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Deciphering molecular consequences of the prohormone convertase 1/3 inhibition in macrophages for application in immunotherapy - Secretomes

ABSTRACT: The prohormone convertase 1/3 (PC1/3) is an enzyme playing an important role in the processing of precursor proteins involved in neuroimmune phenotype of neuroendocrine cells. The expression of PC1/3 is not only restricted to neuroendocrine tissues; recent studies reported a role of PC1/3 in Toll-like receptor immune response in macrophages. Here, using lentivirus strategy, we investigated the consequences of PC1/3 down-regulation with the aim of understanding the intracellular impact of such inhibition and its biological application to cancer therapy. Under sterile conditions, we demonstrated that PC1/3 inactivated macrophages express a M1-like phenotype characterized by filopodia extensions, pro-inflammatory chemokines and cytokines secretion (IL6, CXCL10; TNF) and TLR4 Myd88 dependent signaling activation. Under LPS challenge, PC1/3 KO cells secrete through store-operated calcium entry increase, a cocktail of pro-inflammatory factors including alarmins and chemokines conducting to attract naïve T helper lymphocytes (Th0) which favors cytotoxic response. Tests perform with the secreted factors by the challenged PC1/3 KO cells on breast and ovarian cancer cells (SKBR3, SKOV3) establish that the factors secreted are able to inhibit both viability and resistance to chemotherapies. Under inhibitory conditions using IL-10, the PC1/3 KO cells still continue to produce inflammatory cytokines and anti-tumoral factors. Taken together, these data establish that inhibition of PC1/3 can be used as a potential immune therapy for awaking intra-tumoral macrophages.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Cell Culture, Macrophage

SUBMITTER: Maxence Wisztorski

LAB HEAD: Michel Salzet

PROVIDER: PXD001986 | Pride | 2015-09-15

REPOSITORIES: Pride

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Publications

Molecular Consequences of Proprotein Convertase 1/3 (PC1/3) Inhibition in Macrophages for Application to Cancer Immunotherapy: A Proteomic Study.

Duhamel Marie M Rodet Franck F Delhem Nadira N Vanden Abeele Fabien F Kobeissy Firas F Nataf Serge S Pays Laurent L Desjardins Roxanne R Gagnon Hugo H Wisztorski Maxence M Fournier Isabelle I Day Robert R Salzet Michel M

Molecular & cellular proteomics : MCP 20150901 11

Macrophages provide the first line of host immune defense. Their activation triggers the secretion of pro-inflammatory cytokines and chemokines recruiting other immune cells. In cancer, macrophages present an M2 anti-inflammatory phenotype promoting tumor growth. In this way, strategies need to be develop to reactivate macrophages. Previously thought to be expressed only in cells with a neural/neuroendocrine phenotype, the proprotein convertase 1/3 has been shown to also be expressed in macropha ...[more]

PMID: 26330543

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Project description:Anti-cancer immunotherapy approaches are increasingly coveted. Chimeric antigen receptor (CAR)-T cell therapy has been shown to be an effective treatment for hematological tumors, but the treatment of solid tumors still lacks effectiveness, due to lower intra-tumor infiltration of CAR-T cells and tumor-induced immunosuppression. Macrophages represent a very large proportion of the tumor environment, participate in many aspects to tumor development and therefore represent interesting therapeutic targets. Macrophages can infiltrate solid tumor tissue and interact with almost all cellular components in the tumor microenvironment. In addition, macrophages can also promote a direct anti-tumor response by phagocyting tumor cells. We have developed macrophages expressing a CAR receptor against the HER2 antigen. The CAR receptor possesses an intracellular domain CD3ζ having homology with the protein FcεRI-γ, which once activated by the recognition antibody-antigen, induces the phagocytic activity of macrophages. 72% of macrophages express the CAR after transduction. CAR-M can specifically phagocyte HER2 coated-beads in a much more effective way than WT macrophages. We have then confirmed the capacity of CAR-M to phagocyte HER2+ cancer cell lines. Co-culture of CAR-M with breast cancer tumoroids (HER2+ or HER2-) has also been performed demonstrating their efficacy in a more complex environment. However, in the tumor microenvironment, due to their plasticity, macrophages tend to adopt an anti-inflammatory phenotype losing their anti-tumor activities. We have therefore developed a combined strategy by inhibiting two proprotein convertases, Furin and PC1/3 in CAR-M. The inhibition of furin or PC1/3 induces an increase in pro-inflammatory markers and maintains macrophage activation in the presence of cancer cells. In addition, HER2+ CAR-M with shFurin or shPC1/3 greatly increases the phagocytic activity on Her2+ beads or Her2+ tumors. These enzymes are therefore phenotypic regulators of macrophages. Our strategy is therefore based on a double activation of tumor-infiltrating macrophages. The first one consists in boosting the phagocytic activity of macrophages by having them express a CAR receptor targeting a tumor antigen. The second allows their reprogramming towards a pro- inflammatory phenotype by the inhibition of Furin and/or PC1/3 proprotein convertases

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