Plasma-Derived 3D Culture for Leukemic Proliferation

INTRODUCTION: Acute (AML) and chronic myeloid leukemia (CML) are hematological malignancies that remain incurable despite novel treatments. In order to improve current treatment options as well as clinical efficacy, there remains a need for more complex in vitro models that mimic the intricate human leukemic microenvironment. A bone marrow-derived 3D tissue engineered bone marrow (3DTEBM) model was previously developed to mimic the multiple myeloma pathophysiology, which allowed for patient material survival after one week in culture and recreated drug resistance. This study aimed to use 3D cultures derived from the peripheral blood of AML and CML patients to promote proliferation of leukemic cells and to be used as a drug screening tool for leukemic treatment.

METHODS: 3D cultures derived from AML and CML patients were developed through calcium cross-linking of plasma from peripheral blood samples of AML or CML patients. AML cell lines (K052 and NOMO-1) and CML cell lines (K562 and Ku812) were incorporated in plasma-derived 3D cultures or 2D cultures in co-culture with stromal cell line Hs5, and tested for cell proliferation for 7 days by flow cytometry. In addition, five AML patient samples and five CML patient samples were incorporated in plasma-derived 3D cultures or 2D cultures and tested for cell proliferation for 7 days by flow cytometry. After optimization of cell growth, we tested the sensitivity of AML and CML cell lines to different chemotherapeutic agents, including alisertib (0 - 200 nM) and Ara-C (0 - 200 nM) for AML, and nilotinib (0 - 10 nM) and Cpd22 (0 - 1000 nM) for CML in plasma-derived 3D cultures or 2D cultures in co-culture with stromal cell line Hs5.

RESULTS: We found that AML and CML cell lines demonstrated cell expansion rates of 2 and 2.5 fold and 2.5 and 3.5 fold in 2D cultures for days 3 and 7 compared to day 0, respectively. In contrast, AML and CML cell lines demonstrated higher cell expansion rates of 3.5 and 5 fold and 3.5 and 6 fold in plasma-derived 3D cultures for days 3 and 7 compared to day 0, respectively. In addition, while AML and CML patient samples showed non or very low cell expansion rates (lower than 1.5 fold) after 7 days in 2D cultures compared to day 0, AML and CML patient samples demonstrated cell expansion rates of 3 and 4.5 fold and 4 and 7.5 fold in plasma-derived 3D cultures for days 3 and 7 compared to day 0, respectively.

Furthermore, we tested the effect of the 3D cultures on drug resistance of AML and CML cells. While Ara-C induced IC50 at about 100 nM in both AML cell lines in 2D cultures, only 25-35% of AML cells were killed at the same drug concentration. Alisertib induced IC50 at 100 nM in both AML cell lines in 2D cultures. However, at the same drug concentration only 5-10 % AML cells were killed in plasma-derived 3D cultures. Similarly, while in 2D co-cultures about 50-80% of CML cells were killed using the different concentrations of nilotinib and Cdp22, CML cell lines cultured in plasma-derived 3D cultures showed a significant decrease in sensitivity to the treatments with a 2-fold decrease in the number of cells killed.

CONCLUSIONS: 3D tissue engineered AML and CML-derived cultures supported the growth of the primary AML and CML cells better than classic 2D systems. In addition, it induced significantly more drug resistance in AML and CML cells, compared to classic 2D culture systems. The 3D tissue engineered AML and CML cultures created a more physiologically relevant environment for leukemia cell proliferation, due to recreation of microenvironment cues including interactions with accessory stromal cells, and provided a reliable model for growing myeloid leukemia patient samples, serving as a relevant tool for drug screening, which could improve patient specific drug resistance testing for development of personalized medicine strategies in individual patients.