Cutibacterium acnes-derived extracellular vesicles promote tumor growth in renal cell carcinoma

Abstract

Bacterial flora are present in various parts of the human body, including the intestine, and are thought to be involved in the etiology of various diseases such as multiple sclerosis, intestinal diseases, cancer, and uterine diseases. In recent years, the presence of bacterial 16S rRNA genes has been revealed in blood, which was previously thought to be a sterile environment, and characteristic blood microbiomes have been detected in various diseases. However, the mechanism and the origin of the bacterial information are unknown. In this study, we performed 16S rRNA metagenomic analysis of bacterial DNA in serum extracellular vesicles from five healthy donors and seven patients with renal cell carcinoma and detected Cutibacterium acnes DNA as a characteristic bacterial DNA in the serum extracellular vesicles of patients with renal cell carcinoma. In addition, C. acnes DNA was significantly reduced in postoperative serum extracellular vesicles from patients with renal cell carcinoma compared with that in preoperative serum extracellular vesicles from these patients and was also detected in tumor tissue and extracellular vesicles from tumor tissue-associated microbiota, suggesting an association between C. acnes extracellular vesicles and renal cell carcinoma. C. acnes extracellular vesicles were taken up by renal carcinoma cells to enhance their proliferative potential. C. acnes extracellular vesicles also exhibited tumor-promoting activity in a mouse model of renal cancer allografts with enhanced angiogenesis. These results suggest that extracellular vesicles released by C. acnes localized in renal cell carcinoma tissues act in a tumor-promoting manner.

Keywords: Cutibacterium acnes; 16S rRNA gene; bacterial DNA; extracellular vesicles; renal cell carcinoma.

FIGURE 1

Cutibacterium acnes genomic DNA is enriched in renal cell carcinoma (RCC) patient serum extracellular vesicles (EVs). (A) Taxa bar plots of healthy donors (HD) and RCC serum EVs. Results of diversity analysis of Fisher's α diversity (B), Shannon index (C), and Chao1 (D) of HD and RCC serum EVs. (E) Observed operational taxonomic units (OTUs). Number of the samples is given in parentheses. (F) Heat tree analysis leverages the hierarchical structure of taxonomic classifications to quantitatively (using median abundance) and statistically (using non‐parametric Wilcoxon rank‐sum test) depict taxonomic differences between microbial communities in RCC and HD serum EVs (RCC vs. HD). (G) Distinctive bacterial species between RCC and HD and the percentage of species abundance of each species. LDA: linear discriminant analysis. (H) Percentage of C. acnes abundance. Number of the samples is given in parentheses. Mann–Whitney test (two tailed).

FIGURE 2

16S ribosomal RNA metagenomic analysis of pre‐ and postoperative serum extracellular vesicles (EVs). (A) Taxa bar plots of pre‐ and postoperative serum EVs. Results of diversity analysis of Fisher's α diversity (B), Shannon index (C), and Chao1 (D) of pre‐ and postoperative serum EVs. (E) Observed operational taxonomic units (OTUs). Number of the samples is given in parentheses. (F) Heat tree analysis leverages the hierarchical structure of taxonomic classifications to quantitatively (using median abundance) and statistically (using nonparametric Wilcoxon rank‐sum test) depict taxonomic differences between microbial communities in pre‐ and postoperative serum EVs (pre vs. post). (G) Distinctive bacterial species between pre‐ and postoperative serum EVs and the percentage of species abundance of each species. LDA: linear discriminant analysis. Percentage of C. acnes abundance in serum EVs (H) and serum (I). Number of the samples is given in parentheses. Mann–Whitney test (two tailed).

FIGURE 3

Cutibacterium acnes releases extracellular vesicles (EVs) with its genomic DNA. (A) A representative image of C. acnes culture. (B) Electron microscopic image of C. acnes EVs. Black bars indicate 200 nm. A representative image of three independent experiments is shown. (C) Nanoparticle tracking analysis of C. acnes EVs. A representative image of three independent experiments is shown. (D) PCR targeting 16S rRNA gene of C. acnes in C. acnes EVs. Figures are representative of three independent results. Black arrow indicates 946‐bp amplified region of C. acnes 16S rRNA gene. MQ: Milli‐Q water.

FIGURE 4

Cutibacterium acnes extracellular vesicles (EVs) promote proliferation of renal carcinoma cells. (A) Confocal microscopy analyses of renal cancer cells incubated with PKH67‐labeled C. acnes EVs or E. coli EVs for 24 h. White bars indicate 50 μm. A representative image of three independent experiments is shown. (B–D) Flow cytometry analyses of renal cancer cells: (B) Caki‐2, (C) 786‐O, and (D) RenCa. Cells were incubated with PKH67‐labeled C. acnes EVs or E. coli EVs for 24 h. One‐way ANOVA test (post‐hoc Tukey). (E–G) Proliferation assay of renal cancer cells: (E) Caki‐2, (F) 786‐O, and (G) RenCa. Cells were incubated with C. acnes EVs. Unpaired t‐test. (H–J) Proliferation assay of renal cancer cells: (H) Caki‐2, (I) 786‐O, and (J) RenCa. Cells were incubated with E. coli EVs. Unpaired t‐test.

FIGURE 5

Cutibacterium acnes extracellular vesicles (EVs) promote renal cell carcinoma tumor growth in vivo. PKH26‐labeled PBS (A), C. acnes EVs (B), or E. coli EVs (C) were intraperitoneally administered to mice with RenCa tumors and subjected to immunofluorescence analysis. A representative image of two independent experiments is shown. (D) The effect of C. acnes EVs on RenCa cell tumor growth. Unpaired t‐test. (E) Immunohistochemistry (IHC) of RenCa cell tumor using anti‐Ki67 and anti‐CD31 antibodies. Figure shows a representative image of IHC. Percentage of Ki67‐positive (F) and CD31‐positive (H) cells and percentage of Ki67 (G) and CD31 (I) highly positive cells. Unpaired t‐test. (J) The effect of E. coli EVs on RenCa cell tumor growth. (K) IHC of RenCa cell tumor using anti‐Ki67 and anti‐CD31 antibodies. Figure shows a representative image of IHC. Percentage of Ki67‐positive (L) and CD31‐positive (N) cells and percentage of Ki67 (M) and CD31 (O) highly positive cells.