Reconstructing the human hematopoietic niche in immunodeficient mice: opportunities for studying primary multiple myeloma.

Interactions within the hematopoietic niche in the BM microenvironment are essential for maintenance of the stem cell pool. In addition, this niche is thought to serve as a sanctuary site for malignant progenitors during chemotherapy. Therapy resistance induced by interactions with the BM microenvironment is a major drawback in the treatment of hematologic malignancies and bone-metastasizing solid tumors. To date, studying these interactions was hampered by the lack of adequate in vivo models that simulate the human situation. In the present study, we describe a unique human-mouse hybrid model that allows engraftment and outgrowth of normal and malignant hematopoietic progenitors by implementing a technology for generating a human bone environment. Using luciferase gene marking of patient-derived multiple myeloma cells and bioluminescent imaging, we were able to follow pMM cells outgrowth and to visualize the effect of treatment. Therapeutic interventions in this model resulted in equivalent drug responses as observed in the corresponding patients. This novel human-mouse hybrid model creates unprecedented opportunities to investigate species-specific microenvironmental influences on normal and malignant hematopoietic development, and to develop and personalize cancer treatment strategies.

Targeting EXT1 reveals a crucial role for heparan sulfate in the growth of multiple myeloma.

Expression of the heparan sulfate proteoglycan syndecan-1 is a hallmark of both normal and multiple myeloma (MM) plasma cells. Syndecan-1 could affect plasma cell fate by strengthening integrin-mediated adhesion via its core protein and/or by accommodating and presenting soluble factors via its HS side chains. Here, we show that inducible RNAi-mediated knockdown of syndecan-1 in human MM cells leads to reduced growth rates and a strong increase of apoptosis. Importantly, knockdown of EXT1, a copolymerase critical for HS chain biosynthesis, had similar effects. Using an innovative myeloma xenotransplantation model in Rag-2(-/-)gamma(c)(-/-) mice, we demonstrate that induction of EXT1 knockdown in vivo dramatically suppresses the growth of bone marrow localized myeloma. Our findings provide direct evidence that the HS chains of syndecan-1 are crucial for the growth and survival of MM cells within the bone marrow environment, and indicate the HS biosynthesis machinery as a potential treatment target in MM.

N-cadherin-mediated interaction with multiple myeloma cells inhibits osteoblast differentiation.

BACKGROUND: Multiple myeloma is a hematologic malignancy characterized by a clonal expansion of malignant plasma cells in the bone marrow, which is accompanied by the development of osteolytic lesions and/or diffuse osteopenia. The intricate bi-directional interaction with the bone marrow microenvironment plays a critical role in sustaining the growth and survival of myeloma cells during tumor progression. Identification and functional analysis of the (adhesion) molecules involved in this interaction will provide important insights into the pathogenesis of multiple myeloma. DESIGN AND METHODS: Multiple myeloma cell lines and patients' samples were analyzed for expression of the adhesion molecule N-cadherin by immunoblotting, flow cytometry, immunofluorescence microscopy, immunohistochemistry and expression microarray. In addition, by means of blocking antibodies and inducible RNA interference we studied the functional consequence of N-cadherin expression for the myeloma cells, by analysis of adhesion, migration and growth, and for the bone marrow microenvironment, by analysis of osteogenic differentiation. RESULTS: The malignant plasma cells in approximately half of the multiple myeloma patients, belonging to specific genetic subgroups, aberrantly expressed the homophilic adhesion molecule N-cad-herin. N-cadherin-mediated cell-substrate or homotypic cell-cell adhesion did not contribute to myeloma cell growth in vitro. However, N-cadherin directly mediated the bone marrow localization/retention of myeloma cells in vivo, and facilitated a close interaction between myeloma cells and N-cadherin-positive osteoblasts. Furthermore, this N-cadherin-mediated interaction contributed to the ability of myeloma cells to inhibit osteoblastogenesis. CONCLUSIONS: Taken together, our data show that myeloma cells frequently display aberrant expression of N-cadherin and that N-cadherin mediates the interaction of myeloma cells with the bone marrow microenvironment, in particular the osteoblasts. This N-cadherin-mediated interaction inhibits osteoblast differentiation and may play an important role in the pathogenesis of myeloma bone disease.

