Installment of Automation and Modernized Culture Methods Updates a Drug-Sensitivity Screening Platform and Paves the Way to Next-Generation of Precision Medicine
Authors: Kawabata Kimihito Cojin , Li Chao , Kakinuma Hideaki , Takama Yoshiharu , Tsuji Hayato , Komori Hironobu , Gong Bo , Kimura Yasumasa , Tanomura Hideki , Okamoto Atsushi , Morita Maiko , Takamori Hiroyuki , Fukushima Hidehito , Yokoyama Kazuaki , Konuma Takaaki , Ohno Nobuhiro , Goyama Susumu , Yamazaki Satoshi , Oka Tetsushi , Kudo Gen , Melnick Ari M , Nannya Yasuhito , Imoto Seiya , Takahashi Satoshi 
Published: December 2024

Since the field of drug discovery has seen a rise in the use of primary tumor specimens (PTS) derived from patients with hematological malignancies as live materials in ex vivo drug treatments in late 2010s, they have discovered many intriguing phenotypes of AML cells that can lead to therapeutic insights of this disease category. However, when we consider the fact that current methods in ex vivo drug screenings are not using the most updated culture and cell processing methodology, it is highly expected that modernization of cell processing and analyzing procedures will enable us to detect phenotypes of PTS at a higher resolution.

Herein, we have installed our own updated methods in multimodal categories. Even though the number of primary tumor specimens in the screening cohort is not comparable to the ones in other studies using AML PTS, (24 cases of AML, 3 cases of CML, and 1 case of MPN, while we ongoingly recruit more cells), our approach is characterized by its full-automation both in cell processing and data acquisitions using multicolor flow cytometry, as well as updated ex vivo culture methods after optimization using different culture media. We harvested cells for flow cytometry as of day 0, 3, and 6. In a monotherapy section, 24 compounds (6 conventional chemotherapy drugs, 10 epigenetic inhibitors, 4 kinase inhibitors including a FLT3 inhibitor, 3 signal transduction inhibitors, and Venetoclax) were included, while 3 different combination therapies (IDR+ AraC, Ven + 5-Aza, and Ven + AraC) were performed using 6x6 matrix layouts. Drugs were prepared in 384-well plates using a computationally controlled drug dispenser.

Using those updated systems, we could discover; 1) Those fully-automated high-throughput platform needs their own optimizations regarding seeding numbers, liquid handling methods, and optimal plastic vessels with low inherent variations of results. 2) PTS are well tolerable to up to 6 days of ex vivo culture 3) Choosing optimal time points for each of the drugs ensures more accurate representation of their drug activities. For example, epigenetic inhibitors have tendency to show better results on day 6 rather than day 3. 4) Multicolor flow cytometry provides previously unseen differences in drug activity profiles upon different clusters, which had not been visible from bulk-grade sensitivity profiling, such as colorimetric viability assay. Those specimens were genetically profiled by whole genome/ exome sequencing, as well as other multi-omics analyses (Bulk RNA seq, methylome analysis, and scRNA seq). 5) Optimizations of ex vivo culture methods not only ensure our better quality of these 6-day drug effects, but also extend the possibility of PTS for further applications by allowing leukemia stem cell fractions to be maintained in vitro for longer periods of time. They include long-term culture and ex vivo expansion of leukemic stem cells using the most updated serum-free liquid culture, and further gene modifications using various vectors.

Collectively, our platform for DSS confers next-generation use of primary patient-derived biomaterials so that even a small- to middle-sized group can conduct these complexed assays to test their hypotheses based on primary specimens, which had been possible only in high-volume multi-institutional alliances.