Sabino Ciavarella

Mesenchymal Stem Cells: A New Promise in Anticancer Therapy

Novel cell-based and gene therapies represent promising approaches for the treatment of incurable diseases, including cancer. Following the success of the hematopoietic stem cell-based transplantation, other populations of adult progenitor cells, including mesenchymal stem cells (MSCs), have been identified as powerful therapeutic tools in humans. The intrinsic capability of MSCs to migrate toward injured tissues emphasizes their suitability to deliver anticancer agents for new clinical applications in addition to the tissue repairing capacity. Here, we revisit the experimental history of MSCs, the most exciting features of their biology in keeping with their promising applications in cell-based therapeutic strategies for cancer treatment.

Targeted Therapies in Cancer

Recent advances in understanding the biologic mechanisms underlying cancer development have driven the design of new therapeutic approaches, termed ‘targeted therapies’, that selectively interfere with molecules or pathways involved in tumor growth and progression. Inactivation of growth factors and their receptors on tumor cells as well as the inhibition of oncogenic tyrosine kinase pathways and the inhibition of molecules that control specific functions in cancer cells constitute the main rational bases of new cancer treatments tailored for individual patients. Small-molecule inhibitors and monoclonal antibodies are major components of these targeted approaches for a number of human malignancies. As the studies of the biomolecular features of cancer progress, new exciting strategies have arisen, such as targeting cancer stem cells that drive tumor relapses or the selective induction of apoptosis in malignant cells. This article primarily focuses on the biologic bases of the new cancer drugs and summarizes their mechanisms of action, the clinical evidence of their anti-cancer effectiveness as well as the rationale for their use in clinical practice.

Oversecretion of cytokines and chemokines in lupus nephritis is regulated by intraparenchymal dendritic cells: a review.

Lupus nephritis (LN) occurs in more than one‐third of patients with systemic lupus erythematosus. Its pathogenesis is attributed to the glomerular deposition of immune complexes as well as to imbalance of the cytokine homeostasis. In this context, high production of cytokines and chemokines by dendritic cells (DCs) may concur to LN. In addition, urinary cytokine excretion may reflect the accumulation of DCs within glomeruli. DCs are differentiated in both myeloid and plasmacytoid (p) subsets in relation to their typical antigen and chemokine expression. Both subsets migrate in response to chemotactic stimuli because pDCs are susceptible to IL‐18 expressed by resident glomerular cells. pDCs bear the IL‐18R, and it is conceivable that DCs migrate to the kidney under the attraction of IL‐18. Therefore, the depletion of DCs reflects the inflammation severity in LN, whereas measurement of Th1 cytokines may represent an effective tool for monitoring the onset of LN.

In vitro anti-myeloma activity of TRAIL-expressing adipose-derived mesenchymal stem cells.

Recently, genetically modified mesenchymal stem cells (MSCs) have been exploited to deliver anti‐cancer bio‐drugs directly within the tumour mass. Here, we explored whether adipose‐derived MSCs (AD‐MSCs), engineered to express the pro‐apoptotic ligand TRAIL (also known as TNFSF10), kill multiple myeloma (MM) cells and migrate towards MM cells in vitro. Different MM cell lines were assessed for their sensitivity to recombinant human (rh) TRAIL alone and in combination with the proteasome inhibitor bortezomib, which was shown to enhance the effect of rhTRAIL. TRAIL+‐AD‐MSCs were co‐cultured with bortezomib‐pretreated MM cells and their killing activity was evaluated in presence or absence of caspase inhibition. AD‐MSC migration towards media conditioned by both myeloma cells and myeloma bone fragments was also investigated. Despite moderate MM cell sensitivity to rhTRAIL, TRAIL+‐AD‐MSCs in combination with bortezomib significantly induced myeloma cell death. This effect was associated with caspase‐8 activation and abrogated by capsase inhibition. On the other hand, co‐culture experiments were performed to evaluate whether unmodified AD‐MSCs affect myeloma cell growth in vitro. AD‐MSCs appeared ineffective on myeloma cell growth and showed migratory capacity towards MM cells in vitro. These data emphasize the anti‐myeloma activity of TRAIL‐engineered AD‐MSCs and provide support for a future model of a cell‐based approach against MM.

