Poster BB8 - Vascularized Bioprinted Tissue Model for Studying Cellular Senescence

Introduction: Patients diagnosed with cancer are subjected to aggressive treatment, including surgical interventions followed by chemo and/or radiotherapy. Doxorubicin (DOX), a potent chemotherapeutic drug, is often the first choice for treatment. Unfortunately, this life-saving pharmaceutical intervention in the fight against cancer induces cardiotoxicity and, subsequently, heart failure. Cardiotoxicity often impairs the quality of life or, in some severe cases, could be lethal. Unfortunately, the detailed molecular mechanisms of DOX-induced cardiotoxicity are poorly understood, with gaps in understanding why some people have more significant adverse responses to chemotherapy. As such, there is an urgent need to identify potential biomarkers that could help to better predict the severity of side effects that each individual patient may present when undergoing chemotherapy. Previous studies have shown that DOX and many other chemotherapeutics are strongly associated with cellular senescence. Senescent cells are metabolically active and can acquire a destructive, pro-inflammatory senescence-associated secretory phenotype (SASP), contributing to the pathogenesis of multiple age-related diseases. Importantly, SASP includes proinflammatory cytokines, chemokines, proteases, and a variety of circulating, exosomal microRNAs (miRNAs). The shift in expression levels of different senescence-specific miRNAs takes part in shifting the fate of the cell from pro-apoptotic to senescent, thereby having fatal consequences by inducing the production of pro-inflammatory markers that alter the phenotype of neighboring cells. In this study, the goal is to investigate the effects of senescent human umbilical vein endothelial cells (HUVECs) on 3D cell constructs to further elucidate their biomolecular effects.

Materials and

Methods: Cellink BioX6 bioprinter was used to 3D print vascularized cardiovascular tissue constructs as shown in Figure 1. Alongside, human umbilical vein derived endothelial cells (HUVECs) were seeded and irradiated to induce cellular senescence. Briefly, HUVECs were exposed to 15Gy of radiation for 7 minutes and 28 seconds using a Kimtron x-ray system. Of note, previous studies from this group have shown that HUVECs become 100% senescent seven days following irradiation. Thereafter, bioprinted constructs were co-cultured with non-irradiated or irradiated HUVECs. After seven days, 3D constructs were disintegrated with collagenase and RNA was extracted using Trizol, followed by the addition of chloroform to promote phase separation and ethanol precipitation. cDNA for downstream qRT-PCR was prepared using the TaqMan Advanced microRNA (miRNA) cDNA synthesis kit. The process flowchart is shown in Figure 2.

Results, Conclusions, and Discussions: Result and

Discussion:
A microfluidic-based system has been used to print and maintain 3D bioprinted vascularized tissue constructs. Before performing experiments with DOX, preliminary experiments were performed with control, non-irradiated and senescent, irradiated HUVECs. This was achieved by plating the cells in a 6-well plate, and exposing the experimental group to x-rays. The constructs were then added and co-cultured for seven days prior to lysis. qRT-PCR analysis has been conducted for senescence associated biomarkers such as miR-146a-5p and miR-217. Senescence-associated miRNA miR-146a-5p demonstrated a relative 2.2 fold-increase in expression (Figure 3) while pro-apoptosis miRNA miR-217 exhibited a decrease in expression (Figure 4) in cells co-cultured with old HUVECs when compared with cells co-cultured with young HUVECs.

Conclusion:
The ultimate goal of this research is to establish a novel method to understand how senescence mediated by antineoplastic agents such as DOX can contribute to systemic pathologies such as cardiac damage. Cells within the bioprinted constructs displayed the potential to interact with and be influenced by cultured cells in the same well. Co-culturing of bioprinted tissue construct with different age senescent cells can be utilized to study cellular interactions resembling in vivo parabiosis to understand the impact of senescent cells on healthy cells. Future biomolecular analysis will reveal novel biomarkers to help elucidate how antineoplastic, senescence-inducing agents may have adverse systemic effects in cancer patients, particularly through induction of cardiotoxicity.

Acknowledgements (Optional): The authors would like to acknowledge funding support from National Science Foundation – Grant Number CBET 2317757 and 2317758.