F10 cells (day 0), infused intravenously with cisplatin, oxaliplatin, temozolomide, gemcitabine, or pemetrexed on day 7, and injected with CR108 on days 9, 16 and 23. ( E ) TUNEL/DAPI staining of B16-F10 tumors 48 h after chemotherapy. All drug + CR108 combinations yield broad TUNEL positivity, whereas PBS lesions are largely negative. ( F ) Endpoint tumor mass for B16-F10 (day 30). Each combination markedly inhibits tumor growth ( p < 0.05 to p < 0.001, one-way ANOVA with Tukey post-test). ( G ) Immune-cell composition of 4T1 tumors on day 30. Flow cytometry of single-cell suspensions shows increases in B cells, plasma cells, and migratory dendritic cells (migDCs), the latter most pronounced with gemcitabine and temozolomide. IFN-γ + CD8 + T cells rise significantly with cisplatin, gemcitabine, and oxaliplatin, whereas IFN-γ + CD4 + T cells increase chiefly after gemcitabine. Data are mean ± s.d. ( n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Systemic arm—a single tail-vein injection of one of four chemotherapeutics (oxaliplatin 3 mg kg −1 , gemcitabine 20 mg kg −1 , temozolomide 10 mg kg −1 , or pemetrexed 50 mg kg −1 ; all from MCE).

Techniques: TUNEL Assay, Staining, Injection, Flow Cytometry, Clinical Proteomics