Advanced three-dimensional imaging of the immune system within the melanoma tumor microenvironment holds tremendous implications. The most commonly used syngeneic model for understanding melanoma pathophysiology employs the transplantation of the B16-F10 melanoma cell line into mouse skin and/or hypodermis (Guo et al., 2019Guo D. Lui G.Y.L. Lai S.L. Wilmott J.S. Tikoo S. Jackett L.A. et al.RAB27A promotes melanoma cell invasion and metastasis via regulation of pro-invasive exosomes.Int J Cancer. 2019; 144: 3070-3085Crossref PubMed Scopus (44) Google Scholar; Overwijk and Restifo, 2001Overwijk W.W. Restifo N.P. B16 as a mouse model for human melanoma.Curr Protoc Immunol. 2001; (Chapter 20:Unit 20.1)Crossref PubMed Google Scholar; Potez et al., 2018Potez M. Trappetti V. Bouchet A. Fernandez-Palomo C. Güç E. Kilarski W.W. et al.Characterization of a B16-F10 melanoma model locally implanted into the ear pinnae of C57BL/6 mice.PLoS One. 2018; 13e0206693Crossref PubMed Scopus (16) Google Scholar). However, B16-F10 cells are heavily pigmented, which impedes the penetration of light into solid tumors. In addition, the presence of melanin may induce speckling when two-photon laser light hits melanoma cells, resulting in compromised image quality (Roediger et al., 2008Roediger B. Ng L.G. Smith A.L. Fazekas de St Groth B. Weninger W. Visualizing dendritic cell migration within the skin.Histochem Cell Biol. 2008; 130: 1131-1146Crossref PubMed Scopus (47) Google Scholar). Furthermore, speckling indicates heat injury, which can trigger an inflammatory response, thereby altering physiological antitumor immune response (Li et al., 2012Li J.L. Goh C.C. Keeble J.L. Qin J.S. Roediger B. Jain R. et al.Intravital multiphoton imaging of immune responses in the mouse ear skin.Nat Protoc. 2012; 7: 221-234Crossref PubMed Scopus (138) Google Scholar). All these factors impede the use of B16-F10 cells for certain experimental approaches, particularly microscopic imaging. We reasoned that interference with the melanin synthesis pathway should render B16-F10 cells amelanotic, which consequently would circumvent the shortcomings of their pigmentation. Using an RNA-guided CRISPR/Cas9 system (Adli, 2018Adli M. The CRISPR tool kit for genome editing and beyond.Nat Commun. 2018; 9: 1911Crossref PubMed Scopus (663) Google Scholar), we generated B16-F10 cells lacking the Tyr gene. We then carried out extensive in vitro and in vivo characterization of these cells to validate their utility in furthering our understanding of melanoma cell biology. The workflow for the experimental design is depicted in Figure 1a. Three guide RNAs targeting the mouse Tyr gene were designed, synthesized, and cloned into the pX330 vector (Ran et al., 2013Ran F.A. Hsu P.D. Wright J. Agarwala V. Scott D.A. Zhang F. Genome engineering using the CRISPR-Cas9 system.Nat Protoc. 2013; 8: 2281-2308Crossref PubMed Scopus (5874) Google Scholar). After cloning and validation, B16-F10 cells were transfected with three guide RNA clones and the vector control (Figure 1a and Supplementary Figure S1a). Single-cell clones were generated and screened, and the colonies with loss of melanin were further expanded (Figure 1a and Supplementary Figure S1b). Three clones, one from each guide RNA group (B16-F10-1B, B16-F10-2C, and B16-F10-3C) that had lost visible pigmentation, were selected for further experimentation (Figure 1b, top and Supplementary Figure S1c). Melanin content assay confirmed that the selected clones had lost melanin pigmentation compared with the parental B16-F10 or B16-F10-vector control cell line (Figure 1b, bottom). After the successful generation of amelanotic B16-F10 (amel-B16-F10) cells, we wanted to test whether these cells behaved in a manner comparable with wild-type B16-F10 cells. To test this, growth profiling using a confluency-based cell proliferation assay was performed. Compared with the wild-type and vector cells, clones 2C and 3C showed comparable growth profiles, whereas clone 1B had slight growth impairment (Figure 1c). Investigating