Establishment, functional and genetic characterization of a colon derived large cell neuroendocrine carcinoma cell line

Abstract

Aim: To establish cell line and patient-derived xenograft (PDX) models for neuroendocrine carcinomas (NEC) which is highly desirable for gaining insight into tumor development as well as preclinical research including biomarker testing and drug response prediction.

Methods: Cell line establishment was conducted from direct in vitro culturing of colonic NEC tissue (HROC57). A PDX could also successfully be established from vitally frozen tumor samples. Morphological features, invasive and migratory behavior of the HROC57 cells as well as expression of neuroendocrine markers were vastly analyzed. Phenotypic analysis was done by microscopy and multicolor flow cytometry. The extensive molecular-pathological profiling included mutation analysis, assessment of chromosomal and microsatellite instability; and in addition, fingerprinting (i.e., STR analysis) was performed from the cell line in direct comparison to primary patient-derived tissues and the PDX model established. Drug responsiveness was examined for a panel of chemotherapeutics in clinical use for the treatment of solid cancers.

Results: The established cell line HROC57 showed distinct morphological and molecular features of a poorly differentiated large-cell NEC with KI-67 > 50%. Molecular-pathological analysis revealed a CpG island promoter methylation positive cell line with microsatellite instability being absent. The following mutation profile was observed: KRAS (wt), BRAF (mut). A high sensitivity to etoposide, cisplatin and 5-FU could be demonstrated while it was more resistant towards rapamycin.

Conclusion: We successfully established and characterized a novel patient-derived NEC cell line in parallel to a PDX model as a useful tool for further analysis of the biological characteristics and for development of novel diagnostic and therapeutic options for NEC.

Keywords: Individualized medicine; Large cell neuroendocrine carcinoma; Patient-derived tumor model.

Figure 1

Migratory potential and invasiveness of HROC 57 cells. Invasion and migration of the cell line HROC57 in comparison to the reference cell line HCT116 was analyzed. Cells were subjected to migration assay (Migration, grey bars) and matrigel invasion assay (Invasion, black bars). Values are means ± SEM of n = 3. ^aP < 0.05 vs HCT116.

Figure 2

Expression of human lymphocyte antigen molecules (ß2Microglobulin, HLA-ABC, HLA-DR/DP/DQ, HLA-A2, epithelial cell markers (CD26, CD29, CD44, CD166, CD326), neuroendocrine markers (CD56, Chromogranin A, Synapthophysin, NSE, CD90), proliferation markers (Ki67, CD71) and immune evasion molecules (CD73, CD152, CD275, CD278) were assessed by flow cytometry using a BD FACSARIA II.

Figure 3

Light microscopy of HROC57 after direct establishment (P10) and medium-term in vitro culture (P22). Cell lines were established directly from patients’ tumor material as described in material and methods. Original magnification: × 100.

Figure 4

Sensitivity of HROC57 to conventional chemotherapeutics. HROC57was treated for 72 h with etoposide (A), cisplatin (B) and combination of etoposide and cisplatin (C). Cell viability was measured using the crystal violet assay as described in materials and methods. Values represent the mean absorbance at 570 nm ± SD of n = 4 analyses.

Figure 5

Radiosensitivity of HROC57. HROC57 was irradiated with 50 Gy using a ¹³⁷CS-source. Control cells were not irradiated. Cell viability was measured using the crystal violet assay as described in materials and methods. n = 2 analyses.

Figure 6

Expression of p53 and some of its targets in HROC57 cells. Western Blot analysis of untreated HROC57 cells compared to HCT116 (p53 wildtype) and HCT116 p53^-/- was performed. Acetylation (K382) and phosphorylation (S15) of p53 (A) and expression of its targets p21, survivin and HDAC 1 and 2 (B) were assessed using specific antibodies. HSP90 served as loading control. Numbers indicate densitometric analysis of respective signals normalized to loading control and relative to HCT116 cells.