Hes4: A potential prognostic biomarker for newly diagnosed patients with high-grade osteosarcoma.

BACKGROUND: Prognostic biomarkers for osteosarcoma (OS) at the time of diagnosis are lacking. Necrotic response of OS to preoperative chemotherapy correlates with survival and is determined 3-4 months after diagnosis. The purpose of this study is to identify biomarkers that will stratify patients into good or poor responders to chemotherapy at diagnosis and determine the role of potential biomarkers in OS pathogenesis. PROCEDURE: Because OS may be caused by disruptions of osteogenic differentiation, and the Notch pathway is one regulator of bone development, we examined the link between Notch effectors, OS differentiation, and OS outcome. We probed the R2: Genomics Analysis and Visualization Platform for RNA expression levels of Notch targets in mixed high-grade OS pretreatment biopsies. We used human OS cell lines in vitro and in mice to determine the role of the Notch target hairy/enhancer of split 4 (Hes4) in OS. RESULTS: We found that in OS patients, high expression of Hes4 is correlated with decreased metastasis-free and overall survival. Human OS cells that overexpress Hes4 are more immature and have an increased invasive capacity in vitro. This was not universal to all Notch effectors, as Hes1 overexpression induced opposing effects. When injected into NSG mice, Hes4-overexpressing OS cells produced significantly larger, more lytic tumors and significantly more metastases than did control cells. CONCLUSIONS: Hes4 overexpression promotes a more aggressive tumor phenotype by preventing osteoblastic differentiation of OS cells. Hes4 expression may allow for the stratification of patients into good or poor responders to chemotherapy at diagnosis.

Intracrine VEGF Signaling Mediates the Activity of Prosurvival Pathways in Human Colorectal Cancer Cells.

The effects of vascular endothelial growth factor-A (VEGF-A/VEGF) and its receptors on endothelial cells function have been studied extensively, but their effects on tumor cells are less well defined. Studies of human colorectal cancer cells where the VEGF gene has been deleted suggest an intracellular role of VEGF as a cell survival factor. In this study, we investigated the role of intracrine VEGF signaling in colorectal cancer cell survival. In human colorectal cancer cells, RNAi-mediated depletion of VEGF decreased cell survival and enhanced sensitivity to chemotherapy. Unbiased reverse phase protein array studies and subsequent validation experiments indicated that impaired cell survival was a consequence of disrupted AKT and ERK1/2 (MAPK3/1) signaling, as evidenced by reduced phosphorylation. Inhibition of paracrine or autocrine VEGF signaling had no effect on phospho-AKT or phospho-ERK1/2 levels, indicating that VEGF mediates cell survival via an intracellular mechanism. Notably, RNAi-mediated depletion of VEGF receptor VEGFR1/FLT1 replicated the effects of VEGF depletion on phospho-AKT and phospho-ERK1/2 levels. Together, these studies show how VEGF functions as an intracrine survival factor in colorectal cancer cells, demonstrating its distinct role in colorectal cancer cell survival. Cancer Res; 76(10); 3014-24. ©2016 AACR.

Understanding the role of Notch in osteosarcoma.

The Notch pathway has been described as an oncogene in osteosarcoma, but the myriad functions of all the members of this complex signaling pathway, both in malignant cells and nonmalignant components of tumors, make it more difficult to define Notch as simply an oncogene or a tumor suppressor. The cell-autonomous behaviors caused by Notch pathway manipulation may vary between cell lines but can include changes in proliferation, migration, invasiveness, oxidative stress resistance, and expression of markers associated with stemness or tumor-initiating cells. Beyond these roles, Notch signaling also plays a vital role in regulating tumor angiogenesis and vasculogenesis, which are vital aspects of osteosarcoma growth and behavior in vivo. Further, osteosarcoma cells themselves express relatively low levels of Notch ligand, making it likely that nonmalignant cells, especially endothelial cells and pericytes, are the major source of Notch activation in osteosarcoma tumors in vivo and in patients. As a result, Notch pathway expression is not expected to be uniform across a tumor but likely to be highest in those areas immediately adjacent to blood vessels. Therapeutic targeting of the Notch pathway is likewise expected to be complicated. Most pharmacologic approaches thus far have focused on inhibition of gamma secretase, a protease of the presenilin complex. This enzyme, however, has numerous other target proteins that would be expected to affect osteosarcoma behavior, including CD44, the WNT/ß-catenin pathway, and Her-4. In addition, Notch plays a vital role in tissue and organ homeostasis in numerous systems, and toxicities, especially GI intolerance, have limited the effectiveness of gamma secretase inhibitors. New approaches are in development, and the downstream targets of Notch pathway signaling also may turn out to be good targets for therapy. In summary, a full understanding of the complex functions of Notch in osteosarcoma is only now unfolding, and this deeper knowledge will help position the field to better utilize novel therapies as they are developed.

