Name: assessing tumor microenvironment of metastasis doorway-mediated vascular permeability associated with cancer cell dissemination using intravital imaging and fixed tissue analysis
Uploaded: 2019-06-26T14:00:00
Description: Watch this Scientific Journal Video about Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and F

We would like to thank the Analytical Imaging Facility (AIF) in the Albert Einstein College of Medicine for imaging support. This work was supported by grants from the NCI (P30CA013330, CA150344, CA 100324 and CA216248), the SIG 1S10OD019961-01, the Gruss-Lipper Biophotonics Center and its Integrated Imaging Program, and Montefiore&#8217;s Ruth L. Kirschstein T32 Training Grant of Surgeons for the Study of the Tumor Microenvironment (CA200561).

GSK co-wrote the manuscript, performed imaging for figure 1C and 3B, developed fixed tissue analysis protocol, and analyzed and interpreted all data; JMP co-wrote the manuscript, and performed the surgery and intravital imaging for Figure 1B,2C and 3A; LB &#38; AC performed the surgery and intravital imaging for Figure 2B; RJ performed the surgery and intravital imaging for Figure 2A; JSC co-wrote the manuscript and analyzed and interpreted all data; MHO co-wrote the manuscript and analyzed and interpreted all data; and DE performed the surgery and intravital imaging for Figure 2D, co-wrote the manuscript, developed fixed tissue analysis and intravital imaging protocols, and analyzed and interpreted all data.

All experiments using live animals must be conducted in accordance with animal use and care guidelines and regulations. The procedures described in this study were carried out in accordance with the National Institutes of Health regulations concerning the care and use of experimental animals and with the approval of the Albert Einstein College of Medicine Animal Care and Use Committee (IACUC).
1. Evaluation of &#34;bursting permeability&#34;&#160;using live animal imaging
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Here, we outline two protocols that can be applied to visualize and quantify a specific type of vascular permeability which is present at TMEM doorways and is associated with the disruption of vascular tight and adherens junctions. This type of vascular permeability is transient and controlled by the tripartite TMEM cell complex, as explained above5. The ability to identify and quantify TMEM-associated vascular permeability is crucial for the assessment of a pro-metastatic cancer cell microenviron...

Recent advances in our understanding of cancer metastasis have uncovered that epithelial-to-mesenchymal transition (EMT) and the induction of a migratory/invasive cancer cell subpopulation are not, by themselves, sufficient for hematogenous dissemination1. Indeed, it was previously thought that metastasizing cancer cells intravasate through the entirety of cancer-associated endothelium as the tumor neovasculature is often characterized by low pericyte coverage, and as such, is highly permeable and unstable2,3,4. Although highly suggestive of defective functions within the tumor, vascular modifications during carcinogenesis do not provide evidence per se that tumor cells can penetrate blood vessels easily and in an uncontrolled fashion. Insights from intravital imaging (IVI) studies, in which tumor cells are fluorescently-tagged and the vasculature is labeled via the intravenous injection of fluorescent probes (such as dextran or quantum dots), show that, while tumor vessels are uniformly permeable to low molecular weight dextrans (e.g. 70 kD), high molecular weight dextrans (155 kD) and tumor cells can cross the endothelium only at specialized sites of intravasation which are preferentially located at vascular branch point5,6,7. Immunohistochemical (IHC) analyses using animal models and human patient-derived material have shown that these sites are &#34;doorways&#34;&#160;that specialize in regulating vascular permeability, locally and transiently, providing a brief window of opportunity for migratory/invasive cancer cells to enter the circulation. These doorways are called &#34;Tumor Microenvironment of Metastasis&#34;&#160;or &#34;TMEM&#34;, and, quite expectedly, their density correlates with an increased risk of developing metastatic disease in breast cancer patients8,9,10.
Each TMEM doorway consists of three distinct types of cells: a perivascular macrophage, a tumor cell over-expressing the actin-regulatory protein mammalian enabled (Mena), and an endothelial cell, all in direct physical contact with each other1,5,9,10,11,12,13. The key event for the function of TMEM as an intravasation doorway is the localized release of vascular endothelial growth factor (VEGF) onto the underlying vessel by the perivascular macrophage14. VEGF can disrupt homotypic junctions between endothelial cells15,16,17,18,19, a phenomenon that results in transient vascular leakage, also known as &#34;bursting&#34;&#160;permeability as described in IVI studies 5. TMEM macrophages have been shown to express the tyrosine kinase receptor TIE2, which is required for VEGF-mediated TMEM function and homing of these macrophages to the perivascular niche5,20,21,22. In addition to regulating cancer cell dissemination and metastasis, TIE2+ macrophages have been shown to be central regulators of tumor angiogenesis21,22,23,24,25,26,27,28,29,30,31. As such, TIE2+ macrophages represent a critical constituent of the tumor microenvironment and the main regulator of the metastatic cascade.
To better conceptualize TMEM-mediated vascular permeability (i.e. &#34;bursting&#34;), it is very important to distinguish it from other modes of vascular permeability that are not associated with the dissolution of endothelial cell-cell junctions. In an intact endothelium (one whose tight and adherens junctions are not disrupted), there are three main types of vascular permeability: (a) pinocytosis, which may, or may not, be coupled to transcytosis of the ingested material; (b) transportation of material through endothelial fenestrae; and (c) transportation of material through the paracellular pathway, which is regulated by endothelial tight junctions15,16,17,18,19,32,33,34. Although deregulated in many tumors, the aforementioned modes of vascular permeability have been described mostly in the context of normal tissue physiology and homeostasis, the extremes of which are tissues with either limited permeability (e.g., blood-brain barrier, blood-testis barrier), or abundant permeability (e.g., fenestrated capillaries of the kidney glomerular apparatus)34,35,36,37.
Using multiphoton intravital imaging and multiplexed immunofluorescence microscopy, we are able to distinguish between TMEM-mediated vascular permeability (&#34;bursting&#34;) and other modes of vascular permeability in breast tumors. To achieve this, we perform a single intravenous injection of a high-molecular weight, fluorescently-labeled probe in mice. Spontaneous bursting events can then be captured using intravital imaging in live mice; or alternatively, extravasation of the probe can be quantified by co-localization studies with blood vasculature (e.g. CD31+ or Endomucin+) and TMEM doorways using immunofluorescence microscopy. The protocols presented here describe both of these techniques, which could be used either independently or in conjunction with one another.

