Thrombosis and inflammation are intricately linked in several major clinical disorders, including disseminated intravascular coagulation and acute ischemic events. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) is upregulated by activated platelets in multiple inflammatory diseases; however, the contribution of platelet-derived HMGB1 in thrombosis remains unexplored. Here, we generated transgenic mice with platelet-specific ablation of HMGB1 and determined that platelet-derived HMGB1 is a critical mediator of thrombosis. Mice lacking HMGB1 in platelets exhibited increased bleeding times as well as reduced thrombus formation, platelet aggregation, inflammation, and organ damage during experimental trauma/hemorrhagic shock. Platelets were the major source of HMGB1 within thrombi. In trauma patients, HMGB1 expression on the surface of circulating platelets was markedly upregulated. Moreover, evaluation of isolated platelets revealed that HMGB1 is critical for regulating platelet activation, granule secretion, adhesion, and spreading. These effects were mediated via TLR4- and MyD88-dependent recruitment of platelet guanylyl cyclase (GC) toward the plasma membrane, followed by MyD88/GC complex formation and activation of the cGMP-dependent protein kinase I (cGKI). Thus, we establish platelet-derived HMGB1 as an important mediator of thrombosis and identify a HMGB1-driven link between MyD88 and GC/cGKI in platelets. Additionally, these findings suggest a potential therapeutic target for patients sustaining trauma and other inflammatory disorders associated with abnormal coagulation.

Osteosarcoma (OS) is the most common primary malignancy of bone and patients with metastatic disease or recurrences continue to have very poor outcomes. Unfortunately, little prognostic improvement has been generated from the last 20 years of research and a new perspective is warranted. OS is extremely heterogeneous in both its origins and manifestations. Although multiple associations have been made between the development of osteosarcoma and race, gender, age, various genomic alterations, and exposure situations among others, the etiology remains unclear and controversial. Noninvasive diagnostic methods include serum markers like alkaline phosphatase and a growing variety of imaging techniques including X-ray, computed tomography, magnetic resonance imaging, and positron emission as well as combinations thereof. Still, biopsy and microscopic examination are required to confirm the diagnosis and carry additional prognostic implications such as subtype classification and histological response to neoadjuvant chemotherapy. The current standard of care combines surgical and chemotherapeutic techniques, with a multitude of experimental biologics and small molecules currently in development and some in clinical trial phases. In this review, in addition to summarizing the current understanding of OS etiology, diagnostic methods, and the current standard of care, our group describes various experimental therapeutics and provides evidence to encourage a potential paradigm shift toward the introduction of immunomodulation, which may offer a more comprehensive approach to battling cancer pleomorphism.

