Reagents
Chemicals were acquired from standard commercial suppliers (SigmaAldrich, Merck).
All experiments involving animals were reviewed and approved by the Regierung von Unterfranken, Würzburg (Germany). The experiments were performed in accordance with relevant guidelines and regulations.
Cell culture
LLC and 4T1 cells were obtained from ATCC. AT3 cells were obtained from SigmaAldrich. All cell lines were maintained in DMEM with 10% FBS and penicillin/streptomycin. Cell lines were tested at least every three months for mycoplasma contamination.
Clearing Media and procedures
Animals were injected 20 min prior to sacrifice with 10 µg Alexa-647 labeled CD105 antibody and killed by CO2 asphyxiation [42].
Fixation
Euthanized mice were perfused through the heart with 10 ml PBS, followed by 10 mL of 4% (w/v) PFA in PBS. Organs and tumors were removed and submersion-fixed for 24h at 4°C in 4% (w/v) PFA in PBS. Excess PFA was removed with two changes of PBS for 24h at 4°C.
Decolorization:
CUBIC1 reagent [25]: 25 % urea, 25 % (w/v) N,N,N’,N’-tetrakis(2-hydroxypropyl)ethylenediamine (Quadrol), and 15 % Triton X-100 in water.
For decolorization, organs and tumors were immersed in a 10-20-fold excess of CUBIC1 at 37 °C with shaking for 72 h. The reagent was exchanged for an equal volume of CUBIC1 and incubation was continued for another 72 h. Organs were washed in several changes of PBS for 72 h before further processing.
The procedure for decolorization with Quadrol was performed analogous, only 25 % (w/v) N,N,N’,N’-tetrakis(2-hydroxypropyl)ethylenediamine (Quadrol) in water was substituted for the CUBIC1 reagent.
Hydrogen peroxide decolorization reagent [26]: 20 % (v/v) H2O2 (30%), 20 % (v/v) DMSO, 60 % (v/v) methanol.
For decolorization, organs and tumors were first subjected to a gradual dehydration in methanol (50%, 80% and 100 % MeOH for at least 4h at r.t.) and then placed in a 10- to 20-fold excess of the H2O2-decolorization reagent at 4°C with shaking for 24h. Samples were than washed twice with methanol for 4 h at r.t. and either stored in methanol or rehydrated (80% MeOH (4h), 50% MeOH (4h) and PBS ON) and processed.
Clearing
CUBIC2 reagent [25]: 50 % (w/v) sucrose, 25 % (w/v) urea, 10 % (w/v) 2,2’,2’’-nitrilotriethanol, and 0.1% Triton X-100 in water. Sucrose and urea were dissolved in water by stearing at 37°C. 2,2’,2’’-nitrilotriethanol and Triton X-100 were added. The reagent was stored at 37 °C.
For clearing the organs were first immersed in 20% (w/v) sucrose in PBS for 24 h at 4 °C, and then placed in a 10-20-fold excess of CUBIC2 at 37 °C with shaking for 72 h. The reagent was exchanged for an equal volume of CUBIC2 and incubation was continued for at least another 72 h or until the organs appeared transparent.
iDISCO [26]: For clearing the organs were first dehydrated in THF (50% THF in water (4 h, r.t.), 80% THF in water (4 h, r.t.), 100% THF (ON, r.t.)) and then placed in excess of dichloromethane (DCM) for at least 24 h or until the tissue sank. DCM was then replaced by dibenzylether (DBE) and incubated at r.t. for 24 h after which DBE was exchanged. Tissue sections were kept in DBE until imaging.
Ethyl cinnamate [27]: For clearing the organs were first dehydrated in EtOH (50% EtOH in 10 mM Tris HCl pH 9.0 (4 h, 4 °C), 70% EtOH in 10 mM Tris HCl pH 9.0 (4 h, 4 °C), 90% EtOH in 10 mM Tris HCl pH 9.0 (4 h, 4 °C), 96% EtOH (4 h, 4 °C), 96% EtOH (4 h, 4 °C), 100% EtOH (100 h, 4 °C), 100% EtOH (4 h, 4 °C),) and then placed in excess of ethyl cinnamate (EtCi) for at least 24 h at r.t. after which EtCi was exchanged. Tissue sections were kept in EtCi until imaging.
