Abstract 5798: Ex vivo culturing of primary AML cells facilitates the study of developmental therapeutics
Authors: Patel Satyam , Golla Upendarrao , Sharma Arati , Claxton David 
Published: April 2023

Abstract
Studies with acute myeloid leukemia (AML) cell lines have limited potential as they tend to acquire mutations, thus, do not represent typical leukemias found in patients. Ex vivo culturing of primary human AML patient cells is therefore of interest; however, the long-term culture of these cells has been limited by rapid differentiation and loss of clonogenicity. Previously, different growth factors and stromal cells were implicated in the short-term culturing of primary cells. Thus, we aimed to evaluate culture conditions using a cytokine cocktail, allowing long-term expansion and phenotypic maintenance of primary AML cells in vitro. Different culture conditions were constituted by combination of cytokines (SCF, FLT-3L, TPO, IL-6, IL-3, GCSF, GM-CSF) and small molecules (SR1, UM729) in serum-free StemSpan SFEM media. The ability of different growth mediums to support the expansion and clonogenicity of 6 primary AML cells (collected from blood/bone marrow) over two weeks was assessed using cell counts and colony formation assays. Additionally, cell surface markers (hCD34, hCD45, hCD123, hCD117) were assessed by flow cytometry during the culture period. All tested cultured conditions exhibited successful cell proliferation (4-6-fold expansion) and survival of primary AML cells over two weeks of culture. In addition, the growth media successfully supported 3.5-fold CD34+ stem cell expansion. Growth mediums with early-acting cytokines (SCF, FLT-3L, TPO, and IL-6) and small molecules resulted in phenotypic maintenance of cell surface markers (CD34, CD123) and secondary clonogenicity of primary AML cells over the culture period. The addition of late-acting cytokines (IL-3, GCSF, and GM-CSF) showed a higher growth rate but negatively affected the cell surface markers and clonogenicity. Secondary colony counts (day-9 culture) showed a significant positive correlation with CD34 expression levels. Thus, CD34 levels could serve as a potential surrogate marker for the clonogenicity of primary AML cells. Furthermore, we tested the engraftment ability of two primary AML cells grown in 3 of our promising culture conditions (Day 4, Day 8) in xenograft mice. Flow cytometry analysis on peripheral blood and tissues (spleen and bone marrow) from xenograft mice showed successful engraftment of primary cells cultured for 8 days. On an application note, the optimal condition from our results was used to study the effects of ceramide nanoliposomes (CNL) in AML patient cells. Interestingly, the AML patient cells treated for two weeks with IC15 and IC30 doses of CNL led to cell cycle arrest, reduced CD34 expression, and clonogenicity relative to control cells. The CNL findings from primary cells are different from that of cell lines cultured under the same conditions. Thus, our optimal growth conditions may facilitate researchers to understand AML biology and study various therapeutics under development using primary AML cells.
Citation Format: Satyam Patel, Upendarrao Golla, Arati Sharma, David Claxton. Ex vivo culturing of primary AML cells facilitates the study of developmental therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5798.