Mesangiogenic progenitor cells are forced toward the angiogenic fate, in multiple myeloma

Abstract

Multiple myeloma (MM) progresses mainly in the bone marrow where the involvement of a specific microenvironment plays a critical role in maintaining plasma cell growth, spread, and survival. In active disease, the switch from a pre-vascular/non-active phase to a vascular phase is coupled with the impairment of bone turnover. Previously, we have isolated Mesangiogenic Progenitor Cells (MPCs), a bone marrow population that showed mesengenic and angiogenic potential, both in vitro and in vivo. MPC differentiation into musculoskeletal tissue and their ability of sprouting angiogenesis are mutually exclusive, suggesting a role in the imbalancing of the microenvironment in multiple myeloma. MPCs from 32 bone marrow samples of multiple myeloma and 23 non-hematological patients were compared in terms of frequency, phenotype, mesengenic/angiogenic potential, and gene expression profile. Defective osteogenesis was recorded for MM-derived MPCs that showed longer angiogenic sprouting distances respect to non-hematological MPCs, retaining this capability after mesengenic induction. This altered MPCs differentiation potential was not detected in asymptomatic myelomatous disease. These in vitro experiments are suggestive of a forced angiogenic fate in MPCs isolated from MM patients, which also showed increased sprouting activity. Taking together our results suggest a possible role of these cells in the "angiogenic switch" in the MM micro-environment.

Keywords: angiogenesis; bone marrow microenvironment; mesangiogenic progenitor cells; multiple myeloma; osteogenesis.

Figure 1. Frequency of Pop#8 sub-population in MM patients.

(A) MPCs and haematopoietic progenitors were identified by flow cytometry as CD31^brightCD64^brightCD14^neg (black box) and CD34⁺CD45^dim (purple box), respectively. Malignant PCs were identified as CD138^brightCD45^dim (green box). (B) Frequency of Pop#8 was significantly lower in MM patients as compared to NH patients. (C) Pop#8 frequency negatively correlated with the percentage of PCs and (D) was associated to reduced haematopoiesis.

Figure 2. MPC frequency and differentiative potential in NH and MM patient.

(A) MPC frequency from primary cultures was found to be significantly lower in MM samples. (B) Under mesengenic stimuli no difference was found between MM and NH patient-derived MPCs, neither in growth curves. (C) After 21 days of terminal osteogenic differentiation, a reduced mineralized area was detected in MM patients indicating defective calcium deposition. (D, E) BTZM inhibited MM patient-derived MPC differentiation into P1-MSC at a difference with CLMDZ. (F) Under angiogenic stimuli sprouting from MPC 3D spheroids resulted in longer sprouting distances in MM samples. BTZM impaired sprouting in a dose-dependent manner. (^* p<0.05, ^** p<0.01, ^*** p<0.001)

Figure 3. Angiogenic potential after mesengenic induction.

(A) P1-MSCs from MM patients retained higher sprouting activity under angiogenic stimuli with respect to P1-MSCs from SMM and NH patients. (B) This residual angiogenic potential was lost after the final step of mesengenic differentiation to P2-MSCs. (scale bar = 200 µm)

Figure 4. Gene expression profile of P1-MSCs from NH and MM patients.

(A) Clustering gene expression analysis of samples from NH (blue font) and MM (red font) patients showed up-regulation of MSC-related genes (red dots) and down-regulation of MPC-related genes (green dots). (B) Single gene expression analysis revealed increased expression of DKK1 and EMCN in MM samples (red dots). Specific MPC markers showed increased expression in NH samples (green dots). Five-fold ratio of normalized fold expression was fixed as a threshold (red and green lines).