their migratory behaviors showed that clones 1B and 3C had the most consistent profile when compared with the wild-type B16-F10 and vector control cells (Figure 1d). Because B16-F10-3C cells most closely resembled B16-F10 wild-type and vector cells in terms of their growth and migration kinetics and showed a robust loss of melanin, we selected these cells for further in vivo characterization. To examine the in vivo growth kinetics, we subcutaneously injected mice with amel-B16-F10-3C or mel-B16-F10-vector cells. Measurement of tumor volume revealed no statistical difference between the two groups (Figure 1e). As expected, mel-B16-F10-vector cells generated dark, melanin-laden tumors, whereas amel-B16-F10-3C cells generated amelanotic tumors with visible neoangiogenic vasculature (Supplementary Figure S1d). Because tyrosinase is a prominent melanoma antigen (Rosenberg, 1999Rosenberg S.A. A new era for cancer immunotherapy based on the genes that encode cancer antigens.Immunity. 1999; 10: 281-287Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar), we next investigated whether its deletion would alter the composition of the immune cell infiltrate within melanoma tumors in vivo. Enumeration of various immune cell populations, including that of lymphoid cells, granulocytes, macrophages, monocytes, and dendritic cells, revealed similar immune cell infiltration in amel-B16-F10-3C allograft tumors and mel-B16-F10-vector control tumors (Figure 1f). We also asked whether the ablation of tyrosinase altered the intracellular signaling characteristics of amel-B16-F10-3C melanoma cells. To this end, cGMP measurements were performed. cGMP is a second messenger that is crucial for the maintenance of various cellular processes in melanoma cells (Dhayade et al., 2016Dhayade S. Kaesler S. Sinnberg T. Dobrowinski H. Peters S. Naumann U. et al.Sildenafil potentiates a cGMP-dependent pathway to promote melanoma growth.Cell Rep. 2016; 14: 2599-2610Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The real-time visualization of intracellular cGMP after stimulation with different endogenous hormones demonstrated no difference between the parental and the amel-B16-F10-3C melanoma cells (Figure 1g). Intravital multiphoton microscopy is a powerful tool for the dissection of cellular behavior within native environments (Jain et al., 2016Jain R. Tikoo S. Weninger W. Recent advances in microscopic techniques for visualizing leukocytes in vivo.F1000Res. 2016; 5: F1000Crossref PubMed Google Scholar; Tikoo et al., 2018Tikoo S. Barki N. Jain R. Zulkhernain N.S. Buhner S. Schemann M. et al.Imaging of mast cells.Immunol Rev. 2018; 282: 58-72Crossref PubMed Scopus (11) Google Scholar). However, the presence of melanin pigment potentially interferes with the acquisition of meaningful imaging datasets (Supplementary Figure S2). Intravital imaging of amel-B16-F10-3C tumors implanted into the flanks of Lyz2GFP/+ mice (Faust et al., 2000Faust N. Varas F. Kelly L.M. Heck S. Graf T. Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages.Blood. 2000; 96: 719-726Crossref PubMed Google Scholar), in which myeloid cells express GFP, did not reveal the induction of laser light‒induced speckles even after extended imaging periods (Figure 2a and Supplementary Video S1). Consequently, we did not observe the recruitment of GFPhi neutrophils from the patent vasculature into the tumor periphery (Figure 2a and Supplementary Video S1). Most GFPhi neutrophils displayed random migratory behavior in contrast to the sessile GFPint myeloid cells present within the region (Supplementary Video S1). Similarly, employing mCherry-expressing amel-B16-F10-3C implanted into Csf1r-GAL4/VP16, UAS-ECFP transgenic mice (MacBlue mice [Sauter et al., 2014Sauter K.A. Pridans C. Sehgal A. Bain C.C. Scott C. Moffat L. et al.The MacBlue binary transgene (csf1r-gal4VP16/UAS-ECFP) provides a novel marker for visualisation of subsets of monocytes, macrophages and dendritic cells and responsiveness to CSF1 administration.PLoS One. 2014; 9e105429Crossref