Bone Metastasis Model Development in an Immunocompetent Murine Host - PowerPoint PPT Presentation

Bone Metastasis Model Development in an Immunocompetent Murine Host December 9, 2017 Alexander H. Jinnah MD, Daniel N. Bracey MD PhD, Thomas L. Smith PhD, Bethany A. Kerr PhD

Background • Prevalence of bone metastasis is 280,000 in US • Serious skeletal complications • Hypercalcemia, spinal cord compression, bone pain, pathological fractures • Survival after bone metastasis diagnosis is ~ 2 years Li et al. 2012 Thibaudeau et al. 2014 Holen et al. 2015 Wake Forest Baptist Medical Center 2

Background • Treatment strategies consist of: • Chemotherapy • Immunotherapy Will not cure – will • Radiopharmaceuticals only treat symptoms • Bisphosphonates • Surgery Wake Forest Baptist Medical Center 3

? Lack of an appropriate in vivo models Current Models: 1. Direct injection 2. Intracardiac Injection Wake Forest Baptist Medical Center 4

Purpose To create an in vivo bone metastasis model in an immunocompetent host using a decellularized porcine xenograft Wake Forest Baptist Medical Center 5

Methods • Decellularized xenograft was created using porcine femurs Wake Forest Baptist Medical Center 6

Methods • To determine osteoblast viability and osteoinductive potential - Pre-osteoblasts were seeded on the scaffold • Incubated for 1 week with and without ascorbic acid • Analyzed for gene expression (qPCR) of Alkaline Phosphatase and BMP-7 Cells + Ascorbic Acid Cells No Ascorbic Acid Scaffold + Ascorbic Acid Scaffold No Ascorbic Acid Wake Forest Baptist Medical Center 7

Methods • To determine prostate Ca viability on scaffold – prostate cancer cells were seeded on the scaffold • Incubated for 1 week • Analyzed for gene expression (qPCR) of mTor, SOX-2 and RANKL Prostate Ca Cells Scaffold + Prostate Ca. Cells Wake Forest Baptist Medical Center 8

Methods • To determine osteoblast viability and osteoinductive potential in vivo • Pre-osteoblasts seeded scaffolds and unseeded scaffolds were implanted subcutaneously on the dorsum of a mouse • Explanted scaffolds were analyzed for gene expression (qPCR) of Wnt3a, BMP-2, and OSX • 5 seeded and 5 unseeded were microCT scanned • Histological and Immunohistochemistry Analysis Wake Forest Baptist Medical Center 9

Results Wake Forest Baptist Medical Center

Pre-osteoblasts In-vitro Alkaline Phosphatase BMP-7 BMP-7 80 40 Relative to control Normalized to 18S Gene Expression 10 8 Control 6 + Ascorbic 4 2 0 Scaffold Monolayer • Increased Alkaline phosphatase expression in scaffold Ascorbic acid lead to further increase in expression  • No significant differences seen in BMP-7 Wake Forest Baptist Medical Center 11

Cancer Cells In-Vitro • mTor and sox-2 gene expression were significantly higher in the scaffold than the control monolayer • Suggests cancer cell proliferation Wake Forest Baptist Medical Center 12

Cancer Cells In-Vitro In-Vitro • RANKL gene expression was significantly higher in the scaffold than the control monolayer • Suggests osteoblast activity Wake Forest Baptist Medical Center 13

Pre-osteoblasts In-vivo BMP-2 * 200 100 Relative to control Normalized to 18S Gene Expression 10 cells 8 No cells 6 4 2 0 k wnt3a * 2000 • gene expression was 1000 Relative to control Normalized to 18S Gene Expression significantly greater in all 5 4 cells groups 3 No cells 2 1 0 Wake Forest Baptist Medical Center 14

Pre-osteoblasts In-vivo • MicroCT Scans were performed on 8 of 10 scaffolds due to inadequate pre-implantation scans • 2 more were excluded based on inadequate post- implantation scan or partial destruction of the scaffold • Increase in bone volume and trabecular thickness post- implantation (ns) Trabecular Thickness 5 4 Average Tb.Th Fold change 3 2 Pre Implantation 1 Post Implantation 0 Scaffold Scaffold + OB Wake Forest Baptist Medical Center 15

Histological Analysis • H&E staining demonstrating cellular material accumulating within the scaffold • IHC staining demonstrated that these cells express osteopontin • Pentachrome staining demonstrating new collagen formation within the pores Wake Forest Baptist Medical Center 16

Conclusions Wake Forest Baptist Medical Center

Conclusions • Osteoinductive potential of decellularized porcine xenograft • Decellularized xengoraft is a viable host to osteoblasts in vivo and in vitro and to cancer cells in vitro Future Work • Implant scaffold and a tumor into immunocompetent mouse • PET-CT Wake Forest Baptist Medical Center 18

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