Abstract B058: CD73 siRNA therapy regulates glioblastoma immune microenvironment
Authors: Lenz Gabriela Spies , Azambuja Juliana Hofstätter , Schuh Roselena Silvestri , Michels Luana Roberta , Gelsleichter Nicolly Espindola , Beckenkamp Liziane Raquel , Roliano Gabriela Goncalves , Figueiró Frabricio , Scholl Juliete N. , Sévigny Jean , Wink Márcia R , Teixeira Helder Ferreira , Braganhol Elizandra 
Published: March 2020

Abstract
Glioblastoma (GB) is the most common primary brain tumor, characterized by high aggressiveness and poor prognostic. Although all efforts, current therapy is just palliative. Therefore, new biological targets are needed to treat this invasive cancer. In this regard, immune modulation emerges as a possibility for cancer therapeutic. Literature has shown that the purinergic system can regulate peripheral immune cell functions and adenosine (ADO) plays an important role as an immunosuppressive molecule. CD73 enzyme activity is considered the main extracellular ADO source and it is overexpressed in a variety of tumors, including GB. According to CD73 participation in tumor progression, studies from our group have shown that CD73 knockdown impairs in vitro and in vivo GB growth. Here, we determined the potential of CD73siRNA (CD73 small interference RNA) delivery using the nanotechnology strategy to reduce tumor growth and modulate GB immune microenvironment (GME) in a preclinical immunocompetent GB model. For this purpose, an in vivo experiment was performed. C6 glioma cells were cultivated and implanted in the striatum of Wistar rat brains (male, 60 d) by stereotaxic surgery. Following 5 days of GB implant, the treatment with CD73siRNA complexed to nanoemulsion (NE) via nasal route has started and continuous by 15 days (twice a day). Experimental groups were composed as follows: control group (glioma-bearing animals that receive saline via nasal route) and treated group (CD73siRNA-NE; 10 µg/kg). After 20 days of GB implant surgery, animals were euthanized and the GB histopathological characteristics were analyzed by HE staining; apoptosis cell death analysis and GME composition were determined by flow cytometry using annexin V and specific antibodies for macrophage and microglia staining. In the histopathological analysis, tumors presented GB characteristics, as necrosis, edema, and angiogenesis. The treatment decreased tumor volume by 60% and increased the GME apoptotic cell index at 10%. Also, the drug interfered in GME population cells, the microglia CD11b+CD45low cells (from 3.4 ± 1.9 to 0.2 ± 0.1 %) and macrophages CD11b+CD45high cells (from 1.22 ± 0.3 to 0.4 ± 0.1 %) were practically abolished in the GME. Our data suggest that the lower tumoral volume is due to the higher apoptotic rates, indicating immune participation in cancer cell death. Furthermore, the absence of microglia and macrophages tumor-associated may contribute to the decreased tumor volume, proposing an immune system activation ADO modulated over the GB.
Citation Format: Gabriela Spies Lenz, Juliana Hofstätter Azambuja, Roselena Silvestri Schuh, Luana Roberta Michels, Nicolly Espindola Gelsleichter, Liziane Raquel Beckenkamp, Gabriela Goncalves Roliano, Frabricio Figueiró, Juliete N. Scholl, Jean Sévigny, Márcia R Wink, Helder Ferreira Teixeira, Elizandra Braganhol. CD73 siRNA therapy regulates glioblastoma immune microenvironment [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B058. doi:10.1158/1535-7163.TARG-19-B058