(20e) “Lymphatics-on-a-Chip” to Reconstitute Lymphatic Drainage Function and Lymphedema

Lymphatic vessels (LVs), comprised of lymphatic endothelial cells (LECs) are central for tissue fluid homeostasis, immune surveillance, and tumor metastasis [1]. Given that LVs serve as a route for tumor spreading, LV interactions with tumor cells were studied using tumor secretome analysis, biochemistry, MRI imaging, bioinformatics, and mouse models to understand paracrine loops in lymphatic tumor metastasis and to provide new therapeutic strategies [2-5]. Moreover, novel anti-lymphangiogenic peptides were developed and treated in mouse models, showing reduced metastases in the lungs and the lymph nodes [6-10]. Apart from the lymphatic functions in cancers, more general understanding of lymphatic transport of fluid, lipids, proteins, and immune cells is important in investigation of the vast majority of LV-related diseases, such as lymphedema, obesity, heart failure, autoimmune diseases, inflammation, and infections. All of these disorders are closely linked to poor lymphatic transport.

Among many lymphatic diseases, “Lymphedema (LE)”, featured by abnormal tissue swelling, is the most common lymphatic disorder, influencing 150 million individuals worldwide. In physiological condition, normal LVs properly drain excess interstitial fluid that is leaked from blood capillaries; and the drained lymph fluid travels through the lymphatic system to go back to the blood circulation through the subclavian veins. These recirculating processes maintain interstitial fluid homeostasis in normal tissues. However, any failures in the LV drainage trigger abnormal fluid accumulation in the tissues, causing “swelling”. Currently, mechanisms of LV drainage in normal or LE condition are poorly understood, thus therapeutic options for LE treatment are limited. There is no clinically available drug for LE, and widely practiced conservative therapies, such as massage and compression garments are palliative.

One of the major obstacles to better understanding and curing LE is a lack of appropriate experimental tools for evaluating lymphatic drainage function. Lymphatic drainage is the most crucial lymphatic function, directly linked to LE and influenced by numerous biological factors. Although animal models have contributed to major discoveries in the field, isolating and controlling biological factors that may contribute to LE in the animal models is challenging. Majorities of in vitro models that have attempted to solve these problems were two-dimensional (2D) cell culture models on a plastic dish or a transwell, which have not successfully recapitulated lymphatic cell behaviors in 3D environment as in in vivo settings. As such, detailed, mechanistic investigation of LE has remained limited. Thus, 3D biomimetic vascular models would be useful to study blood and lymphatic biology in many different contexts [11-13].

To study lymphatic drainage function and LE pathogenesis in 3D in vitro, we built a new biomimetic “human lymphatics-on-a-chip" model system by fabricating a microfluidics-based device that includes a poly-dimethyl siloxane (PDMS) housing and two parallel micro-channels within 3D collagen matrix. Employing physiological luminal flow in the micro-channels, human primary dermal lymphatic endothelial cells (LECs) seeded in the channels formed lumenized and perfusable LVs within 3D collagen, exhibiting physiologically relevant LV structure and function. For example, interstitial flow containing lymph molecules, such as albumins, fatty acids, phospholipids, and particles, demonstrated that LVs much better drain the lymph molecules than blood vessels (BVs) do. Surprisingly, we observed that LVs exhibited dilated lumens under interstitial flow, however, BVs were squeezed in the same condition. Immunostaining revealed that LVs have weak tight junctions and jagged adherens junctions with specialized portal-like structures to promote fluid transport. The dynamic LV lumens under interstitial pressure, weaker junctions, and preformed portals have been reported in mouse models and human lymphatic tissues, so we concluded that we have a functionally relevant LV model in 3D.

Based on the normal lymphatics-on-a-chip model, inflammatory cytokine treatment and Podoplanin (Pdpn) deficiency significantly impaired lymphatic drainage function by abnormally tightening LV junction. This is a notable finding because most of the inflammation conditions cause vessel leakages by loosening vascular junction. Mechanistically, we discovered that one of the integrin families is a new therapeutic target for LE. This integrin is normally inactivated, however, becomes highly activated in LE conditions, including both cytokine treated- and Pdpn deficient groups. The activated integrin dramatically tightens lymphatic junction, delays fluid, lipid, cell transport through the LVs, and ultimately impairs lymphatic drainage rate. Employing multiple inhibitors of this integrin and the integrin-related pathways, we could normalize pathological lymphatic junction, restoring almost 100% of lymphatic drainage function. Now, we are in validating these findings in mouse LE models.