HuMab-7D8, a monoclonal antibody directed against the membrane-proximal small loop epitope of CD20 can effectively eliminate CD20 low expressing tumor cells that resist rituximab-mediated lysis.

BACKGROUND: Incorporation of the chimeric CD20 monoclonal antibody rituximab in the treatment schedule of patients with non-Hodgkin's lymphoma has significantly improved outcome. Despite this success, about half of the patients do not respond to treatment or suffer from a relapse and additional therapy is required. A low CD20-expression level may in part be responsible for resistance against rituximab. We therefore investigated whether the CD20-expression level related resistance to rituximab could be overcome by a new group of CD20 mAbs (HuMab-7D8 and ofatumumab) targeting a unique membrane-proximal epitope on the CD20 molecule. DESIGN AND METHODS: By retroviral transduction of the CD20 gene into CD20-negative cells and clonal selection of transduced cells a system was developed in which the CD20-expression level is the only variable. These CD20 transduced cells were used to study the impact of rituximab and HuMab-7D8 mediated complement-dependent cytotoxicity. To study the in vivo efficacy of these mAbs an in vivo imaging system was generated by retroviral expression of the luciferase gene in the CD20-positive cells. RESULTS: We show that HuMab-7D8 efficiently killed CD20(low) cells that are not susceptible to rituximab-induced killing in vitro. In a mouse xenograft model, we observed a comparable increase in survival time between HuMab-7D8 and rituximab-treated mice. Most significantly, however, HuMab-7D8 eradicated all CD20-expressing cells both in the periphery as well as in the bone marrow whereas after rituximab treatment CD20(low) cells survived. CONCLUSIONS: Cells that are insensitive to in vitro and in vivo killing by rituximab as the result of their low CD20-expression profile may be efficiently killed by an antibody against the membrane-proximal epitope on CD20. Such antibodies should, therefore, be explored to overcome rituximab resistance in the clinic.

Correlation of high-resolution X-ray micro-computed tomography with bioluminescence imaging of multiple myeloma growth in a xenograft mouse model.

Multiple myeloma (MM) is an incurable B-cell neoplasia in which progressive skeletal lesions are a characteristic feature. Earlier we established an animal model for human MM in the immune-deficient RAG2(-/-)gammac(-/-) mouse, in which the growth of luciferase-transduced MM cells was visualized using noninvasive bioluminescence imaging (BLI). This model appeared well suited to study disease progression and response to therapy by identifying the location of various foci of MM tumor growth scattered throughout the skeleton and at subsequent time points the quantitative assessment of the tumor load by using BLI. We report here on the corresponding high-resolution X-ray micro-computed tomographic (micro-CT) analysis to study skeletal defects in the mice with full-blown MM. Several anatomical derangements were observed, including abnormalities in geometry and morphology, asymmetrical bone structures, decreased overall density in the remaining bone, loss of trabecular bone mass, destruction of the inner microarchitecture, as well as cortical perforations. Using the combination of BLI, micro-CT imaging, and immune-histopathological techniques, we found a high correlation between the micro-CT-identified lesions, exact tumor location, and infiltration leading to structural lesions and local bone deformation. This confirms that this animal model strongly resembles human MM and has the potential for studying the biology of MM growth and for preclinical testing of novel therapies for MM and for repair of MM-induced bone lesions.

A bioluminescence imaging based in vivo model for preclinical testing of novel cellular immunotherapy strategies to improve the graft-versus-myeloma effect.

