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

Summary

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.

Abstract

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 naïve or pharmacologically treated animals, which is of paramount importance for pre-clinical trials of agents that prevent cancer cell dissemination via TMEM.

Introduction

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 unstable^2,3,4. Although highly suggestive of defective ....

Protocol

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 "bursting permeability" using live animal imaging

1. Transplantation of syngeneic breast tumors into mouse hosts with fluorescent macrophages
   1. Generate pieces of tumor tissue suitable ....

Results

The experimental procedures described in this protocol article are briefly summarized and illustrated in Figure 1A-C.

To measure TMEM-mediated vascular permeability ("bursting activity") 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.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
Anti-rabbit IgG (Alexa 488)	Life Technologies Corporation	A-11034
Anti-rat IgG (Alexa 647)	Life Technologies Corporation	A-21247
Bovine Serum Albumin	Fisher Scientific	BP1600-100
Citrate	Eng Scientific Inc	9770
Cover Glass Slips	Electron Microscopy Sciences	72296-08
Cyanoacrylate Adhesive	Henkel Adhesive	1647358
DAPI	Perkin Elmer	FP1490
Dextran-Tetramethyl-Rhodamine	Sigma Aldrich	T1287
DMEM/F12	Gibco	11320-033
Endomucin (primary antibody)	Santa Cruz Biotechnology	sc-65495
Enrofloxacin	Bayer	84753076 v-06/2015
Fetal Bovine Serum	Sigma Aldrich	F2442
Fish Skin Gelatin	Fisher Scientific	G7765
Insulin Syringe	Becton Dickinson	309659
Isofluorane	Henry Schein	NDC 11695-6776-2
Matrigel	Corning	CB40234	Artificial extracellular matrix
Needle (30 G)	Becton Dickinson	305128
Phosphate Buffered Saline	Life Technologies Corporation	PBS
Polyethylene Tubing	Scientific Commodities Inc	BB31695-PE/1
Pulse Oximeter	Kent Scientific	MouseOx
Puralube Vet Ointment	Dechra	NDC 17033-211-38
Quantum Dots	Life Technologies Corporation	Q21561MP
Rubber	McMaster Carr	1310N14
TMR (primary antibody)	Invitrogen	A6397
Tween-20	MP Biologicals	TWEEN201
Xylene	Fisher Scientific	184835