Modeling lymphangiogenesis: Pairing in vitro and in vivo metrics.

Measuring lymphangiogenesis in vivo is an essential step in research because the native microenvironment with its 3D structure, support cells, fluid flow, and circulating proteins are challenging to recapitulate in vitro as described above. Lymphatic system research commonly uses lower order vertebrate animal models (i.e. zebrafish and mice), though higher order vertebrate animal models, such as sheep, are useful in better mimicking human lymphatic function due to the similarity in size. Though zebrafish do not recapitulate all elements of the human lymphatic system (i.e. lymph nodes) they share cellular and molecular characteristics of the early development of lymphatic vessels and the thoracic duct in vertebrates that closely parallels lymphangiogenesis in mammals 75 , 76 . The ability to externally fertilize zebrafish embryos and ease of genetic modification makes them a higher throughput model organism 76 . Their transparency, aided by expression of the green fluorescent protein (GFP) transgene that produces GFP in endothelial cells allows for in vivo optical imaging of the vascular systems. Studies focus on the development and measurement of the thoracic duct as the duct length can translate to broader lymphatic system defects 76 , 77 . Likewise, mouse models are used for their small size, cost efficiency, and similarities to the human genome 76 . They also can be genetically modified to visualize the lymphatic system. Researchers have added fluorescent tags to lymphatic vessels via the Prox1 gene in Prox1-EGFP or Prox1-tdTomato mice 78 – 80 . Another tactic has been to modify the mouse’s genetic code so that EGFP-luciferase is co-expressed with the Vegfr3 gene to monitor lymphangiogenesis in vivo 81 . Within mice, scientists commonly utilize the ear, cornea, hind leg, and tail as locations to visualize the lymphatic system and its development in situ ( Figure 2 ). Mouse ears have a many lymphatic vessels close to the surface and can be used as dermal sheets or in sponge assays 76 , 77 . The mouse ear sponge assay simultaneously examines the proliferation, migration, and tubulogenesis of LECs in a gelatin sponge implanted into the upper mouse ear 82 . The mouse cornea is a unique location to observe lymphangiogenesis since it is avascular, providing an environment to observe lymphangiogenesis without interference from the native network 76 , 77 . This model was first described by Cao et al in 2011 83 but has been modified to look at inflammation-induced lymphangiogenesis by placing a suture in the cornea 84 . Another form of visualizing lymphangiogenesis, specifically invasion, utilizes an implanted collagen window in a mouse tail, as created by Boardman and Swartz in 2003 18 . A collagen dermal equivalent is used as a scaffold in an excised section midway up the tail to visualize cell migration and proliferation in an acellular and regenerative format 85 . A similar technique is performed outside of the tail, as seen with the Matrigel plug assay, where Matrigel is subcutaneously injected and then excised later to visualize the vascularization of the overlaying skin 86 . Researchers have also implanted chronic windows to use intravital microscopy to visualize native lymphangiogenesis in a number of tissues and contexts 87 , 88 In addition to the models and techniques used to visualize the growth of new lymphatic vessels, in vivo studies also examine lymphatic drainage to probe the functionality of new and existing lymphatic vessels. This can be monitored by injecting Evans blue into the mouse’s tail or rear footpad to observe the drainage of the hind leg 89 , 90 . Micro-lymphangiography is another in situ technique that uses fluorescent isothiocyanate (FITC) or a macromolecule homolog injected intradermally in the tail to reveal the hexagonal fluid channels and network of functional lymphatic capillaries 18 , 85 , 90 . Alternative methods to image the lymphatic system and its dynamics include microbubbles with ultrasound 89 , microbead injection and tracking 91 , lymphoscintigraphy 92 , 93 and other imaging technologies, recently reviewed 94 . With any in vivo model, histology and immunofluorescence can be used to demonstrate how LEC-specific positive staining denotes the luminal structures of vessels while maintaining the native tissue organization. Due to the multiple cell types in tissues, it is crucial to make a distinction between lymphatic and non-lymphatic endothelial structures using colocalization of the standard LEC markers (LYVE-1, Prox-1, and podoplanin) with CD31 (PECAM-1) to differentiate blood vessel endothelial cells 85 , and F4/80 to mark macrophages 85 , 95 . The colocalization of LEC markers can also distinguish the maturity of vessels with CC chemokine ligand 21 (CCL21) for initial lymphatic capillaires 96 and alpha-smooth muscle actin to denote collecting vessels 91 , 97 . Researchers have also used transmission electron microscopy (TEM) to visualize matrix remodeling and LEC migration using ultrathin sections stained with uranyl acetate and lead citrate 77 . Multiple metrics have been used to determine if lymphangiogenesis is indeed occurring as described in Figure 2 . Researchers can monitor the expression of VEGF-C and VEGF-D in vivo using the same protein analysis methods mentioned earlier 80 , 95 . For instance, one can measure the activation of VEGFR-3 to denote the initiation of lymphangiogenesis 18 , 95 . Quantifying the cell density of positively stained LECs (i.e. proliferating LECs using co-stains with BrdU) or the percentage of the fluorescent area using the LEC-specific markers can display LEC activation and increase in lymphatic vessels 85 . Counting the number of LEC+ lumens, the lymphatic vessel density (count over a specific area), and the area of lymphatic vessel coverage identifies the creation of new lymphatic vessels in tissues showing the cross-section 77 , 95 , 98 . In models where the 3D lymphatic network can be visualized, studies denote the increase in lymphatic vessels or tubulogenesis by quantifying the nodes (points where multiple vessels meet) and bifurcations or branches (number of additional vessels connected to a selected vessel) 76 , 77 , 84 . Lymphatic hyperplasia can be quantified by measuring the diameters of lymphatic vessels as well as the cross-sectional area of each vessel to signal the start of the lymphangiogenesis initiated by increased levels of VEGF-C from proliferating LECs 8 , 9 , 77 , 84 , 91 . Any model used to capture human physiology and disease has drawbacks and weaknesses. While in vivo animal models allow for experiments in native tissue environments 99 , they are still limited by how the animal anatomy and physiology are related to those in humans. For example, much lymphatic drainage is driven by the physical movements of individuals, as muscle contraction drives lymphatic flows. Animal models are subject to different types of motions, weights, and length scales which contribute to different fluid flow rates and frequencies, in addition to the inherent differences in lymphatic number and distribution 100 . Additionally, many lymphatic dysfunctions are more prevalent in aging populations, and many in vivo studies are done in relatively young mice, and thus results can vary greatly 101 . Regardless, animal studies have resulted in a number of therapeutic strategies translated to humans, but as the lymphatics are explored in more detail and across more pathologies and tissues, the careful consideration of differences between human and animal models is increasingly important 102 .

In this review, we focus on articles with both in vitro and in vivo components of measuring lymphangiogenesis. to highlight the correlation of the results within single studies. Defining which aspects of lymphangiogenesis each study investigated can inform future experimental design by matching potential model outcomes. The following sections summarize articles that meet this criterion and place them in the context of healthy anatomical locations and related pathologies. Focusing on lymphatic development is important to understanding how lymphangiogenesis occurs in the embryo and regenerates to remedy pathologies like lymphedema. Detry et al explored the step-by-step mechanism of lymphangiogenesis by using injury models in vivo and bridged the gap across experimental platforms using an ex vivo LRA 103 . In vitro and in vivo, LEC sprouting can be characterized by the elongation of cells into thin structures that probe the tissue microenvironment, the formation of vacuoles, and the remodeling of a chemotactic matrix that promotes tubulogenesis and migration. In this case, the selection of the LRA allows for sprouting lymphangiogenesis in vitro, showing vessel formation from an existing lymphatic vessel. Therefore, the in vitro and in vivo models demonstrate comparable results 103 . In more basic models, the lack of 3D structures or pre-existing vessels could limit whether the results correlate to in vivo work. Lymphedema is a disorder where fluid accumulates in a tissue due to a damaged or blocked lymphatic system that can be congenital or secondary to injury. Studies aim to stimulate lymphangiogenesis as a therapeutic. ADSCs are of interest due to their pro-lymphangiogenic abilities. Dai et al found that a Podoplanin+ subset of ADSCs (~11.3%) can be differentiated into LECs (shown by distinct cobblestone morphology and secretion of LEC factors) by indirect co-culture with primary LECs. In vivo, the Podoplanin+ ADSCs were able to relieve limb lymphedema in mice 104 . Beyond ADSCs, there is also interest in mesenchymal stem cells (MSCs). Lee et al found that hypoxic media from MSCs aided lymphangiogenesis, shown in vitro by proliferation, migration, and tube formation, and in vivo by LYVE-1 staining and lymphangiography 105 . In these cases, positive impacts in vitro correlated with functional outcomes and edema relief in vivo; these studies focus on LECs in a regenerative capacity and not on recapitulating the disordered state. In contrast to lymphedema treatments, where lymphangiogenesis is stimulated, treatments for cornea-related inflammation aim to inhibit lymphangiogenesis as it creates a hostile environment for transplants. Here, studies often focus on pharmacological inhibitors, such as Nintedanib 106 and Toluquinol 76 . Yuen et al used 3D cultures for tube formation assays alongside corneal neovascularization to explore Angiopoietin-2’s role in inflammatory lymphangiogenesis and how its suppression could inhibit lymphangiogenesis 84 . The use of the cornea model makes these findings applicable to the broader field of lymphatics research as it is a good model