MSACL 2023 Abstract

Application of LC-MS/MS to Assess the Chemosensitivity of Breast Cancer Spheroids for an in vitro to in vivo Extrapolation Analysis Ramisa Fariha (1), Zahra Ahmed (1), Jad Hamze (2),Emma Rothkopf (1),Oluwanifemi David Okoh (1), Anubhav Tripathi (1) (1) Center for Biomedical Engineering, School of Engineering, Brown University, 182 Hope Street, Providence, RI- 02912, USA (2) Department of Biology and Medicine, Brown University, Providence, RI- 02912, USA Ramisa Fariha, Master of Science in Biomedical Engineering (Presenter) Brown University

Presenter Bio: Hailing from the small town called Narayanganj in Bangladesh, I am a second-year Ph.D. candidate at the Tripathi Lab for Diagnostics and Microfluidics at Brown University. I pursued my undergraduate education in Biomedical Engineering at Penn State University, where I was the first international student to be the recipient of Freshman of the Year award, as well as graduated as one of the Top 20 Most Active Female Engineers on campus. Upon graduating, I worked briefly in R&D at ACell Inc. (now a part of Integra LifeSciences) in Columbia, MD. After that, I pursued my master’s in Biomedical Engineering at Brown University, working in the Lee Lab for Biomedical Optics and the Morgan lab for Tissue Engineering. I joined the Tripathi lab as a Ph.D. student, leading all LC-MS/MS-based studies. Currently, my team and I design, develop and optimize LC-MS/MS study protocols for automation adaptability. Outside the lab, I am an advocate for international students, students with special needs and females in STEM fields. I am also the founder and President of South Asian Scholars in STEM at Brown. Owing to my contributions, I was awarded the Graduate Student Contribution to Community Life award in 2020.

Introduction:
While 2D in vitro cell culture models have been used over the years, 3D cell culture technique is becoming increasingly popular due to its ability to replicate the in vivo tumor environment better. Out of the existing 3D cell culture techniques, MicrotissuesTM has garnered attention due to its scaffold-free nature and high-throughput ability. However, traditional cell culture assessment techniques are unable to fully characterize this 3D platform due to limitations of quantifying trace amounts of absorption taking place within these molds.

Objectives:
Our study expands the use of LC-MS/MS to quantify the absorption of Paclitaxel, a chemotherapeutic, by the 3D MicrotissuesTM molds, and its subsequent impact on MCF7 breast cancer tissue viability.

Methods:
MicrotissuesTM molds were made using 2% agarose and MCF7 cells were allowed to self-assemble to tissue spheroids over DIV= 3. Drug adhesion was initially monitored for the empty wells and molds. Following the initial observation trend and drug absorption, the spheroids were treated with the actual concentration (measured after absorption by mold) versus the concentration it should have been receiving for 12 and 24-hours intervals to assess the impact of the minute change on cellular viability.

Results:
By optimizing the sample preparation technique, our strategy utilizes cell culture media to accurately quantify the drug adhesion to the MicrotissuesTM molds, with LoD= 0.03 μM, optimized for cellular microenvironment with relevance for extrapolation to patient application. The overall assay exhibits linearity of R2>0.99 inclusive of both MRMs, and a percent coefficient of variation that is less than or equal to 10%. We have tested and validated the findings against a traditional live-dead assay for the spheroids. In addition to manual plating with cell culture supernatant testing, we have exhibited the adaptability of our protocol on the JANUS G3 liquid handling workstation for rapid sample preparation, in addition to the fast three minutes total run time per sample analysis. This makes our protocol adaptable for clinical usage, expanding its application to personalized cancer therapies.

Conclusion:
Overall, our work plays an important role in the in vitro to in vivo extrapolation study of chemotherapeutics for breast cancer and how LC-MS/MS can provide a viable and highly sensitive and quantitative method for analysis.