Long-Lived Human Lymphatic Endothelial Cells to Study Lymphatic Vessel/Tumor Coculture

Long-Lived Human Lymphatic Endothelial Cells to Study Lymphatic Biology and Lymphatic Vessel/Tumor Coculture in a 3D Microfluidic Model

The lymphatic system is essential in maintaining tissue fluid homeostasis as well as antigen and immune cell transport to lymph nodes. Moreover, lymphatic vasculature plays an important role in various pathological processes, such as cancer. Fundamental to this research field are representative in vitro models. Here they present a microfluidic lymphatic vessel model to study lymphangiogenesis and its interaction with colon cancer organoids using a newly developed lymphatic endothelial cell (LEC) line. They generated immortalized human LECs by lentiviral transduction of human telomerase (hTERT) and BMI-1 expression cassettes into primary LECs. Immortalized LECs showed an increased growth potential, reduced senescence, and elongated lifespan with maintenance of typical LEC morphology and marker expression for over 12 months while remaining nontransformed. Immortalized LECs were introduced in a microfluidic chip, comprising a free-standing extracellular matrix, where they formed a perfusable vessel-like structure against the extracellular matrix. A gradient of lymphangiogenic factors over the extracellular matrix gel induced the formation of luminated sprouts. Adding mouse colon cancer organoids adjacent to the lymphatic vessel resulted in a stable long-lived coculture model in which cancer cell-induced lymphangiogenesis and cancer cell motility can be investigated. Thus, the development of a stable immortalized lymphatic endothelial cell line in a membrane-free, perfused microfluidic chip yields a highly standardized lymphangiogenesis and lymphatic vessel–tumor cell coculture assay.

Graphical Abstract

The lymphatic vasculature performs vital tasks to maintain tissue fluid homeostasis: Blunt-ended capillary networks take up extravasated fluid and macromolecules from the interstitial compartment.The lymphatic capillaries also take up lymphocytes and antigen-presenting cells and provide a route for these cells to traverse the body. Lymphatic capillaries drain into larger collecting lymphatics, which end up in the thoracic duct to eventually return the fluid, macromolecules, and immune cells to the blood circulation.

Nontransformed immortalized LECs retain expression of lymphatic markers and form 3D microvasculature in fibrin bead assay. (A) LECs were immortalized using lentiviral hTERT and BMI-1 expression cassettes. Nonimmortalized and immortalized LECs (imLECs) show typical endothelial cobblestone phenotype. (B) imLECs retain protein expression of lymphatic markers LYVE-1 and podoplanin until passage 25 (6 months). (C) Box plot of RNA gene expression imLECs on a 2Log scale. (R2 platform data set Exp. LEC (immortalized) – Kranenburg −15). Naïve LECs (Exp. LEC (immortalized) – Kranenburg −15) show similar expression of lymphatic markers. HEK-293T cells (Exp HEK293 Apoptosis - Palmer −30) express comparatively low levels of LEC markers. (D) Naïve LECs and imLECs were grown in Ibidi glass-bottom eight-well slides and stained for lymphatic markers Prox-1, podoplanin, LYVE-1, and neuropilin-2. The two LEC cell lines show comparable expression of lymphatic markers. Negative controls were performed without incubation with primary antibodies. (E) Quantification of the number of cells positive for lymphatic marker staining, compared between naïve LECs and imLECs. In total, per lymphatic marker, five fields of view were counted, each containing around 50 cells per field of view. No significant difference was observed between the amounts of positive stained cells of all lymphatic markers. (F) Fibrin bead assay to assess three-dimensional lymphangiogenesis.Naïve and imLECs show formation of small sprouts at day 1. In the following days, sprouting expands for both cell types. Only imLECs could sustain sprouts up to day 14, whereas naïve LECs had progressively regressed (data not shown). Bar graphs depicting quantification of sprout surface area and sprout length show lymphangiogenesis by the naïve and imLECs for days 1 and 4, whereas only imLECs continued to proliferate afterward. Surface area and length were measured by tracing lymphatic structures using Fiji. (G) At day 14, imLECs had formed extensive viable microvascular networks (triangles). GFP-tag of the BMI-1 vector allowed fluorescent imaging under the EVOS XL microscope. Dashed lines mark lymphatic microvessels.

