Evaluation of the autocrine and paracrine effects of IFN gene therapy with adenovirus and its relationship with IFNAR1 in melanoma and microenvironment of the murine model

Author(s)

Igor de Luna Vieira

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina (FM/SBD)
• Defense date: 2021-07-02

Examining board members

Bryan Eric Strauss; Luciana Nogueira de Sousa Andrade; Fernando Moreira Simabuco

• Advisor: Bryan Eric Strauss

Abstract

Cancer is a complex disease, often without cure, which is present in all populations and at all ages. Cancer generates enormous economic and emotional losses, and its current treatments are expensive and, typically, ineffective in advanced cases. Melanoma is an aggressive skin cancer in its evolution and often deadly for the hosts that present metastatic disease since few effective treatments are available. Faced with this situation, it is necessary to create and develop new approaches with greater efficacy in the treatment of cancer. Interferon beta (IFN) is an immunostimulatory cytokine with a known anti-tumor effect, but its systemic application is toxic. Our group proposes the use of gene therapy using adenoviral vectors that carry the therapeutic transgene IFN (Ad-mIFN), thus providing the production of IFN in a localized and transient way, which should avoid the systemic toxic effect. Our studies have shown that, in murine models, gene therapy with Ad-mIFN in vitro results in cell death and, in the in situ model, delays the progression of melanoma tumors. The research performed in this thesis aimed to study the autocrine and paracrine effects in the microenvironment and tumor cells of melanoma after IFN gene therapy and the role of the IFNAR1 receptor. To investigate the influence of IFN signaling on autocrine and paracrine effects, we performed IFNAR1 knockout on B16 and microenvironment cells (tEnd, NIH3T3) using the CRISPR / Cas9 technology. The results showed that cells transduced by Ad- mIFN require the production and release of IFN for activation of the IFN/ pathway, by binding with the IFNAR1/IFNAR2 receptors and finally inducing cell death, a mechanism known as autocrine. The cells transduced by Ad-mIFN also induce a paracrine effect by inhibiting nearby cells, by a mechanism similar to the autocrine but directed at other nearby cells. The cells with IFNAR1 knockout were blocked from receiving the autocrine and paracrine effects of Ad-mIFN and recombinant IFN protein (rIFN), however even without IFNAR1 they continue to produce the paracrine effect. These data were observed in tumor cells and endothelial and fibroblast cells. Another in vitro study of therapy with Ad-mIFN and Ad-p19 in angiogenesis identified an endothelial cell line unresponsive to treatment with Ad-mIFN and the IFN protein alone, however these cells suffered inhibition when tested for the paracrine effect provided by the melanoma cell line transduced with Ad-mIFN. The observation of this phenomenon suggests the existence of factors other than IFN, produced by melanoma cells transduced with Ad-mIFN, which would be involved in the paracrine effect. Gene expression assays of the melanoma lineage transduced by Ad-mIFN showed a significant increase in TRAIL, which may be a candidate for involvement in the paracrine effect. Together, these results indicate that the presence of IFNAR1 is not essential for the production of the factors necessary to induce the paracrine effect, however the presence of IFNAR1 is necessary for the cell to suffer this effect. In the future, IFNAR1-deficient strains can be evaluated in in vivo assays to reveal the role of IFNAR1 in the autocrine and paracrine effects between tumor cells and their microenvironment after treatment, including with Ad-mIFN (AU)

• FAPESP's process: 16/18197-3 - Investigation of the direct and paracrine angiogenic effects after gene transfer mediated by adenoviral vectors carrying the Interferon-beta in murine melanoma
• Grantee: Igor de Luna Vieira
• Support Opportunities: Scholarships in Brazil - Doctorate