Establishment and Characterization of 5-Fluorouracil-Resistant Human Colorectal Cancer Stem-Like Cells: Tumor Dynamics under Selection Pressure

Abstract

5-Fluorouracil (5-FU) remains the gold standard of first-line treatment for colorectal cancer (CRC). Although it may initially debulk the tumor mass, relapses frequently occur, indicating the existence of cancer cells that are therapy-resistant and are capable of refueling tumor growth. To identify mechanisms of drug resistance, CRC stem-like cells were subjected to long-term 5-FU selection using either intermittent treatment regimen with the IC50 drug dose or continuous treatment regimen with escalating drug doses. Parental cancer cells were cultivated in parallel. Real-time PCR arrays and bioinformatic tools were used to investigate gene expression changes. We found the first method selected for cancer cells with more aggressive features. We therefore transplanted these cancer cells or parental cells in mice, and again, found that not only did the 5-FU-selected cancer cells generate more aggressive tumors with respect to their parental counterpart, but they also showed a different gene expression pattern as compared to what we had observed in vitro, with ID1 the top upregulated gene. We propose ID1 as a stemness marker pervasively expressed in secondary lesions emerging after completion of chemotherapy.

Keywords: cancer stem cells; chemoresistance; colorectal cancer.

Figure 1

Establishment of 5-FU resistant human colorectal cancer (CRC) stem-like cells. (A) Line graphs showing cell viability (%) of Tu11, Tu14, Tu27, Tu28, and Tu42 CRC stem-like cells (P stands for passage) treated with vehicle or serial dilutions (10–250 µM) of 5-FU. Data are expressed as mean percentage (± SD) of cell numbers relative to control culture. (B–D) Line graphs showing cell viability (%) of consecutive passages of Tu27, Tu28, and Tu42 CRC stem-like cells treated with vehicle or serial dilutions (10–500 µM) of 5-FU. Data are expressed as mean percentage (± SD) of cell numbers relative to control culture. (E) Line graph showing the expression intensity of PTEN/MMAC1/TEP1 in short-term feeder-expanded Tu11, Tu14, Tu27, Tu28, and Tu42 CRC stem cells obtained using three different microarray probes (204054_at; 217492_s_at; 204053_x_at).

Figure 2

Features of the human colorectal cancer (CRC) stem-like cell line Tu42 under low 5-FU doses. (A) Merged images of phase contrast (PhC) and Hoechst-stained nuclei (DNA) of parental (top) or 20 µM 5-FU resistant (center) Tu42 cells. Boxed areas are shown on the right at higher power and indicate the presence of distinct large foci in response to low-dose 5-FU. Immunofluorescence staining for Wheat germ agglutinin (WGA), Laminin B1 (LMNB1), SF2, Fibrillarin (FBR), Nucleostemin (NS), Nucleophosmin (NPM), or PML in 20 µM 5-FU resistant Tu42 cells (bottom) suggested high nucleus-to-cytoplasm ratio, the presence of nuclear buds, and identified the previously observed foci as nucleolar structures. Nuclei were counterstained using Hoechst. Pictures are representative of three independent experiments. (B) Immunofluorescence staining for Ki-67 or TRA-2-49 of parental (top) or 20 µM 5-FU resistant (bottom) Tu42 cells indicated the accumulation of Ki-67 in nucleolar regions of resistant cells and the upregulation of TRA-2-49 in the same cells with respect to parental cells. Nuclei were counterstained using Hoechst. Pictures are representative of three independent experiments. (C) Merged images of phase contrast (PhC), EpCAM, and Mucin2 (Muc2), or PhC, EpCAM, and Villin of parental (top) or 20 µM 5-FU resistant (bottom) Tu42 cells indicated that resistant cells downregulate EpCAM, Muc2, and Villin, as compared to parental cells. Pictures are representative of three independent experiments. (D) Bright field (BF) images of live unstained parental (top left) or 20 µM 5-FU resistant (top right) Tu42 cells forming organoids in Matrigel, and immunofluorescence staining for EpCAM/Muc2 (center) or EpCAM/Villin (bottom) in their respective frozen sections indicated resistant cells reacquire EpCAM expression in three-dimensional (3D) cell culture but fail to generate organoids with a central lumen. Pictures are representative of three independent experiments.

