Soft tissue sarcomas (STS) are rare, aggressive malignancies derived from connective tissues such as muscle and fat. Undifferentiated pleomorphic sarcoma (UPS) is one of the most common STS in adults. UPS is an aggressive, highly metastatic sarcoma, and is resistant to chemotherapy. New therapies for UPS are desperately needed. STS have an immune desert tumour immune microenvironment (TIME), characterized by a paucity of tumour infiltrating lymphocytes and subsequent resistance to immunotherapies such as immune checkpoint inhibitors. Strategies capable of creating an immune-rich, inflamed TIME may improve immunotherapy efficacies for sarcoma. Activation of the STING (stimulator of interferon genes) receptor can induce potent innate and adaptive immune responses within immunogenic solid tumours. However, this approach has never been attempted in immune-inert sarcomas. Purpose: To determine the therapeutic anti-tumour effects of STING activation in UPS tumours. We have developed an inducible, immune-competent mouse model of UPS. We evaluated intra-tumoural injection of the murine STING receptor agonist, DMXAA, into UPS-bearing immune-competent mice. DMXAA was injected into palpable UPS tumours of the hindlimb. Tumour volume and bioluminescence imaging was recorded bi-weekly. DMXAA treated UPS tumours were also evaluated for necrosis and immune infiltration at defined time points. UPS tumours developed necrosis and lymphocytic infiltration 72 hours after DMXAA treatment. A single intra-tumoural dose of DMXAA into UPS tumours resulted in durable cure in 50% of mice. All survivors rejected a re-challenge of the UPS tumours in both the contralateral hindlimb and lung, suggesting adaptive immunity. The therapeutic effects of DMXAA were mitigated in lymphocyte deficient Rag2 knockout mice. STING therapy is a promising immunotherapeutic opportunity for immune-inert sarcomas. Our data warrants further preclinical investigations in other sarcoma models and in combination with other immune-based therapies

Impaired bone healing biology secondary to soft tissue deficits and chemotherapy contribute to non-union, fracture and infection following limb salvage surgery in Osteosarcoma patients. Approved bone healing augments such as recombinant human bone morphogenetic protein-2 (rhBMP-2) have great potential to mitigate these complications. rhBMP-2 use in sarcoma surgery is limited, however, due to concerns of pro-oncogenic signalling within the tumour resection bed. To the contrary, recent pre-clinical studies demonstrate that BMP-2 may induce Osteosarcoma differentiation and limit tumour growth. Further pre-clinical studies evaluating the oncologic influences of BMP-2 in Osteosarcoma are needed. The purpose of this study is to evaluate how BMP-2 signalling affects Osteosarcoma cell proliferation and metastasis in an active tumour bed. Two Osteosarcoma cell lines (143b and SaOS-2) were assessed for proliferative capacity and invasion. 143b and SaOS-2 cells were engineered to upregulate BMP-2. In vitro proliferation was assessed using a cell viability assay, motility was assessed with a scratch wound healing assay, and degree of osteoblastic differentiation was assessed using qRT-PCR of Osteoblastic markers (CTGF, ALP, Runx-2 and Osx). For in vivo evaluation, Osteosarcoma cells were injected into the intramedullary proximal tibia of immunocompromised (NOD-SCID) mice and local tumour growth and metastases were assessed using weekly bioluminescence imaging (BLI) and tumour volume measurements for 4–6 weeks. At the experimental end point we assessed radiographic tumour burden using ex-vivo micro-CT, as well as tibial and pulmonary gross and histologic pathology. SaOS-2 was more differentiated than 143b, with increased expression of Runx-2 (p = 0.009), Osx (p = 0.004) and ALP (p = 0.035). BMP-2 upregulation did not stimulate an osteoblast differentiation response in 143b, but stimulated an increase in Osx expression in SaOS-2 (p = 0.002). BMP-2 upregulation in 143b cells resulted in increased proliferation in vitro (p = 0.014), faster in vitro wound healing (p = 0.03), significantly increased tumour volume (p = 0.001) with enhanced osteolysis detected on micro-CT, but did not affect rates of lung metastasis (67% vs. 71%, BMP-2 vs. Control). BMP-2 over-expression in SaOS-2 cells reduced in vitro proliferation when grown in partial osteogenic-differentiation media (p < 0.001), had no effect on in vitro wound healing (p = 0.28), reduced in vivo SaOS-2 tumour burden at 6 weeks (photon counts, p < 0.0001), decreased tumour-associated matrix deposition as assessed by trabecular thickness (p = 0.02), and did not affect rates of lung metastasis (0% vs. 0%). Our results indicate BMP-2 signalling incites a proliferative effect on a poorly differentiated Osteosarcoma cell line (143b), but conditionally reduces proliferative capacity and induces a partial differentiation response in a moderately-differentiated Osteosarcoma cell line (SaOS-2). This dichotomous effect may be due to the inherent ability for Osteosarcoma cells to undergo BMP-2 mediated terminal differentiation. Importantly, these results do not support the clinical application of BMP-2 in Osteosarcoma limb salvage surgery due to the potential for stimulating growth of poorly differentiated Osteosarcoma cells within the tumour bed. Additional studies assessing the effects of BMP-2 in an immune-competent mouse model are ongoing