of neo-lymphangiogenesis while matching the simplicity of in vitro models due to limited crosstalk with the tissue microenvironment. However, overall, research on lymphatics is beginning to move toward more complex in vitro modeling via microfluidic devices 56 . These models could be leveraged to develop in vitro studies that more accurately replicate the characteristics of lymphedema and other lymphatic-related disorders. Many models of lymphangiogenesis focus on dermal lymphatics, as they are well-studied, easy to access and derive from human tissues. Thus, dermal LECs have been the standard for in vitro cell culture. Gordon et al investigated the impact of macrophages on dermal lymphatic vessel development 107 . Macrophages, an immune cell responsible for phagocytosis of pathogens, can cause and alleviate inflammation at a site of injury and regulate angiogenesis and lymphangiogenesis 109 . During development, a subset of LYVE-1+ macrophages colocalizes to embryonic lymphatic vasculature, sometimes even incorporating into vessels. However, transdifferentiation of these macrophages was disproven as they do not express Prox1. In PU.1 knockout mice that lack macrophages, lymphatic vessels were hyperplastic, with large vessel diameters and enhanced proliferation, but no change in branching ( Figure 3B , C ). These results suggest that macrophages can negatively regulate LEC proliferation. However, in vitro, macrophages directly co-cultured with LECs enhance proliferation ( Figure 3D ), while macrophage conditioned media did not, suggesting that direct crosstalk between macrophages and LECs is necessary for positive impacts on proliferation in vitro 107 . Macrophages require a broad spectrum of cues to regulate their pro- or anti-inflammatory behavior, and in vitro, these cues are not easily recapitulated. Gordon et al also d that macrophage transdifferentiation is potentially easier to observe in vitro. Here, it is necessary to comment that simplified in vitro systems may not always recapitulate findings seen in vivo, especially when complex multicellular interactions are involved. Lymphangiogenesis during wound healing is a longtime focus of study in the skin. For direct comparison of wound healing assays in vivo and in vitro, studies have paired the in vivo ear biopsy wound margin closing assay with in vitro scratch and tube (or chord) formation assays 110 . In patients with diabetes, therapeutic strategies for improving wound healing are particularly important. Wu et al examined lymphangiogenesis in type 2 diabetes in vitro and in vivo 108 . Epsins 1 and 2 were identified as potential therapeutic targets due to their roles in regulating the spatial availability of VEGFR-2 and VEGFR-3. In diabetic mice, epsin deficiency with VEGF-C supplementation led to enhanced corneal lymphangiogenesis in vivo, shown by increased vessel density and branching ( Figure 3E , F ), and in vitro, shown by increased proliferation, migration, and tube formation ( Figure 3G ). Further, epsin deficiency relieved tail edema in diabetic mice 108 . Therefore, positive impacts in vitro correlated with functional outcomes in vivo in this study of diabetic wound healing. The involvement of dermal lymphatics in melanoma progression and metastasis is also well studied with in vitro assays of LEC proliferation, tube formation, scratch assays, LEC migration out of spheroids, and LRAs, and in vivo with lymph node and tumor volume, lymphangiography, and quantification of lymphatic vessel density and diameter 111 – 113 . Typically, in vitro outcomes are matched to in vivo outcomes. However, in vitro studies usually look at how drugs may inhibit LEC migration and proliferation, separate from the melanoma microenvironment, and then compare those outcomes to in vivo lymphangiogenesis. Though it is useful to surmise pharmacological impacts on LECs, these in vitro models do not allow for study of melanoma microenvironment-lymphatic crosstalk or metastasis. Development of complex melanoma-lymphatic models is underway 114 , 115 . Adipose tissue is the master regulator of metabolism in the body. Adipocytes intrinsically support vascularization in vitro 116 , 117 and thus could be harnessed for lymphatic regeneration. Many lymphatic-related diseases are interconnected with adipose tissue dysfunction. Lipedema, for example, is a disorder characterized by dysfunctional drainage of adipose tissue lymphatics; this is an understudied and underdiagnosed disease, but work is underway to address it. In obesity, there is conflicting evidence on how obesity and high-fat diets may interact to impair lymphatics 93 , 117 . More complex in vitro models of obesity have recently been created with a focus on hypoxia and mechanical changes in obese adipose tissue. These models could be modified in the future to include a lymphatic component 118 , 119 . One major primary adipose tissue in which lymphatics have been better studied is the breast, composed mainly of adipose tissue. Lymphatic research in the breast primarily examines inflammation in the breast cancer microenvironment. Preclinical cancer research has benefitted from xenografts where human tumor cells are injected into animals to model certain disease states like inflammatory breast cancer and its metastasis to lymph nodes 120 – 122 . For example, by inoculating mice with metastatic MDA-MB-231 and