Profiling of antibodies against prostate cancer xenograft-released proteins

The present study describes a novel xenograft-based biomarker discovery platform and proves its usefulness in the discovery of novel serum markers for prostate cancer (PCa). By immunizing immuno-competent mice with serum from nude mice bearing PCa xenografts, an antibody response against xenograft-derived antigens was elicited. By probing protein microarrays with serum from immunized mice, several PCa-derived antigens were identified, of which a subset was successfully retrieved in serum from mice bearing PCa xenografts and validated in human serum samples of PCa patients. In conclusion, this novel method allows for the identification of low abundant cancer-derived serum proteins, circumventing dynamic range and host-response issues in standard patient cohort proteomics comparisons. To perform a large-scale identification of antibodies generated against human PCa-derived proteins in the serum of immunized mice, sera from mice immunized with either depleted serum, full serum or both from PC346 and PC339-bearing mice as well as preimmune serum and serum from mice immunized with normal mouse serum were incubated onto ProtoArrays. These ProtoArrays contain approximately 8,000 partial and full-length human proteins, expressed as N-terminal glutathione S-transferase (GST) fusion proteins. To detect antibodies bound to spotted proteins, ProtoArrays were developed using a fluorescent labeled secondary antibody. Before being used for immunization, serum from xenografted mice was not treated (full) or depleted for most abundant proteins (depleted). Two arrays were hybridized with pre-immune serum and one array with serum from an immune competent mouse that was immunized with serum from a nude mouse. Six arrays were performed using serum from immune competent mice that were immunized with serum from xenograft-bearing nude mice.