Figure 1.
Experimental mouse metastatic breast cancer model.
(A) Schematic of lentiviral construct comprising a fusion reporter gene (Luciferase-2 and enhanced GFP) under the control of the ubiquitin promoter, used to establish the imageable metastatic mammary carcinoma cell line 4T1-GL. (B) FACs analysis of GFP fluorescence, comparing the stable cell line 4T1-GL at passage 2 and passage 12 (resp. P2 and P12) to wild-type 4T1 cells (4T1-WT). (C) Metastatic tumor growth in the lungs as monitored non-invasively by Bioluminescence (BLI) imaging, following a systemic injection of 1×106 4T1-GL cells via the tail vein (n = 7). (D) Biodistribution of metastatic cells, 12 days after systemic injection (n = 7) in the following organs: Lungs, Liver, Heart, Kidneys Spleen, Bone marrow, and corresponding quantification of BLI signal per organ (n = 7). (E) CTCs in 100 µL blood samples of mice (n = 7) at various times from day 0 (immediately after injection) to 12 days after injection and corresponding signal quantification. Positive BLI signals correspond to <20 CTCs/100 uL of blood.
Figure 2.
Miniature mountable intravital microscopy system design for in vivo CTCs imaging in awake animals.
(A) Computer-assisted design of an integrated microscope, shown in cross-section. Blue and green arrows mark illumination and emission pathways, respectively. (B) Image of an assembled integrated microscope. Insets, filter cube holding dichroic mirror and excitation and emission filters (bottom left), PCB holding the CMOS camera chip (top right) and PCB holding the LED illumination source (bottom right). The wire bundles for LED and CMOS boards are visible. Scale bars, 5 mm (A,B). (C) Schematic of electronics for real-time image acquisition and control. The LED and CMOS sensor each have their own PCB. These boards are connected to a custom, external PCB via nine fine wires (two to the LED and seven to the camera) encased in a single polyvinyl chloride sheath. The external PCB interfaces with a computer via a USB (universal serial bus) adaptor board. PD, flash programming device; OSC, quartz crystal oscillator; I2C, two-wire interintegrated circuit serial communication interface; and FPGA, field-programmable gate array. (D) Schematic of the miniature mountable intravital microscopy system and corresponding images. The miniature microscope is attached to a dorsal skinfold window chamber via a lightweight holder. (E) mIVM imaging of cells in suspension in a glass-bottom 96-well plate. 4T1-GL cells; 4T1-GL cells that have been transiently transfected with the Luc2-eGFP DNA to enhance their fluorescence (4T1-GL-tt); 4T1-GL cells that have been labeled with the bright green fluorescent CFSE dye (4T1-GL-CFSE). (F) Quantification of the cell to background green fluorescence for the three cell types described in (E) for n = 3 field of view, average ±standard deviation. Fig. 2 (A), (B), (C) reprinted by permission from Macmillan Publishers Ltd: Nature Methods (Ghosh, K. K. et al. Miniaturized integration of a fluorescence microscope. Nat Meth 8, 871–878 (2011)), copyright 2011.
Figure 3.
In vivo CTCs imaging using miniature mountable intravital microscopy (mIVM) method.
(A, B, C) In vivo imaging of CTCs using the mIVM after systemic injection of FITC-dextran for vessel labeling followed by injection of 1×106 4T1-GL labeled with CFSE. (A) Raw image from the miniature microscope. (B) Image processed by our MATLAB algorithm for detection of CTCs and vessel edges. (C) Computing of CTCs trajectories within the blood vessel. (D) Quantification of the speeds of CTCs over time as imaged in Movie S1, and (E) corresponding average speeds per CTC, plotted as box and whiskers where the box extends from the 25th to 75th percentiles and the whiskers extend from the minimum to the maximum speed values measured. (F) For the slowest CTC – CTC2 on (D, E) – details of the speed of the cell over time (red curve) and the corresponding location of the cell relative to the vessel edge (blue curve).
Figure 4.
Imaging of circulating tumor cells in an awake, freely behaving animal using the mIVM.
(A) Photograph of the animal preparation: Following tail-vein injection of FITC-dextran for vessel labeling and subsequent injection of 1×106 4T1-GL labeled with CFSE, the animal was taken off the anesthesia and allowed to freely behave in its cage while CTCs were imaged in real-time. (B) mIVM image of the field of view containing two blood vessel, Vessel 1 of 300 µm diameter and Vessel 2 of 150 µm diameter. (C, D) Quantification of number of CTCs events during 2h-long awake imaging, using a MATLAB image processing algorithm, in Vessel 1 (C) and Vessel 2 (D). (E, F) Computing of CTC dynamics: average CTC frequency (Hz) as computed over non-overlapping 1 min windows for Vessel 1 (E) and Vessel 2 (F) and (G) Second-order smoothing (10 neighbor algorithm) of the data presented in (E, F).