Intravital imaging of metastatic behavior through a mammary imaging window

Abstract

We report a technique to evaluate the same tumor microenvironment over multiple intravital imaging sessions in living mice. Individual tumor cells expressing photoswitchable proteins in an orthotopic mammary carcinoma are optically marked and followed for extended times through a Mammary Imaging Window (MIW). We demonstrate, for the first time, that two distinct microenvironments in the same orthotopic mammary tumor affect differently the invasion and intravasation of tumor cells. KeywordsTumor microenvironment; photoswitching; photoactivatable fluorescent proteins; migration; invasion; intravasation; intravital imaging; metastasis; mammary imaging window The early steps of metastasis are characterized by tumor cells invading the stroma (invasion) and entering the blood (intravasation)1 , 2. Short term tracking of individual cells inside fluorescent tumors by intravital imaging has revealed dramatic heterogeneity in tumor cell invasion and intravasation3. However, long term tracking of individual cells is required to quantify these behaviors and to determine the fates of cells in specific tumor microenvironments. Imaging techniques that rely on surgical dissection to expose the imaging site have limitations for long-term experiments such (1) tissue dehydration, impaired thermoregulatory control and/or animal survival upon surgical dissection, (2) possible effects of prolonged anesthesia exposure, and (3) a limited field of view. These limitations can be overcome by studying tumors through a dorsal skinfold chamber4. The use of dorsal skinfold chambers, however, limits the experiments to tumor-models based on cell lines, and for many tumors a non-orthotopic environment. For example, invasion and intravasation of breast tumor cells is highly dependent on the specific local microenvironment5 which may not exist in the Correspondence to: John Condeelis, condeeli@aecom.yu.edu; Jeffrey E. Segall, segall@aecom.yu.edu; Jacco van Rheenen, j.vanrheenen@niob.knaw.nl. 4 DK and BG contributed equally Author contributions: DK, BG, and JvR, JES, JC contributed equally to this manuscript, and they conceived of and designed the study, performed the imaging, and analysis and drafted the manuscript. JW advised and trained in intravital imaging, VVV provided materials and advice. To image orthotopic (in the natural, mammary gland environment) breast tumors intravitally at high resolution for prolonged times, we developed a MIW that can be placed on top of the mammary gland of a mouse (Fig. 1a). The protocol for these animal studies was approved by the Institutional Animal Care and Use Committee for the Albert Einstein College of Medicine. The MIW consists of two plastic rings which form a mount for a glass coverslip. The mount has holes which facilitate suturing into the skin, whereas the glass coverslip assures the optimal working distance and refraction index for high resolution imaging (for more details on the equipment used for imaging, see Supplementary Fig. 1 online). While surgical dissection of the skin overlaying t...

Monomeric red fluorescent proteins with a large Stokes shift

“…We previously observed that tumor cell migration and invasion in vivo occurs more frequently near blood vessels (3,31). Therefore, we examined tumor cell Golgi-nucleus alignments as a function of blood vessel distance.…”

“…2P microscopy and intravital imaging procedure were preformed as described previously (5). The primary tumor was surgically exposed as described (31). Tumor cell polarization analysis was performed at 870 nm; and Z-stacks were captured at 2 μm steps over a Z-axis distance of 40 μm.…”

Three-dimensional microfluidic model for tumor cell intravasation and endothelial barrier function

“…Despite progress in characterizing these vessels (18,19), it is not clear whether tumor cells require abnormal blood vessels to intravasate and how tumor cells and other factors in the tumor microenvironment (e.g., macrophages) contribute to vessel remodeling. Although in vivo imaging studies in animals have demonstrated the highly dynamic interaction between tumor and endothelial cells (9,10,12,20), quantitation of, or control over, vessel permeability and tumor microenvironmental factors is challenging. On the other hand, studies employing transwell inserts (21) that allow for manipulation of tumor-endothelial interactions do not enable visualization of intravasation events in real-time or precise control over cell-cell distance and growth-factor gradients.…”

Optical imaging for the new grammar of drug discovery

“…breast imaging) and minimally invasive imaging devices promise the translation of such tools into clinic [5]. Live cell and intravital microscopy (IVM) are other powerful optical imaging technologies, which are closing the gap between in vitro and in vivo assays by providing molecular information in high resolution [14,15]. Ideally, a comprehensive preclinical imaging platform consists of live cell microscopy, IVM and macroscopic imaging for whole animal imaging (fluorescence and bioluminescence).…”