PubMed Scopus (44) Google Scholar]), in which monocytes and macrophages express CFP, we were able to visualize both myeloid and tumor cells within the deep tumor tissue (Figure 2b and Supplementary Video S2). To formally prove that pigmented cells induce the aforementioned imaging artifacts, we injected a mixture of nonfluorescent amel-B16-F10-3C:mCherry+mel-B16-F10-parental (90%:10%) cells into Lyz2GFP/+ mice. Intravital imaging of deep tumor tissue highlighted the rapid speckling of mCherry+ mel-B16-F10-parental melanoma cells, followed by tumor cell lysis (marked by the loss of mCherry signal) (Figure 2c and Supplementary Video S3). Similar to mCherry+ tumor cells, we also observed lysis of GFP-expressing myeloid cells present in the vicinity of the injury foci (Figure 2c and Supplementary Video S3). Other than mCherry+ tumor cells, we observed the presence of GFP-expressing myeloid cells containing mCherry+ vesicles within the tumor periphery (Supplementary Figure S3). Intravital imaging of these regions resulted in rapid speckling, followed by extensive tissue damage and the recruitment of GFPhi neutrophils from either the interstitium or through the patent vasculature (Figure 2d and Supplementary Video S4). These observations confirm that the presence of melanin not only results in heat-induced lysis of cells but also alters immune cell migration. Finally, amel-B16-F10 tumors enabled us to reconstruct the three-dimensional tumor microenvironment using light-sheet fluorescence microscopy and chemical clearing methodologies (Kubota et al., 2017Kubota S.I. Takahashi K. Nishida J. Morishita Y. Ehata S. Tainaka K. et al.Whole-body profiling of cancer metastasis with single-cell resolution.Cell Rep. 2017; 20: 236-250Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). To this end, we injected amel-B16-F10-3C or mel-B16-F10-parental cells into the flanks of Lyz2GFP/+ mice (Faust et al., 2000Faust N. Varas F. Kelly L.M. Heck S. Graf T. Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages.Blood. 2000; 96: 719-726Crossref PubMed Google Scholar) crossed with mT/mG mice (Muzumdar et al., 2007Muzumdar M.D. Tasic B. Miyamichi K. Li L. Luo L. A global double-fluorescent Cre reporter mouse.Genesis. 2007; 45: 593-605Crossref PubMed Scopus (1892) Google Scholar). mT/mG is a transgenic reporter strain wherein all cells endogenously express mTomato, with the highest expression observed in vascular endothelial cells enabling imaging of blood vessels. Light-sheet fluorescence microscopy imaging sessions were not very informative in pigmented tumors because the lasers could not penetrate beneath the surface, thereby revealing minimal information about the architecture of the vasculature and the localization of myeloid cells (Figure 2e, left and Supplementary Figure S4 and Supplementary Video S5). In stark contrast, the imaging of the amelanotic tumors revealed the localization of neutrophils (Supplementary Video S6) and their relationship to blood vessels within the melanoma tumor milieu in great detail (Figure 2e, right and Supplementary Video S7). In addition, by employing light-sheet fluorescence microscopy, we could also visualize various components of the tumor microenvironment, such as the endothelium and immune cells at a higher resolution (Figure 2f, inset). These experiments established that the amelanotic tumors are highly compatible with advanced imaging modalities, such as intravital multiphoton microscopy and light-sheet fluorescence microscopy, and therefore are an excellent tool for the investigation of the melanoma microenvironment in vivo. Data generated in this study are available from the corresponding authors on request. All experiments described in this manuscript have been performed in accordance with the ethics protocols approved by the animal ethics committee; Sydney Local Health District; and Animal ethics committee, The University of Sydney (Australia). Shweta Tikoo: http://orcid.org/0000-0001-5850-8075 Rohit Jain: http://orcid.org/0000-0002-4905-462X Florence Tomasetig: