Bacterial Iron Siderophore Drives Tumor Survival and Ferroptosis Resistance in a Biofilm-Tumor Spheroid Coculture Model

Abstract

Interactions between tumoral cells and tumor-associated bacteria within the tumor microenvironment play a significant role in tumor survival and progression, potentially impacting cancer treatment outcomes. In lung cancer patients, the Gram-negative pathogen Pseudomonas aeruginosa raises questions about its role in tumor survival. Here, a microfluidic-based 3D-human lung tumor spheroid-P. aeruginosa model is developed to study the bacteria's impact on tumor survival. P. aeruginosa forms a tumor-associated biofilm by producing Psl exopolysaccharide and secreting iron-scavenging pyoverdine, which is critical for establishing a bacterial community in tumors. Consequently, pyoverdine promotes cancer progression by reducing susceptibility to iron-induced death (ferroptosis), enhancing cell viability, and facilitating several cancer hallmarks, including epithelial-mesenchymal transition and metastasis. A promising combinatorial therapy approach using antimicrobial tobramycin, ferroptosis-inducing thiostrepton, and anti-cancer doxorubicin could eradicate biofilms and tumors. This work unveils a novel phenomenon of cross-kingdom cooperation, where bacteria protect tumors from death, and it paves the way for future research in developing antibiofilm cancer therapies. Understanding these interactions offers potential new strategies for combatting cancer and enhancing treatment efficacy.

Keywords: Pseudomonas aeruginosa; biofilm; ferroptosis; pyoverdine; tumor microenvironment.

Figure 1

Establishment of 3D‐human lung tumor spheroid‐P. aeruginosa interactions model. a) Schematic diagram of microfluidics chip for culturing 3D‐lung tumor spheroid‐tumor‐associated P. aeruginosa coculture model. b) Population number of intratumor P. aeruginosa in lung tumor spheroid over time. c) Representative images of 3D‐lung tumor spheroid‐tumor‐associated P. aeruginosa coculture model over time. Scale bar: 100 µm. d) Relative GFP fluorescence levels of intra‐tumor P. aeruginosa in lung tumor spheroid over time. e) Relative Deep‐Red fluorescence levels of lung tumor spheroids in tumor‐bacterial coculture model over time. f) Relative PI fluorescence levels of lung tumor spheroids in tumor‐bacterial coculture model over time. The means and s.d. from triplicate experiments from 3 independent trials were shown. ***p < 0.001.

Figure 2

Biofilms are important for bacterial colonization in tumors. a) Representative images of 3D‐lung tumor spheroid‐tumor‐associated P. aeruginosa PAO1/p _cdrA ‐gfp coculture model over time. Scale bar: 200 µm. b) Relative intracellular c‐di‐GMP levels of tumor‐associated P. aeruginosa in the tumor‐bacterial co‐culture model, as quantified by ELISA. c) CFU assay of intratumor wild‐type PAO1 and its exopolysaccharide mutants in lung tumor spheroid showed exopolysaccharides were important for bacterial colonization within tumors. d) Representative images comparing wild‐type PAO1 and exopolysaccharide‐deficient mutants in the tumor‐bacterial co‐culture model. Scale bar: 200 µm. e) Relative GFP fluorescence levels of intratumor P. aeruginosa PAO1 and its mutants in the tumor‐bacterial coculture model. f) Relative deep‐red fluorescence levels of lung tumor spheroids in a tumor‐bacterial coculture model. g) Relative PI fluorescence levels of lung tumor spheroids in a tumor‐bacterial coculture model. The means and s.d. from triplicate experiments from 3 independent trials were shown. ***p < 0.001.

Figure 3

P. aeruginosa is important in the suppression of ferroptosis in tumor cells. a) Representative images of tumors treated with exogenous iron or DIPY, where exogenous iron addition could cause the death of tumor, while exogenous DIPY treatment could reduce the death of tumor cells. Scale bar: 200 µm. b) Relative PI fluorescence levels of lung tumor spheroids after exogenous iron or DIPY treatment. c) Relative transferrin receptor expression by tumor cells after exogenous iron or DIPY treatment. d) Relative ROS levels within tumor cells after exogenous iron or DIPY treatment. e) Representative images of 3D‐lung tumor spheroid‐tumor‐associated P. aeruginosa coculture model after exogenous iron treatment. Scale bar: 200 µm. f) Relative PI fluorescence levels of lung tumors in the tumor‐bacterial coculture model after exogenous iron treatment. g) Relative transferrin receptor expression by lung tumors in the tumor‐bacterial co‐culture model after exogenous iron treatment. h) Relative ROS levels within lung tumor cells in the tumor‐bacterial co‐culture model after exogenous iron treatment. The means and s.d. from triplicate experiments from 3 independent trials were shown. ***p < 0.001.

