Characterization of an In Vitro model of MGUS Progression to Multiple Myeloma
Authors: Lipe Brea , Wise Amanda , Lin Tara L. , Aljitawi Omar S. , Koestler Devon , Graw Stefan , Godwin Andrew 
Published: October 2019

Abstract
Introduction: Multiple Myeloma (MM) is an incurable cancer characterized by a pre-malignant clonal phase of disease called monoclonal gammopathy of undetermined significance (MGUS). Most patients with MGUS do not develop overt MM and the biology underlying this potential transformation is unclear. Investigations to prevent the development of MM from MGUS are limited by the relative infrequency of MGUS progression. Unfortunately, MGUS cells have historically proven difficult to grow in vitro because of slow rates of proliferation and difficulty in sustaining cell cultures. We present evidence of an in vitro model that generates MM-like plasma cells from patients diagnosed with only MGUS. We further present gene expression patterns of primary patient cells versus the induced MM cells to provide guidance as to important initiating events within our model.
Methods: We collected a CD38+ cell fraction and a mononuclear (CD38-) fraction from 4 patients with MGUS using a Miltenyl Biotec column Separator. The CD38- fraction was grown in RPMI with 10% FBS and 1% sodium pyruvate with or without a polyglycolic acid/ poly L-lactic acid 90/10 (PLGA) copolymer scaffold to create 3D culture conditions. The mononuclear layer from healthy donors and the MM cell line, U266, were grown as controls. We then analyzed the initial CD38+ fraction, the initial CD38- fraction, and the CD38- fraction grown in media or 3D co-culture by flow cytometry for expression of kappa, lambda, CD38, CD138, CD45, CD19, and CD56. Gene expression analysis was performed using RNA-sequencing data from the CD38+, CD38-, cultured CD38-, and control cells. Expression of the top 100 ranked differentially expressed genes, which demonstrated the largest variation, were further analyzed using the nCounter® Analysis System (NanoString Technologies).
Results: The CD38- fraction from MGUS patients grew into an adherent layer of elongated cells, consistent with bone marrow stromal cells. After several months, the stromal cells were noted to change shape and new, round cells were observed budding off from the stromal layer. Over time, the stromal layer disappeared and the round plasmacytoid cells remained. Characterization of these round cells revealed them to be plasma cells by IHC and flow cytometry. When comparing these in vitro generated cells to the initial CD38+ fraction removed from patients, the new cells showed the re-emergence of CD38 and CD138, increased expression of CD56 and CD19, and decreased expression of CD45. Gene expression analysis revealed 3 distinct populations of cells. The initial CD38- fraction separated with the healthy mononuclear layer. The initial CD38+ fraction clustered independently while the grown plasma cells clustered with the U266 cells. Analysis of the differential gene expression patterns revealed differences in the expression of immunoglobulin genes, as well as alterations in expression of extracellular matrix and cell adhesion markers including PAI-1, MMP2, COL1A2, and GREM1; and alterations in expression of mitochondrial genes.
Conclusion: To our knowledge, this is the first in vitro simulation of disease progression from MGUS to MM. Our model induced the growth of plasma cells with an aggressive phenotype as assayed by flow cytometry. The gene expression profile further demonstrates gene expression patterns from our induced plasma cells consistent with MM versus MGUS. The alterations in extracellular matrix proteins as seen in our induced plasma cells are consistent with an epithelial to mesenchymal type transition implicated in disease progression, metastasis, and bone lesions. Additionally, the alterations in mitochondrial gene expression have been implicated in early disease progression in colon cancer and MM. These findings provide further evidence that our model simulates disease transformation and the expression data suggest possible pathways that may be important in myeloma disease progression that can be further evaluated in vivo.

Disclosures
No relevant conflicts of interest to declare.