Immunogenic cell death in a combined synergic gene- and immune-therapy against cancer.

It was previously demonstrated that engineered mesenchymal stem cells (MSCs) which express a high level of a very efficient modified gene CYP2B6* (CYP2B6TM-RED) acting as a suicide gene (MSC-2B6*) in combination with cyclophosphamide (CPA) constitute a powerful cell/gene therapy approach for solid tumors. In murine models, this combination led to tumor eradication and triggered a durable immune response against tumoral cells, which prevented recurrence and metastasis. The first goal, in this work, was to determine whether the mechanism of tumor cell death caused by CPA metabolites could explain the appearance of this anti-tumor immune response. In vitro, CPA metabolites produced by MSC-2B6* were able to induce immunogenic cell death (ICD) of tumor cells. Indeed, all ICD characteristic events were clearly identified: calreticulin translocation, LC3II expression and release of ATP and HMGB1. The second goal was to determine the respective roles of the direct cytotoxicity of CPA metabolites and the immune anti-tumor response due to ICD of tumor cells during tumor eradication. In vivo, the early inhibition of ICD (with anti-HMGB1 antibodies) or the depletion of CD8+T lymphocytes (with anti-CD8 antibodies) prevented tumor eradication by CPA metabolites and tumor regrowth occurred, despite CPA treatment. In conclusion, the full eradication of the tumors depends on the association of cytotoxic CPA metabolites triggering the ICD of tumor cells and an anti-tumor immune response. The absence of one or the other of these effects prevents the complete eradication of tumors.

Engineered mesenchymal stem cells as vectors in a suicide gene therapy against preclinical murine models for solid tumors.

Gene-directed enzyme pro-drug therapy (GDEPT) consists of expressing, in tumor cells, a suicide gene which converts a pro-drug into cytotoxic metabolites, in situ. In a previous work, we demonstrated that the combination of the suicide gene CYP2B6TM-RED (a fusion of a triple mutant of CYP2B6 with NADPH cytochrome P450 reductase) and cyclophosphamide (CPA) constituted a powerful treatment for solid tumors. In this work, we investigated the use of mesenchymal stem cells (MSCs) as cellular vehicles for the delivery of our suicide gene. MSCs were genetically engineered ex-vivo to stably express CYP2B6TM-RED. Ex vivo and in vivo investigations showed that MSCs expressing CYP2B6TM-RED were able 1) to bioactivate CPA and produce local cytotoxic metabolites in tumor sites and 2) to destroy neighboring tumor cells through a bystander effect. Intratumoral injections of CYP2B6TM-RED-MSCs and CPA completely eradicated tumors in 33% of mice without recurrence after 6months. Rechallenge experiments demonstrated an efficient immune response. These data suggest that MSCs expressing CYP2B6TM-RED with CPA could represent a promising treatment for solid tumors to test in future clinical trials.

Mesenchymal stem cells as cellular vehicles for prodrug gene therapy against tumors.

Gene-directed enzyme prodrug therapy (GDEPT) consists of targeted delivery to tumor cells of a suicide gene responsible for the in situ conversion of a prodrug into cytotoxic metabolites. One of the major impediments of GDEPT is to target specifically the tumor cells with the suicide gene. Among gene delivery methods, mesenchymal stem cells (MSCs) have emerged recently as potential cellular vehicles for gene delivery. MSCs are particularly suited for gene transduction. They exhibit remarkable migratory property towards tumors and their metastases and they are weakly immunogenic. This review will summarize the current knowledge about MSCs engineered to express different suicide genes (cytosine deaminase, thymidine kinase, carboxylesterase, cytochrome P450) to elicit a significant antitumor response against brain tumors, ovarian, hepatocellular, pancreatic, renal or medullary thyroid carcinomas, breast or prostate cancer and pulmonary metastases. The potential side effects of these MSC-based tumor therapies will also be considered to highlight certain aspects that need to be improved prior to clinical use.