Nanocapsule Delivery of IL-12.

Interleukin(IL)-12 is a protein that activates T cells and macrophages to kill tumor cells. However, despite this cytokine showing strong antitumor activity in preclinical settings, translation to patients has been slowed by toxic side effects, poor distribution to peripheral tissues, and improper dosing regimens. Osteosarcoma (OS) is an aggressive primary tumor of bone that has shown particular responsiveness to recombinant (r)IL-12 in preclinical models. Poly(lactic-co-glycolic) acid (PLGA) nanospheres, an FDA-approved drug delivery vector, may be a viable delivery vector for transporting biologically active IL-12 to tissues without disturbing normal homeostasis. In this chapter, we explore the potential for using IL-12-loaded nanospheres (IL-12-NS, <1 µm in diameter) to treat cancer, describe the synthesis process, and examine a typical protein release profile while providing insight and future directions of nanoscale tumor immunotherapeutics.

Applying Osteosarcoma Immunology to Understand Disease Progression and Assess Immunotherapeutic Response.

Osteosarcoma, the most common malignant bone tumor in children and adolescents, remains a complicated disease to treat; no new treatments have been developed in more than three decades. Due to the importance of the immune system in osteosarcoma disease progression, immunotherapeutic strategies have been explored to potentially improve long-term survival. However, most immunotherapeutics have not reached the level of success hoped would occur in this disease. Understanding the immune system in osteosarcoma will be key to optimizing treatments and improving patient outcomes. Therefore, immunophenotyping can be used as a very powerful tool to help better understand the complexity of the immune response seen in osteosarcoma and in the use of immunotherapy in this malignancy. This book chapter will provide an overview of the known immune responses seen in this disease and potential developments for the future of immunophenotyping. Indeed, it appears that being able to track the immune system throughout the disease and treatment of patients with osteosarcoma could allow for a personalized approach to immunotherapy.

Posterior Hip Precautions Do Not Impact Early Recovery in Total Hip Arthroplasty: A Multicenter, Randomized, Controlled Study.

BACKGROUND: Posterior hip precautions have been routinely prescribed to decrease dislocation rates. The purpose of this study was to determine whether the absence of hip precautions improved early recovery after total hip arthroplasty via the posterolateral approach. METHODS: Patients undergoing total hip arthroplasty via the posterolateral approach at 3 centers were enrolled. Patients meeting the selection criteria were randomized to standard hip precautions (SHP) or no hip precautions (NHP) for 6 weeks following surgery. HOOS Jr, Health State visual analog score, and rate of pain scores were recorded preoperatively and in subsequent postoperative visits; dislocation episodes were also noted. Standard statistical analysis was performed. RESULTS: From 2016 to 2017, 159 patients were randomized to SHP and 154 patients were randomized to NHP. Controlling for the center at which the surgery was performed, the only difference in outcome scores between the 2 groups was at 2 weeks; the NHP group had a lower HOOS Jr score when compared to the SHP group (P = .03). There was no difference in outcome scores at any other time points when compared to preoperative assessments. In the SHP group, there were 2 recorded dislocations (1.3%) and 1 in the NHP group (0.7%; P = .62). CONCLUSION: In this multicenter, randomized, controlled study, the absence of hip precautions in the postoperative period did not improve subjective outcomes which may be explained by the self-limiting behavior of NHP patients. Furthermore, with the numbers available for the study, there was no difference in the rate of dislocation between the 2 groups.

A novel co-culture model of murine K12 osteosarcoma cells and S. aureus on common orthopedic implant materials: 'the race to the surface' studied in vitro.

Infection is a major cause of orthopedic implant failure. There are few studies assessing both tissue cell and bacterial adherence on common orthopedic implant materials in a co-culture environment. An in vitro co-culture model was created using K12 osteosarcoma cells and Staphylococcus aureus in a medium incubated over metal disks for 48 h. The results showed that, in the presence of S. aureus, there were fewer osteosarcoma cells attached to the disks for all substrata tested. There were significantly more osteosarcoma cells adhering to the cobalt chrome than the stainless steel and titanium disks. Overall, in the presence of osteosarcoma cells, there were more bacteria adhering to the disks for all the substrata tested, with significantly more bacteria adhering to the stainless steel disks compared to cobalt chrome and titanium disks. Scanning electron microscopy verified that osteosarcoma cells and bacteria were adherent to the metal disks after incubation for 48 h. Furthermore, the observation that more bacteria were in the co-culture than in the control sample suggests that the osteosarcoma cells serve as a nutrient source for the bacteria. Future models assessing the interaction of osteogenic cells with bacteria on a substratum would be improved if the model accounted for the role of the immune system in secondary bone healing.

