Monitoring Angiotropic Extravascular Migratory Metastasis In Vitro.

The mechanism of cancer cell migration from the primary tumor toward secondary sites is not fully understood. In addition to intravascular cellular migration, angiotropic extravascular migratory metastasis (EVMM) has been recognized as a metastatic pathway involving tumor cells crawling along the abluminal vascular surface to distant sites. A very simple in vitro 3D assay is described here, which is based on a previous in vitro angiogenesis assay. The assay involves monitoring single fluorescence-tagged migrating cancer cells in the presence of vascular structures in real time. This coculture assay represents a quantitative approach for monitoring the migration processes of cancer cells along vessels, demonstrating phenotypic switching and migration dynamics. This protocol can be used for molecular analyses and can also be adapted for screening of therapeutic agents to block cancer metastasis.

Hematogenous metastasis and tumor dormancy as concepts or dogma? The continuum of vessel co-option and angiotropic extravascular migratory metastasis as an alternative.

It has been accepted for many years that tumor cells spread via the circulation to distant sites. The latency period between treatment and tumor recurrence has been attributed to dormant cells in distant organs that emerge and grow as metastatic tumors. These processes are accepted with an incomplete demonstration of their existence. Challenging such a well-established accepted paradigm is not easy as history as shown. An alternative or co-existing mechanism involving tumor cell migration along the outside of the vessels and co-option of the blood vessel has been studied for over 25 years and is presented. Several lines of data support this new mechanism of tumor spread and metastatic growth and is termed angiotropic extravascular migratory metastasis or EVMM. This slow migration along the outside of the vessel wall may explain the latency period between treatment and metastatic tumor growth. The reader is asked to be open to this possible new concept in how tumors spread and grow and the reason for this latency period. A full understanding of how tumors spread and grow is fundamental for the targeting of new therapeutics.

Matrigel: history/background, uses, and future applications.

Basement membranes are thin sheets of extracellular matrix with many diverse roles in the body. Those in normal tissue are also highly insoluble and resist attempts to extract and characterize their components. A mouse tumor, the EHS tumor, has provided large amounts of basement membrane material, which has facilitated the structural and functional characterization of its components. An extract of the tumor, known as Matrigel, contains components which reconstitute into a solid gel at 37°. This solid basement membrane matrix has been used in both cell culture and in vivo. Matrigel has been utilized in some 12,000-plus publications for a variety of studies with embryonic, normal, and stem or malignant cells. Evidence presented in this Commentary suggests that Matrigel isolated from tumors grown in diverse hosts could exert unique effects that could be helpful in analyzing the causes of various pathologies and for screening possible therapeutic agents.

Purinergic Signaling Involvement in Thymosin ß4-mediated Corneal Epithelial Cell Migration.

Purpose: This study aimed to determine the effect of thymosin beta 4 (Tß4) on human corneal epithelial cell migration and the downstream signaling pathways. Tß4 has a role in tissue development, cell migration, inflammation, and wound healing. A previous study showed that Tß4 directly binds to F0-F1 ATP synthase. Other studies reported the role of extracellular ATP and purinergic receptors in cell migration with several cell types. Despite advancing to the clinical stage for treatment of eye disorders, the effect of Tß4 on human corneal epithelial cell (HCEC) migration and proliferation and the precise downstream signaling pathway(s) have not been identified. Methods: Various concentrations of Tß4 were tested in vitro on human corneal epithelial cell proliferation using the CCK-8 Kit and on cell migration using the gap closure migration assay. Additionally, ATP levels at various time points were determined using the ATP Lite One-Step Kit. The Fluo 8 NO Wash Calcium Assay Kit was used to measure the intracellular Ca2+ concentration after treatment with various concentrations of Tß4. P2X7 inhibitors were tested on ATP signaling and migration. Total- and phospho-ERK1/2 levels were determined in western blot. Results: Tß4 enhanced HCEC proliferation and migration in a dose- and time-dependent manner. Moreover, these functions were related to increased extracellular ATP levels, intracellular Ca2+ influx, and ERK1/2 phosphorylation. Tß4-mediated HCEC migration was inhibited by specific P2X7 purinergic receptor antagonists suggesting the role of this receptor in Tß4-mediated human corneal epithelial cell migration. Conclusions: These results suggest that Tß4-mediated HCEC proliferation and migration are associated with increased ATP levels, P2X7 R-mediated Ca2+ influx, and the ERK1/2 signaling pathway. This study begins to describe the mechanisms for Tß4-mediated corneal healing and regeneration.