BACKGROUND: The development and preclinical testing of novel immunotherapy strategies for multiple myeloma can benefit substantially from a humanized animal model that enables quantitative real-time monitoring of tumor progression. Here we have explored the feasibility of establishing such a model in immunodeficient RAG2(-/-)gammac(-/-) mice, by utilizing non-invasive bioluminescent imaging for real-time monitoring of multiple myeloma cell growth. DESIGN AND METHODS: Seven multiple myeloma cell lines, marked with a green fluorescent protein firefly luciferase fusion gene, were intravenously injected into RAG2(-/-)gammac(-/-) mice. Tumor localization and outgrowth was monitored by bioluminescent imaging. The sensitivity of this imaging technique was compared to that of free immumoglobulin light chain -based myeloma monitoring. Established tumors were treated with radiotherapy or with allogeneic peripheral blood mononuclear cell infusions to evaluate the application areas of the model. RESULTS: Five out of seven tested multiple myeloma cell lines progressed as myeloma-like tumors predominantly in the bone marrow; the two other lines showed additional growth in soft tissues. In our model bioluminescent imaging appeared superior to free light chain-based monitoring and also allowed semi-quantitative monitoring of individual foci of multiple myeloma. Tumors treated with radiotherapy showed temporary regression. However, infusion of allogeneic peripheral blood mononuclear cells resulted in the development of xenogeneic graft-versus-host-disease and a powerful cell dose-dependent graft-versus-myeloma effect, resulting in complete eradication of tumors, depending on the in vitro immunogenicity of the inoculated multiple myeloma cells. CONCLUSIONS: Our results indicate that this new model allows convenient and sensitive real-time monitoring of cellular approaches for immunotherapy of multiple myeloma-like tumors with different immunogenicities. This model, therefore, allows comprehensive preclinical evaluation of novel combination therapies for multiple myeloma.

Spatial distribution and survival of human and goat mesenchymal stromal cells on hydroxyapatite and ß-tricalcium phosphate.

The combination of scaffolds and mesenchymal stromal cells (MSCs) is a promising approach in bone tissue engineering (BTE). Knowledge on the survival, outgrowth and bone-forming capacity of MSCs in vivo is limited. Bioluminescence imaging (BLI), histomorphometry and immunohistochemistry were combined to study the fate of gene-marked goat and human MSCs (gMSCs, hMSCs) on scaffolds with different osteoinductive properties. Luciferase-GFP-labelled MSCs were seeded on hydroxyapatite (HA) or ß-tricalcium phosphate (TCP), cultured for 7 days in vitro in osteogenic medium, implanted subcutaneously in immunodeficient mice and monitored with BLI for 6 weeks. The constructs were retrieved and processed for histomorphometry and detection of luciferase-positive cells (LPCs). For gMSCs, BLI revealed doubling of signal after 1 week, declining to 60% of input after 3 weeks and remaining constant until week 6. hMSCs showed a constant decrease of BLI signal to 25% of input, indicating no further expansion. Bone formation of gMSCs was two-fold higher on TCP than HA. hMSCs and gMSCs control samples produced equal amounts of bone on TCP. Upon transduction, there was a four-fold reduction in bone formation compared with untransduced hMSCs, and no bone was formed on HA. LPCs were detected at day 14, but were much less frequent at day 42. Striking differences were observed in spatial distribution. MSCs in TCP were found to be aligned and interconnected on the surface but were scattered in an unstructured fashion in HA. In conclusion, the spatial distribution of MSCs on the scaffold is critical for cell-scaffold-based BTE.

Enhanced engraftment of human cells in RAG2/gammac double-knockout mice after treatment with CL2MDP liposomes.

OBJECTIVE: The ability of human cells to repopulate the bone marrow of nonobese diabetic immunodeficient mice (NOD/SCID) is commonly used as a standard assay to quantify the primitive human hematopoietic stem cell population. We studied the applicability of the immunodeficient RAG2(-/-)gammac(-/-) double-knockout mouse for this purpose. METHODS: RAG2(-/-)gammac(-/-) mice and NOD/SCID mice were injected intravenously (i.v.) with umbilical cord blood-derived CD34(+) cells and engraftment was quantified by determining the human CD45+ cell chimerism in bone marrow at several time points. RAG2(-/-)gammac(-/-) were pretreated with total-body irradiation and depleted of macrophages in liver, spleen, and bone marrow by i.v. injection of clodronate diphosphonate containing liposomes. RESULTS: We demonstrated that the frequency of chimerism and the level of engraftment in macrophage-depleted RAG2(-/-)gammac(-/-) largely resemble that in NOD/SCID mice. Also similar is the multilineage differentiation pattern in the two mouse strains at 7 weeks after transplantation, with a prominent outgrowth in RAG2(-/-)gammac(-/-) of CD19+ cells (88% +/- 10%). Cells of other lineages were clearly less frequent: 9% +/- 2% myeloid cells and 0.1% +/- 0.1% erythroid cells. As for immature progenitors, 6% +/- 1% of the human cells express the CD34 antigen and 0.4% +/- 0.1% have the CD34+,CD33,38,71(-) phenotype. The presence of human committed progenitors (i.e., CFU-GM/BFU-E) was evident. The persistence of human cells at 4 months after transplantation shows that the RAG2(-/-)gammac(-/-) support long-term maintenance of human hematopoiesis. CONCLUSION: Our findings indicate that macrophage-depleted RAG2(-/-)gammac(-/-) are a suitable model for studying human hematopoiesis including multipotential stem cells, and long-term repopulation.

