Fluorescence‐guided surgery improves outcome in an orthotopic osteosarcoma nude‐mouse model

In order to develop a model for fluorescence‐guided surgery (FGS), 143B human osteosarcoma cells expressing red fluorescent protein (RFP) were injected into the intramedullary cavity of the tibia in nude mice. The fluorescent areas of residual tumors after bright‐light surgery (BLS) and FGS were 10.2 ± 2.4 mm² and 0.1 ± 0.1 mm², respectively (p < 0.001). The BLS‐treated mice and BLS + cisplatinum (CDDP)‐treated mice had significant recurrence. In contrast, the FGS mice and FGS + CDDP mice had very little recurring tumor growth. Disease‐free survival (DFS) in the BLS‐, BLS + CDDP‐, FGS‐, and FGS + CDDP‐treated mice was 12.5%, 37.5%, 75.0%, and 87.5%, respectively. The FGS‐treated mice had a significantly higher DFS rate than the BLS‐treated mice (p = 0.021). The FGS + CDDP‐treated mice had significantly higher DFS rate than the BLS + CDDP‐treated mice (p = 0.043). Although chemotherapy significantly reduced multiple metastases (p = 0.033), there was no significant correlation between FGS and lung metastasis. FGS significantly reduced the recurrence of the primary tumor but did not reduce lung metastasis. The combination of FGS and adjuvant CDDP reduced tumor recurrence and prevented multiple metastases. FGS and adjuvant chemotherapy should be performed as early as possible in the disease to prevent both recurrence and metastatic development. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1596–1601, 2014.     

Fluorescence‐guided surgery of human prostate cancer experimental bone metastasis in nude mice using anti‐CEA DyLight 650 for tumor illumination

The present report demonstrates efficacy of fluorescence‐guided surgery (FGS) to resect and prevent recurrence of experimental skeletal metastasis in a nude‐mouse model of human prostate cancer. Green fluorescent protein (GFP)‐expressing PC‐3 human prostate cancer cells were injected into the intramedullary cavity of the tibia in 25 nude mice. One week after implantation, monoclonal antibodies, specific for carcinoembryonic antigen (CEA), labeled with DyLight 650, were injected into the tail vein of 13 mice. Thirteen mice underwent FGS and 12 mice underwent bright‐light surgery (BLS). Weekly GFP fluorescence imaging of the mice was performed to observe tumor recurrence. The extent of residual tumor after BLS was 13‐fold greater than after FGS (p < 0.001). Time‐course imaging visualized rapid growth of the residual tumor in the BLS group, whereas the FGS group showed only slight tumor growth and significantly improved disease‐free survival of the treated mice. Our study demonstrated that FGS significantly reduced residual tumor as well as the recurrence of experimental prostate‐cancer bone metastasis. The present results suggest that FGS will be effective for resection of skeletal metastases in selected patients with prostate cancer. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:559–565, 2016.     

Fluorescence-guided surgery of prostate cancer bone metastasis

Background

The aim of this study is to investigate the effectiveness of fluorescence-guided surgery (FGS) of prostate cancer experimental skeletal metastasis.

Materials and methods

Green fluorescent protein-expressing PC-3 human prostate cancer cells (PC-3-green fluorescent protein) were injected into the intramedullary cavity of the tibia in 32 nude mice. After 2 wk, 16 of the mice underwent FGS; the other 16 mice underwent bright-light surgery (BLS). Half of BLS and FGS mice (8 mice in each group) received zoledronic acid (ZOL). Weekly fluorescence imaging of the mice was performed. Six weeks after surgery, metastases to lung and inguinal lymph node were evaluated by fluorescence imaging.

Results

The percentage of residual tumor after BLS and FGS was 9.9 ± 2.2% and 0.9 ± 0.3%, respectively (P < 0.001). FGS reduced recurrent cancer growth compared with BLS (P < 0.005). Although FGS alone had no significant effect on inguinal lymph node metastases, lung metastasis or disease-free survival (DFS), ZOL in combination with FGS significantly increased DFS (P = 0.01) in comparison with the combination of BLS and ZOL. ZOL reduced lymph node metastases (P = 0.033) but not lung metastasis.

