Abstract 3222: Disulfiram equivalent to doxorubicin in reducing quantitative osteosarcoma metastatic tumor burden in a validated orthotopic mouse model
Authors: Fourman Mitchell S. , Mahjoub Adel , Crasto Jared A. , Mandell Jonathan , Hirsch David C. , Tebbets Jessica , Watters Rebbeca , Weiss Kurt R. 
Published: July 2017

Introduction: The five-year survival of patients with osteosarcoma (OS) with lung metastases is as low as 15%. Our group has shown that disulfiram, a FDA-approved aldehyde dehydrogenase inhibitor, inhibit osteosarcoma proliferation and metastasis in vitro. Here we compare the quantitative effects of disulfiram and doxorubicin on metastatic OS burden in a orthotopic mouse model using near-infrared indocyanine green (ICG) angiography.

Methodology: In an IACUC-approved protocol, 60 immunocompetent balb/c mice were given tibial trans-physeal injections of 500K K7M2 mouse OS cells into their left hindlimbs. Legs were amputated 4 weeks after OS injection, and mice were euthanized with ex vivo lung retrieval 10 weeks after OS injection. Animals in the doxorubicin group (n=20) were administered 2 mg/kg retro-orbitally each week, starting 2 weeks after OS injection to simulate the start of therapy only after a clinical tumor was palpable. Disulfiram (n=20) 50 mg/kg was administered retro-orbitally daily, also starting 2 weeks after OS injection. Controls (n=20) received no therapy. Quantitative near-infrared imaging of the hindlimb and lungs was performed by injecting 20 uL of 25 mg/cc ICG retro-orbitally 24 hours prior to amputation and lung salvage, respectively. ICG reliably extravasates specifically into tumor mass when injected 24 hours prior to fluorescence measurements. Fluorescence analysis was performed using Novadaq SPY (Novadaq, Bonita Springs, FL) and NIH ImageJ (Bethesda, MD). Statistical analysis was performed using Prism 6.0 (GraphPad, LaJolla CA) using Fisher's Exact Test and a one-way analysis of variance with Tukey's post-test as indicated. Significance was defined as p &lt; .05. All numbers are represented as mean ± standard deviation, and are described in arbitrary perfusion units (APU).

Results: Both disulfiram (2.4 ± 1.7 APU) and doxorubicin (0.8 ± 1.7 APU)-treated animals demonstrated a significantly decreased metastatic tumor burden compared to untreated controls (6.4 ± 3.4 APU, p &lt; .01). This finding was independent of hindlimb fluorescence (p &gt; .05). No significant differences were noted between the doxorubicin and disulfiram groups using Tukey's post-test (p = .76). Nineteen of 20 control animals developed metastatic disease, compared to nine of 19 surviving disulfiram-treated and two of 12 surviving doxorubicin-treated (both p &lt; .01) animals. No animals died prematurely in the control group, while one animal died in the disulfiram group (5% mortality) and eight animals died in the doxorubicin group (40% mortality, p &lt; .05 compared to controls).

Conclusion: In our model of metastatic OS, disulfiram appears to have potent anti-metastatic properties. It may also be better tolerated by hosts. Molecular analysis of disulfiram and doxorubicin-treated primary and metastatic tumors is ongoing, which can help us understand the mechanism behind disulfiram's anti-tumor effect.

Citation Format: Mitchell S. Fourman, Adel Mahjoub, Jared A. Crasto, Jonathan Mandell, David C. Hirsch, Jessica Tebbets, Rebbeca Watters, Kurt R. Weiss. Disulfiram equivalent to doxorubicin in reducing quantitative osteosarcoma metastatic tumor burden in a validated orthotopic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3222. doi:10.1158/1538-7445.AM2017-3222