Tumourigenic canine osteosarcoma cell lines associated with frizzled-6 up-regulation and enhanced side population cell frequency

Abstract

An increased serum alkaline phosphatase concentration is known to be associated with a negative prognosis in canine and human osteosarcoma. To expand upon previous studies regarding the biological relevance of increased serum alkaline phosphatase as a negative prognostic factor, xenogeneic heterotopic transplants were performed using six canine primary osteosarcoma cell lines generated from patients with differing serum alkaline phosphatase concentrations (three normal and three increased). Three of the six cell lines were capable of generating tumours and tumour formation was independent of the serum alkaline phosphatase status of the cell line. Microarray analysis identified 379 genes as being differentially expressed between the tumourigenic and non-tumourigenic cell lines. Frizzled-6 was upregulated to the greatest extent (7.78-fold) in tumourigenic cell lines compared with non-tumourigenic cell lines. Frizzled-6, a co-receptor for Wnt ligands has been associated with enhanced tumour-initiating cells and poor prognosis for other tumours. The increased expression of frizzled-6 was confirmed by quantitative reverse transcription polymerase chain reaction (QPCR) and Western blot analysis. Additionally, the tumourigenic cell lines also had an increase in the percentage of side population cells compared with non-tumourigenic cell lines (5.89% versus 1.58%, respectively). There were no differences in tumourigenicity, frizzled-6 or percentage of side population cells noted between osteosarcoma cell lines generated from patients of differing serum alkaline phosphatase concentration. However, to our knowledge this is the first study to identified frizzled-6 as a possible marker of osteosarcoma cell populations with enhanced tumourigenicity and side population cells. Future work will focus on defining the role of frizzled-6 in osteosarcoma tumourigenesis and tumour-initiating cells.

Keywords: cell signalling; comparative oncology; mouse models; oncology; tumour biology.

Figure 1

(A) Photomicrograph of tumour histology from mouse receiving UWKOS3. The lesion is representative of the three tumour-forming cell lines. (B) Representative histopathology section of the canine primary osteosarcoma lesion from which UWKOS3 was generated.

Figure 2

Hierarchical clustering of six canine osteosarcoma cell lines on the top-112 significant genes differentially expressed between tumourigenic and non-tumourigenic lines.

Figure 3

Expression of frizzled-6 mRNA in canine osteosarcoma cell lines relative to a canine normal osteoblast cell line.

Figure 4

Western blot analysis for frizzled-6 expression in tumourigenic (UWKOS1 and UWKOS7) and non-tumourigenic (UWKSO2, UWKOS6 and UWKOS8) cell lines. The tumourigenic UWKOS3 cell line is not included in this image, however, frizzled-6 expression has been confirmed via Western blot analysis in this cell line as well.

Figure 5

Frequency of side population cells as determined by Hoechst-exclusion assay. The flow cytometry images depict representative findings from tumourigenic (top row) and non-tumourigenic (bottom row) cell lines without (left column) and with verapamil treatment (right column). Bar graph to far right represents the frequency of side population cells (average ± SEM) in non-tumourigenic (n = 3) and tumourigenic (n = 3) canine primary osteosarcoma cell lines.