In vitro induction of alkaline phosphatase levels predicts in vivo bone forming capacity of human bone marrow stromal cells.

One of the applications of bone marrow stromal cells (BMSCs) that are produced by ex vivo expansion is for use in in vivo bone tissue engineering. Cultured stromal cells are a mixture of cells at different stages of commitment and expansion capability, leading to a heterogeneous cell population that each time can differ in the potential to form in vivo bone. A parameter that predicts for in vivo bone forming capacity is thus far lacking. We employed single colony-derived BMSC cultures to identify such predictive parameters. Using limiting dilution, we have produced sixteen single CFU-F derived BMSC cultures from human bone marrow and found that only five of these formed bone in vivo. The single colony-derived BMSC strains were tested for proliferation, osteogenic-, adipogenic- and chondrogenic differentiation capacity and the expression of a variety of associated markers. The only robust predictors of in vivo bone forming capacity were the induction of alkaline phosphatase, (ALP) mRNA levels and ALP activity during in vitro osteogenic differentiation. The predictive value of in vitro ALP induction was confirmed by analyzing "bulk-cultured" BMSCs from various bone marrow biopsies. Our findings show that in BMSCs, the additional increase in ALP levels over basal levels during in vitro osteogenic differentiation is predictive of in vivo performance.

Brief report: Autologous stem cell transplantation restores immune tolerance in experimental arthritis by renewal and modulation of the Teff cell compartment.

OBJECTIVE: Autologous stem cell transplantation (ASCT) induces long-term drug-free disease remission in patients with juvenile idiopathic arthritis. This study was undertaken to further unravel the immunologic mechanisms underlying ASCT by using a mouse model of proteoglycan-induced arthritis (PGIA). METHODS: For initiation of PGIA, BALB/c mice received 2 intraperitoneal injections of human PG in a synthetic adjuvant on days 0 and 21. Five weeks after the first immunization, the mice were exposed to total body irradiation (7.5 Gy) and received (un)manipulated bone marrow (BM) grafts from mice with PGIA. Clinical scores, T cell reconstitution, (antigen-specific) T cell cytokine production, and intracellular cytokine expression were determined following autologous BM transplantation (ABMT). RESULTS: ABMT resulted in amelioration and stabilization of arthritis scores. BM grafts containing T cells and T cell-depleted grafts provided the same clinical benefit, with similar reductions in PG-induced T cell proliferation and the number of PG-specific autoantibodies. In vivo reexposure to PG did not exacerbate disease. Following ABMT, basal levels of disease-associated proinflammatory cytokines (interferon-γ [IFNγ], interleukin-17 [IL-17], and tumor necrosis factor α [TNFα]) were reduced. In addition, restimulation of T cells with PG induced a strong reduction in disease-associated proinflammatory cytokine production. Finally, although the remaining host T cells displayed a proinflammatory phenotype following ABMT, IFNγ, IL-17, and TNFα production by the newly reconstituted donor-derived T cells was significantly lower. CONCLUSION: Taken together, our data suggest that ABMT restores immune tolerance by renewal and modulation of the Teff cell compartment, leading to a strong reduction in proinflammatory (self antigen-specific) T cell cytokine production.

Human regulatory T cells do not suppress the antitumor immunity in the bone marrow: a role for bone marrow stromal cells in neutralizing regulatory T cells.

