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By directly injecting engineered dying (necroptotic) cells into tumors, researchers have successfully triggered the immune system to attack cancerous cells at multiple sites within the body and reduce tumor growth, in mice. The study was published in Science Immunology, June 21, 2019. This novel strategy presents as a potential treatment approach that could be used, in combination with existing immunotherapies, to "boost" the body's ability to fight cancer, in future.



We had the pleasure of speaking with Andrew Oberst, corresponding author of the study to learn more about the type of cells used in the study, the study design and limitations, how necroptotic cell death within the tumor leads to antitumor immunity, and the team’s plans for future studies.



Laura Lansdowne (LL): Could you tell us more about the type of dying cells used in the study?



Andrew Oberst (AO): We started by using dying tumor cells; that is, we created tumors in mice using either melanoma or lung carcinoma tumor cells, then introduced dying cells of the same type to those tumors. We found that when we introduced necroptotic (but not apoptotic) cells to these tumors, it activated the immune system to attack the tumor, reducing tumor growth and prolonging survival. What was surprising to us though was that we also saw a reduction in tumor growth when we introduced necroptotic cells of a different type–murine fibroblasts–to the tumors. This was surprising because these fibroblasts don't have any of the antigens that are present in the tumor. This indicated to us that the necroptotic cells were acting on the tumor microenvironment (TME), rather than directly priming antigen-specific T cells.



LL: Could you give us an overview of the study design and any limitations?



OA: We used murine flank tumor models to study the immune response to necroptotic cells within the TME in vivo. We first did this by injecting necroptotic tumor cells or fibroblasts; in these cases, we activated the key necroptosis-inducing kinase RIPK3 in these cells in vitro, then injected them into the tumors so that they would undergo cell death in vivo. Next, we wanted to find a way to directly trigger necroptosis within tumor cells themselves, in unmodified tumors. To do this, we engineered a form of the RIPK3 kinase that was constitutively active, so that just expression of this kinase was enough to rapidly induce necroptosis. We packaged a gene encoding this suicide enzyme into adeno-associated viruses (AAVs) and delivered them to tumors. We found that this approach also led to activation of antitumor immune responses.



A limitation of our study design is that it relies exclusively on transplanted flank tumors (tumor cells injected under the skin of mice, which then grow into tumors). These models are widely used, and are useful because they are easy to work with and yield rapid results; however, they are also much less complex and generally easier to "cure" than autochthonous tumor models. So, while our study provides a good proof-of-concept, significant additional work is needed to verify these ideas in models that more closely mimic human disease.



LL: How does necroptotic cell death within the tumor microenvironment (TME) lead to antitumor immunity?

AO: Our data indicate that necroptotic cells within the TME produce cytokines and chemokines that recruit and activate phagocytes (macrophages and dendritic cells) within the TME. Activation of these cells leads them to take up more material from the surrounding tumor, and to more readily present tumor-derived antigens to local CD8+ T cells. These activated CD8+ T cells can then control the tumor, and are also able to act systemically, controlling tumors at distant sites as well. What's interesting and unexpected is that the necroptotic cells act on the TME generally; they don't need to carry any tumor-associated antigen, but rather "turn on" tumor-resident phagocytes which then promote antitumor immune responses.



LL: Are there any plans you could share regarding future studies, to validate your findings?



AO: Our development of AAVs that directly induce necroptosis opens up possibilities for the delivery of these genes to unmodified, autochthonous tumors; that's something we'd like to explore using more physiologically relevant tumor models. Furthermore, our data indicate that it's the production of cytokines and chemokines upon RIPK3 activation that is immunogenic, not necroptotic cell death per se. Given that, understanding how cells balance inflammatory transcription vs. cell death upon RIPK3 activation, what controls these decisions, and how different primary or tumor cell types engage these pathways will be important if we want to translate this work into additional tumor settings.



Reference: A.G. Snyder, et al. Intratumoral activation of the necroptotic pathway components RIPK1 and RIPK3 potentiates antitumor immunity. Science Immunology (2019) DOI: http://immunology.sciencemag.org/lookup/doi/10.1126/sciimmunol.aaw2004



Andrew Oberst was speaking to Laura Elizabeth Lansdowne, Science Writer for Technology Networks.







