Candy Lee

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.

Rna Interference Characterization of Proteins Discovered by Proteomic Analysis of Pancreatic Cancer Reveals Function in Cell Growth and Survival

Objectives There is a clear need for better therapeutics and diagnostics for pancreatic cancer. We aimed to discover plasma membrane-associated proteins overexpressed in pancreatic cancer using quantitative proteomics and apply RNA interference (RNAi) to uncover proteins associated with cancer cell survival. Methods Cell surface glycoproteins from 5 pancreatic cancer cell lines were isolated, and differential analyses were performed using mass spectrometry and the “normoid” cell line Hs766T as the comparator. For validation, immunohistochemistry was performed on tissues from 10 independent patients and 2 normal donors. Correlation of protein and mRNA expression level was determined, and functional activity characterized using RNAi. Results Integrin &bgr;6, CD46, tissue factor, and a novel protein, chromosome 14 open reading frame 1, were identified as overexpressed on pancreatic cancer cell lines. Immunohistochemistry demonstrated the 4 targets were overexpressed in 20% to 70% of primary pancreatic tumor specimens. Small interfering RNA knockdown resulted in a reduction of cellular proliferation by inhibiting DNA synthesis, blocking S-phase progression or induction of apoptosis. Conclusions By combining a mass spectrometry identification platform and an RNAi validation platform, we have identified a panel of cell surface glycoproteins that not only are overexpressed, but also play a functional role in pancreatic tumor cell survival.

Generation of a conditionally immortalized myeloid progenitor cell line requiring the presence of both interleukin-3 and stem cell factor to survive and proliferate.

Summary. The H‐2Κb temperature‐sensitive (ts) A58 transgenic (Immorto) mouse has been used previously to generate conditionally immortalized cells from a number of tissues. The present study aimed to investigate characteristics of primitive myeloid precursor cells derived from H‐2Κb‐tsA58 bone marrow. Cell populations were enriched for granulocyte/macrophage progenitors by centrifugal elutriation, and were cultured in the presence and absence of cytokines at the permissive and restrictive temperatures for the A58 oncogene. Cells derived from H‐2Κb‐tsA58 mice required both A58 activation and the growth factors, stem cell factor (SCF) and interleukin‐3 (IL‐3), for long‐term cell survival and growth; cells were maintained for > 300 d in culture under these conditions. IL‐3‐ and SCF‐dependent clonal cell lines were derived with a phenotype (lin–, Sca‐1+, CD34+, ER‐MP 58+, ER‐MP 12+, ER‐MP 20–) characteristic of primitive myeloid progenitors. These cells differentiated on addition of granulocyte/macrophage colony‐stimulating factor (GM‐CSF) or macrophage colony‐stimulating factor (M‐CSF) and acquired mature cell morphology with some upregulation of differentiation markers. In conclusion, the A58 oncogene can immortalize haemopoietic progenitor cells. These cells require two cytokines for growth, IL‐3 and SCF; as such, they constitute a useful resource for the study of synergistic interactions between growth factors. The ability to develop monocytic cell characteristics also permits the investigation of cytokine‐mediated early haemopoietic progenitor cell development.