weakly metastatic MCF-7 tumor cells, Pathak et al showed that the tumor microenvironment, specifically the integrity of the ECM and stroma, were key factors of lymph node metastasis 121 . They demonstrated that the different levels of invasiveness of the breast cancer tumor are also reflected in the morphometric analysis of the surrounding lymphatic vessels and their functionality using immunohistochemistry with LYVE-1 and a MRI contrast agent, respectively 121 . However, it’s important to note that xenograft models do not have a fully functioning immune system, and thus differ from both syngeneic models and humans in ways that likely affect and are affected by lymphatics 123 . Tumors with more fluid drainage (seen by contrast-enhanced magnetic resonance imaging) would also have more and larger lymphatic vessels, which correlated with more metastasis to lymph nodes 121 . In vivo tumor studies established the contribution of the lymphatic system to cancer metastasis and are the cornerstone of translating in vivo work to the clinical setting. More recently, lymphatic research associated with breast cancer has investigated how chemotherapies interact with lymphatics and metastasis. Nandi et al explored the role of prostaglandin E2 (PGE2) in regulating inflammatory lymphangiogenesis 23 . PGE2 inactivates anti-tumor immune cells 124 , supports lymphangiogenesis by upregulating VEGF-C and D 125 , 126 , and stimulates tumor cells to migrate and invade 127 , 128 . Similar results were observed in vitro and in vivo, and notably, the authors used breast cancer conditioned media on LECs in vitro, contributing to the translatability of the results 23 . In contrast, chemotherapies have been used to treat tumor metastasis by inhibiting tumor lymphangiogenesis. Studies have shown how therapeutics like paclitaxel, docetaxel, and platinum therapy may promote lymphangiogenesis by the upregulation of VEGFR-3 and VEGF-C expression with blockade of this pathway reducing pro-lymphangiogenic outcomes both in vitro and in vivo 47 , 129 , 130 . Larrieu-Lahargue et al show how disrupting fibroblast growth factor receptor signaling can have similar inhibitory effects on breast cancer-associatedlymphangiogenesis and metastasis 131 . They noted decreased VEGFR-3+ ( Figure 4A , B ) and Podoplanin+ vessel density in the tumors expressing a dominant negative FGFR (FGFR-2DN) reflecting similar outcomes from 3D tube formation assays ( Figure 4C ). Across these studies, it seems that modeling the tumor microenvironment with 3D co-cultures is important to translation in vivo and should become standard practice to address the complexities of breast cancer pathophysiology 47 , 132 . In the cardiovascular system, lymphangiogenesis for regeneration post-myocardial infarction (MI) is the primary pathological focus. After MI, impaired lymphatics lead to edema, inflammation, and fibrosis 134 . Interestingly, an increased number of lymphatic vessels is observed post-MI, but the remodeling of the collecting vessels causes impaired fluid transport. In short, even though there are more vessels after MI, they are dysfunctional 135 . Zhang et al combined CD34+VEGFR-3+ endothelial progenitor cells with a VEGF-C releasing hydrogel and observed increased lymphangiogenic behaviors in vitro using tube formation and migration assays. In vivo echocardiography and area of lymphatic vessels both indicated similar positive results 136 . When M2b macrophages are colocalized with LECs, they promote increased vessel count in vivo, and increased tube formation, proliferation, and migration in vitro 137 . Additionally, VEGF-C and VEGFR-3 expression were higher when macrophage supernatant was introduced, ultimately showing that macrophages can aid in the treatment of myocardial fibrosis after MI 137 . A study by Bizou et al further touched on how LYVE-1+ macrophages could specifically contribute to lymphangiogenesis after MI 138 . In the lung, tumor-associated lymphangiogenesis, matrix remodeling, and metastasis have been a focus. Here, inhibition of lymphangiogenesis has been explored via matrix metalloproteinase inhibitors 139 and other pharmacological inhibitors 140 . Commonly, these inhibitors exhibit similar results on LECs in vitro, with negative impacts on proliferation and migration, and in vivo, with decreased intensity of LYVE-1 expression 140 . Interestingly, MSCs were shown by Maertens et al to benefit tumor lymphangiogenesis in lung cancer 141 . Zhang et al demonstrated how epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) inhibit VEGF-C secretion by tumor cells in both in vitro and in vivo studies, thereby decreasing LEC proliferation and lymphatic vessel density in non-small lung cancer tumors 142 . Ultimately, studies here focus on inhibiting tumor-associated lymphangiogenesis in the lung to prevent metastasis and increase survival. Lung tumors can also be studied through the lens of inflammatory lymphangiogenesis. In this regard, TNF- α has mixed impacts on LECs: alone, it inhibits proliferation, but when combined with angiopoietin-2, it promotes it 67 , 143 . Interestingly, Ji et al report that in vivo, blocking VEGFR-3 in mice in vivo prevented TNF- α stimulated lymphangiogenesis ( Figure 4D ). In vitro, TNF- α induced morphological changes in both human LECs and murine LECs, including elongation and formation of spindle-like