The endothelial cell spheroid assay provides a suitable in vitro model to study (lymph) angiogenesis and test pro- and anti-(lymph) angiogenic factors or drugs. Usually, the extent of cell invasion, observed through optical microscopy, is measured. The present study proposes the spatial distribution of migrated cells as a new descriptor of the (lymph) angiogenic response. The utility of this novel method rests with its capacity to locally characterise spheroid structure, allowing not only the investigation of single and collective cell invasion but also the evolution of the spheroid core itself. Moreover, the proposed method can be applied to 2D-projected spheroid images obtained by optical microscopy, as well as to 3D images acquired by confocal microscopy. To validate the proposed methodology, endothelial cell invasion was evaluated under different experimental conditions. The results were compared with widely used global parameters. The comparison shows that our method prevents local spheroid modifications from being overlooked and leading to the possible misinterpretation of results.

BackgroundAbnormal lymphatic vessel formation (lymphangiogenesis) is associated with different pathologies such as cancer, lymphedema, psoriasis and graft rejection. Lymphatic vasculature displays distinctive features than blood vasculature, and mechanisms underlying the formation of new lymphatic vessels during physiological and pathological processes are still poorly documented. Most studies on lymphatic vessel formation are focused on organism development rather than lymphangiogenic events occurring in adults. We have here studied lymphatic vessel formation in two in vivo models of pathological lymphangiogenesis (corneal assay and lymphangioma). These data have been confronted to those generated in the recently set up in vitro model of lymphatic ring assay. Ultrastructural analyses through Transmission Electron Microscopy (TEM) were performed to investigate tube morphogenesis, an important differentiating process observed during endothelial cell organization into capillary structures.ResultsIn both in vivo models (lymphangiogenic corneal assay and lymphangioma), migrating lymphatic endothelial cells extended long processes exploring the neighboring environment and organized into cord-like structures. Signs of intense extracellular matrix remodeling were observed extracellularly and inside cytoplasmic vacuoles. The formation of intercellular spaces between endothelial cells led to tube formation. Proliferating lymphatic endothelial cells were detected both at the tips of sprouting capillaries and inside extending sprouts. The different steps of lymphangiogenesis observed in vivo are fully recapitulated in vitro, in the lymphatic ring assay and include: (1) endothelial cell alignment in cord like structure, (2) intracellular vacuole formation and (3) matrix degradation.ConclusionsIn this study, we are providing evidence for lymphatic vessel formation through tunneling relying on extensive matrix remodeling, migration and alignment of sprouting endothelial cells into tubular structures. In addition, our data emphasize the suitability of the lymphatic ring assay to unravel mechanisms underlying lymphangiogenesis.

It is now well accepted that multipotent Bone-Marrow Mesenchymal Stem Cells (BM-MSC) contribute to cancer progression through several mechanisms including angiogenesis. However, their involvement during the lymphangiogenic process is poorly described. Using BM-MSC isolated from mice of two different backgrounds, we demonstrate a paracrine lymphangiogenic action of BM-MSC both in vivo and in vitro. Co-injection of BM-MSC and tumor cells in mice increased the in vivo tumor growth and intratumoral lymphatic vessel density. In addition, BM-MSC or their conditioned medium stimulated the recruitment of lymphatic vessels in vivo in an ear sponge assay, and ex vivo in the lymphatic ring assay (LRA). In vitro, MSC conditioned medium also increased the proliferation rate and the migration of both primary lymphatic endothelial cells (LEC) and an immortalized lymphatic endothelial cell line. Mechanistically, these pro-lymphangiogenic effects relied on the secretion of Vascular Endothelial Growth Factor (VEGF)-A by BM-MSC that activates VEGF Receptor (VEGFR)-2 pathway on LEC. Indeed, the trapping of VEGF-A in MSC conditioned medium by soluble VEGF Receptors (sVEGFR)-1, -2 or the inhibition of VEGFR-2 activity by a specific inhibitor (ZM 323881) both decreased LEC proliferation, migration and the phosphorylation of their main downstream target ERK1/2. This study provides direct unprecedented evidence for a paracrine lymphangiogenic action of BM-MSC via the production of VEGF-A which acts on LEC VEGFR-2.