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assessing tumor microenvironment of metastasis doorway-mediated vascular permeability associated with cancer cell dissemination using intravital imaging and fixed tissue analysis

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary sites. Recently, we established that cancer cell dissemination in primary breast cancer and at metastatic sites in the lung occurs only at doorways called Tumor MicroEnvironment of Metastasis (TMEM). TMEM doorway number is prognostic for distant recurrence of metastatic disease in breast cancer patients. TMEM doorways are composed of a cancer cell which over-expresses the actin regulatory protein Mena in direct contact with a perivascular, proangiogenic macrophage which expresses high levels of TIE2 and VEGF, where both of these cells are tightly bound to a blood vessel endothelial cell. Cancer cells can intravasate through TMEM doorways due to transient vascular permeability orchestrated by the joint activity of the TMEM-associated macrophage and the TMEM-associated Mena-expressing cancer cell. In this manuscript, we describe two methods for assessment of TMEM-mediated transient vascular permeability: intravital imaging and fixed tissue immunofluorescence. Although both methods have their advantages and disadvantages, combining the two may provide the most complete analyses of TMEM-mediated vascular permeability as well as microenvironmental prerequisites for TMEM function. Since the metastatic process in breast cancer, and possibly other types of cancer, involves cancer cell dissemination via TMEM doorways, it is essential to employ well established methods for the analysis of the TMEM doorway activity. The two methods described here provide a comprehensive approach to the analysis of TMEM doorway activity, either in na&#239;ve or pharmacologically treated animals, which is of paramount importance for pre-clinical trials of agents that prevent cancer cell dissemination via TMEM.

The experimental procedures described in this protocol article are briefly summarized and illustrated in Figure 1A-C.
To measure TMEM-mediated vascular permeability (&#34;bursting activity&#34;) and to reduce experimental noise from other modes of vascular permeability (i.e. transcellular and paracellular, as explained in the introduction), we performed intravenous (i.v.) injection of high molecular weight probes, such as 155 kDa Dextran, conjugat...

Watch this Scientific Journal Video about Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and F

Assessing Tumor Microenvironment of Metastasis Doorway-Mediated Vascular Permeability Associated with Cancer Cell Dissemination using Intravital Imaging and Fixed Tissue Analysis

We describe two methods for assessing transient vascular permeability associated with tumor microenvironment of metastasis (TMEM) doorway function and cancer cell intravasation using intravenous injection of high-molecular weight (155 kDa) dextran in mice. The methods include intravital imaging in live animals and fixed tissue analysis using immunofluorescence.

The most common cause of cancer related mortality is metastasis, a process that requires dissemination of cancer cells from the primary tumor to secondary ...

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