Understanding the Biology of Bone Sarcoma from Early Initiating Events through Late Events in Metastasis and Disease Progression.

The two most common primary bone malignancies, osteosarcoma (OS), and Ewing sarcoma (ES), are both aggressive, highly metastatic cancers that most often strike teens, though both can be found in younger children and adults. Despite distinct origins and pathogenesis, both diseases share several mechanisms of progression and metastasis, including neovascularization, invasion, anoikis resistance, chemoresistance, and evasion of the immune response. Some of these processes are well-studies in more common carcinoma models, and the observation from adult diseases may be readily applied to pediatric bone sarcomas. Neovascularization, which includes angiogenesis and vasculogenesis, is a clear example of a process that is likely to be similar between carcinomas and sarcomas, since the responding cells are the same in each case. Chemoresistance mechanisms also may be similar between other cancers and the bone sarcomas. Since OS and ES are mesenchymal in origin, the process of epithelial-to-mesenchymal transition is largely absent in bone sarcomas, necessitating different approaches to study progression and metastasis in these diseases. One process that is less well-studied in bone sarcomas is dormancy, which allows micrometastatic disease to remain viable but not growing in distant sites - typically the lungs - for months or years before renewing growth to become overt metastatic disease. By understanding the basic biology of these processes, novel therapeutic strategies may be developed that could improve survival in children with OS or ES.

Buckysomes: New Nanocarriers for Anticancer Drugs.

Buckysomes, liposome-like vesicles comprised of dendritic C60 subunits that self-assemble into unilamellar vesicles, are unique nanovectors that have utility in drug delivery. We have prepared paclitaxel-embedded buckysomes (PEBs) and examined biodistriubition profiles with commercially available formulations of the drug. As compared to Abraxane, an albumin-bound formulation of paclitaxel, PEBs showed higher tissue accumulation in the liver and the kidney at 45 and 60 minutes and in the lungs at 30 minutes, making them suitable drug-delivery carriers for short-term therapy to the mentioned organs. These buckysomes can be further functionalized to specifically deliver paclitaxel to the tumor site.

Longitudinal evaluation of mouse hind limb bone loss after spinal cord injury using novel, in vivo, methodology.

Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse, aberrant neuronal signaling and hormonal changes. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies. Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis. An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (naïve) mice (13% decrease, p < 0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p < 0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current "gold standard" for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI (9). Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.

Aged plasma transfusion increases mortality in a rat model of uncontrolled hemorrhage.

INTRODUCTION: Recent data have associated improved survival after hemorrhagic shock with the early use of plasma-based resuscitation. Our laboratory has shown that FFP5 has decreased hemostatic potential compared with freshly thawed plasma (FFP0). We hypothesized that FFP5 would increase bleeding and mortality compared with FFP0 in a rodent bioassay model of uncontrolled liver hemorrhage. METHODS: Hemostatic potential of plasma was assessed with the Calibrated Automated Thrombogram (CAT) assay. Rats underwent isovolemic hemodilution by 15% of blood volume with the two human plasma groups (FFP0 and FFP5) and two controls (sham and lactated Ringers). A liver injury was created by excising a portion of liver resulting in uncontrolled hemorrhage. Rats that lived for 30 minutes after liver injury were resuscitated to their baseline blood pressure and followed for 6 hours. Hemostasis was assessed by thromboelastography. RESULTS: Hemostatic potential of FFP5 decreased significantly in all areas measured in the CAT assay as compared with FFP0 (p < 0.01). In the FFP5 group, overall survival was 54%, compared with 100% in the FFP0 and sham group (p = 0.03). For animals that survived 30 minutes and were resuscitated, there was no difference in bleeding and/or coagulopathy between groups. Irrespective of treatment, animals that died after resuscitation demonstrated increased intraperitoneal fluid volume (14.85 mL ± 1.9 mL vs. 7.02 mL ± 0.3 mL, p < 0.001). CONCLUSION: In this model of mild preinjury hemodilution with plasma, rats that received FFP5 had decreased survival after uncontrolled hemorrhage from hepatic injury. There were no differences in coagulation function or intraperitoneal fluid volume between the two plasma groups.