Abstract
Immunotherapy has demonstrated great therapeutic potential by activating the immune system to fight cancer. However, little is known about the specific dynamics of interactions that occur between tumor and immune cells. In this protocol we describe a novel method to visualize the interaction of tumor and immune cells in the lung of live mice, which can be applied to other organs. In this protocol fluorescent-labeled tumor cells are transferred to recipient mice expressing fluorescently tagged immune cells. Tumor-immune cell interactions in the lung are then imaged by confocal or two photon microscopy. Analysis of tumor interactions with immune cells using this protocol should aid in a better understanding of the importance of these interactions and their role in developing immunotherapies.
Keywords: In vivo imaging, Cancer, Immunology, Lung, Phagocytosis
Background
A number of immunotherapies have demonstrated great promise in treating cancer. Understanding the spatial temporal resolution of how these tumor-immune interactions occur is important for enhancing and developing new immunotherapies. In this protocol we describe a novel method to directly visualize tumor-immune cell interactions in vivo in mouse lung. This protocol is initially described in our work examining the interactions of patrolling monocytes and tumor cells in the mouse lung (Hanna et al., 2015). This fluorescent microscopy protocol uses the vacuum imaging ring to stabilize and image the lung, which was initially described by Looney and colleagues (Thornton et al., 2012). In this protocol fluorescent-labeled tumor cells are transferred to recipient mice expressing fluorescently tagged immune cells. Tumor-immune cell interactions in the lung are then imaged by confocal or two photon fluorescent microscopy using the vacuum imaging ring. This protocol allows for the addition of other immune cell markers by intravenous (IV) injection of fluorescently labeled antibodies, and is adaptable to image tumor-immune cell interactions in other organs. Quantitative information such as the localization, engulfment of tumor material, timing, speed and frequency of these immune cell interactions can be collected using this protocol. This protocol should aid in helping to better understand the specific immune-tumor cell interactions that are important to developing better immunotherapies in the future.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Three dimensional reconstructions and drift correction of lung images were performed using Imaris software (version 7.1.1 x 64). Detailed instructions on the use of Imaris software can be found at: http://www.bitplane.com/download/manuals/ReferenceManual6_1_0.pdf. Images were smoothed by median filtering at kernel size 3 x 3 pixels. When needed, motion artifacts caused by breathing were corrected with rigid body registration using an ImageJ plug-in (McArdle et al., 2014; McArdle et al., 2015). Imaris software was also used to automatically process 3D video data by detecting cells in each fluorescence channel, then creating tracks by linking the detected cells over time (using the Imaris 'Track spots over time' function). Tracks were manually edited to improve accuracy. Data for all experiments were analyzed with Prism software. Unpaired t-tests and two-way analysis of variance were used for comparison of experimental groups. P values of less than 0.05 were considered significant. The data appeared to be normally distributed with similar standard deviation and error observed between and within experimental groups.
Notes
Acknowledgments
This protocol is adapted from an earlier protocol describing live imaging of the lung (Thornton et al., 2012). This work was supported by NIH grants R01 HL118765 and R01 CA202987 (both to C.C.H.), American Heart Association Scientist Development Grant 125SDG12070005 (to R.N.H.), the La Jolla Institute for Allergy and Immunology Board of Directors Fellowship (to R.N.H.).
References
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