Conclusions

FGS significantly reduced recurrence of experimental prostate cancer bone metastasis compared with BLS. The combination of FGS and ZOL increased DFS over BLS and ZOL. ZOL inhibited lymph node metastasis but not lung metastasis.     

Fluorescence-guided surgery of human colon cancer increases complete resection resulting in cures in an orthotopic nude mouse model

BackgroundWe inquired if fluorescence-guided surgery (FGS) could improve surgical outcomes in fluorescent orthotopic nude mouse models of human colon cancer.MethodsWe established fluorescent orthotopic mouse models of human colon cancer expressing a fluorescent protein. Tumors were resected under bright light surgery (BLS) or FGS. Pre- and post-operative images with the OV-100 Small Animal Imaging System (Olympus Corp, Tokyo Japan) were obtained to assess the extent of surgical resection.ResultsAll mice with primary tumor that had undergone FGS had complete resection compared with 58% of mice in the BLS group (P = 0.001). FGS resulted in decreased recurrence compared with BLS (33% versus 62%, P = 0.049) and lengthened disease-free median survival from 9 to >36 wk. The median overall survival increased from 16 wk in the BLS group to 31 weeks in the FGS group. FGS resulted in a cure in 67% of mice (alive without evidence of tumor at >6 mo after surgery) compared with only 37% of mice that underwent BLS (P = 0.049).ConclusionsSurgical outcomes in orthotopic nude mouse models of human colon cancer were significantly improved with FGS. The present study can be translated to the clinic by various effective methods of fluorescently labeling tumors.     

Fluorescence-guided Surgery with a Fluorophore-conjugated Antibody to Carcinoembryonic Antigen (CEA), that Highlights the Tumor,Improves Surgical Resection and Increases Survival in Orthotopic Mouse Models of Human Pancreatic Cancer

Background

We have developed a method of distinguishing normal tissue from pancreatic cancer in vivo using fluorophore-conjugated antibody to carcinoembryonic antigen (CEA). The objective of this study was to evaluate whether fluorescence-guided surgery (FGS) with a fluorophore-conjugated antibody to CEA, to highlight the tumor, can improve surgical resection and increase disease-free survival (DFS) and overall survival (OS) in orthotopic mouse models of human pancreatic cancer.

Methods

We established nude-mouse models of human pancreatic cancer with surgical orthotopic implantation of the human BxPC-3 pancreatic cancer. Orthotopic tumors were allowed to develop for 2 weeks. Mice then underwent bright-light surgery (BLS) or FGS 24 h after intravenous injection of anti-CEA-Alexa Fluor 488. Completeness of resection was assessed from postoperative imaging. Mice were followed postoperatively until premorbid to determine DFS and OS.

Results

Complete resection was achieved in 92 % of mice in the FGS group compared to 45.5 % in the BLS group (p = 0.001). FGS resulted in a smaller postoperative tumor burden (p = 0.01). Cure rates with FGS compared to BLS improved from 4.5 to 40 %, respectively (p = 0.01), and 1-year postoperative survival rates increased from 0 % with BLS to 28 % with FGS (p = 0.01). Median DFS increased from 5 weeks with BLS to 11 weeks with FGS (p = 0.0003). Median OS increased from 13.5 weeks with BLS to 22 weeks with FGS (p = 0.001).

Conclusions

FGS resulted in greater cure rates and longer DFS and OS using a fluorophore-conjugated anti-CEA antibody. FGS has potential to improve the surgical treatment of pancreatic cancer.     

Proteomic identification of 14‐3‐3ϵ as a linker protein between pERK1/2 inhibition and BIM upregulation in human osteosarcoma cells

