Bacteriolytic therapy can generate a potent immune response against experimental tumors

Abstract

When spores of the anaerobic bacterium Clostridium novyi-NT are systemically injected into animals, they germinate exclusively within the hypoxic regions of cancers. The germinated bacteria destroy adjacent tumor cells but spare a rim of well oxygenated tumor cells that subsequently expand. Surprisingly, we found that approximately 30% of mice treated with such spores were cured of their cancers despite the viable tumor rim initially remaining after spore germination. The mechanism underlying this effect was shown to be immune-mediated, because cured animals rejected a subsequent challenge of the same tumor. Similar effects were observed in rabbits with intrahepatic tumors. It was particularly notable that the induced immune response, when combined with the bacteriolytic effects of C. novyi-NT, could eradicate large established tumors.

Figures

Fig. 1.

Tumor inflammation after i.v. injection with C. novyi-NT spores. Untreated tumors (a-c) and tumors treated with C. novyi-NT spores (d-f) were examined histopathologically after hematoxylin/eosin staining. Extensive areas of necrosis (N) were present within CT26 tumors 24 h after systemic injection of spores, and a ring of inflammation surrounded the tumors (e). The boxed areas in a and d are magnified in b and e, respectively, showing neutrophilic infiltrate in the treated lesions (e) but only tumor cells in the untreated control (b). The great majority of the cells in d and e were inflammatory cells (white arrow), with only a few nests of tumor cells remaining (red arrows). Gram stains (c and f) showed that the necrotic regions of only the treated lesions were filled with vegetative C. novyi-NT bacteria (white arrow).

Fig. 2.

Serum cytokines 24 h after treatment with C. novyi-NT. Il-6, MIP-2, G-CSF, TIMP-1, and KC were up-regulated. Antibodies to each cytokine were spotted twice on each array. Levels of tumor necrosis factor α and 26 other cytokines did not change significantly. Positive (+) controls are circled in red. Very similar patterns to those observed (Right and Left) were depicted in this figure in the sera of two treated mice and two control mice, respectively.

Fig. 3.

Rechallenge experiments. (a) Mice with the indicated tumors cured by treatment with C. novyi-NT or by surgical excision were challenged with a s.c. injection of 5 × 106 tumor cells. The responses were compared to those in mice that had not previously been injected with tumor cells (“Control”). (b) Lymphocytes were purified from naive mice or from mice that were cured of their CT26 tumors by C. novyi-NT treatment. Forty-eight hours after i.v. injection of the lymphocytes, the mice were challenged with a s.c. injection of 5 × 105 tumor cells. The y axes in both a and b represent the fraction of animals that formed tumors after tumor challenge. The number (n) of mice used in each group is indicated.

Fig. 4

Photographs of mice used for challenge experiments. A mouse surgically cured of a CT26 tumor was not resistant to a challenge of CT26 cells in the opposite flank (a), whereas a mouse cured with C. novyi-NT was resistant (b). (c) CT26 tumors and RENCA tumors formed in naive mice when the corresponding cells were injected into one flank. (d) When mice cured of their RENCA tumors with C. novyi-NT were similarly injected, only CT26 tumors formed.

Fig. 5

C. novyi-NT germinates within VX2 tumors. (a) Small regions of necrosis (N) were found with the VX2 tumors of a control rabbit. (b) In rabbits treated with C. novyi-NT for 4 days, the necrotic regions enlarged, and the tumor was largely replaced with inflammatory cells, as indicated in the high-power view (d) of the boxed region in b. (f) Pico Green staining showed that the necrotic regions of the treated lesions were filled with vegetative C. novyi-NT.

Fig. 6.

Serial CT scans of VX2 tumors. Arterial-dominant axial high-resolution CT images at the level of the primary tumor are shown in representative cases at the indicated days. Nine days after tumor implantation, there was peripheral enhancement of the tumor in an untreated rabbit (Left, yellow arrows). Twenty-one days later (day 30), the tumor had grown considerably in size, and prominent peripheral enhancement could still be observed. Six days later (day 36), the tumor had replaced most of the normal liver in this untreated rabbit. By 15 days after tumor implantation in another rabbit (Right), the tumor had established and exhibited peripheral enhancement (yellow arrow). This animal was treated on day 22, and 12 days later (day 34), there was diminished peripheral enhancement and gas appeared within the tumor (red arrow). By day 77 (55 days after treatment), there was no peripheral enhancement, and the gas had completely resolved. The small hyperintense foci (green arrow) on day 77 represent regions of calcification. This rabbit remains alive and healthy at 650 days after tumor inoculation. The yellow bar in the day 15 image (Right) represents 5 cm.

Fig. 7.

PET/CT images of lesions after C. novyi-NT therapy. (Left) Fused PET/CT images of an untreated rabbit. Fourteen days after implantation of VX2 cells, there was intense uptake of FDG in the tumor. Twenty-two days after implantation, the region incorporating FDG was much larger and contained a central area of decreased uptake, suggesting a necrotic focus. Continued tumor growth and progressive FDG uptake were evident on the images at days 34 and 51, and the animal died on day 52 with widespread FDG-positive metastatic disease in the mediastinum. (Right) Analogous images of a rabbit treated with C. novyi-NT. There was intense uptake of FDG 14 days after tumor implantation, similar to that in the untreated rabbit. An i.v. injection of C. novyi-NT spores was administered on day 15. Seven days later (day 22), two lesions were visible. One had considerably decreased uptake of FDG, suggesting C. novyi-NT germination (blue arrow), and the other, which appeared to be budding from the first, exhibited intense FDG uptake (yellow arrow). Twelve days later (day 34), both lesions had less FDG uptake (yellow and blue arrows). Seventeen days later (day 51), the first two lesions were resolving (yellow and blue arrows), but a new FDG-incorporating lesion was evident in the abdominal wall (red arrow). The FDG uptake in the abdominal wall lesion was substantially decreased by day 86 (red arrow). On day 455, the animal was alive and healthy, and there was no abnormal FDG uptake in the liver or elsewhere.

Fig. 8.

Working model. C. novyi-NT spores localize in hypoxic/anoxic areas of tumors, where they germinate and cause lysis of tumor cells. The resulting inflammation, as well as the oxygen present in adjacent areas, inhibits the proliferation and spread of C. novyi-NT. However, the inflammation may also directly contribute to tumor destruction through the release of reactive oxygen species, proteases, pore-forming agents, and tumoricidal cytokines. In addition, the inflammatory response stimulates a specific cellular antitumor immune response that constrains subsequent tumor growth.