Nanosphere pharmacodynamics improves safety of immunostimulatory cytokine therapy.

Systemic administration of interleukin (IL)-12 induces potent anti-tumor immune responses in preclinical cancer models through the systemic activation of effector immune cells and release of proinflammatory cytokines. IL-12-loaded PLGA nanospheres (IL12ns) are hypothesized to improve therapeutic efficacy and thwart unwanted side effects observed in previous human clinical trials. Through the investigation of peripheral blood and local tissue immune responses in healthy BALB/c mice, the immune-protective pharmacodynamics of IL12ns were suggested. Nanospheres increased pro-inflammatory plasma cytokines/chemokines (IFN-γ, IL-6, TNF-α, and CXCL10) without inducing maladaptive transcriptomic signatures in circulating peripheral immune cells. Gene expression profiling revealed activation of pro-inflammatory signaling pathways in systemic tissues, the likely source of these effector cytokines. These data support that nanosphere pharmacodynamics, including shielding IL-12 from circulating immune cells, depositing peripherally in systemic immune tissues, and then slowly eluting bioactive cytokine, thereafter, are essential to safe immunostimulatory therapy.

Single-cell RNA-seq reveals intratumoral heterogeneity in osteosarcoma patients: A review.

While primary bone malignancies make up just 0.2% of all cancers, osteosarcoma (OS) is the third most common cancer in adolescents. Due to its highly complex and heterogeneous tumor microenvironment (TME), OS has proven difficult to treat. There has been little to no improvement in therapy for this disease over the last 40 years. Even the recent success of immunotherapies in other blood-borne and solid malignancies has not translated to OS. With frequent recurrence and lung metastases continuing to pose a challenge in the clinic, recent advancements in molecular profiling, such as single-cell RNA sequencing (scRNA-seq), have proven useful in identifying novel biomarkers of OS tumors while providing new insight into this TME that could potentially lead to new therapeutic options. This review combines the analyses of over 150,000 cells from 18 lesions ranging from primary, recurrent, and metastatic OS lesions, revealing distinct cellular populations and gene signatures that exist between them. Here, we detail these previous findings and ultimately convey the intratumoral heterogeneity that exists within OS tumor specimens.

Optimizing the synthesis of interleukin-12-loaded PLGA nanospheres (rmIL-12ns) via ultrasonication for treatment of metastatic osteosarcoma.

Clinical trials exploring bolus intravenous delivery of interleukin-12 (IL-12) for treatment of solid tumors ultimately failed due to lack of clinical response and severe dose-limiting toxicities. The present study was conducted to evaluate whether recombinant murine IL-12 (rmIL-12) could be successfully encapsulated within Poly (D, l-lactide-co-glycolide) (PLGA) nanospheres (rmIL-12ns) for safe and effective systemic delivery at pharmacologic scale. Optimal fabrication of rmIL-12ns occurs with dichloromethane as the organic solvent and emulsion formation via ultrasonication at 50% power (250 W sonicator) for 10 s (50W10s). We then determined whether utilization of synthesis modifiers including fetal bovine serum (FBS), magnesium hydroxide [Mg(OH)2 ], trehalose, or the surfactants polysorbate 80 and Span 60 alone or in combination could increase the encapsulation efficiency (EE) and/or modify the burst elution profile characteristic of the 50W10s rmIL-12ns formulation. The greatest EEs compared to the unmodified formulation were measured with modifications containing the surfactants polysorbate 80 and Span 60 (surfactant: 28.3 ± 6.10%, p = 0.29 and Surf/FBS: 85.4 ± 2.19%, p = 0.039). The Surf/FBS formulation was further modified for in vivo murine injection by substituting FBS with mouse serum albumin (MSA). The resulting Surf/MSA rmIL-12ns were then characterized before delivery at three doses (0.1, 1, and 10 mg rmIL-12ns) in our established murine model of metastatic osteosarcoma to assess efficacy. Preliminary results suggested no evidence of disease with delivery of the 0.1 mg dose in 75% of mice (3 of 4) versus a nontreated historical control (2 of 34).

A Novel Protocol for Nasal Decolonization Using Prolonged Application of an Alcohol-Based Nasal Antiseptic Reduces Surgical Site Infections in Total Joint Arthroplasty Patients: A Retrospective Cohort Study.

