Biophysical quantification of reorganization dynamics of human pancreatic islets during co-culture with adipose-derived stem cells.

Islet transplantation is a potential therapy for type 1 diabetes, but it is expensive due to limited pancreas donor numbers and the variability in islet quality. The latter is often addressed by co-culture of harvested islets with stem cells to promote in vitro remodeling of their basement membrane and enable expression of angiogenic factors for enhancing vascularization. However, given the heterogeneity in islet size, shape and function, there is a need for metrics to assess the reorganization dynamics of single islets over the co-culture period. Based on shape-evolution of individual multi-cell aggregates formed during co-culture of human islets with adipose derived stem cells and the pressures required for their bypass through microfluidic constrictions, we present size-normalized biomechanical metrics for monitoring the reorganization. Aggregates below a threshold size exhibit faster reorganization, as evident from rise in their biomechanical opacity and tightening of their size distribution, but this size threshold increases over culture time to include a greater proportion of the aggregates. Such biomechanical metrics can quantify the subpopulation of reorganized aggregates by distinguishing them versus those with incomplete reorganization, over various timepoints during the co-culture.

Platinum Chemotherapy Induces Lymphangiogenesis in Cancerous and Healthy Tissues That Can be Prevented With Adjuvant Anti-VEGFR3 Therapy.

Chemotherapy has been used to inhibit cancer growth for decades, but emerging evidence shows it can affect the tumor stroma, unintentionally promoting cancer malignancy. After treatment of primary tumors, remaining drugs drain via lymphatics. Though all drugs interact with the lymphatics, we know little of their impact on them. Here, we show a previously unknown effect of platinums, a widely used class of chemotherapeutics, to directly induce systemic lymphangiogenesis and activation. These changes are dose-dependent, long-lasting, and occur in healthy and cancerous tissue in multiple mouse models of breast cancer. We found similar effects in human ovarian and breast cancer patients whose treatment regimens included platinums. Carboplatin treatment of healthy mice prior to mammary tumor inoculation increased cancer metastasis as compared to no pre-treatment. These platinum-induced phenomena could be blocked by VEGFR3 inhibition. These findings have implications for cancer patients receiving platinums and may support the inclusion of anti-VEGFR3 therapy into treatment regimens or differential design of treatment regimens to alter these potential effects.

An Ex Vivo Tissue Culture Model for Anti-angiogenic Drug Testing.

Angiogenesis, defined as the growth of new blood vessels from existing ones, plays a key role in development, growth, and tissue repair. Its necessary role in tumor growth and metastasis has led to the creation of a new category of anti-angiogenic cancer therapies. Preclinical development and evaluation of potential drug candidates require models that mimic real microvascular networks. Here, we describe the rat mesentery culture model as a simple ex vivo assay that offers time-lapse imaging of intact microvascular network remodeling and demonstrate its application for anti-angiogenic drug testing.

Lysophosphatidic acid does not cause blood/lymphatic vessel plasticity in the rat mesentery culture model.

Understanding the mechanisms behind endothelial cell identity is crucial for the goal of manipulating microvascular networks. Lysophosphatidic acid (LPA) and serum stimulation have been suggested to induce a lymphatic identity in blood endothelial cells in vitro. The objective of this study was to determine if LPA or serum induces blood-to-lymphatic vessel phenotypic transition in microvascular networks. The rat mesentery culture model was used to observe the effect of stimulation on blood and lymphatic microvascular networks ex vivo. Vascularized mesenteric tissues were harvested from adult Wistar rats and cultured with LPA or 10% serum for up to 5 days. Tissues were then immunolabeled with PECAM to identify blood vessels and LYVE-1 or Prox1 to identify lymphatic vessels. We show that while LPA caused capillary sprouting and increased vascular length density in adult microvascular networks, LPA did not cause a blood-to-lymphatic phenotypic transition. The results suggest that LPA is not sufficient to cause blood endothelial cells to adopt a lymphatic identity in adult microvascular networks. Similarly, serum stimulation caused robust angiogenesis and increased lymphatic/blood vessel connections, yet did not induce a blood-to-lymphatic phenotypic transition. Our study highlights an understudied area of lymphatic research and warrants future investigation into the mechanisms responsible for the maintenance of blood and lymphatic vessel identity.

Laser Direct-Write Onto Live Tissues: A Novel Model for Studying Cancer Cell Migration.

Investigation into the mechanisms driving cancer cell behavior and the subsequent development of novel targeted therapeutics requires comprehensive experimental models that mimic the complexity of the tumor microenvironment. Recently, our laboratories have combined a novel tissue culture model and laser direct-write, a form of bioprinting, to spatially position single or clustered cancer cells onto ex vivo microvascular networks containing blood vessels, lymphatic vessels, and interstitial cell populations. Herein, we highlight this new model as a tool for quantifying cancer cell motility and effects on angiogenesis and lymphangiogenesis in an intact network that matches the complexity of a real tissue. Application of our proposed methodology offers an innovative ex vivo tissue perspective for evaluating the effects of gene expression and targeted molecular therapies on cancer cell migration and invasion. J. Cell. Physiol. 231: 2333-2338, 2016. © 2016 Wiley Periodicals, Inc.

Macrophages: An Inflammatory Link Between Angiogenesis and Lymphangiogenesis.

