Solid tumors provide niche-specific conditions that lead to preferential growth of Salmonella

Abstract

Therapeutic attenuated strains of Salmonella Typhimurium target and eradicate tumors in mouse models. However, the mechanism of S. Typhimurium for tumor targeting is still poorly understood. We performed a high-throughput screening of single-gene deletion mutants of S. Typhimurium in an orthotopic, syngeneic murine mammary model of breast cancer. The mutants under selection in this system were classified into functional categories to identify bacterial processes involved in Salmonella accumulation within tumors. Niche-specific genes involved in preferential tumor colonization were identified and exemplars were confirmed by competitive infection assays. Our results show that the chemotaxis gene cheY and the motility genes motAB confer an advantage for colonization of Salmonella within orthotopic syngeneic breast tumors. In addition, eutC, a gene belonging to the ethanolamine metabolic pathway, also confers an advantage for Salmonella within tumors, perhaps by exploiting either ethanolamine or an alternative nutrient in the inflamed tumor environment.

Keywords: 4T1; Salmonella Typhimurium; ethanolamine; high-throughput; mammary cancer.

Figure 1. Pathways enriched for genes under selection during growth in tumor

A ranked list of all mutants, based on their Limma t scores for selection relative to wild type, was used as an input for Gene Set Enrichment Analysis [50] (http://www.broadinstitute.org/gsea/index.jsp). A network of gene sets enriched at a false discovery rate (FDR) < 25% was plotted using cytoscape [82]. Circle size is proportionaly to the number of genes corresponding to mutants under selection in each data set. The thickness of the connecting lines indicates the number of significantly selected genes shared by related ontologic categories. Blue nodes represent gene sets with a significant number of genes where the corresponding mutants confer a growth disadvantage relative to wild-type bacteria.

Figure 2. The chemotaxis and motility genes cheY and motAB confer an advantage during colonization of solid tumor tissue in vivo

A 1:1 ratio of either the ΔcheY A. or ΔmotAB B. mutant to wild-type S. Typhimurium 14028s was inoculated IP in regular (left) or tumor-bearing (right) BALB/c mice. After 48 hours, bacteria were recovered from tissue and competitive index (CI) values were calculated as mean ratios of mutant to wild type, normalized to the input ratio and converted logarithmically. Thus, a value of zero indicates that the two strains in comparison are equally fit, a value < 0 indicates that the tested strain was outcompeted by the wild type and a value > 0 indicates that the tested strain outcompeted the wild type. Error bars denote standard error. Statistical significance was determined using a two-tailed Student's t test (** P < 0.005, *** P < 0.001).

Figure 3. A gene in the ethanolamine degradation cluster confers an advantage for Salmonella growth in the tumor environment that does not require SPI-1 mediated host cell invasion

A. 1:1 ratio of the ΔeutC mutant to wild-type S. Typhimurium 14028s was inoculated IP in regular A. or tumor- bearing B. BALB/c mice. C. A 1:1 ratio of a ΔssaD ΔinvA ΔeutC mutant and a ΔssaD ΔinvA mutant was inoculated IT in tumor-bearing BALB/c mice. After 48 hours, bacteria were recovered from the designated tissues and CI values were calculated as mean ratios of the two mutants, normalized to the input ratio and converted logarithmically. A value of zero indicates that the two strains in comparison are equally fit, a value < 0 indicates that the tested strain was outcompeted by the wild type and a value > 0 indicates that the tested strain outcompeted the wild type. Error bars denote standard error. Statistical significance was determined using a two-tailed Student's t test (* P < 0.05).