BackgroundThe lymphatic system controls tissue homeostasis by draining protein-rich lymph to the vascular system. Lymphangiogenesis, the formation of lymphatic vessels, is a normal event in childhood but promotes tumor spread and metastasis during adulthood. Blocking lymphangiogenesis may therefore be of therapeutic interest. Production of adenosine is enhanced in the tumor environment and contributes to tumor progression through stimulation of angiogenesis. In this study, we determined whether adenosine affects lymphangiogenesis.MethodsLymphatic endothelial cells (HMVEC-dLy) were cultured in presence of adenosine and their proliferation, migration and tube formation was assessed. Gelatin sponges embedded with the stable analogue of adenosine 2-chloro adenosine were implanted in mice ear and lymphangiogenesis was quantified. Mice were intravenously injected with adenoviruses containing expression vector for 5′-endonucleotidase, which plays a major role in the formation of adenosine.ResultsIn vitro, we observed that adenosine decreased the proliferation of lymphatic endothelial cells, their migration and tube formation. However, in vivo, gelatin sponges containing 2-chloro adenosine and implanted in mice ear displayed an elevated level of lymphangiogenesis (2.5-fold, p<0.001). Adenovirus-mediated over-expression of cytosolic 5′-nucleotidase IA stimulated lymphangiogenesis and the recruitment of macrophages in mouse liver. Proliferation of lymphatic endothelial cells was enhanced (2-fold, p<0.001) when incubated in the presence of conditioned medium from murine macrophages.ConclusionWe have shown that adenosine stimulates lymphangiogenesis in vivo, presumably through a macrophage-mediated mechanism. This observation suggests that blockade of adenosine receptors may help in anti-cancer therapies.

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Introduction: Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of death from cancer, with a 5-year survival of less than 8%. To date, the therapeutic approaches are chemotherapy regimens. Despite promising data from various preclinical models, new immunotherapeutic agents like checkpoint inhibitors (CPI), vaccines, oncolytic viruses, or TGFβ inhibitors do not show a benefit in the overall survival of patients. Thus, especially in PDAC and in respect to the immune system, there is an urgent need for sufficient preclinical systems that can mirror a human patient scenario. Therefore, we developed a human-based pipeline of whole explant tissue culture using PDAC to predict responses of the tumor to different types of treatments in a personalized manner. Material and Methods: Human cancer tissue is taken into culture after surgery for several days. We tested different timings and technical variations (e.g., thickness of explants, medium changing protocols) to find superior conditions for tissue viability. In culture, the explants can be treated with (pre-) clinical-grade drugs and subsequently processed for quality controls via evaluation of the tissue integrity (on H&E slides) and cytokine stability. Also, histologic analyzes (e.g., cancer cells, T cells, B cells, macrophages) as well as multiplex cytokine analyzes are carried out. This combined approach enables us to study the impact of various drugs on the tissue in terms of tumor cell death, changes in the infiltration and activation of lymphocytes and macrophage polarization, etc. Results In vivo features of the tissue and the matrix architecture as well as the cytokine signatures are well maintained in the explants. Our model is reproducible among different explants of the same tissue in terms of infiltrating cells and cytokine expression. In addition, we were able to achieve an overview of the cytokine signature of PDAC in patients and to distinguish between clusters of patients by identifying different immune landscapes, for example, immunologically hot and cold subtypes. Discussion: Our model is usable as a predictive system for the response of PDAC to different types of treatments and seems to be a reliable and sufficient preclinical extension alternative to mouse models, single-cell experiments, or organoid models. The whole context of the tissue is from human origin and we aim to expand our platform for multiscreening of drug responses for personalized decision-making. Furthermore, we described the tumor microenvironment in detail, unraveling the complex cytokine signature of PDAC, and subsequently identified different clusters of immune landscapes, which may be of prognostic value. Citation Format: Azaz Ahmed, Dyke Ferber, Meggy Suarez-Carmona, Bénédicte Lenoir, Rosa Klotz, Thilo Hackert, Nathalia Giese, Inka Zörnig, Dirk Jäger, Niels Halama. Establishment of a human PDAC explant culture model for treatment prediction and characterization of the tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C02.