Follow-up of bone lesions in an experimental multiple myeloma mouse model: description of an in vivo technique using radiography dedicated for mammography.

The evolution of bone lesions in transplantable C57BL/KaLwRjj 5T mouse myeloma (MM) has been followed in vivo. Mice were anaesthetised and a radiograph of the pelvis and hind legs was performed by a radiograph dedicated for mammography. This is the first description of an in vivo technique under experimental conditions whereby the development of bone lesions owing to the MM growth was demonstrated

[89Zr]Oxinate4 for long-term in vivo cell tracking by positron emission tomography

Purpose 111In (typically as [111In]oxinate3) is a gold standard radiolabel for cell tracking in humans by scintigraphy. A long half-life positron-emitting radiolabel to serve the same purpose using positron emission tomography (PET) has long been sought. We aimed to develop an 89Zr PET tracer for cell labelling and compare it with [111In]oxinate3 single photon emission computed tomography (SPECT). Methods [89Zr]Oxinate4 was synthesised and its uptake and efflux were measured in vitro in three cell lines and in human leukocytes. The in vivo biodistribution of eGFP-5T33 murine myeloma cells labelled using [89Zr]oxinate4 or [111In]oxinate3 was monitored for up to 14 days. 89Zr retention by living radiolabelled eGFP-positive cells in vivo was monitored by FACS sorting of liver, spleen and bone marrow cells followed by gamma counting. Results Zr labelling was effective in all cell types with yields comparable with 111In labelling. Retention of 89Zr in cells in vitro after 24 h was significantly better (range 71 to >90 %) than 111In (43–52 %). eGFP-5T33 cells in vivo showed the same early biodistribution whether labelled with 111In or 89Zr (initial pulmonary accumulation followed by migration to liver, spleen and bone marrow), but later translocation of radioactivity to kidneys was much greater for 111In. In liver, spleen and bone marrow at least 92 % of 89Zr remained associated with eGFP-positive cells after 7 days in vivo. Conclusion [89Zr]Oxinate4 offers a potential solution to the emerging need for a long half-life PET tracer for cell tracking in vivo and deserves further evaluation of its effects on survival and behaviour of different cell types

Murine 5T multiple myeloma cells induce angiogenesis in vitro and in vivo

Multiple myeloma is a B cell malignancy. Recently, it has been demonstrated that bone marrow samples of patients with multiple myeloma display an enhanced angiogenesis. The mechanisms involved seem to be multiple and complex. We here demonstrate that the murine 5T multiple myeloma models are able to induce angiogenesis in vitro by using a rat aortic ring assay and in vivo by determining the microvessel density. The rat aortic rings cultured in 5T multiple myeloma conditioned medium exhibit a higher number of longer and more branched microvessels than the rings cultured in control medium. In bone marrow samples from 5T multiple myeloma diseased mice, a statistically significant increase of the microvessel density was observed when compared to bone marrow samples from age-matched controls. The angiogenic phenotype of both 5T multiple myeloma cells could be related, at least in part, to their capacity to produce vascular endothelial growth factor. These data clearly demonstrate that the 5T multiple myeloma models are good models to study angiogenesis in multiple myeloma and will allow to unravel the mechanisms of neovascularisation, as well as to test new putative inhibitors of angiogenesis

Antitumour and antiangiogenic effects of Aplidin® in the 5TMM syngeneic models of multiple myeloma

Aplidin® is an antitumour drug, currently undergoing phase II evaluation in different haematological and solid tumours. In this study, we analysed the antimyeloma effects of Aplidin in the syngeneic 5T33MM model, which is representable for the human disease. In vitro, Aplidin inhibited 5T33MMvv DNA synthesis with an IC50 of 3.87 nM. On cell-cycle progression, the drug induced an arrest in transition from G0/G1 to S phase, while Western blot showed a decreased cyclin D1 and CDK4 expression. Furthermore, Aplidin induced apoptosis by lowering the mitochondrial membrane potential, by inducing cytochrome c release and by activating caspase-9 and caspase-3. For the in vivo experiment, 5T33MM-injected C57Bl/KaLwRij mice were intraperitoneally treated with vehicle or Aplidin (90 μg kg−1 daily). Chronic treatment with Aplidin was well tolerated and reduced serum paraprotein concentration by 42% (P<0.001), while BM invasion with myeloma cells was decreased by 35% (P<0.001). Aplidin also reduced the myeloma-associated angiogenesis to basal values. This antiangiogenic effect was confirmed in vitro and explained by inhibition of endothelial cell proliferation and vessel formation. These data indicate that Aplidin is well tolerated in vivo and its antitumour and antiangiogenic effects support the use of the drug in multiple myeloma

Targeting angiogenesis in multiple myeloma by the VEGF and HGF blocking DARPin® protein MP0250: a preclinical study