Despite advancements in multimodality chemotherapy, conventional cytotoxic treatments still remain ineffective for a subset of patients with aggressive metastatic or multifocal osteosarcoma. It has been shown that pERK1/2 inhibition enhances chemosensitivity to doxorubicin and promotes osteosarcoma cell death in vivo and in vitro. One of the pro‐apoptotic mechanisms is upregulation of Bim by pERK1/2 inhibitors. To this end, we examined proteomic changes of 143B human osteosarcoma cells with and without treatment of PD98059, pERK1/2 inhibitor. Specifically, we identified 14‐3‐3? protein as a potential mediator of Bim expression in response to inhibition of pERK1/2. We hypothesized that 14‐3‐3? mediates upregulation of Bim expression after pERK1/2 inhibition. We examined the expression of Bim after silencing 14‐3‐3? using siRNA. The 14‐3‐3? gene silencing resulted in downregulation of Bim expression after PD98059 treatment. These data indicate that 14‐3‐3? is required for Bim expression and that it has an anti‐cancer effect under pERK1/2 inhibition in 143B cells. By playing an essential role upstream of Bim, 14‐3‐3? may potentially be a coadjuvant factor synergizing the effect of pERK1/2 inhibitors in addition to conventional cytotoxic agents for more effective osteosarcoma treatments. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:848–854, 2014.

     

Bone morphogenetic protein‐2 promotes osteosarcoma growth by promoting epithelial‐mesenchymal transition (EMT) through the Wnt/β‐catenin signaling pathway

The correlation between BMP‐2 and osteosarcoma growth has gained increased interest in the recent years, however, there is still no consensus. In this study, we tested the effects of BMP‐2 on osteosarcoma cells through both in vitro and in vivo experiments. The effect of BMP‐2 on the proliferation, migration and invasion of osteosarcoma cells was tested in vitro. Subcutaneous and intratibial tumor models were used for the in vivo experiments in nude mice. The effects of BMP‐2 on EMT of osteosarcoma cells and the Wnt/β‐catenin signaling pathway were also tested using a variety of biochemical methods. In vitro tests did not show a significant effect of BMP‐2 on tumor cell proliferation. However, BMP‐2 increased the mobility of tumor cells and the invasion assay demonstrated that BMP‐2 promoted invasion of osteosarcoma cells in vitro. In vivo animal study showed that BMP‐2 dramatically enhanced tumor growth. We also found that BMP‐2 induced EMT of osteosarcoma cells. The expression levels of Axin2 and Dkk‐1 were both down regulated by BMP‐2 treatment, while β‐catenin, c‐myc and Cyclin‐D1 were all upregulated. The expression of Wnt3α and p‐GSK‐3β were also significantly upregulated indicating that the Wnt/β‐catenin signaling pathway was activated during the EMT of osteosarcoma driven by BMP‐2. From this study, we can conclude that BMP‐2 significantly promotes growth of osteosarcoma cells (143B, MG63), and enhances mobility and invasiveness of tumor cells as demonstrated in vitro. The underlying mechanism might be that BMP‐2 promotes EMT of osteosarcoma through the Wnt/β‐catenin signaling pathway. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1638–1648, 2019.     

Quantifying intra‐osseous growth of osteosarcoma in a murine model with radiographic analysis

The orthotopic murine osteosarcoma model is an excellent representation of the human condition as mice develop rapid growth of ‘primary’ tumor with subsequent lung metastasis. Currently, monitoring tumor growth relies on measuring pulmonary metastases occurring four weeks post injection. Studies show that amputation of the tumor‐bearing limb is required before pulmonary metastases are detectable due to rapid growth causing morbidity. Thus, a method measuring ‘primary’ tumor growth independent of metastasis is required. We hypothesized that serial radiography would allow for longitudinal quantification of ‘primary’ osteosarcoma growth and explored this idea by utilizing the tibial orthotopic model. Tumor growth was monitored weekly by radiography and calipers, and results were compared with µCT and histology. We found that radiographs demonstrate extra and intra‐osseous tumor growth by displaying lytic and blastic lesions and the surrounding radio‐opaque area enlarged significantly (p < 0.0001) allowing for quantification. Additionally, radiographs proved more precise than indirect caliper measurements (intra‐observer error ±6.64%: inter‐observer error ±15.84%). Therefore, we determined that radiography provides accurate, longitudinal quantification of ‘primary’ osteosarcoma tumor that can be performed serially in the same mouse, does not require introduction of bioluminescence to the host or cell, and is more precise than the current caliper method. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1957–1962, 2011     

Evaluation of P‐glycoprotein (Pgp) expression in human osteosarcoma by high‐throughput tissue microarray

