3D Tissue-Engineered Bone Marrow Cultures Induce Drug Resistance, De-Differentiation and Cytokine Expression Changes in Multiple Myeloma
Authors: De La Puente Pilar , Muz Barbara , Azab Feda , King Justin , Vij Ravi , Azab Abdel Kareem 
Published: October 2019

Abstract
INTRODUCTION:
Multiple myeloma (MM) is a hematological malignancy that remains incurable because most patients will eventually relapse or become refractory to the treatments. Although the treatments have improved, the major problem in MM is the resistance to therapy. The discrepancy between in vitro efficacy and clinical outcomes can be attributed to several limitations of the classic tissue culture drug screening models including: (1) most of the in vitro models use MM cell line cultures and neglect the vital role of the bone marrow (BM) microenvironment in MM progression, which promotes drug resistance. (2) The BM niche is a three-dimensional (3D) structure with a gradient of both oxygen and drug concentration as a function of distance from blood vessels. The classic two-dimensional (2D) in vitro tissue culture system cannot mimic oxygen and drug gradients in culture wells, making all cells highly oxygenated. Therefore, 2D cultures cannot accurately predict drug sensitivity in different parts of the BM niche due to lack of accurate effects throughout various tissue depths. The goal of this study is to develop an in vitro model that will allow for better evaluation of interactions of MM cells and their microenvironment in the BM niche in a 3D system and how these interactions may affect MM progression and drug resistance.
METHODS:
The 3D tissue-engineered bone marrow (3DTEBM)was formed through calcium cross-linking of BM supernatants from MM patients. MM cell lines (MM1s, H929 and RPMI) and BM microenvironment (BMM) components, including MM-derived BM stromal cells, HUVECs, and ECM component (fibronectin), were incorporated in 3DTEBM or 2D cultures. We tested by flow cytometry the growth of MM cell lines with and without BMM in 2D vs 3DTEBM cultures at 3 and 7 days. The effect of 3DTEBM and 2D cultures with and without BMM on cytokine expression was determined at day 3 by human cytokine antibody arrays. In addition, the effect of 3DTEBM and 2D cultures with and without BMM on CD markers expression was tested at day 3 by flow cytometry. Finally, drug uptake and hypoxia levels by MM cells in 3DTEBM of different depth of tissue and 2D cultures was determined using flow cytometry and immunohistochemistry, respectively. Then, drug resistance of MM cell lines with and without BMM in 2D vs 3DTEBM cultures after 24h drug treatment were analyzed by flow cytometry.
RESULTS:
We found that MM cells doubled within 3 days in 2D and 3DTEBM cultures with and without BMM, compared to day 0. In contrast, while in the 3DTEBM induced about 3-5-fold increase in 7 and 14 days, the 2D cultures showed lower growth. In addition, 3DTEBM expressed more cytokines than 2D media in absence of cells, and incorporation of MM cells and BMM induced higher cytokine expression in 3DTEBM than in 2D cultures of SDF-1, IL1-α, TNF-α, TNF-β, MIP-1-δ, PARC, angiopoietin, eotaxin 3 and osteoprotegin. MM cells expressed loss of the plasma cells markers (CD38, CD56, and CD138), mildly reduced or no changes of B cells markers (CD19, CD20 and CD22), and increased of the stem cell marker CD34 in 3DTEBM compared to 2D without BMM. Finally, we found that MM cells in 3DTEBM exhibited 2-fold less drug uptake than MM cells grown in 2D cultures, and drug uptake of MM cells grown in the 3DTEBM was inversely correlated with the depth of the tissue; with increasing tissue depth the drug uptake by MM cells decreased. Accordingly, MM cells in the 3DTEBM exhibited higher hypoxia levels (MFI of PIM) compared to MM cells grown in 2D cultures, and anti-HIF-1α staining revealed that the cells at the lower area were more hypoxic that the cells at the upper area of the 3DTEBM. Therefore, we found that 3DTEBM cultures induced drug resistance in MM cells; for the same drug concentration about 50 and 35% of the MM cells were killed in 2D, and about 15 and 5% of the cells were killed in 3DTEBM without and with BMM, respectively.
CONCLUSIONS:
Our results suggest that 3DTEBM cultures promoted tumor growth, enhanced cytokine expression, and induced de-differentiation of MM cells, and that a stem-cell-like phenotype might be developing. The 3DTEBM recreated hypoxia and drug gradients by reproducing tissue-specific structural features, so the 3DTEBM cultures induced drug resistance in MM cells more accurately than 2D. Therefore, the 3DTEBM is a promising model for the study of multiple myeloma biology, for in vitro examination of anti-myeloma drugs, tumor microenvironment and interactions between myeloma cells and the BMM.

Disclosures
No relevant conflicts of interest to declare.