Angiotropism, pericytic mimicry and extravascular migratory metastasis: an embryogenesis-derived program of tumor spread.

Intravascular dissemination of tumor cells is the accepted mechanism of cancer metastasis. However, the phenomenon of angiotropism, pericyte mimicry (PM), and extravascular migratory metastasis (EVMM) has questioned the concept that tumor cells metastasize exclusively via circulation within vascular channels. This new paradigm of cancer spread and metastasis suggests that metastatic cells employ embryonic mechanisms for attachment to the abluminal surfaces of blood vessels (angiotropism) and spread via continuous migration, competing with and replacing pericytes, i.e., pericyte mimicry (PM). This is an entirely extravascular phenomenon (i.e., extravascular migratory metastasis or EVMM) without entry (intravasation) into vascular channels. PM and EVMM have mainly been studied in melanoma but also occur in other cancer types. PM and EVMM appear to be a reversion to an embryogenesis-derived program. There are many analogies between embryogenesis and cancer progression, including the important role of laminins, epithelial-mesenchymal transition, and the re-activation of embryonic signals by cancer cells. Furthermore, there is no circulation of blood during the first trimester of embryogenesis, despite the fact that there is extensive migration of cells to distant sites and formation of organs and tissues during this period. Embryonic migration therefore is a continuous extravascular migration as are PM and EVMM, supporting the concept that these embryonic migratory events appear to recur abnormally during the metastatic process. Finally, the perivascular location of tumor cells intrinsically links PM to vascular co-option. Taken together, these two new paradigms may greatly influence the development of new effective therapeutics for metastasis. In particular, targeting embryonic factors linked to migration that are detected during cancer metastasis may be particularly relevant to PM/EVMM.

RGN-259 (thymosin ß4) improves clinically important dry eye efficacies in comparison with prescription drugs in a dry eye model.

This study evaluated the clinical activity of RGN-259 (thymosin ß4) in comparison with cyclosporine A (CsA), diquafosol (DQS), and lifitegrast (LFA) in a murine model of dry eye. The model was NOD.B10-H2b mice in a 30-40% humidified environment together with daily scopolamine hydrobromide injections for 10 days. After desiccation stress, all drugs were evaluated after 10 treatment days. RGN-259 increased tear production similar to that in the DQS- and LFA-treated mice while CsA was inactive. RGN-259 improved corneal smoothness and decreased fluorescein staining similar to that of LFA group while CsA and DQS were inactive. Corneal epithelial detachment was reduced by RGN-259, and DQS and LFA showed similar activity but the CsA was inactive. RGN-259 increased conjunctival goblet cells and mucin production comparable to that seen with CsA, while DQS and LFA were inactive. RGN-259 reduced the over-expression of inflammatory factors comparable to that with CsA and LFA, while DQS was inactive. RGN-259 increased mucin production comparable to that observed with CsA, while DQS and LFA were inactive. In conclusion, RGN-259 promoted recovery of mucins and goblet cells, improved corneal integrity, and reduced inflammation in a dry eye mouse model and was equal to or more effective than prescription treatments.

Imaging of Angiotropism/Vascular Co-Option in a Murine Model of Brain Melanoma: Implications for Melanoma Progression along Extravascular Pathways.

Angiotropism/pericytic mimicry and vascular co-option involve tumor cell interactions with the abluminal vascular surface. These two phenomena may be closely related. However, investigations of the two processes have developed in an independent fashion and different explanations offered as to their biological nature. Angiotropism describes the propensity of tumor cells to spread distantly via continuous migration along abluminal vascular surfaces, or extravascular migratory metastasis (EVMM). Vascular co-option has been proposed as an alternative mechanism by which tumors cells may gain access to a blood supply. We have used a murine brain melanoma model to analyze the interactions of GFP human melanoma cells injected into the mouse brain with red fluorescent lectin-labeled microvascular channels. Results have shown a striking spread of melanoma cells along preexisting microvascular channels and features of both vascular co-option and angiotropism/pericytic mimicry. This study has also documented the perivascular expression of Serpin B2 by angiotropic melanoma cells in the murine brain and in human melanoma brain metastases. Our findings suggest that vascular co-option and angiotropism/pericytic mimicry are closely related if not identical processes. Further studies are needed in order to establish whether EVMM is an alternative form of cancer metastasis in addition to intravascular cancer dissemination.