Survival of osteosarcoma patients is currently limited by the development of metastases and multidrug resistance (MDR). A well‐established cause of MDR involves overexpression of P‐glycoprotein (Pgp) in tumor cells. However, some discrepancies still exist as to the clinical significance of Pgp in osteosarcoma. We sought to elucidate further whether the Pgp expression correlated with clinical behavior in a series of patients with osteosarcoma via high‐throughput tissue microarray (TMA) analysis. Immunohistochemical analysis of Pgp expression in a TMA of 114 specimens with a retrospective review of 70 osteosarcoma patients admitted to the Massachusetts General Hospital (MGH) was performed. High Pgp expression was correlated with metastasis development and poor response to pre‐operative chemotherapy in osteosarcoma patients. Eighteen of the fifty‐seven patients initially admitted with primary osteosarcoma showed high Pgp expression. Among these 18 patients with high Pgp expression, 13 of 18 (72%) patients eventually developed metastases. There was no significant clinical relevance between Pgp expression and osteosarcoma survival. These results support that high expression of Pgp is important, but cannot be assigned as, an individual predictor in the development of human osteosarcoma. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1606–1612, 2016.     

Bisphosphonates Inhibit Osteosarcoma‐Mediated Osteolysis Via Attenuation of Tumor Expression of MCP‐1 and RANKL

Osteosarcoma is the most common primary malignant tumor of bone and accounts for around 50% of all primary skeletal malignancies. In addition to novel chemotherapies, there is a need for adjuvant therapies designed to inhibit osteosarcoma proliferation and tumor‐induced osteolysis to attenuate tumor expansion and metastasis. As such, studies on the efficacy of bisphosphonates on human osteosarcoma are planned after feasibility studies determined that the bisphosphonate zoledronic acid (ZOL) can be safely combined with conventional chemotherapy. However, the molecular mechanisms responsible for, and means of inhibiting, osteosarcoma‐induced osteolysis are largely unknown. We establish that osteosarcoma growth directly correlates with tumor‐induced osteolysis and activation of osteoclasts in vivo. In vitro, tumor cells were determined to expresses surface, but not soluble, receptor activator of NF‐κB ligand (RANKL) and stimulated osteoclastogenesis in a manner directly proportional to their malignant potential. In addition, an aggressive osteosarcoma cell line was shown to secrete monocyte chemoattractant protein‐1 (MCP‐1), resulting in robust monocyte migration. Because MCP‐1 is a key cytokine for monocyte recruitment and surface‐bound RANKL strongly supports local osteoclastogenesis, we suggest that high levels of these signaling molecules are associated with the aggressive potential of osteosarcoma. Consistent with these findings, abundant expression of RANKL/MCP‐1 was observed in tumor in vivo, and MCP‐1 plasma levels strongly correlated with tumor progression and osteolysis. ZOL administration directly attenuates osteosarcoma production of RANKL/MCP‐1, reducing tumor‐induced bone destruction. In vivo, these findings also correlated with significant reduction in osteosarcoma growth. ZOL attenuates tumor‐induced osteolysis, not only through direct inhibition of osteoclasts, but also through direct actions on tumor expression of osteoclast activators. These data provide insight regarding the effect of ZOL on osteosarcoma essential for designing the planned upcoming prospective randomized trials to determine the efficacy of bisphosphonates on osteosarcoma in humans. © 2014 American Society for Bone and Mineral Research.     

The orally bioavailable met inhibitor PF‐2341066 inhibits osteosarcoma growth and osteolysis/matrix production in a xenograft model

Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents. Ninety percent of patients who present with metastatic and 30% to 40% of patients with nonmetastatic disease experience relapse, creating an urgent need for novel therapeutic strategies. The Met receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), are important for mitosis, motility, and cell survival. Upregulation of Met/HGF signaling via receptor overexpression, amplification, or mutation drives the proliferation, invasiveness, and metastasis of a variety of cancer cells, including OS, prompting the development of Met/HGF inhibitors. OS cells depend on Met overexpression because introduction of dominant‐negative Met inhibits in vivo tumorigenicity. Despite the importance of Met/HGF signaling in the development and maintenance of OS, the potential efficacy of pharmacologic Met inhibition in OS has been addressed only in in vitro studies. PF‐2341066 is an orally bioavailable, selective ATP‐competitive Met inhibitor that showed promising results recently in a phase I clinical trial in non–small cell lung cancer (NSCLC) patients. We tested the ability of PF‐2341066 to inhibit malignant properties of osteosarcoma cells in vitro and orthotopic xenograft growth in vivo. In vitro, PF‐2341066 inhibited osteosarcoma behavior associated with primary tumor growth (eg, proliferation and survival) as well as metastasis (eg, invasion and clonogenicity). In nude mice treated with PF‐2341066 via oral gavage, the growth and associated osteolysis and extracortical bone matrix formation of osteosarcoma xenografts were inhibited by PF‐2341066. PF‐2341066 may represent an effective new systemic therapy for localized and potentially disseminated osteosarcoma. © 2011 American Society for Bone and Mineral Research.     

Outcome Following Local‐Regional Recurrence in Women with Early‐Stage Breast Cancer: Impact of Biologic Subtype

Local‐regional recurrence (LRR) after breast‐conserving therapy (BCT) can result in distant metastasis and decreased disease‐free survival (DFS). This study examines factors associated with DFS following LRR. The initial population included 2,233 consecutive women who underwent BCT from 1998 to 2007. Biologic subtype was approximated using a combination of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and tumor grade. Cumulative incidence of DFS after LRR was calculated. The association of clinical, pathologic, and treatment parameters with DFS was evaluated using a Cox regression model. At a median follow‐up of 105 months, 82 patients (3.7%) had a LRR. Of these, 66 (80%) were in‐breast and 16 (20%) involved the ipsilateral lymph nodes. Twenty patients subsequently developed distant metastases. Five‐year DFS after initial recurrence was 69.6% for the overall cohort. On univariate analysis, triple‐negative disease (ER/PR/HER2 negative, TNBC) was associated with reduced DFS (HR = 3.8; 95% CI: 1.8–8.1; p < 0.001). Other factors associated with reduced DFS were larger tumor size (HR = 1.3; 95% CI: 1.03–1.6; p = 0.02), shorter interval from initial diagnosis to LRR (HR = 0.98 per month; 95% CI: 0.97–0.99; p = 0.02), and no salvage surgery (HR = 0.2; 95% CI: 0.09–0.5; p = 0.001). On multivariate analysis, TNBC remained the most significant factor associated with reduced DFS (HR = 4.8; 95% CI: 2.25–10.4; p < 0.001). Compared to women with luminal A disease, those with TNBC had significantly worse DFS (37.5% versus 88.3% at 5 years; p < 0.001). Women with TNBC who developed LRR were at high risk of subsequent recurrence. Efforts should be targeted toward both preventing initial recurrence and decreasing subsequent metastasis.     

Role of integrin‐linked kinase in osteosarcoma progression

Although integrin‐linked kinase (ILK) has been suggested to play a role in the tumorigenesis of a number of human epithelial carcinomas, little is known of its role in musculoskeletal sarcoma. The authors studied ILK expression by immunohistochemistry using osteosarcoma prechemotherapy specimens from 56 patients, and investigated the prognostic implications of the findings obtained. It was found that ILK overexpression was significantly correlated with the presence of distant metastasis (p = 0.008) and that it was an independent prognostic factor for both poor overall survival and poor event‐free survival (p = 0.015 and 0.010, respectively). During a transfection experiment conducted by transfecting osteosarcoma cells with ILK siRNA, VEGF concentrations were measured using an ELISA kit, and then compared with those of untransfected controls to evaluate its angiogenic effects. In addition, apoptotic percentages were measured by Annexin‐V flow cytometry, and invasive properties were evaluated by measuring the numbers of non‐migrating cells in a Boyden chamber. It was found that ILK downregulation significantly decreased angiogenesis, increased apoptosis, and decreased invasiveness of osteosarcoma cells. These results show that ILK is a promising prognostic factor in osteosarcoma and a novel potential therapeutic target for the treatment of osteosarcoma. © 2013 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 31:1668–1675, 2013     

Metastatic tumor cells detection and anti‐metastatic potential with vesicular stomatitis virus in immunocompetent murine model of osteosarcoma

