Oncology/Endocrine
A Model of Primary Culture of Colorectal Cancer and Liver Metastasis to Predict Chemosensitivity

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Background

Prediction of chemosensitivity is a major goal of modern oncology. The aim of this study was to establish a simple and effective model of primary culture of colorectal cancer fragments and to test whether it allows prediction of chemosensitivity.

Methods

Colorectal cancer fragments (primary tumors or liver metastases) of 94 consecutive and previously untreated patients were obtained, prepared, and cultured in polyHEMA. For each fragment cultured, a proliferative index (PI) was calculated after immunostaining at d 0 and after 7 d in culture with media alone or supplemented for 24h with the topoisomerase I inhibitor metabolite SN-38. The correlation between in vitro response (decrease in PI after exposure to the drug) and in vivo response (RECIST criteria) was studied in a subset of patients who had measurable metastases and were treated with a topoisomerase I inhibitor.

Results

PolyHEMA allowed three-dimensional culture of tumor fragments up to 7 d without fibroblastic invasion and with a slight but significant decrease of PI (59% at d 0 versus 51% after 7 d in culture, P < 0.001). In vitro drug efficacy was tested in 67 fragments, the mean PI after culture with SN-38 dropped to 22% (P < 0.001). In a subset of 12 patients, there was no statistically significant correlation between in vitro and in vivo response (P = 0.13).

Conclusion

Primary culture in polyHEMA was easy to perform successfully in 71% of cases. On this model, the antiproliferative effect of SN-38 could be measured and results correlated to clinical data.

Introduction

Colorectal carcinoma is one of the most prevalent cancers in western countries 1, 2. Nearly 50% of patients suffering from colorectal cancer develop distant metastasis and receive systemic chemotherapy [3]. The number of effective drugs administered in this indication has grown rapidly in the recent years, and besides the classical antimitotic agents (fluorouracil, topoisomerase-I inhibitors, platinum analogs), we now found monoclonal antibodies (anti-EGF-R, anti VEGF-R), antiangiogenic agents, peptide combination, defense modifiers, and vaccines. However, these anticancer agents, often used in combination, are effective in 60% of cases so far, meaning that many patients are treated for some period with an ineffective drug with some degree of toxicity 4, 5, 6, 7, 8. A major challenge for medical oncology is, therefore, to predict tumor response or resistance to a given regimen in order to prescribe the most appropriate drug for each patient.

Tumor response and drug toxicity depend on numerous factors, including patient metabolism, pharmacogenetics [9], tumor environment, and molecular alterations found in the tumor [10]. In some cancer types, such as breast cancer, gene expression profile can help to identify sensitive and resistant tumors to a given drug [11]. In colorectal cancer, response to a given drug for a given patient is difficult to assess because patients frequently receive several drugs simultaneously or have received successively several lines of treatment. This drawback could be bypassed if tumor sensitivity could be tested in an appropriate and reproducible in vitro model. However, primary explant cultures of tumor fragments have been, to date, very disappointing for this purpose. Primary culture of tumor fragments are often contaminated by rapid growth of normal fibroblast, the tumor architecture is lost as well as the cell-cell interaction and in vitro models do not permit to apply the drug to be tested on the tumor cells only for a desirable period.

Poly-2-hydroxyethylmetacrylate (PolyHEMA) has been used to preserve three-dimensional organization of tumors [12]. This compound is also interesting because a drug washout process is permitted by the use of a liquid medium [13].

The aim of this study was to establish a model of primary culture of colorectal cancer fragments using PolyHEMA, to measure the efficacy of an antimitotic drug on cell proliferation in this model, and to correlates the results obtained in vitro to the chemosensitivity in vivo observed in the same patients.

Section snippets

Generation and Culture of HT29 Nodules

The cell line HT29 issued from a human colon tumor was purchased at the ATCC (Rockville, MD). Cells were routinely maintained in monolayer with Dulbecco modified Eagle medium supplemented by 10% heat activated fetal calf serum, antibiotics and glutamine at 37 °C under humidified 5% CO2 atmosphere. In order to form the nodules, trypsin-dispersed cells (2.5 mL) were seeded in 6-well plates previously pre-coated with poly-2-hydroxyethylmetacrylate (PolyHEMA) (Sigma, Paris, France) according to our

PolyHEMA Coating: A Well Adapted Method for Primary Culture of Human Tumors

On noncoated surface of culture dish, HT29 human colon cancer cells were growing as a monolayer, whereas on polyHEMA-coated surface, tumor cells organized in three dimension nodules (Fig. 1). As human tumors are composed of a wide spectrum of genotypes and phenotypes, we then tested various human cancer cell lines with different genetic characteristics. Tumor cell growth was evaluated by colorimetric assay WST-1, and all the tumor cell lines tested (i.e., A427, Mia-PaCa2, DU145, PC3) were able

Discussion

In the treatment of metastatic colorectal cancer, each therapeutic line which fails delays and decreases the chances of success. An ex vivo model allowing to test on a small tissue sample several chemotherapeutic agents could help to avoid the prescription of ineffective drugs. In the present study, we evaluated the feasibility and the relevance of culture of tumor fragments in order to predict chemosensitivity or chemoresistance. Using polyHEMA coated surface, we have established a simple and

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