structures, and induced proliferation and migration 133 . Blocking TNFR-1 abolished TNF- α ’s impact on LECs, but blocking VEGFR-3 did not, proving that the impacts of TNF- α on LECs do not depend on VEGFR-3 in vitro ( Figure 4E ). In this study, VEGFR-3 blocking did not yield the same results in vitro and in vivo, pointing to the idea that more crosstalk, cells, or pathways are involved. For complex disease processes like inflammation, in vitro models often cannot recapitulate what is seen in vivo. For this reason, it is necessary to develop more immune-competent models that can replicate systemic or localized inflammation in vitro for more translatable results 40 . Though the abdomen is home to the specialized gut lymphatic vessels (lacteals), studies here primarily focus on lymphatic function and drug transport rather than lymphangiogenesis. Thus we refer the interested reader to a recent review 144 . As often noted in this review, lymphangiogenesis can be beneficial or detrimental to organ function, and thus there is a careful balance needed. In the kidney, lymphangiogenesis can be associated with fibrosis and renal disease 145 . Kinashi et al studied how connective tissue growth factor (CTGF) regulates fibrosis-associated renal lymphangiogenesis using a tube formation assay in vitro compared to Masson’s Trichrome staining and immunohistochemistry for VEGF-C, CTGF, and LYVE-1 in vivo 146 . Here, the in vitro assays allowed for isolated observation of how CTGF regulated LECs via supplementation or gene silencing. A wound healing assay is also effective in modeling the lymphangiogenic migratory activity seen in mice with unilateral ureteral obstructions 95 . In contrast, immunosuppressive drugs are used during kidney transplants to prevent lymphangiogenesis, negatively impacting organ function as perivascular lymphatic density is correlated with glomerular filtration rate 147 , 148 . Huber et al showed that immunosuppressive drugs, even at low concentrations, negatively impact lymphangiogenesis in vitro by decreasing LEC proliferation and migration and in vivo by decreasing the number of lymphatic vessels developed in a Matrigel plug assay 148 . Chen et al took a similar approach for pancreatic cancer by using foretinib as a lymphangiogenic inhibitor and visualizing lymphatic vessel density in vivo and in vitro with tube formation and spheroid sprouting 149 . Simple in vitro assays are idea for testing drugs and isolated cytokines and often translate in vivo. The lymphatic system can be found throughout the reproductive tract, and lymphangiogenesis is an integral part of normal and pathologic physiology. Svingen et al portrayed how lymphatic vessels develop in the gonads of genetically modified PROX-1-EGFP mouse embryos 79 . It is enlightening to note how lymphangiogenesis occurs differently in the testes, where lymphatic vessels do not invade the whole organ and in the ovaries, where lymphatic vessels form a complex network throughout the organ that sprout from pre-existing lymphatic vessels. This sex difference provides a foundation to observe the different molecular pathways that govern neo-lymphangiogenesis versus sprouting lymphangiogenesis. Women’s health has historically been understudied; therefore, the rest of this section highlights lymphatic research in obstetrics and gynecology to shed light on this essential area of research. During the development of the placenta, lymphatics are required for regulating fluid homeostasis and possibly immune cell trafficking at the maternal-fetal interface in the uterus. Lymphangiogenesis associated with pregnancy can be characterized as lymphatic vessel hyperplasia 96 , 150 . Placental cytotrophoblasts stimulate LEC migration in vitro as seen by chemotaxis towards cytotrophoblast conditioned media in a tissue culture insert assay 96 . Thus, there are unique intercellular interactions in the reproductive tract, though more complex models would offer better mechanistic insight. Lymphangiogenesis is also associated with the gynecological malignancy endometriosis, an estrogen-driven disease where endometrial tissue is found outside the uterus. Endometriosis affects 10–15% of females of reproductive age, and there is no cure 98 . The pathophysiology is still undetermined, though some hypothesize that it originates and disseminates through the lymphatic system. Researchers have investigated how VEGF-C regulation can be leveraged as a potential therapeutic 98 , 151 . Inflammation caused by neighboring immune and support cells is a significant focus of the study of this disease. Li et al showed lymphatic invasion into endometriotic lesions by the amount of LYVE-1+ cell expression in vivo comparing to using tube formation and migration assays to denote lymphatic vessel development in vitro 151 . They found that VEGF-C is a potential diagnostic biomarker, and treatment of Lenvatinib can possibly alleviate endometriosis by reducing the size of endometriotic lesions and decreasing lymphangiogenesis 151 . One drawback to some of the current studies is the reliance on immunohistochemistry, and in many lymphatic vessels require further distinguishment from immune cells with additional LEC markers. Regardless, the structure and function of the lymphatic system in the reproductive tract and development of in vitro models of this tissue will greatly advance this field of