Tumor models and treatment
Tumor engraftment: LLC (1x106 cells in matrigel/PBS 1:1) tumors were generated by subcutaneous injection in the dorsal region of female C57Bl/6J mice. 4T1 (1x105 cells in PBS) breast adenocarcinomas were generated by injection of cells into the inguinal mammary fat pad of female Balb/c mice. EMT6 (1x106 cells in PBS) breast adenocarcinomas were generated by injection of cells into the inguinal mammary fat pad of female Balb/c mice. AT3 (1x106 cells in PBS) breast adenocarcinomas were generated by injection of cells into the inguinal mammary fat pad of female C57Bl/6J mice.
All animals in the individual experiments were of the same age and sex. For each experiment tumor bearing mice were randomly assigned to the different treatment groups just prior to the start of treatment.
Miles Assay for vascular permeability
Anesthetized mice were injected retroorbital with 50 µL of an 2% (w/v) solution of Evans Blue in 0.9 sterile saline. Animals were sacrificed 30 min later, perfused with PBS through the left ventricle, before organs were removed and stored at -80°C until later processing. At least three samples from different parts of individual tumor were harvested to account for the strong heterogeneity in this tissue. Extravasated Evans Blue was extracted by adding 9 parts (w/v) of formamide to the tissue samples and incubation at 60°C for 24 h. Absorption was measured at 620 nm and 740 nM. After correcting values for co-extracted hemoglobin (A620nm (corrected) = A620nm - (1.426 x A740nm + 0.030). Organ concentrations were calculated by comparison with a standard curve [43].
IHC and IF staining of tumor sections
H&E, IHC and IF staining was performed using standard techniques on formalin fixed paraffin embedded sections. Tissues for quantitative evaluation were processed in parallel. For quantification whole tissue sections were imaged on a Keyence BD 6000 microscope with an automated stage using a Nikon 10x objective. The individual images were stitched using the Keyence Analyzer software, to obtain a virtual slide. The whole virtual slide was used for quantification using the ImageJ software package (rsbweb.nih.gov/ij/).
Immunofluorescence images were acquired on a Nikon A1 laser-scanning confocal microscope using a Nikon 20x objective. Image processing and quantification was performed using the ImageJ software package (rsbweb.nih.gov/ij/).
Antibodies used for IHC, IF or WB: Cleaved Caspase-3 (Cell Signaling Technology Cat# 9661, RRID:AB_2341188), Carbonic Anhydrase IX (Santa Cruz Biotechnology Cat# sc-25599, RRID:AB_2066539)), CD31 (Santa Cruz Biotechnology Cat# sc-28188, RRID:AB_2267979), CD34 (Abcam Cat# ab8158, RRID:AB_306316), Ki67 (Abcam Cat# ab16667 RRID:AB_302459).
LSFM Image Acquisition
LSFM Setup
A custom-built LSFM setup tailor-made for organ imaging was used: a customized fiber-coupled laser combiner (BFI OPTiLAS GmbH, Groebenzell, Germany) provided the required excitation lines of 491, 532, 642, and 730 nm. For laser beam collimation two objectives (RMS10X-PF Thorlabs, Bergkirchen, Germany) for VIS and 730 nm, were used. A DCLP 660 dichroic beam splitter (AHF Analysentechnik, Tübingen, Germany) combined the two beam paths. A following telescope (BEX 1x-4x 017052-202-26, Jenoptik, Jena, Germany) served to adjust beam diameter. Alternating dual-side illumination was realized by a two-axis galvanometer scanner (6210H; Cambridge Technologies, Bedford, MA, USA) in combination with a theta lens (VISIR f. TCS-MR II; Leica, Mannheim, Germany) which finally created a virtual light sheet that was additionally pivot scanned by a single-axis resonant scanner system (EOP-SC, 20-20x20-30-120; Laser2000, Wessling, Germany) to minimize shadowing artifacts. The light sheet was projected onto the sample via a 200 mm tube lens (TTL200,Thorlabs, Bergkirchen, Germany) and a lens objective (Nikon CFI60 TU Plan Epi 5×/0.15, Edmund Optics, York, United Kingdom). The objective on the detection side (HCX APO L 20×/0.95 IMM; Leica, Mannheim, Germany) placed on a piezo positioning system (P-611.1 and E-665, PI, Karlsruhe, Germany) for focus correction collected the fluorescence perpendicularly to the light sheet, and, in combination with an infinity-corrected 1.3x tube lens (model 098.9001.000; Leica, Mannheim, Germany), projected the image into a scientific complementary metal oxide semiconductor (sCMOS) camera (Neo 5.5; Andor, Belfast, United Kingdom) (2,560 by 2,160 pixels, 16.6-mm-by-14.0-mm sensor size, 6.5-μm pixel size). The fluorescence was spectrally filtered by typical emission filters (AHF Analysentechnik, Tübingen, Germany) according to the use of the following fluorophores: BrightLine HC 525/50 (Alexa Fluor 488 or autofluorescence), BrightLine HC 580/60 (Alexa Fluor 532), HQ697/58 (Alexa Fluor 647), BrightLine HC 785/62 (Alexa Fluor 750). Filters were part of a motorized filter wheel (MAC 6000 Filter Wheel Emission TV 60 C 1.0× with MAC 6000 controller; Zeiss, Göttingen, Germany) placed in the collimated light path between detection objective and tube lens.
