Abstract 3951: Colony lysate arrays for proteomic profiling of drug-persistent cancer cell subpopulations
Authors: Kume Kohei , Nishizuka Satoshi S. 
Published: July 2017

Clonal heterogeneity of cancer cells is one of the key properties by which to understand relapse after anticancer drug therapy. However, a very limited number of technologies are available for individually profiling these heterogeneous drug-resistant subpopulations. Therefore, the development of techniques to monitor molecular changes in the subpopulations responsible for drug-resistance, namely, colony-forming drug-tolerant persisters (DTPs), is needed in order to understand the occurrence of cancer relapse. To profile DTPs at the molecular level in relation to the colony-forming function, this study aimed to develop colony lysate array (CoLA) for the quantitative measurement of protein levels in individual colonies.

To investigate multiple parameters that may affect the emergence of DTP, DTPs derived from 5 cancer cell lines grown in the presence of 6 doses of 4 different drugs were collected. A set of &gt;2,000 colony lysates was printed onto a nitrocellulose-coated glass slide using a microarrayer, which has been used for the production of reverse-phase protein arrays (RPPAs). Each resulting CoLA slide was stained with a specific antibody from the list of 44 functionally-diverse proteins. Immunostained slides were then scanned using an ordinary optical flatbed scanner for quantifying the spot intensity.

To evaluate whether the CoLA system produced biologically relevant data relating to the DTPs, the epithelial/non-epithelial protein relationship between E-cadherin and vimentin was examined. With the proteomic profiling of &gt;2,000 DTPs, E-cadherin was negatively correlated with vimentin (r = -0.74, P &lt; 0.0001) and positively correlated with CK-8 (r = 0.86, P &lt; 0.0001), suggesting that DTPs may flexibly transit their biological entity in response to drug administration. A set of hierarchical clustering of DTPs displays proteomic patterns in each dose, allowing the comparison of proteomic changes in a dose-response manner. For example, the “cisplatin-induced cluster” demonstrated similar proteomic patterns regardless of the drug dose. In contrast to cisplatin, the “gefitinib-induced cluster” indicated that the proteomic patterns were similar at drug concentrations from 0 to 0.1 μM; however, those at 1 and 10 μM were distinct from those at lower concentrations. Individual protein analyses of the DTPs revealed that the level of STAT3 decreased while that of OCT4A increased according to the gefitinib concentrations.

An RPPA-modified method, CoLA, for individual colony profiling has been successfully established. The results of validation studies suggest that CoLA can be a tool for understanding the emergence mechanism of DTPs. The molecular characteristics of DTPs may reflect the entity of cancer cell subpopulations that contribute primarily to post-chemotherapeutic cancer relapse. Further technical development will enhance understanding of the occurrence of cancer relapse.

Citation Format: Kohei Kume, Satoshi S. Nishizuka. Colony lysate arrays for proteomic profiling of drug-persistent cancer cell subpopulations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3951. doi:10.1158/1538-7445.AM2017-3951