Abstract 5120: Using flow-proteometric platform to analyze individual signaling complexes in tumor tissue
Authors: Chou Chao-Kai , Lee Heng-Huan , Tsou Pei-Hsiang , Chen Chun-Te , Hsu Jung-Mao , Yamaguchi Hirohito , Wang Ying-Nai , Hsu Jennifer L. , Lee Jin-Fong , Kameoka Jun , Hung Mien-Chie 
Published: August 2015

Abstract
Cellular signaling complexes, which are made up of mostly proteins and nucleic acids, play a major role in signal transduction by carrying and delivering messages that coordinate basic biological functions. These processes are mainly relayed through protein-protein and protein-nucleic acid interactions. Delineating the underlying mechanisms of cancer signal transduction, which is often deregulated, relies highly on the analysis of specific protein-protein and protein-nucleic acid interactions. Conventional methods for detecting protein-protein and protein-nucleic acid interactions mostly rely on cells collected from two-dimensional cell cultures, which are very different from the natural cellular environment in organs or tissues. Therefore, we developed a multiplex flow-proteometric platform that can analyze individual signaling complexes directly from tissue to enable us to accurately acquire the information on in vivo signal transduction. To demonstrate that single signaling complexes can be directly detected in lysates from fluorescent-labeled frozen tissue sections, we selected STAT3, p300, and genomic DNA as targets to detect and quantify individual complexes in xenograft tumor tissues. Endogenous STAT3 and p300 in frozen tumor tissue were immunolabeled with A488 and QD605, respectively, and with TOTO3 to label genomic DNA. All the detected events were presented in a 3D fluorescence plot, revealing seven different types of events. A minimal number of non-specific control IgG events were detected, and no interactions events were observed, indicating low background noise. Among all of the detected STAT3 events, on average 7.04% interacted with both p300 and genomic DNA in the same complex, 2.99% interacted with p300 only, and 15.23% interacted with DNA only. Thus, in the frozen xenograft tissue section, around 22% of STAT3 (15.23% STAT3−DNA + 7.04% STAT3−p300 −DNA) interacted with DNA and 7.04% of STAT3 interacted with p300 when bound on DNA to activate gene transcription. Looking at the data from the standpoint of p300, on average 5.88% of total p300 protein molecules interacted with STAT3 and genomic DNA, 2.5% with STAT3 only, and 3.06% with DNA only. All these data simultaneously obtained in each experiment not only identified STAT3, p300, and DNA in a single complex but also quantified the distribution of lone proteins and population complexes, which cannot be analyzed by conventional methods. Thus, we expect that this technique may reveal new aspects of molecular interaction in tissue.
Citation Format: Chao-Kai Chou, Heng-Huan Lee, Pei-Hsiang Tsou, Chun-Te Chen, Jung-Mao Hsu, Hirohito Yamaguchi, Ying-Nai Wang, Jennifer L. Hsu, Jin-Fong Lee, Jun Kameoka, Mien-Chie Hung. Using flow-proteometric platform to analyze individual signaling complexes in tumor tissue. [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 5120. doi:10.1158/1538-7445.AM2015-5120