Image Acquisition
Cleared organ and samples were placed in the light path within the EtCi-filled objective chamber using a clamp-holder. Stacks were acquired in increments of 1 μm by imaging each plane in two color channels (BrightLine HC 525/50 (autofluorescence), and HQ697/58 (Alexa Fluor 647)) sequentially. Typically stacks of 1,024 x 1,024 x 400-700 voxels with a voxel size of 0.5 x 0.5 x 1 µm3 were acquired. Hardware components for image acquisition (laser, camera, filter wheel, stage, focus correction) were controlled by IQ 2.9 software (Andor, Belfast United Kingdom). Images were saved as tagged image files (TIF), processed and analyzed as described below.
Image Analysis
Image pre-processing
Acquired raw tiff-image stacks were loaded into the Fiji-distribution of ImageJ (https://fiji.sc/). As stack parameters, were routinely not correctly read-in from image meta data, they were manually corrected. The stack was processed using Fiji’s 3D-median filter by replacing each pixel value with the median of two neighboring pixel values. The processed stack was saved as singled tif file and converted using the Imaris file converter in an Imaris-9.8-file. Converted files were read into Imaris (Version 9.8.0, Oxford Instruments, Abington, UK).
3D-Reconstruction
The vascular network was reconstructed in the software package Imaris. A surface was created with standardized settings (Suppl. Table 2), although threshold levels for the used fluorescent signal had to be individually adjusted to compensate for varying staining and background intensities in the different organs. The generated surface was cleaned up by filtering fragments smaller than 2000 vx. Tabular results of the calculated surface parameters were exported in form of spreadsheets.
Tracing
The pre-processed fluorescence signal proofed in some organs too clouded with background for successful tracing of the vasculature. Therefore, the fluorescence signal was cleaned by masking using the previously reconstructed surface: the fluorescence signal outside the vessel surface was set to zero to remove background and small artifacts. Using the filament creation tool, the thereby generated new masked channel was utilized to trace the blood vessel signal. Standardized settings were used for generating traces (Suppl. Table 3). The imaging procedure by restricting the analyzable volume to a small FOV of high resolution, resulted necessarily in vessels cut-off at the rim of the FOV. These partial vessel segments would falsify the data and were therefore removed before statistical analysis. Tabular results of the calculated vessel parameters were exported in form of spreadsheets.
(Side note: the “filament tool” was developed for analysis of dendritic trees. Accordingly, the results are described with unusual terms: “Dendrites” for individual segments between two bifurcations/branching points; “Filaments” for connected networks of individual segments (“Dendrites”)).
Distance transformation
The Matlab-based (MathWorks, Natick, MA) distance transformation routine in Imaris was used to first visualize distance of individual voxels to the surface of nearest vessel structure. This generated a new 8-bit channel in the 3D-stack in which distance to the vessel surface was decoded by a grey value in the range of 0-255. The channel was exported to Fiji. Using the histogram of the 3D-stack allowed export of the visual data into numerical values for further analysis. For visualization the grey values of the channel were spectral color-coded.
Statistical Analysis
Statistical analysis and PCA was done using the R environment (https://www.r-project.org/). Differences between two groups were analyzed using an unpaired, two-tailed Student’s T-test. In parallel the samples were tested for significant variation of variance, and if necessary a Welch correction was included in the statistical analysis. All statistical tests were performed between sets of individual biological replicates.