Macrophages are immune cells that normally seek out and engulf potential threats including viruses, bacteria, and cancer cells. However, the tumor microenvironment switches macrophages converging at the tumor site from their tumor-killing state to a tumor-promoting state. Researchers at Harvard University’s Wyss Institute for Biologically Inspired Engineering and the John A. Paulson School of Engineering and Applied Sciences (SEAS) have now created cytokine-secreting “backpacks” for macrophages that keep these key immune cells in a tumor-killing state for up to five days after they arrive at a tumor site. Initial tests showed that these backpack-carrying macrophages slowed tumor growth and reduced metastasis in mice with an aggressive form of breast cancer.

“This study speaks to the beauty of macrophages—they are highly adaptable cells that respond very strongly to stimuli in their environment, but this can also be a problem when they receive a stimulus that tells them to do something that’s actually harmful to the body, like helping cancer grow or metastasize,” said first author C. Wyatt Shields, PhD, who completed the study as a postdoctoral fellow in the lab of Wyss core faculty member Samir Mitragotri, PhD. “We have shown that it’s possible to provide a sustained stimulus via these backpack particles to keep macrophages in their desired state, and we hope that this technique could one day be used to treat a variety of conditions related to immune dysfunction.” Shields is now an assistant professor at the University of Colorado, Boulder.

The researchers describe development of the macrophage backpack technology in Science Advances, in a paper titled, “Cellular backpacks for macrophage immunotherapy.”

Macrophages can assume many roles in the body, such as defending against pathogens, aiding in wound healing, and regulating tissue homeostasis. The cells rely on soluble cues in the tissue environment to direct their polarization into different phenotypes. Macrophages could thus feasibly be harnessed for the development of anticancer immunotherapies that might work where existing approaches such as chimeric antigen receptor (CAR) T cell treatments aren’t effective. “… the phenotypic plasticity of macrophages makes them excellent candidates for addressing a range of diseases,” the team wrote.

However, when tissues become dysfunctional, macrophages can develop phenotypes that promote, rather than act to correct disease pathogenesis. “In the case of cancer, tumor-associated macrophages (TAMs) typically adopt M2 (tumor-promoting) phenotypes due to the immunosuppressive microenvironment of solid tumors, which is associated with tumor growth, angiogenesis, chemotherapy resistance, and metastasis,” the scientists pointed out.

Efforts to extract macrophages from the body, force them into their tumor-killing state by polarizing them with proinflammatory cytokines, and then reintroduce them into the body to fight cancer, have thus failed in the clinic, as the tumor environment switches them back to their protumor M2 state. “… a major hurdle that has slowed the adoption of macrophages in cancer immunotherapy is their tendency to shift to protumoral phenotypes once injected into the body,” the authors continued. “Thus, for macrophage-based therapies to induce robust therapeutic effects in the clinic, strategies must be developed to control phenotypes of adoptively transferred macrophages in vivo.”

Previous work by the Mitragotri lab had showed that, surprisingly, small, disc-shaped particles can stick to and hitch a ride on macrophages for several days without triggering an “eat me” phagocytic response, and so could offer an opportunity for influencing macrophage behavior. For their newly reported work, the team developed a class of soft backpacks that can regulate the phenotype of macrophages in vivo.

The backpacks are constructed of two layers of the biocompatible polymer poly(lactic-co-glycolic) acid (PLGA) with a filling of polyvinyl alcohol (PVA) and the cytokine interferon gamma (IFNγ) sandwiched between them. IFNγ is known to be a potent stimulator of the proinflammatory response in macrophages, and has been shown to reduce the size of some tumors. To finish off the backpacks, a final cell-adhesive layer was added to help them stick to their macrophage mounts. “Backpacks were prepared from biodegradable materials that enable facile preparation, long-term storage, and simple metabolic clearance, all of which are favorable for clinical translation,” the scientists commented. “Furthermore, injected macrophages were allogeneic, which reduces the time scale of preparing cell transfers from weeks [i.e., for CAR T cell therapy to several hours].