Background: Current nasal decolonization strategies utilize pre-operative agents without consideration for short-term re-colonization or de novo colonization. Many strategies utilize an antibiotic-based agent, raising concerns of limited gram-negative antimicrobial coverage and the emergence of resistant bacterial strains. This study evaluated the clinical utility of a non-antibiotic, alcohol-based nasal decolonization agent in decreasing surgical site infection (SSI) rates after total joint arthroplasty. Patients and Methods: We retrospectively compared an 18-month cohort of elective primary total joint arthroplasty patients treated peri-operatively with an alcohol-based sanitizer to historical controls. The alcohol-based agent was administered pre-operatively the day of surgery and for two weeks after surgery. Patients were followed for 90 days and assessed for signs or symptoms of SSI. Patient and caregiver compliance was recorded. There were 779 patients included in the experimental group and 647 included in the historical control group. Results: Patients receiving alcohol-based nasal decolonization had a lower rate of SSI compared with controls not receiving nasal decolonization (0.64% [5/779] vs. 1.55% [10/647]; p = 0.048; odds ratio, 2.43). Utilization of an alcohol-based nasal sanitizer in the pre-operative and prolonged post-operative setting decreased infection rates by 41.3% in our elective total joint arthroplasty setting. Conclusions: When used pre- and post-operatively, alcohol-based nasal decolonization of bacteria in patients undergoing total joint arthroplasty led to a substantial decrease in SSIs.

Synthesis, Characterization, and In Vivo Cytokinome Profile of IL-12-Loaded PLGA Nanospheres.

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 × 109 ± 4.45 × 108 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-1ß 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.

A case report on a novel use of intraoperative Intrabeam™ radiation therapy for a recurrent malignant peripheral nerve sheath tumor with sciatic nerve involvement.

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNST) are sarcomas that arise from peripheral nerves. They generally have a poor prognosis which is exacerbated by high local recurrence rates. This case report discusses the treatment of a patient with a MPNST with local recurrence. This case report is novel due to the use of intraoperative Intrabeam™ (Zeiss, Dublin, CA) radiation therapy use in the protection of neurovascular structures such as the sciatic nerve. CASE PRESENTATION: The patient was a 65-year-old male who noticed a right posterior thigh mass slowly increasing in size over two months. A planned positive margin wide-resection excision was performed due to sciatic nerve abutment. The mass was determined to be a MPNST via postoperative pathology with positive margins along the sciatic nerve. The patient began adjuvant radiation therapy to the upper and lower thigh fields over a period of three months. Thirty-two months later, the patient was found to have a hypermetabolic mass with venous congestion and hyperemia at the prior surgical site which was confirmed by core needle biopsy to be local recurrence of the MPNST. Re-excision of the tumor was planned and performed followed by intraoperative Intrabeam™ radiation therapy. At two years of follow-up, the patient was doing well with minimal pain in his right buttock region with no new or recurrent neurological deficits. Radiologic imaging was negative for local recurrence of the MPNST. CONCLUSION: We believe this case report demonstrates a novel treatment strategy for sarcoma management. The unique use of intraoperative Intrabeam™ radiation therapy, which had not previously been used for this indication, may be efficacious in cases involving neurovascular structures. In this case, focal radiation from the intraoperative Intrabeam™ radiation device was used in a way to affect the recurrent tumor yet protect the sciatic nerve.

Randomized Trial of Postoperative Venous Thromboembolism Prophylactic Compliance: Aspirin and Mobile Compression Pumps.

BACKGROUND: Aspirin, as a routine venous thromboembolism (VTE) prophylaxis, is approved along with pneumatic compression pumps by the American College of Chest Physicians. We assessed compliance of aspirin and pump use after total joint arthroplasty. METHODS: A randomized trial of aspirin alone or aspirin/mobile compression pumps after total joint arthroplasty was performed. Aspirin and pump compliance, VTE events, and satisfaction with pump use were collected. Compliance was assessed through an internal device monitor and drug log book. Patients were also contacted 90 days postoperatively for reported symptomatic VTEs. RESULTS: Each group had 40 patients and greater than 94% compliance with aspirin use, with no difference between groups (P = 0.55). Overall pump compliance during the first 14 days after hospital discharge was 51% (SD ± 33), which was significantly worse than aspirin compliance at 99% (SD ± 4.1) (P < 0.0001). Only 10 patients were compliant (>20 hr/d) with recommended pump use throughout the entire recommended period. There was no notable association between aspirin compliance and VTE within 90 days. There was no notable association between pump compliance and VTE at 90 days. However, average pump use compliance was 20% in patients with VTE and 54% in patients without VTE within 90 days. With the numbers available in this compliance study, there was no significant difference (P = 0.11). DISCUSSION: Aspirin compliance was notably greater than pump compliance. In this study, we found that pump compliance was not associated with lower VTE risk. In fact, no increased risk was recognized in patients with an average pump usage of >50%. Further study is warranted to define the duration of pump use required for clinical significance. The recommended use of compression pumps should continue to be examined.