http://orcid.org/0000-0002-6827-9453 Kathy On: http://orcid.org/0000-0002-8556-1856 Brendon Martinez: http://orcid.org/0000-0001-6026-812X Celine Heu: http://orcid.org/0000-0001-6976-9924 Daniel Stehle: http://orcid.org/0000-0001-9086-2306 Peyman Obeidy: http://orcid.org/0000-0002-1957-3154 Dajiang Guo: http://orcid.org/0000-0001-5856-5854 Jonathan N. Vincent: http://orcid.org/0000-0002-3232-2149 Adam J.L. Cook: http://orcid.org/0000-0002-5735-522X Ben Roediger: http://orcid.org/0000-0002-4593-091X Robert Feil: http://orcid.org/0000-0002-7335-4841 Renee M. Whan: http://orcid.org/0000-0002-6664-6344 Wolfgang Weninger: http://orcid.org/0000-0003-3133-8699 BR is presently an employee at Novartis Institutes for Biomedical Research. The remaining authors state no conflict of interest. We would like to acknowledge the core flow cytometry and imaging facility at The Centenary Institute of Cancer Medicine and Cell Biology (Sydney, Australia). We would also like to acknowledge various funding bodies, especially the National Health and Medical Research Council , Cancer Australia (CanToo), Centenary Institute of Cancer Medicine and Cell Biology, and The University of Sydney (Australia) internal funding schemes. All experiments described in this manuscript have been performed in accordance with the ethics protocols approved by the animal ethics committee; Sydney Local Health District; and Animal ethics committee, The University of Sydney. Illustrations have been created using BioRender (biorender.com). Conceptualization: ST, RJ, WW; Data Curation: ST, RJ, FT, KO, BM, CH, DS, PO, JNV, BR; Formal Analysis: ST, RJ, FT, KO, BM, CH, DS, PO, BR; Funding Acquisition: ST, WW; Investigation: ST, RJ, FT, KO, BM, CH, DS, PO, DG, JNV, AJLC, BR; Supervision: ST, RJ, WW; Writing - Original Draft Preparation: ST, RJ, WW; Writing - Review and Editing: ST, RJ, FT, KO, BM, CH, DS, PO, DG, JNV, AJLC, BR, RF, RMW, WW https://www.jidonline.org/cms/asset/f7ca45f0-7c05-48f2-840b-d2af67ab4734/mmc1.mp4Loading ... Download .mp4 (4.13 MB) Help with .mp4 files Supplementary Video 1https://www.jidonline.org/cms/asset/43b2f88d-03de-44ce-bb95-d0a808293af0/mmc2.mp4Loading ... Download .mp4 (3.43 MB) Help with .mp4 files Supplementary Video 2https://www.jidonline.org/cms/asset/031dd6f2-edb7-4cbc-b30f-b3b7bf3d7092/mmc3.mp4Loading ... Download .mp4 (0.62 MB) Help with .mp4 files Supplementary Video 3https://www.jidonline.org/cms/asset/faf6922e-aa27-4f77-932f-a73b00924ba1/mmc4.mp4Loading ... Download .mp4 (3.07 MB) Help with .mp4 files Supplementary Video 4https://www.jidonline.org/cms/asset/8a8ffc42-343f-4069-b144-ccdae96e93ae/mmc5.mp4Loading ... Download .mp4 (2.49 MB) Help with .mp4 files Supplementary Video 5https://www.jidonline.org/cms/asset/9013956b-e2fb-494b-b309-dc60567585dc/mmc6.mp4Loading ... Download .mp4 (2.43 MB) Help with .mp4 files Supplementary Video 6https://www.jidonline.org/cms/asset/f10670ed-c075-4111-b2c8-434c71ad587d/mmc7.mp4Loading ... Download .mp4 (8.22 MB) Help with .mp4 files Supplementary Video 7 The various transgenic mice strains employed in this study were bred and maintained within the Animal Facility at the Centenary Institute (Camperdown, Australia). All experimental mice were maintained under specific pathogen-free conditions at 22–26 °C with 12-hour light and/or dark cycle. mT/mG mice (Muzumdar et al., 2007Muzumdar M.D. Tasic B. Miyamichi K. Li L. Luo L. A global double-fluorescent Cre reporter mouse.Genesis. 