Page 47 - 2021_09-Haematologica-web

3D co-culture model of CLL cells within BM microenvironment
mainly found in the lymph nodes, and/or sheltered in vaguely characterized niches in the BM.3-5 CLL cells accu- mulating within tissues tend to spill over into the circulat- ing blood where they acquire a more resting phenotype, indicating that the most clinically relevant events occur in tissues. This feature also underlines the importance of the host tissues in CLL which conceivably contribute to disease progression and ultimately to treatment resistance.3,5,6
Cytoskeleton regulation is clearly implicated in the dynamic behavior of CLL cells, contributing to the homing and trafficking in and out of tissues, also during treatment. In particular, we previously reported that the activated status of the cytoskeletal protein hematopoietic lineage cell-specific protein 1 (HS1) defines a distinct signaling pathway and cytoskeletal activity in CLL, while also having prognostic implications, with the active and inactive forms of HS1 cor- relating with a favorable or adverse prognosis, respectively.7- 9 In parallel, we demonstrated that downregulation of HS1 expression interferes with secondary lymphoid organ (lymph nodes and spleen) infiltration by CLL cells and leads to increased BM homing associated with impaired cytoskele- tal activity.9,10 More recently, HS1 has been found to associate with ROR1 in enhancing CLL cell migration,11 further under- lining its potential clinical significance.
New targeted therapies, namely kinase inhibitors, have multiple modes of action, including the mobilization of leukemic cells from tissues into the bloodstream, where CLL cells lose the protective effect exerted by the microen- vironment, eventually becoming more susceptible to cell apoptosis.12-14 Effectively, the use of the BTK inhibitor ibru- tinib for CLL treatment has been a game-changer in the management of patients with this disease,14 although it is not curative and patients may relapse after several years of response.15 Inhibition of VLA-4-dependent adhesion of CLL cells to stroma and stromal components has been pro- posed as an explanation for the lymphocytosis induced by ibrutinib treatment,16 while other studies suggest a role of ibrutinib in modulating migration of CLL cells to chemokine gradients, in particular through CXCR4.17
However, a major limitation of investigating tissue reten- tion and egress (or mobilization) in CLL originates from the lack of suitable in vitro models for recreating the close inter- actions between leukemic cells and the microenvironment. Calissano et al. first showed a relationship between in vivo CLL cell kinetics and the expression of CD38, a protein involved in CLL cell retention and trafficking.4 More recent- ly, Pasikowska et al. reported differences between lymph node-derived CLL cells versus PB-derived cells by taking advantage of an in vitro system that models trans-endothe- lial migration,18 while Chen et al. demonstrated the dynam- ic expression of CXCR4 following BTK inhibition in vivo in a CLL mouse model.17 Despite these advances, none of the existing models is suitable for deeply characterizing what is happening to human CLL cells in the tissues.
In order to partially overcome this limitation, we have exploited, and adapted to CLL, a three-dimensional (3D) co-culture model, already thoroughly validated for multiple myeloma, which is able to reproduce malignant cell- microenvironment interactions.19 This 3D model is based on the integrated use of cell-repopulated scaffolds and a rotating bioreactor. This combination enables reciprocal interactions to be established between tumoral and non- tumoral compartments inside the scaffolds and to promote CLL cell survival. Moreover, CLL cells can be recovered from both inside and outside the scaffolds, counted and
characterized for expression of lineage markers and of mol- ecules putatively involved in their mobilization, providing the possibility to elucidate this mechanism, also in response to mobilizing agents, particularly ibrutinib. As a proof-of-principle, we here provide evidence of HS1 mod- ulation in the presence of the drug, ultimately regulating CLL cell tissue homing and egress. Moreover, we report that this innovative 3D model is able to reliably reproduce the events occurring in vivo during homing and migration, thus potentially contributing to better understanding the pathogenic mechanisms leading to the dissemination and homing of CLL cells, particularly in response to treatment.
Methods
Study subjects and ethics statement
Patients with CLL were diagnosed according to the updated National Cancer Institute Working Group guidelines.20 PB samples were obtained after informed consent from patients who were (i) either untreated or off treatment for at least 6 months; or (ii) under ibrutinib treatment. The study was approved by the “San Raffaele” Hospital ethics committee under the protocol VIVI-CLL entitled: “In vivo and in vitro characterization on CLL”; and the CERTH ethics committee in response to the application entitled “Molecular and functional studies of B cell malignancies”.
The clinical and biological characteristics of the patients with CLL who provided samples for the experiments are reported in Online Supplementary Table S1.
Scaffold preparation
Scaffolds were populated as described by Belloni et al.19 and adapted to CLL cells. Briefly: scaffold discs were cut from SpongostanTM sheets (Ethicon, Inc. USA) using a sterile 4 mm2 biopsy punch and then pre-seeded with BM-derived stromal cells HS5 (200,000/scaffold) in 96-well suspension culture plates (Greiner bio-one, Germany). Scaffolds were then transferred to 10 mL High Aspect Ratio Vessels (HARV) in 1 mL TCM (DMEM cul- ture medium supplemented with 10% v/v fetal bovine serum) and cultured overnight in the RCCSTM bioreactor at the lower speed (rpm). Twenty-four hours later, CLL cells were added to the ves- sels, using the optimal ratio of CLL cells to stromal cells estab- lished in preliminary experiments (MEC1 cells=2x106, primary CLL cells =3x10^6). After 5 h, vessels were filled with growth medium (RPMI1640 culture medium supplemented with 10% v/v or 20% fetal bovine serum for MEC1 or primary CLL cells, respec- tively). At the end of the culture period, cells outside and inside the scaffold were recovered from the scaffolds by means of lib- erase (Roche) (25 μg/mL) treatment for further analysis (see Online Supplementary Methods). The cells outside and inside the scaffold were counted using the trypan blue exclusion test for viability, which showed that more than 90% of the cells were viable. Alternatively, scaffolds were formalin-fixed for IF or lysed with 100 μL RIPA buffer for western blotting analysis (see Online Supplementary Methods).
Bioreactor RCCSTM
The 3-D dynamic culture was performed using the RCCSTM bioreactor RCCS-4DQ equipped with four rotating 10 mL-HARV culture vessels, which work as culture chambers (Synthecon Inc., USA).19
Vessels are provided with a gas exchange membrane made of silicon rubber, which allows optimal diffusion of O2. The bioreac- tor was kept inside an incubator, in a humidified atmosphere, at 37°C with 95% air and 5% CO2. During the experimental proce-
haematologica | 2021; 106(9)
2335