Establishment and molecular characterisation of seven novel soft-tissue sarcoma cell lines

Abstract

Background: Soft-tissue sarcomas (STS) are a diverse group of malignancies that remain a diagnostic and therapeutic challenge. Relatively few reliable cell lines currently exist. Rapidly developing technology for genomic profiling with emerging insights into candidate functional (driver) aberrations raises the need for more models for in vitro functional validation of molecular targets.

Methods: Primary cell culture was performed on STS tumours utilising a differential attachment approach. Cell lines were characterised by morphology, immunocytochemistry, proliferation assays, short tandem repeat (STR) and microarray-based genomic copy number profiling.

Results: Of 47 STS cases of various subtypes, half formed adherent monolayers. Seven formed self-immortalised cell lines, including three undifferentiated pleomorphic sarcomas, two dedifferentiated liposarcomas (one of which had received radiotherapy), a leiomyosarcoma and a myxofibrosarcoma. Two morphologically distinct yet genetically identical variants were established in separate cultures for the latter two tumours. All cell lines demonstrated genomic and phenotypic features that not only confirm their malignant characteristics but also confirm retention of DNA copy number aberrations present in their parent tumours that likely include drivers.

Conclusions: These primary cell lines are much-needed additions to the number of reliable cell lines of STS with complex genomics available for initial functional validation of candidate molecular targets.

Figure 1

Representative phase-contrast micrographs of STS primary cell lines.(A) Shef-UPS 01-spindle-shaped cells at passage 69 without distinct colony formation in culture. (B) Shef-UPS 03 cultures at passage 35 showing homogeneous cultures of long spindle-shaped cells growing in loose colonies. (C) Shef-DDLPS 01 cultures at passage 71 showing pleomorphic cells without distinct colony formation. (D) Shef-DDLPS 02 cultures at passage 22 composed of a combination of spindle-shaped cells (white arrows) and round, histiocyte-like cells (black arrows), both growing in distinct colonies. (E, F) Cells derived from Shef-LMS 01 growing in separate cultures designated as Ws (passage 69) and w1 (passage 56), respectively. (G, H) Morphologically distinct cells derived from Shef-MFS 01 growing in separate adherent cultures designated as w1 (passage 35) and w2 (passage 31), respectively. Scale bars=100 μm. DDLPS=dedifferentiated liposarcoma; LMS=leiomyosarcoma; MFS=myxofibrosarcoma.

Figure 2

Genomic copy number profile comparisons of seven soft-tissue sarcoma (STS) primary cell lines (shown on the left) with their parent tumours.Individual cell lines and passage number at which genomic DNA was extracted are shown to the left of the corresponding autosome ideograms. The overlaid red and blue lines represent the moving average of log₂ ratios of the cultured cells and parent tumour tissue, respectively. Deviations above and below the horizontal baseline represent amplifications and deletions, respectively. Relative amplitude of deviation shows the log₂ ratio and represents DNA copy number at the corresponding genomic locus. Note the close similarity and/or near-identical breakpoints in the moving average patterns in each case over the majority of the genome. Copy number analysis was performed on the Agilent 4 × 180K DNA microarray platform and data were analysed using Agilent Genomic Workbench Software v.6.0 (Agilent Technologies).

Figure 3

Selected chromosome copy number profile comparisons of soft-tissue sarcoma (STS) primary cell lines with their parent tumours.Ideograms of specific chromosomes are as shown at the top of each panel with the corresponding regions at the bottom. The overlaid red and blue lines represent the moving average of log₂ ratios (vs normal genomic DNA) of the cultured cells and parent tumour tissue, respectively. Deviations above and below the horizontal baseline represent amplifications and deletions, respectively. Amplitude of deviation shows the relative log₂ ratio and represents relative DNA copy number. Note the close similarity and/or near-identical breakpoints in the moving average patterns in each case. Copy number analysis was performed on the Agilent 4 × 180K DNA microarray platform and data analysed using Agilent Genomic Workbench Software v.6.0 (Agilent Technologies).