study and our understanding of gynecological pathologies. Overall, the highlighted articles show that in vitro and in vivo results often correspond to one another. However the assays and analyses done in either context vary from lab to lab, organ to organ, and pathology to pathology. In studies of lymphatic development, in vitro assays often mimic well with neo-lymphangiogenesis, as they do not require a pre-existing lymphatic network. In wound healing and lymphatic regeneration, often harnessed for pathologies like lymphedema and type 2 diabetes, the outcomes of in vitro and in vivo studies correspond with therapeutic outcomes, such as edema relief or improved wound healing. Similarly, in the cardiothoracic system and abdomen, most outcomes pair well and aid in alleviating symptoms of related pathologies. In the reproductive tract, lymphangiogenic outcomes in vivo do not reflect in vitro metrics at times due to immune cell interaction during inflammation or pregnancy. In tumor-related lymphangiogenesis, in vitro studies focusing on how anti-cancer drugs may impact LECs often predict results in vivo, but the complexity of the tumor microenvironment can weaken this cross-validation. The field needs to develop more complex in vitro models that recapitulate the in vivo microenvironment to yield more translatable outcomes and improve what is known about the physiology of lymphangiogenesis for some complex tissues and pathologies ( Figure 5 ). One strategy for developing in vitro models of lymphangiogenesis that are comparable to in vivo models is microfluidic devices. These “on-chip” systems allow for precise control of the microenvironment and the addition of perfusion. Lee et al developed a perfusable tumor-lymphatic network-on-chip and tested anti-VEGFR3 therapies as well as demonstrated natural killer cell trans-lymphatic endothelial migration 54 . Selahi et al replicated the alignment of LECs and muscle cells in a lymphangion-on-chip model, a phenomenon only previously observed in vivo 62 . Frenkel et al developed a perfusable LEC network that can be co-cultured with tumor organoids 30 . The potential of perfusable lymphatic networks for the study of lymphangiogenesis is an exciting development in the field, allowing more functional analyses to be conducted. Further, Cho et al, Kim et al and Pisano et al developed lymphatic models with interstitial flow, providing even more physiologically relevance by replicating drainage 55 , 56 , 63 . Another critical gap in the literature is a lack of in vitro models of the lymph node that allow for the study of lymphangiogenesis. The lymph node is responsible for immunosurveillance and adaptive immunity. In cancer research, tumor crosstalk and chemotherapies can cause lymphangiogenesis in the lymph node 47 , 152 , 153 . As many tumors metastasize to the lymph node and evade the immune system, it is essential to study. Lymph node lymphangiogenesis is also impacted by immunization, inflammation, and immune cell crosstalk 154 , 155 . In vitro models for the study of lymph node lymphangiogenesis can provide insight into autoimmunity and cancer metastasis, as well as the potential to screen therapeutics and vaccines. The development of microphysiological lymph node models is underway 156 , and applicable ex vivo models are also available 157 – 159 . Further, a recent in vivo imaging technique using microbubbles and ultrasound can aid in visualizing lymph node vasculature and flow dynamics so that these vital data can be translated to microfluidics 89 . Specifically, within the context of this review, the correspondence of in vitro to in vivo outcomes depends on the metrics evaluated and the tissue or pathology of interest. Some metrics of lymphangiogenesis typically require more physiological context to interpret. One example is proliferation, which can translate to remodeled hyperplastic vessels or new vessel generation 8 , 9 , 107 , 137 , 142 , 148 . Therefore, interpreting in vitro LEC proliferation results can be complex - relating to pathological or beneficial outcomes. One recommendation is that proliferation assays be paired with another metric, specifically an assay that allows for lymphangiogenesis from an existing lymphatic vessel as seen in vivo, to provide more informative results. Additionally, many in vitro studies focus on LECs alone with the addition of drugs of interest. Translation of drug delivery dosages and frequencies is a perennial challenge for all in vitro to in vivo studies, and the same is true when studying lymphangiogenesis. Furthermore, the necessity of supporting cells in the microenvironment to impact LEC behavior is vital to consider in the local tissue environment once the drug is present. At times these other cells (i.e. fibroblasts, immune cells, tumor cells) behave differently depending on the in vitro model, where growth factors, scaffolding, and other tissue microenvironment factors may be causing discrepancies that in turn affect LECs. For example, tumor cells express different levels of VEGF-C and VEGF-D in vitro versus in vivo, two factors that directly regulate lymphangiogenesis 160 . Cell-mediated changes were shown by by Gordon et al, where macrophages promoted LEC proliferation in vitro but suppressed it in vivo 107 . VEGF-C is known to indirectly recruit proteolytically active macrophages in vivo, aiding LEC