A Novel Ex Vivo Tumor Spheroid-Tissue Model for Investigating Microvascular Remodeling and Lymphatic Blood Vessel Plasticity.

Biomimetic tumor microenvironment models bridge the gap between in vitro and in vivo systems and serve as a useful way to address the modeling challenge of how to recreate the cell and system complexity associated with real tissues. Our laboratory has developed an ex vivo rat mesentery culture model, which allows for simultaneous investigation of blood and lymphatic microvascular network remodeling in an intact tissue environment. Given that angiogenesis and lymphangiogenesis are key contributors to the progression of cancer, the objective of this study was to combine tissue and tumor spheroid culture methods to establish a novel ex vivo tumor spheroid-tissue model by verifying its use for evaluating the effects of cancer cell behavior on the local microvascular environment. H1299 or A549 tumor spheroids were formed via hanging drop culture and seeded onto rat mesenteric tissues harvested from adult male Wistar rats. Tissues with transplanted spheroids were cultured in serum-free media for 3 to 5 days. PECAM, NG2, CD11b, and αSMA labeling identified endothelial cells, pericytes, immune cells, and smooth muscle cells, respectively. Time-lapse imaging confirmed cancer cell type specific migration. In addition to increasing PECAM positive capillary sprouting and LYVE-1 positive endothelial cell extensions indicative of lymphangiogenesis, tumor spheroid presence induced the formation of lymphatic/blood vessel connections and the formation of hybrid, mosaic vessels that were characterized by discontinuous LYVE-1 labeling. The results support the application of a novel tumor spheroid microenvironment model for investigating cancer cell-microvascular interactions.

Lymphatic/blood vessel plasticity: motivation for a future research area based on present and past observations.

The lymphatic system plays a significant role in homeostasis and drainage of excess fluid back into venous circulation. Lymphatics are also associated with a number of diseases including lymphedema, tumor metastasis, and various lymphatic malformations. Emerging evidence suggests that lymphatics might have a bigger connection to the blood vascular system than originally presumed. As these two systems are often studied in isolation, several knowledge gaps exist surrounding what constitutes lymphatic vascular plasticity, under what conditions it arises, and where structures characteristic of plasticity can form. The objective of this review is to overview current structural, cell lineage-based, and cell identity-based evidence for lymphatic plasticity. These examples of plasticity will then be considered in the context of potential clinical and surgical implications of this evolving research area. This review details our current understanding of lymphatic plasticity, highlights key unanswered questions in the field, and motivates future research aimed at clarifying the role and therapeutic potential of lymphatic plasticity in disease.

The Microvascular-Lymphatic Interface and Tissue Homeostasis: Critical Questions That Challenge Current Understanding.

Lymphatic and blood microvascular networks play critical roles in the clearance of excess fluid from local tissue spaces. Given the importance of these dynamics in inflammation, tumor metastasis, and lymphedema, understanding the coordinated function and remodeling between lymphatic and blood vessels in adult tissues is necessary. Knowledge gaps exist because the functions of these two systems are typically considered separately. The objective of this review was to highlight the coordinated functional relationships between blood and lymphatic vessels in adult microvascular networks. Structural, functional, temporal, and spatial relationships will be framed in the context of maintaining tissue homeostasis, vessel permeability, and system remodeling. The integration across systems will emphasize the influence of the local environment on cellular and molecular dynamics involved in fluid flow from blood capillaries to initial lymphatic vessels in microvascular networks.

A Challenge for Engineering Biomimetic Microvascular Models: How do we Incorporate the Physiology?

The gap between in vitro and in vivo assays has inspired biomimetic model development. Tissue engineered models that attempt to mimic the complexity of microvascular networks have emerged as tools for investigating cell-cell and cell-environment interactions that may be not easily viewed in vivo. A key challenge in model development, however, is determining how to recreate the multi-cell/system functional complexity of a real network environment that integrates endothelial cells, smooth muscle cells, vascular pericytes, lymphatics, nerves, fluid flow, extracellular matrix, and inflammatory cells. The objective of this mini-review is to overview the recent evolution of popular biomimetic modeling approaches for investigating microvascular dynamics. A specific focus will highlight the engineering design requirements needed to match physiological function and the potential for top-down tissue culture methods that maintain complexity. Overall, examples of physiological validation, basic science discoveries, and therapeutic evaluation studies will emphasize the value of tissue culture models and biomimetic model development approaches that fill the gap between in vitro and in vivo assays and guide how vascular biologists and physiologists might think about the microcirculation.

