Oscillatory Shear Stress Modulates Lymphatic Progenitor Cells Maturation Into Lymphatic Vessels With Anti-Cancer Phenotypes

Introduction: Lymphedema is a chronic condition characterized by the swelling of tissues caused by the failure lymphatic valves. Lymphatic valves provide a one-way flow of lymph that aids intrinsic pumping in the lymphatic system. Our research group have previously reported that human pluripotent stem cells (hPSCs) can be differentiated into lymphatic endothelial cells (LECs) to form lymphatic capillaries (1,2). However, these lymphatic vessels lack lymphatic valve that is required for lymphatic pumping. Since oscillatory shear stress (OSS) has been shown to regulate lymphatic valve morphogenesis in vivo, we hypothesize that physiological OSS can induce LECs maturation and morphogenesis into lymphatic valves (Figure 1).
Furthermore, tumor-induced lymphangiogenesis from growth factors can promote metastasis in cancer and promote immune cell evasion from primary tumor. However, some cancer types benefit from lymphangiogenesis as it can promote immune cell activation (3). Thus, investigation of how mechanically-induced lymphangiogenesis in LECs can respond to cancer will be of interest due to contradicting studies (Figure 2).

References:
1. Alderfer L, Wei A, Hanjaya-Putra D. Lymphatic Tissue Engineering and Regeneration. Journal of Biological Engineering. 2018;12(1):32.
2. Alderfer L, Russo E, Archilla A, Coe B, Hanjaya-Putra D. Matrix stiffness primes lymphatic tube formation directed by vascular endothelial growth factor-C. The FASEB Journal. 2021;35(5):e21498.
3. Fankhauser M, Broggi MAS, Potin L, et al. Tumor lymphangiogenesis promotes T cell infiltration and potentiates immunotherapy in melanoma. Science Translational Medicine. 2017;9(407):.

Materials and

Methods: Human juvenile LECs and hiPSC-derived LECs (differentiated using our previously reported step-wise protocols) were cultured onto ibidi μ-Slide I 0.6 Luer channel slides (4). Slides were coated with 40-60 μg/mL fibronectin. OSS (at 0.5 dyne/cm2 and 10 dyne/cm2) were applied to all LECs with the ibidi Pump System for 48 hours (see Figure 3). For oscillatory flow, the pump changed flow directions every 4 seconds. Immunostaining was conducted and imaging was done on a revolve fluorescence microscope and confocal microscope. Image analysis was conducted on ImageJ utilizing the directionality and circularity plugins. Real-time PCR was conducted with human angiogenesis array panels. Statistical analyses were unpaired t-tests with P = 0.05 conducted on GraphPad Prism V10.

References:
4. Kusuma S, Shen Y-I, Hanjaya-Putra D, et al. Self-organized vascular networks from human pluripotent stem cells in a synthetic matrix. Proceedings of the National Academy of Sciences. 2013;110(31):12601–12606.

Results, Conclusions, and Discussions:

Results:
In response to physiological OSS, juvenile LECs and differentiated LECs (dLECs) acquire cellular alignment with “zipper-like” junctions as indicated by the expression of VE-cadherin that resemble lymphatic valves. Both low and high OSS influenced a cuboidal morphology in both juvenile LECs and dLECs which is a characteristic of lymphangiogenesis (Figure 4).

The essential differential gene expression of Prox1 in response to differing OSS for lymphatic valve development was present in both groups of LECs. OSS-dependent gene expression responses in FOXC2 and CDH5 (as well as other lymphangiogenesis and lymphatic related genes) were also evident in both LECs and dLECs (Figure 5 and Figure 6).

Both low and high OSS were also found to influence the expression of genes related to cancer immunity and tumor growth in the lymphatic system (Figure 7). There are also OSS-dependent releases of tumor-inflammatory cytokines and chemokines related to LEC-driven T-cell/immune cell trafficking. These genes and cytokines have been previously associated with LECs and certain cancer types (5).

Discussion:
The morphological properties and genetic expressions obtained from exposing hiPSC-derived LECs to OSS flow rates demonstrates its efficiency in maturing into lymphatic-valve forming cells. It is important to note that the juvenile LECs are in a more mature state which may impact its response to OSS compared to dLECs. Thus, demonstrating the importance of varying lymphangiogenesis stages and their ability to form into lymphatic valves.

It was also identified that low and high OSS significantly changed the expression of genes that can promote tumor growth and T-cell de-cumulation. Furthermore, a release of both inflammatory and immune-cell trafficking cytokines in response to varying OSSs was found in juvenile LECs and dLECs. Thus, these findings show that the differing 'lymphangiogenic' states and use of OSS can impact the response of the lymphatic system to cancer.

Conclusion:
OSS promotes lymphatic valve phenotypes in hiPSC-derived LECs for lymphatic tissue engineering. Furthermore, OSS-dependent lymphangiogenesis can possibly regulate the lymphatic’s system response to cancer in LECs and differentiated LECs (Figure 8).

References:
5. Stacker SA, Williams SP, Karnezis T, et al. Lymphangiogenesis and lymphatic vessel remodelling in cancer. Nat Rev Cancer. 2014;14(3):159–172.

Acknowledgements (Optional): We acknowledge support from the University of Notre Dame through “Advancing Our Vision” Initiative in Stem Cell Research,
Harper Cancer Research Institute – American Cancer Society Institutional Research Grant (IRG-17-182-04), American Heart
Association through Career Development Award (19-CDA-34630012 to D.H.-P), National Science Foundation (2047903 to D.H-P.), National
Institutes of Health (1R35-GM-143055-01 to D.H.-P.), and GEM Fellowship to N.K.L.