University of Pittsburgh researchers have designed cancer-fighting nanoparticles that can deliver a chemotherapy drug in combination with a novel immunotherapy. The new strategy uses a short interfering RNA (siRNA) to silence the gene for a scramblase, Xkr8, that the researchers discovered was involved in immunosuppression. Combined with an existing chemotherapy drug and packaged into tiny nanoparticles, the dual treatment shrank tumors in mouse models of colon cancer and pancreatic cancer, and increased the antitumor immune response.

“There are two innovative aspects of our study: the discovery of a new therapeutic target and a new nanocarrier that is very effective in selective delivery of immunotherapy and chemotherapeutic drugs,” said senior author Song Li, MD, PhD, professor of pharmaceutical sciences in the Pitt School of Pharmacy and UPMC Hillman Cancer Center investigator. “I’m excited about this research because it’s highly translational. We don’t know yet whether our approach works in patients, but our findings suggest that there is a lot of potential.”

Li and colleagues reported on their development in Nature Nanotechnology, in a paper titled, “Targeting Xkr8 via nanoparticle-mediated in situ co-delivery of siRNA and chemotherapy drugs for cancer immunochemotherapy,” in which they concluded, “Targeting Xkr8 in combination with chemotherapy may represent a novel strategy for the treatment of various types of cancer.”

Chemotherapy is a pillar of cancer treatment, but residual cancer cells can persist and cause tumor relapse. This process involves a lipid called phosphatidylserine (PS), which is usually found inside the tumor cell membrane’s inner layer but migrates to the cell surface in response to chemotherapy drugs. On the surface, PS acts as an immunosuppressant, protecting the remaining cancer cells from the immune system. As the investigators noted in their paper, “PS has long been implicated in immunosuppression.”

The Pitt researchers’ studies found that treatment with chemotherapy drugs fluorouracil and oxoplatin (FuOXP) led to increased levels of Xkr8, a protein that controls distribution of PS on the cell membrane. This finding suggested that blocking Xkr8 would prevent cancer cells from shunting PS to the cell surface, allowing immune cells to mop up cancer cells that lingered after chemotherapy. In an independent study that was recently published in Cell Reports, Yi-Nan Gong, PhD, assistant professor of immunology at Pitt, had also identified Xkr8 as a novel therapeutic target to boost anti-tumor immune response.

For their newly reported research, Li and his team designed an siRNA that shuts down production of Xkr8. The team then packaged the siRNA and FuOXP together into dual-action nanoparticles (NPs). “Since no small molecule inhibitors of Xkr8 are available, we developed a new nanocarrier, PMBOP-CP, for co-delivery of murine siXkr8 and FuOXP,” the scientists explained.

The next step was targeting them to tumors. Nanoparticles are typically too large to cross intact blood vessels in healthy tissue, but they can reach cancer cells because tumors sometimes have poorly developed vessels with holes that allow them passage. But this tumor-targeting approach is limited because many human tumors do not have large enough holes for nanoparticles to pass through.

“Like a ferry carrying people from one side of the river to the other, we wanted to develop a mechanism that allows nanoparticles to cross intact blood vessels without relying on holes,” said Li. To develop such a ferry, the researchers decorated the surface of the nanoparticles with chondroitin sulfate (CS) and PEG. These compounds help the nanoparticles target tumors and avoid healthy tissue by binding to cell receptors common on both tumor blood vessels and tumor cells and prolonging the length of time they remain in the bloodstream. The resulting PMBOP-CP nanoparticles were loaded with the siXkr8 and FuOXP, and tested in mouse models of cancer.

When injected into mice, about 10% of the nanoparticles made their way to their tumor—a significant improvement over most other nanocarrier platforms. A previous analysis of published research found that, on average, only 0.7% of nanoparticle doses reach their target. The dual-action nanoparticles dramatically reduced the migration of immunosuppressing PS to the cell surface, when compared to nanoparticles containing the chemo drug FuOXP alone.

Next, the researchers tested their platform in mouse models of colon and pancreatic cancer. Animals treated with nanoparticles containing both FuOXP and siRNA had better tumor microenvironments with more cancer-fighting T cells and fewer immunosuppressive regulatory T cells than animals that received placebo or FuOXP doses. “Co-delivery of siXkr8 and FuOXP led to substantial improvement in the tumor immune microenvironment and enhanced antitumor activity,” the team further noted. As a result, mice that received the siRNA-FuOXP nanoparticles showed a dramatic decrease in tumor size compared to animals that received those carrying just one therapy.

“Targeting Xkr8 in combination with chemotherapy may represent a novel and effective immunochemotherapy for the treatment of various types of cancers,” the researchers concluded. “In addition to enhanced delivery of both types of therapeutics to tumors, this strategy has the advantage of selectively delivering siXkr8 to those tumor cells that are exposed to chemo drugs. Consequently, our co-delivery approach is particularly effective in antagonizing the Xkr8 mRNA that is induced in situ by a co-delivered chemotherapeutic drug.”

According to Li, the study also pointed to the potential of combining the FuOXP-siRNA nanoparticles with another type of immunotherapy called checkpoint inhibitors. Immune checkpoints such as PD-1 act like brakes on the immune system, but checkpoint inhibitors work to release the brakes and help immune cells to fight cancer.

The researchers found that FuOXP nanoparticles with or without siRNA increased PD-1 expression. But when they added a PD-1 inhibitor drug, the combination therapy had drastic improvements in tumor growth and survival in a murine pancreatic cancer model, Panc02. “FuOXP NPs with or without co-loaded siXkr8 also caused significant upregulation of PD-1 expression in CD8+ T cells in the Panc02 model, suggesting potential for a combination therapy with anti-PD-1 antibody,” the investigators reported. “Indeed, combination of FuOXP/siXkr8 NPs with anti-PD-1 led to a drastic improvement in therapeutic efficacy as evident from significant inhibition of tumor growth and prolongation of survival time in a relatively advanced tumor model.”

With their sights set on translating their novel therapy to the clinic, the team is now looking to validate their findings with additional experiments and further evaluate potential side effects.

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