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. 2012;7(12):e52485.
doi: 10.1371/journal.pone.0052485. Epub 2012 Dec 31.

Establishment, characterization and chemosensitivity of three mismatch repair deficient cell lines from sporadic and inherited colorectal carcinomas

Claudia Maletzki  1 , Saskia Stier, Ulrike Gruenert, Michael Gock, Christiane Ostwald, Friedrich Prall, Michael Linnebacher
Affiliations

Establishment, characterization and chemosensitivity of three mismatch repair deficient cell lines from sporadic and inherited colorectal carcinomas

Claudia Maletzki et al. PLoS One. 2012.

Abstract

Background: Colorectal cancer (CRC) represents a morphologic and molecular heterogenic disease. This heterogeneity substantially impairs drug effectiveness and prognosis. The subtype of mismatch repair deficient (MMR-D) CRCs, accounting for about 15% of all cases, shows particular differential responses up to resistance towards currently approved cytostatic drugs. Pre-clinical in vitro models representing molecular features of MMR-D tumors are thus mandatory for identifying biomarkers that finally help to predict responses towards new cytostatic drugs. Here, we describe the successful establishment and characterization of three patient-derived MMR-D cell lines (HROC24, HROC87, and HROC113) along with their corresponding xenografts.

Methodology: MMR-D cell lines (HROC24, HROC87, and HROC113) were established from a total of ten clinicopathological well-defined MMR-D cases (120 CRC cases in total). Cells were comprehensively characterized by phenotype, morphology, growth kinetics, invasiveness, and molecular profile. Additionally, response to clinically relevant chemotherapeutics was examined in vitro and in vivo.

Principal findings: Two MMR-D lines showing CIMP-H derived from sporadic CRC (HROC24: K-ras(wt), B-raf(mut), HROC87: K-ras(wt), B-raf(mut)), whereas the HROC113 cell line (K-ras(mut), B-raf(wt)) was HNPCC-associated. A diploid DNA-status could be verified by flow cytometry and SNP Array analysis. All cell lines were characterized as epithelial (EpCAM(+)) tumor cells, showing surface tumor marker expression (CEACAM(+)). MHC-class II was inducible by Interferon-γ stimulation. Growth kinetics as well as invasive potential was quite heterogeneous between individual lines. Besides, MMR-D cell lines exhibited distinct responsiveness towards chemotherapeutics, even when comparing in vitro and in vivo sensitivity.

Conclusions: These newly established and well-characterized, low-passage MMR-D cell lines provide a useful tool for future investigations on the biological characteristics of MMR-D CRCs, both of sporadic and hereditary origin. Additionally, matched patient-derived immune cells allow for comparative genetic studies.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Scheme of the experimental protocol for cell line establishment either fresh from surgery specimens ( = patient-derived cell line) of following xenografting in nu/nu mice ( = xenopatient-derived cell line).
Established cell lines were comprehensively characterized and routinely cyropreserved together with xenograft-tissue, immune cells, serum, and normal as well as primary tumor tissue in a biobank. This procedure leads to generation of individualized, patient-derived tumor models available for functional analyses.
Figure 2
Figure 2. Morphology of primary MMR-D tumors and their corresponding xenografts.
HE-histology representing maintenance of HROC24 tumor morphology following xenografting. The pan-cytokeratin staining is strongly positive, consistent with the tumors’ epithelial origin (left upper and lower panel). HE-histology of HROC50 tumors (right upper and lower panel).
Figure 3
Figure 3. SNP Array 6.0 for assessment of CIN in MMR-D cell lines.
Analysis was performed according to manufacturer’s instructions. (A) HROC24P cells, (B) HROC87X cells, and (C) HROC113P cells.
Figure 4
Figure 4. Light microscopy of MMR-D CRC cell lines both directly after establishment (P4) and following long-term in vitro culture (P40).
(A) Morphology of patient-derived cell lines HROC24P and HROC113P. (B) Morphology of HROC24X and HROC87X. Both cell lines were established from xenopatients as described in material & methods. Original magnification x100.
Figure 5
Figure 5. In vitro growth kinetic, ploidy analysis & invasiveness of MMR-D tumor cells.
(A) Growth curve of HROC24P, HROC87X and HROC113P cells in culture. The results shown are the mean population doubling times ± standard deviation. Results were calculated from three independent assays each performed in duplicates. (B) Exemplary DNA histograms of MMR-D tumor cells compared to normal cells (PBMC). All cells were classified as diploid. (C) Tumor invasiveness was analysed using a matrigel-based assay. Quantification of cellular invasiveness was estimated by MTT assay. Data are expressed as percentage invasion versus HCT116 cells ( = internal positive control). All experiments were repeated at least three times.
Figure 6
Figure 6. In vivo tumorigeneicity & response to selected chemotherapeutics.
NMRI nu/nu mice either received s.c. injections of established MMR-D cell lines (5 Mio. cells/mouse; A–C) or were s.c grafted with HROC29 and HROC71 tumor fragments (D, E). For analyzing in vivo drug response, mice with established (A) HROC24, (B) HROC87 or (C) HROC113 tumors were treated with irinotecan, paclitaxel or gemcitabine (i.p.; 20 mg/kg bw each, n = 6−7 mice per group). HROC71 and HROC29 xenografts were given gemcitabine (i.p.; 20 mg/kg bw, n = 5−6 mice per group). Therapeutic regimens consisted of six injections in total, applied twice a week. Control animals received equivalent volumes of saline (n = 6−7). Values of are given as mean ± SEM. *p<0.05 vs. control, U-Test; #p<0.05 vs. control, t-test.
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Grants and funding

This work was substantially supported by grant number 2006/A29 from the Else-Kröner Fresenius Stiftung to ML and by grant number 108919 from the Deutsche Krebshilfe (http://www.northgermantumorbank-crc.de). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.