MM is a malignancy of antibody-secreting plasma cells, where B-cell plasmacytomas stimulate osteoclast activity, and hence bone resorption, resulting in progressive osteolytic lesions . Based on studies concerning the pathogenic role of autoantibodies in MM diseases, recent advances in this field suggest a more central role for B cells in the maintenance of the disease process beyond their roles as precursors for (auto)antibody-producing plasma cells . Particularly, a number of surface molecules and subsequent downstream signaling pathways are involved in the regulation of MM-related bone destroying events, in which bone resorption and formation are no longer balanced as a consequence of the increased activity of osteoclasts, but rather the osteoblast activity is reduced, leading to an uncoupled, or severely imbalanced, bone remodeling process . Clinical data have shown that MM patients with advanced bone lesions might show a reduction of bone formation markers, such as alkaline phosphatase and osteocalcin, together with increased bone resorption markers, such as receptor activator of nuclear factor κB ligand (RANKL) and C-terminal cross-linked telopeptide of type I collagen . Similarly, marked osteoblastopenia and reduced bone formation have also been reported in murine models of MM bone disease . These studies demonstrate that MM cells suppress osteoblast formation and differentiation, and consequently inhibit bone formation.
Recent mounting evidence indicates that MM cells suppress osteoblastogenesis through contact-dependent cell–cell interaction [7, 34] and the production of osteoblast-inactivating factors including Wnt inhibitors, such as dickkopf-1  and secreted frizzled-related protein 2 , and cytokines, such as CCL3 (also known as macrophage inflammatory protein-1 alpha) , hepatocyte growth factor, and IL-3/6 . Osteolytic lesions in MM are only observed adjacent to intramedullary plasma cell foci or plasmacytomas, supporting the idea that MM cells might secrete factors that promote the activation of osteoclasts and the inactivation of osteoblast function to replace bone loss . More effective approaches to cure MM-related bone disease, in addition to the correction of osteoblast function, should therefore be reflected in therapeutic modalities aimed at inducing MM cell death.
Researchers in the stem cell field are working to translate the knowledge gained from stem cell biology and function into therapeutic breakthroughs and applications. It is well known that osteoblasts originate primarily from MSCs and are responsible for bone matrix synthesis through the secretion of collagen, which forms strands called osteoid . Osteoclast activity is regulated through the expression of cytokines, such as receptor activator of RANKL, which activates osteoclast differentiation, and osteoprotegerin, which acts as a decoy receptor and inhibits RANKL . Based on this knowledge, MSC-based cytotherapy has established a novel concept for the treatment of MM-related bone disease . Recently, Li and colleagues demonstrated that both systemic and intrabone cytotherapeutic strategies were effective and clinically applicable for treating MM-related bone disease , where weekly systemic injections of MSCs restrained MM disease progression through the ability of MSCs to traffic to myelomatous bone and survive for a short period of time . Intrabone injections of MSCs, however, not only inhibited tumor growth in the bone with active MM but also effectively promoted bone formation during disease, remission and delayed MM relapse. Although this study provides a proof-of-concept for the use of MSC cytotherapy to treat large, unhealed, osteolytic lesions and for the systemic inhibition of MM bone disease, the mechanisms of action by which MSC cytotherapy stimulates bone formation and inhibits MM-induced bone tumor growth, are, however, only partially understood . Whether MSCs inhibit or promote cancer growth has developed into a controversy reflected in concern over the use of MSCs, which exhibit a propensity to home to tumors. Once resident in the tumor microenvironment, these cells support tumor growth and spread , although the ability of cultured MSCs to support long-term growth of primary MM cells is often limited and not reproducible [40, 41]. Therefore, understanding the in vivo milieu in which MSCs either inhibit or enhance MM cell survival and metastasis is crucial both to safely develop MSCs as a therapeutic tool and to advance our understanding of the role of tumor stroma in carcinogenesis [16, 17]. Moreover, we still do not have a general consensus of what defines these MSCs; the polarization of MSCs into a proinflammatory or an immunosuppressive phenotype showing reversed effects on tumor growth has been observed [17, 42]. Similarly, in our present study, Fas-Lhigh MSCs showed significant inhibition, while Fas-Lnull MSCs showed promoted MM growth, suggesting that the levels of Fas-L expression in MSCs determine, at least in part, the effect of MSCs on cancer growth.
