Identification of Three Molecular and Functional Subtypes in Canine Hemangiosarcoma through Gene Expression Profiling and Progenitor Cell Characterization

Abstract

Canine hemangiosarcomas have been ascribed to an endothelial origin based on histologic appearance; however, recent findings suggest that these tumors may arise instead from hematopoietic progenitor cells. To clarify this ontogenetic dilemma, we used genome-wide expression profiling of primary hemangiosarcomas and identified three distinct tumor subtypes associated with angiogenesis (group 1), inflammation (group 2), and adipogenesis (group 3). Based on these findings, we hypothesized that a common progenitor may differentiate into the three tumor subtypes observed in our gene profiling experiment. To investigate this possibility, we cultured hemangiosarcoma cell lines under normal and sphere-forming culture conditions to enrich for tumor cell progenitors. Cells from sphere-forming cultures displayed a robust self-renewal capacity and exhibited genotypic, phenotypic, and functional properties consistent with each of the three molecular subtypes seen in primary tumors, including expression of endothelial progenitor cell (CD133 and CD34) and endothelial cell (CD105, CD146, and αvβ3 integrin) markers, expression of early hematopoietic (CD133, CD117, and CD34) and myeloid (CD115 and CD14) differentiation markers in parallel with increased phagocytic capacity, and acquisition of adipogenic potential. Collectively, these results suggest that canine hemangiosarcomas arise from multipotent progenitors that differentiate into distinct subtypes. Improved understanding of the mechanisms that determine the molecular and phenotypic differentiation of tumor cells in vivo could change paradigms regarding the origin and progression of endothelial sarcomas.

Comparative oncology: what dogs and other species can teach us about humans with cancer

“…Gorden et al [95] performed genome-wide expression profiling of a small number of primary canine haemangiosarcomas rstb.royalsocietypublishing.org Phil. Trans.…”

“…B 370: 20140231 (N ¼ 6 golden retrievers, N ¼ 1 Rottweiler, N ¼ 1 golden retriever  Great Pyrenees, N ¼ 1 Portuguese water dog) and determined three distinct haemangiosarcoma subtypes associated with: angiogenesis (group 1), inflammation (group 2) and adipogenesis (group 3) [95]. The authors investigated haemangiosarcoma cell lines and discovered expression of multiple and distinct markers for early endothelial, haematopoietic and myeloid cells along with several phagocytic and adipogenic functional experiments [95], further supporting the multipotent potential and origin of this blood vessel sarcoma. Genome-wide DNA copy number profiling of canine haemangiosarcoma has also been performed using high-resolution oaCGH [96].…”

A double blinded, placebo-controlled pilot study to examine reduction of CD34+/CD117+/CD133+ lymphoma progenitor cells and duration of remission induced by neoadjuvant valspodar in dogs with large B-cell lymphoma

“…Lymphoma cells were maintained in short-term culture as described 6, 20, 21 . COSB hemangiosarcoma cells were maintained as adherent cultures as described 22 .…”

“…RNA prepared from biopsies obtained at diagnosis (Day 0) and on the fourth day of neoadjuvant treatment for enrolled dogs (Day 4) was quantified and assessed for quality as described 11, 22 . Briefly, total RNA was quantified using a fluorimetric RiboGreen assay and the total RNA integrity was assessed using capillary electrophoresis in the Agilent BioAnalyzer 2100 to generate RNA Integrity Numbers (RIN).…”

“…Samples passed a QC step if they contained >1 µg with a RIN >8. Next-generation RNA sequencing (RNAseq, 50-bp paired-end) with HiSeq 2000 (Illumina) was done at the University of Minnesota Genomics Center (UMGC) in 14-paired (pre- and post-treatment) samples and two additional pre-treatment samples as described 22 . A minimum of ten million read-pairs was generated for each sample.…”