After the discovery of several markers that are relatively specific for lymphatic endothelial cells (LECs), including VEGFR3, LYVE-1, Prox-1, and podoplanin, lymphatic research quickly expanded to yield numerous insights in (patho)physiological processes that include vascular development, inflammation, and cancer metastasis. Lately, organ-specific roles for lymphatic vasculature have been discovered that include cardiac, dermal, intestinal, brain lymphatics, and liver lymphatics.

imLECs form a perfused vessel model. (A, B) Schematics of 3-lane OrganoPlate showing the triple lane configuration: a top perfusion channel (medium), an ECM (collagen) matrix channel, and a bottom perfusion channel (medium containing lymphangiogenic mix). imLECs are added to the top perfusion channel and start forming a monolayer tube. (C, D) imLECs form a leak-tight monolayered vessel, capable of retaining FITC-labeled dextran. (E) Barrier integrity assay was performed using 20 kDa FITC-dextran. A mean Papp value of 1.093 × 10–5 with a standard deviation of 2.526 × 10–6 was calculated on the basis of fluorescence ratios (n = 19 chips and a cell-free negative control).

In cancer progression, (peri)tumor lymphatics are far from the passive channels used by metastasizing tumor cells for dissemination as was once believed. Instead, as part of the tumor microenvironment, lymphatics show a complex and active role in cancer migration and progression.In response to factors secreted by tumor cells and other cells of the tumor microenvironment, many changes happen in the lymphatic system geared toward tumor spread. For instance, lymphangiogenesis—new lymphatic vessels developing from pre-existing vessels—occurs, as well as lymphatic vessel hyperplasia, and increased flow in tissue-draining lymphatics. Further downstream, remodeling of lymph nodes happens, allowing for a more immune tolerogenic environment. Moreover, in a complex interplay of chemokines and receptors, tumor cells are believed to mimic immune cells by upregulating receptors such as CXCR4 and CCR7 on their cell membranes. Immune cells possessing these receptors use chemokine gradients (CXCL12, CCL21) to migrate to lymphatic vessels. In research on melanoma and breast cancer, tumor cells appear to mimic this immune cell-lymphatic vessel interaction for cancer cell migration and lymphatic metastasis.

imLECs form a perfused lymphangiogenesis model. (A) Sprouts are formed once lymphangiogenesis is induced (from day 3 onward) by adding (lymph)angiogenic factors to the bottom channel, creating a gradient. (B) imLEC microvascular network formation over time (days 1–18). Extensive sprout formation crossing the collagen matrix toward the bottom perfusion channel at day 10. Sprout surface area and maximum sprout length were analyzed for days 3–10. One chip was divided into three fields of view, in which surface area was measured by freehand tracing using Fiji and 10 randomly chosen sprouts were measured for length using the Fiji freehand line tool. One way ANOVA analysis for sprout surface area and maximum sprout length, p value < 0.0001 and p value < 0.0001, respectively, was performed. (C) Still 3D image of imLEC culture stained for Actin and DAPI showing the monolayer vessel lumen and extending sprouts into the collagen matrix at day 10. (D) Time-lapse images of FITC-dextran dye added to 10-day imLEC tube with a formed microvascular network. Dye is observed within the sprouts (arrows), whereas dye is released at the sprout tips (triangles). (E) Maximum projection confocal image of imLEC culture stained for occludin and CD31 showing the monolayer vessel and extending sprouts into the collagen matrix at day 10. (F) Maximum projection confocal image of imLEC culture stained for PROX-1, LYVE-1, actin, and DAPI illustrating the main imLEC vessel and formed sprouts at day 10.

For investigating lymphatic biology such as lymphangiogenesis, in vivo (live imaging) and in vitro models are used. Well-characterized two-dimensional in vitro assays are used for studying lymphatic biology such as proliferation, adhesion, and scratch assays. However, a three-dimensional assay would allow for a more representative model for lymphatic vessels. Standardized three-dimensional models include lymphatic ring assays where thoracic duct fragments are embedded in an extracellular matrix (ECM) gel and tubulogenesis assays in which LECs form capillary structures in an ECM gel where LECs can be seeded directly on top or into the gel. Moreover, LECs can be adhered to onto microcarrier beads and embedded in ECM gels, called bead assays.