Primary Mechanisms of Thymosin ß4 Repair Activity in Dry Eye Disorders and Other Tissue Injuries.

Dry eye disorders are becoming more common due to many causes, including an aging population, increased pollution, and postrefractive surgery. Current treatments include artificial tears; gels; lubricants; tear duct plugs; and anti-inflammatory agents such as steroids, doxycycline, and cyclosporine. For more severe forms of the disease, serum tears and scleral contact lenses are employed. Despite these therapies, successful resolution of the problem is limited because none of these treatments fully addresses the underlying causes of dry eye to promote ocular surface repair. Thymosin ß4 (Tß4), a small, naturally occurring protein, promotes complete and faster corneal healing than saline alone or prescription agents (doxycycline and cyclosporine) in various animal models of eye injury. In human trials, it improves both the signs and symptoms of moderate to severe dry eye with effects lasting beyond the treatment period. This review will cover the multiple activities of Tß4 on cell migration, inflammation, apoptosis, cytoprotection, and gene expression with a focus on mechanisms of cell migration, including laminin-332 synthesis and degradation, that account for this paradigm-shifting potential new treatment for dry eye disorders. We will also speculate on additional mechanisms that might promote eye repair based on data from other tissue injury models. Such studies provide the rationale for use of Tß4 in other types of eye disorders beyond dry eye. Finally, we will identify the gaps in our knowledge and propose future research avenues.

In vitro microtumors provide a physiologically predictive tool for breast cancer therapeutic screening.

Many anti-cancer drugs fail in human trials despite showing efficacy in preclinical models. It is clear that the in vitro assays involving 2D monoculture do not reflect the complex extracellular matrix, chemical, and cellular microenvironment of the tumor tissue, and this may explain the failure of 2D models to predict clinical efficacy. We first optimized an in vitro microtumor model using a tumor-aligned ECM, a tumor-aligned medium, MCF-7 and MDA-MB-231 breast cancer spheroids, human umbilical vein endothelial cells, and human stromal cells to recapitulate the tissue architecture, chemical environment, and cellular organization of a growing and invading tumor. We assayed the microtumor for cell proliferation and invasion in a tumor-aligned extracellular matrix, exhibiting collagen deposition, acidity, glucose deprivation, and hypoxia. We found maximal proliferation and invasion when the multicellular spheroids were cultured in a tumor-aligned medium, having low pH and low glucose, with 10% fetal bovine serum under hypoxic conditions. In a 7-day assay, varying doses of fluorouracil or paclitaxel had differential effects on proliferation for MCF-7 and MDA-MB-231 tumor spheroids in microtumor compared to 2D and 3D monoculture. The microtumors exhibited a tumor morphology and drug response similar to published xenograft data, thus demonstrating a more physiologically predictive in vitro model.

Angiotropism, pericytic mimicry and extravascular migratory metastasis in melanoma: an alternative to intravascular cancer dissemination.

For more than 15 years, angiotropism in melanoma has been emphasized as a marker of extravascular migration of tumor cells along the abluminal vascular surface, unveiling an alternative mechanism of tumor spread distinct from intravascular dissemination. This mechanism has been termed extravascular migratory metastasis (EVMM). During EVMM, angiotropic tumor cells migrate in a 'pericytic-like' manner (pericytic mimicry) along the external surfaces of vascular channels, without intravasation. Through this pathway, melanoma cells may spread to nearby or more distant sites. Angiotropism is a prognostic factor predicting risk for metastasis in human melanoma, and a marker of EVMM in several experimental models. Importantly, analogies of EVMM and pericytic mimicry include neural crest cell migration, vasculogenesis and angiogenesis, and recent studies have suggested that the interaction between melanoma cells and the abluminal vascular surface induce differential expression of genes reminiscent of cancer migration and embryonic/stem cell state transitions. A recent work revealed that repetitive UV exposure of primary cutaneous melanomas in a genetically engineered mouse model promotes metastatic progression via angiotropism and migration along the abluminal vascular surface. Finally, recent data using imaging of melanoma cells in a murine model have shown the progression of tumor cells along the vascular surfaces. Taken together, these data provide support for the biological phenomenon of angiotropism and EVMM, which may open promising new strategies for reducing or preventing melanoma metastasis.