Postdischarge Opioid Use after Total Hip and Total Knee Arthroplasty.

BACKGROUND: As America's third highest opioid prescribers, orthopedic surgeons have contributed to the opioid abuse crisis. This study evaluated opioid use after primary total joint replacement. We hypothesized that patients who underwent total hip arthroplasty (THA) use fewer opioids than patients who underwent total knee arthroplasty (TKA) and that both groups use fewer opioids than prescribed. METHODS: A prospective study of 110 patients undergoing primary THA or TKA by surgeons at an academic center during 2018 was performed. All were prescribed oxycodone 5 mg, 84 tablets, without refills. Demographics, medical history, and operative details were collected. Pain medication consumption and patient-reported outcomes were collected at 2 and 6 weeks postoperatively. Analysis of variance was performed on patient and surgical variables. RESULTS: Sixty-one patients scheduled for THA and 49 for TKA were included. THA patients consumed significantly fewer opioids than TKA patients at 2 weeks (28.1 tablets vs 48.4, P = .0003) and 6 weeks (33.1 vs 59.3, P = .0004). Linear regression showed opioid use decreased with age at both time points (P = .0002). A preoperative mental health disorder was associated with higher usage at 2 weeks (58.3 vs 31.4, P < .0001) and 6 weeks (64.7 vs 39.2, P = .006). Higher consumption at 2 weeks was correlated with worse outcome scores at all time points. CONCLUSIONS: TKA patients required more pain medication than THA patients, and both groups received more opioids than necessary. In addition, younger patients and those with a preexisting mental health disorder required more pain medication. These data provide guidance on prescribing pain medication to help limit excess opioid distribution.

Programmed Multidrug Delivery Based on Bio-Inspired Capsule-Integrated Nanocoatings for Infected Bone Defect Treatment.

Infection and delayed wound healing are two major serious complications related to traumatic injuries and cause a significant burden to patients and society. Most currently available drug delivery materials typically carry a single drug, lack protection from drug loading, and face challenges in on-demand and precisely controlled drug release. Here, we report a flower (Cirsium arvense)-inspired capsule-integrated multilayer nanofilm (FICIF), synthesized using a layer-by-layer self-assembly, for programmed multiple drug co-delivery for trauma (open fracture as an example) treatments. Our approach allows polypeptide multilayer nanofilms and innovative impregnated capsules to assemble hierarchical reservoirs with specific drug binding sites, shielding protection capability, and ordered packing structures. The resultant FICIF nanocarriers enable sustained and on-demand co-delivery of a unique immune-tuning cytokine (interleukin 12p70) and a growth factor (bone morphogenetic protein 2) in clinical use, resulting in extraordinary anti-infection (3 orders of magnitude improved bacterial killing) and bone regeneration (5 times enhanced bone healing) in treating infected rat femur fractures. The successful synthesis of these biomimetic high-performance delivery nanocoatings is expected to serve as a source of inspiration for the development of biomaterials for various clinical applications.

Portable compression devices in total joint arthroplasty: poor outpatient compliance.

BACKGROUND: Aspirin and mechanical compression devices are approved means of venous thromboembolism (VTE) prophylaxis after total joint arthroplasty. Prior studies of mechanical compression pumps after joint arthroplasty have been limited to the inpatient setting. The purpose of this study was to evaluate outpatient compliance and utilization factors in a rural population after elective hip or knee arthroplasty. METHODS: Utilization for portable pneumatic compression pumps after joint arthroplasty was prospectively recorded (hours). Compliance was defined as the recommended 20 hours per day. A questionnaire 2 weeks postoperatively assessed factors that may contribute to noncompliance. Patients were followed up for 90 days postoperatively to record VTE events. RESULTS: Data were collected for 115 joint arthroplasty patients (50 hips, 65 knees). Postdischarge day one had the highest average usage at 13.2 hours/day (66.0%, range 0%-100%), but this number fell to 4.8 hours/day (24.0, range 0%-100%) by day 14. Patient compliance (>20 hours use/day) was highest on postdischarge day one at 40 patients (34.7%). By postdischarge day 14, patient compliance fell to 17 patients (14.8%). Difficulty using the pumps (P = .027) and pump-associated heat (P = .009) were significantly associated with patient compliance. A deep vein thrombosis and nonfatal pulmonary embolism were recorded in 2 separate patients. CONCLUSIONS: This study demonstrated poor outpatient compliance with portable pneumatic compression devices. Poor compliance was related to pump heat and difficulty with pump use. Even with poor compliance, a low incidence of VTE events was observed.

