Abstract LB-7: Protein crosstalk in tumor microenvironment: direct identification of intercellular protein transfer in retinoblastoma after topotecan or radiation treatment

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Current work on the tumor microenvironment has shown that crosstalk between cancer and normal cells can be of significant importance. Here we show direct intercellular communication between different cancer cell populations can provide insights into the tumor microenvironment and the bystander effect. Retinoblastoma is the primary intraocular cancer in childhood and resistance to current treatments such as chemotherapy or radiation often results in enucleation. We seek to improve retinoblastoma treatments by elucidating the diversity of and communication between retinoblastoma tumors. Here we provide the first direct evidence of intercellular protein transfer between retinoblastoma cells after topotecan or radiation treatment using stable isotope labeling and tandem mass spectrometry-based protein identification. Retinoblastoma Y79 cells were grown in media containing 13C, 15N lysine and arginine analogues (Y79S) so that all the proteins had full incorporation of these stable isotopes. The Y79S cells were untreated, treated with 0.02 micromolar topotecan, or treated with 4 Gy of radiation before being co-cultured with either Y79 or CHLA215 retinoblastoma cells for 48 hr. Protein populations within each cell type and the concentrated media were identified using mass spectrometry-based sequencing. Direct intercellular transfer was monitored using the unique 13C, 15N protein tags. Changes in the tumor microenvironment and the protein communication between retinoblastoma cells with chemotherapy and radiation treatment were monitored using a transwell co-culture system. As expected, irradiation resulted in a significant decrease in Y79S cell number and viability. Additionally, irradiated Y79S cells caused a significant decrease in the viability of the non-irradiated Y79 bystander cells. Studies of the proteins directly transferred from Y79S to the other retinoblastoma cells identified heat shock protein 90 (HSP90) in both the topotecan and radiation studies. Significantly, time point data provided insights into the protein transfer dynamics. We show significant protein transfer by 24 hours with a decrease in isotopically labeled proteins by 48 hours. We are currently working on elucidating the functional roles of HSP90 and the other proteins transferred during chemotherapy and radiation treatment. In summary, we have shown the first evidence for direct protein crosstalk between retinoblastoma cell populations in response to chemotherapy and radiation. We anticipate that this work will lead to greater insights into how tumor heterogeneity contributes to treatment response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-7. doi:1538-7445.AM2012-LB-7