Background Epigenetics is the study of changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence. It is widely accepted that cancer has genetic and epigenetic origins. The idea of epigenetic reprogramming of cancer cells by an embryonic microenvironment possesses potential interest from the prospect of both basic science and potential therapeutic strategies. Chick embryo extract (CEE) has been used for the successful expansion of many specific stem cells and has demonstrated the ability to facilitate DNA demethylation.Questions/purposes The current study was conducted to compare the status of DNA methylation in highly metastatic and less metastatic osteosarcoma cells and to investigate whether CEE may affect the epigenetic regulation of tumor suppressor genes and thus change the metastatic phenotypes of highly metastatic osteosarcoma cells. Methods K7M2 murine OS cells were treated with CEE to determine its potential effect on DNA methylation, cell apoptosis, and invasion capacity. Results Our current results suggest that the methylation status of tumor suppressor genes (p16, p53, and E-cadherin) is significantly greater in highly metastatic mouse ostoesarcoma K7M2 cells in comparison with less metastatic mouse osteosarcoma K12 cells. CEE treatment of K7M2 cells caused demethylation of p16, p53, and E-cadherin genes, upregulated their expression, and resulted in the reversion of metastatic phenotypes in highly metastatic osteosarcoma cells. Conclusions CEE may promote the reversion of metastatic phenotypes of osteosarcoma cells and can be a helpful tool to study osteosarcoma tumor reversion by epigenetic reprogramming. Clinical Relevance Demethylation of tumor suppressor genes in osteosarcoma may represent a novel strategy to diminish the metastatic potential of this neoplasm. Further studies, both in vitro and in vivo, are warranted to evaluate the clinical feasibility of this approach as an adjuvant to current therapy.

BackgroundOsteosarcoma (OS) is the most common primary bone tumor, with a propensity to metastasize to the lungs. Five-year survival for metastatic OS is below 30%, and has not improved for several decades despite the introduction of multi-agent chemotherapy. Understanding OS cell migration to the lungs requires an evaluation of the lung microenvironment. Here we utilized an in vitro lung cell and OS cell co-culture model to explore the interactions between OS and lung cells, hypothesizing that lung cells would promote OS cell migration and survival. The impact of a novel anti-OS chemotherapy on OS migration and survival in the lung microenvironment was also examined.MethodsThree human OS cell lines (SJSA-1, Saos-2, U-2) and two human lung cell lines (HULEC-5a, MRC-5) were cultured according to American Type Culture Collection recommendations. Human lung cell lines were cultured in growth medium for 72 h to create conditioned media. OS proliferation was evaluated in lung co-culture and conditioned media microenvironment, with a murine fibroblast cell line (NIH-3 T3) in fresh growth medium as controls. Migration and invasion were measured using a real-time cell analysis system. Real-time PCR was utilized to probe for Aldehyde Dehydrogenase (ALDH1) expression. Osteosarcoma cells were also transduced with a lentivirus encoding for GFP to permit morphologic analysis with fluorescence microscopy. The anti-OS efficacy of Disulfiram, an ALDH-inhibitor previously shown to inhibit OS cell proliferation and metastasis in vitro, was evaluated in each microenvironment.ResultsLung-cell conditioned medium promoted osteosarcoma cell migration, with a significantly higher attractive effect on all three osteosarcoma cell lines compared to basic growth medium, 10% serum containing medium, and NIH-3 T3 conditioned medium (p <0.05). Lung cell conditioned medium induced cell morphologic changes, as demonstrated with GFP-labeled cells. OS cells cultured in lung cell conditioned medium had increased alkaline phosphatase staining.ConclusionsLung endothelial HULEC-5a cells are attractants for OS cell migration, proliferation, and survival. The SJSA-1 osteosarcoma cell line demonstrated greater metastatic potential than Saos-2 and U-2 cells. ALDH appears to be involved in the interaction between lung and OS cells, and ALP may be a valuable biomarker for monitoring functional OS changes during metastasis.

Given the histologic localization of ICG to only the tumor bed, we envision the clinical use of ICG angiography as an intraoperative margin and tumor detector. Such a tool may be used as an alternative to intraoperative histology to confirm negative primary tumor margins or as a valuable tool for debulking surgeries in vulnerable anatomic locations. Because we have demonstrated the successful preservation of ICG in frozen tumor samples, future work will focus on blinded validation of this modality in observational human trials, comparing the ICG fluorescence of harvested tissue samples with fresh frozen pathology.

