Abstract 1709: Quantification of biologically relevant vascular phenotypes in human prostate cancer: automated image analysis using hyperplexed immunofluorescence
Authors: Sevinsky Chris , Santamaria-Pang Alberto , Zhang Jingyu , Lowes Christina , Sangurdekar Dipen , Falcon Beverly , Li Qing , Pytowski Bronek , Benjamin Laura , Graff Jeremy , Ginty Fiona , Nasir Aejaz , Uhlik Mark T. 
Published: August 2015

Abstract
Although anti-angiogenic therapy has emerged as a leading modality in treating human cancer, further improvements in the duration and frequency of clinical response of various human cancers remain important clinical needs. Maturity of the tumor vasculature has been identified as one of the major determinants of response of cancers to anti-angiogenic treatments. We have developed and optimized the application of a quantitative, high throughput, hyper-plexed, fluorescence imaging technology to refine our understanding of the complexity of vascular phenotypes in various human malignancies. The technology was used to quantify tumor blood vessels and expression of thirteen proteins of known roles in blood vessel biology in single sections from archival primary tumor tissues from 64 prostate cancer patients. CD31 was used to segment vascular objects in each image. CD31 and CD34 endothelial cell staining, SMA pericyte staining, and collagen IV basement membrane staining were used to classify detected vessels using K-means cluster analysis. Segmented vessels were clustered into 2-20 cluster sets, and the reproducibility of vessel classification of each cluster set was determined using the consensus clustering algorithm. A six cluster set that reflected biologically relevant tumor vascular subsets with high consensus clustering concordance was selected for further analysis. Clusters consistent with different stages of vessel development were obtained, including clusters with CD34 high/SMA low and CD34 low/SMA high profiles, reflecting immature and mature vascular phenotypes. Additional clusters representing phenotypes consistent with transitional vascular developmental stages were also identified. Nine additional proteins involved in angiogenesis were also quantified in each vessel and expression profiles for each cluster were determined. The enrichment of blood vessel clusters was then analyzed for each patient. This revealed differential patterns of vascular maturity phenotypes in the prostate cancer tissues analyzed. We have demonstrated that high-throughput, quantitative characterization of vascular maturity phenotypes is feasible in human cancer tissue specimens. Such immunofluorescence hyper-plex profiling of vascular-related proteins has the potential to illuminate the complex biology of tumor angiogenesis and to enable novel approaches for patient tailoring in clinical trials of anti-angiogenic therapeutics.
Citation Format: Chris Sevinsky, Alberto Santamaria-Pang, Jingyu Zhang, Christina Lowes, Dipen Sangurdekar, Beverly Falcon, Qing Li, Bronek Pytowski, Laura Benjamin, Jeremy Graff, Fiona Ginty, Aejaz Nasir, Mark T. Uhlik. Quantification of biologically relevant vascular phenotypes in human prostate cancer: automated image analysis using hyperplexed immunofluorescence. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1709. doi:10.1158/1538-7445.AM2015-1709