Abstract So far, therapeutic vaccination with allogeneic cancer cells has been only marginally effective. A reason for failure could stem from the fact that vaccine cells are usually cultured in air (pO2=20 kPa), while tumors in situ are often highly hypoxic. Hence, it is possible that vaccine cells expressed antigens differently than tumor cells in situ. We postulate that cells grown at low pO2 provide a better antigen match to tumors in situ and could be more effective vaccines. We tested the former hypothesis by comparing prostate cancer (CaP) cells propagated at pO2 = 2 kPa and 20 kPa. To identify potential tumor-associated antigens, we prepared CaP cell lysates, resolved them by 2D electrophoresis and immunoblotting using spontaneous antibodies from plasma derived from CaP patients and control subjects. Antibody-labeled spots were analyzed by MALDI-TOF mass spectrometry. Among identified molecules, we selected hypoxia-regulated HSP70 and hnRNP L and hypoxia-independent HSP60 and determined by ELISA the frequency of plasma samples reacting with these molecules two standard deviations above their reaction with normal sera. Frequency of HSP60-reactive plasma was 1/76 in healthy controls (HC; 1.3%), while it was 7/54 in CaP patients (13.0%; p<0.05). The corresponding values were 1/14 in colorectal cancer (7.1%), 1/10 in lung cancer (LC; 10.0%), 1/20 in renal cell carcinoma (5.0%) and 0/17 in rheumatoid arthritis (RA; 0%), not different from healthy controls. These data suggest that CaP patients develop humoral immunity to HSP60. Levels of autoantibodies to HSP70 in CaP patients (2/38, 5.3%) did not differ from HC (2/54, 3.7%) while they did differ from HC in LC (2/10, 20.0%) and RA (3/17, 17.7%). Similarly, frequency of autoantibodies to hnRNP L did no differ between HC (3/49, 6.1%) and CaP patients (2/38, 5.3%). Further, we evaluated the expression of hnRNP L protein in native CaP tissue by Western blot. In contrast to the levels of autoantibodies in plasma, hnRNP L antigen was present at twice the levels in CaP tissue (N=8) than in control benign prostate tissue (N=4; p<0.05). Overall, our results suggest that it is worthwhile to pursue evaluating of HSP60-specific autoantibodies as a CaP-associated marker. Currently, we are analyzing HSP60 and hnRNP L (hypoxia-regulated) protein and transcript expression by immunohistochemistry and PCR, respectively, in a large patient cohort. This approach could validate the use of hypoxia-sensitive molecules as effective prognostic biomarkers and help develop better therapy for CaP and, possibly, other tumors. Support: DOD PC094680 and PCF Creativity Award (both to CRG). Citation Format: Tangeng Ma, Abdelouahid Elkhattouti, Farhad Kosari, R. Jeffrey Karnes, John C. Cheville, George Vasmatzis, Stanimir Vuk-Pavlović, Christian R. Gomez. Hypoxia as a tool for identifying prostate cancer-associated antigens. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4056. doi:10.1158/1538-7445.AM2013-4056