Phenotypic subpopulations of metastatic colon cancer stem cells: genomic analysis.
BACKGROUND
Human cancer is characterized by high heterogeneity in gene expression, varieties of differentiation phenotypes and tumor-host interrelations. Growing evidence suggests that tumor-initiating, or cancer stem cells (CSCs), may also represent a heterogeneous population. The present study was undertaken to isolate and characterize the different phenotypic subpopulations of metastatic colon cancer and to develop a working colon CSC model for obtaining highly tumorigenic and clonogenic cells in sufficient numbers.


METHODS
Different phenotypic cell subpopulations were isolated based on differential levels and patterns of expression of several stemness markers, including CD133, CD44, CD166 and CD49b. Stemness properties of isolated cells were tested by analysis of their ability to form floating colonospheres in vitro, to induce tumors in NOD/SCID mice after transplantation at relatively low cell numbers, and to produce progenitors of different phenotypes.


RESULTS
The metastatic colon cancer HCT116 cell line, which expressed a majority of known CSC markers, closely resembling the patterns of expression in exfoliated peritoneal cells from several metastatic colon cancer patients, was selected as a reference material. Genome-wide microarray analysis (Affymetrix; DAVID) of CD133(high) CSC-enriched versus CSC-depleted cell populations revealed 4,351 differentially expressed genes with an overrepresentation of those responsible for apoptosis resistance, regulation of cell cycle, proliferation, stemness and developmental pathways. Simultaneous analysis of 84 stem cell- and metastasis-related genes with corresponding PCR arrays identified genes differentially expressed in several colon CSC phenotypic populations versus bulk tumor cells, and in relation to each other. It was found that colonospheres induced by tumorigenic cells with the highest expression of CD133 and those which were induced by CD133/CD44-negative cells possessed profoundly different stem cell-related gene expression profiles.


CONCLUSIONS
The proposed approaches allow for reliable isolation and propagation of highly tumorigenic and clonogenic cells of different phenotypes. Genomic analysis of several candidate CSC phenotypic populations may contribute to the identification of novel targets for colon cancer stem cell-targeted drug development and treatment.
Botchkina
Inna L
IL
Department of Surgery/Surgical Oncology, HSC-18, 050, SBUMC, Stony Brook, NY 11794, USA.
Rowehl
Rebecca A
RA
Rivadeneira
David E
DE
Karpeh
Martin S
MS
Crawford
Howard
H
Dufour
Antoine
A
Ju
Jingfang
J
Wang
Yuan
Y
Leyfman
Yan
Y
Botchkina
Galina I
GI
D000071916:AC133 Antigen; D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism; D003110:Colonic Neoplasms / Q000235:genetics* / Q000473:pathology; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D015894:Genome, Human; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D016688:Mice, Inbred NOD; D008819:Mice, Nude; D016513:Mice, SCID; D014411:Neoplastic Stem Cells / Q000473:pathology*; D020411:Oligonucleotide Array Sequence Analysis; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D018874:Spheroids, Cellular / Q000473:pathology; D014407:Tumor Cells, Cultured
D000071916:AC133 Antigen; D015703:Antigens, CD; D014408:Biomarkers, Tumor; D006023:Glycoproteins; C000606023:PROM1 protein, human; D010455:Peptides; C000606024:Prom1 protein, mouse