Lymphoblast cells secrete putative protein factors in response to high-dose radiation causing anti-tumor effect in lung cancer cells

High-dose ionizing radiation is known to induce non-targeted effects through bystander, abscopal, or immunological factors. As a complex niche, tumor microenvironment (TME) consists of several types of cells including cancer cells, immune cells, cancer-associated fibroblasts, endothelial cells etc. During the focal irradiation of the tumors, these cells may get exposed and release specific/unique factors, resulting in enhanced anti-tumor effects. The aim of this study was to identify the protein factors secreted by irradiated lymphoblasts and investigate their bystander/abscopal effects on lung cancer cells.Lymphoblasts (GM3798) were irradiated (10 Gy) at the density of 1x106/mL. After 24 h, conditioned medium (CM) from radiation exposed (L-RCM) and un-exposed (L-CCM) flasks was collected and centrifuged to remove debris. H460 cells pre-plated on the microelectronic sensors of the 16-well plate were either incubated with L-CCM or L-RCM and their growth was continuously monitored by real time-cell electronic sensing system. Furthermore, lymphoblasts were grown in serum-free media for 24 h and following irradiation, 200 mL of the L-RCM and L-CCM was concentrated and fractionated by ultrafiltration (10, 30, 50 and 100KDa fractions) to identify the protein factors secreted by the cells. The effects of different fractions on A549 cell survival were assessed by colony forming assay. To obtain a proteomic comparison of >100 KDa fractions of L-RCM vs. L-CCM, following 1D PAGE, each gel slice was subjected to tryptic digestion and protein identification by mass spectrometry.We found that L-RCM exerted significant growth inhibition of H460 lung tumor cells compared to L-CCM. Based on these results, we analyzed the secretory proteome profile of high-dose irradiated lymphoblasts. The protein factors responsible for 10 Gy-specific cell killing of A549 cells were found to reside in the 100 KDa fraction in a manner proportional to protein concentration. 277 proteins were identified with confidence in the protein fraction retained by the >100 KDa fractions, representing both large proteins and protein complexes. A number of statistically reliable proteins were found only in the 10 Gy RCM fraction. The highest eMPAI score was assigned to HLA-DQB2 protein involved in immune response of CD4+ T lymphocytes.Our findings strongly suggest that high-dose radiation to TME can facilitate the release of secretory factors that can render a robust anti-tumor effect through bystander/abscopal signaling. Furthermore, identification of secretory proteins in response to high-dose radiation by the use of systematic and high through-put proteomics will help in identifying biomarkers in cancer patients. These proteins will serve as a marker for tailoring the use of adjuvants such as conventional subsequent radiation or chemotherapy or biological modifying drugs.Citation Format: Kamila Rawojc, Anthony Yeung, Mansoor M. Ahmed, Seema Gupta. Lymphoblast cells secrete putative protein factors in response to high-dose radiation causing anti-tumor effect in lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2920.