Multiple myeloma mesenchymal stromal cells: Contribution to myeloma bone disease and therapeutics

Table 1 Minimal criteria for mesenchymal stromal cell definition (International Society for Cellular Therapy)

Adherence to plastic surfaces in standard culture conditions
	Positive (> 95% +)	Negative (< 2% +)
Immunophenotype	CD105	CD45
	CD73	CD34
	CD90	CD14 or CD11b
		CD79a or CD19
		HLA-DR
In vitro differentiation to osteoblasts, adipocytes and chondroblasts (demonstrated by appropriate staining of cell cultures)

Table 2 Main similarities and differences between bone marrow mesenchymal stromal cells from myeloma patients and mesenchymal stromal cells from healthy donors

Study	Assay	Description
Similarities
Adipogenic differentiation	Oil O Red staining	Both pMSCs and dMSCs showed accumulation of lipid-rich vacuoles[109,194]
Chondrogenic differentiation	Toluidine Blue staining	Chondrogenic differentiation potential was similar between pMSCs and dMSCs[109]
Differences
Chromosomic alterations	CGH arrays, FISH	pMSCs did not carry the genomic abnormalities detectable in their correspondent myeloma cells[111,194,195]. Only pMSCs showed several non-recurrent chromosomal gains and losses (> 1 Mb size) and “hot-spot” regions with discrete (< 1 Mb) genomic alterations[195]
Gene expression profiling	Gene expression microarray	Among 145 differentially expressed genes between pMSCs and dMSCs, 46% accounted for tumor-microenvironment cross-talk. Functional assignment revealed their implication in tumor-support (e.g., GDF15), angiogenesis (e.g., ANGPTL4, PAI1, SCG2), and contribution to bone disease (e.g., NPR3, WISP1, EDG2)[111]. Even a distinct transcriptional pattern was found associated to the occurrence of bone lesions in pMSCs[113]
Immunophenotype	Flow cytometry	Although few significant differences in cell surface marker expression were found between dMSCs and pMSCs, the latter expressed reduced VCAM1 and fibronectin[196], and higher ICAM1[197] compared to dMSCs
Bone formation markers	qPCR, WB	Expression of bone formation markers (i.e., osteocalcin and osteopontin), master transcription factors of osteogenic differentiation (i.e., Runx2/Cbfa1 and Osterix) and TAZ (a Runx2/Cbfa1 transcriptional co-activator) was lower in pMSCs than in dMSCs[109]
Expression and secretion of growth factors/cytokines/ chemokines	RT-PCR, ELISA	Compared to dMSCs, pMSCs showed increased expression of IL-1β[111], IL-3[112], IL-6[111,112,194,198], IL-10[199], BAFF[199], GDF15[111,198], TNFα[112], TGFβ1[112,198], DKK1[111,121,198], RANKL[112], AREG[111], and decreased expression of TGFβ2, TGFβ3 and FasL[112]
Senescence profile	β-gal staining, propidium iodide DNA staining, qPCR	pMSCs showed an early senescence state compared to dMSCs, as assessed by increased expression of senescence-associated β-galactosidase, increased cell size and accumulation of cells in S phase[198]
Immunoability	Co-cultures of MSCs and lymphocytes or PBMCs	pMSCs exhibited reduced efficiency to suppress T-cell proliferation compared to that of dMSCs[112,194,198]
Angiogenic potential	qPCR, ELISA, tube formation assay	Angiogenic factors (bFGF, HGF and VEGF) were elevated in the CM of pMSCs compared to dMSCs. Besides, CM from pMSCs significantly promoted proliferation, chemotaxis and capillary formation of HUVECs compared to dMSCs[200]
Controversial points
Proliferation rate	Cell density, CFU-F	Whereas some studies did not find differences in CFU-F number and cell density between dMSCs and pMSCs[111], others found a deficient proliferative potential in pMSCs which could be partly explained by the reduced expression of receptors for several growth factors[121]
ALP expression and activity	BCIP-NBT staining and pNPP hydrolysis	ALP expression/activity did not differ between MSCs from both origins[111], whereas other authors found it was significantly reduced in pMSCs compared to dMSCs, with lowest levels in pMSCs from patients with bone lesions[110]
Matrix mineralization	Alizarin Red and Von Kossa staining	Some groups have reported a significative reduction of matrix mineralization by pMSCs relative to dMSCs[110,111,198], although others have not observed those differences[121,194]
Hematopoietic stem cell support	Long-term co-cultures	Some authors reported that the ability to support the growth of hematopoietic stem cells did not differ between dMSCs and pMSCs[111,194], whilst others found that pMSCs better supported CD34+ progenitor expansion[198]

pMSCs: Mesenchymal stromal cells from myeloma patients; dMSCs: Mesenchymal stromal cells from healthy donors; CGH: Comparative genomic hybridization; FISH: Fluorescence in situ hybridization; qPCR: Quantitative PCR; WB: Western blot; RT-PCR: Reverse transcription-PCR; PBMC: Peripheral blood mononuclear cells; CM: Conditioned media; HUVECs: Human umbilical vein endothelial cells; CFU-F: Colony forming unit-fibroblast assay; BCIP-NBT: Bromo-chloro-indolyl-phosphate and nitro blue tetrazolium staining; pNPP: p-nitrophenyl phosphate; ALP: Alkaline phosphatase; IL-3: Interleukin-3; TNFα: Tumor necrosis factor α; TGFβ1: Transforming growth factor β1; DKK1: Dickkopf-1; RANKL: Receptor activator of NFκB ligand; NFκB: Nuclear factor-κB.

Table 3 Therapeutic targets, bone anabolic drugs and preclinical/clinical studies in the context of myeloma bone disease or other bone diseases

Drug	Mechanism of action	Signaling pathway	Cell target	Preclinical studies	Phase of clinical trials
BHQ880	Neutralizing anti-DKK1 antibody	Wnt	MSC, MMPC	[122,123,125]	II[126,127]
Romosozumab (AMG785)	Neutralizing anti-sclerostin antibody		MSC	[79,83]	II (postmenopausal osteoporosis)[129]
LiCl	GSK3β inhibitor		MSC, MMPC	[131]	NA
DAPT	γ-secretase inhibitor	Notch	MSC, MMPC	[110,142]	NA
GSI15			OC, MMPC	[139]
Bortezomib and second generation PIs	Proteasome inhibitor	UPR	MSC, OC, MMPC	[179,180,201]	Bortezomib and carfilzomib: Approved Oprozomib: I/II Ixazomib: III
RAP-011 (mouse) Sotatercept/ACE-011 (human)	Decoy receptor neutralizing activin A	BMP	MSC, OC, MMPC	[61,62]	II[153,154]
SB431542	TGFβ inhibitor		MSC	[150]	NA
Ki26894			MSC
MLN3897	CCR1 antagonists	CCL3	MSC, OC, MMPC	[58,160]	NA
CCX721 (mouse) CCX354-C (human)			OC, MMPC	[157]	II (rheumatoid arthritis)[161]

MSC: Mesenchymal stromal cell; MMPC: Multiple myeloma plasma cell; OC: Osteoclast; NA: Not applicable; DKK1: Dickkopf-1; GSK3β: Glycogen synthase kinase 3β; DAPT: N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; TGFβ: Transforming growth factor β; UPR: Unfolded protein response; BMP: Bone morphogenetic protein; CCR1: Chemokine (C-C Motif) receptor 1; CCL3/MIP1α: Macrophage inflammatory protein 1-α.