Recent findings suggest that the overexpression of growth differentiation factor 15 in bone marrow MSCs occurs widely in patients with MM, and tumor microenvironment-derived growth differentiation factor 15 is a key survival and chemoprotective factor for MM cells, indicating that the behavior of MSCs might be principally determined by the surrounding environment . The two side effects of MSCs on MM cells identified from previous studies are therefore basically acceptable. To further clarify the role of healthy MSCs in MM metastasis and apoptosis, we examined whether outside-infused MSCs would have therapeutic effects on MM cells in mice under co-culture conditions. The data obtained in the present study are clearly inconsistent with some reports, which have indicated that murine and human MSCs promote breast and coronal cancer growth and metastasis [44, 45]. Interestingly, Ma and colleagues showed that human umbilical cord MSCs significantly inhibited the growth of breast cancer cells in vitro and in vivo. Furthermore, the ability of cytotherapy through placenta-derived adherent cells to impact bone remodeling and increase bone formation in nonmyelomatous SCID-rab mice has been demonstrated . Intralesional mesenchymal cell cytotherapy also resulted in inhibiting growth of H929 MM cells and primary MM cells categorized through global gene expression as high risk. Moreover, placenta-derived adherent cells had no effect on the subcutaneous growth of H929 MM cells in SCID mice, and did not confer a growth advantage to MM cells co-cultured with placenta-derived adherent cells or supportive MSCs . Recently, adipose-derived MSCs, engineered to express the pro-apoptotic ligand TRAIL (also known as TNFSF10), killed MM cells and migrated towards MM cells in vitro. These results, together with data in the present study, support the idea that certain phenotypes of MSCs exhibit inhibitory effects on MM cells, such that the anti-myeloma activity of MSCs can be harnessed or enhanced, for example, via gene-modified approaches.
It has been well recognized that MSC therapy potentially offers novel therapeutic modalities that are translatable for clinical treatment of a large variety of pathological conditions or diseases . This development is also true for clinically managing and combating cancer, as MSCs play a central role in the pathogenesis and progression of tumors [5, 50]. MSC administration thus reduces solid tumor growth in mice due to an inhibition of tumor cell proliferation, probably resulting from deep modifications of the tumor angiogenesis, regardless of the tumor model and mode of MSC injection . Clinically, current evidence suggests that cytotherapy markedly increases the proportion of MSCs in bone of MM patients, at least for a short period of time [5, 15]. As deduced from in vitro studies, during this short time, the injected MSCs probably interact with endogenous osteoblast precursors and secrete factors that induce their differentiation into bone-building osteoblasts, while simultaneously directly interactions with osteoclast precursors to secrete factors that attenuate the formation of bone-resorbing osteoclasts . Notably, similar to osteoblasts, MSCs might produce a high level of decorin protein, which inhibits osteoclast formation and promotes osteoblast differentiation . Following the identification of the potential for MSCs to enhance engraftment of hematopoietic stem cells, increase osteoblast activity and suppress osteoclast activity , MSCs recruited hematopoietic elements that inhibit inflammatory conditions typically associated with MM growth in bone . Along with recent findings in this field [17, 42], it is speculated that MM progression is restrained, directly and indirectly, through anti-inflammatory factors produced by the injected MSCs or endogenous cells recruited to myelomatous bone after cytotherapy. The findings that MSCs express high levels of anti-inflammatory and antineoplastic factors, such as SERPINF1 and decorin, support this concept . Decorin also attenuates MM cell growth . Although certain soluble factors produced by MSCs might mediate part of their therapeutic activities, cytotherapy at a remote site (subcutaneous) was found to have no effect on MM bone disease or growth , suggesting that MSCs must be present in bone marrow to elicit their antimyeloma effects. Indeed, only MSCs injected directly into bone might efficiently induce an antimyeloma environment. Systemically injected MSCs significantly promote bone formation or restrain MM growth because relatively few of those MSCs can transmigrate and traffic to bone . Recent results, however, also suggest that MSCs might be attracted to bone through MM cells or conditions induced through MM or melphalan treatment. More importantly, MSCs might be cleared in various tissues, but exhibit higher survival rates in the implanted bone or lymph nodes and therefore could be detected in these tissues at 2 to 3 days after intravenous or intracardiac injections, respectively . The accumulation of MSCs in lymph nodes, however, might partially explain their immunomodulatory properties. In fact, evidence suggests that intravenously injected MSCs might localize in the lymph nodes of experimental mouse models of autoimmunity [53, 54]. This body of work might also explain the fewer numbers and smaller sizes of cancroid pearls in the neck and root tail of the MSC-treated MM mice in the present study.
