Page 171 - Haematologica-April 2018

3D reconstructed myeloma microenvironments
leukemia (PCL) despite several lines of therapy (Figure 7B). At progression, a BM biopsy was performed and MM cells were cultured in bioreactor (Figure 7B). MM cell proliferation was confirmed by the high frequency of Ki67+ cells at IHC analysis of the scaffold, which mir- rored that observed in the BM biopsy (Figure 7C) and was paralleled by β2-microglobulin levels in super- natants (Figure 7A). Given the unique proliferative behavior of the patient’s MM cells, we compared genom- ic changes occurring over time in vivo and ex vivo. FISH analysis performed on MM cells retrieved at the end of 3D culture identified cells carrying the 13q14.3 deletion; no deletions of ATM or p53 loci could be detected (Figure 7D, upper panels), nor IGH rearrangements, indicating the absence of the t(4;14) translocation. Cells expressing the 13q14.3 deletion represented 46% of all MM cells at the beginning of culture and became 70% after 14-day 3D culture (Figure 7E), suggesting that this clone prefer- entially proliferated in bioreactor. Accordingly, few spontaneous metaphases, which are extremely rare in MM samples, could be observed in 3D culture and they all presented the deletion (Figure 7D, lower panel). Notably, at diagnosis, 96% of MM cells carried the 13q14.3 deletion and 46% co-expressed the t(4;14) IGH/FGFR3 translocation, consistent with the reported frequent association between the two cytogenetic lesions.33-35 After an initial tumor burden reduction in response to VTD, MM cells with the 13q14.3 deletion, but not those also carrying the t(4;14) translocation, pro- gressively expanded in vivo, ultimately representing the whole population in PCL cells (Figure 7E).
Discussion
The availability of suitable models that recapitulate the complex tumor-host interplay is central to understanding cancer biology and developing appropriate treatments. This is especially true in MM,36 where MM-BM interac- tions are crucial to disease progression and responsiveness to drugs.7,8 We here show that our ex vivo 3D co-culture model in bioreactor meets the requirements of recapitulat- ed MM-BM dialogue, permanence and survival of primary MM cells for an extended time period, thereby also incor- porating the temporal dimension. The model relies on the integrated use of a gelatin scaffold seeded with tumor cells and stroma and the RCCSTM bioreactor technology. Scaffolds are a key component for the reconstitution of MM microenvironment as they provide cells with mechanical support;37-39 we selected a gelatin biomaterial that allows morphological investigations and also mimics 3D ultrastructure of MM BM. Stroma is required to achieve ex vivo cell seeding efficiency and to re-create tumor-stroma contacts and signaling, both with MM cell lines and primary MM cells. Overall, the construct repro- duces the tumor-stroma 3D 'dynamic reciprocity'40 which is lost in conventional 2D co-culture. Both MM cells and stroma retain the expression of lineage specific markers as well as their specialized functions, including the release of β2-microglobulin, cytokines and growth factors, thus recapitulating the profile of the native BM.29,31
The model was established with MM cell lines and suc- cessfully applied to primary MM cells. Notably, primary MM cells from patients survived for up to seven days when cultured inside scaffolds. We used as stroma pri-
mary allogeneic BMSC from MM patients which possess the repertoire of adhesion molecules and have been previ- ously validated for the reconstruction of an MM BM niche.12 Given the high flexibility of our model, the contri- bution of additional cellular elements of MM microenvi- ronment can be addressed. As an example, the engage- ment of intimate EC-MM contacts inside the scaffold, documented by morphological and functional analyses, indicates that the system is suitable to elicit and study dynamic EC-MM targetable interactions. Moreover, the feasibility of co-culturing MM cells and bone-differentiat- ed BMSC underscores the potential to fulfill the unmet need for a model to study the relationship between MM progression and bone disease.41 Finally, development of repopulated scaffolds could be further exploited to address the contribution of circulating MM cells in humanized in vivo scaffold-mouse models.42
Multiple myeloma-BM functional interactions and down-stream signaling are promoted inside our surrogate microenvironment. Indeed, higher levels of pAkt, pSTAT and survivin are more appreciable in 3D than in 2D culture with both MM cell lines and primary MM cells, mimick- ing the activation of pro-survival signaling pathways in MM cells from patients.25,43,44 Akt pathway is crucial for MM survival and drug resistance, and has been proposed as a promising target for future molecular-based therapies.43 Tumor-stroma interactions are considered a major determinant in drug resistance in MM via the release of soluble factors and cell-to-cell adhesion. Our data on the protective effect of stroma against borte- zomib-induced apoptosis are consistent with the induc- tion of CAM-DR; in this regard, experiments conducted with the L-VCAM transfectant indicate that the system can be exploited to model and elucidate specific molecular interactions. The additional contribution by 3D culture further supports the importance of tissue architecture per se in drug resistance. The achievement of a construct where MM cells survive via the establishment of proper 3D interactions with a compliant microenvironment is a prerequisite to test the impact of drugs in a relevant human context. Accordingly, the impact of bortezomib on primary MM cells and their microenvironment could be assessed by means of FACS and IHC analyses, and also through determination of specialized functions in super- natants. In particular, the decrease of Ang-2 can monitor the cytotoxic effect of the drug on EC,45 while variations in IL-6 and MMP-2/-9 activities may result from disrup- tion of MM-stroma interplay.7,9
The dissection of clonal dynamics during disease pro- gression and in response to therapy is increasingly emerg- ing as a central issue of MM investigation.34,35 In a patient with high-risk MM, sequential BM sampling allowed the evolutionary path to be defined along the clinical course. Two sub-clones co-existing at diagnosis initially respond- ed to first-line therapy; subsequently, only one evolved over time. Significantly, the bioreactor culture could antic- ipate the expansion of the same clone. These data suggest that, in selected cases, the model can be exploited to mon- itor the dynamics of clones inside the whole MM cell pop- ulation, and possibly to identify new potential targets.
Altogether, our findings indicate that 3D dynamic cul- ture of reconstructed human MM microenvironments in RCCSTM bioreactor may represent an important platform for drug testing and the study of tumor-stroma molecular interactions.
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