2007; 45: 593-605Crossref PubMed Scopus (2136) Google Scholar) were purchased from The Jackson Laboratory (Bar Harbor, ME). Unless specified, mice aged between 12 and 18 weeks and of either sex were used for experimentation. All experiments described in this manuscript have been performed in accordance with the ethics protocols approved by the animal ethics committee; Sydney Local Health District; and Animal ethics committee, The University of Sydney (Australia). All experiments were carried out as per the approved Standard Operating Procedures and flow cytometry biosafety protocols at the Centenary Institute. Individual strains were employed as per the experimental requirements and have been listed throughout the manuscript. B16-F10 parental and vector control melanoma cell lines and amelanotic B16-F10 melanoma Tyr−/− CRISPR clones 1B, 2C, and 3C were cultured in DMEM-GlutaMAX (Gibco, Waltham, MA) supplemented with 10% HyClone fetal bovine serum (GE Healthcare Life Sciences, Marlborough, MA), 1 mM sodium pyruvate (Gibco), and ×1 Pen Strep (Gibco) and incubated at 37 °C and 5% carbon dioxide (CO2). Approximately 10,000 cells per well were seeded into 24-well plates with DMEM-GlutaMAX media (supplemented with 10% fetal calf serum, 1 mM sodium pyruvate, and ×1 Pen Strep). B16-F10 parental, B16-F10 vector, and amel B16-F10 melanoma cells were seeded in triplicate. Cells were left to adhere to the bottom of each well for approximately 4–6 hours at 37 °C and 5% CO2. Plates were then imaged using the IncuCyte ZOOM live cell analysis system (Essen Bioscience, Newark, United Kingdom) for 84 hours, where a total of nine images were taken of each well every hour. For each well, the IncuCyte ZOOM software calculates the percentage cellular confluency on the basis of the corresponding images taken. Raw data obtained from three independent experiments were normalized and graphed using the Prism software (GraphPad, GraphPad Software, San Diego, CA) to obtain an average growth curve for each cell line. In vitro migration assay is an assay used to determine the dynamic migratory behavior of a cell. Approximately 10,000 cells per well were seeded into 24-well plates, wherein B16-F10 parental, B16-F10 vector, and amel B16-F10 melanoma cells were plated in triplicate. Cells were left to adhere for approximately 4–6 hours at 37 °C and 5% CO2. Plates were then imaged overnight using the IncuCyte ZOOM live cell analysis system. A total of four images were taken of each well every 5 minutes. Images taken by the IncuCyte ZOOM system within the first 3 hours were used for analysis. All images were exported and converted into eight-bit image stacks on FIJI (Schindelin et al., 2012Schindelin J. Arganda-Carreras I. Frise E. Kaynig V. Longair M. Pietzsch T. et al.Fiji: an open-source platform for biological-image analysis.Nat Methods. 2012; 9: 676-682Crossref PubMed Scopus (26921) Google Scholar). From each eight-bit image stack, 20 cells were randomly selected and tracked using the semiautomatic tracker of the CellTrackerGUI program on MATLAB (MathWorks, Natick, MA) (Piccinini et al., 2015Piccinini F. Kiss A. Horvath P. CellTracker (not only) for dummies.Bioinformatics. 2015; 32: 955-957Crossref PubMed Scopus (65) Google Scholar). Average speed (μm/minute) was calculated on the basis of the individual tracks generated for each selected cell. The overall average speed (μm/minute) for each cell line was calculated from three independent experiments. For melanin content assays, approximately 50,000 cells per well were seeded into six-well plates and incubated at 37 °C and 5% CO2 for 3 days. On day 2, 20 nM of α-melanocyte‒stimulating hormone was added into each well and incubated overnight. The next day, cells were lysed with 200 μl 1 M sodium hydroxide and passed through a 26G needle to obtain a homogenous cell lysate. Each sample (100 μl aliquots each) were transferred to a 96-well plate, and absorbance readings at 405 nm were obtained using the FLUORstar Omega plate reader (BMG Labtech, Ortenberg, Germany). Similarly, 50,000 cells per well seeded into separate six-well plates for the bicinchoninic acid assay were also incubated at 37 °C and 5% CO2 for 3 days. On day 3, cells were trypsinized and pelleted using a desktop centrifuge set at maximum speed for 5 minutes. Pellets were washed twice with Hank’s Buffered Salt Solution (Gibco) and incubated with 100 μl RIPA buffer on ice for 15 minutes. Samples were then centrifuged at 12,000g and 4 °C for 15 minutes, and the supernatant was collected and diluted with double distilled water (1:5 dilution). Diluted sample (10 μl each) and BSA standards (2 