Figure 3

Differential morphology, gene expression profiles and biological functions in 5-FU resistant Tu42 colorectal cancer (CRC) stem-like cells as compared to parental cells. (A) Phase contrast images of parental (P) or 5-FU resistant Tu42 cells selected over an 8-month period by either intermittent treatment with the IC50 drug dose (R1; two pulses of 100 µM 5-FU were given) or continuous treatment with escalating drug doses (R2; six doses were given, starting with the 1/20 IC50 dose up to the IC50 dose). (B) List of the top up- or downregulated genes in 5-FU resistant Tu42 cells as compared to parental cells obtained by real-time PCR arrays. (C) Biological functions arranged in alphabetical order predicted by IPA to be activated (red) or inhibited (green) in 5-FU resistant Tu42 cells as compared to parental cells. (D) Bar graphs show fold regulation (positive/negative fold changes calculated from two technical replicates) of genes involved in DNA damage and cell cycle, apoptosis, stemness, invasion, metastasis and angiogenesis, mTOR pathway, other pathways, metabolism, and chromatin remodeling in 5-FU resistant Tu42 cells as compared to parental cells (grey bars: R1 cells versus parental cells; black bars: R2 cells versus parental cells). (E) Representative amplification plots of EIF4B, EGF, and MYCN in parental and 5-FU resistant Tu42 cells. ΔRn is plotted against PCR cycle number. (F) Flow cytometry histograms showing expression of CD24 and CD227 (Muc1) in parental (black histograms) and R1 (red histograms) cells (grey histograms indicate unstained control). Histograms are representative of three independent experiments. (G) Scatter dot plot with mean and SEM showing percentages of phospho-S6RP (pS6RP-S235/236 and pS6RP-S240/244) positive cells/colony generated by parental, R1, or R2 cells. Each dot represents a colony. Statistical significance, calculated using unpaired t-test with Welch’s correction, is shown (ns, p > 0.05; **, p ≤ 0.01; ***, p ≤ 0.001). Representative pS6RP-S235/236 and pS6RP-S240/244 stained colonies are shown at the right of each graph. Data are representative of three independent experiments.

Figure 4

Differential histology, gene expression profiles and biological functions in R1 cells versus parental cells in vitro and in vivo. (A) PFA-fixed, paraffin-embedded serial sections of parental or R1 cell-derived tumor xenografts stained with hematoxylin and eosin (H&E) or alcian blue (AB). Characteristics of R1 cell-derived tumor xenograft include: a pseudostratified glandular epithelium with marked nuclear atypia and cellular overlapping and few mucinous goblet cells. Parental cell-derived tumor xenograft shows a lesser degree of architectural abnormalities including glands with low-grade cytologic dysplasia and abundant goblet cells. Pictures are representative of five histological sections/tumor. (B) Graph shows fold regulation (positive fold changes) of de novo expressed genes in R1 cell- versus parental cell-derived tumor xenograft. (C) List of the top up- or downregulated genes in R1 cell- versus parental cell-derived tumor xenograft obtained by real-time PCR arrays. (D) Biological functions ranked by p-value (from smallest or strongest to largest or weakest) predicted by IPA to be activated (red) or inhibited (green) in R1 cell- versus parental cell-derived tumor xenograft. (E) Venn diagrams show the lists of diverging and overlapping genes (for each of the following categories: DNA damage and cell cycle; apoptosis; stemness; invasion, metastasis and angiogenesis; mTOR pathway; other pathways; metabolism and chromatin remodeling) whose expression was altered (≥2.0 or ≤−2.0) in R1 cells as compared to parental cells in vitro and in vivo. Upregulated genes are in red, downregulated genes are in green. Graphs on the right of each Venn diagram show differential regulation of selected genes in vitro (grey bars) and in vivo (black bars).

Figure 5

ID1-expressing cell enrichment is a feature of 5-FU resistant Tu42 cell-derived tumor xenograft. (A) PFA-fixed, paraffin-embedded serial sections of parental or R1 cell-derived tumor xenografts stained with ID1, ID2, ID3, ID4, or BMP4 antibodies (DAB, brown color). Pictures are representative of five histological stains per tumor. (B) Dot plot with line in the mean showing ID1 expression obtained from quantile normalized microarray datasets of short-term feeder-expanded normal small or large intestinal stem cells (SiSCs and LiSCs, respectively) and CRC stem cells (CoCSCs). Each dot represents one sample. (ns. p > 0.05; ** p ≤ 0.01).

Figure 6

Potential activated pathways and targets in R1-cell derived tumor xenograft. Solid arrows indicate connections that are well established. Dashed arrows indicate less well established and/or more controversial. Light red color denotes detected upregulation.