PURPOSE: The aim of this paper was to compare protein content of chaperone-rich cell lysate (CRCL) anti-cancer vaccines prepared from human tumours of different histological origins to evaluate the uniformity of their protein content. MATERIALS AND METHODS: Clinical grade CRCL was prepared under Good Manufacturing Practice (GMP) conditions from surgically resected human tumours (colorectal cancer, glioblastoma, non-small cell lung cancer, ovarian cancer). Protein samples were separated by SDS-PAGE and slices cut from gels for protease digestion followed by mass spectrometry analysis. Proteins were identified, and the content assessed by gene ontogeny/networking programmatic computation. CRCL preparations were also analysed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). RESULTS: We identified between 200 and 550 proteins in the various CRCL preparations. Gene ontogeny analysis indicated that the vaccines showed clear relationships, despite different tumour origins. A total of 95 proteins were common to all the CRCLs. Networking analyses implicated heat shock proteins in antigen processing pathways, and showed connections to the cytoskeletal network. We found that CRCL vaccines showed a particulate structure by NTA, and TEM revealed an extended fence-like structural network in CRCL, with regions that were microns in size. CONCLUSIONS: We conclude that it is feasible to prepare and characterise CRCL from a variety of different tissue sources; a substantial portion of the protein content is identical among the different CRCLs, while the overall compositions also suggest high overlaps in functional categories. The protein content indicates the presence of antigens and implies a potential structure, which we believe may play a role in CRCL's ability to stimulate innate antigen presenting cell activation.
PURPOSE: The aim of this paper was to compare protein content of chaperone-rich cell lysate (CRCL) anti-cancer vaccines prepared from humantumours of different histological origins to evaluate the uniformity of their protein content. MATERIALS AND METHODS: Clinical grade CRCL was prepared under Good Manufacturing Practice (GMP) conditions from surgically resected humantumours (colorectal cancer, glioblastoma, non-small cell lung cancer, ovarian cancer). Protein samples were separated by SDS-PAGE and slices cut from gels for protease digestion followed by mass spectrometry analysis. Proteins were identified, and the content assessed by gene ontogeny/networking programmatic computation. CRCL preparations were also analysed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). RESULTS: We identified between 200 and 550 proteins in the various CRCL preparations. Gene ontogeny analysis indicated that the vaccines showed clear relationships, despite different tumour origins. A total of 95 proteins were common to all the CRCLs. Networking analyses implicated heat shock proteins in antigen processing pathways, and showed connections to the cytoskeletal network. We found that CRCL vaccines showed a particulate structure by NTA, and TEM revealed an extended fence-like structural network in CRCL, with regions that were microns in size. CONCLUSIONS: We conclude that it is feasible to prepare and characterise CRCL from a variety of different tissue sources; a substantial portion of the protein content is identical among the different CRCLs, while the overall compositions also suggest high overlaps in functional categories. The protein content indicates the presence of antigens and implies a potential structure, which we believe may play a role in CRCL's ability to stimulate innate antigen presenting cell activation.