(598a) Intracellular Invasion of Salmonella Drives Tumor Colonization of the Bacteria in vitro

Salmonella colonize tumors at ratios greater than ten thousand to one over any other organ in the body [1]. Unlike small molecule drugs, which disperse into tumors through passive diffusion, Salmonella can actively penetrate into tumors using flagella. The bacteria can also be genetically modified to produce DNA, RNA and peptide therapies. These characteristics make the bacteria well suited to deliver targeted and patient specific cancer therapies.

However, the mechanisms governing tumor colonization of Salmonella are not completely understood. While motility and chemotaxis influence tumor colonization [2–5], the bacteria also have the ability to intracellularly invade cancer cells. The dependence of intracellular invasion on tumor colonization has not yet been elucidated. In addition, bacterial motility and intracellular invasion have cross regulatory mechanisms that make each phenomenon dependent on one another. This study, therefore, aimed to test two interrelated hypotheses: (1) Overexpressing the master motility regulator, flhDC, in Salmonella increases dispersion and colonization as well as intracellular invasion in tumor masses and (2) Upregulating flagellar dependent motility increases intracellular invasion, which, is the main driver of Salmonella tumor colonization in vitro. Salmonella utilize the transcription factor complex, flhDC, to regulate flagellar synthesis and thus, motility. Flagellar synthesis also influences type three secretion system (T3SS) synthesis, which, Salmonellause to intracellularly invade epithelial cells.

To determine whether flagellar-dependent motility could increase bacterial intratumoral dispersion and intracellular invasion, flhDC was overexpressed within Salmonella (flhDC overexpressing Salmonella, or, foS) using the arabinose inducible pBAD system. A microfluidic tumor-on-a-chip model was used to determine the dynamics of foS colonization and intracellular invasion of tumor tissue in vitro. This tumor-on-a-chip model recapitulates in vivo nutrient gradients and cancer cell heterogeneity present within tumors. In order to quantify tumor colonization and intracellular invasion dynamics of foS within the in vitro tumor masses, the arabinose inducible flhDC gene circuit was coupled with constitutively expressed red fluorescent protein, DsRed, as well as pSSEJ induced green fluorescent protein (GFP) gene circuits. The plac promoter drove constitutive DsRed expression while the intracellularly active pSSEJ promoter only drove GFP expression within intracellular Salmonella. Time lapse microscopy was used to determine the dynamics of tumor colonization and intracellular invasion of Salmonella. When foS was grown in the presence of the inducer, arabinose, aqueous motility increased 33% (P<0.05). In the tumor-on-a-chip model, flhDC overexpression in Salmonella increased distal tumor colonization and intracellular invasion of cancer cells ten-fold and eight-fold, respectively (P<0.05), compared to Salmonella only. These results illustrate that overexpressing flhDC in Salmonella increases both intratumoral dispersion and intracellular invasion of the bacteria.

In order to isolate which factor, either motility or intracellular invasion, was the main driver of tumor colonization, a ΔSipB, non-intracellularly invading, strain of Salmonella was created. This strain was transformed with the same DsRed/GFP reporter system to assess bacterial tumor colonization and intracellular invasion dynamics. Not surprisingly, the ΔSipB strain intracellularly invaded cancer cells in the tumor-on-a-chip model five-fold less than Salmonella alone (P<0.05). However, the ΔSipB colonized tumor tissue five-fold less than Salmonella alone (P<0.05). In order to isolate the dependence of motility on tumor colonization, flhDC was overexpressed in the ΔSipB, non-intracellularly invading Salmonella, and administered to the tumor-on-a-chip along with ΔSipB Salmonella as a control. Overexpressing flhDC did not alter tumor colonization in any way when compared to ΔSipB Salmonella alone. These results demonstrate that intracellular invasion into cancer cells, independent of bacterial motility, drives tumor colonization of Salmonella.

Since Salmonella are complex microbes with several colonization mechanisms, it is important to understand which of these most influences bacterial survival and growth within tumors. This study has demonstrated that bacterial motility by itself does not influence tumor colonization. However, motility strongly influences the ability of Salmonella to intracellularly invade tumor cells, which, greatly increases tumor colonization in vitro. This study has provided a provided a foundational framework to investigate how motility and intracellular invasion could influence bacterial tumor colonization in vivo.

1. Forbes NS, Munn LL, Fukumura D, Jain RK. Sparse initial entrapment of systemically injected Salmonella typhimurium leads to heterogeneous accumulation within tumors. Cancer Res. 2003;63(17):5188–93.

2. Toley BJ, Forbes NS. Motility is critical for effective distribution and accumulation of bacteria in tumor tissue. Integr Biol. 2012;4:165.

3. Zhang M, Forbes NS. Trg-deficient Salmonella colonize quiescent tumor regions by exclusively penetrating or proliferating. J Control Release. 2015;199:180–9.

4. Kasinskas RW, Forbes NS. Salmonella typhimurium lacking ribose chemoreceptors localize in tumor quiescence and induce apoptosis. Cancer Res. 2007;67(7):3201–9.

5. Kasinskas RW, Forbes NS. Salmonella typhimurium specifically chemotax and proliferate in heterogeneous tumor tissue in vitro. Biotechnol Bioeng. 2006;94(4):710–21.