A Novel Three-Dimensional Co-Culture System to Study Leukemia in the Bone Marrow Microenvironment
Authors: Salman Zeena , Balandrán-Juárez Juan Carlos , Pelayo Rosana , Guzman Monica L. 
Published: October 2019

Abstract
The need for novel therapies in acute leukemia has been motivated by sub-optimal 5-year survival rates of 25.4% in acute myeloid leukemia (AML) and approximately 70% in acute lymphoblastic leukemia (ALL). While these rates are higher in the pediatric population, novel approaches are necessary in all age groups to improve outcomes.
Pre-clinical studies of novel therapeutics using in vitro and in vivo methods remain suboptimal with frequent lack of correlation with clinical outcomes at the bedside. Recent evidence has shown that human leukemia xenografts into immunodeficient mice yield variable results, indicating that treatment using these methods is not replicable. When using in vitro cell culture methods, the well-documented protective effects of the bone marrow (BM) microenvironment (BMME) on leukemia are not mimicked. Furthermore, these techniques cannot be used to investigate the effects of novel agents on leukemia stem cells (LSC) and their mobilization, which is important in the ablation of leukemia. Thus, we explored a novel 3-dimensional co-culture system to study the effects of drugs on leukemia cells in the presence of stroma in an environment more similar to that of human leukemia in the BMME.
We generated a 3-dimensional (3D) spheroid co-culture system using human stromal cell line (HS-5) cells or human mesenchymal stromal cells (hBMSC) from primary AML or ALL BM. To evaluate the dynamics of the 3D system, we labeled the stroma cells with GFP and the leukemia cells with mCherry. We observed rapid homing to the center of the 3D stroma. We evaluated ROS levels, proliferation status, hypoxia and expression of key niche proteins such as CXCL12 in leukemia cells found outside and inside the 3D system. These methods were compared to similar treatments in leukemia cell monolayer culture and 2-dimensional co-culture systems.
We treated this system with various drugs such as cytarabine, doxorubicin, TG02 (a multi-kinase inhibitor with LSC mobilization effects), and plerixafor; we then harvested cells from the outer and inner layers and evaluated these separately by multi-parameter flow cytometry for viability and mobilization of LSCs in relation to the stroma and xenotransplant assays.
Our studies reveal that the 3D culture system has lower ROS internally, suggesting a similarly hypoxic environment to BMME. Our studies also reveal that, when treated with cytarabine, AML cells closest to the stromal center of the spheroid remain protected, with higher viability compared to those farther from stroma, and even more so than leukemia cells in a 2-D bilayer with stroma or in a monolayer. A lower CXCL12 level was also observed in the stroma of leukemic BM compared to healthy BM within the co-culture system.
This culture method possesses many of the characteristics of leukemia cells within the bone marrow niche and should be considered for future in vitro pre-clinical drug testing to model the tumor within its microenvironment.

Disclosures
No relevant conflicts of interest to declare.