ABOVE: Salmonella invade a human epithelial cell. Flickr, NIAID

As tumors grow, they skillfully evade the body’s immune response. Cancer cells multiply quickly, forming a dense mass of tissue and vasculature that becomes increasingly difficult for immune cells to infiltrate, and they begin to pump out molecules that suppress immune cell function.

In some cancer patients, just outside the tumor, many immune cells function normally. Dendritic cells capture antigens on the tumor’s surface and launch an immune cascade, marking cancer cells for destructionbut often not at the rate necessary to halt tumor growth.

In a paper published January 20 in Nature Biomedical Engineering, scientists report using a combination of modified Salmonella bacteria and radiation to enhance the body’s natural immune response against tumors in mice. The researchers injected the Salmonella into tumors to capture antigens and shuttle them out, making the antigens accessible to immune cells.

“I thought the study was really innovative,” Andrew Redenti, a graduate student in biomedical engineering at Columbia University who was not involved in the study but coauthored a commentary to accompany it in the journal, tells The Scientist. “Salmonella has been used in many different contexts to try and deliver things to tumors, but I've never seen it used to take things out of tumors and bring them to the immune system.”

Salmonella is one of many vectors commonly used to boost the body’s immune response to cancer. These so-called cancer vaccines or in situ vaccines—typically small molecules, RNA, or attenuated viruses—are injected directly into tumors. But this is the first study to report trying to move antigens out. 

See “In Situ Vaccination: A Cancer Treatment a Century in the Making

Following radiation treatment, tumor cells produce massive amounts of antigens. But they remain trapped within the mass, unable to reach healthy immune cells just microns away. Study coauthor Jinhui Wu, a biomedical engineer at Nanjing University in China, writes in an email to The Scientist that he wondered whether “antigens released by the tumor can be grabbed by these bacteria and then transported around the tumor to become a mobile vaccine.” 

To find out, the researchers first modified VNP200009, an attenuated strain of Salmonella, by coating it with positively charged nanoparticles. Since most antigens are negatively charged, the researchers hypothesized that this modification would help Salmonella to shepherd antigens out of the tumor.

This attenuated strain of Salmonella has previously been shown to be effective at penetrating and accumulating in the tangle of cells inside a tumor. But before they tried it out on treating cancer in the body, the researchers tested whether their modified version would take up and transfer antigens to immune cells—mainly T cells, which mark cancer cells for destruction. They started by loading up the positively charged Salmonella with ovalbumin, a negatively charged protein sometimes produced by cancer cells. In culture, they saw the modified Salmonella travel through agar to reach dendritic cells. The dendritic cells, upon encountering the bacteria, did what they’re supposed to do in response to a foreign substance: they changed shape and bound the ovalbumin, ready to recruit anti-ovalbumin T cells. 

Next, the researchers performed a similar experiment in tumor-ridden mice. They injected the antigen-shuttling Salmonella, which were already loaded with ovalbumin as cargo, into the center of a tumor. They then looked at the dendritic cells from the mice and again saw that the dendritic cells had bound to the ovalbumin, meaning that the bacteria had carried the ovalbumin out of the tumor. 

“The fact that they were able to show that a protein, the ovalbumin protein, gets to the periphery of the tumor with this bacteria was surprising,” says Anusha Kalbasi, a tumor immunologist and radiation oncologist with the University of California, Los Angeles (UCLA), David Geffen School of Medicine and the UCLA Jonsson Comprehensive Cancer Center. “I don’t think I’ve seen that before.”

The last step was to see how well the modified bacteria could treat cancer in mice. 

The researchers used a combination of radiotherapy and an injection of nanoparticle-studded bacteria to treat tumors in three mouse models of cancer: colon cancer, melanoma, and breast cancer. They compared this treatment to a combination of radiotherapy and uncoated, attenuated Salmonella (which are negatively charged), radiotherapy and Salmonella coated with neutrally charged particles, radiotherapy alone, and saline. They also examined the effects of each type of bacteria without radiotherapy. Treatment with radiotherapy and the Salmonella loaded with positively charged nanoparticles inhibited the growth of tumors in mice with all three kinds of cancer, and in some mice, the tumors shrank or even disappeared. The treatment enhanced the therapeutic effect of radiotherapy, improving the mice’s chances of survival compared with radiotherapy alone, and was more effective than neutrally or negatively charged bacteria combined with radiotherapy. 

Mice with colon cancer that received nanoparticle-coated Salmonella and radiotherapy also lived up to 100 days longer than mice that received any other therapy. The researchers didn’t document how long the mice with breast cancer and melanoma lived past 80 days.

In mice with all three kinds of cancer, the researchers also saw increased antitumor T cell activation in animals that received the radiotherapy and positively charged bacteria, compared with other treatment groups.

If there is a way to break through the limitations of the immune microenvironment, it will greatly promote the development of in situ vaccines.

—Jinhui Wu, Nanjing University

Treatment with positively charged bacteria also enhanced the effect of an immunotherapy called PD-L1 therapy, which tells the immune system to attack the PD-L1 proteins cancer cells use to trick the immune system into leaving them alone. Treatment with PD-L1 antibodies, positively charged bacteria, and radiotherapy inhibited the growth of tumors in 10 out of 10 animals—significantly more than any other single or combination therapy the researchers tested. 

In addition, the researchers investigated the effect of their therapy combination on cancer metastasis. Tumors with mutations that are typically responsible for breast cancer metastasize aggressively throughout the body, while those responsible for colon cancer and melanoma do not. In mice with breast cancer, the rate of metastasis decreased in animals treated with both positively charged bacteria and radiotherapy compared with those given other treatments, as measured by the number of metastatic tumors in the lungs. 

Since immune cells can’t function properly inside the tumor, the study authors suggest that once bacteria shuttle antigens out, healthy immune cells from the periphery come and attack the tumor. Wu suggests that the principle of carrying proteins out of the tumor may help improve other cancer vaccines and therapies in the future. “[I]f there is a way to break through the limitations of the immune microenvironment, it will greatly promote the development of in situ vaccines,” he says. 

Although the researchers did not see the same effects in the mice that received radiotherapy and an injection of attenuated bacteria that lacked nanoparticles, both the researchers and other experts who spoke with The Scientist stress that the modifications to the bacteria may not fully explain why the immune system was activated. 

Kalbasi, for example, argues that the shuttling effect of the bacteria may not be the only reason they can combat tumors. “It could very well be that just the bacteria being in there is eliciting a specific type of immune response,” Kalbasi says. 

Wu concedes that the bacteria themselves may be eliciting an immune response, saying they “also likely act as immune adjuvants.”

Kalbasi says that, to show more convincingly that the bacteria are indeed shuttling antigens out the tumor, he’d like to see more imaging experiments in mice showing that bacteria can pick up antigens and bring them out. “To be able to see the bacteria, without being loaded beforehand, pick up a specifically labeled tumor protein, and taking that protein from the inside to the outside of the tumor, would be a beautiful way of describing what they’re proposing,” Kalbasi says. 

In the future, both Redenti and Kalbasi say they hope to see more direct comparisons between different types of cancer vaccine vectors to find what works best for patients. Redenti explains that “it’s important to look at what the merits and pros and cons of each one are.”