Integrated in vitro and in silico lymphangiogenesis : Development and use of emerging tools to generate mechanistic insights into lymphangiogenic sprouting

Lymphangiogenesis (LA) is the formation of new lymphatic vessels by lymphatic endothelial cells (LECs) from pre-existing vessels. This process begins with lymphangiogenic sprouting, where LECs migrate and proliferate to extend the lymphatic network. In adulthood, the lymphatic system remains largely inactive, except under pathological conditions triggering lymphatic remodeling. Chronic inflammation and cancer are major drivers of LA activation, where lymphatic changes sustain immune activation, disease progression or facilitate tumor metastasis. Conversely, insufficient LA contributes to dysfunctions like lymphedema, where impaired fluid drainage leads to tissue swelling and fibrosis. Understanding the mechanisms regulating pathological LA is crucial for developing strategies to modulate this process depending on the condition. Deciphering its molecular and cellular regulators could reveal new therapeutic targets, which remain largely unexplored in clinical applications.
Biological processes have long been studied through in vitro and in vivo models, each with intrinsic limitations in faithfully replicating physiological complexity. Emerging technologies are now transforming biomedical research by providing complementary and more integrated approaches. In silico modeling enables the numerical representation of multiscale phenomena, providing mechanistic predictions while minimizing experimental constraints. Ensuring the biological relevance of these computational approaches requires experimental validation, typically conducted using conventional in vitro models but further refined through advanced platforms like microphysiological systems for greater precision. The symbiotic integration of in silico and in vitro methods enhances modeling accuracy and deepens insights into physiological complexity, fostering a more predictive approach to the investigation of biological mechanisms.
This doctoral work aimed to integrate and apply in vitro and computational approaches to better understand the biological dysfunctions of the LA process in pathological conditions. This involved a combination of in silico techniques, including ordinary differential equations (ODEs) and agent-based modeling, as well as inference-based approaches. In vitro functional studies, using both traditional and microfluidic-based setups, complemented these simulations.
The first stage of this research involved developing an intracellular ODE model to represent LEC membrane receptor interactions and trafficking (VEGFR-2 and VEGFR-3). Multiple biological experiments were conducted on LECs to gather data for both the creation and validation of this in silico model. Computational optimization and parameter sensitivity analysis were performed to estimate and analyze the model features. The parameters related to heterodimerization and internalization showed the greatest sensitivity, emphasizing the importance of these processes in achieving the desired LEC sprouting behavior following stimulation.
While this ODE model provided valuable insights into receptor dynamics, it remained focused on well-characterized pathways. To gain a broader perspective on the molecular regulators of LA at the (intra-)cellular level, a complementary data-driven approach was undertaken. Available microarray data from LECs stimulated with a panel of molecules derived from the tumor microenvironment were processed and integrated to perform a network inference analysis. Multiple inference algorithms were applied to construct a gene regulatory network (GRN) from these multi-perturbed transcriptomic data. The graphical study of this inferred GRN revealed the central role of the migratory signaling pathway DLL4-NOTCH1, which has received limited attention in the context of LA but is well recognized for its involvement in angiogenic sprouting.
The DLL4-NOTCH1 pathway was then further characterized in LECs using several in vitro approaches, including a microfluidic chip specifically adapted to study lymphangiogenic sprouting, combined with advanced imaging and computational analysis tools. This chip, consisting of three parallel channels — a LEC channel, a collagen gel channel to be invaded, and a medium channel — enabled the generation of growth factor gradients and interstitial flow. The in vitro experiments confirmed the presence and functionality of the DLL4-NOTCH pathway in LECs, although its activation and sprouting-related behavior appears to differ from that observed in blood endothelial cells (BECs). These differences are likely related to the heterogeneity of receptors present on the membranes of LECs and BECs.
To investigate the impact of receptor heterogeneity at the membrane, a pre-existing multiscale mathematical model of BEC migration during angiogenesis was adapted for lymphatic vessels. Key modifications included coupling this agent-based model with relevant information from the previously developed in silico intracellular model and incorporating LA-specific actors. This adapted framework successfully captures the different combinations of pro-lymphangiogenic factors used to stimulate LECs and the receptors subsequently activated. It highlighted the individual but also combined impacts of each signaling pathway, associated with specific dimer/monomer activation, on the migratory phenotype. Model outcomes suggested that the DLL4-NOTCH1 pathway, driven by VEGFR-2, is not strictly necessary for lymphatic sprouting but acts as a regulator of the VEGFR-3-related migratory pathway, ensuring a more organized process. This emphasizes the importance of both VEGFR-2/VEGFR-3 heterodimers and VEGFR-3 homodimers in LECs.
This work highlights the successful integration of in vitro experiments and in silico modeling accross multiple scales, from intracellular receptor interactions to multicellular sprouting dynamics, providing a robust framework to study lymphangiogenesis and its underlying mechanisms, with potential applications in therapy development and targeted interventions.