Immature dendritic cells in multiple myeloma are prone to osteoclast-like differentiation through interleukin-17A stimulation

Interleukin 17A (IL17A), a cytokine involved in allergy, inflammation and osteoclastogenesis, was investigated in multiple myeloma (MM) to assess its role in the osteoclast (OC)‐like activity of marrow immature dendritic cells (iDCs). Comparing nine MM patients with control subjects affected by monoclonal gammopathy of undetermined significance, we found high IL17A expression in the marrow plasma of MM patients in parallel with its deposits within the stromal matrix. Increased expression of the IL17A receptor (IL17RA) was also found in primary myeloma iDCs, which underwent OC‐like transdifferentiation after IL17A stimulation. To assess the role of IL17A, we measured the activity of the IL17/IL17RA pathway in IL17A‐transdifferentiated iDCs and the expression of functional OC genes by Western blotting and real‐time polymerase chain reaction. These cells showed increased RNA transcription of genes enrolled in the maturation of OCs, while NFATC1 and FOS were induced by IL17A, independently of NFKB1 phosphorylation. Moreover, the concurrent phosphorylation of the Lip isoform of CEBPB and the down‐regulation of MAFB supported the activation of IL17RA pathway in OC‐like transdifferentiated iDCs that was apparently unrelated to TNFRSF11A signalling. These data emphasize the involvement of iDCs in MM hyperactive osteoclastogenesis and suggest that their bone resorption activity is also regulated, at least in vitro, by IL17RA.

Bone-Resorbing Cells in Multiple Myeloma: Osteoclasts, Myeloma Cell Polykaryons, or Both?

Myeloma bone disease (MBD) leads to progressive destruction of the skeleton and is the most severe cause of morbidity in multiple myeloma. Its pathogenetic mechanisms are not fully understood, though the current evidence points to osteoclast (OC) hyperactivity coupled with defective osteoblast function unable to counteract bone resorption. OCs are generated in bone marrow by myeloid progenitors through increased levels of receptor activator of nuclear factor kappaB ligand and M-CSF, whose intracellular pathways propagate signals that activate sequential transcription factors, resulting in the production of major OC enzymes that drive specific functions such as acidification and degradation of the bone matrix. Osteolytic lesions, however, are not characterized by massive OC content, whereas malignant plasma cells, which are usually present in a high number, may occur as large multinucleated cells. The possibility that myeloma cells fuse and generate polykaryons in vivo is suggested by the in vitro formation of multinuclear cells that express tartrate-resistant acid phosphatase and produce pits and erosive lacunae on experimental osteologic substrates. Further, the detection in vivo of polykaryons with chromosome translocations typical of myeloma cells lends support to the view that myeloma polykaryons may act as functional OCs and participate in the skeletal destruction by resorbing bone.

Bendamustine overcomes resistance to melphalan in myeloma cell lines by inducing cell death through mitotic catastrophe

Melphalan has been a mainstay of multiple myeloma (MM) therapy for many years. However, following treatment with this alkylator, malignant plasma cells usually escape both apoptosis and cell cycle control, and acquire drug-resistance resulting in tumor progression. Bendamustine is being used in MM patients refractory to conventional DNA-damaging agents, although the mechanisms driving this lack of cross-resistance are still undefined. Here, we investigated the molecular pathway of bendamustine-induced cell death in melphalan-sensitive and melphalan-resistant MM cell lines. Bendamustine affected cell survival resulting in secondary necrosis, and prompted cell death primarily through caspase-2 activation. Also, bendamustine blocked the cell cycle in the G2/M phase and induced micronucleation, erratic chromosome spreading and mitotic spindle perturbations in melphalan-resistant MM cells. In these cells, both Aurora kinase A (AURKA) and Polo-like kinase-1 (PLK-1), key components of the spindle-assembly checkpoint, were down-regulated following incubation with bendamustine, whereas levels of Cyclin B1 increased as a consequence of the prolonged mitotic arrest induced by the drug. These findings indicate that, at least in vitro, bendamustine drives cell death by promoting mitotic catastrophe in melphalan-resistant MM cells. Hence, activation of this alternative pathway of cell death may be a novel approach to the treatment of apoptosis-resistant myelomas.

Immature dendritic cells from patients with multiple myeloma are prone to osteoclast differentiation in vitro.