Recent studies have revealed that exogenously injected MSCs were not detectable in vivo for long periods of time; the majority of these cells disappeared within 3 to 5 weeks [15, 55]. Clinically, this phenomenon might be advantageous because it limits the duration of the intervention, and these observations support the notion that most of their activities are mediated through the touch-and-go mechanisms of bystander cells, although proof of such evanescence is thus far not well defined [5, 56]. In support of using allogeneic MSCs for MM, Li and colleagues recently demonstrated that intralesionally injected human placenta mesenchymal cells exert similar therapeutic effects in SCID-rab mice . Together, these studies raise an intriguing possibility: if we could understand how MSCs induce MM cell death, then perhaps we could exogenously manipulate MSCs to effectively manage MM and saved a large number of lives. An important, yet unelucidated, question raised by our study is whether a majority of MSCs transmigrate to the myelomatous bone to kill MM cells after intravenous injection, or traffic to lymph nodes to exert inhibitory effects on MM cells via the secretion of anti-inflammatory factors.
The potential role of molecules involved in altered B-cell longevity, particularly those involved in apoptosis (for example, Fas/Fas-L modulators), and those that might alter activation thresholds of B cells in the development of autoimmunity, might contribute to the clinical management of MM [27, 31]. Unfortunately, however, we still know relatively little about this issue. Recently, a number of studies have reported the effects of the Fas/Fas-L pathway on fluoride-induced or melatonin-induced cell apoptosis [57, 58]. In the present study, it was shown that MSCs counterattack MM cells using the same mechanism as observed in other cancer cells; namely, Fas-mediated apoptosis. Fas and Fas-L are co-expressed on primary MSCs that might kill co-cultured MM cells. Co-expression of Fas and Fas-L by MSCs calls into question the functionality of the extrinsic apoptosis pathway in these cells. Although Mazar and colleagues detected Fas expression on MSCs, stimulation of Fas with different concentrations of anti-Fas antibody did not result in any apoptotic response . At this stage, we were not able to identify the exact molecular mechanism of Fas-mediated pathway inactivation in MSCs, but we might narrow this process to events between Fas protein trimerization and caspase-8 activation. As previously demonstrated, caspase-8 deficiency resulted in the inhibition of apoptosis of Jurkat cells, and blocking with Fas Fc protein prevented bone marrow MSC-induced apoptosis in 80% . Other studies have shown that the transformation of the intracellular domain of Fas protein expressed in human MSCs prevents the trimerization of the receptor and blocks the activation of apoptotic pathway activation . MM cells might thus be susceptible to the induction of apoptosis through MSCs.
Briefly, we showed that MSCs act directly on MM cells, inhibiting proliferation. MSC-induced apoptosis in MM cells is evidenced by an increase in the Annexin/7AAD-positive cell population. Most of this effect can be attributed to the Fas/Fas-L pathway. These results confirm and extend previous reports [60, 61]. In our MM model, the activation of both caspase-3 and caspase-8 was observed, suggesting that two main pathways of procaspase activation – the intrinsic mitochondrial pathway and the extrinsic death receptor pathway – are both involved in MSC-induced apoptosis of MM. Having determined the effects of MSCs activated through aspirin (with highly activated Fas-L) in the MM model mice, these MSCs resulted in a more effective clinical outcome compared with MSCs from the gld mice. We thus expected that MSCs with high Fas-L expression would be extremely effective in inhibiting MM growth and metastasis. These results are consistent in principle with those of another study, showing that infused MSCs moved immediately to the tumor site . However, the positive TUNEL reactions within MSCs from the gld mice were much lower than the others, suggesting that MSCs without Fas-L have no capacity to kill MM cells.