The investigational drug MP0250 is a multi-specific DARPin® molecule that simultaneously binds and neutralizes VEGF and HGF with high specificity and affinity. Here we studied the antiangiogenic effects of the MP0250 in multiple myeloma (MM). In endothelial cells (EC) isolated from bone marrow (BM) of MM patients (MMEC) MP0250 reduces VEGFR2 and cMet phosphorylation and affects their downstream signaling cascades. MP0250 influences the secretory profile of MMEC and inhibits their in vitro angiogenic activities (spontaneous and chemotactic migration, adhesion, spreading and capillarogenesis). Compared to anti-VEGF or anti-HGF neutralizing mAbs, MP0250 strongly reduces capillary network formation and vessel-sprouting in a Matrigel angiogenesis assay. MP0250 potentiates the effect of bortezomib in the same in vitro setting. It significantly reduces the number of newly formed vessels in the choriollantoic membrane assay (CAM) and the Matrigel plug assay. In the syngeneic 5T33MM tumor model, MP0250 decreases the microvessel density (MVD) and the combination MP0250/bortezomib lowers the percentage of idiotype positive cells and the serum levels of M-protein. Overall results define MP0250 as a strong antiangiogenic agent with potential as a novel combination drug for treatment of MM patients

Epigenetic modifiers: anti-neoplastic drugs with immunomodulating potential

Cancer cells are under the surveillance of the host immune system. Nevertheless, a number of immunosuppressive mechanisms allow tumors to escape protective responses and impose immune tolerance. Epigenetic alterations are central to cancer cell biology and cancer immune evasion. Accordingly, epigenetic modulating agents (EMAs) are being exploited as anti-neoplastic and immunomodulatory agents to restore immunological fitness. By simultaneously acting on cancer cells, e.g. by changing expression of tumor antigens, immune checkpoints, chemokines or innate defense pathways, and on immune cells, e.g. by remodeling the tumor stroma or enhancing effector cell functionality, EMAs can indeed overcome peripheral tolerance to transformed cells. Therefore, combinations of EMAs with chemo- or immunotherapy have become interesting strategies to fight cancer. Here we review several examples of epigenetic changes critical for immune cell functions and tumor-immune evasion and of the use of EMAs in promoting anti-tumor immunity. Finally, we provide our perspective on how EMAs could represent a game changer for combinatorial therapies and the clinical management of cancer

RPL5 on 1p22.1 is recurrently deleted in multiple myeloma and its expression is linked to bortezomib response

Chromosomal region 1p22 is deleted in 6520% of multiple myeloma (MM) patients, suggesting the presence of an unidentified tumor suppressor. Using high-resolution genomic profiling, we delimit a 58 kb minimal deleted region (MDR) on 1p22.1 encompassing two genes: ectopic viral integration site 5 (EVI5) and ribosomal protein L5 (RPL5). Low mRNA expression of EVI5 and RPL5 was associated with worse survival in diagnostic cases. Patients with 1p22 deletion had lower mRNA expression of EVI5 and RPL5, however, 1p22 deletion status is a bad predictor of RPL5 expression in some cases, suggesting that other mechanisms downregulate RPL5 expression. Interestingly, RPL5 but not EVI5 mRNA levels were significantly lower in relapsed patients responding to bortezomib and; both in newly diagnosed and relapsed patients, bortezomib treatment could overcome their bad prognosis by raising their progression-free survival to equal that of patients with high RPL5 expression. In conclusion, our genetic data restrict the MDR on 1p22 to EVI5 and RPL5 and although the role of these genes in promoting MM progression remains to be determined, we identify RPL5 mRNA expression as a biomarker for initial response to bortezomib in relapsed patients and subsequent survival benefit after long-term treatment in newly diagnosed and relapsed patients

Chemokine receptor CCR2 is expressed by human multiple myeloma cells and mediates migration to bone marrow stromal cell-produced monocyte chemotactic proteins MCP-1, -2 and -3

The restricted bone marrow (BM) localisation of multiple myeloma (MM) cells most likely results from a specific homing influenced by chemotactic factors, combined with the proper signals for growth and survival provided by the BM microenvironment. In analogy to the migration and homing of normal lymphocytes, one can hypothesise that the BM homing of MM cells is mediated by a multistep process, involving the concerted action of adhesion molecules and chemokines. In this study, we report that primary MM cells and myeloma derived cell lines (Karpas, LP-1 and MM5.1) express the chemokine receptor CCR2. In addition, we found that the monocyte chemotactic proteins (MCPs) MCP-1, -2 and -3, three chemokines acting as prominent ligands for CCR2, are produced by stromal cells, cultured from normal and MM BM samples. Conditioned medium (CM) from BM stromal cells, as well as MCP-1, -2 and -3, act as chemoattractants for human MM cells. Moreover, a blocking antibody against CCR2, as well as a combination of neutralizing antibodies against MCP-1, -2 and -3, significantly reduced the migration of human MM cells to BM stromal cell CM. The results obtained in this study indicate the involvement of CCR2 and the MCPs in the BM homing of human MM cells. (C) 2003 Cancer Research UK