Impaired bone healing biology secondary to soft tissue deficits and chemotherapy contribute to non-union, fracture and infection following limb salvage surgery in Osteosarcoma patients. Approved bone healing augments such as recombinant human bone morphogenetic protein-2 (rhBMP-2) have great potential to mitigate these complications. rhBMP-2 use in sarcoma surgery is limited, however, due to concerns of pro-oncogenic signalling within the tumour resection bed. To the contrary, recent pre-clinical studies demonstrate that BMP-2 may induce Osteosarcoma differentiation and limit tumour growth. Further pre-clinical studies evaluating the oncologic influences of BMP-2 in Osteosarcoma are needed. The purpose of this study is to evaluate how BMP-2 signalling affects Osteosarcoma cell proliferation and metastasis in an active tumour bed. Two Osteosarcoma cell lines (143b and SaOS-2) were assessed for proliferative capacity and invasion. 143b and SaOS-2 cells were engineered to upregulate BMP-2. In vitro proliferation was assessed using a cell viability assay, motility was assessed with a scratch wound healing assay, and degree of osteoblastic differentiation was assessed using qRT-PCR of Osteoblastic markers (CTGF, ALP, Runx-2 and Osx). For in vivo evaluation, Osteosarcoma cells were injected into the intramedullary proximal tibia of immunocompromised (NOD-SCID) mice and local tumour growth and metastases were assessed using weekly bioluminescence imaging and tumour volume measurements for 4–6 weeks. At the experimental end point we assessed radiographic tumour burden using ex-vivo micro-CT, as well as tibial and pulmonary gross and histologic pathology. SaOS-2 was more differentiated than 143b, with significantly increased expression of the Osteoblast markers Osx (p = 0.004) and ALP (p = 0.035). BMP-2 upregulation did not stimulate an osteoblast differentiation response in 143b, but stimulated an increase in Osx expression in SaOS-2 (p = 0.002). BMP-2 upregulation in 143b cells resulted in increased proliferation in vitro (p = 0.014), faster in vitro wound healing (p = 0.03), significantly increased tumour volume (p = 0.001) with enhanced osteolysis detected on micro-CT, but did not affect rates of lung metastasis (67% vs. 71%, BMP-2 vs. Control). BMP-2 over-expression in SaOS-2 cells reduced in vitro proliferation when grown in osteogenic-differentiation media (p < 0.001), had no effect on in vitro wound healing (p = 0.28), reduced in vivo SaOS-2 tumour burden at 6 weeks (photon counts, p < 0.0001), decreased tumour-associated matrix deposition as assessed by trabecular thickness (p = 0.02), but did not affect rates of lung metastasis (0% vs. 0%). Our results indicate BMP-2 signalling incites a proliferative effect on a poorly differentiated Osteosarcoma cell line (143b), but conditionally reduces proliferative capacity and induces a partial differentiation response in a moderately-differentiated Osteosarcoma cell line (SaOS-2). This dichotomous effect may be due to the inherent ability for Osteosarcoma cells to undergo BMP-2 mediated terminal differentiation. Importantly, these results do not support the clinical application of BMP-2 in Osteosarcoma limb salvage surgery due to the potential for stimulating growth of poorly differentiated Osteosarcoma cells within the tumour bed. Additional studies assessing the effects of BMP-2 in an immune-competent mouse model are ongoing

