In Vitro, In Vivo, and In Silico Models of Lymphangiogenesis in Solid Malignancies

Abstract

Lymphangiogenesis (LA) is the formation of new lymphatic vessels by lymphatic endothelial cells (LECs) sprouting from pre-existing lymphatic vessels. It is increasingly recognized as being involved in many diseases, such as in cancer and secondary lymphedema, which most often results from cancer treatments. For some cancers, excessive LA is associated with cancer progression and metastatic dissemination to the lymph nodes (LNs) through lymphatic vessels. The study of LA through in vitro, in vivo, and, more recently, in silico models is of paramount importance in providing novel insights and identifying the key molecular actors in the biological dysregulation of this process under pathological conditions. In this review, the different biological (in vitro and in vivo) models of LA, especially in a cancer context, are explained and discussed, highlighting their principal modeled features as well as their advantages and drawbacks. Imaging techniques of the lymphatics, complementary or even essential to in vivo models, are also clarified and allow the establishment of the link with computational approaches. In silico models are introduced, theoretically described, and illustrated with examples specific to the lymphatic system and the LA. Together, these models constitute a toolbox allowing the LA research to be brought to the next level.

Keywords: cancer; computational models; in silico methods; in vitro models; in vivo models; lymphangiogenesis; lymphatic endothelial cells; metastatic dissemination.

Figure 1

Lymphatic system organization and lymphatic marker location. The lymph first collected by lymphatic capillaries displaying button-like junctions transits in pre- and collecting lymphatic vessels characterized by zipper-like junctions and valves, then in lymph nodes (LNs), and finally returns to the bloodstream via the subclavian veins. In some cancers, the primary tumor promotes lymphangiogenesis by secreting pro-lymphangiogenic vascular endothelial growth factors such as VEGF-C and-D, which interact with the specific lymphatic vascular endothelial growth factor receptors VEGFR-2 and -3. Cancer cells can use either the blood circulation or the lymphatic route to disseminate to distant organs through the LNs. The membrane VEGFR-3, LYVE-1 (lymphatic vessel endothelial hyaluronan receptor), and podoplanin, as well as the nuclear Prox1 (prospero homeobox protein 1), are the main markers of LECs.

Figure 2

Summary of the in vitro and in vivo lymphangiogenesis (LA) assays according to their level of complexity. The first level of complexity refers to 2D in vitro cultures of isolated LECs and is used for investigating any individual step of the lymphangiogenic process (proliferation, migration, invasion, etc.) and morphogenesis. Three-dimensional in vitro static cultures increase the level of complexity and enable the study of the biological mechanisms underlying the whole process of LA. The third degree of complexity relates to in vitro 3D cultures, including flow and engineered constructs. In vivo mouse and zebrafish models stand for the highest level of complexity.

Figure 3

The mathematical modeling pipeline in biology. The in silico procedure is divided into 4 distinct steps, including literature, modeling, simulation, and validation. During this entire modeling process, the symbiotic approach comparing in vitro and in vivo experimental data with computer outputs is used.

Figure 4

Summary of the in silico models developed in the context of the lymphatic system and the process of lymphangiogenesis. In the context of lymphatics, in silico models of lymphatic flow, drainage, and biomechanics were first developed. Tumor lymphangiogenesis was then studied with hybrid multiscale computational approaches, as well as the interactions between different populations of cells (tumoral, lymphatic, immune, and stromal cells). The effects of a permeable interstitium and a chemokine gradient on the lymphatic network were investigated through in silico techniques. Blood and lymphatic vessel interactions were studied with hybrid mathematical models. The well-known equations of Hodgkin–Huxley were used to mathematically investigate lymphatic electrophysiology. Skin wound healing was lastly modeled and studied with differential equations [173,174,176,177,178,179,180,181,182,183,184,185,186,187,188,189,193,194,195,196,206].