In conclusion, our “lymphatics-on-a-chip” model recapitulates physiological lymphatic drainage function and LV structure in nature. Moreover, the “lymphatics-on-a-chip” provides an important in vitro platform for revealing previously unappreciated disease mechanisms of LE. Together, these data suggest that the “lymphatics-on-a-chip” may serve as a new platform for mechanistic studies and drug screening for lymphatics associated disorders.

REFERENCES (*Equal contribution)

[1] Lee E, Pandey NB, Popel AS, “Crosstalk between cancer cells and blood endothelial and lymphatic endothelial cells in tumour and organ microenvironment”, Expert Rev Mol Med 17:e3, 2015

[2] Lee E, Pandey NB, Popel AS, “Lymphatic endothelial cells support tumor growth in breast cancer”, Sci Rep, 4:5853, 2014

[3] Lee E, Fertig EJ, Jin K, Sukumar S, Pandey NB, Popel AS, “Breast cancer cells condition lymphatic endothelial cells within pre-metastatic niches to promote metastasis”, Nat Commun, 5:4715, 2014

[4] Lee E, Pandey NB, Popel AS, “Pre-treatment of mice with tumor-conditioned media accelerates metastasis to lymph nodes and lungs: a new spontaneous breast cancer metastasis model”, Clin Exp Metastasis, 31(1):67-79, 2014

[5] Fertig EJ*, Lee E*, Pandey NB, Popel AS, “Analysis of gene expression of secreted factors associated with breast cancer metastases in breast cancer subtypes”, Sci Rep, 5:12133, 2015

[6] Lee E*, Rosca EV*, Pandey NB, Popel AS, “Small peptides derived from somatotropin domain-containing proteins inhibit blood and lymphatic endothelial cell proliferation, migration, adhesion and tube formation”, Int J Biochem Cell Biol, 43:1812-21, 2011

[7] Lee E*, Kim YS*, Bae SM, Kim SK, Jin S, Chung SW, Lee M, Jeon OC, Park RW, Kim IS, Byun Y, Kim SY, “Polyproline-type helical-structured low-molecular weight heparin (LMWH)-taurocholate conjugate as a new angiogenesis inhibitor”, Int J Cancer, 124: 2755-65, 2009

[8] Lee E, Koskimaki JE, Pandey NB, Popel AS, “Inhibition of lymphangiogenesis and angiogenesis in breast tumor xenografts and lymph nodes by a peptide derived from transmembrane protein 45A”, Neoplasia, 15(2):112-24, 2013

[9] Koskimaki JE*, Lee E*, Chen W, Rivera CG, Rosca EV, Pandey NB, Popel AS, “Synergy between a collagen IV mimetic peptide and a somatotropin-domain containing peptide as angiogenesis and lymphangiogenesis inhibitors”, Angiogenesis, 16(1):159-70, 2013

[10] Lee E, Lee SJ, Koskimaki JE, Han Z, Pandey NB, Popel AS, “Inhibition of breast cancer growth and metastasis by a biomimetic peptide”, Sci Rep, 4:7139, 2014

[11] Choi, D., Park, E., Jung, E., Seong, Y.J., Yoo, J., Lee, E., Hong, M., Lee, S., Ishida, H., Burford, J., Peti-Peterdi, J., Adams, R.H., Srikanth, S., Gwack, Y., Chen, C.S., Vogel, H.J., Koh, C., Wong, A., Hong, Y.K., “Laminar flow downregulates Notch activity to promote lymphatic sprouting”, J Clin Invest, doi:10.1172/JCI87442. 2017

[12] Lee E, Song HHG, Chen CS, “Biomimetic on-a-chip platforms for studying cancer metastasis”, Curr Opin Chem Eng, 11:20-7, 2016

[13] Nguyen DHT*, Lee E*, Alimperti SA, Wong A, Eyckmans J, Stanger BZ, Chen CS, “Pancreatic ductal adenocarcinoma replaces endothelium during tissue invasion”, In revision 2017

ACKNOWLEDGEMENTS

This work was supported in part by grants from the National Institutes of Health (EB00262, UH3EB017103, UC4DK104196). Esak Lee acknowledges financial support from LE&RN postdoctoral grant from Lymphatic Education and Research Network (LE&RN), and BU-CTSI grant (TL1TR001410) from the National Center for Advancing Translational Sciences at the National Institutes of Health.