mg/ml), obtained from the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific,Waltham, MA), were plated into a 96-well plate. Using bicinchoninic acid reagents from the same kit, samples and standards were incubated with 200 μl of reagent A:reagent B (1:49) for 30 minutes at 37 °C. Plates were read at 562 nm using the FLUORstar Omega plate reader, and absorbance readings obtained were used to calculate the total protein content of each 100 μl cell sample. The melanin content of B16-F10 parental, B16-F10 vector, amel B16-F10 melanoma, and E0771 cells is expressed as a ratio of the absorbance over total protein content (μg) per sample. Therefore, melanin content assays and bicinchoninic acid assays for all cell lines were conducted on the same day. Vector control cells or 2.5 × 105 amel B16-F10 cells were injected into the flanks of C57BL/6J mice (purchased from Australian BioResources, Moss Vale, Australia). Tumors were allowed to grow under appropriate monitoring in accordance with the Sydney Local Health District‒approved protocols. Once palpable, tumors were measured regularly as per the approved guidelines. Tumor growth measurements were graphed, using the Prism software (GraphPad). Whole tumors were excised from the mice in ×1 PBS (Gibco). Tumors were washed twice with ×1 PBS before being cut into small pieces and were digested in PBS containing 10% fetal bovine serum, 1 mg/ml collagenase type IV (Sigma Aldrich, St Louis, MO), and 2% v/v DNase (Sigma Aldrich) for 1 hour at 37 °C. Digested tumor tissue was filtered through a 100-μm cell strainer, topped up with FACS wash (×1 PBS + 2% fetal bovine serum [HyClone, catalog number sh30084.03] + 0.2% EDTA [Sigma Aldrich] + 0.2% sodium azide [Sigma Aldrich]), and centrifuged at 524g for 5 minutes at 4 °C. Red blood cells were lysed with 5 ml of ammonium‒chloride‒potassium lysing buffer (Gibco) for 5 minutes at room temperature, followed by neutralization with FACS wash and centrifugation at 524g for 5 minutes at 4 °C. Cells were resuspended in 10 ml FACS wash and filtered through a nylon filter before being overlayed onto 3 ml Ficoll (GE Healthcare Life Sciences, 17-1440-03). Cell/Ficoll mixture was centrifuged at 931g for 1 hour at room temperature with brakes off. Buffy layer was subsequently removed and washed several times with FACS wash before being transferred to a 96-well U Bottom plate to be stained for flow cytometry using antibodies listed in the Supplementary Table S1. To monitor intracellular cGMP signals in living melanoma cells, cells at 30–40% confluency were transfected with an expression plasmid encoding the Förster resonance energy transfer‒based cGMP sensor, cGi500 (Russwurm et al., 2007Russwurm M. Mullershausen F. Friebe A. Jäger R. Russwurm C. Koesling D. Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach.Biochem J. 2007; 407: 69-77Crossref PubMed Scopus (104) Google Scholar). Förster resonance energy transfer and/or cGMP imaging of the cells was performed 48 hours after transfection as described previously (Thunemann et al., 2013Thunemann M. Wen L. Hillenbrand M. Vachaviolos A. Feil S. Ott T. et al.Transgenic mice for cGMP imaging.Circ Res. 2013; 113: 365-371Crossref PubMed Scopus (52) Google Scholar). Background-corrected F480 and F535 signals were used to calculate the F480/F535 ratio, that is, R. The presented ΔR/R traces (indicating the relative cGMP concentration change) were obtained by normalization to the baseline recorded for 2–5 minutes at the beginning of each experiment. For intravital imaging of the melanoma tumors, we used a custom-made stage. Briefly, mice were anesthetized using a combination of ketamine (100 mg/kg of body weight) and xylazine (10 mg/kg of body weight), with repeated doses being administered as per the requirement. Hair was removed from the surgical area (flank) of the anesthetized mouse using commercial hair removal cream, Nair (Church & Dwight, New York, NY). The skin was cleaned extensively to remove any residual cream. From this point onward, all procedures were performed on