Etablierung kolorektaler Xenomodelle, korrespondierender Zelllinien und autologer B-LCLs

Abstract

Model systems of cancer have consistently been used to qualify new anticancer agents for study in human clinical trials. Among the most frequently used models are xenografts of human tumors grown in immuno-deficient mice. For this system, retrospective studies of preclinical and clinical correlations are available. Together with autologous cell lines of tumor and normal origin, such models are still useful and continue to make contributions to critical clinical development choices.

A series of 40 fresh colorectal tumor biopsies was included. Biopsies were minced under sterile conditions and taken into culture at 37 °C, 5 % CO₂ and 95 % humidity using Quantum 263. Outgrowing cells were re-cultured. In 9 cases, additional biopsies were cut into 3 × 3 × 3 mm pieces and immediately transplanted under the skin of NOD-SCID-mice. For establishment of B-LCLs, peripheral blood lymphocytes were isolated and infected with EBV. Outgrowing cells were phenotyped using FACS.

Five out of the nine xenografts were engrafted successfully (55.6 %). Additionally, four permanent cell lines could be established out of those five xenografts (80 %). In all cases, a subsequent passage in new animals was possible with only minimal changes in growth rates and morphological characteristics. A special freezing protocol was developed, allowing for transient cryoconservation of tumor pieces. One additional permanent tumor cell lines was established exclusively in vitro. In summary, five tumor cell lines could be established (5/40 = 12.5 %). Four of them display a chromosomal-instable phenotype, one has typical characteristics of a sporadic micosatellite-instable tumor. This line (HROC24) does not express MLH1, most likely because of promoter-methylation and it shows instability in 3/5 mikrosatellite markers of the Bethesda-Panel (exclusively dinucleotide markers). The corresponding tumor (G2pT2N0L0V0M0R0) was an adenocarcinoma with a mucinous component and low level of tumor budding. Karyotyping revealed a normal set of chromosomes. Additionally, we found mutations in Braf-, and in p53, whereas APC and ras were wildtype. It displayed an elevated resistance towards chemotherapeutics, expression of CD326 and total loss of MHC class I expression. The rate of successful establishment of B-LCL was 14/15 (93 %).

Taken together, several sets of complete autologous sets of xenografts, tumor cell lines and B-LCLs were successfully established for future use in research projects dealing with morphology, molecular biology, tumor immunology and therapeutical interventions.