Endothelial-to-Osteoblast Conversion Generates Osteoblastic Metastasis of Prostate Cancer

Abstract

Prostate cancer (PCa) bone metastasis is frequently associated with bone-forming lesions, but the source of the osteoblastic lesions remains unclear. We show that the tumor-induced bone derives partly from tumor-associated endothelial cells that have undergone endothelial-to-osteoblast (EC-to-OSB) conversion. The tumor-associated osteoblasts in PCa bone metastasis specimens and patient-derived xenografts (PDXs) were found to co-express endothelial marker Tie-2. BMP4, identified in PDX-conditioned medium, promoted EC-to-OSB conversion of 2H11 endothelial cells. BMP4 overexpression in non-osteogenic C4-2b PCa cells led to ectopic bone formation under subcutaneous implantation. Tumor-induced bone was reduced in trigenic mice (Tie2^cre/Osx^f/f/SCID) with endothelial-specific deletion of osteoblast cell-fate determinant OSX compared with bigenic mice (Osx^f/f/SCID). Thus, tumor-induced EC-to-OSB conversion is one mechanism that leads to osteoblastic bone metastasis of PCa.

Keywords: bone metastasis; endothelial-to-osteoblast conversion; osteoblast; paracrine factors; prostate cancer; proteomics.

Figure 1. Ectopic bone in PCa-118b xenograft originates from the mouse host

(A) Consecutive sections of subcutaneous tumors generated from PCa-118b or C4-2b cells were stained with Goldner’s Trichome or hematoxylin & eosin. T, tumor. Scale bar, 50 μm. (B) β-galactosidase expression in a Col-βGal/SCID mouse. (i) 2.3 kb Col1α1-driven β-gal construct in Col-βGal/SCID mouse. (ii) Left, β-galactosidase activity in lysates prepared from indicated tissues of a 4 day-old Col-βGal/SCID pup. Right, detection of β-galactosidase activity using X-gal as a substrate in the skull, rib, and hind limb of a 4 day-old Col-βGal/SCID mouse. (iii) X-gal staining of a femur from an adult Col- βGal/SCID mouse. Right, higher magnification of the boxed area on the left. Arrows, osteocytes; arrowheads, osteoblasts. Scale bar, 250 μm. (C) PCa-118b tumors in a Col-βGal/SCID mouse. (i) Radiograph of a calcified subcutaneous PCa-118b tumor (circled) in a Col-βGal/SCID mouse. (ii) X-gal staining of a PCa-118b tumor containing immature bone. Immature bone showed lighter eosin staining compared to the mature bone. (iii) X-gal staining of a PCa-118b tumor containing mature bone. Right, higher magnification of the boxed area on the left. Scale bar, 250 μm. (D) qRT-PCR for the expression of mouse or human osteocalcin from RNA prepared from PCa-118b whole tumor or isolated PCa-118b cells. (E) Diagram illustrating the mouse origin of osteoblasts in the tumor-induced bone. Osteoblasts observed in PCa-118b xenograft are from mouse cells that are recruited into the tumor.

Figure 2. Osteoblasts in tumor-associated bone co-express endothelial cell and osteoblast markers

(A) Primary mouse osteoblasts isolated from 2–4 day old newborn calvaria were immunostained with antibodies against Tie2 and osteocalcin. Nuclei were stained with DAPI. Primary mouse osteoblasts expressed osteocalcin but not Tie2. In contrast, mouse endothelial cells expressed Tie2 but not osteocalcin. Scale bar, 50 μm(see also Figure S1). Representative sections of PCa-118b tumor co-stained with (B) osteocalcin and Tie-2 antibodies or (C) EpCAM and osteocalcin. Areas containing bone or tumor are shown. Boxed areas are enlarged in insets. B, bone; T, tumor. (D) Procedure for enrichment of different cell populations from a PCa-118b tumor. First cell isolate, tumor cell enriched population; second cell isolate, osteoblast enriched population. Immunostaining of first and second cell isolates with (E) antibody against EpCAM or (F) antibodies against Tie2 and osteocalcin (see also Figure S2A). (G) Immunostaining of osteocalcin (OCL) and Tie2 in human PCa bone metastasis specimens. Upper, a specimen from laminectomy. Lower, a specimen from needle biopsy (see also Figures S3 and S4). (H) Diagram illustrating that some of tumor-associated endothelial cells are likely converted to osteoblasts based on the results from the expression of cell-specific markers. Scale bars, 25 μm.