Sarcomas are associated with a high incidence of lung metastasis, which leads to a high‐risk of cancer death. This study was performed to explore the pre‐clinical theranostic potential of a novel fully functional recombinant vesicular stomatitis virus carrying imaging gene Katushka (rVSV‐K), as virotherapy and circulating tumor cells (CTCs) detection in the syngeneic mouse model of osteosarcoma with spontaneous pulmonary metastases. Recombinant VSV‐K was generated and evaluated in vitro on human and murine osteosarcoma cells. Spontaneous osteosarcoma metastases were established in immune‐competent mice by implanting subcutaneously syngeneic osteosarcoma LM8 cells. The vector was injected into the tumor‐bearing mice via jugular vein either once or repeatedly. To assess effectiveness, primary tumor growth and development of lung metastasis as well as survival were evaluated. We found that rVSV‐K efficiently replicated in and killed all osteosarcoma cell lines in time‐dependent manners. Both single or repeated systemic injections of the virus did not inhibit the growth of the primary tumor, but the repeated administration could effectively suppress the development of lung metastases and was likely responsible for the observed increase in survival. Furthermore, we demonstrated, for the first time, that CTCs in blood samples from syngeneic osteosarcoma‐bearing mice were successfully detected by utilizing rVSV‐K ex vivo. Our results show that repeated systemic injections of rVSV‐K are an effective anti‐metastatic agent against osteosarcoma in immune‐competent mice and this virus to be a useful tool for detection of osteosarcoma CTCs, suggesting that further development of future viral‐based theranostic approach in patients with osteosarcoma is warranted. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2562–2569, 2018.

     

Expression and Clinical Significance of High‐Mobility Group AT‐hook 2 (HMGA2) in Osteosarcoma

ObjectiveAlthough high‐mobility group AT‐hook 2 (HMGA2) has been shown to have crucial roles in the pathogenesis and metastasis of various malignancies, its expression and significance in osteosarcoma remain unknown. Here we evaluate the expression, clinical prognostic value, and overall function of HMGA2 in osteosarcoma.MethodsSixty‐nine osteosarcoma patient specimens within a tissue microarray (TMA) were analyzed by immunohistochemistry for HMGA2 expression. Demographics and clinicopathological information including age, gender, tumor location, metastasis, recurrence, chemotherapy response, follow‐up time, and disease status were also collected. After validation of expression, we determined whether there was a correlation between HMGA2 expression and patient clinicopathology. HMGA2 expression was also evaluated in osteosarcoma cell lines and patient tissues by Western blot, we analyzed the expression of HMGA2 in the human osteosarcoma cell lines MG63, 143B, U2OS, Saos‐2, MNNG/HOS, and KHOS. HMGA2‐specific siRNA and clonogenic assays were then used to determine the effect of HMGA2 inhibition on osteosarcoma cell proliferation, growth, and chemosensitivity.ResultsHMGA2 expression was elevated in the osteosarcoma patient specimens and human osteosarcoma cell lines. HMGA2 was differentially expressed in human osteosarcoma cell lines. Specifically, a relatively high expression of HMGA2 was present in KHOS, MNNG/HOS, 143B and a relatively low expression was in MG63, U2OS as well as Saos‐2. HMGA2 expression is correlated with metastasis and shorter overall survival. High HMGA2 expression is an independent predictor of poor osteosarcoma prognosis. There was no significant correlation between HMGA2 expression and the age, gender, or tumor site of the patient. HMGA2 expression is predominantly within the nucleus. The expression of HMGA2 also directly correlated to neoadjuvant chemoresistance. There was a significant reduction of HMGA2 expression in the siRNA transfection group. After the use of siRNA, the proliferation of osteosarcoma cells is decreased and the chemosensitivity of osteosarcoma cells is significantly increased.ConclusionOur study supports HMGA2 as a potential prognostic biomarker and therapeutic target in osteosarcoma.     