Aim. Multispecies biofilms are associated with difficult periprosthetic joint infections (PJI), particularly if they have different antibiotic sensitivities. We aimed to determine if we could generate and kill a multispecies biofilm consisting of a Gram negative and Gram positive pathogen in-vitro with antibiotic loaded calcium sulfate beads containing single or combination antibiotics. Methods. To establish whether we could co-culture mixed species biofilms various combinations of Pseudomonas aeruginosa (PA), Enterococcus faecalis (EF), Staphylococcus aureus (SA) and Enterobacter faecalis (EF) were grown together on 316L stainless steel coupons and agar plates. Based on this screen we focused on PA + EF and challenged them with high purity calcium sulfate beads (Stimulan Rapid Cure) loaded with vancomycin (V), alone tobramycin (T) alone or vancomycin and tobramycin in combination (V+T). Bioluminescence, light imaging, plate count, confocal microscopy and scanning electron microscopy were used to quantify growth. Results. On 316LSS the V loaded bead reduced both EF and PA by approximately 2 logs compared to unloaded control beads. A T alone loaded bead eliminated PA from the dual species biofilm and caused a 2-log reduction in EF. The V+T-beads reduced PA by 9-logs and EF by 8.3 logs. In terms of total CFUs V+T beads reduced the bioburden by 8.4 logs compared to V or T alone. which resulted in 2.1 and 2.6 log reductions respectively. (* P<0.05, *** P<0.001). On agar PA dominated the culture for the unloaded and V loaded beads. However, when challenged with a T loaded bead both species were able to coexist and a zone of killing was generated in both species in the multispecies biofilms. However, this zone was smaller and included more tolerant variants than the zone generated by V+T-loaded beads. Conclusions. There were species proportion differences between biofilms grown on agar and 316LSS demonstrating the importance of growth conditions on species interactions. Antibiotics against strains with differing sensitivities can shift species interactions. High purity calcium sulfate beads containing tobramycin a broad-spectrum Gram positive and negative antibiotic vancomycin, a Gram-positive targeted antibiotic killed a larger percentage of a multispecies in an in-vitro biofilm than either single gram-specific antibiotic alone, demonstrating the advantage of using combination antibiotics for treating multispecies biofilms

Bone metastases are the most common cause of cancer-related pain and often lead to other complications such as pathological fractures and spinal cord compression. Bisphosphonates (BP) are a class of potent anti-resorptive agents commonly prescribed to retard osteoporosis progression. Interestingly, BP may have indirect anti-tumour properties through negative effects on macrophages, osteoclasts, endothelial cells and their ability to suppress matrix metalloproteinase (MMP) activity. Currently, the use of bisphosphonates for cancer therapy is generally restricted to high dose systemic delivery. The purpose of this study was to investigate the effects of direct local delivery of Zoledronate at the metastatic site in a mouse model of breast cancer metastasis to bone. Seven days following intra-tibial inoculation with MDA-MB-231 (N = 1× 105) breast cancer cells in athymic mice, the experimental group (N = 11) was treated by direct infusion of 2µg of Zoledronate into the tibial lesion (three times/week for three weeks) and compared to vehicle-treated mice (N = 5). The formation of bone metastases and growth of the lesions were followed up by weekly bioluminescence imaging. In a subsequent experiment, a comparison of the effects of local versus systemic delivery of Zoledronate on the formation of osteolytic bone metastases was carried in athymic mice (N = 15). Seven days following intra-tibial inoculation with MDA-MB-231 breast cancer cells, the systemic group (N = 5) was treated with Zoledronate (0.025mg/kg) once per week for four weeks and compared to systemic delivery of vehicle (N = 4). Following treatment, the mice were sacrificed, and micro-CT images of the right tibia were obtained. Bone volume to tissue volume ratio (BV/TV%) was determined using µ-CT biomarkers. The first experiment showed a statistically significant increase in mean bone volume/tissue volume ratio% (BV/TV%) in the treated group (7.0±1.54%) as compared to the control group (3.8±0.48%) (P <0.001, 95%CI=1.9–4.3). This corresponded to a net increase of 84.21% in response to Zoledronate treatment. Comparison between the local and systemic effects of Zoledronate also revealed a significant increase in the BV/TV% in the locally treated group (6.69±0.62%) when compared to the cohort administered systemic bisphosphonate treatment (4.03±0.44%) (P<0.001, 95%CI=1.24–3.20), corresponding to a net increase of 66.0%. These preliminary results suggest that high dose sustained release of Zoledronate can lead to a significant inhibition of tumor-induced osteolysis. Moreover, comparison between local and systemic delivery revealed that the effect of local bisphosphonate administration exceeds the benefits of systemic delivery in terms of osteolysis inhibition. Lastly, the noted effects of Zoledronate local delivery triggers the need for further assessment of its anti-tumour activity

Aims

Delayed postoperative inoculation of orthopaedic implants with persistent wound drainage or bacterial seeding of a haematoma can result in periprosthetic joint infection (PJI). The aim of this in vivo study was to compare the efficacy of vancomycin powder with vancomycin-eluting calcium sulphate beads in preventing PJI due to delayed inoculation.