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01.12.2016 | Primary research | Ausgabe 1/2015 Open Access

Cancer Cell International 1/2015

Comprehensive characterization of the patient-derived xenograft and the paralleled primary hepatocellular carcinoma cell line

Zeitschrift:
Cancer Cell International > Ausgabe 1/2015
Autoren:
Phyllis F. Y. Cheung, Chi Wai Yip, Linda W. C. Ng, Kwok Wai Lo, Chit Chow, Kui Fat Chan, Tan To Cheung, Siu Tim Cheung
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s12935-016-0322-5) contains supplementary material, which is available to authorized users.

Abstract

Background

Hepatocellular carcinoma (HCC) is an aggressive cancer with high mortality and morbidity worldwide. The limited clinically relevant model has impeded the development of effective HCC treatment strategy. Patient-derived xenograft (PDX) models retain most of the characteristics of original tumors and were shown to be highly predictive for clinical outcomes. Notably, primary cell line models allow in-depth molecular characterization and high-throughput analysis. Combined usage of the two models would provide an excellent tool for systematic study of therapeutic strategies. Here, we comprehensively characterized the novel PDX and the paralleled primary HCC cell line model.

Methods

Tumor tissues were collected from HCC surgical specimens. HCC cells were sorted for in vivo PDX and in vitro cell line establishment by the expression of hepatic cancer stem cell marker to enhance cell viability and the rate of success on subsequent culture. The PDX and its matching primary cell line were authenticated and characterized in vitro and in vivo.

Results

Among the successful cases for generating PDXs and primary cells, HCC40 is capable for both PDX and primary cell line establishment, which were then further characterized. The novel HCC40-PDX and HCC40-CL exhibited consistent phenotypic characteristics as the original tumor in terms of HBV protein and AFP expressions. In common with HCC40-PDX, HCC40-CL was tumorigenic in immunocompromised mice. The migration ability in vitro and metastatic properties in vivo echoed the clinical feature of venous infiltration. Genetic profiling by short tandem repeat analysis and p53 mutation pattern consolidated that both the HCC40-PDX and HCC40-CL models were derived from the HCC40 clinical specimen.

Conclusions

The paralleled establishment of PDX and primary cell line would serve as useful models in comprehensive studies for HCC pathogenesis and therapeutics development for personalized treatment.
Zusatzmaterial
Additional file 1: Figure S1. PDX and cell line establishment from fresh HCC tumor specimens. Experimental workflow of establishing PDXs and primary cell lines from HCC patients was illustrated. Fresh tumor tissues were collected from 24 HCC patients and subject to enzymatic digestion by collagenase to release disaggregated cells. Only cells with viability >70% (11 out of 24 cases) were subject to subsequent cell sorting for GEP-expressing cells. For in vivo PDX establishment, GEP-enriched cells were inoculated with 50% matrigel (v/v) subcutaneously at the dorsal region of the trunk of NOD/SCID mice (11 cases). 4 out of 11 cases generated xenograft tumors. NOD/SCID mice inoculated with cells from HCC40 (10 weeks post-inoculation) was shown, while the lower panel shows the corresponding xenograft tumor (HCC40-PDX). For in vitro primary cell line establishment, cells were either seeded onto gelatin-coated plate in hepatocyte culture medium (HCM) (11 cases), or ultra-low attachment plate in serum-free, stem cell-promoting medium for spheroid formation (3 cases). 3 out of 11 cases generated cells that could attach and grow within 1 month. The phase contrast microscopy image showed the cells derived from patient HCC40. For spheroid culture, spheroids formed in all 3 cases after 1-month culture. Spheroids derived from patient HCC40 were shown. The spheroids were dissociated to disaggregated cells, which were then seeded onto culture plate in serum-supplemented medium to induce differentiation of cells to grow into adherent monolayer. The adherent cells grew from spheroids derived from patient #40 was designated as HCC40-CL.
Literatur
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