Coculture with colon cancer organoid enables studying tumor cell–LEC interaction. (A) Murine colon cancer organoids were added to matrigel and inserted into the central channel. After gelation, imLECs were seeded into the perfusion channel. (B) Cocultures with imLECs and two distinct colon cancer organoid lines, generated from a spontaneously metastasizing murine colorectal cancer model, shown from day 2–9. The organoids show sustained growth in the model as well as concomitant maintenance of the main imLEC vessel. (C) Sprout surface area and maximum sprout length of the imLECs were analyzed for the two different cocultures. One chip was divided into three fields of view, in which the surface area was measured by freehand tracing using Fiji and 10 randomly chosen sprouts were measured for length using the Fiji freehand line tool. Statistical analysis was performed by unpaired t test for maximum sprout length between cocultures at day 5 (p value = 0.5173), day 9 (p value = 0.1421), as well as for sprout surface area at day 5 (p value = 0.1933) and day 9 (p value= 0.0262). (D, E) imLECs retained VE-cad, Prox-1, and CD31 expression in the presence of the colon cancer organoid lines. The colon cancer organoids closely interacted with the imLEC vessel (arrows). The coculture model can be used to investigate sprout formation/lymphangiogenesis (triangles). (F) Still 3D image of CRC organoids invading part of the LEC main vessel compartment (arrows). The coculture model allows for highly standardized investigation of tumor cell–LEC interaction, such as the effect on sprout formation or tumor cell invasion.

Unfortunately, there are two distinct limitations to these 3D assays. First, primary LECs have a limited life span. Mainly, the cordlike structures formed by primary LECs regress within 24–38 h. Recently, scaffolding approaches such as alternating LEC and fibroblast sheets resulted in a longer-lived lymphatic microvascular network.

Commonly, primary LECs are used that are either isolated or commercially purchased. These primary LECs have a limited lifespan of 12–15 population doublings, after which they start to deteriorate.Moreover, experimental results obtained with such cultures are prone to variation caused by batch effects and passage number. Previously, hTERT has been described to immortalize human dermal microvascular endothelial cells (HDMVECs), a mixed population of cell types with both blood and lymphatic endothelial traits.Cells were able to proliferate up to 40 passages and formed tubes in 3D collagen gels, whereas expression levels of LEC markers podoplanin decreased.To generate a pure LEC line with long life span, we generated a novel human immortalized LEC (imLEC) line using lentiviral transduction of human telomerase (hTERT) and BMI-1 expression cassettes into primary LECs. Generated imLECs showed an increased growth potential and elongated lifespan with maintenance of typical LEC morphology and marker expression for more than 53 passages/12 months while remaining nontransformed.

Second, for LECs, the addition of flow, gradients, and mechanical strain has been supported by previous research.

Since the establishment of microfluidic approaches, several microfluidic lymphatic models have been used. Most of these employed commercially available (nonimmortalized) LECs from different suppliers on matrix gels in microfluidic devices, with or without continuous flow and partly with use of permeable membranes that may interfere with biological processes. Viable lymphatic tubes and sprouting in these models could be observed only between 3 and 7 days after seeding. In the reported setups aside from the Swartz model the chips are experimental in nature and do not provide throughput that would be required for routine experimentation. Here, employing a 3-lane OrganoPlate that allows patterning of an extracellular matrix gel and adjacent growth of a vessel-like structure, they created a microfluidic lymphatic vessel culture, verified its barrier function and showed it capable of sustained lymphangiogenesis. To illustrate our further intentions for using this model for studying lymphatic endothelial cell–cancer cell interaction in the future, they created a coculture model using mouse-derived colon cancer organoids. In addition to using a long-lived LEC line, the 3-lane OrganoPlate uses a throughput of 40 chips per plate that would thus allow for routine experimentation. Moreover, the assay makes no use of artificial membranes, preventing its interference in sprouting, imaging, and cross talk between cell types.

  1. Long-Lived Human Lymphatic Endothelial Cells to Study Lymphatic Biology and Lymphatic Vessel/Tumor Coculture in a 3D Microfluidic Model Nicola Frenkel, Susanna Poghosyan, Carmen Rubio Alarcón, Silvia Bonilla García, Karla Queiroz, Lotte van den Bent, Jamila Laoukili, Inne Borel Rinkes, Paul Vulto, Onno Kranenburg, and Jeroen Hagendoorn ACS Biomaterials Science & Engineering 2021 7 (7), 3030-3042 DOI: 10.1021/acsbiomaterials.0c01378