Rate of surface contamination in the operating suite during revision total joint arthroplasty.

BACKGROUND: This study estimated operating room surface contamination rates during aseptic vs septic total joint arthroplasty and evaluated the similarity between clinically infecting organisms and those isolated from contaminated surfaces. METHODS: Patients undergoing total hip and knee revision arthroplasties were identified, and surface and tissue samples were collected. Cases were classified aseptic or septic based on Musculoskeletal Infection Society criteria for prosthetic joint infection. Positive surface cultures were compared with intraoperative tissue cultures. Positive cultures were speciated and tested for antimicrobial sensitivity. RESULTS: Samples were collected from 31 aseptic and 18 septic cases. Patients had similar demographics and time to explantation. Surface contamination rates for septic revisions were greater than those for aseptic revisions (77% vs 13%). During septic revisions, when intraoperative tissue cultures were positive, the surgical field was contaminated in 14 of 15 cases. The kappa correlation statistic for positive surgical cultures matching the surface sample was 0.9 (95% confidence interval: 0.78-1). CONCLUSIONS: Septic revisions had a significantly higher rate of surgical field contamination than aseptic revisions. Cultures suggest that bacteria contaminating the septic revision surgical field likely originated from the infected joint. Although this observation seems obvious, it is an important piece of information when discussing best practices during a single-stage exchange revision. Further clinical studies will demonstrate the use of a preparation and reset period during a single-stage revision to remove contaminated surfaces.

Unicentric epithelioid hemangioendothelioma of the calcaneus: a case report and review of literature.

BACKGROUND: This review of the literature combined with a clinical case will allow the illustration of a favorable outcome of this variable low grade malignancy, display a role for limb salvage surgery with intralesional treatment, and offer a clinical example of epithelioid hemangioendothelioma, a rare malignancy. CASE PRESENTATION: The case report presents a case of solitary epithelioid hemangioendothelioma (EHE) of the calcaneus in a 60-year-old male. Primary vascular tumors of the bone are rare; however, EHE is one of the most common primary malignant vascular tumors to occur in bone. A review of the literature found few cases that involved the calcaneus; those cases found that involved the calcaneus were either part of a multifocal or metastatic disease process. Our case presents a 45-month clinical follow-up of solitary EHE in the calcaneus treated with surgical excision by curettage and cementing. CONCLUSION: This case has clinical follow-up greater than 2 years post-operatively and could be a guide for treatment of a rare disorder with a substantial paucity of literature.

Osseointegrated Transcutaneous Device for Amputees: A Pilot Large Animal Model.

Traditional above-the-knee amputation prosthetics utilize a stump-socket interface that is well-known for skin/socket problems, sitting difficulty, disuse osteopenia, and increased work of ambulation. As a result, we evaluated a novel osseointegrated transcutaneous implant in a large animal. The implant was designed to promote osseointegration at the bone-implant interface and minimize complications. As proof of concept, four Dorset sheep underwent a two-stage surgery for forelimb placement of an osseointegrated transcutaneous implant utilizing Compress® technology (Biomet, Inc., Warsaw, IN). Two sheep received a long anchor plug (90 mm long x 9 mm in diameter) and two received a short anchor plug (46 mm long x 9 mm in diameter). Sixteen weeks after the initial surgery, the operative limbs, along with the attached implant, underwent radiographic and histological analysis for osseointegration. Periprosthetic fractures occurred in the two animals that received the longer internal prosthesis; one healed with splinting and the other animal underwent a second surgical procedure to advance the amputation site more proximal. No fractures occurred in the shorter internal prosthesis group. There was no histological evidence of infection and none of the transcutaneous adapters failed. Bone-implant osseointegration was demonstrated in two of three limbs that underwent histological analysis. This unique implant demonstrated osseointegration without transcutaneous adapter failure, all while displaying minimal infection risk from the outside environment. Although it involved short-term follow-up in a limited number of animals, this pilot study provides a platform for further investigation into the valid concept of using Compress® technology as an endo-exo device.

Highly metastatic K7M2 cell line: A novel murine model capable of in vivo imaging via luciferase vector transfection.

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.

Using the Spleen as an In Vivo Systemic Immune Barometer Alongside Osteosarcoma Disease Progression and Immunotherapy with α-PD-L1.

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.

Osteosarcoma Overview.

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.