A biomimetic pancreatic cancer on-chip reveals endothelial ablation via ALK7 signaling

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, lethal malignancy that invades adjacent vasculatures and spreads to distant sites before clinical detection. Although invasion into the peripancreatic vasculature is one of the hallmarks of PDAC, paradoxically, PDAC tumors also exhibit hypovascularity. How PDAC tumors become hypovascular is poorly understood. We describe an organotypic PDAC-on-a-chip culture model that emulates vascular invasion and tumor-blood vessel interactions to better understand PDAC-vascular interactions. The model features a 3D matrix containing juxtaposed PDAC and perfusable endothelial lumens. PDAC cells invaded through intervening matrix, into vessel lumen, and ablated the endothelial cells, leaving behind tumor-filled luminal structures. Endothelial ablation was also observed in in vivo PDAC models. We also identified the activin-ALK7 pathway as a mediator of endothelial ablation by PDAC. This tumor-on-a-chip model provides an important in vitro platform for investigating the process of PDAC-driven endothelial ablation and may provide a mechanism for tumor hypovascularity.R01 EB000262 - NIBIB NIH HHS; TL1 TR001410 - NCATS NIH HHS; UC4 DK104196 - NIDDK NIH HHS; UH3 EB017103 - NIBIB NIH HHSPublished versio

Synergy between a collagen IV mimetic peptide and a somatotropin-domain derived peptide as angiogenesis and lymphangiogenesis inhibitors

Angiogenesis is central to many physiological and pathological processes. Here we show two potent bioinformatically-identified peptides, one derived from collagen IV and translationally optimized, and one from a somatotropin domain-containing protein, synergize in angiogenesis and lymphangiogenesis assays including cell adhesion, migration and in vivo Matrigel plugs. Peptide-peptide combination therapies have recently been applied to diseases such as human immunodeficiency virus (HIV), but remain uncommon thus far in cancer, age-related macular degeneration and other angiogenesis-dependent diseases. Previous work from our group has shown that the collagen IV-derived peptide primarily binds β1 integrins, while the receptor for the somatotropin-derived peptide remains unknown. We investigate these peptides’ mechanisms of action and find both peptides affect the vascular endothelial growth factor (VEGF) pathway as well as focal adhesion kinase (FAK) by changes in phosphorylation level and total protein content. Blocking of FAK both through binding of β1 integrins and through inhibition of VEGFR2 accounts for the synergy we observe. Since resistance through activation of multiple signaling pathways is a central problem of anti-angiogenic therapies in diseases such as cancer, we suggest that peptide combinations such as these are an approach that should be considered as a means to sustain anti-angiogenic and anti-lymphangiogenic therapy and improve efficacy of treatment

NOVEL ROLE OF LYMPHATIC AND BLOOD VASCULATURES IN BREAST CANCER GROWTH AND METASTASIS AND PEPTIDE AGENTS WITH ANTI-LYMPHANGIOGENIC AND ANTI-ANGIOGENIC ACTIVITY