Matrigel: from discovery and ECM mimicry to assays and models for cancer research.

The basement membrane is an important extracellular matrix that is found in all epithelial and endothelial tissues. It maintains tissue integrity, serves as a barrier to cells and to molecules, separates different tissue types, transduces mechanical signals, and has many biological functions that help to maintain tissue specificity. A well-defined soluble basement membrane extract, termed BME/Matrigel, prepared from an epithelial tumor is similar in content to authentic basement membrane, and forms a hydrogel at 24-37°C. It is used in vitro as a substrate for 3D cell culture, in suspension for spheroid culture, and for various assays, such as angiogenesis, invasion, and dormancy. In vivo, BME/Matrigel is used for angiogenesis assays and to promote xenograft and patient-derived biopsy take and growth. Studies have shown that both the stiffness of the BME/Matrigel and its components (i.e. chemical signals) are responsible for its activity with so many different cell types. BME/Matrigel has widespread use in assays and in models that improve our understanding of tumor biology and help define therapeutic approaches.

Could pericytic mimicry represent another type of melanoma cell plasticity with embryonic properties?

We hypothesize that the interaction between angiotropic melanoma cells and the abluminal vascular surface can induce or sustain embryonic and/or stem cell migratory properties in these tumor cells. As a result, such angiotropic melanoma cells may migrate along the abluminal vascular surface, demonstrating pericytic mimicry. Through these cellular interactions, melanoma cells may migrate toward secondary sites.

Pilot study on "pericytic mimicry" and potential embryonic/stem cell properties of angiotropic melanoma cells interacting with the abluminal vascular surface.

The interaction of tumor cells with the tumor vasculature is mainly studied for its role in tumor angiogenesis and intravascular metastasis of circulating tumor cells. In addition, a specific interaction of tumor cells with the abluminal surfaces of vessels, or angiotropism, may promote the migration of angiotropic tumor cells along the abluminal vascular surfaces in a pericytic location. This process has been termed extravascular migratory metastasis. The abluminal vascular surface may also provide a vascular niche inducing or sustaining stemness to angiotropic tumor cells. This pilot study investigated if angiotropic melanoma cells might represent a subset population with pericytic and embryonic or stem cell properties. Through microarray analysis, we showed that the interaction between melanoma cells and the abluminal surface of endothelial cells triggers significant differential expression of several genes. The most significantly differentially expressed genes have demonstrated properties linked to cancer cell migration (CCL2, ICAM1 and IL6), cancer progression (CCL2, ICAM1, SELE, TRAF1, IL6, SERPINB2 and CXCL6), epithelial to mesenchymal transition (CCL2 and IL6), embryonic/stem cell properties (CCL2, PDGFB, EVX1 and CFDP1) and pericytic recruitment (PDGFB). In addition, bioinformatics-based analysis of the differentially expressed genes has shown that the most significantly enriched functional groups included development, cell movement, cancer, and embryonic development. Finally, the investigation of pericyte/mesenchymal stem cells markers via immunostaining of human melanoma samples revealed expression of PDGFRB, NG2 and CD146 by angiotropic melanoma cells. Taken together, these preliminary data are supportive of the "pericytic mimicry" by angiotropic melanoma cells, and suggest that the interaction between melanoma cells and the abluminal vascular surface induce differential expression of genes linked to cancer migration and embryonic/stem cell properties.

Laminin-111-derived peptides and cancer.

Laminin-111 is a large trimeric basement membrane glycoprotein with many active sites. In particular, four peptides active in tumor malignancy studies have been identified in laminin-111 using a systematic peptide screening method followed by various assays. Two of the peptides (IKVAV and AG73) are found on the α1 chain, one (YIGSR) of the ß1 chain and one (C16) on the γ1 chain. The four peptides have distinct activities and receptors. Since three of the peptides (IKVAV, AG73 and C16) strongly promote tumor growth, this may explain the potent effects laminin-111 has on malignant cells. The peptide, YIGSR, decreases tumor growth and experimental metastasis via a 32/67 kD receptor while IKVAV increases tumor growth, angiogenesis and protease activity via integrin receptors. AG73 increases tumor growth and metastases via syndecan receptors. C16 increases tumor growth and angiogenesis via integrins. Identification of such sites on laminin-111 will have use in defining strategies to develop therapeutics for cancer.

