Normalizing Lymphatic Drainage Potentiates Immunotherapy in Breast Cancer

Introduction: Triple-negative breast cancer (TNBC) forms solid tumors that are characterized by high interstitial fluid pressure (IFP), which promotes tumor progression by modulating angiogenesis, matrix metalloproteinase activity, and cancer cell motility. High tumor IFP further compromises cancer immunotherapies by keeping T lymphocytes from infiltrating into tumors. Leaky blood vessels (BVs), combined with impaired lymphatic drainage, contribute to the formation of high IFP, in addition to the solid stress formed by the tumor extracellular matrix (ECM). To reduce tumor IFP, researchers tried to decrease solid stress and normalize hyperpermeable BVs in tumors, but how lymphatic vessels (LVs) are impaired in the tumor microenvironment is poorly understood.

Materials and

Methods: Here, we created a three-dimensional (3D) bioengineered model of vascularized tumors to study blood and lymphatic vessel junctions, fluid permeability, and drainage in TNBC. The device consisted of a central chamber to hold collagen ECM and either a single channel or two parallel channels, both with a diameter of 250 μm, fully embedded in the ECM bulk. On the single-channel device, the hollow channel was seeded with human primary blood endothelial cells (BECs), which was surrounded by ECM containing either normal human mammary epithelial cell line (MCF-10A) or malignant human TNBC cell line (MDA-MB-231). We measured the diffusive permeability of the engineered BVs by introducing fluorescently labeled 70 kDa Dextran into the vessel lumen. To examine LV drainage, we created two-channel devices. One of the channels was seeded with human primary lymphatic endothelial cells (LECs) and added with a minimal volume of media to maintain cell viability, while the counterpart acellular channel was introduced with excessive media to create an interstitial fluid pressure gradient and the consequential interstitial fluid flow towards the engineered LV. We also created a 2D biomimicry of the endothelial monolayers using micropatterned polyacrylamide gel substrates with tunable stiffness to quantify the traction force generated by BVs and LVs in the normal and TNBC conditions using traction force microscopy.

Results, Conclusions, and Discussions: Using the 3D device, we revealed that BV junctions became loosened in the TNBC condition, exhibiting increased permeability, while LV junctions became tightened, exhibiting lower fluid drainage, compared to normal breast epithelial conditions. TNBC tumor conditions also elevated the traction force of both BECs and LECs, leading to a striking reorganization of their junctions. This elevated contractility, however, posed differential effects on lymphatic and blood junctions, thereby resulting in abnormal junction formation and subsequent permeabilities on tumor blood and lymphatic vessels. Blocking rho-associated protein kinases reduced the contractility of both blood and lymphatic endothelial cells and reconstructed vessel structural integrity and functionality in vitro. Injection of Rho kinase blockade to the breast tumor-bearing mice decreased tumor IFP, increased CD8+ T cell infiltration, and enhanced tumor suppression in synergistic with anti-PD-L1 immunotherapy. Together, our study provides new insights into understanding the physical interaction of endothelial, tumor, and immune cells, evidence of rho-kinases as a target for cancer immunotherapy, as well as a promising way to increase the efficacy of immunotherapies in cancer patients.

Acknowledgements (Optional): This work was supported by NIH grants (CA279560, HL165135). Y.P. was supported by the International Foundation for Ethical Research Fellowship. This work was performed in part at the Cornell NanoScale Facility, a member of the National Nanotechnology Coordinated Infrastructure, which is supported by the National Science Foundation (Grant NNCI-2025233). This work was performed in part at the Cornell Biotechnology Resource Center imaging facility (NIH S10OD025049).