hree-dimensional spheroids are more representative of tumors than cell-cultured monolayers. As in tumors, gradients of oxygen, nutrients and wastes are found in spheroid cultures but not in classical cultured monolayers. On the other hand, cell-based assays on the latter are hardly applicable to spheroid cultures. Such is the case for zymogram assays, which are classically used to measure MMP-2 and MMP-9 activities, and for immunoblots to measure the phosphorylation of proteins involved in ligand-induced intracellular signaling in normal and tumor cells. In this study we used two renal cancer cell lines as models, the first derived from a pediatric rhabdoid tumor and the second from an adult clear cell renal cell carcinoma. Using these two cell lines, we successfully developed a simple inexpensive assay to measure MMP-2 and MMP-9 activities in spheroids established in the presence
of methylcellulose. After washing, 1 to 5 spheroids were pooled and stimulated with collagen I for 24 h before analysis. MMP-2 and MMP-9 activities were measured in supernatants using a standard but enhanced zymogram assay. Both pro-MMP-9 and MMP-2 activities were detected in spheroids established from both cell lines. In contrast with our previous data using classical cultures monolayers, collagen I stimulation decreased pro-MMP-9 activity without affecting MMP-2 activity. On the other hand, we could not accurately measure AKT intracellular signaling pathways from spheroids stimulated with collagen I. Finally, we adapted our 3D protocol to analyze the MAPK/ERK pathway in kidney tumor cells following induction by EGF. In conclusion, this zymogram assay for analyzing MMP-2 and MMP-9 activities in spheroids paves the way for novel experimentations in tumor biology.

The biological components of tumor tissue are now relatively well known. They include for instance tumor cells, immune cells and blood capillaries. However, the 3D organization of these tissues is poorly known and the bio-architectural parameters defining the internal structure of a tumor remains unknown. In a paper published in Communications Biology, our team dissected the organization of a hepatoblastoma tumor using “Serial Block face-scanning electron microscopy” volumetric imaging technology, deep/machine learning, applied mathematics and infographics. This pilot study shows for the first time that blood capillaries and bile canaliculus-like structures influence the size, planar alignment and orientation of tumor cells within the tissue. This work paves the way for a whole new field of research called Onconanotomy.

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The aim of this axis of research is to understand energy and lipid metabolic adaptation of liver cancer cells and the crosstalk with innate immunity in the tumoral microenvironment. We have particular interest of assessing the benefit of available hypolipidemic drugs (statins and ati-PCSK9) to remodel lipid metabolism as a potential approach to treat liver cancers. Our ongoing results show profound anti-tumoral effects of these drugs in cell models as well as in in vivo models.
We also study the metabolic pathway of tryptophan/kynurenin/NAD and its impact in modulating immunosuppression of macrophages.

Figure: Crosstalk between metabolism and innate immunity within the classically activated M1 (antitumor-like macrophages) and the alternately activated M2 (protumor-like macrophages) (left) and overview of lipid metabolism in the liver (right).

Several of our projects aim at testing and validating the efficacy of anticancer agents, alone or in combination, in the treatment of childhood cancers. To validate the efficacy of these compounds, we use several animal models including the mouse, the chicken embryo, the Xenopus embryon and the Zebra fish. A first patent was filed in September 2021 following the discovery of the efficacy of the GSK126 + Statin combination in the treatment of infiltrating brainstem glioma (DIPG or also known as K3K27-altered Diffuse Midline glioma).

Figure: Synergistic effect GSK126 and inhibitors of cholesterol biosynthesis pathway enzymes on DMG tumor development in mice.

https://academic.oup.com/view-large/figure/340497140/vdac018f0006.jpg

Introduction: Hepatoblastoma (HB) is the most frequent liver cancer in children, typically occurring before the age of five. Thanks to the combination of chemotherapy and surgery, the 5-year survival rate following diagnosis is approximately 83%. Today, the main challenge is the efficient treatment of high-risk patients, particularly those presenting with lung metastasis or experiencing relapse. To better study HB and validate new therapeutic options, various animal models have been developed in mice, chick and zebrafish. However, none of these models fully recapitulates the complexity and juvenile context of the disease, as observed in very young patients.

Methods: To account for the young age of patients and better mimic the hepatic microenvironment in which HBs develop, we established an innovative orthotopic xenograft model of HB in juvenile mice, which also generates lung metastases. Eleven-day-old immunocompromised mice were injected intrahepatically with Huh6 cells. Tumor progression was monitored through bioimaging and confirmed post-euthanasia by direct examination of the liver and lungs using microscopic imaging and immunohistochemistry. To further validate the model, some implanted mice were treated with cisplatin, and the response of HB cells to this DNA intercalating agent was assessed.

Conclusion: This neonatal orthotopic xenograft model of HB in mice reproduces lung metastases and exhibits sensitivity to cisplatin. It fully mimics the developmental progression of this pediatric tumor and clearly surpasses existing models in adult mice, paving the way for more robust basic research investigations and preclinical studies in whole animals.

Left panel: Representative images of immunohistochemistry-labeled murine liver (on the left) and lung (on the right) tissue sections stained with hematoxylin-eosin-saffron (HES) or beta-catenin antibody, as indicated. Magnification and scale are as shown on right and in corresponding image, respectively. NL: normal liver; T: tumor; M: metastasis.

Right Panel: Cisplatin inhibits HB development in vivo using an orthotopic xenograft tumor model in juvenile mice. a) Representative images of day-43 mice with tumors treated with vehicle or cisplatin, analyzed by bioluminescence live imaging. b) Graph represents tumor volume kinetics in mice repeatedly treated with either vehicle (n = 3) or cisplatin (4 mg/kg, n = 4). Two way-ANOVA, ***p<0.001; Sidak’s multiple comparisons post-test. Arrow indicates treatment starting point in mice aged 22 days. **p<0.01; ****p<0.0001

https://doi.org/10.1159/000546028