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. 2009 Mar 1;69(5):1951-7.
doi: 10.1158/0008-5472.CAN-08-2023. Epub 2009 Feb 24.

Chemoresistant colorectal cancer cells, the cancer stem cell phenotype, and increased sensitivity to insulin-like growth factor-I receptor inhibition

Nikolaos A Dallas  1 , Ling Xia, Fan Fan, Michael J Gray, Puja Gaur, George van Buren 2nd, Shaija Samuel, Michael P Kim, Sherry J Lim, Lee M Ellis
Affiliations

Chemoresistant colorectal cancer cells, the cancer stem cell phenotype, and increased sensitivity to insulin-like growth factor-I receptor inhibition

Nikolaos A Dallas et al. Cancer Res. .

Abstract

5-Fluorouracil (5FU) and oxaliplatin are standard therapy for metastatic colorectal cancer (CRC), but the development of chemoresistance is inevitable. Because cancer stem cells (CSC) are hypothesized to be chemoresistant, we investigated CSC properties in newly developed chemoresistant CRC cell lines and sought to identify targets for therapy. The human CRC cell line HT29 was exposed to increasing doses of 5FU (HT29/5FU-R) or oxaliplatin (HT29/OxR) to achieve resistance at clinically relevant doses. Western blotting and flow cytometry were done to determine molecular alterations. The insulin-like growth factor-I receptor (IGF-IR) monoclonal antibody (mAb) AVE-1642 was used to inhibit signaling in vitro and in vivo using murine xenograft models. HT29/5FU-R and HT29/OxR showed 16- to 30-fold enrichment of CD133(+) cells and 2-fold enrichment of CD44(+) cells (putative CRC CSC markers). Resistant cells were enriched 5- to 22-fold for double-positive (CD133(+)/CD44(+)) cells. Consistent with the CSC phenotype, resistant cells exhibited a decrease in cellular proliferation in vitro (47-59%; P < 0.05). Phosphorylated and total IGF-IR levels were increased in resistant cell lines. HT29/5FU-R and HT29/OxR cells were approximately 5-fold more responsive to IGF-IR inhibition relative to parental cells (P < 0.01) in vitro. Tumors derived from HT29/OxR cells showed significantly greater growth inhibition in response to an IGF-IR mAb than did parental cells (P < 0.05). Chemoresistant CRC cells are enriched for CSC markers and the CSC phenotype. Chemotherapy-induced IGF-IR activation provided for enhanced sensitivity to IGF-IR-targeted therapy. Identification of CSC targets presents a novel therapeutic approach in this disease.

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Figures

Figure 1
Figure 1. Chemoresistant cell lines are enriched for tumor stem cell markers
(A) Western blotting demonstrated that expression of the CRC stem cell markers CD133 and CD44 was higher in the chemoresistant cell lines HT29/5FU-R and HT29/OxR than in the parental HT29 human CRC cell line. Vinculin and β-actin levels are provided as loading controls (B) Flow cytometric analysis demonstrated that both chemoresistant cell lines were enriched for cells that expressed CD133 and CD44 when compared with the parental cell line. 36% of HT29/5FU-R cells and 57% of HT29/OxR cells expressed CD133 relative to 2% of HT29 cells. Similarly, 95% of chemo-resistant cells expressed CD44 relative to only 43% of parental cells. Cytometric analysis plots using isotype control antibodies are provided as staining controls. (C) When cells were labeled with both markers, flow cytometric analysis demonstrated ~5-fold enrichment of double-positive cells in the HT29/5FU-R cell line and ~22-fold enrichment in the oxaliplatinresistant cell line compared with the parental HT29 cell line. SCC = Side scatter.
Figure 2
Figure 2. Chemoresistant cell lines are enriched for tumor stem cell phenotype
(A) Chemoresistant cells proliferate at a significantly slower rate than do parental cells by MTT analysis. (B) Parental cells demonstrate sensitivity to both 5FU and oxaliplatin after exposure to drug for 72 hours, with only 40% and 58% of cells remaining, respectively, relative to untreated cells. In contrast, HT29/5FU-R cells are resistant to 5FU as expected; however, they also demonstrate an increase in resistance to oxaliplatin relative to parental cells. Similarly, oxaliplatin-resistant cells are resistant to oxaliplatin but also are resistant to 5FU. (C) Cells were plated in an ultra-low-attachment 96-well plate in the absence of serum, and 14 days later the number of viable sphere-forming cells was assayed by MTT. Chemoresistant cells demonstrated a 2- to 3-fold increase in colonosphere-forming cells relative to parental cells. (D) In a soft agar assay, 5FU- and oxaliplatin-resistant cells formed significantly more colonies larger than 50 µm in diameter under anchorage-independent growth conditions when compared to HT29 cells. These findings are consistent with the described CSC phenotype. Error bars represent standard error of the mean, and asterisks denote p<0.05. HPF=High Power Field. OD = optical density.
Figure 3
Figure 3. Effect of IGF-1R inhibition on chemoresistant cells
(A) Analysis of whole cell lysates from parental and resistant cells demonstrated an increase in both phosphorylation and total levels of IGF-1R in chemoresistant cells relative to parental cells. (B) Cells were treated with control or IGF-1R targeting antibodies, and the cell number relative to that of the control treatment was analyzed. There was a significantly greater decrease in cell number in the chemoresistant cells treated with AVE-1642 than in the parental cells (48%-54% vs. 13%; p<0.05).
Figure 4
Figure 4. Effect of IGF-1R inhibition on in vivo tumor growth, proliferation, and apoptosis
Mice were subcutaneously injected with 1×106 HT29 or HT29/5FU-R cells in one experiment or HT29 or HT29/OxR cells in a second and treated with control IgG or AVE1642 twice weekly. The final tumor masses were measured and compared between mice bearing tumors from each cell line. Relative to control-treated mice, HT29/5FU-R-(A) and HT29/OxR-derived tumors (B) showed significantly greater growth inhibition than did HT29-derived tumors. TUNEL staining revealed significantly greater apoptosis in response to AVE1642 in tumors derived from HT29/5FU-R- (C) and HT29/OxRd-erived tumors (D) than in tumors derived from parental cells. Asterisks denote p<0.05 and error bars represent standard error of the mean.
Figure 5
Figure 5. Effect of IGF-1R inhibition on in vivo tumor characteristics
Immunohistochemical analysis of tumors was conducted, and multiple tumor fields were evaluated per group. Representative images for all groups from both experiments are presented. H&E staining revealed similar subcutaneous tumor morphology among all groups of tumors. Ki67 staining demonstrated decreased proliferative cells in tumors treated with AVE1642; however, differences in reduction of proliferative cells between parental and resistant-cell-derived tumor sections were not significant. TUNEL staining revealed significantly greater apoptosis in response to AVE1642 in tumors derived from chemoresistant cells than in tumors derived from parental cells (p<0.05).
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