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3D co-culture model of CLL cells within BM microenvironment
zomib,19 we customized, for CLL, a 3D BM model based on scaffolds within a bioreactor. The model allows us to analyze in parallel CLL cells retained inside the recon- structed BM microenvironment and those recovered from outside. We were able to define BM stromal cells as the minimal requirement to support CLL cell retention and homing inside the scaffolds, paving the way for future improvements aimed at testing the individual contribu- tion of additional types of cells in this process. The obser- vation that BM stromal cells support the retention of a consistent albeit variable fraction of primary CLL cells among different patients indicates that CLL compartmen- talization in a bioreactor is not a random phenomenon. The demonstration of differential HS1 expression and activation status of CLL cells inside and outside the scaf- folds, along with a similar modulation of CXCR4 expres- sion, indicates a potential molecular basis for this process that does indeed mirror the events taking place in vivo in the BM and PB.
Kinase inhibitors, such as the BTK inhibitor ibrutinib, influence the kinetics of leukemic cell recirculation and, interestingly, mobilize CLL cells more efficiently from the lymph nodes than from the BM, suggesting a specific tis- sue-dependent effect.25 Taking advantage of our 3D BM model, we here provide evidence that CLL cells mobilized from the scaffolds upon exposure to ibrutinib are mainly those with active HS1 and suggest that ibrutinib may exert its mobilizing effect through HS1 activation. It remains to be elucidated whether ibrutinib affects HS1 activation on CLL cells directly or indirectly in the scaf- fold. We have evidence that ibrutinib does not affect HS1 activation in the absence of BM-derived stromal cells (data not shown), suggesting a specific role exerted by the microenvironment, possibly toward HS1 downregulation, following direct contact with BM-derived stromal cells. Accordingly, we observed that HS1 undergoes activation during the first weeks of ibrutinib treatment in patients. We may then infer that CLL cells with low/inactive HS1 preferentially home to the BM niche where they encounter a protective microenvironment against the mobilization effect promoted by ibrutinib. This further indicates that our 3D model may reproduce the events occurring in vivo under ibrutinib treatment and may help to understand the slower clearance of the BM in patients.25 In conclusion, we here present and validate a reproducible
3D BM model to aid understanding of the mechanisms underlying CLL tissue retention and mobilization, but also capable of predicting patient-specific efficacy of CLL mobilizing agents,23 as schematically summarized in Figure 7. This may serve in the future as a precision med- icine tool to test these and other drugs acting through sim- ilar mechanisms in a more suitable system than the tradi- tional 2D models from which the dynamic effects of treat- ments cannot be inferred. Moreover, it represents the first step towards the development of new and more complex 3D in-vitro models mimicking different microenviron- ments such as lymph nodes.
Disclosures
PG and KS have received honoraria and research funding from AbbVie and Janssen not related to this project. The other authors have no conflicts of interest to disclose.
Contributions
CS wrote the manuscript. CS, FB, DB, FVS and LP per- formed the experiments and analyzed the data. LS, PG and KS provided patients' and clinical information. MP and LB per- formed immunohistochemistry. DZ performed image stream analysis. VRC helped in the microscopy. EF, VRC, PG, MP and KS revised the manuscript.
Acknowledgments
We thank Alembic for helpful suggestions and technical support.
Funding
This project was supported in part by: the Associazione Italiana per la Ricerca sul Cancro AIRC (Special Program on Metastatic Disease – 5 per mille #21198); My first grant AIRC (#17006) (principal investigator CS). The research leading to these results received funding from AIRC under IG 2018 - ID. 21332 project (principal investigator CS). Roche per la ricerca 2016. Leukemia Research Foundation grant 2018; GCH-CLL project funded by ERA NET TRANSCAN-2 Joint Transnational Call for Proposals 2014 (JTC 2014) and project #179 NOVEL funded by ERA-NET TRANSCAN-2 JTC 2016; by the European Commission/DG Research and Innovation. CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades and the Pro CNIC Foundation, and it is a Severo Ochoa Center of Excellence (SEV- 2015-0505). VRC acknowledges the support of FEDER "Una manera de hacer Europa".
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