Paul A. Moore

Expansion of CD133+ colon cancer cultures retaining stem cell properties to enable cancer stem cell target discovery

Background:Despite earlier studies demonstrating in vitro propagation of solid tumour cancer stem cells (CSCs) as non-adherent tumour spheres, it remains controversial as to whether CSCs can be maintained in vitro. Additional validation of the CSC properties of tumour spheres would support their use as CSC models and provide an opportunity to discover additional CSC cell surface markers to aid in CSC detection and potential elimination.Methods:Primary tumour cells isolated from 13 surgically resected colon tumour specimens were propagated using serum-free CSC-selective conditions. The CSC properties of long-term cultured tumour spheres were established and mass spectrometry-based proteomics performed.Results:Freshly isolated CD133+ colorectal cancer cells gave rise to long-term tumour sphere (or spheroids) cultures maintaining CD133 expression. These spheroid cells were able to self-renew and differentiate into adherent epithelial lineages and recapitulate the phenotype of the original tumour. Relative to their differentiated progeny, tumour spheroid cells were more resistant to the chemotherapeutic irinotecan. Finally, CD44, CD166, CD29, CEACAM5, cadherin 17, and biglycan were identified by mass spectrometry to be enriched in CD133+ tumour spheroid cells.Conclusion:Our data suggest that ex vivo-expanded colon CSCs isolated from clinical specimens can be maintained in culture enabling the identification of CSC cell surface-associated proteins.

Identification and Characterization of Angiogenesis Targets through Proteomic Profiling of Endothelial Cells in Human Cancer Tissues

Genomic and proteomic analysis of normal and cancer tissues has yielded abundant molecular information for potential biomarker and therapeutic targets. Considering potential advantages in accessibility to pharmacological intervention, identification of targets resident on the vascular endothelium within tumors is particularly attractive. By employing mass spectrometry (MS) as a tool to identify proteins that are over-expressed in tumor-associated endothelium relative to normal cells, we aimed to discover targets that could be utilized in tumor angiogenesis cancer therapy. We developed proteomic methods that allowed us to focus our studies on the discovery of cell surface/secreted proteins, as they represent key antibody therapeutic and biomarker opportunities. First, we isolated endothelial cells (ECs) from human normal and kidney cancer tissues by FACS using CD146 as a marker. Additionally, dispersed human colon and lung cancer tissues and their corresponding normal tissues were cultured ex-vivo and their endothelial content were preferentially expanded, isolated and passaged. Cell surface proteins were then preferentially captured, digested with trypsin and subjected to MS-based proteomic analysis. Peptides were first quantified, and then the sequences of differentially expressed peptides were resolved by MS analysis. A total of 127 unique non-overlapped (157 total) tumor endothelial cell over-expressed proteins identified from directly isolated kidney-associated ECs and those identified from ex-vivo cultured lung and colon tissues including known EC markers such as CD146, CD31, and VWF. The expression analyses of a panel of the identified targets were confirmed by immunohistochemistry (IHC) including CD146, B7H3, Thy-1 and ATP1B3. To determine if the proteins identified mediate any functional role, we performed siRNA studies which led to previously unidentified functional dependency for B7H3 and ATP1B3.

Immune modulator CD70 as a potential cisplatin resistance predictive marker in ovarian cancer

OBJECTIVE We have used mass-spectrometry (MS) based proteomics platform to identify cell surface proteins over-expressed on a cisplatin resistant derivative of an ovarian cancer cell line A2780. METHODS Membrane associated glycoproteins from A2780 and its cisplatin resistant derivative cell line, A2780cis, were processed for liquid chromatography (LC)-MS based analysis. The expression of proteins found at elevated levels in A2780cis cell line was confirmed using flow cytometry and Taqman analysis. The expression of these proteins was further evaluated in unrelated ovarian cancer cell lines using MS analysis and flow cytometry. Immunohistochemical (IHC) analysis was performed using ovarian tumor tissues to evaluate the protein density on the cell surface. Monoclonal antibodies were used in an alamar blue proliferation assay to examine the cytotoxic effects on cell proliferation in resistant cell lines. RESULTS Six proteins were identified by LC-MS as being over-expressed on cell surface of A2780cis cell line. Mass spectrometry and flow cytometry confirmed the over-expression of CD49f, CD70 and Her-2/neu in other cisplatin resistant ovarian cancer cell lines. Immunohistochemical analysis revealed that only CD70 was expressed at moderate levels in ovarian tumors. When cisplatin resistant ovarian cell lines A2780cis and SKOV-3 were treated with antibody against CD70, there was a significant decrease in cell proliferation. CONCLUSION Using a MS based proteomics approach we have shown that expression of CD70 is associated with cisplatin resistance in ovarian cancer cell lines. Follow-up examination of these tumor cell line findings in clinical tumor specimens with available pathology staging and cisplatin treatment history is warranted.

RNAi Validation of Pancreatic Cancer Antigens Identified by Cell Surface Proteomics

Pancreatic cancer is one of the most aggressive malignancies that is often diagnosed at a late stage following tumor metastasis. Given the low response rate to current therapies, there is a clear and urgent need for new therapeutic and diagnostic approaches. Proteomics is an emerging tool that offers the opportunity to discover novel antigens elevated in cancer. Considering the large number of marketed drugs that target plasma membrane proteins, and given their amenability to both small molecule and antibody-based technologies, an approach that combines a cell membrane labeling strategy with proteomic (LC-MS/MS) methodology would enable novel cell surface antigens that can act as novel biomarkers and/or targets for drug development to be identified. One major consideration when selecting targets for therapeutics is whether or not the antigen plays a functional role in a disease context. RNA interference (RNAi) permits the downregulation of any given gene, and therefore allows the identification of functionality in the target disease. As such, this tool permits the selection of targets prior to investment in a therapeutic antibody program or an extensive small molecule–screening program. In this chapter, we describe the utility of the cell membrane labeling strategy and LC-MS/MS method that was developed at Celera. Subsequent application of RNAi allowed us to assign functionality to cell surface antigens we identified that were upregulated in pancreatic cancer. By using a combination of a proteomics and an RNAi platform, we demonstrate that it is possible to discover novel therapeutic and diagnostic targets for this devastating disease.