3D Model Characterization by 2D and 3D Imaging in t(14;18)-Positive B-NHL: Perspectives for In Vitro Drug Screens in Follicular Lymphoma

Abstract

Follicular lymphoma (FL) is an indolent B cell lymphoproliferative disorder of transformed follicular center B cells, which accounts for 20–30 percent of all non-Hodgkin lymphoma (NHL) cases. Great advances have been made to identify the most relevant targets for precision therapy. However, no relevant models for in vitro studies have been developed or characterized in depth. To this purpose, we generated a 3D cell model from t(14;18)-positive B-NHL cell lines cultured in ultra-low attachment 96-well plates. Morphological features and cell growth behavior were evaluated by classical microscopy (2D imaging) and response to treatment with different drugs was evaluated by a high-content analysis system to determine the robustness of the model. We show that the ultra-low attachment (ULA) method allows the development of regular, spherical and viable ULA-multicellular aggregates of lymphoma cells (MALC). However, discrepancies in the results obtained after 2D imaging analyses on drug-treated ULA-MALC prompted us to develop 3D imaging and specific analyses. We show by using light sheet microscopy and specifically developed 3D imaging algorithms that 3D imaging and dedicated analyses are necessary to characterize morphological properties of 3D models and drug effects. This study proposes a new method, but also imaging tools and informatic solutions, developed for FL necessary for future preclinical studies.

Development and Characterization of 3D Hybrid Spheroids for the Investigation of the Crosstalk Between B-Cell Non-Hodgkin Lymphomas and Mesenchymal Stromal Cells

“…In the current study, we observed that B-NHL cells are grown within an ultra-low attachment plate or a hanging drop method aggregating into loose clumps of cells instead of 3D structures, which was previously described. 59 Such methods may be helpful in drug studies; however, they do not support cell–cell and microenvironment interactions. Here, we observed that the lymphoma cells form tight spheroids with agarose gel; however, the cells disintegrate when transferred with a pipette for further examination.…”

A novel 3D culture model recapitulates primary FL B-cell features and promotes their survival

“…Lymphomas develop as complex cell structures including tumor cells and their microenvironment within mechanically constrained LN. Previous lymphoma spheroid models incorporating only malignant B cells 17,18,32 suggest that the 3D cell architecture is a key feature for the regulation of lymphoma growth and therapeutic response. LN are highly dynamic structures expanding and becoming mechanically stiff under immune cell recruitment and proliferation, whereas immune response resolution is associated with LN contraction and return to a baseline of mechanical softness 33 .…”

“…Multicellular spheroids or tumor organoids represent promising models allowing high-throughput screening of anti-cancer drugs in versatile systems mixing several cell types and ECM components. While 3D culture models are increasingly developed for solid cancer 15 , their transfer to lymphoma modeling is still limited and includes: i) multicellular aggregates of lymphoma cells, which are obtained using the hanging drop and ultra-low attachment method, and are useful for testing drug efficacy but do not account for the effect of cell-cell and cell-ECM interactions [16][17][18] , ii) 3D lymphoma organoid models integrating lymphoid-like stromal cells or integrin-specific binding peptides recapitulating more accurately lymphoma microenvironment, but difficult to handle and low-throughput 19 , iii) a DLBCL-on-chip model utilizing lymphoma and TME murine cells, enabling the in vitro modeling of DLBCL niche and associated vasculature 20 . However, modeling the interactions of the TME with primary lymphoma B cells in 3D cocultures remains a challenge.…”

Production d’organoïdes tumoraux 3D par la technologie des capsules cellulaires TCC

“…Les mono-cultures 3D de cellules encapsulées (primaires de patients ou de lignées), permettent de créer des modèles très variés reproduisant des tissus 3D simples : avec la TCC, de nombreuses cellules de tumeurs solides ont été déjà été encapsulées avec succès (Fig 1B existent sont des agrégats multicellulaires difficiles à manipuler et à imager [24][25][26]. L'encapsulation permet de les faire pousser dans un environnement clos, équivalent à un mini-bioréacteur, qui facilite leur manipulation mais également permet de les imager en petits groupes, ce qui n'est généralement pas évident pour ce type de cellules.…”