MSCs from patients with hematological disorders
MSCs from the enrolled patients were expanded in culture to address the experimental questions posed in this manuscript. The morphology of Passage 3 MSCs from patients and healthy donors were similar (Figure 1A-Representative images). Similarly, they exhibited comparable phenotypes for the following cell surface markers: CD105+, CD73+ and CD45- (Figure 1B). The patients’ MSCs were multipotent, based on differentiation into osteogenic cells and neurons, as described (31) (Figure 1C).
MHC-II expression on patients’ MSCs
MSCs can mediate both anti- and pro-inflammatory responses, depending on the microenvironment (5, 13, 14, 32, 33). Proinflammatory functions include antigen presentation (13). We therefore examined the patients’ MSCs for MHC-II/HLA-DR by flow cytometry (Figure 1D). A subset of MSCs from healthy donors express MHC-II (7). MHC-II was not detected on MSCs from PV, MDS and CML BM whereas the two AML samples showed different MHC-II. This section indicated variation among patients’ MSCs with respect to MHC-II expression and highlights AML MSCs with MHC-II.
Third party patients’ MSCs in immune suppression
MSCs from AML patients show heterogeneity with respect to MHC-II expression whereas those from other patients analyzed in this study exhibit low MHC-II (Figure 1). Unlike macrophages in which IFNg is proportional to MHC-II expression, in MSCs, while low IFNγ induces MHC-II, high levels decrease MHC-II (31). In cases of high IFNg such as an inflammatory milieu caused by GvHD, MSCs can be licensed as veto cells (10, 34). We therefore asked if patients’ MSCs can be licensed as immune suppressor cells, similar to healthy MSCs. This was addressed by adding patients’ MSCs as third party cells in 2-way MLR.
First, we verified allogeneic difference between the responder and stimulator PBMCs in MLR assay without MSCs (Figure 2A, open bar). As third party cells in the 2-way MLR studies, healthy MSCs decreased the response by 4 folds, consistent with their reported veto property (10, 34). Similar studies with patients’ MSCs varied depending on the source - except for AML MSCs, which enhanced the proliferative response whereas other patients’ MSCs significantly (p<0.05) resulted in decreased stimulation indices (S.I.) (Figures 2A and 2B).
Patients’ MSCs within an inflammatory milieu caused by mitogen
We next asked if patients’ MSCs also suppress mitogenic response. This was addressed in PHA-stimulated PBMCs containing MSCs. Control cultures containing PBMCs and healthy MSCs without PHA (media) resulted in >5-fold S.I. (Figure 2C, far left bar). This was expected because MSCs were not added to an inflammatory milieu and the PBMCs responded to the allogeneic difference of healthy MSCs. Similar analyses with MSCs from MDS patient showed 2-fold S.I. which was significantly (p<0.05) less than the decreased by healthy MSCs (Figure 2C, left group). The reduced ability of MDS MSCs to elicit an allogeneic response was in line with undetectable MHC-II (Figure 1).
Mitogenic response using PHA alone showed S.I., ~20 (Figure 2C, open bar). After adding healthy MSCs to the PHA-stimulated PBMCs, the S.I. was reduced by ~50% and with MDS MSCs, by ~90% (Figure 2C, right group). Next, we tested AML and CML MSCs in PHA-stimulated PBMCs (Figure 2D). Control included PHA-stimulated cultures without MSCs (media) (open bar). MSCs from healthy individuals and from CML and MDS patients showed significantly (p<0.05) reduced S.I. Similarly, MSCs from AML and CML reduced the S.I. (right group). In summary, regardless of MHC-II expression, MSCs from AML, CML and MDS enhanced the immune suppressive licensing ability in the presence of PHA, as compared to healthy MSCs.
MSC distribution in BM biopsies from myelofibrosis (MF) patients
We noted enhanced immune suppressive licensing of MSC from AML, MDS and MPD patients (Figure 2). The question is whether MSC dysfunction is secondary to the respective hematological malignancies. Since patients with myeloproliferative neoplasms (MPNs) in particular MF, can transition to AML (35), we asked if MSCs are increased in MF BM. Particularly, increased MSCs in the BM of MF patients may contribute to enhanced angiogenesis and fibroblasts in MF BM and may contribute to the risk of these patients transitioning to leukemia (36). Indeed, increased angiogenesis in MF patients is in line with the role of MSCs in angiogenic processes – production of angiogenic factors, differentiation into endothelial cells and supporting the architecture of blood vessels as pericytes (37, 38).
