Osterix-Cre marks distinct subsets of CD45- and CD45+ stromal populations in extra-skeletal tumors with pro-tumorigenic characteristics

Abstract

Cancer-associated fibroblasts (CAFs) are a heterogeneous population of mesenchymal cells supporting tumor progression, whose origin remains to be fully elucidated. Osterix (Osx) is a marker of osteogenic differentiation, expressed in skeletal progenitor stem cells and bone-forming osteoblasts. We report Osx expression in CAFs and by using Osx-cre;TdTomato reporter mice we confirm the presence and pro-tumorigenic function of TdTOSX+ cells in extra-skeletal tumors. Surprisingly, only a minority of TdTOSX+ cells expresses fibroblast and osteogenic markers. The majority of TdTOSX+ cells express the hematopoietic marker CD45, have a genetic and phenotypic profile resembling that of tumor infiltrating myeloid and lymphoid populations, but with higher expression of lymphocytic immune suppressive genes. We find Osx transcript and Osx protein expression early during hematopoiesis, in subsets of hematopoietic stem cells and multipotent progenitor populations. Our results indicate that Osx marks distinct tumor promoting CD45- and CD45+ populations and challenge the dogma that Osx is expressed exclusively in cells of mesenchymal origin.

N-cadherin in osteolineage cells modulates stromal support of tumor growth

“…Cells with osteogenic capacity are present in the circulation of animals and humans [51] , and recent data suggest that circulating osteocalcin positive cells may represent an early marker of bone metastasis [52] . In related work with other collaborators, we also find Osx+ cells in peripheral blood, but these cells are hematopoietic in origin [41] , [42] , and ontogenetically unrelated to the tumor associated TdT Osx + cells described here, which were selected for not expressing CD45. In fact, our transcriptomic analysis show that relative to the TdT Osx – population, tumor associated TdT Osx + cells have a mesenchymal, osteogenic signature, as they up-regulate genes involved in collagen matrix production and processing, as well as osteoblast-specific markers.…”

“…The larger subcutaneous tumor growth and the apparent increased severity of metastatic disease in cKO mice prompted us to ask whether Ncad deletion in extra-skeletal cells may be involved in modulating tumorigenesis. Indeed, in a parallel study using the Ai9:Osx-cre reporter mouse we found that a proportion of cells in the stromal component of tumors generated by subcutaneous inoculation of breast (BO1) and melanoma (B16-10) cells express red fluorescence (TdT Osx +) [41] , [42] . We confirmed the presence of TdT Osx + cells in the stromal component (GFP– CD45– population) of orthotropic BO1 tumors grown in either WTe mice or in Cdh2 cKO mice, with no significant differences between the two genotypes ( Fig.…”

Constitutive activation of NF-κB inducing kinase (NIK) in the mesenchymal lineage using Osterix (Sp7)- or Fibroblast-specific protein 1 (S100a4)-Cre drives spontaneous soft tissue sarcoma

“…FSP1-Cre (S100a4-Cre) [31] mice on a FVB/n background were a gift from Dr. Gregory Longmore [32]. Osx-Cre;TdT mice were generated by crossing the following lines from The Jackson Laboratory, USA: B6.Cg-Tg(Sp7-tTA,tetO-EGFP/Cre)1Amc/J (catalog #006361) and B6.Cg-Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze /J (TdT) (catalog #007909) [33]. All parental lines were maintained separately due to strain differences.…”

“…Both the Osx-Cre and FSP1-Cre drivers have been previously shown to have off-target effects outside of the mesenchymal lineage, most notably in cells of hematopoietic origin [32,33,44,45,47,48]. To this end, we assayed CD45 expression.…”

“…The calcium binding protein S100, a marker that is highly expressed in melanomas, was completely absent in our tumors, and this result argues against a melanocytic derivation. Although expression of both Osx-Cre and FSP1-Cre alleles has been observed in CD45+ cells [32,33,44,45,48], the absence of CD45 in the tumor cells indicates that a hematopoietic origin is unlikely. In further support of this conclusion, expression of this NT3 transgene in various hematopoietic cells did not result in malignancy [51][52][53].…”