Abstract B10: Effects of vascular normalization when combining miR-200b with radiation

Abstract

Abstract Introduction: Lung cancer is the leading cause of cancer-related deaths worldwide, and patients usually die from metastatic disease. Radiation is a mainstay treatment in lung cancer, but its efficacy is largely dependent on tumor perfusion. We recently found that miR-200b is a novel AngiomiR capable of inhibiting metastasis and tumor angiogenesis while inducing vascular normalization. We are investigating whether vascular normalization with miR-200b can enhance therapeutic efficacy of radiation in a metastatic model of lung cancer. Methods: Using lung cancer cell lines, we assessed direct effects of miR-200b on radio-sensitivity using clonogenic assays and gamma-H2AX indices. Using an orthotopic lung cancer model (344SQ) in athymic nude mice, we determined peri-vascular biodistribution of intravenously administered chitosan nanoparticles (CH) containing Cy3-labled miRNA. Using the same model, a randomized therapeutic experiment was performed as follows (n=10 mice/group): 1) NC miR-CH, 2) miR-200b-CH, 3) NC miR-CH + radiation, or 4) miR-200b-CH + radiation. Radiation groups received four fractions of 4 Gy (16 Gy total). Following two weeks of treatment, mice were necropsied, and tumor burden was annotated and subsequently analyzed for microvessel density (MVD), pericyte coverage (CD31/Desmin) and tumor hypoxia (pimonidazole staining). Results: Following a single delivery of CH nanoparticles containing Cy3-labled miRs, we found that 37% of tumor vessels had Cy3 signal within 12.5 uM, 60% within 25 uM, and 82% within 50 uM. Compared to NC miRNA, we found that miR-200b overexpression in conjunction with radiation did not diminish colony formation or increase gamma-H2AX indices, suggesting miR-200b likely has little role in directly sensitizing cancer cells to radiation. In an orthotopic model of lung cancer, compared to NC miR-CH, while delivery of miR-200b-CH alone or treatment with NC miR-CH plus radiation showed no differences in metastasis, the combination of miR-200b-CH and radiation led to a 58% reduction in aggregate metastatic disease (p<0.05). Compared with the NC miR-CH treated group, MVD was significantly reduced by miR-200b-CH (27%), NC miR-CH plus radiation (50%) and miR-200b-CH plus radiation (77%), all p<0.0001. Dual treatment with miR-200b-CH and radiation had an additive anti-angiogenic effect compared with miR-200b-CH or NC miR-CH plus radiation alone, p<0.0001. Consistent with these dramatic reductions in MVD was an associated increase in the area of tumor hypoxia (ATH). Compared to NC miR-CH (16% ATH), while miR-200b-CH treatment did not significantly affect hypoxia (20% ATH, p=0.26), both radiation treatment groups had significantly increased tumor hypoxia (NC miR-CH plus radiation, 25% ATH, p<0.01; miR-200b-CH plus radiation, 41% ATH, p<0.0001). Again, dual treatment had a significantly additive effect compared to either treatment alone (miR-200b-CH v. miR-200b-CH plus radiation, p<0.01; NC-miR-CH plus radiation v. miR-200b-CH plus radiation, p=0.027). Intriguingly, compared with NC miR-CH treatment, only the miR-200b-CH delivery groups led to significant increases (40-50%) in pericyte coverage (both p<0.05). Discussion: Our work demonstrates that CH nanoparticle delivery of miR-200b can effectively target tumor vasculature. The combination of miR-200b delivery with radiation effectively inhibited lung cancer metastasis and was significantly associated with a marked decrease in MVD and increase in pericyte coverage. These additive effects appear to be independent of miR-200b directly radio-sensitizing cancer cells. However, we found that the profound anti-angiogenic effects of combination treatment (miR-200b-CH plus radiation) led to a 2.5 fold increase in tumor hypoxia. Our findings suggest that vascular normalization induced by miR-200b can be exploited to create an optimal “treatment window” for combination with radiation therapy. Citation Format: Salma Azam, Seth Miller, Trent Waugh, Vivek Somasundaram, Mitchell Smith, Adrienne D. Cox, Chad V. Pecot. Effects of vascular normalization when combining miR-200b with radiation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr B10.