We performed immunohistochemistry for CD31 (endothelial cells) and CD105 (MSCs or endothelial cells) with tissues from BM biopsies of patients with fibrosis (MF and other hematological disorder) or non-hematological disorders (Figure 3 and Table 2). There were few areas in which the red fluorescence (CD31) was in contact with green fluorescence (CD105), suggesting pericytes around blood vessels. In all cases including non-hematological disorder, there were single areas of green (CD105) with wide-spread CD31, indicative of increased angiogenesis in MF BM. A summary of the staining is summarized in the lower section (Figure 3), showing increased MSCs in patients with MF and idiopathic myelofibrosis (IMF) and to a lesser extend for slides from non-hematological disorders.
Loss of contact inhibition by AML MSCs
Flow cytometry for MHC-II on MSCs from patients’ BM indicated that AML MSCs, as compared to the other sources, showed varied MHC-II (Figure 1D). Regardless of MHC-II, (change this to a comma and delete the period) unlike healthy and other sources of MSCs, AML MSCs failed to exert veto property as third party cells in 2-way MLR (Figure 2A). Yet, they were able to inhibit the proliferation of PBMCs stimulated with the mitogen, PHA (Figure 1D). We therefore focused on AML MSCs due to their ability to support chemoresistance (39).
We asked if the patients’ MSCs at passage 5 showed evidence of transformation. All MSCs were seeded at the same time and then observed for time to achieve confluence. We noted rapid confluence by AML MSCs (Figure 4 – top right panel). Unlike AML MSCs, which showed evidence of transformation, MDS MSCs grew slowly with no evidence of transformation (Figure 4 third row). Others studies using a large cohort of AML MSCs observed no evidence of disease mutation but reported on increased clonogenic potential (40). This report is in line with the loss of contact differentiation, expected for healthy MSCs.
Phenotype of AML MSCs
Due to the loss of contact by AML MSCs, we asked if these cells are still phenotypically MSCs. We addressed this question with a panel of markers that are known to be present or absent on MSCs (Figure 5A). The latter included hematopoietic cells, myeloid and lymphoid markers and the former, CD90, CD44, CD73, CD105. In addition, we also evaluated for MHC-II expression. We evaluated AML MSCs for their ability to differentiate into osteogenic cells (Figure 5B). The results indicated phenotypic and functional MSCs, despite the loss of contact inhibition.
CD74-NFĸB axis in MSC function
MSCs expanded from AML patients MSCs were less efficient as veto cells, implying their inability to suppress T-cell activation (Figure 2). As compared to the other patients, MSCs from AML expressed MHC-II (Figures 1 and 5A). We therefore focused on the surface glycoprotein CD74 for the following reasons: it serves as the receptor for migration inhibitory factor (MIF) to mediate cancer progression (41); CD74 serves as the invariant chain for MHC-II and could induce intracellular signal transduction via second messengers to promote cell proliferation, noted for AML MSCs (Figure 5A). Thus, CD74 expression on AML MSCs may explain the increased proliferation as well as MHC-II expression. Indeed, similar to healthy subjects, flow cytometry indicated CD74 on AML MSCs (Figure 6A).
CD74 can induce intracellular signaling via NFĸB, ERK, and PI(3)K pathways to promote cell proliferation (42). Since NFĸB can also maintain MSC multipotency, we asked if inhibiting its activity could affect the function of MSCs (43). We therefore used a model of breast cancer cells due to their ability to interact with MSCs (44). Specifically, we asked if inhibiting NFĸB activity would interfere with their ability to interact with MSCs. We inhibited NFĸB activity in MSCs with a pharmacological Rel A inhibitor or with Rel A siRNA. Control MSCs were incubated with vehicle or scramble siRNA. After 24 h, MSCs were incubated with fluorescence labeled MDA-MB-231 breast cancer cells. After 48 h, we assessed cancer cell adhesion to MSCs by fluorescence scanning. We noted significant (p<0.05) decreases when NFĸB was inactivated (Figure 6B). In summary, the results support the involvement of CD74-NFĸB axis in MSC function.
Enhanced sensitivity by bortezomib and azacytidine on AML blasts
We isolated CD34+/CD38- cells from the blood of four patients with hematologic malignancies (CML, MDS transitioned to AML, AML, MF transitioned to AML). We cultured the cells in the presence of azacytidine and/or bortezomib for 48 h and then accessed cell viability. Each of the four patient cells tested resulted in <5% viable cells as compared to a range of 56-93% after 3- and 5-day exposure with single drug (Table 3). The resistance to single drug occurred despite enhanced cell proliferation. The results indicated that bortezomib seems to prepare the patient’s CD34+CD38- cells to azacytidine treatment.