migration, highlighting the spectrum of roles macrophages play in lymphangiogenesis 161 . Interestingly, this phenomenon of macrophage or monocyte transdifferentiation into LECs could offer an interesting in vitro system for both cell sourcing and lymphatic study. For example, in the melanoma tumor microenvironment, LYVE-1+ macrophages mimic sprouting lymphatic vessels 162 . andin renal fibrosis, M1 macrophages can transdifferentiate into LECs 163 . These vascular-associated macrophages are thought to be derived from endothelial progenitor cells 109 . Inflammation is key to causing this transdifferentiation 164 , 165 , and harnessing this phenomenon in vitro, described by Zhigeng et al, could be a promising source of LECs for studying pathologies with lymphatic involvement 166 . Moreover, in Ji et al’s study on TNF-alpha in ovarian cancer, blocking VEGFR3 did not suppress TNF-alpha’s effects on LECs in vitro, but it did suppress TNF-alpha related lymphangiogenesis in vivo 133 . In breast cancer, platinum agents induce lymphangiogenic effects in vitro and in vivo alone. However, taxanes, another class of breast cancer chemotherapy, require the presence of tumor cells to induce the same effects 47 , 129 . These studies directly demonstrate that the complex in vivo environment provides signals that impact lymphangiogenesis and supporting cells and that these cues may not be recapitulated in vitro without careful model design and cell sourcing. LEC migration is impacted by ECM composition, alignment, and dimensionality 77 , 167 . Detry et al showed that sprouting fails to occur when a LRA is performed in Matrigel instead of collagen and that excessive or insufficient collagen impairs LEC sprouting 77 . Further, a key ECM element that is often missing from in vitro lymphangiogenesis assays is hyaluronan 168 . Cell migration in 2D differs significantly from 3D. In 2D assays, such as scratch assays, there is potential for single or collective cell migration 44 . In 3D, proteolytic degradation of the matrix must occur. As mentioned above, macrophages are recruited in vivo to aid matrix remodeling during lymphangiogenesis 161 . Support cells often aid in proteolytic degradation during lymphangiogenesis 168 . Thus, it is essential to design in vitro models that approach the complexity of the tissue microenvironment so that in vitro results can translate to in vivo outcomes. In contrast, in regenerative applications, where the goal is induction or regrowth of lymphatics, in vitro results typically translate to functional relief of edema. Without the added complexity of the tumor, LEC behavior seems more predictable. This is also seen in studies that simply aim to examine how anti-cancer drugs can suppress LEC migration and proliferation. When selecting an in vitro assay, it is necessary to consider the underlying hypotheses, desired therapeutic outcomes, and the complexity of the native environment that needs to be replicated when interpreting results between in vitro and in vivo. Furthermore, there is a distinct gap between studies that neglect fluid flow and those that account for it. Even low interstitial fluid flow velocity still affect lymphangiogenic characteristics including, but not limited to, junction integrity, protein expression, cell-cell interactions, and sprouting 169 . We recommend that experiments measuring sprouting and maturation use fluid flow to mimic the forces in vivo. Previous research has shown that interstitial fluid flow can guide lymphangiogenesis by enhancing morphogenic effects of ECM-bound growth factors (like VEGF-C) and inducing the expression of essential genes required for lymphatic vessel development 18 , 56 , 85 , 91 . The direction of lymph flow has been shown to guide fluid channeling, LEC migration, and functional vessel formation 18 . Sweet et al reveal that though fluid flow is not required for the primary formation of lymphatic vessels, it is necessary for its remodeling into mature collecting vessels by using knockdown experiments in vitro and in vivo 91 . Fluid accumulation and stretch on the cells cause shear stress, making LECs proliferate, elongate, and sprout 170 , 171 . Adding interstitial flow can be as easy as calculating the necessary pressure head for a tissue culture insert 45 – 48 or using a radial flow chamber 172 , and shear stress experiments can be performed on parallel plate flow chamber 91 . These modifications can enhance the physiological relevance of lymphatic study in vitro. As lymphangiogenesis has been overlooked in the past compared to other physiological players, many current studies rely on the fact that drugs already approved for their anti-angiogenic effects also tend to hinder lymphangiogenesis 173 . Preclinical models have played a vital role in uncovering anti-lymphangiogenic therapies, such as antibodies targeting the VEGF-C/VEGFR-2/3 axes, leading to recent clinical trials 174 . As preclinical models of lymphangiogenesis carefully increase in complexity and relevance, their ability to uncover and explore potential therapeutics will lead to immense progress toward treating lymphatic-involved pathologies. Further, the development of models and knowledge surrounding lymphatics in other tissues (such as gynecological tissues) and diseases, will offer even more opportunity for novel models and discoveries surrounding these important vessels.

