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

Bone Metastasis Model Development in an Immunocompetent Murine Host

Alexander H. Jinnah MD, Daniel N. Bracey MD PhD, Thomas L. Smith PhD, Bethany A. Kerr PhD

December 9, 2017

Wake Forest Baptist Medical Center

• 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

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Background

Li et al. 2012 Thibaudeau et al. 2014 Holen et al. 2015

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• Treatment strategies consist of:
• Chemotherapy
• Immunotherapy
• Radiopharmaceuticals
• Bisphosphonates
• Surgery

Background

Will not cure – will

• nly treat symptoms

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?

Lack of an appropriate in vivo models Current Models:

1. Direct injection 2. Intracardiac Injection

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Purpose

To create an in vivo bone metastasis model in an immunocompetent host using a decellularized porcine xenograft

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Methods

• Decellularized xenograft was created using

porcine femurs

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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

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Cells + Ascorbic Acid Cells No Ascorbic Acid Scaffold + Ascorbic Acid Scaffold No Ascorbic Acid

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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

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Prostate Ca Cells Scaffold + Prostate Ca. Cells

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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

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Results

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• Increased Alkaline phosphatase expression in scaffold
• Ascorbic acid lead to further increase in expression
• No significant differences seen in BMP-7

Pre-osteoblasts In-vitro

BMP-7

Scaffold Monolayer 2 4 6 8 10 40 80

Control + Ascorbic

Gene Expression Relative to control Normalized to 18S

Alkaline Phosphatase BMP-7

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Cancer Cells In-Vitro

• mTor and sox-2 gene expression were significantly higher in the scaffold

than the control monolayer

• Suggests cancer cell proliferation

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In-Vitro

• RANKL gene expression was

significantly higher in the scaffold than the control monolayer

• Suggests osteoblast activity

Cancer Cells In-Vitro

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• gene expression was

significantly greater in all groups

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Pre-osteoblasts In-vivo

BMP-2

k 2 4 6 8 10 100 200

cells No cells

*

Gene Expression Relative to control Normalized to 18S

wnt3a

1 2 3 4 5 1000 2000

cells No cells

*

Gene Expression Relative to control Normalized to 18S

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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

Scaffold Scaffold + OB 1 2 3 4 5

Pre Implantation Post Implantation Average Tb.Th Fold change

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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

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Conclusions

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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

• Implant scaffold and a tumor into immunocompetent

mouse

• PET-CT

Future Work

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