Up to 90% of deaths from breast cancer are a result of metastasis. Metastasis is a very complex process that has been difficult to fully understand as it involves diverse parenchymal and stromal cells and their secreted factors in the tumor and organ microenvironment. Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer for which current therapeutic options are very limited. This dissertation investigates molecular mechanisms of TNBC growth and metastasis by focusing on lymphatic and blood vasculatures in the tumor and organ microenvironment. Crosstalk between TNBC cells and lymphatic or blood endothelial cells reveals under-investigated roles of the microenvironment in pre-metastatic niche formation in distant organs as well as tumor progression in primary sites. The dissertation also focuses on development of novel anti-lymphangiogenic and anti-angiogenic peptides to treat TNBC. For investigating cancer metastasis and testing anti-metastatic therapeutic agents, efficient and reproducible spontaneous metastasis models are needed. Conventional spontaneous breast cancer metastasis models require a long period of observation after establishment of primary tumors to see significant metastatic progression. The dissertation demonstrates that pre-treatment of animals with tumor-conditioned media (TCM) prepared from TNBC cells accelerates spontaneous metastasis in the corresponding TNBC animal models. The TCM contains all the factors secreted by TNBC cells; thus the injection of TCM conditions pre-metastatic niche. An inguinal breast tumor model facilitated by TCM showed robust thoracic metastasis in the lymph nodes (LN) and the lungs, compared to the serum-free media (SFM) treated control group. The TCM-induced metastasis model was further investigated by focusing on molecular crosstalk between lymphatic endothelial cells (LEC) and TNBC cells, as the pre-metastatic organs in TCM-treated animals showed highly enhanced lymphangiogenesis. The dissertation shows that LEC within pre-metastatic niches are educated by TNBC cells to accelerate metastasis in the lungs and the LN. LEC within these organs, educated by tumor secretion secretes a chemokine, CCL5 that is not secreted by either physiological LEC or TNBC cells, directing CCR5-positive TNBC cell dissemination into the tissues. Moreover, tumor-educated LEC promote angiogenesis in these organs by secreting VEGFA, allowing tumor extravasation in the lungs and colonization in the LN. Mechanistically, interleukin-6 (IL6) secreted by the TNBC cells activates Stat3 phosphorylation, causing the formation of a pStat3-pc-Jun-pATF-2 ternary complex, inducing HIF-1α expression in LEC, and ultimately resulting in expression of CCL5 and VEGF. Additional crosstalk between TNBC cells and LEC shows that LEC promote TNBC cell proliferation and induce pericyte recruitment in primary tumor microenvironment by expressing EGF and PDGF-BB, thus promoting tumor growth. Surprisingly, microvascular endothelial cells (MEC) showed an opposite effect by suppressing tumor growth. Motivated by the knowledge that angiogenesis supports tumor growth and metastasis and lymphangiogenesis actively conditions pre-metastatic niches and promotes breast tumor metastasis, novel anti-lymphangiogenic and anti-angiogenic peptides were developed to target tumor growth and metastasis in TNBC. Endogenous peptides derived from proteins containing a conserved somatotropin domain were screened for inhibition of angiogenesis and lymphangiogenesis using in vitro proliferation, migration, adhesion and tube formation assays with blood and lymphatic endothelial cells. A short 14-mer peptide derived from transmembrane protein 45A human shows the most potent multimodal inhibition of angiogenesis and lymphangiogenesis in breast tumor xenografts and tumor-conditioned lymph nodes. Mechanistically, the peptide blocks vascular endothelial growth factor receptors 2 and 3 (VEGFR2/3) and downstream proteins by binding to neuropilin 1/2 (NRP1/2) and inhibiting VEGFR2/3 and NRP1/2 complex formation in the presence of VEGFA/C. A mimetic 20-mer peptide derived from Collagen IV shows synergy with the somatotropin-derived peptide as inhibitors of lymphangiogenesis in vitro and in vivo. The collagen-derived peptide was further optimized, and was evaluated in vitro in lymphangiogenesis and angiogenesis cell assays, and in animal experiments including TNBC breast tumor xenograft and TCM-induced distant metastasis models. In summary, this dissertation investigates molecular mechanisms of breast cancer metastasis, proposing novel roles of lymphatic endothelial cells in pre-metastatic organs and primary tumor microenvironment; identifies key molecules regulating metastatic dissemination and colonization as well as tumor growth. The dissertation also explores several novel anti-angiogenic and anti-lymphangiogenic peptides to effectively treat TNBC tumor growth and metastasis

Tissue-Engineered Models for Glaucoma Research

Glaucoma is a group of optic neuropathies characterized by the progressive degeneration of retinal ganglion cells (RGCs). Patients with glaucoma generally experience elevations in intraocular pressure (IOP), followed by RGC death, peripheral vision loss and eventually blindness. However, despite the substantial economic and health-related impact of glaucoma-related morbidity worldwide, the surgical and pharmacological management of glaucoma is still limited to maintaining IOP within a normal range. This is in large part because the underlying molecular and biophysical mechanisms by which glaucomatous changes occur are still unclear. In the present review article, we describe current tissue-engineered models of the intraocular space that aim to advance the state of glaucoma research. Specifically, we critically evaluate and compare both 2D and 3D-culture models of the trabecular meshwork and nerve fiber layer, both of which are key players in glaucoma pathophysiology. Finally, we point out the need for novel organ-on-a-chip models of glaucoma that functionally integrate currently available 3D models of the retina and the trabecular outflow pathway

Blood and Lymphatic Vasculatures On-Chip Platforms and Their Applications for Organ-Specific In Vitro Modeling