The Oncolytic Adenovirus Δ24‐RGD in Combination With Cisplatin Exerts a Potent Anti‐Osteosarcoma Activity

Osteosarcoma is the most common malignant bone tumor in children and adolescents. The presence of metastases and the lack of response to conventional treatment are the major adverse prognostic factors. Therefore, there is an urgent need for new treatment strategies that overcome both of these problems. Our purpose was to elucidate whether the use of the oncolytic adenovirus Δ24‐RGD alone or in combination with standard chemotherapy would be effective, in vitro and in vivo, against osteosarcoma. Our results showed that Δ24‐RGD exerted a potent antitumor effect against osteosarcoma cell lines that was increased by the addition of cisplatin. Δ24‐RGD osteosarcoma treatment resulted in autophagy in vitro that was further enhanced when combined with cisplatin. Of importance, administration of Δ24‐RGD and/or cisplatin, in novel orthotopic and two lung metastatic models in vivo resulted in a significant reduction of tumor burden meanwhile maintaining a safe toxicity profile. Together, our data underscore the potential of Δ24‐RGD to become a realistic therapeutic option for primary and metastatic pediatric osteosarcoma. Moreover, this study warrants a future clinical trial to evaluate the safety and efficacy of Δ24‐RGD for this devastating disease. © 2014 American Society for Bone and Mineral Research.     

PEDF‐derived synthetic peptides exhibit antitumor activity in an orthotopic model of human osteosarcoma

Pigment epithelium‐derived factor (PEDF) is one of the most potent inhibitors of angiogenesis, and has recently been demonstrated to have an important multifunctional role in tumor growth, invasion, and metastasis. However, relatively little is known of mechanisms through which PEDF exerts its antitumor activity. Therefore, with the aim of identifying potential functional epitopes specifically against osteosarcoma, we evaluated the bioactivity of four 25‐mer synthetic PEDF‐derived peptides (termed StVOrth‐1, ‐2 ‐3, and ‐4) against a human osteosarcoma cell line, SaOS‐2. We found that StVOrth‐2 (residues 78–102) predominantly inhibited tumor cell proliferation, while StVOrth‐3 (residues 90–114) markedly increased cellular adhesion to collagen type‐1, with StVOrth‐4 (residues 387–411) demonstrating most significant inhibition of Matrigel invasion. Furthermore, we show that StVOrth‐1 (residues 40–64), ‐2 and ‐3 induce osteoblastic differentiation, evidenced by increased mineralized nodule formation. Interestingly, although no peptide inhibited angiogenesis in the tube formation assay, StVOrth‐3 and ‐4 markedly suppressed VEGF expression. We further tested the activity of StVOrth‐2 and StVOrth‐3 in vivo, in an orthotopic model of osteosarcoma and found that both peptides significantly inhibited primary tumor growth and the development of pulmonary metastases. Together these results provide greater insight into the potential mechanisms through which PEDF exerts its antitumor function. Furthermore, this raises the possibility of developing short PEDF fragments as lead compounds for the treatment of osteosarcoma. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:1671–1680, 2007     

KIR‐Ligand Mismatches Are Associated With Reduced Long‐Term Graft Survival in HLA‐Compatible Kidney Transplantation

Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system with the ability to detect HLA class I disparities via killer‐cell immunoglobulin‐like receptors (KIR). To test whether such KIR‐ligand mismatches contribute to the rejection of human solid allografts, we did a retrospective cohort study of 397 HLA‐DR‐compatible kidney transplantations and determined the KIR and HLA genotypes of recipients and the HLA genotypes of donors. In transplantations compatible for HLA‐A, HLA‐B and HLA‐DR (n = 137), in which a role for T cells and HLA antibodies in rejection was minimized, KIR‐ligand mismatches were associated with an approximately 25% reduction in 10‐year death‐censored graft survival (p = 0.043). This effect was comparable to the effect of classical HLA‐A and HLA‐B incompatibility, and in HLA‐A,‐B‐incompatible transplantations (n = 260) no significant additional effect of KIR‐ligand mismatches was observed. Multivariate Cox regression analysis confirmed the effect of KIR‐ligand mismatching as an independent risk factor in HLA‐A,‐B,‐DR‐compatible transplantations (hazard ratio 2.29, range 1.03–5.10, p = 0.043). This finding constitutes the first indication that alloreactive NK cells may thwart the success of HLA‐compatible kidney transplantations, and suggests that suppression of NK‐cell activity can improve the survival of such kidney grafts.