Shields and his co-authors mixed macrophages with their backpack particles in vitro, and found that about 87% of the cells picked up one to four backpacks on their surfaces, which remained there for at least five days without being consumed. The backpacks secreted IFNγ for at least 60 hours.

The researchers then tested the macrophages for various markers that indicated whether they were in a proinflammatory (M1) state that combats tumors, or in the anti-inflammatory, M2 state. They found that macrophages carrying IFNγ backpacks expressed three M1-associated factors much more strongly than macrophages with blank backpacks or macrophages in the presence of free IFNγ, while their expression of M2-associated factors did not change significantly. The increased M1 expression profile also lasted longer than that of either of the control groups, suggesting that the IFNγ backpacks could induce a lasting shift to the M1 state.

The team tested their backpack-carrying macrophages by injecting the cells into the tumors of mice with an aggressive form of metastatic breast cancer, to see if the backpacks could maintain the macrophages in their M1 state. Their results showed that the macrophages carrying IFNγ backpacks expressed M1 indicators for at least 48 hours, and relevant expression levels were significantly higher than that of injected cells with blank backpacks or with free IFNγ. They also found that mice treated with the IFNγ backpack therapy had significantly fewer metastatic nodules and smaller tumors than control mice, and lived longer.

More detailed analyses showed that not only did the backpack-carrying macrophages stay in their M1 state, they actually helped other tumor-associated macrophages revert from an anti-inflammatory M2 state back to an M1 state, effectively activating them to fight the tumors. Encouragingly, this result was achieved with a dose of IFNγ that is 100-fold lower than the maximum total dose used in other studies, and the mice did not display any signs of toxicity from the treatment. “Future studies will investigate the optimal loading of IFNγ into backpacks and their release kinetics to enhance this therapeutic efficacy against solid tumors. In addition, future work can combine backpacks with adjuvant therapies to enhance therapeutic effects,” the team noted.

“Macrophages can make up roughly 50% of the mass of a tumor. If we are able to switch them into their M1 state and sustain that activation, it could massively reduce the size of tumors and give both the immune system and treatments like chemotherapy better access to the cancer cells themselves,” said Shields.

In addition to triggering a sustained proinflammatory response against tumors, the team’s backpack approach could feasibly be used to shift macrophages into an anti-inflammatory state in patients who are suffering from diseases such as rheumatoid arthritis, Crohn’s disease, or lupus, which are associated with excess inflammation.

Mitragotri’s lab is continuing to explore different applications of the technology, including loading different agents into the backpacks and testing their ability to bind and control other types of cells. “While the backpacks described here bind to macrophage surfaces, other designs can be conceived to attach to other circulatory cells with higher chemotactic sensitivity,” the scientists wrote. “Also, a range of immunomodulatory payloads can be considered, including those that facilitate adaptive immune responses toward a backpack-based vaccine or promote anti-inflammatory phenotypes to aid in tissue regeneration or repair for autoimmune diseases.”

“Improving drug delivery is a hot topic in biomedical research these days, and cells are one of the best vehicles for that because they can navigate through the body’s defensive barriers and reach their target with high precision,” said Mitragotri, who is the Hiller professor of bioengineering and Hansjörg Wyss professor of biologically inspired engineering at SEAS. “The biggest challenge with introducing a living entity into the body for treatment is figuring out how to control it after it’s injected, so that it does what you want it to do. This study is a very strong proof-of-concept for a new way of controlling cells in vivo, and we think it could provide a versatile platform for treating numerous different conditions.”

“Samir Mitragotri and his team are continuously inventing new ways to overcome the many barriers in the body that hamper drug efficacy,” added Wyss Institute founding director Donald Ingber, MD, PhD, who also is the Judah Folkman professor of vascular biology at Harvard Medical School and Boston Children’s Hospital, and professor of bioengineering at SEAS. “This study harnesses nature itself by using patients’ own macrophages to deliver the drugs that are required to induce these cells to kill cancer cells. It represents an exciting new breakthrough in the Institute’s approach to develop bioinspired therapeutics that will provide better and safer treatments for a wide range of human diseases.”

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