Thymosin ß4 stabilizes hypoxia-inducible factor-1α protein in an oxygen-independent manner.

The small actin-binding protein thymosin ß4 (Tß4) is understood to stimulate angiogenesis. Previously, we reported that Tß4 induces angiogenesis by increasing vascular endothelial growth factor (VEGF) expression, but the mechanism underlying how Tß4 upregulates VEGF expression remain unknown. To identify the mechanism of VEGF induction by Tß4, we measured VEGF promoter activity and analyzed the effect of Tß4 on VEGF RNA stability. The Tß4 peptide had no effect on either VEGF promoter activity or VEGF RNA stability. We focused on the possibility that Tß4 may indirectly induce VEGF expression via hypoxia-inducible factor (HIF)-1α. We determined that Tß4 increased the stability of HIF-1α protein under normoxic conditions. These data suggest that Tß4 indirectly induces VEGF expression by increasing the protein stability of HIF-1α in an oxygen-independent manner.

Increased initiation and growth of tumor cell lines, cancer stem cells and biopsy material in mice using basement membrane matrix protein (Cultrex or Matrigel) co-injection.

This protocol requires 2-4 h and presents a method for injecting tumor cells, cancer stem cells or dispersed biopsy material into subcutaneous or orthotopic locations within recipient mice. The tumor cells or biopsy are mixed with basement membrane matrix proteins (CultrexBME or Matrigel) at 4 °C and then injected into recipient animals at preferred anatomical sites. Tumor cells can also be co-injected with additional cell types, such as fibroblasts, stromal cells, endothelial cells and so on. Details are given on appropriate cell numbers, handling and concentration of the basement membrane proteins, recipient animals, injection location and techniques. This procedure enables the growth of tumors from cells or biopsy material (tumor graft) with greater efficiency of take and growth, and with retention of the primary tumor phenotype based on histology. Co-injection with additional cell types provides more physiological models of human cancers for use in drug screening and studying cancer biology.

Thymosin ß4: a multi-functional regenerative peptide. Basic properties and clinical applications.

INTRODUCTION: Thymosin ß(4), a low molecular weight, naturally-occurring peptide plays a vital role in the repair and regeneration of injured cells and tissues. After injury, thymosin ß(4), is released by platelets, macrophages and many other cell types to protect cells and tissues from further damage and reduce apoptosis, inflammation and microbial growth. Thymosin ß(4) binds to actin and promotes cell migration, including the mobilization, migration, and differentiation of stem/progenitor cells, which form new blood vessels and regenerate the tissue. Thymosin ß(4) also decreases the number of myofibroblasts in wounds, resulting in decreased scar formation and fibrosis. AREAS COVERED: This article will cover the many thymosin ß(4) activities that directly affect the repair and regeneration cascade with emphasis on its therapeutic uses and potential. Our approach has been to evaluate the basic biology of the molecule as well as its potential for clinical applications in the skin, eye, heart and brain. EXPERT OPINION: The considerable advances in our understanding of the functional biology and mechanisms of action of thymosin ß(4) have provided the scientific foundation for ongoing and projected clinical trials in the treatment of dermal wounds, corneal injuries and in the regeneration and repair of heart and CNS tissue following ischemic insults and trauma.

The potent peptide antagonist to angiogenesis, C16Y, and cisplatin act synergistically in the down-regulation of the Bcl-2/Bax ratio and the induction of apoptosis in human ovarian cancer cells.