Angiogenesis and lymphangiogenesis often occur in response to tissue injury or in the presence of pathology (e.g., cancer), and it is these types of environments in which macrophages are activated and increased in number. Moreover, the blood vascular microcirculation and the lymphatic circulation serve as the conduits for entry and exit for monocyte-derived macrophages in nearly every tissue and organ. Macrophages both affect and are affected by the vessels through which they travel. Therefore, it is not surprising that examination of macrophage behaviors in both angiogenesis and lymphangiogenesis has yielded interesting observations that suggest macrophages may be key regulators of these complex growth and remodeling processes. In this review, we will take a closer look at macrophages through the lens of angiogenesis and lymphangiogenesis, examining how their dynamic behaviors may regulate vessel sprouting and function. We present macrophages as a cellular link that spatially and temporally connects angiogenesis with lymphangiogenesis, in both physiological growth and in pathological adaptations, such as tumorigenesis. As such, attempts to therapeutically target macrophages in order to affect these processes may be particularly effective, and studying macrophages in both settings will accelerate the field's understanding of this important cell type in health and disease.

Printing cancer cells into intact microvascular networks: a model for investigating cancer cell dynamics during angiogenesis.

While cancer cell invasion and metastasis are dependent on cancer cell-stroma, cancer cell-blood vessel, and cancer cell-lymphatic vessel interactions, our understanding of these interactions remain largely unknown. A need exists for physiologically-relevant models that more closely mimic the complexity of cancer cell dynamics in a real tissue environment. The objective of this study was to combine laser-based cell printing and tissue culture methods to create a novel ex vivo model in which cancer cell dynamics can be tracked during angiogenesis in an intact microvascular network. Laser direct-write (LDW) was utilized to reproducibly deposit breast cancer cells (MDA-MB-231 and MCF-7) and fibroblasts into spatially-defined patterns on cultured rat mesenteric tissues. In addition, heterogeneous patterns containing co-printed MDA-MB-231/fibroblasts or MDA-MB-231/MCF-7 cells were generated for fibroblast-directed and collective cell invasion models. Printed cells remained viable and the cells retained the ability to proliferate in serum-rich media conditions. Over a culture period of five days, time-lapse imaging confirmed fibroblast and MDA-MB-231 cell migration within the microvascular networks. Confocal microscopy indicated that printed MDA-MB-231 cells infiltrated the tissue thickness and were capable of interacting with endothelial cells. Angiogenic network growth in tissue areas containing printed cancer cells was characterized by significantly increased capillary sprouting compared to control tissue areas containing no printed cells. Our results establish an innovative ex vivo experimental platform that enables time-lapse evaluation of cancer cell dynamics during angiogenesis within a real microvascular network scenario.

An ex vivo model for anti-angiogenic drug testing on intact microvascular networks.

New models of angiogenesis that mimic the complexity of real microvascular networks are needed. Recently, our laboratory demonstrated that cultured rat mesentery tissues contain viable microvascular networks and could be used to probe pericyte-endothelial cell interactions. The objective of this study was to demonstrate the efficacy of the rat mesentery culture model for anti-angiogenic drug testing by time-lapse quantification of network growth. Mesenteric windows were harvested from adult rats, secured in place with an insert, and cultured for 3 days according to 3 experimental groups: 1) 10% serum (angiogenesis control), 2) 10% serum + sunitinib (SU11248), and 3) 10% serum + bevacizumab. Labeling with FITC conjugated BSI-lectin on Day 0 and 3 identified endothelial cells along blood and lymphatic microvascular networks. Comparison between day 0 (before) and 3 (after) in networks stimulated by 10% serum demonstrated a dramatic increase in vascular density and capillary sprouting. Growing networks contained proliferating endothelial cells and NG2+ vascular pericytes. Media supplementation with sunitinib (SU11248) or bevacizumab both inhibited the network angiogenic responses. The comparison of the same networks before and after treatment enabled the identification of tissue specific responses. Our results establish, for the first time, the ability to evaluate an anti-angiogenic drug based on time-lapse imaging on an intact microvascular network in an ex vivo scenario.

An angiogenesis model for investigating multicellular interactions across intact microvascular networks.

Developing therapies aimed at manipulating microvascular remodeling requires a better understanding of angiogenesis and how angiogenesis relates to other network remodeling processes, such as lymphangiogenesis and neurogenesis. The objective of this study was to develop an angiogenesis model that enables probing of multicellular and multisystem interactions at the molecular level across an intact adult microvascular network. Adult male Wistar rat mesenteric windows were aseptically harvested and cultured in serum-free minimum essential media. Viability/cytotoxicity analysis revealed that cells remain alive for at least 7 days. Immunohistochemical labeling at 3 days for platelet endothelial cell adhesion molecule (PECAM), neuron-glial antigen 2 (NG2), lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), and class III ß-tubulin identified endothelial cells, pericytes, lymphatics, and nerves, respectively. Media supplemented with bFGF or VEGF induced an increase in endothelial cell sprouting off existing vessels. Endothelial cell sprouting in both growth factor groups was inhibited by targeting pericytes with NG2 functional blocking antibody. VEGF caused an increase in the number of lymphatic/blood endothelial cell connections compared with media alone or bFGF groups. Finally, the comparison of the same network before and after angiogenesis stimulated by the supplement of media with 20% serum identified the ability of disconnected endothelial segments to reconnect to nearby vessels. The results establish a novel in situ angiogenesis model for investigating the location of capillary sprouting within an intact network, the role of pericytes, lymphatic/blood endothelial cell interactions, and the fate of specific endothelial cell segments. The rat mesentery culture system offers a unique tool for understanding the complex dynamics associated with angiogenesis in an intact adult tissue.