Figure 4

P. aeruginosa pyoverdine is important in suppressing ferroptosis‐led death in tumor cells. a) Representative images of 3D‐lung tumor spheroid‐tumor‐associated P. aeruginosa PAO1/p _pvdA ‐gfp coculture model. Scale bar: 200 µm. b) Relative GFP fluorescence levels of PAO1/p _pvdA ‐gfp in a tumor‐bacterial coculture model. c) Relative pyoverdine fluorescence levels of PAO1/p _pvdA ‐gfp in a tumor‐bacterial coculture model. d) CFU assay of intra‐tumor wild‐type PAO1 and its pyoverdine mutant in lung tumor spheroid showed pyoverdine was not important for bacterial colonization within tumors. e) Representative images of 3D‐lung tumor spheroid‐tumor‐associated P. aeruginosa PAO1 and its pyoverdine mutants, all tagged with p _lac ‐gfp. Scale bar: 200 µm. f) Relative PI fluorescence levels of lung tumors cultured with P. aeruginosa PAO1 and its pyoverdine mutants in the tumor‐bacterial coculture model. g) Representative images of 3D‐lung tumor spheroid‐tumor‐associated pyoverdine mutant bacteria treated with exogenous pyoverdine addition. Scale bar: 200 µm. h) Relative PI fluorescence levels of lung tumors cultured with pyoverdine mutant bacteria treated with exogenous pyoverdine addition in the tumor‐bacterial coculture model. The means and s.d. from triplicate experiments from 3 independent trials were shown. ***p < 0.001. n.s: not significant.

Figure 5

P. aeruginosa pyoverdine modulates iron levels in tumor spheroids. a) Intracellular iron concentration in tumor spheroids containing tumor‐associated P. aeruginosa PAO1 and its pyoverdine mutant. b) Representative images of 3D‐lung tumor spheroid‐tumor‐associated P. aeruginosa PAO1/p _pvdA ‐gfp coculture model treated with exogenous iron or DIPY. Scale bar: 100 µm. c) Relative GFP fluorescence levels of PAO1/p _pvdA ‐gfp in tumor‐bacterial co‐culture model treated with exogenous iron or DIPY. d) Relative pyoverdine fluorescence levels of PAO1/p _pvdA ‐gfp in tumor‐bacterial coculture model treated with exogenous iron or DIPY. e) Intracellular iron concentration in tumor spheroids containing tumor‐associated P. aeruginosa PAO1 treated with exogenous iron or DIPY. f) Representative images of 3D‐lung tumor spheroid‐tumor‐associated P. aeruginosa PAO1/p _lac ‐gfp coculture model treated with exogenous iron or DIPY. Scale bar: 100 µm. g) Relative PI fluorescence levels of lung tumors cultured with tumor‐associated P. aeruginosa PAO1 in the tumor‐bacterial coculture model. h) CFU assay of intra‐tumor wild‐type PAO1 in lung tumor spheroids treated with exogenous iron or DIPY. The means and s.d. from triplicate experiments from 3 independent trials were shown. ***p < 0.001.

Figure 6

Pyoverdine enables tumor epithelial‐to‐mesenchymal transition (EMT) progression. a) Representative images of CD44 expression by 3D‐lung tumor spheroid cultivated with tumor‐associated P. aeruginosa PAO1 or pyoverdine mutant. Scale bar: 100 µm. b) Relative CD44 expression by tumor cells cultivated with tumor‐associated P. aeruginosa PAO1 or pyoverdine mutant. c) Representative images of CD133 and CD166 expression by 3D‐lung tumor spheroid cultivated with tumor‐associated P. aeruginosa PAO1 or pyoverdine mutant. Scale bar: 100 µm. d) CD133⁺/CD166⁺ ratio of tumor cells cultivated with tumor‐associated P. aeruginosa PAO1 or pyoverdine mutant. e) Representative images of CD133 and CD166 expression by 3D‐lung tumor spheroid treated only with exogenous pyoverdine. Scale bar: 100 µm. f) CD133⁺/CD166⁺ ratio of tumor cells treated only with exogenous pyoverdine. The means and s.d. from triplicate experiments from 3 independent trials were shown. ***p < 0.001.

Figure 7

Triple treatment of thiostrepton (TST), tobramycin (TOB) and doxorubicin (DOX) can eliminate tumor‐bacterial coculture. a) Scheme of TST‐TOB‐DOX triple treatment on tumor and tumor‐associated P. aeruginosa. TST could induce b) ROS and c) transferrin receptor expression in tumors. d) CFU assay of intratumor wild‐type PAO1 in lung tumor spheroids treated with TST‐TOB‐DOX monotherapies or combinatorial therapy. e) Representative images of 3D‐lung tumor spheroid‐tumor‐associated P. aeruginosa co‐culture model treated with TST‐TOB‐DOX monotherapies or combinatorial therapy. Scale bar: 100 µm. f) Relative GFP fluorescence levels of tumor‐associated P. aeruginosa PAO1 in the tumor‐bacterial coculture model after TST‐TOB‐DOX monotherapies or combinatorial therapy. g) Relative pyoverdine fluorescence levels of tumor‐associated P. aeruginosa PAO1 in the tumor‐bacterial coculture model after TST‐TOB‐DOX monotherapies or combinatorial therapy. h) Relative PI fluorescence levels of lung tumors cultured with tumor‐associated P. aeruginosa PAO1 in the tumor‐bacterial coculture model after TST‐TOB‐DOX monotherapies or combinatorial therapy. The means and s.d. from triplicate experiments from 3 independent trials were shown. ***p < 0.001.