Persistent enhancement of bacterial motility increases tumor penetration

Abstract

Motile bacteria can overcome the transport limitations that hinder many cancer therapies. Active bacteria can penetrate through tissue to deliver treatment to resistant tumor regions. Bacterial therapy has had limited success, however, because this motility is heterogeneous, and within a population many individuals are non-motile. In human trials, heterogeneity led to poor dispersion and incomplete tumor colonization. To address these problems, a swarm-plate selection method was developed to increase swimming velocity. Video microscopy was used to measure the velocity distribution of selected bacteria and a microfluidic tumor-on-a-chip device was used to measure penetration through tumor cell masses. Selection on swarm plates increased average velocity fourfold, from 4.9 to 18.7 μm/s (P < 0.05) and decreased the number of non-motile individuals from 51% to 3% (P < 0.05). The selected phenotype was both robust and stable. Repeating the selection process consistently increased velocity and eliminated non-motile individuals. When selected strains were cryopreserved and subcultured for 30.1 doublings, the high-motility phenotype was preserved. In the microfluidic device, selected Salmonella penetrated deeper into cell masses than unselected controls. By 10 h after inoculation, control bacteria accumulated in the front 30% of cell masses, closest to the flow channel. In contrast, selected Salmonella accumulated in the back 30% of cell masses, farthest from the channel. Selection increased the average penetration distance from 150 to 400 μm (P < 0.05). This technique provides a simple and rapid method to generate high-motility Salmonella that has increased penetration and potential for greater tumor dispersion and clinical efficacy.

Keywords: bacterial cancer therapy; microfluidic tumor-on-a-chip; motility; salmonella; tumor penetration.

Figure 1

Swarm plate selection and tumor-on-a-chip device. A) Motile bacteria were selected by inoculating droplets of liquid culture at the center of swam plates (left). After incubation, bacteria were extracted from the outer ring (right). B) Microfluidic device used to measure bacterial penetration. Tumor tissue was contained within six 1000×300×150 μm chambers, alternately staggered on either side of a central flow channel. The illustration shows the first three chambers.

Figure 2

Selection of motile Salmonella. (A) Motile bacteria were selected on soft-agar swarm plates. A small ring (arrow) was present directly after inoculation (0 hours, left) that spread to the outer edge of the plate by 36 hours (right). Bacteria were extracted from this outer edge (arrow). (B) Fluorescence images of two bacteria (arrows) from control and selected populations at 0.54, 1.08, and 1.62 seconds. Motility was determined by measuring locations in sequential images. Scale bars are 50 μm. (C) Velocity distributions of three populations: unselected controls (n=231), bacteria after one selection (n=214), and bacteria after two selections (n=247). One and two selections decreased the percentage in the 0–5 μm/sec range (**, P<0.01), and increased the percentage in the 15–20 μm/sec range (*, P<0.05). (D) The growth profiles of control and selected Salmonella in liquid medium were equivalent. (E) Average population velocities after one (*, P<0.05) and two (**, P<0.01) selections were greater than controls. (F) The percentage of nonmotile bacteria (velocity < 2.5 μm/sec) decreased after two selections compared to controls (*, P<0.05) and one selection (**, P<0.01).

Figure 3

Selection of wild-type Salmonella. (A) Velocity distributions of Salmonella, strain SL1344, after one and two selections on swarm plates. Selection decreased the percentage of bacteria in the 0–5 μm/sec range (**, P<0.01), and increased the percentage in the 15–20, 20–25, and 25–30 μm/sec ranges (*, P<0.05). (B) Average aqueous velocities increased significantly after selection (*, P<0.05). (C) The percentage of non-motile bacteria (velocity < 2.5 μm/sec) decreased after selection (*, P<0.05).

Figure 4

Motile phenotype was preserved after cryopreservation and regrowth. (A) Average velocities of subcultured Salmonella, strain VNP20009, were at least as fast as the population after one selection (*, P<0.05) and significantly greater than the control population (***, P≤ 0.001). (B) The percentage of non-motile individual bacteria was as least as small as after one selection (***, P<0.001) and significantly less than the control population (*, P≤ 0.05). (C,D) Average velocities (C) and percentages of non-motile individual (D) of subcultured Salmonella, strain SL1344, were comparable to the population after one selection and significantly different from controls (*, P<0.05).

Figure 5

Selection increased Salmonella penetration into tumor tissue. (A, B) Merged fluorescent and transmitted microcopy images of control (A) and selected (B) Salmonella (green) in tumor tissue (grey). Images were acquired at 8, 10, and 15 hours after administration of 105 CFU/mL of bacteria for one hour and flushing with fresh media. Scale bars are 200 μm. (C, D) Bacterial density profiles (n=3) as a function of normalized distance into tissue at 8, 10 and 15 hours, for control (C) and selected (D) Salmonella. For control bacteria, the density in the front 40% of the tissue significantly increased between 8 and 15 hours (*, P<0.05). For selected bacteria, the density in the rear 20% of the tissue significantly increased between 8 and 15 hours (*, P<0.05).

Figure 6

Bacterial growth and penetration in tumor tissue. (A) Total bacterial density in tissue, determined from fluorescence images, increased at similar rates for control and selected Salmonella. (B) At 15 hours, the density of selected bacteria in the proximal region was significantly less than control bacteria (***, P<0.001). In the distal region, the density of selected bacteria was significantly greater than controls (*, P<0.05). The proximal and distal regions are defined as the 30% of tissue closest to the channel and the 30% of tissue farthest from the channel, respectively. (C) Average location of bacteria within tumor tissue relative the edge bordering the flow channel. With time, selected bacteria penetrated deeper into tissue. At 13 and 15 hours, the average penetration depth of selected bacteria was significantly greater than control bacteria (*, P<0.05).