a heating pad to maintain the core body temperature, and all surgical procedures were performed with sterilized instruments. To expose the underlying tumor, a lateral incision was generated extending from the lower abdomen to the neck regions. Utilizing blunt dissection methodology, the skin was separated from the surrounding tissue and placed on a temperature-controlled stage. The skin was immobilized utilizing surgical sutures, and a chamber was placed around the tumor to superfuse with the buffer. Any exposed tissue was covered with a sterile cotton-gauze sheet, which was kept warm with saline buffer. The tissue and core body temperatures of the mouse were kept constant throughout the procedure. The exposed tumor was imaged using the LaVision BioTec TriMScope (LaVision BioTec, Bielefeld, Germany) as described later. LaVision BioTec TriMScope (LaVision BioTec) equipped with a ×20 (numerical aperture [NA] 0.95) water immersion objective was employed to perform intravital multiphoton microscopy. Both Ti-Sapphire femtosecond laser (Mai Tai HP; Spectra-Physics, Santa Clara, CA, 720–1,050 nm, <140 fs, 90 MHz) and an APE optical parametric oscillator system (APE, Berlin, Germany) (tuning range 1,050–1,400 nm) were used to illuminate the sample. The fluorescence data were collected using four external non-descanned dual-channel reflections and/or fluorescence detectors. Imaging was performed for a 270 × 270 μm region with 500 × 500 pixel resolution and 6‒12 z steps depending on the experimental design. Nontoxic dye Evans blue (Sigma Aldrich) conjugated to serum albumin (Sigma Aldrich) was injected intravenously to highlight blood vessels in some experiments (Vennin et al., 2017Vennin C. Chin V.T. Warren S.C. Lucas M.C. Herrmann D. Magenau A. et al.Transient tissue priming via ROCK inhibition uncouples pancreatic cancer progression, sensitivity to chemotherapy, and metastasis.Sci Transl Med. 2017; 9: eaai8504Crossref PubMed Scopus (154) Google Scholar). All settings for independent experiments were optimized to visualize the cells present within the individual field of view. Tissues were dissected from perfused mice (4% paraformaldehyde, Sigma Aldrich) and immersed in 4% paraformaldehyde/PBS ×1 at 4 °C until processing. Subsequently, all further steps were performed at 37 °C, with gentle orbital shaking. Tumor samples were cleared following the previously published CUBIC L/R procedure (Kubota et al., 2017Kubota S.I. Takahashi K. Nishida J. Morishita Y. Ehata S. Tainaka K. et al.Whole-body profiling of cancer metastasis with single-cell resolution.Cell Rep. 2017; 20: 236-250Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar; Susaki et al., 2014Susaki E.A. Tainaka K. Perrin D. Kishino F. Tawara T. Watanabe T.M. et al.Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.Cell. 2014; 157: 726-739Abstract Full Text Full Text PDF PubMed Scopus (757) Google Scholar). Samples were rinsed three times for 6 hours with ×1 PBS. For Figure 2e, whole-tumor samples were incubated for 7 days into approximately 10 ml of CUBIC reagent L (10 weighted % Triton X-100, 10 weighted % N-buthyldiethanolamine, Water). The clearing solution was replaced every 2–3 days. Samples were then washed three times for 6 hours with ×1 PBS. To obtain a higher resolution, the refractive index of the sample was matched by incubating the sample in 25 ml of CUBIC reagent R (45 weighted % antipyrine, 30 weighted% nicotinamide, Water, pH 7) for a minimum of 2 days before imaging. To enhance the visualization of blood vessels (Figure 2f) within the core of amelanotic tumors, we sectioned the parent tumor into 20 × 5 mm sections (Supplementary Figure S5) that were individually cleared as described earlier and imaged. Cleared tumor samples were glued onto the end of a glass capillary for immersion inside the Zeiss imaging chamber (Zeiss, Oberkochen, Germany). The chamber was filled with the CUBIC reagent R (RI:1.51, pH 7) to match the refractive index of the sample. Images were acquired with a Light-sheet Z.1 microscope (Zeiss) using