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. 2018 May 3;9(1):1787.
doi: 10.1038/s41467-018-04150-7.

A retinoic acid-dependent stroma-leukemia crosstalk promotes chronic lymphocytic leukemia progression

Diego Farinello  1 , Monika Wozińska  1 , Elisa Lenti  1 , Luca Genovese  1 , Silvia Bianchessi  1 , Edoardo Migliori  1 , Nicolò Sacchetti  1 , Alessia di Lillo  1 , Maria Teresa Sabrina Bertilaccio  1   2 , Claudia de Lalla  3 , Roberta Valsecchi  1 , Sabrina Bascones Gleave  4 , David Lligé  4 , Cristina Scielzo  1 , Laura Mauri  5 , Maria Grazia Ciampa  5 , Lydia Scarfò  1 , Rosa Bernardi  1 , Dejan Lazarevic  6 , Blanca Gonzalez-Farre  7 , Lucia Bongiovanni  8 , Elias Campo  7 , Andrea Cerutti  4 , Maurilio Ponzoni  8   9 , Linda Pattini  10 , Federico Caligaris-Cappio  1   11 , Paolo Ghia  1   9 , Andrea Brendolan  12
Affiliations

A retinoic acid-dependent stroma-leukemia crosstalk promotes chronic lymphocytic leukemia progression

Diego Farinello et al. Nat Commun. .