Cisplatin is one of the most potent antitumor agents for ovarian cancer, but has also been implicated in normal tissue cytotoxicity. We examined the effect of cisplatin alone and in combination with C16Y, a newly-identified anti-angiogenic peptide from the NH2-terminal domains of the γ-chain of laminin-1, on the modulation of Bcl-2/Bax expression and induction of apoptosis in ovarian cancer cells (OVACAR3). C16Y did not elicit cell death of human umbilical vein endothelial cells (HUVECs). Cisplatin exerted a lethal effect with an EC50 of 10 µM in OVACAR3s. In the presence of 25 or 50 µg/ml of C16Y (a range which has no effect against HUVECs), the EC50 for cisplatin in OVACAR3s decreased to 3.5 and 2.0 µM, respectively. Using fluorescence-activated cell sorting (FACS) analysis of DNA stained OVACAR3s and terminal deoxynucleotide tranferase-mediated dUTP nick end-labeling (TUNEL), we found that even at concentrations of 1 and 3 µM cisplatin, C16Y at 10 and 25 µg/ml increased the incidence of apoptosis in OVACAR3s by 3-5-fold. Each drug had some measurable effect on Bax protein expression. Furthermore, Bcl-2 protein expression levels were markedly reduced by C16Y alone and cisplatin alone in a dose-dependent manner. The combination of C16Y and cisplatin resulted in a further dramatic reduction in Bcl-2, underscoring the pronounced synergy produced by cisplatin and C16Y together. On the other hand, C16Y did not activate any other signal transduction pathways that usually culminate in the activation of apoptosis, such as the p53, p21waf1, p73, ERK1/2 or PI3-AKT pathways. These observations suggest that the suppression of the Bcl-2/Bax ratio may play an important role in mediating the synergistic effect of cisplatin and C16Y on the induction of apoptosis in OVACAR3 cells.

Multiple uses of basement membrane-like matrix (BME/Matrigel) in vitro and in vivo with cancer cells.

Significant advances in our understanding of cancer cell behavior, growth, and metastasis have been facilitated by studies using a basement membrane-like extracellular matrix extract, also known as Matrigel. The basement membrane is a thin extracellular matrix that is found in normal tissues and contacts epithelial and endothelial cells, smooth muscle, fat, Schwann cells, etc. It is composed of mainly laminin-111, collagen IV, heparan sulfate proteoglycan, entactin/nidogen, and various growth factors (fibroblast growth factor, transforming growth factor beta, epidermal growth factor, etc.). Most tumors of epithelial origin produce significant amounts of basement membrane matrix and interact with it particularly during metastasis. Cancer cells metastasize via degradation of the vessel basement membrane matrix to extravasate into the blood stream and colonize distant sites. This review will focus on the interaction of cancer cells and cancer stem cells with the basement membrane-like matrix and the various uses of this interaction to accelerate tumor growth in vivo and to develop in vitro assays for invasion, morphology, and dormancy. Such assays and methods have advanced our understanding of the process of cancer progression, the genes and pathways that are involved, the potential of various therapeutic agents, the effects of neighboring cells, and the role of stem cells.

Thymosin ß4 expression in human tissues and in tumors using tissue microarrays.

Thymosin ß4 has been reported to play the key roles in tumor growth, metastasis, and angiogenesis. Although the importance of thymosin ß4 in angiogenesis and metastasis is known, few studies to show the expression patterns of thymosin ß4 in human tissues including tumors have been conducted. The comparisons of the expression of thymosin ß4 between the normal and tumor tissues are also needed to study the role of thymosin ß4 in tumor formation. Using tissue microarray analysis, we compared the expression patterns of thymosin ß4 in the normal human tissues and in the tumors to screen certain tumors and upregulated the expression of thymosin ß4 by tumorigenesis. Thymosin ß4 was highly expressed in the hepatic cells in the normal adult liver, duct, and acinar cells in pancreas, and muscle cells in the heart and also expressed highly in certain tumor cells, including osteosarcoma, colon adenocarcinoma, esophageal squamous cell carcinoma, kidney and urinary bladder transitional carcinoma, lung cancer, and liver cancer. Comparing the thymosin ß4 expression between normal and tumors, thymosin ß4 was upregulated specifically in osteosarcoma, colorectal carcinoma, and esophageal cancer. To confirm the over-expression of thymosin ß4 in these tumors, we analyzed expression of thymosin ß4 with each additional microarray of osteosarcoma, colorectal carcinoma, and esophageal cancer. The significant increased expression of thymosin ß4 was observed in osteosarcoma and in colorectal cancer. These results suggest that the expression of thymosin ß4 is highly related with tumorigenesis of certain tumors including the osteosarcoma and colorectal cancers.