Functional Characterization and Drug Response of Freshly Established Patient-Derived Tumor Models with CpG Island Methylator Phenotype.

Patient-individual tumor models constitute a powerful platform for basic and translational analyses both in vitro and in vivo. However, due to the labor-intensive and highly time-consuming process, only few well-characterized patient-derived cell lines and/or corresponding xenografts exist. In this study, we describe successful generation and functional analysis of novel tumor models from patients with sporadic primary colorectal carcinomas (CRC) showing CpG island methylator phenotype (CIMP). Initial DNA fingerprint analysis confirmed identity with the patient in all four cases. These freshly established cells showed characteristic features associated with the CIMP-phenotype (HROC40: APCwt, TP53 mut, KRAS mut; 3/8 marker methylated; HROC43: APC mut, TP53 mut, KRAS mut; 4/8 marker methylated; HROC60: APCwt, TP53 mut, KRASwt; 4/8 marker methylated; HROC183: APC mut, TP53 mut, KRAS mut; 6/8 marker methylated). Cell lines were of epithelial origin (EpCAM+) with distinct morphology and growth kinetics. Response to chemotherapeutics was quite individual between cells, with stage I-derived cell line HROC60 being most susceptible towards standard clinically approved chemotherapeutics (e.g. 5-FU, Irinotecan). Of note, most cell lines were sensitive towards "non-classical" CRC standard drugs (sensitivity: Gemcitabin > Rapamycin > Nilotinib). This comprehensive analysis of tumor biology, genetic alterations and assessment of chemosensitivity towards a broad range of (chemo-) therapeutics helps bringing forward the concept of personalized tumor therapy

Secretion profile, growth kinetics and invasive potential of CIMP⁺ cell lines.

<p>(<b>A</b>) Cytokine secretion pattern was quantified by ELISA. Cytokine concentrations were determined by comparison with a standard curve generated from serial dilutions of individual standards. Quantitative analysis of CEA, CA19-9 and IL8 secretion after one, three and six days of culture, respectively. (<b>B</b>) Growth kinetics of cells, counted every 24 hours for five consecutive days using a Neubauer chamber. (<b>C</b>) Cellular invasiveness was examined using a Matrigel®-based Boyden chamber assay. Quantification of cellular invasiveness was estimated by MTT assay. Data are expressed as percentage invasion versus HCT116 cells (= internal positive control). (<b>A-C</b>) Results show the mean + standard deviation of three independent experiments.</p

Response to Aza-based drug combinations.

<p>Representative quantitative analysis of HROC40 (left graph) and HROC183 cells (right graph) treated with Aza, standard drugs or combinations thereof. Cells received two (upper panel) or four (lower panel) treatment cycles in concentrations as indicated in the material and methods section. Cytotoxicity was quantified upon crystal violet staining and measurement at 570 nm (reference wavelength: 620 nm). Results show the mean + standard deviation of three independent experiments.</p

Mutational profile of CIMP⁺ cell lines.

<p>wt–wildtype, mut–mutated, ex–exon, cd–codon.</p><p>Mutational profile of CIMP⁺ cell lines.</p

Tumor histology.

<p>H & E sections of (<b>A</b>) HROC40-PDX and (<b>B</b>) its primary. Note the invasive edge towards the right. Principal morphological features are retained in the PDX. β-catenin immunohistochemistry of (<b>C</b>) HROC60-PDX and (<b>D</b>) its primary. Note nuclear β-catenin translocation at the invasive edge of both tumors.</p

Methylation marker and molecular classification of CIMP⁺ cell lines.

<p>+–methylated;—–not methylated.</p><p>Methylation marker and molecular classification of CIMP⁺ cell lines.</p

Drug response of CIMP⁺ cell lines.

<p>Drug response of CIMP⁺ cell lines.</p

Clinical and pathological characteristics of patients as well as cell line establishment protocol.

<p>f–female, m–male, B-LCL–B lymphoid cell line, +–positive,—–negative</p><p>Clinical and pathological characteristics of patients as well as cell line establishment protocol.</p

Immunosuppressive phenotype of CIMP⁺ cell lines.

<p>Assessment of immune evasion markers was conducted by multi-color flow cytometry using fluorochrom-labeled mAbs as given on the x-axis. Results are given as the mean % of positive cells + standard deviation of three independent experiments.</p