The human circulatory system is divided into two complementary and different systems, the cardiovascular and the lymphatic system. The cardiovascular system is mainly concerned with providing nutrients to the body via blood and transporting wastes away from the tissues to be released from the body. The lymphatic system focuses on the transport of fluid, cells, and lipid from interstitial tissue spaces to lymph nodes and, ultimately, to the cardiovascular system, as well as helps coordinate interstitial fluid and lipid homeostasis and immune responses. In addition to having distinct structures from each other, each system also has organ-specific variations throughout the body and both systems play important roles in maintaining homeostasis. Dysfunction of either system leads to devastating and potentially fatal diseases, warranting accurate models of both blood and lymphatic vessels for better studies. As these models also require physiological flow (luminal and interstitial), extracellular matrix conditions, dimensionality, chemotactic biochemical gradient, and stiffness, to better reflect in vivo, three dimensional (3D) microfluidic (on-a-chip) devices are promising platforms to model human physiology and pathology. In this review, we discuss the heterogeneity of both blood and lymphatic vessels, as well as current in vitro models. We, then, explore the organ-specific features of each system with examples in the gut and the brain and the implications of dysfunction of either vasculature in these organs. We close the review with discussions on current in vitro models for specific diseases with an emphasis on on-chip techniques

Inhibition of Lymphangiogenesis and Angiogenesis in Breast Tumor Xenografts and Lymph Nodes by a Peptide Derived from Transmembrane Protein 45A

Angiogenesis, the formation of new blood vessels from preexisting blood vessels, is a process that supports tumor growth and metastatic dissemination. Lymphangiogenesis also facilitates metastasis by increasing dissemination through the lymphatic vessels (LVs). Even after treatment with antiangiogenic agents, breast cancer patients are vulnerable to LV-mediated metastasis. We report that a 14-amino acid peptide derived from transmembrane protein 45A shows multimodal inhibition of lymphangiogenesis and angiogenesis in breast cancer. The peptide blocks lymphangiogenic and angiogenic phenotypes of lymphatic and blood endothelial cells induced by tumor-conditioned media prepared from MDA-MB-231 breast cancer cells. The peptide delays growth of MDA-MB-231 tumor xenografts and normalizes tumor-conditioned lymph nodes (LNs). These studies demonstrate the antilymphangiogenic and antiangiogenic potential of the peptide against primary tumors and premetastatic, tumor-conditioned regional LNs. Mechanistically, the peptide blocks vascular endothelial growth factor receptors 2 and 3 (VEGFR2/3) and downstream proteins by binding to neuropilin 1/2 (NRP1/2) and inhibiting VEGFR2/3 and NRP1/2 complex formation in the presence of VEGFA/C

Angiogenesis Interactome and Time Course Microarray Data Reveal the Distinct Activation Patterns in Endothelial Cells

<div><p>Angiogenesis involves stimulation of endothelial cells (EC) by various cytokines and growth factors, but the signaling mechanisms are not completely understood. Combining dynamic gene expression time-course data for stimulated EC with protein-protein interactions associated with angiogenesis (the “angiome”) could reveal how different stimuli result in different patterns of network activation and could implicate signaling intermediates as points for control or intervention. We constructed the protein-protein interaction networks of positive and negative regulation of angiogenesis comprising 367 and 245 proteins, respectively. We used five published gene expression datasets derived from in vitro assays using different types of blood endothelial cells stimulated by VEGFA (vascular endothelial growth factor A). We used the Short Time-series Expression Miner (STEM) to identify significant temporal gene expression profiles. The statistically significant patterns between 2D fibronectin and 3D type I collagen substrates for telomerase-immortalized EC (TIME) show that different substrates could influence the temporal gene activation patterns in the same cell line. We investigated the different activation patterns among 18 transmembrane tyrosine kinase receptors, and experimentally measured the protein level of the tyrosine-kinase receptors VEGFR1, VEGFR2 and VEGFR3 in human umbilical vein EC (HUVEC) and human microvascular EC (MEC). The results show that VEGFR1–VEGFR2 levels are more closely coupled than VEGFR1–VEGFR3 or VEGFR2–VEGFR3 in HUVEC and MEC. This computational methodology can be extended to investigate other molecules or biological processes such as cell cycle.</p></div

Normalized protein level measurement of VEGFR1, VEGFR2 and VEGFR3 to GAPDH on HUVEC and MEC in (A) and (B), respectively.

<p>Normalized protein level measurement of VEGFR1, VEGFR2 and VEGFR3 to GAPDH on HUVEC and MEC in (A) and (B), respectively.</p

List of genes in the angiome that are annotated as positive and negative regulators of angiogenesis shown in (A) and (B), respectively.

<p>List of genes in the angiome that are annotated as positive and negative regulators of angiogenesis shown in (A) and (B), respectively.</p

Five VEGF-treated time-course microarray datasets with different experimental conditions on endothelial cells.

<p>Five VEGF-treated time-course microarray datasets with different experimental conditions on endothelial cells.</p