PURPOSE: Regulatory T cells (Tregs) are potent tools to prevent graft-versus-host disease (GVHD) induced after allogeneic stem cell transplantation or donor lymphocyte infusions. Toward clinical application of Tregs for GVHD treatment, we investigated the impact of Tregs on the therapeutic graft-versus-tumor (GVT) effect against human multiple myeloma tumors with various immunogenicities, progression rates, and localizations in a humanized murine model. EXPERIMENTAL DESIGN: Immunodeficient Rag2(-/-)γc(-/-) mice, bearing various human multiple myeloma tumors, were treated with human peripheral blood mononuclear cell (PBMC) alone or together with autologous ex vivo cultured Tregs. Mice were analyzed for the in vivo engraftment, homing of T-cell subsets, development of GVHD and GVT. In additional in vitro assays, Tregs that were cultured together with bone marrow stromal cells were analyzed for phenotype and functions. RESULTS: Treatment with PBMC alone induced variable degrees of antitumor response, depending on the immunogenicity and the growth rate of the tumor. Coinfusion of Tregs did not impair the antitumor response against tumors residing within the bone marrow, irrespective of their immunogenicity or growth rates. In contrast, Tregs readily inhibited the antitumor effect against tumors growing outside the bone marrow. Exploring this remarkable phenomenon, we discovered that bone marrow stroma neutralizes the suppressive activity of Tregs in part via production of interleukin (IL)-1ß/IL-6. We furthermore found in vitro and in vivo evidence of conversion of Tregs into IL-17-producing T cells in the bone marrow environment. CONCLUSIONS: These results provide new insights into the Treg immunobiology and indicate the conditional benefits of future Treg-based therapies.

Prospective isolation of mesenchymal stem cells from multiple mammalian species using cross-reacting anti-human monoclonal antibodies.

Mesenchymal stem cells (MSCs) of human and nonhuman mammalian species are often studied for various applications in regenerative medicine research. These MSCs can be derived from human bone marrow (BM) and identified by their ability to form fibroblast-like colony forming units that develop into stromal like cells when expanded in culture. These cells are characterized by their spindle-shaped morphology, their characteristic phenotype (CD73(+), CD90(+), CD105(+), CD45⁻, and CD34⁻), and their ability to differentiate into cells of the osteogenic, adipogenic, and chondrogenic lineages. However, the identification and purification of MSCs from nonhuman mammalian species is hampered by the lack of suitable monoclonal antibodies (mAb). In this report, primary BM and cultured BM-derived MSCs of human and monkey, goat, sheep, dog, and pig were screened for cross-reactivity using a panel of 43 mAb, of which 22 react with either human BM mononuclear cells or cultured human MSCs. We found 7 mAb with specificity for CD271, MSCA-1 (W8B2 antigen), W4A5, CD56, W3C4 (CD349), W5C4, and 58B1, which showed interspecies cross-reactivity. These mAb proved to be useful for prospective sorting of MSCs from the BM of the 6 mammalian species studied as well as for the characterization of their cultured offspring. Flow sorting with the cross-reacting mAb resulted in up to 2400-fold enrichment of the clonogenic cell fraction (fibroblast-like colony forming units). This study provides an important contribution for the comparative prospective isolation of primary BM-MSCs and the characterization of cultured MSCs from multiple mammalian species for preclinical research.

Bone-forming capacity of mesenchymal stromal cells when cultured in the presence of human platelet lysate as substitute for fetal bovine serum.

In tissue engineering, strategies are being developed to repair large bone defects by combining biomaterials and bone marrow-derived multipotent mesenchymal stromal cells (MSCs). For expansion of MSCs under good manufacturing practice conditions, human platelet lysate (PL) can serve as substitute for fetal bovine serum (FBS) in culture media. We compared the in vivo bone-forming capacity of passage 3 MSCs cultured with either PL or FBS for nine different human donors. We also tested the growth kinetics, antigen expression profile, and the multilineage differentiation capacity in vitro of these MSCs. The in vivo bone-forming capacity was determined by seeding culture-expanded MSCs onto biphasic calcium phosphate scaffolds. Hybrid constructs were implanted subcutaneously in nude mice, retrieved after 6 weeks, and analyzed using histomorphometry. PL-supplemented cultures resulted in significantly larger colonies, shorter culture time period, and higher population doublings between P1 and P3 compared to FBS-containing cultures. No differences were observed in antigen expression profiles or differentiation capacities into the osteoblastic, chondrogenic, and adipogenic lineages, qualitatively. In vivo bone formation with PL-supplemented cultures of MSCs was demonstrated in 9/9 donors versus 6/9 for FBS-supplemented cultures. These results warrant the use of PL for ex vivo expansion of human MSCs for bone tissue engineering applications.