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.