OBJECTIVE Recent studies demonstrated that the interactions of immature dendritic cells (iDCs) with myeloma cells enhance the clonogenic capacity of tumor cells while iDCs undergo osteoclast (OC) transformation. Here, we investigated these interactions as well as iDC behavior in terms of both migration and OC differentiation. MATERIALS AND METHODS We studied 12 patients with multiple myeloma (MM) and 5 with monoclonal gammopathy of undetermined significance. Chemokine receptors, tumor-mediated chemotaxis, and a proliferation-inducing ligand (APRIL) expression were investigated in iDCs, whereas receptor activator of nuclear factor κB ligand (RANKL) and transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) levels were measured in primary plasma cells. Furthermore, cocultures of myeloma cells with autologous iDCs were installed to verify OC differentiation of these cells. Finally, the role of RANK/RANKL in such OC differentiation was investigated by inhibiting this molecular pathway. RESULTS Peripheral and marrow iDCs from MM showed high CXCR4 expression and were augmented in bone marrow of MM patients with respect to monoclonal gammopathy of undetermined significance. Also, iDCs expressed APRIL, whereas RANKL and TACI were upregulated by malignant cells. The cellular contact of myeloma cells with iDCs enhanced the clonogenic effect on tumor growth, whereas iDCs were directly primed to undergo OC transformation. These iDCs, indeed, exerted typical bone resorption that was abrogated by disabling the RANK/RANKL pathway signals. By contrast, plasma cells from monoclonal gammopathy of undetermined significance patients were ineffective in transforming autologous iDCs. CONCLUSIONS Our results emphasize the marrow cross-talk of iDCs with myeloma cells as an additional mechanism that upregulates osteoclastogenesis in MM, and suggest that such a RANKL-mediated OC differentiation of iDCs observed in vitro may also occur in vivo.

Cell Fusion and Hyperactive Osteoclastogenesis in Multiple Myeloma

Multiple myeloma (MM) is a hematologic malignancy whose progression may account for uncontrolled osteoclastogenesis promoted by the malignant plasma cells within the marrow microenvironment. Osteoclasts are multinucleated cells derived from the fusion of myeloid progenitors such as monocytes/macrophages, in response to specific differentiation factors released within the marrow niche, that are significantly deregulated in MM. In this malignancy DC-STAMP, a major fusogen protein enrolled by pre-osteoclasts, is highly expressed by peripheral macrophages, whereas dendritic cells and myeloma plasma cells show high fusogenic susceptibility and under specific conditions transdifferentiate to osteoclasts. In particular, the malignant plasma cells, besides altered ploidy, expression of cancer stem cell phenotype and high metastasizing capability, are able to express phenotypic markers of osteclasts, namely the proteolytic enzymes for the bone matrix, and to activate the β3 transcriptional pathway leading to ERK1/2 phosphorylation and initiation of the bone resorbing activity. Thus, based on the imbalanced osteoclast formation and activity that involve cells constitutively uncommitted to osteoclast differentiation, both homotypic and heterotypic cell fusions in myeloma marrow microenvironment represent a major pathogenetic event that drives the development and progression of the skeleton devastation typical of the myeloma bone disease.

A Peculiar Molecular Profile of Umbilical Cord-Mesenchymal Stromal Cells Drives Their Inhibitory Effects on Multiple Myeloma Cell Growth and Tumor Progression

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are under intensive investigation in preclinical models of cytotherapies against cancer, including multiple myeloma (MM). However, the therapeutic use of stromal progenitors holds critical safety concerns due to their potential MM-supporting activity in vivo. Here, we explored whether MSCs from sources other than BM, such as adipose tissue (AD-MSCs) and umbilical cord (UC-MSCs), affect MM cell growth in comparison to either normal (nBM-MSCs) or myelomatous marrow MSCs (MM-BM-MSCs). Results from both proliferation and clonogenic assays indicated that, in contrast to nBM- and MM-BM-MSCs, both AD and particularly UC-MSCs significantly inhibit MM cell clonogenicity and growth in vitro. Furthermore, when co-injected with UC-MSCs into mice, RPMI-8226 MM cells formed smaller subcutaneous tumor masses, while peritumoral injections of the same MSC subtype significantly delayed the tumor burden growing in subcutaneous plasmocytoma-bearing mice. Finally, both microarrays and ELISA revealed different expression of several genes and soluble factors in UC-MSCs as compared with other MSCs. Our data suggest that UC-MSCs have a distinct molecular profile that correlates with their intrinsic anti-MM activity and emphasize the UCs as ideal sources of MSCs for future cell-based therapies against MM.