a PCO Edge 5.5 sCMOS camera. GFP and tdTomato fluorophores were excited with 488 and 561 nm lasers, respectively. Data were collected using 505–545 nm (for GFP) and 575–616 nm (for tdTomato) bandpass filters. Each sample was imaged from either one or two angles at 0° or 180°. Each angle was acquired as a tile region (a mosaic) where each tile is acquired with the same z-stack parameters and a 20% overlap as well as a 30-second delay before the next tile position to allow the sample to set back into its original position. Each z-plan frame was sequentially acquired from left and right illumination. At ×5, images were collected through an air Plan Neofluar ×5/ / NA 0.16 objective (Zeiss, 4908000084), and the light-sheet image was formed through two illumination objectives (LSFM 5X/NA 0.1). At ×20, samples were washed three times and immersed for 2 days into 25 ml of CUBIC reagent 2 (RI 1.48) (Susaki et al., 2014Susaki E.A. Tainaka K. Perrin D. Kishino F. Tawara T. Watanabe T.M. et al.Whole-brain imaging with single-cell resolution using chemical cocktails and computational analysis.Cell. 2014; 157: 726-739Abstract Full Text Full Text PDF PubMed Scopus (757) Google Scholar). Images were collected with an immersion Plan Neofluar ×20/NA 1 objective and corrected for a refractive index 1.48 (Zeiss, 4915000049), and the illumination objectives were LSFM ×10/NA 0.2. The software used for acquisition and dual side fusion was Zen Black 2014 SP1 (version 9.2.6.54). Mosaic tile region stitching, multi-view fusion, and three-dimensional rendering were done with Arivis, version4D (version 2.12.5 64). All imaging was performed and analyzed at the Biomedical Imaging Facility, Mark Wainwright Analytical Centre, University of New South Wales (Sydney, Australia). Statistical analyses were performed using Prism software (GraphPad Software). Data is presented as Mean ± S.E.M. Data for Figure 1 b–d was analyzed using one-way ANOVA followed by Tukey's multiple comparison test. Data presented in Figure 1e was analyzed using paired two-tailed t-test. Data presented in Figure 1f was analyzed employing two-way ANOVA followed by Bonferroni multiple comparison test. Statistical difference was assumed if P < 0.05.Supplementary Figure S2Intravital imaging of amelanotic 3C and parental B16-F10 clones. Intravital imaging of amel-B16-F10-3C tumors in mT/mG mice (top), highlighting the presence of mTom+ cells and tumor vasculature. Intravital imaging of parental tumors (bottom) highlights the inability to obtain meaningful data owing to intense speckling mediated by the melanin pigment. These bright speckles result in automatic switching off of ultrasensitive PMTs to avoid photodamage. Data are representative of a single imaging session; multiple sessions were avoided to prevent significant and/or permanent damage to the PMTs. Bar = 50 μm. PMT, photomultiplier tubes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S3Intravital imaging of B16-F10 melanoma tumors. Image from Figure 2d, left panel, with GFP signal enhanced to highlight the presence of mCherry+ vesicles within GFP+ cells (white arrowheads). Bar = 30 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S4Light-sheet microscopy of parental B16-F10 melanoma tumors. The images provide an alternate view of mel-B16-F10-parental tumor in Figure 2e, highlighting the inability to visualize either GFP+ cells or mTom+ tumor vasculature within these tumors. mTom, mTomato.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Figure S5Methodology for tumor processing. Macroscopic images showing the various stages in sample processing for light-sheet imaging of tumor vasculature. The grid represents 5 × 5 mm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Supplementary Table S1Flow Cytometry AntibodiesAntigenClone(s)B220RA3-6B2CD3e145-2C11CD11bM1/70CD4530-F11CD64X54-5/7.1.1Fc Block2.4G2F4/80BM8Ly6CHK1.4Ly6G1A8MHC IIM5/114.15.2Abbreviation: MHC II, major histocompatibility complex II. Open table in a new tab Abbreviation: MHC II, major histocompatibility complex II.