Combination bacteriolytic therapy for the treatment of experimental tumors

Abstract

Current chemotherapeutic approaches for cancer are in part limited by the inability of drugs to destroy neoplastic cells within poorly vascularized compartments of tumors. We have here systematically assessed anaerobic bacteria for their capacity to grow expansively within avascular compartments of transplanted tumors. Among 26 different strains tested, one (Clostridium novyi) appeared particularly promising. We created a strain of C. novyi devoid of its lethal toxin (C. novyi-NT) and showed that intravenously injected C. novyi-NT spores germinated within the avascular regions of tumors in mice and destroyed surrounding viable tumor cells. When C. novyi-NT spores were administered together with conventional chemotherapeutic drugs, extensive hemorrhagic necrosis of tumors often developed within 24 h, resulting in significant and prolonged antitumor effects. This strategy, called combination bacteriolytic therapy (COBALT), has the potential to add a new dimension to the treatment of cancer.

Figures

Figure 1

Typical human colorectal metastases. Extensive areas of necrosis, such as one one indicated by arrows, are intermixed with areas of viable tumor cells. Similar large areas of necrosis were observed in each of the metastatic lesions from 20 different patients chosen at random from pathologic archives.

Figure 2

Distribution of anaerobic bacteria within tumors. Mice bearing s.c. B16 tumors were intravenously injected through the tail vein with 5 × 107 live B. longum bacteria or wild-type C. novyi spores. Mice that received injections of B. longum were given i.p. injections of lactulose daily for 5 days to increase bacterial growth (16) and then killed for analysis of tumor colonization. Mice with C. novyi were killed the day after injection for analysis. Gram-stains revealed that a large number of B. longum bacteria was concentrated within a few colonies, whereas C. novyi was dispersed throughout the poorly vascularized portions of the tumors. (A and B) High- and low-power views of representative B. longum experiment, showing bacteria (stained deep blue) clustered within a colony. (C) C. novyi experiment, showing dispersion of bacteria throughout the necrotic region of the tumor. (D) High-power view, showing invasion of C. novyi bacteria into surrounding viable tumor cells (stained purple) on the Left.

Figure 3

Elimination of the lethal toxin gene from C. novyi. Following heat shock, PCR was performed on DNA from colonies to identify those which had lost the lethal toxin gene on the phage episome. Agarose gel electrophoresis of the PCR products with two independent primer sets (ToxA and ToxB) shows results from a C. novyi clone (C. novyi-NT) that had lost the gene and another clone (indicated as “C. novyi”) that retained the gene. Controls were provided by primer sets (PlcA and PlcB) specific for the C. novyi phospholipase C gene demonstrating the integrity of the DNA templates in all reactions.

Figure 4

Distribution of C. novyi-NT bacteria after i.v. injection of spores. (A) Hematoxylin/eosin (H&E) stain of a typical HCT116 tumor xenograft from a mouse not injected with bacteria, showing some necrosis. (B) H&E stain of a tumor 24 h after i.v. injection of 5 × 107 C. novyi-NT spores, showing much more extensive necrosis.

Figure 5

Hemorrhagic necrosis following COBALT. (A) HCT116 tumor-bearing mouse 24 h after i.v. injection with 5 × 107 C. novyi-NT spores. Slight swelling associated with edema is seen at the tumor site. D10 (0.3 mg/kg) was then given intravenously (time = 0), and followed 24 h later with MMC (4 mg/kg). A black spot indicating hemorrhagic necrosis is evident near the center of the tumor at 0.3 days. The area of hemorrhagic necrosis gradually expanded over the next day (time = day 1). Swelling at the tumor site then resolved and the necrotic tumor mass shrunk and gradually dissolved (time = days 2–30). (B) Selected mice 5 weeks after treatment with a single dose of D10 plus MMC (Upper) or with a single dose of COBALT (Lower). Of the eight mice treated with COBALT in this experiment, four were apparently cured of their tumor, and three of these are shown (see text and Fig. 6A).

Figure 6

Quantification of the effects of COBALT. (A) HCT116 colorectal cancer cells were grown as xenografts in nude mice. When the tumors were ≈700 mm3 in size, the animals were injected intravenously with 5 × 107 C. novyi-NT spores (time 0), followed by i.v. injection with D10 (0.3 mg/kg) at 24 h and i.p. injection with MMC (4 mg/kg) at 48 h. Control groups were given no treatment or treated with D10 plus MMC without spores. Each group consisted of six to ten mice. Animals were killed when their tumors exceeded 10% of body weight. In the experiment shown, seven of eight mice treated with a single dose of COBALT developed a striking hemorrhagic necrosis of their tumors within 24 h after administration of D10. Four of these seven mice were cured, whereas three of the mice died three days after treatment, perhaps from tumor lysis syndrome (see Discussion). One mouse developed less extensive necrosis and its tumor eventually regrew. Only mice that survived treatment were used to obtain the data plotted in the graph. (B) Mice were treated as in A, except that MMC was not used and treatments were given once every 2 weeks. (C) B16 melanoma cells were grown as s.c. syngeneic tumors in C57BL/6 mice. When the tumors were ≈700 mm3 in size, the animals were injected intravenously with 5 × 107 C. novyi-NT spores (time 0), followed by i.p. injection with CTX (100 mg/kg) at 6 h and i.v. injection with D10 (0.3 mg/kg) at 24 h. Other groups were given no treatment, CTX plus D10, or spores plus D10. Each group consisted of at least ten mice and the treatments were repeated at weekly intervals. Four mice died after the first dose of COBALT and only those mice that survived treatment were used to obtain the data plotted in the graph. Animals were killed when their tumors exceeded 10% of body weight.