Commensal bacteria and fungi differentially regulate tumor responses to radiation therapy

Abstract

Studies suggest that the efficacy of cancer chemotherapy and immunotherapy is influenced by intestinal bacteria. However, the influence of the microbiome on radiation therapy is not as well understood, and the microbiome comprises more than bacteria. Here, we find that intestinal fungi regulate antitumor immune responses following radiation in mouse models of breast cancer and melanoma and that fungi and bacteria have opposite influences on these responses. Antibiotic-mediated depletion or gnotobiotic exclusion of fungi enhances responsiveness to radiation, whereas antibiotic-mediated depletion of bacteria reduces responsiveness and is associated with overgrowth of commensal fungi. Further, elevated intratumoral expression of Dectin-1, a primary innate sensor of fungi, is negatively associated with survival in patients with breast cancer and is required for the effects of commensal fungi in mouse models of radiation therapy.

Keywords: T cells; bacteria; dectin-1; fungi; immunotherapy; macrophages; microbiome; mycobiome; radiation; tumor immunology.

Fig. 1.. Bacterial depletion reduces the tumor response to radiation.

Orthotopic E0771 mammary tumors (A-D) were treated with antibiotics (Abx) for one week prior to being treated with localized kV radiation (16 Gy) (RT). Both individual tumors (A, B), mean tumor burden ± SEM (C) and survival (D) for the indicated treatments were assessed every 3 days until endpoint. Antibiotics (Abx) were ampicillin, imipenem, cilastatin and vancomycin. Mean tumor burden/animal ± SEM and survival were also assessed in subcutaneous B16 melanomas (E, F) treated with antibiotics (Abx) for one week prior to being treated with localized kV irradiation (16 Gy). Antibiotics (Abx) were ampicillin, imipenem, cilastatin and vancomycin. E0771 mammary tumors were harvested at one week following RT and stained for bromodeoxyuridine (BrdU) and cleaved caspase 3 (CC3) to assess for proliferation and cell death respectively (G, H, left panels). Slides were scanned using the Aperio slide scanner and analyzed using the ScanScope nuclear algorithm included in the Aperio software package (G, H, right panels) to quantitate the number of BrdU (I) and CC3 (J) positive cells. Significance was determined by two-way ANOVA with post-hoc testing for tumor growth, Log-Rank test for the Kaplan-Meier survival curves and Student’s t-test with Welch’s correction for the immunohistochemistry (IHC). DNA from fecal pellets obtained from Abx treated mice were examined by quantitative PCR for bacterial 16S or fungal 18S ribosomal DNA and the fold change from untreated mice with standard error was calculated (K). Numbers (n) for each experiment are listed on the figure and are pooled data from at least two independent experiments. For all figures, significance is shown as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 2.. Fungal dysbiosis enhances the tumor response to radiation.

Orthotopic E0771 mammary tumors (A-D) were treated with antifungals (AF) for one week prior to being treated with localized kV irradiation (16 Gy). Both individual tumors (A, B), mean tumor burden ± SEM (C) and survival (D) were treated with their indicated treatments and assessed every 3 days until endpoint. The antifungal (AF) was fluconazole with similar results observed using 5-fluorocytosine (Supplemental Figure S2). Subcutaneous B16 melanomas (E, F) were treated with antifungals (AF) for one week prior to being treated with localized kV irradiation (16 Gy). Total tumor burden/animal was assessed every 3 days until endpoint. Mean tumor burden ± SEM (E) and survival (F) with the indicated treatments is shown. DNA from fecal pellets obtained from AF treated mice were examined by quantitative PCR for bacterial 16S and fungal 18S ribosomal DNA and the fold change from untreated mice with standard error was calculated (G). E0771 tumors from mice were harvested at one week following RT and stained for bromodeoxyuridine (BrdU) and cleaved caspase 3 (CC3) to quantitate the number of BrdU (H) and CC3 (I) positive cells to assess for proliferation and cell death respectively. Significance was determined by two-way ANOVA with post-hoc testing for tumor growth, Log-Rank test for the Kaplan-Meier survival curves and Student’s t-test with Welch’s correction for the IHC. Numbers (n) for each experiment are listed on the figure and are pooled data from at least two independent experiments. For all figures, significance is shown as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 3.. Depletion of bacteria, but not fungi, decreases antitumor immunity.

