Abstract C223: Targeting hypoxia for more effective immunotherapy of prostate cancer.
Authors: Gomez Christian R. , Schreiber Claire A. , Knutson Gaylord J. , Charlesworth Cristine , Vuk-Pavlovic Stanimir 
Published: June 2012

Abstract
In early clinical trials, vaccination with allogeneic cancer cells enhanced the overall survival in some patients with malignancies. Because the administered vaccine cells were cultured at pO2=20 kPa, it is unclear whether they provided an adequate antigen match to tumor cells in situ where pO2 is generally much lower. Thus, we postulate that hypoxically grown vaccine cells will provide a better antigen match to tumors in situ. We are testing this hypothesis by studying the effects of hypoxia on prostate cancer (CaP) cells. LnCaP and VCaP cells were cultured at pO2=2 kPa or 20 kPa and analyzed by 2-D gel electrophoresis. The difference in pO2 affected the proteome mostly quantitatively (i.e., by change in spot intensity). Using a threshold of fivefold change, we found that hypoxia decreased the levels of thirteen proteins and increased of four in VCaP cells. These results are in line with the reports showing that hypoxia affects expression only of a small fraction of total cellular protein and that the content of total protein is not altered significantly. To determine if the hypoxia-dependent changes affected immune reactivity of the cells, we took advantage of the spontaneous autoantibodies against tumor-associated antigens (TAAs). Hence, we compared the binding to lysates of LnCaP and VCaP cells of antibodies pooled from CaP patients (n=25), healthy controls (n=25), rheumatoid arthritis (n=17), colorectal cancer (n=10) and lung cancer (n=10). CaP patient sera plasma reacted with numerous spots, some even in control groups and thus considered nonspecific. CaP sera plasma specifically bound to seven spots; four were hypoxia-specific. All selected spots were excised from the gel, trypsin-digested, and identified by MALDI-TOF mass spectrometry as heat shock 70 kDa protein 4; protein disulfide isomerase A3; heterogeneous nuclear ribonucleoprotein L and leucine-rich repeat-containing protein 47. With the exception of the latter, the proteins have been identified or validated as TAAs before, but apparently none in CaP. CaP patient antibodies bound to lysates of LnCaP cells grown at pO2=2 kPa, but less than to such VCaP cell lysates. Our results confirmed the findings that hypoxia affects the proteome mostly quantitatively. Lysates of hypoxic CaP cells revealed novel potential TAAs suggesting the potential immunologic relevance of antigens in the hypoxic proteome. Thus, pO2 control can provide a tool for the development of more effective immunotherapy for CaP and possibly other cancers.
Support: DOD PC094680 (CRG), PCF Creativity Award (CRG), Mayo Clinic Prostate SPORE 5P50CA091956 (SV-P); Mrs. Adelyn L. Luther, Singer Island, Florida and Mayo Clinic Cancer Center (SV-P).
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C223.