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To develop 3D bioPRINTed osteoinductive constructs that deliver CHEMOtherapeutics within large bone defects that are surgically created when removing bone tumours.

Periodic Reporting for period 1 - PRINT-CHEMO (To develop 3D bioPRINTed osteoinductive constructs that deliver CHEMOtherapeutics within large bone defects that are surgically created when removing bone tumours.)

Reporting period: 2019-07-01 to 2021-06-30

Osteosarcoma is the most commonly diagnosed bone tumour with most of these cases being in children and adolescents. Each year over 3.4 million new cases of osteosarcoma are diagnosed in the world. Osteosarcoma predominantly initiates in the metaphysis of long bones, such as the distal femur, proximal tibia, and proximal humerus. Over 50% of these tumours are relatively resistant to radiation therapy, due to the molecular aberration of the tumour. The current gold standard for treatment is tumour resection and adjuvant chemotherapy, with a 5-year survival rate of 61.6% in patients aged 0-24 years old. Approximately one-third of patients diagnosed with osteosarcoma are expected to relapse, with only 15% of these patients surviving the disease a second time. Despite the clinical urgency and significant advances in treatment seen in other malignancies, no major improvements in patient outcome have been achieved since the 1970s.

As metastatic disease is the most critical clinical factor that influences malignant progression and mortality in osteosarcoma, most of the research to date has focused on tumour elimination and prevention of metastases, with little attention to bone repair or salvation. Yet the current gold-standard therapy involves extensive surgical intervention from total limb reconstruction to amputation. The belief being that growth factor delivery might exert local (residual cancer cells following resection) and systemic tumour-promoting effects. As a result, there is limited research that focuses on bone regeneration post-resection, which is further compromised following chemotherapy. Therefore, the overall aim of this proposal was to understand the paradoxical relationship between tumour elimination and bone regeneration. We aim to achieve this in two parts. First, by developing and validating a 3D spheroid model mimicking the osteosarcoma tumour microenvironment. Once validated we aim to use our in vitro model we aim to screen potential regenerative cues to evaluate the best option to aid in regeneration even in presence of chemotherapeutics. Secondly, the best options tested in vitro will then be evaluated in vivo in an osteosarcoma orthotopic mouse model to establish which therapy could potentially aid in regeneration without cause tumour growth.
Work Package 1: Understanding the divergent relationship between tumour elimination and bone regeneration in Osteosarcoma
Recently, there has being increasing interest in the use of 3D cultures to study the interactions between tumour cells and other cellular or acellular components of the tumour microenvironment as the results better correlate with results seen in vivo. Specifically, studies have shown that spheroid cells displayed more chemoresistance to chemotherapeutics than conventional monolayer cells. Furthermore, studies have shown that drug sensitivity of tumour cells might be affected by microenvironmental factors, including the presence of Mesenchymal Stromal Cells. Therefore, we developed a 3D spheroid model of early and late-stage osteosarcoma, consisting of a direct co-culture of both osteosarcoma cells and MSCs. As MSCs are an integral player in osteosarcoma progression, but also are well known to play an important role in bone regeneration, it was vital that these were included in the model. We validated the clinical relevancy of the model using FDA-approved chemotherapeutic Doxorubicin. Following validation with Doxorubicin, we were able to use this model to further understand the paradoxical relationship between tumour elimination and bone regeneration. Specifically, our model validated that osteogenic supplements have stimulatory effect on the stromal cells, but minimal effect of cancer cell growth. However, when these osteogenic supplements are delivered along with chemotherapeutics this stimulatory effect is completely abolished. Taken together, we have developed and validated a model that mimics the vital relationship between stromal and osteosarcoma cells as well as models their response to chemotherapeutics and regenerative cues, providing vital information that can inform the design of future therapies for these young patients. This work is currently under review in Advanced Healthcare and Materials.

Work package 2: Investigate the tumour promotive/regenerative potential of BMP-2 delivery in an immunocompetent orthotopic model for osteosarcoma.
The results generated from WP1 clearly demonstrate that there is a fine balance with regenerating the damaged tissue without causing tumour recurrence. With this in mind, I first established an orthotopic model for osteosarcoma which metastasises to the lung. Next, we conducted our in vivo study to understand the effect BMP-2 delivery alone or in combination with Doxorubicin would have on tumour progression and bone formation. The results from this study show that BMP-2 delivery did not accelerate tumour progression in a pre-clinical osteosarcoma orthotopic model, when compared to non-treated or doxorubicin alone groups. Interestingly, when treated locally with a hydrogel containing BMP-2 alone ectopic bone formation was observed surrounding the tibia However, when treated with the same hydrogel and concentration of BMP-2 in combination with systemic chemotherapeutics this stimulatory effect was significantly diminished (see Figure 1). Taken together, these results indicate that although BMP-2 delivery does not exert any tumour-promoting effects as previously feared, it is not an effective therapy to aid with the regeneration, while the patient is undergoing chemotherapy.
Due to the young age of initial diagnosis, the management of osteosarcoma is a challenging and costly exercise, which has a significant socioeconomic cost, it is estimated to be €14.7 billion in Europe in the last 18 years. Despite the resonating clinical urgency for newer and more effective treatment options, thus far, no major changes in treatment and outcome have been achieved since the 1970s. Furthermore, as the global oncology drugs market is expected to grow from $80.92 billion in 2020 to $84.38 billion in 2021, the discovery of druggable targets and development of innovative therapies for inhibiting metastatic progression could have significant economic implications. The results gained from this proposal will provide pharmaceutical companies a cost-effective platform for testing potential new drugs or combinations.

I have set up a hastag on twitter for the project #PRINTCHEMO and will begin heighten awareness of medical research and MSCA actions in the public through it.

I have taken part in European Researcher night and was spotlighted #meettheresearchers.

I was highlighted in a series on women in bioprinting - online article about my work.

I was also awarded New Investigator Recognition Award from the Orthopeadic Research Society.
Figure 1: Investigating the therapeutic potential of BMP-2 delivery