A spheroid model that recapitulates the protective role of the lymph node microenvironment and serves as a platform for drug testing in chronic lymphocytic leukemia

Abstract

Chronic lymphocytic leukemia (CLL) B cells are characterized by a propensity to undergo rapid apoptosis when cultured in vitro, underscoring the importance of the tissue microenvironment for disease survival. One of the major limitations in studying the role of the microenvironment in tumor development and drug response is the inadequacy of conventional two-dimensional (2D) in vitro assays to physiologically reconstruct the complex spatial organization and interactions of cells in their natural lymphoid niches. To overcome this limitation, we developed a novel in vitro 3D lymph node-like spheroid model of the leukemic microenvironment by culturing human CLL cells with fibroblastic reticular cells (FRCs). FRCs are a key structural component of secondary lymphoid organs and are emerging as crucial players in tissue homeostasis and immune responses. Our results demonstrate that CLL spheroids maintain the physiological cellular ratio between FRCs and leukemic cells over time and protect tumor cells from apoptosis by mimicking the protective effects of the microenvironment. This was further demonstrated by venetoclax treatment that showed reduced apoptosis in 3D compared to a 2D setting. Importantly, the spheroids promote a gene expression profile more aligned with that of CLL cells in lymphoid tissues. The spheroid model provides a straightforward, quick-to-use platform for investigating drug efficacy under conditions that better replicate the natural lymph node microenvironment. This 3D lymph node-like spheroid model could serve as a valuable tool for studying tumor biology and the protective effects of the stromal microenvironment, and for testing therapeutic strategies in a more clinically relevant setting.

Figure 1

Dynamic of spheroids formation, cells positioning, and dye perfusion. (A) Experimental design of spheroids formation. Human leukemic cells from chronic lymphocytic leukemia (CLL) patients were mixed or not along with a murine splenic fibroblastic reticular cell (FRC) line YFP⁺ or a human lymph node‐derived FRC line in a rat tail Collagen I solution. Droplets of the resulting cell suspensions were spotted on the lids of Petri dishes and placed in an incubator at 37°C to allow collagen polymerization. After 20 min, droplets were flushed into the medium by gentle pipetting, and CLL alone or CLL + FRCs spheroids were cultured for 2–4 days. (B, C) Representative bright field images of spheroids with CLL cells alone (upper panel) or with murine (B) or human (C) FRCs (lower panel), at different incubation periods, 24, 48, and 96 h, after spheroids preparation. Scale bar: 300 μm. (D, E) Confocal images of spheroids stained for YFP or PDPN (green) and CD45 (red) to detect murine or human FRCs and leukemic cells, respectively, and nuclei (blue) were stained with DAPI. Three time points after culture (24, 48, and 96 h) are shown. Scale bar: 300 μm. (F) Representative fluorescence image of CLL + murine/human FRC spheroids perfused with CMTPX Cell Tracker^TM, after 12 h of incubation with the dye. 4',6‐diamidino‐2‐phenylindole (DAPI) staining (in blue) was performed to stain nuclei. Scale bars: 150 and 50 μm.

Figure 2

Spheroids support chronic lymphocytic leukemia (CLL) viability. (A) Analysis of the CLL B cell number in spheroids over time at two different time points (24 and 96 h), for both culture settings (CLL alone and CLL + murine fibroblastic reticular cells [FRCs]). A pool of three spheroids was prepared and digested to obtain a cell suspension. Then, cells were placed in the Bürker chamber and manually counted. Trypan Blue was added to exclude dead cells. Data are representative of CLL cells from 7 to 11 different patients cultured for 24–96 h, respectively. (B) Graph showing the differences in the cell ratio between the 2D and 3D conditions in the presence of murine FRCs. The ratio is calculated as the total number of CLL cells over the total number of FRCs at two different time points (7 and 10 days). Data are plotted as mean ± SD of four independent experiments/CLL samples. (C, D) Test of cell viability at short‐term (C) and long‐term (D) spheroid culture. Graphs show cell viability as a percentage of Annexin‐V/propidium iodide (PI) double negative cells at 4 days (C, D) and at 7–10 days of culture (D). Data are representative of 12 independent experiments for short‐term culture (C) and 5 independent experiments for long‐term culture (D). Data are represented as mean ± SD.

Figure 3

Spheroids protect from drug‐induced apoptosis. (A) Experimental scheme of spheroids treatment with drugs. After 24 h from spheroids generation, chronic lymphocytic leukemia (CLL) alone or CLL + murine or human fibroblastic reticular cell (FRC) spheroids were distributed in a 24‐multiwell ultra‐low attachment plate (3 spheroids/well) and were cultured for 96 h in presence of drug (ibrutinib or venetoclax) or vehicle (dimethyl sulfoxide [DMSO]). Then, spheroids were collected, randomly distributed in tubes (10 spheroids/tube), and digested with Liberase^TM to obtain a single cell suspension. Next, samples were stained for Annexin‐V and propidium iodide (PI) and analyzed by flow cytometry. (B, C) Graphs represent CLL cell viability at 72 h post‐treatment with venetoclax in 2D (left) and 3D (right) conditions of spheroids generated with murine FRCs (B) or human FRCs (C). Double negative CLL cells for Annexin‐V/PI staining are shown as percentages of total cells. Data are representative of 13 samples and are plotted as mean ± SD. (D, E) Representative confocal microscope images of spheroids prepared with CLL B cells and murine FRCs (D) or human FRCs (E) treated for 72 h with venetoclax or vehicle (DMSO). Antibodies against YFP (D) protein or PDPN (E) (green), to mark the FRC population, and hematopoietic superficial marker CD45 (red), for CLL cells, were used. 4',6‐diamidino‐2‐phenylindole (DAPI) staining (blue) was performed to stain nuclei. Scale bar: 100 μm.

Figure 4

Gene expression profile of chronic lymphocytic leukemia (CLL) cells from 3D or 2D culture conditions. (A) Experimental scheme of spheroids formation, CLL isolation, and flow cytometry gating strategy. (B) Principal component analysis of 5000 most variable genes (log 10 rpkm) in bulk RNA‐seq samples from 3D and 2D spheroids. (C) Volcano plot of differentially expressed genes (DEGs) in 3D versus 2D spheroids. Significantly upregulated genes in the 3D spheroids are highlighted in red, downregulated in blue (|log FC| > 1, adjusted P‐value < 0.05). Nonsignificant genes are displayed in gray. (D) Dot plot of selected significant (adjusted P‐value < 0.05) Gene Ontology (GO) Biological Process (BP) pathways for upregulated and downregulated DEGs. (E) Hierarchically clustered heatmap (Z score) of apoptotic and antiapoptotic gene expression in 3D and 2D spheroids.