Within this review, we have discussed models and metrics of lymphangiogenesis both in vitro and in vivo. In vitro models of lymphangiogenesis correlate best with in vivo data in applications such as wound healing, where lymphatics play a beneficial role. LEC behavior is altered by an extensive range of supporting cells in the tumor microenvironment and other pathological states. Thus, these cues can be more challenging to recapitulate in vitro. We recommend developing in vitro models with physiologically relevant microenvironments and fluid flow, while carefully considering underlying hypotheses or outcomes to be tested, to facilitate the correlation of lymphangiogenesis in vitro and in vivo.

The lymphatic system is responsible for maintaining fluid homeostasis in the body and aiding immune cell trafficking from lymph nodes to injury sites. A healthy lymphatic system is integral to overall health, and its disruption is apparent in many disorders. The lymphatic system mirrors the blood capillaries, reaching every part of the body except the epithelium, sclera, cartilage, brain parenchyma, and intima of blood vessels. There are specialized lymphatic vessels in the meninges surrounding the central nervous system (meningeal lymphatics), and the small intestines (lacteals). The lymphatics within tissues are comprised of initial and collecting lymphatics. The initial lymphatics are thin-walled cylinders made up of lymphatic endothelial cells (LECs) that lack a basement membrane to allow the filtering of the interstitial fluid from tissues. These lymphatic capillaries empty into larger mature collecting lymphatic vessels surrounded by pericytes and smooth muscle cells that transport lymph (the interstitial fluid that entered the lymphatic system) using one-way valves. These mature vessels drain and filter lymph through the various lymph nodes before entering the thoracic duct to empty into the venous circulation. Lymphangiogenesis is the formation of new lymphatic vessels. The process may start from LEC progenitor cells during embryonic development (considered neo-lymphangiogenesis henceforth) or extend from an existing lymphatic vessel (sprouting lymphangiogenesis). LECs initiating from an existing vessel proliferate and migrate into the extracellular matrix (ECM) after activation. Proteins in the vascular endothelial growth factor (VEGF) family that promote vascular genesis also direct LEC behavior. VEGF-C is the central protein that modulates LEC maintenance, migration, and proliferation with VEGF-D as a close paralog that aids in generating lymphatic vessel growth 1 , 2 . VEGF-C primarily binds with the tyrosine kinase receptor VEGF receptor- 3 (VEGFR-3) that is expressed most abundantly on LECs, and activates downstream molecular pathways that regulate LEC migration and proliferation 3 , 4 . VEGF-C can also activate VEGFR-2 to form a heterodimer with VEGFR-3 to promote lymphatic vessel development 5 , 6 . The proliferating cells begin to sprout in the surrounding ECM to form naïve lymphatic capillaries, which undergo remodeling for maturation 7 . Enhanced proliferation of LECs in the presence of excess VEGF-C is suggested to cause lymphatic hyperplasia, which can be part of normal physiological lymphangiogenesis but also contributes to a number of pathological states 8 , 9 . Though the human lymphatic system was anatomically defined and received its name in the 16 and 17 th centuries, 21 st -century research aims to characterize its function and development in healthy and pathological tissues 10 . For example, in a diseased or disordered state, the lymphatic system can be blocked, leading to fluid accumulation and fibrosis seen as secondary lymphedema. A report has suggested that patients with head and neck cancers have a 75% prevalence rate for lymphedema after resection surgeries and radiation therapies 11 . Likewise, the lymphatic system can facilitate tumor metastasis to lymph nodes and other organs. Lymph node metastasis is correlated with poor prognosis in a variety of cancers 12 – 14 . Therefore, modulating lymphangiogenesis as a therapeutic approach is often investigated as enhancing it can relieve fluid buildup in the case of cancer-associated lymphedema, while inhibiting it pre-clinically can slow the metastasis of malignancies. In order to study and understand lymphangiogenesis and the lymphatic system, researchers use a variety of in vitro and in vivo models to simultaneously examine multiple elements of lymphatic physiology. Here, we aim to describe how lymphangiogenesis can be studied across different test environments, specifically in vitro and in vivo, and how outcomes in these contexts correlate. In this review, the ‘hallmarks’ of lymphangiogenesis refer to the phenomena (e.g., sprouting), while ‘metrics’ are how the hallmarks are quantified (e.g., number of new vessels). Only articles that have both in vitro and in vivo experiments are discussed to better pair results across diseases and tissuesto inform how lymphangiogenesis is studied and how to interpret results seen in different contexts by examining a breadth of normal and disease states.