利用新型腺相关病毒介导疗法靶向三阴性乳腺癌中的Notch信号传导以增强免疫调节

Triple-negative breast cancer (TNBC) is a group of aggressive breast cancers with a higher mortality rate. TNBC patients have a higher rate of mortality due to metastasis or local recurrences, which are chemo- and radio-resistant. Notch signaling has emerged as an important factor in TNBC. There is strong evidence for Notch signaling involvement in TNBC. Recent studies suggest that Cancer Stem cells (CSCs) that emerge after chemotherapy or targeted agents in TNBC are often Notch-dependent. Notch inhibitors, including Gamma Secretase Inhibitors (GSIs), are quite effective in preclinical models of TNBC because they eliminate CSCs that are resistant to chemotherapy. Unfortunately, this approach has had minimal success in clinical trials due to its intestinal toxicity and adverse effects on immune cells. To avoid these toxicities, we aim to use a recombinant adeno-associated virus (r-AAV) vector encoding a soluble Notch1 decoy (r-AAV-Notch1D) to block Notch signaling intratumorally in TNBC. r-AAVs are non-replicating viruses and, therefore, are a safe platform for in vivo gene delivery. We found that r-AAV-GFP transduced both human and mouse TNBC cells. Our r-AAV constructs contain a C1QTNF3-derived collagen domain (CD) that facilitates the trimerization and stabilization of secreted fusion proteins. We purified these proteins using His-Tag affinity chromatography and found that they significantly reduced mammosphere growth in human TNBC cells and reduced Notch intracellular domain (NICD) expression. Intratumoral injections of the r-AAV-Notch1D virus significantly reduced syngeneic mouse TNBC tumor (C0321) growth and increased the tumor-infiltrating lymphocytes, including CD3 and CD8. Notably, the r-AAV-Notch1D virus significantly enhanced the efficacy of immunotherapy (a-PD1) in mediating the anti-tumor effect in the syngeneic TNBC mouse model. We performed RNA sequencing to identify tentative target(s) for r-AAV-Notch1D. We found that Hmox1, Timp3, Il33, Adam8, Ero1l, and Saa3 genes were upregulated, on the other hand Gm11408, Gm9844, Gm11407, Gm8430, Rnu3a, and Per1 genes were downregulated upon the treatment. From our Gene Ontology analysis, we found that r-AAV-Notch1D treatment enhanced hexosaminidase activity, carbohydrate catabolic process, covalent chromatin modification, histone modification, and histone acetylation. r-AAV-Notch1D treatment also altered several cellular pathways, including MicroRNAs in cancer, Ferroptosis, Porphyrin metabolism, Mineral absorption, and the Cytosolic DNA-sensing pathway. Given the diverse biological functions of these pathways, further study is warranted to elucidate their roles in TNBC. Overall, our findings indicate that the r-AAV-Notch1D virus is a promising approach for treating TNBC, alone or in combination with immunotherapy, and is a candidate for translational application.