Figure 3. Effects of BMP4 or TGFβ2 on EC-to-OSB conversion in vitro and in vivo

(A) 2H11 endothelial cells were incubated in differentiation medium with indicated concentrations of BMP4 or TGFβ2 for 7 days. Left, the culture was stained with alkaline phosphatase substrate. Middle, cell lysates were incubated with p-nitrophenylphosphate and the OD₄₀₅ measured. Right, alkaline phosphatase activity was normalized with the protein concentration. (B) qRT-PCR using primers for mouse osteocalcin on RNA from 2H11 cells treated with indicated concentrations of BMP4 or TGFβ2 for 2 or 3 days. The relative level of osteocalcin RNA induced in 2H11 cells was compared to that of mouse calvaria (PMO). (C) Alizarin Red S staining or (D) von Kossa staining of 2H11 cells in differentiation medium treated with BMP4 or TGFβ2 for 21 days. Right panels, quantification of the stainings using image J. (E) Western blot of 2H11 cell lysates prepared as in (B) using Cad11 or actin antibody. Right, quantification of Cad11 levels relative to actin. (F) Characterization of C4-2b-vector, C4-2b-BMP4, and C4-2b-TGFβ2 cells. Left, RT-PCR using primers specific for BMP4 or TGFβ2. GAPDH message was used as a control. Middle, Western blot for the expression of TGFβ2 protein in the conditioned media. Right, ELISA for the expression of BMP4 in the conditioned media. (G) Histological analyses of C4-2b-vector, C4-2b-BMP4, and C4-2b-TGFβ2 tumors by H&E and Goldner’s trichrome staining. Right, quantification of mineralized area in tumors based on Goldner’s trichrome staining. Scale bars, 50 μm. (H) von Kossa staining of C4-2b-vector, C4-2b-BMP4, and C4-2b-TGFβ2 tumors and its quantification (right) in tumor sections. Scale bars, 50 μm. (I) Representative immunohistochemistry images of primary prostate cancer or bone biopsies stained for BMP4. Scale bar, 200 μm. Densitometry analyses of BMP4 expression in human prostate cancer specimens were quantified by Aperio ImageScope software. Data are presented as mean plus SEM. p value was determined by t-test. (J) Diagram illustrating that tumor secreted BMP4 is one of the factors produced by tumor cells that induce EC-to-OSB conversion to form ectopic bone.

Figure 4. Co-localization of osteocalcin and tdTomato in osteoblasts of tumor-induced bone in Tie2^Cre/Rosa^tdTomato mouse

In vivo lineage tracing of tumor-induced osteoblasts in Tie2^Cre/Rosa^tdTomato mice. TRAMP-BMP4 cells (1 million) were injected into right femur of either Rosa-tdTomato or Tie2^Cre/Rosa^tdTomato mice for seven weeks. Mice were subjected to radiograph analysis (A, left panel). Femurs were collected for micro CT (A, right panel). (B) Histological analyses. Scale bars are at 50 μm of x400 magnification (top panel) and at 50 μm of x200 magnification (bottom panel). (C) Immunostaining of osteocalcin. Confocal images of the co-expression of Tie2 (tdTomato) and osteocalcin (green). Scale bars are at 100 μm of x400 magnification. Arrowheads point to the tumor-induced osteoblasts.

Figure 5. Role of OSX in EC-to-OSB conversion

(A) Left, qRT-PCR for the levels of OSX messages in BMP4 or TGFβ2-treated 2H11 cells. Right, western blot of lysates from BMP4 or TGFβ2-treated 2H11 cells with anti-OSX antibody. (B) qRT-PCR for OSX mRNA in 2H11-vector, 2H11-shOSX#1, and 2H11-shOSX#5 cells (left panel). When compared to 2H11-vector cells, osteocalcin expression induced by BMP4 was significantly reduced in 2H11-shOSX#1 and 2H11-shOSX#5 cells in mRNA (middle panel) and protein levels (right panel). (C) (i) qRT-PCR for OSX mRNA in 2H11, 2H11-GFP, and 2H11-OSX cells. (ii) Upper, qRT-PCR of osteocalcin mRNA upon incubation in differentiation medium with BMP4 for 7 days. Lower, Western blot of OSX protein in 2H11-GFP and 2H11-OSX cells cultured in differentiation medium with BMP4 for 7 days. (iii) Upper, alkaline phosphatase activity in 2H11, 2H11-GFP, and 2H11-OSX cells. Lower, alkaline phosphatase activity in 2H11-GFP and 2H11-OSX cells cultured in differentiation medium with BMP4 for 7 days. (D) Diagram illustrating that both BMP4 and OSX are necessary for EC-to-OSB conversion.

Figure 6. Tumor-induced ectopic bone in C4-2b-BMP4 tumors is significantly reduced in mice with endothelial-specific deletion of OSX

(A) Design and generation of bigenic OSX^f/f/B6^scid/scid and endothelial-specific OSX knockout trigenic Tie2^cre/OSX^f/f/B6^scid/scid mice. Upper right, mouse genotypes were determined by PCR. Lower right, qPCR for Cre or OSX in genomic DNA prepared from microvascular endothelial cells isolated from the lungs of bigenic and trigenic mice using CD31 magnetic beads. (B) H&E and Goldner’s trichome staining of C4-2b-BMP4 tumors generated in male trigenic and bigenic mice. Right, quantification of ectopic bone in tumors. Scale bars, 100 μm. (C) C4-2b-BMP4 tumors generated in female trigenic and bigenic mice. Right, quantification of ectopic bone in tumors. n, number of tumor samples in each group. Scale bars, 50 μm.

Figure 7. Tumor-induced ectopic bone in PCa-118b tumors is significantly reduced in mice with endothelial-specific deletion of OSX

(A) H&E and Goldner’s trichome staining of PCa-118b tumor generated in male bigenic OSX^f/f/B6^scid/scid and trigenic Tie2^cre/OSX^f/f/B6^scid/scid mice described in Fig. 5A. Right, quantification of ectopic bone in tumors in males. n, number of tumor samples. (B) H&E and Goldner’s trichome staining of PCa-118b tumor generated in female bigenic and trigenic mice. Right, quantification of ectopic bone in tumors in females. n, number of tumor samples in each group. (C) Quantification of tumor weight of PCa-118b tumors in male bigenic and trigenic mice. (D) Model of PCa-induced bone formation through endothelial-to-osteoblast conversion. PCa cells secrete factors, e.g. BMP4, which induce OSX expression in endothelial cells (or other stromal cells), leading to EC-to-OSB conversion. The EC-to-OSB conversion is one mechanism accounting for the characteristic osteoblastic bone lesion of PCa. Scale bars, 50 μm.