Despite tumors' best efforts to induce angiogenesis, blood and oxygen usually are in short supply at a tumor's core. From an oxygenation standpoint, tumors consist of an anaerobic core that is more or less coated by a layer of aerobic tissue.

Anoxic regions are difficult to target with the conventional arsenal of radiation and chemotherapy. But the environment they create also has been used against them by scientists, who have sicced anaerobic bacteria onto that core to germinate within it and kill the tumor cells.

In 2001, Bert Vogelstein and his colleagues at the Johns Hopkins Medical Institutions in Baltimore first reported success in treating tumors with such anaerobic bacterial therapy. In a one-two punch, Vogelstein's group used the bacterium Clostridium novyi to destroy the anoxic core of the tumor, and chemotherapy and radiation to destroy its oxygenated rim. Together, the treatment was called combination bacteriolytic therapy or COBALT (See BioWorld Today, Nov 29, 2001.)

Now, the group reports that the CO can be removed out of COBALT and still get a cure in about a third of cases. The findings are reported in the Oct. 19, 2004, issue of the Proceedings of the National Academy of Sciences in a paper titled "Bacteriolytic therapy can generate a potent immune response against experimental tumors." The data also illustrated how a researcher's choice of animal model can have unintended consequences.

"Most early experiments were done in nude mice, but they don't have T cells, so you would not see the immune response we saw," said Nishant Agrawal, postdoctoral researcher at Johns Hopkins University and first author of the paper. The fact that nude mice would not mount an immune response to the tumors also is the very feature that makes nude mice attractive for studying transplanted tumors in the first place. But studies using different animal models would show greater effects of C. novyi than expected from the nude mouse studies, and when Agrawal began doing experiments on rabbits, "our controls would be cured."

Those miracle-cure controls, who received just C. novyi instead of C. novyi plus chemotherapy and radiation, alerted the researchers that though it needs anaerobic conditions to survive, the bacterium had to somehow be affecting the aerobic outer ring of the tumor as well as its anaerobic core.

From Nude Mice To Rabbits To Eventually Humans

In the experiments described in the PNAS paper, the researchers first injected immunocompetent mice with cancer cells to induce subcutaneous tumors. The mice were then injected with C. novyi. Histopathological evaluation of the tumors showed that there was a massive inflammatory response directly after C. novyi injection. Bacterial spores germinated in the anaerobic tumor cores, destroying them.

In two-thirds of mice, the aerobic outer ring of the tumors survived the bacterial assault, promptly generating new tumors. However, in one-third, the tumor disappeared completely and for the duration of the study (over two months), meaning that injecting the anaerobic bacterium had managed to affect the aerobic outer ring, where C. novyi itself cannot survive.

To show that C. novyi's effects on the aerobic part of tumors were due to a specific immune response, rather than a nonspecific effect of either activating the immune system or reducing the size of the tumor, the researchers investigated whether being cured of one tumor via C. novyi conferred protective immunity against further exposure to that tumor. Like a person who has measles once and becomes immune but can still catch the flu, mice who had been cured of one type of tumor using C. novyi were resistant to subsequent injections of the same type of cancer cells, but not of different types. Mice that had been cured of the first tumor through surgery had no such protection, and developed tumors when they were injected again with the same type of cancer cells.

In a separate set of experiments, the scientists essentially vaccinated na ve mice with either CD4+ (helper T cells) or CD8+ (killer T cells) from mice that had been successfully treated with C. novyi for their induced tumors.

"CD4+ cells are pivotal in potentiating the immune response, but CD8+ cells actually kill the tumor cells," Agrawal told BioWorld Today. Pretreatment with killer but not helper T cells prevented mice from developing tumors after they were injected with cancer cells.

Agrawal and his colleagues then went on to test C. novyi's effectiveness in rabbits. There, they injected tumor cells into the liver to mimic metastatic colon cancer.

In rabbits, as in mice, C. novyi treatment led to a specific immune response. Being cured of one tumor by C. novyi protected against further injections of the same types of cancer cells, but not different types. When the researchers followed the course of one rabbit's disease using in vivo imaging, they saw the animal developing a series of metastases over the course of several months after a single injection of cancer cells. However, each of the metastases disappeared after some time, even though there was only one injection of C. novyi 15 days after the injection of cancer cells. Agrawal interpreted that to mean that "even though you are killing only a fraction of the tumor with your original C. novyi injection, eventually C. novyi plus the immune system make it disappear completely."

The group intends to take their findings into the clinic. The scientists are in preliminary discussions with the FDA; they have had a pre-investigational new drug application meeting and are currently conducting toxicity studies, which they hope to present to the FDA early next year if all goes well.

"We have to be cautious, but we are hopeful because it has worked in these different animal models and we know that the wild-type C. novyi infects humans," Agrawal said. He added that, as a clinician, the focus on ultimately bringing knowledge to the clinic is very important to him.

"If this is not somehow going to benefit patients, what's the point?" he said.