Abstract 325: Multiple myeloma in a physiologically relevant Me-HA -3D Hydrogel: Discovering new phenotypes of drug resistance
Authors: Narayanan Bhagavathi A. , Duan Bin , Narayanan Narayanan K. , Butcher Jonathan Butcher , Mazumder Amitabha 
Published: August 2015

Abstract
Background: Extracellular matrix (ECM) characteristics, including stiffness, geometry, chemistry, and spatial interaction with the neighboring cells and soluble factors are key components for cancer cell survival in a pathogenic tissue microenvironment (TME). However, poor performance of 2D in vitro systems and animal models for metastatic cancer types demands physiologically relevant well controlled 3D platforms to quantitatively assess (a) metastatic cancer stem cell survival (b) oncogenic mechanisms and (c) drug resistant phenotypes and (d) new drug efficacy. Advances in the “state of the art” 3D platforms with difference in matrix stiffness and viscoelastic properties are showing promising results in identifying changes in drug resistant phenotypes, cell behavior and gene expression profiles. Preliminary findings by us and others using 3D matrices for human multiple myeloma (MM) reveal promising results on cancer /stromal cell interactions. In this study we have shown that Hyaluronic acid (HA) based 3D hydrogel support human metastatic MM cancer cells survival and thus reveal new oncogenic mechanisms of drug resistant cancer stem cells.
Experimental approaches: Bone marrow derived CD138 positive cells and BMSCs were isolated from MM patient samples (with IRB approval) using magnetically labeled CD138 MicroBeads (autoMACS Pro Starting Kit). Bone marrow stromal cells (BMSCs) were cultured as described by us earlier. Cell viability measurements and quantitative RT-PCR for c-myc and other related gene targets were performed using total RNA. Me-HA-3D hydrogels were created containing either fibronectin/collagen/laminin or ME-HA mixture of Matrigel/fibronectin/laminin in different ratios. Cells were grown as surface seeded or encapsulated in Me-HA gels, and analyzed between 4 and 21 days in the conditioned medium. Cell proliferation analysis was performed by both MTS and live dead cell assays using CFSE stain.
Results: The overall cell viability by surface coating and in encapsulated gel was found to be higher than 80% for up to 21 days in all the conditions including changes in the UV cross linking time and gel stiffness. Colony forming units (CFU) of the mononuclear cells were higher (27%) at 21 days compared to the 4 days 3% (p&gt;0.001). Spheroids were found on the surface of the hydrogels with PC-3 cells as well as inside the encapsulated hydrogels specifically for MM, and were higher in numbers (&gt;30%) compared to that in surface coating (5%) p&gt;0.001. qRT-PCR analysis determined c-myc, cyclin D1, E-cadherin, and ZEB-1 showed&gt;4 fold increase (p&gt;0.001) in encapsulated cells compared to that in cells transfected with siRNA for c-myc, suggesting c-myc mediated cell survival in 3D.
Summary: Me-HA hydrogels are found to be suitable platform for the first time to identify new metastatic cancer phenotypes types and to investigate drug resistance mechanisms.
Citation Format: Bhagavathi A. Narayanan, Bin Duan, Narayanan K. Narayanan, Jonathan Butcher Butcher, Amitabha Mazumder. Multiple myeloma in a physiologically relevant Me-HA -3D Hydrogel: Discovering new phenotypes of drug resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 325. doi:10.1158/1538-7445.AM2015-325