Osteosarcoma is rare and little improvement in survival rates has occurred in the last 25 years despite modern chemotherapeutic treatment. Bioluminescent cell lines for the modeling of osteosarcoma have shown success in tracking metastases in vivo, but commonly use adenoviral vectors to transfect the native cell line with bioluminescent reporters. The purpose of this study was to develop an orthotopic model for metastatic osteosarcoma capable of in vivo monitoring of metastatic and primary tumor burden in an immunocompetent mouse and compare that model to its wild type pathogenesis. K7M2 cells were transfected using a plasmid vector and were stable after 12 weeks. Thirty-four female BALB/c mice aged 4-5 weeks underwent orthotopic implantation of either wild type (n = 12) or transfected (n = 22) K7M2 cells in the proximal tibia. Mice were monitored for tumor growth and weekly In Vivo Imaging System (IVIS) imaging was performed to monitor for pulmonary metastasis. Although tumors developed sooner in the wild type group, no significant differences were seen compared to Transfected Group 1 in rate of inoculation, growth rates after first detection, metastatic rate, and time between inoculation and death. This study establishes a new murine model for metastatic osteosarcoma using the K7M2-wt cell line transfected with a non-viral plasmid luciferase vector. The benefits of this preclinical model include an intact immune system and orthotopically driven metastatic disease; this model appears comparable to its wild type counterpart. In the future, the model may be used to examine promising immunomodulatory therapies using bioluminescence in vivo. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Indications for immunotherapies are still unclear, and there is a great need for real-time patient immune status monitoring. In this study, we confirmed that the local and systemic immune profiles of an orthotopic osteosarcoma model with or without luciferase transfection were statistically equivalent. Next, we used flow cytometry to describe systemic immune cell populations influenced by osteosarcoma disease progression. When compared to vehicle-inoculated sham mice, it was found that tumor-bearing mice had significant immunophenotype disturbances at approximately 11 weeks after inoculation (at which time 90% of primary tumor-bearing mice have fulminant pulmonary metastases). Percent populations of natural killer cells and T regulatory cells were increased in the spleens of tumor-bearing mice (p < 0.0083) compared to shams. Additionally, T lymphocytes from spleens of tumor-bearing mice showed increased Tim-3/PD-1 exhaustion status (p < 0.0083). There were also increases in the percent populations of myeloid cells and overall M1/M2 macrophage marker expression on tumor-bearing mice spleens versus controls (p < 0.00714). Finally, treatment with 20 μg α-PD-L1 decreased T-cell exhaustion back to sham status, with a corresponding increase in CTLA-4 expression on cytotoxic T cells in the majority of mice tested. Checkpoint inhibition also increased splenic monocyte maturation and returned macrophage M1/M2 marker expression back to sham status. These data suggest that cancer induces systemic immune dysregulation and that these changes may be elucidated and utilized for treatment purposes by sampling the systemic immune environment via the spleen. In addition, treatment with the checkpoint inhibitor α-PD-L1 may neutralize but not overcome the systemic immunological changes induced by a progressing malignancy.

We report the successful encapsulation and elution of recombinant murine IL-12 (rmIL-12) from poly(lactide-co-glycolic) acid (PLGA) nanospheres (IL-12-NS) synthesized using the double emulsion/solvent evaporation (DESE) technique with microsphere depletion through ultracentrifugation. Images obtained with scanning electron microscopy (SEM) showcased a characteristic spherical shape with a mean particle diameter of 138.1 ± 10.8   nm and zeta potential of − 15.1 ± 1.249   mV . These values suggest minimal flocculation when in solution, which was reflected in an in vivo biodistribution study that reported no observed morbidity/mortality. Encapsulation efficiency (EE) was determined to be 0.101 ± 0.009 % with average particle concentration obtained per batch of 1.66 × 10 9 ± 4.45 × 10 8 particles/mL. Disparate zeta (ζ) potentials obtained from both protein-loaded and protein-unloaded batches suggested surface adsorption of protein, and confocal microscopy of BSA-FITC-loaded nanospheres confirmed the presence of protein within the polymeric shell. Furthermore, elution of rmIL-12 from IL-12-NS at a concentration of 500 million particles/mL was characterized using enzyme-linked immunosorbent assay (ELISA). When IL-12-NS was administered in vivo to female BALB/c mice through retroorbital injection, IL-12-NS produced a favorable systemic cytokine profile for tumoricidal activity within the peripheral blood. Whereas IFN-γ nadir occurred at 72 hours, levels recovered quickly and displayed positive correlations postburst out to 25 days postinjection. IL-12-NS administration induced proinflammatory changes while prompting minimal counterregulatory increases in anti-inflammatory IL-10 and IL-4 cytokine levels. Further, while IL-6 levels increased to 30 folds of the baseline during the burst phase, they normalized by 72 hours and trended negatively throughout the sill phase. Similar trends were observed with IL-1β and CXCL-1, suggesting a decreased likelihood of progression to a systemic inflammatory response syndrome-like state. As IL-12-NS delivers logarithmically lower amounts of IL-12 than previously administered during human clinical trials, our data reflect the importance of IL-12-NS which safely create a systemic immunostimulatory environment.