Fig. Ext Data 4

a Schematic of our in vivo zebrafish xenograft assay to study immune cell?cancer cell interactions in situ. Firstly, human cancer cells were cultured and modified as desired, for example, by expressing a fluorescent marker to label the cells. Secondly, cells were harvested and injected near the common cardinal vein (CCV) into the yolk of zebrafish larvae (violet arrow). Then, xenografts were imaged on our self-driving, multiresolution microscope, and subsequent analysis allowed visualization and quantification of cell spreading, cell?cell interactions, and cell morphological changes. b Low-resolution mSPIM images captured the distribution of macrophages (Tg(mpeg1:EGFP), top: magenta, bottom: grayscale image) in the entire zebrafish larvae after xenografting U-2 OS osteosarcoma cells (pVimentin-PsmOrange label, top: white, bottom: bright white). For tissue context, zebrafish also expressed the vascular marker Tg(kdrl:Hsa.HRAS-mCherry) (top: cyan). The image highlights how macrophages clustered around sites with cancer cells (Supplementary Movie 7). c In contrast, zebrafish without xenografts (control) displayed a uniform distribution of macrophages (Tg(mpeg1:EGFP), top: magenta, bottom: grayscale image) across the entire zebrafish embryo (top: vascular marker Tg(kdrl:Has.HRAS-mCherry in cyan). d The self-driving feature of the microscope enabled high-resolution imaging of selected cancer colonies in the zebrafish tail over many hours by keeping it in focus. Frequently, we observed how zebrafish macrophages (Tg(mpeg1:EGFP), top: magenta, bottom: gray) attached to the U-2 OS cancer cells (pVimentin-PsmOrange label, top and bottom: green), and phagocytosed them (Supplementary Movie 7, 8). Scale bar lengths are as follows: b,c 500 ?m; d 50 ?m.