Bacterial delivery of Staphylococcus aureus α-hemolysin causes regression and necrosis in murine tumors

Abstract

Bacterial therapies, designed to manufacture therapeutic proteins directly within tumors, could eliminate cancers that are resistant to other therapies. To be effective, a payload protein must be secreted, diffuse through tissue, and efficiently kill cancer cells. To date, these properties have not been shown for a single protein. The gene for Staphylococcus aureus α-hemolysin (SAH), a pore-forming protein, was cloned into Escherichia coli. These bacteria were injected into tumor-bearing mice and volume was measured over time. The location of SAH relative to necrosis and bacterial colonies was determined by immunohistochemistry. In culture, SAH was released and killed 93% of cancer cells in 24 hours. Injection of SAH-producing bacteria reduced viable tissue to 9% of the original tumor volume. By inducing cell death, SAH moved the boundary of necrosis toward the tumor edge. SAH diffused 6.8 ± 0.3 µm into tissue, which increased the volume of affected tissue from 48.6 to 3,120 µm(3). A mathematical model of molecular transport predicted that SAH efficacy is primarily dependent on colony size and the rate of protein production. As a payload protein, SAH will enable effective bacterial therapy because of its ability to diffuse in tissue, kill cells, and expand tumor necrosis.

Figure 1

Plasmids developed in this study. (a) Plasmid that produces S. aureus α-hemolysin after induction with l-arabinose. (b) Control plasmid that produces ZsGreen.

Figure 2

SAH expression and in vitro toxicity. (a) Distribution of bacterially-produced SAH in cultures of EC-SAH. Half of the cultures were induced with 0.2% l-arabinose for 4 hours and half were not induced. The cytoplasmic fraction was isolated by mechanical lysis. The released fraction represents the SAH in the culture supernatant. (b) SAH in the supernatant from bacterial cultures of EC-SAH and EC-ZsG. (c) Supernatant from induced EC-SAH cultures decreased survival of MCF-7 cells compared to untreated (PBS) controls (*P < 0.05). The decrease was comparable to 500 ng/ml of pure SAH (*P < 0.05). Supernatant from uninduced EC-SAH did not affect survival compared to PBS controls. (d) MCF7 cells treated with supernatant from EC-SAH cultures. Arrows indicate internal rearrangement of organelles and loss of membrane integrity. (e) Supernatant from EC-SAH cultures decreased the survival fraction of MCF7 cells in a time dependent manner (*P < 0.001). Values are normalized to untreated controls at each time point. (f) Long-term treatment with supernatant from EC-SAH cultures decreased the survival fraction of MCF7 cells (*P < 0.001). Values are normalized to untreated controls at 24 hours. PBS, phosphate-buffered saline; SAH, Staphylococcus aureus α-hemolysin.

Figure 3

Tumor response to bacterially-delivered SAH. (a) Tumor volume in treated mice. Protein expression was induced on day 2 (arrow). On day 5, tumor volume in EC-SAH-treated mice was less than before induction and less than EC-ZsG-treated controls (*P < 0.05). Volumes are normalized by values on day 0. (b) H&E staining of control and EC-SAH-treated tumors. Regions of necrosis (N) and viable (V) tissue are indicated. Bars = 5 mm. (c) The fraction of viable tissue in treated tumors was significantly less than control tumors at the time of sacrifice (*P < 0.05). (d) Total volume and volume of viable tissue in EC-SAH-treated and EC-ZsG-treated control tumors. The volume of viable tissue at the end of the study (final) was less than the initial tumor volume (*P < 0.01). H&E, hematoxylin and eosin; SAH, Staphylococcus aureus α-hemolysin.

Figure 4

Location of SAH and necrosis in tumor tissue. (a) Band of SAH (brown, white arrows) in 4T1 tumor in a BALB/c mouse. Bar = 200 µm. (b) Ring of SAH (false-colored in yellow, white arrows) around the periphery of a 4T1 tumor. Bar = 4 mm. (c) Transition zone between proliferating cells and necrosis (white arrows). Bar = 4 mm. (d) H&E stained tumors showing transition from viable (e) to necrotic cell types (f, pyknosis; g, karyorrhexis; and h, karyolysis) in EC-SAH-treated and EC-ZsG-treated (control) mice. Bars = 500 μm. (e–h) High-resolution micrographs of (e) proliferating cells, (f) pyknosis (arrows indicate nuclear condensation), (g) karyorrhexis (arrows indicate nuclear fragmentation), and (h) karyolysis (arrows indicate nuclear dissolution). Bar = 25 μm. (i) The distance from the tumor edge to regions of pyknosis and karyolysis was shorter in EC-SAH-treated mice compared to EC-ZsG-treated (control) mice (*P < 0.05). The location of karyolysis in EC-SAH-treated mice corresponded to the location of SAH (1.31 mm; dashed line). H&E, hematoxylin and eosin; SAH, Staphylococcus aureus α-hemolysin.

Figure 5

Diffusion of SAH from bacterial colonies in tumors. (a) Colonies of EC-SAH (red) in a 4T1 tumor and associated SAH (green). White arrows indicate areas of colocalization between bacteria and SAH. Bars = 50 µm. (b) Diffusion of SAH (green) was quantified as the distance (r_SAH) of SAH penetration into tissue away from the outer edge (r₀) of bacterial colonies (red). Colocalized pixels appear as yellow. Bar = 25 µm. (c) Distribution of SAH diffusion distances from bacterial colonies (n = 4,743). The average diffusion distance was 6.8 ± 0.3 µm. (d) SAH intensity as a function of colony size. SAH intensity was normalized by the minimum detectable level. A logarithmic fit (red line) was added as an aid to the eye. (Inset) Larger colonies produced more SAH (*P < 0.01). (e) SAH diffusion distance and colony size were inversely related. (Inset) The average distance from small colonies (r₀ < 5 µm) was greater than intermediate (5 < r₀ < 10 µm) and large (r₀ > 10 µm) colonies (*P < 0.001). SAH, Staphylococcus aureus α-hemolysin.

Figure 6

Mathematical analysis of SAH diffusion from bacterial colonies. (a) SAH concentration profiles as a function of time and distance from the center of bacterial colonies, as predicted by a mathematical model of SAH production and diffusion. SAH concentrations are normalized by the minimum detectable level. The steady-state profile describes the concentration as time becomes very large. The radii r₀ and r_SAH are the colony edge (2.26 µm) and the average diffusion distance (9.06 µm), respectively. (b) The concentration at r = r_SAH quickly approaches steady state, being equal to 94 and 99% of the maximum value at 1 and 24 hours, respectively. (c) Effect of increasing M (dimensionless SAH production) on the concentration profile at 24 hours. Increasing M is proportional to increasing m, the SAH production rate. (d) Effect of increasing M on the volume of cells with a concentration of SAH greater than the minimum detectable level. (e) Larger colonies had lower predicted SAH production (M). Data were obtained from measured values of SAH diffusion distance (r_SAH) and colony radius (r₀; Figure 5e). (f) The measured effective volume of SAH (squares) had a non-linear relationship with colony size. For colonies with radii smaller than 2.4 µm, the effective volume decreased with increasing size. The effective volume increased with increasing size for colonies larger than 2.4 µm. The theoretical effective volume (solid line) is based on the approximate steady-state relationship between colony radius and SAH diffusion distance: . M, dimensionless SAH production; SAH, Staphylococcus aureus α-hemolysin.