A Novel ex vivo Method for Investigating Vascularization of Transplanted Islets.

Revascularization of transplanted pancreatic islets is critical for survival and treatment of type 1 diabetes. Questions concerning how islets influence local microvascular networks and how networks form connections with islets remain understudied and motivate the need for new models that mimic the complexity of real tissue. Recently, our laboratory established the rat mesentery culture model as a tool to investigate cell dynamics involved in microvascular growth. An advantage is the ability to observe blood vessels, lymphatics, and immune cells. The objective of this study was to establish the rat mesentery tissue culture model as a useful tool to investigate islet tissue integration. DiI-labeled islets were seeded onto adult rat mesentery tissues and cultured for up to 3 days. Live lectin labeling enabled time-lapse observation of vessel growth. During culture, DiI-positive islets remained intact. Radial lectin-positive capillary sprouts with DiI labeling were observed to form from islets and connect to host networks. Lectin-positive vessels from host networks were also seen growing toward islets. PECAM and NG2 labeling confirmed that vessels sprouting from islets contained endothelial cells and pericytes. Our results introduce the rat mesentery culture model as a platform for investigating dynamics associated with the initial revascularization of transplanted islets.

Viewing stromal vascular fraction de novo vessel formation and association with host microvasculature using the rat mesentery culture model.

OBJECTIVE: The objective of the study is to demonstrate the innovation and utility of mesenteric tissue culture for discovering the microvascular growth dynamics associated with adipose-derived stromal vascular fraction (SVF) transplantation. Understanding how SVF cells contribute to de novo vessel growth (i.e., neovascularization) and host network angiogenesis motivates the need to make observations at single-cell and network levels within a tissue. METHODS: Stromal vascular fraction was isolated from the inguinal adipose of adult male Wistar rats, labeled with DiI, and seeded onto adult Wistar rat mesentery tissues. Tissues were then cultured in MEM + 10% FBS for 3 days and labeled for BSI-lectin to identify vessels. Alternatively, SVF and tissues from green fluorescent-positive (GFP) Sprague Dawley rats were used to track SVF derived versus host vasculature. RESULTS: Stromal vascular fraction-treated tissues displayed a dramatically increased vascularized area compared to untreated tissues. DiI and GFP+ tracking of SVF identified neovascularization involving initial segment formation, radial outgrowth from central hub-like structures, and connection of segments. Neovascularization was also supported by the formation of segments in previously avascular areas. New segments characteristic of SVF neovessels contained endothelial cells and pericytes. Additionally, a subset of SVF cells displayed the ability to associate with host vessels and the presence of SVF increased host network angiogenesis. CONCLUSIONS: The results showcase the use of the rat mesentery culture model as a novel tool for elucidating SVF cell transplant dynamics and highlight the impact of model selection for visualization.

An Ex Vivo Tissue Culture Method for Discovering Cell Dynamics Involved in Stromal Vascular Fraction Vasculogenesis Using the Mouse Mesentery.

Stromal vascular fraction (SVF), isolated from adipose tissue, identifies as a rich cell source comprised of endothelial cells, endothelial progenitor cells, pericytes, smooth muscle cells, fibroblasts, and immune cells. SVF represents a promising therapeutic heterogonous cell source for growing new blood microvessels due to its rich niche of cells. However, the spatiotemporal dynamics of SVF within living tissues remain largely unknown. The objective of this chapter is to describe a protocol for culturing SVF on mouse mesentery tissues in order to aid in the discovery of SVF dynamics and associated vessel growth over time. SVF was isolated from the inguinal adipose from adult mice and seeded onto mesentery tissues. Tissues were then cultured for up to 5 days and labeled with endothelial cell and pericyte markers. Representative results demonstrate the observation of SVF-derived vasculogenesis characterized by de novo vessel formation and subsequent vessel connection.