Irradiated E0771 mammary tumors were harvested from mice and dissociated at one week following radiation (RT). CD45⁺ cells were magnetically isolated and the resulting CD45⁺ cells stained with fluorescent antibodies as described in Materials and Methods and analyzed by flow cytometry. (A) Total leukocytes (CD45⁺ cells), (B) CD11b⁺F4/80⁺ macrophages, (C) CD4⁺ T cells and (D) CD8⁺ T cells were assessed following antibiotic or antifungal treatment. Both activated CD69⁺CD8⁺ T cells (E), CD25⁺CD4⁺ T cells (F) and CD11b⁺F4/80⁺MHCII⁺ macrophages (G) and immunosuppressive PD-1⁺CD8⁺ T cells (H), PD-1⁺CD4⁺ T cells (I) and CD11b⁺F4/80⁺CD206⁺ macrophages (J) were examined. Tissue sections were also examined for Granzyme B (GzmB) expressing CD8⁺ cells (K) and (L). Significance was determined by Student’s t-test with Welch’s correction. Antibiotics (Abx) were ampicillin, imipenem, cilastin and vancomycin. Fluconazole was the antifungal agent (AF) used for these experiments. n=5 per group.

Fig. 4.. Antibiotic treatment leads to overgrowth of specific fungi.

DNA from fecal samples was obtained from mice treated with antibiotics (Abx) or antifungals (AF). 16S and ITS sequencing was then performed to identify the types of bacteria (A, C, E, G) and fungal (B, D, F, H) species respectively. Samples were analyzed at the genus level for alpha diversity (Fisher’s alpha index) for both bacterial (A) and fungi (B). Data were analyzed by linear discriminant analysis effect size (LEfSe) method (Segata et al., 2011), and the results displayed as bacterial (C) and fungal (D) cladograms. Microbial prevalence was examined at the order level and pooled for analysis (E, F) or not pooled for visualization of individual samples (G, H). Sequencing data were then examined at the genus level for Saccharomyces (I) and Candida (J). The percentage of ITS transcripts for each genus was log2 transformed and plotted. Antibiotics (Abx) were ampicillin, imipenem, cilastatin and vancomycin. Fluconazole was the antifungal (AF) used for these experiments. Numbers (n) for each experiment are listed on the figure and are pooled data from at least three independent experiments.

Fig. 5.. Fungal-free mice show enhanced efficacy of RT whereas overgrowth of fungus leads to reduced efficacy.

(A) Experimental timeline for SPF mice with orthotopic E0771 mammary tumors treated with cefoperazone (Abx) and then colonized by gavage with Candida. Tumor growth (B) and survival (C) were assessed every three days following radiation (RT). (G) Experimental timeline for antifungal treatment (fluconazole) to suppress colonized C. albicans. Tumor growth (H) and survival (I) were assessed every three days following RT. (M) Experimental timeline for orthotopic E0771 tumors implanted in either normal specific-pathogen free (SPF) mice or germ-free mice colonized with fungal-free altered Schaedler flora (ASF). Tumor growth (N) and survival (O) were assessed every three days following RT. For all three experiments, the tumor infiltrating lymphocyte populations were analyzed in a separate group of mice at one week following RT by flow cytometry for PD-1 expression on cytotoxic CD8⁺ T cells (D, J, P). Cell proliferation (BrdU) (E, K, Q) and apoptosis (cleaved caspase 3, CC3) (F, L, R) were also assessed by quantitative immunohistochemistry as described in the Methods. Significance was determined by two-way ANOVA with post-hoc testing for tumor growth, Log-Rank test for the Kaplan-Meier survival curves and Student’s t-test with Welch’s correction for the IHC. Numbers (n) for each experiment are listed on the figure and are pooled data from at least three independent experiments. For all figures, significance is shown as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 6.. Dectin-1 (CLEC7A) predicts for survival in breast cancer and mediates the effect of fungal dysbiosis on RT.

Human triple-negative breast cancer (TNBC) samples were stained for Dectin-1 (A, B) and the number of positive cells quantitated (C). Expression dataset for all breast cancers from the Cancer Genome Atlas (TCGA) was analyzed for levels of Dectin-1. Dectin-1 levels were then normalized against CD11b and CD11c expression (D) and the elevated normalized Dectin-1 levels divided into high (red line) and low (green line) cohorts. Survival for these cohorts was then compared on Kaplan-Meier plots (E). Orthotopic E0771 mammary tumors were then placed in Dectin-1−/− mice and treated with antifungals (AF) for one week prior to being treated with localized kV irradiation (16 Gy). Tumor growth (G, H) and survival (I, J) were assessed every three days following radiation (RT). Significance was determined by two-way ANOVA with post-hoc testing for tumor growth and Log-Rank test for the Kaplan-Meier survival curves. Numbers (n) for each experiment are listed on the figure and are pooled data from three independent experiments. For all figures, significance is shown as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.