Abstract 4228: Imaging and targeting of hypoxic microenvironments in prostate cancer
Authors: Krishnamachary Balaji , Dore-Savard Louis , Bharti Santosh Kumar , Wildes Flonne , Mironchik Yelena , Black Margaret E. , Bhujwalla Zaver M. 
Published: July 2016

Abstract
Cancer cells display an adaptive response to hypoxia through the activation of several genes mediated by the binding of hypoxia inducible factors (HIFs) to hypoxia response elements (HRE) in the promoter region of target gene that results in their increased transcription [1]. HIFs promote key steps in tumorigenesis, including angiogenesis, metabolism, proliferation, metastasis, and differentiation [1]. Bacterial or yeast cytosine deaminase (yCD) converts the nontoxic prodrug 5-fluorocytosine (5-FC) to the anti-cancer drug 5-fluorouracil (5-FU) that is widely used in cancer treatment [2]. Using a lentivirus approach, we established controlled expression of yCD by HRE in prostate cancer cells (PC-3). These cells also report on HIF-1α expression with regulated luciferase (Luc) expression, allowing detection of hypoxia, and the generation of 5-FU from 5-FC by yCD in the presence of hypoxia. Transduction efficiency and reporter activity in response to hypoxia was evaluated by performing luciferase assays, and bioluminescence imaging (BLI) of cells in vitro or in vivo using a Xenogen IVIS Spectrum system. Cell viability in vitro in response to hypoxia in the presence of 5-FC was assessed by MTS assay. In vivo studies were performed by inoculating 2×10⁁6 PC-3-HRE-Luc cells and PC-3-HRE-yCD+Luc cells on either flank of 5-week-old male severely combined immune deficient (SCID) mice. BLI was performed once tumors reached ∼200mm3 followed by 5-FC injection through the tail vein (200mg/kg) and intraperitoneally (250mg/kg). BLI was performed 3 days after the first 5-FC injection and continued through the treatment protocol. At the end of the treatment protocol, tumors were excised, and a part of the tumor was processed for immunohistochemistry. Bioluminescence was detected in both PC3-HRE-Luc and PC-3-HRE-yCD+Luc cells only in response to the hypoxia mimetic cobalt chloride or hypoxia (1% O2) confirming the regulation of luciferase by hypoxia and activation of CD. Expression of yCD and its ability to convert the prodrug 5-FC to 5-FU, with increased cell kill was evident under hypoxia. In vivo, engineered PC-3-HRE-yCD+Luc cells reported hypoxia, and showed significant reduction of hypoxic regions and tumor volume. Morphologically, PC-3-HRE-yCD+Luc tumors exhibited extensive necrosis. We are currently evaluating the effects of eliminating hypoxic cancer cells on distant metastasis as well as on aggressive subpopulations such as cancer stem cells in the primary tumor.
References: [1] Philip, B., et al., Carcinogenesis, 2013. 34(8):1699-707.,[2] Longley DB, et al., Nat Rev Cancer, 2003. 3: 330-38.
Acknowledgements: This work was supported by NIH R01CA136576 and P50 CA103175. We thank Mr. Gary Cromwell for technical assistance
Citation Format: Balaji Krishnamachary, Louis Dore-Savard, Santosh Kumar Bharti, Flonne Wildes, Yelena Mironchik, Margaret E. Black, Zaver M. Bhujwalla. Imaging and targeting of hypoxic microenvironments in prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4228.