Establishment and characterization of cell lines from chromosomal instable colorectal cancer

Abstract

Aim: To generate novel tumor models for preclinical validation of biomarkers that allow drug response prediction and individual therapeutic decisions.

Methods: Cell line establishment was conducted by both direct in vitro culturing and in vivo xenografting followed by in vitro culturing procedure. A comprehensive characterization was subsequently performed. This included quality control, consisting of the confirmation of human and colorectal cancer (CRC) origin by DNA fingerprint and epithelial cell adhesion molecule (EpCAM) staining, as well as mycoplasma and human virus testing. Phenotypic analysis was done by light microscopy and multicolor flow cytometry. Histopathological examination (β-catenin and cytokeratin staining) was conducted in direct comparison to parental tumor tissues. Extensive molecular-pathological profiling included mutation analysis for CRC-associated driver mutations, assessment of chromosomal and microsatellite instability, and the grade of CpG island methylation. Additionally, an array-based comparative genomic hybridization analysis was performed. Drug responsiveness was assessed for a panel of classical and novel substances in clinical use for the treatment of solid cancers. Finally, tumorigenicity of the cell lines was tested by xenografting into immunocompromised nude mice.

Results: Herein we describe the establishment of three ultra-low passage cell lines from two individual patients suffering from sporadic CRC. One cell line was derived directly from an early stage case (HROC18), whereas two cell lines could be established both direct from patient material and after xenografting from a late stage tumor (HROC32). All cell lines were free of contaminating mycoplasma and viruses. Molecular-pathological analysis allowed all cell lines to be classified as chromosomal instable (CIN(+)). They were aneuploid, with CpG island promoter methylation and microsatellite instability being absent. The following mutational profile was observed both in the cell lines and the parental tumor tissue: HROC18: APC(mut), p53(mut), K-ras(wt); HROC32: APC(wt), p53(mut), K-ras(mut). All cell lines were characterized as epithelial (EpCAM(+)) cells, showing distinct morphology and migration speed, but comparable growth kinetics. The cell lines showed different patterns of response towards clinically approved and novel drugs, with HROC18 being more resistant than HROC32 cells. Finally, in vivo tumorigenicity was demonstrated.

Conclusion: We successfully established and characterized novel ultra-low passage patient-derived CRC models as useful instruments for analyzing biological characteristics associated with the CIN(+) phenotype.

Keywords: Colorectal cancer; Drug response; Individualized medicine; Molecular characterization; Patient-derived tumor model; Translational research.

Figure 1

Representative histology of original patients’ tumors HROC18 and HROC32. HE histology, β-catenin, and cytokeratin (MNF116) staining as described in material and methods. Original magnification × 40.

Figure 2

Light microscopy of tumor cell lines after direct establishment (P5), and following short- (P30) and long-term in vitro culture (P60). Cell lines were established directly from patients’ tumor material and following xenografting in murine recipients as described in material and methods. Original magnification × 100.

Figure 3

Mycoplasma polymerase chain reaction, ploidy status, and secretion profile of tumor cells. A: Displayed are the results for 16S-rRNA-gene-based polymerase chain reaction for mycoplasma. All three lines were found free of contaminating mycoplasma. Samples amplified in the absence of tumor DNA served as a negative control, whereas a highly contaminated cell line was applied as a positive control, run in duplicates; B: Cytokine secretion pattern as determined by enzyme-linked immunosorbent assays. Cytokine concentrations were determined by comparison with a standard curve generated from serial dilutions of individual standards. Quantitative analysis of carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), and interleukin (IL) 8 secretion after three and five days of culture, respectively. Results show the mean ± SD of three independent experiments.

Figure 4

Single nucleotide polymorphism array 6.0 for assessment of chromosomal instability in cell lines. Analysis was performed according to manufacturer’s instructions. A: HROC18 cells; B: HROC32P cells.

Figure 5

Wound healing assays were performed with HROC18 and HROC32P cells, respectively. Representative images of migrating tumor cells around the wound area at d0, d2, d5, and d9 after wounding are shown. d: Day.

Figure 6

In vivo tumorigenicity. Female NMRI Foxn1^nu mice were challenged subcutaneously with 5 × 10⁶ tumor cells. Monitoring of tumor growth was performed for a total of 80 d. Both cell lines exhibited low tumorigenic potential in vivo, as can be depicted from the tumor growth curve. Values are given as mean tumor diameter ± SD.