Abstract

In chronic lymphocytic leukemia (CLL), the non-hematopoietic stromal microenvironment plays a critical role in promoting tumor cell recruitment, activation, survival, and expansion. However, the nature of the stromal cells and molecular pathways involved remain largely unknown. Here, we demonstrate that leukemic B lymphocytes induce the activation of retinoid acid synthesis and signaling in the microenvironment. Inhibition of RA-signaling in stromal cells causes deregulation of genes associated with adhesion, tissue organization and chemokine secretion including the B-cell chemokine CXCL13. Notably, reducing retinoic acid precursors from the diet or inhibiting RA-signaling through retinoid-antagonist therapy prolong survival by preventing dissemination of leukemia cells into lymphoid tissues. Furthermore, mouse and human leukemia cells could be distinguished from normal B-cells by their increased expression of Rarγ2 and RXRα, respectively. These findings establish a role for retinoids in murine CLL pathogenesis, and provide new therapeutic strategies to target the microenvironment and to control disease progression.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Leukemic cells induce retinoid-signaling in the stromal microenvironment. a Heat map of deregulated genes and their annotation analysis for the Illumina microarray. Black bars represent genes belonging to the collection of the MatrisomeDB atlas (see lists); enrichment significance was computed according to the hypergeometric distribution. b Experimental design of RA-reporter cells cultured with murine Eμ-TCL1 CLL or control splenic B cells (left). RA-signaling was measured by quantifying the β-gal activity (absorbance after ONPG staining) (left) after co-cultures, and treatments with vehicle (DMSO) or with BMS493 (right). Data are representative of six independent experiments. The mean of triplicates and ±SD are shown, ***p < 0.001
Fig. 2
Fig. 2
Inhibition of the RA-signaling pathway in stromal cells affects multiple biological processes. a Experimental design of RNA-seq analysis performed on mRNA obtained from stromal cells treated with either vehicle (DMSO) or BMS493, and most down-regulated gene-signatures (bottom). Validation of mRNA expression levels of selected stromal gene-signatures after vehicle (DMSO) or BMS493 treatment. Data are representative of four independent experiments. The mean of triplicates and ±SD are shown, *p < 0.05, **p < 0.01, ***p < 0.001. b Interpolation of gene expression profiles between microarray and RNA-seq data. c Quantification of murine Eμ-TCL1 CLL cells adherence to stromal cells after BMS493 or vehicle (DMSO) treatment. Data are representative of three independent experiments with three different leukemia preparations. The mean of triplicates and ±SD are shown, *p < 0.05. d Aggregation of murine stroma (mSSC) and murine Eμ-TCL1 CLL cells in spheroid assay. The percentage of aggregation results from the number of murine CLL cells that contribute to spheroid formation after treatment with BMS493 or vehicle (DMSO). Data are representative of one out of four independent experiments. The mean of triplicates and ±SD are shown, **p < 0.01
Fig. 3
Fig. 3
Leukemic cells induce Cxcl13 expression in stromal cells through retinoid-signaling. a qPCR analysis of Cxcl13 expression from a murine stromal cell line (mSSC) cultured with murine Eμ-TCL1 CLL cells or control splenic B cells, and after treatment with DMSO or BMS493. Expression of Rarβ after BMS493 treatment was used as control. The mean of triplicates and ±SD are shown, * p < 0.05, ** p < 0.01, ***p < 0.001. b Representative qPCR analysis of Cxcl13 expression from a murine stromal cell line (mSSC) cultured in vitamin A deficient media and treated with vehicle (DMSO), retinoic acid (RA), or BMS493. Expression of Rarβ after BMS493 treatment was used as control. qPCR data are representative of one out of three independent experiments. The mean of triplicates and ±SD are shown, ***p < 0.001. c Representative confocal images of the spleen from wild type mice treated with either DMSO (left) or BMS493 (right). Tissue sections were stained for laminin (ECM) (gray) and CXCL13 (red). Yellow arrowheads indicate the outer follicular region. Images are representative of one out of three mice analyzed. Scale bars = 50 μm
Fig. 4
Fig. 4
Leukemia development is associated with increased CXCL13 in stromal cells. a Representative confocal mosaic images of the spleen. Tissues were isolated from mice with low (<5%), intermediate (10–30%), and high (>50%) leukemia infiltration and stained for CXCL13 (gray) to visualized follicular stromal cells. Graphs indicate the frequency of the different CXCL13 patterns (normal, #1, #2, and #3). Scale bars = 200 μm. b Representative confocal images of spleen sections from Rag2−/−γc−/− mice injected with leukemic cells, and stained three-weeks after with CD35 (red) or CXCL13 (green) to visualize stromal cells. Each staining is representative of three mice analyzed. Scale bars = 50 μm. c Representative confocal images of the spleen from Pdgfrαgfp/+ mice injected with EdU to assess proliferation at different stages of leukemia development. Tissues were stained for GFP (green) to visualize PDGFRα+ cells and CXCL13 (red). Graphs represent: (i) the percentage of EdU+PDGFRα+ proliferating stromal cells (middle); (ii) the number of CXCL13+PDGFRα+ stromal cells (left); and (iii) the number (density) of PDGFRα+ stromal cells per field (right). Each count represents the mean ± SD of cells from seven to ten fields analyzed for each tissue. ***p < 0.001. Scale bars = 50 μm. Each staining is representative of one out of three to five mice analyzed
Fig. 5
Fig. 5
Targeting RA-signaling controls disease progression and prolongs survival. a FACS analysis of control mice (n = 3) or VAD (n = 3) injected with leukemic cells. The table summarizes the percentage of murine Eμ-TCL1 CLL cells in spleen and BM for each mice analyzed. b Survival curve for Eμ-TCL1 mice fed with vitamin A deficient (VitA–) or control (VitA+) diet. Quantification of RA-precursors retinol and retinyl palmitate in the liver of moribund Eμ-TCL1 VitA+ or VitA– fed mice. The mean of triplicates and ±SD are shown, *p < 0.05, **p < 0.01 and ***p < 0.001. c Analysis of leukemia progression over time in wild type mice transplanted with leukemic cells and treated with vehicle (DMSO) or BMS493. Flow cytometry analysis of leukemia cell infiltration in different tissues at 20 or 30 days after the indicated treatment, *p < 0.05, **p < 0.01 and ***p < 0.001. d Survival curve of wild-type mice transplanted with Eμ-TCL1 CLL cells and treated with vehicle (DMSO) or BMS493, *p < 0.05. Representative ultrasound images and echographical measurement of the spleen size performed at day 35 of disease progression
Fig. 6
Fig. 6
Increased expression of RA-nuclear receptors in human primary CLL cells correlates with bad prognosis. a qPCR analysis of Rarγ2 expression in Eμ-TCL1 CLL and control B cells. Data are representative of one out of five mice analyzed. The mean of triplicates and ±SD are shown. *p < 0.05. b Relative RXRα expression data of purified B-cells from healthy donors and CLL patients (b, left) from public repository, and expression of RXRα in CLL patients with different genomic aberrations (b, right). ***p < 0.001. c Correlation analysis of RXRα expression from purified CLL cells from patients with different IGHV mutational status, CD38 and ZAP70 expression
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