IntroductionThe overall survival rate of patients with osteosarcoma (OS) and pulmonary metastases has remained stagnant at 15–30% for several decades. Disulfiram (DSF) is an FDA-approved aldehyde dehydrogenase inhibitor that reduces the metastatic phenotype of OS cells in vitro. Here we evaluate its in vivo efficacy, as compared to doxorubicin chemotherapy, in a previously-validated orthotopic model of metastatic OS.ResultsAll treatment groups displayed a significantly reduced quantitative OS metastatic burden compared with controls. The metastatic burden of Lo DSF-treated animals was equivalent to the DXR group. Ninety-five percent of control animals displayed evidence of metastatic disease, which was significantly greater than all treatment groups.DiscussionDisulfiram treatment resulted in a reduced burden of OS metastatic disease compared with controls. This was statistically-equivalent to doxorubicin. No additive effect was observed between these two therapies.Materials and MethodsOne-hundred twenty immunocompetent Balb/c mice received proximal tibia paraphyseal injections of 5 × 105 K7M2 murine OS cells. Therapy began three weeks after injection: saline (control), low-dose disulfiram (Lo DSF), high-dose disulfiram (Hi DSF), doxorubicin (DXR), Lo DSF + DXR, and Hi DSF + DXR. Transfemoral amputations were performed at 4 weeks. Quantitative metastatic tumor burden was measured using near-infrared indocyanine green (ICG) angiography.

Background Surgical resection with negative margins is the foundation of extremity sarcoma management. Failure to achieve negative surgical margins can result in local recurrence (LR), a potentially devastating complication. Indocyanine green (ICG) is a US FDA-approved fluorophore previously used to guide carcinoma resections. We investigated the potential of ICG as an intraoperative guide during experimental sarcoma resection. Methods Fifty 6-week-old immunocompetent Balb/c female mice received left proximal tibia paraphyseal injections of 5 × 10 5 K7M2 murine osteosarcoma cells. Animals were separated into two groups ( n = 25 each): (1) ICG-assisted surgical resection; and (2) no ICG-assisted resection. Resections were performed 4 weeks after primary tumor engraftment. All animals received 7.5 ug ICG via retro-orbital injection 12 h prior to surgery. ICG fluorescence measurements and clinical evaluations were performed 4 weeks after resection to detect LR. Results Eleven of 25 animals from each group developed gross tumors. Four weeks after resection, group 1 had 0/11 tumor recurrences, while group 2 had recurrences in 9/11 (81.8%) experimental mice ( p < 0.0002) (Fig. 2 ). There was a 100% NPV in group 1, and no tumor recurrence with fluorescence-free margins after the primary surgery. Group 2 had a 100% positive predictive value for the development of an LR if any fluorescent signal was present at the surgical margin after resection. Conclusion Intraoperative ICG guidance led to reliably negative surgical margins and a diminished LR rate. Given the benign safety profile of ICG and its prior clinical success, these results could be immediately translatable to the clinical realm.

Aldehyde dehydrogenase (ALDH) is a cancer stem cell marker. Retinoic acid has antitumor properties, including the induction of apoptosis and inhibition of proliferation. Retinal, the precursor of retinoic acid, can be oxidized to retinoic acid by dehydrogenases, including ALDH. We hypothesized that retinal could potentially be transformed to retinoic acid with higher efficiency by cancer stem cells, due to the higher ALDH activity. We previously observed that ALDH activity is greater in highly metastatic K7M2 osteosarcoma (OS) cells than in nonmetastatic K12 OS cells. We also demonstrated that ALDH activity correlates with clinical metastases in bone sarcoma patients, suggesting that ALDH may be a therapeutic target specific to cells with high metastatic potential. Our current results demonstrated that retinal preferentially affected the phenotypes of ALDH-high K7M2 cells in contrast to ALDH-low K12 cells, which could be mediated by the more efficient transformation of retinal to retinoic acid by ALDH in K7M2 cells. Retinal treatment of highly metastatic K7M2 cells decreased their proliferation, invasion capacity, and resistance to oxidative stress. Retinal altered the expression of metastasis-related genes. These observations indicate that retinal may be used to specifically target metastatic cancer stem cells in OS.

Cases: Long-bone fractures in patients with Klippel-Trénaunay syndrome (KTS), a rare disorder of the venous, lymphatic, and capillary system, are difficult to treat with many complications. Two patients diagnosed with KTS presented with closed femoral shaft fractures after low-energy falls. Conservative treatment, open reduction internal fixation, and intramedullary nailing resulted in painful nonunions. Ultimately, both patients achieved pain relief and the ability to ambulate after en bloc resection and reconstruction. Conclusions: These cases demonstrate the challenges in achieving bony union when treating long-bone fractures in KTS. The feasibility of undergoing extensive resection and reconstruction to regain function is best approached with a multidisciplinary team.

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