The intersection of IVM observation with RF hyperthermia treatment will likely be a subset of the feasible applications of each. Investigators should be advised that testing may be necessary to determine whether in situ imaging is possible under the desired treatment modality and setup limitations. Figure 1 shows a schematic of the overall RF-IVM procedure with data analysis methods.
All experiments were performed with the approval of the Institutional Animal Care and Use Committee of the Baylor College of Medicine and established protocols followed.
Experimental design and controls
Due to heterogeneity within the tumor microenvironment both within different regions of the same tumor (intra-tumoral) and across different tumors within one or more animals (inter-tumoral), it is recommended to maintain the same microenvironment structures (e.g., microvasculature, tissue, and cells) within the microscope field of view for the duration of the experiment.
To achieve this, the IVM video of a specific region of the tumor microenvironment can be recorded continuously before, during, and after NP intravenous injection, followed by RF field exposure, where the video of the same region continues during and after the RF field is turned off. Limitations on the ability to maintain self-consistent imaging are addressed in the discussion section. With this methodology, changes in the biodistribution of different drugs and NPs can be self-referenced to earlier time points, including post-to-pre-RF field exposure. Furthermore, continuous video provides insight into kinetics beyond what is possible with end-point analysis alone.
To compare biodistribution and kinetics between tumor and normal tissue, in this study, we took advantage of the presence of microvasculature with normal features adjacent to that with tumor-like features within the same IVM video. Various types and stages of tumor vasculature in murine mammary carcinoma at the microenvironment level have previously been characterized morphologically and compared to normal vasculature by the visual inspection of IVM images (Boucher et al. 1996). In addition to the comparison of normal and tumor-like vasculature adjacent at the microenvironment level, the entire procedure with continuous video can be repeated in a separate region of normal tissue within the same animal and of the same organ or tissue type as that of the tumor.
Materials
Excitation and emission spectra of tracer and NPs should be well characterized and care should be taken to separate the emission spectra for each reagent. In the current study, Alexafluor-647 Bovine Serum Albumin (BSA; 10 mg/mL in PBS, Thermo Fisher Scientific) was used as a vascular flow tracer, and water soluble carboxylic acid functionalized cadmium selenide/zinc sulfide core/shell nanocrystal quantum dots (QDs; NN Labs, USA) with a diameter of 10 nm and a zeta potential of −20 mV were used as a model NP to demonstrate NP perfusion into the tumor microenvironment. QD absorption/emission spectra are presented in Additional file 1: Figure S1.
RF-IVM setup
Figure 2 shows the RF-IVM setup prior to positioning of the anesthetized mouse. Panel A shows the main components of the setup: the RF amplifier (1) level with the microscope stage and positioned just lateral to the objective lens of the confocal microscope (2). Easily connectable anesthesia tubing (3) is located behind the microscope stage. The position of the mouse stage (arrow) located directly under the objective and to the left of the RF amplifier is controlled by an XYZ servo-controller (4). The RF amplifier (heretofore referred to as the portable RF device) should be connected to a computer to control RF output power and to a power supply cooled by a chiller (not shown).
The portable RF device is positioned (shown in Fig. 2, Panel B), such that it is centered vertically with the objective lens and centered horizontally between the tip of the objective lens and the mouse stage. The cross-hair center of the RF transmission antenna (TX in Panel C) will be adjusted to the exact position of the mouse tumor during the procedure. The receiving antenna (RX in Panel C) is positioned against the mouse stage directly opposite and in line with the TX. Yellow arrows in Panel C show the grounding wire connecting the copper bed of the mouse stage to a grounding wire connected to the RX. The red arrow in Panel C points to a glass coverslip that will locate the tumor in the z-direction. Note that connecting arm to the coverslip, positioned by a manual XYZ positioner here is optional and must be non-heating and non-interacting with the RF field. Alternatively, the coverslip can be placed on the surface of the exposed mouse tumor. Be aware that all XYZ components that extend into the RF field (the volumetric space connecting the squares of the transmission and receiving antennas) must be pre-tested to characterize their RF heating profiles and potential to spark at a range of RF power wattages prior to running the experiment. Be certain to test to a power higher than that planned for use. Ungrounded metal must never be exposed to the RF field.
Water-immersion objective lens (16X used here) should be selected for magnification beyond 10X. Position RF transmission and receiving heads (TX and RX RF antennas) on either side of the microscope, so that centers line up. Precision adjustments are made during the procedure to select the field of view.
Animal characterization and preparation
Female Balb/c mice are housed in the standard temperature and lighting conditions with free access to food and water. Acclimated mice were injected with the 4T1 cells (105 cells/50 μL) into the left inguinal mammary fad pad as previously described by Pulaski and Ostrand-Rosenberg (2001). Because of the mammary gland development, care was taken to develop the tumor (3–5 mm average diameter) in 9–12 week old mice.
Animals were anesthetized by isoflurane inhalation in oxygen (the anesthesia level was maintaining by adjustment of the isoflurane concentration, 0.8–2.2 %). Each mouse was kept on a warming pad and/or under a heating lamp to maintain body temperature during and after imaging. Mouse body temperature was monitored by fiber optic probe.
Procedure
Preparation of mice for tail vein injection and skin flap non-survival surgery
The first step requires mouse anesthetization in a chamber with 2 % isofluorane. Next, the animal should be transferred to the customized heated stage (~40 °C) fitted with a nosecone connected to the anesthesia line and the mouse’s nose secured to the line with tape. The stage is customized to fit under a microscope objective lens and between the TX and RX antennas of a custom-built portable RF device. In additional, the stage must be covered with a sheet of copper tape in position, where the mouse will lie. When working with animals with fur, e.g., Balb/c mice, be certain to remove all hair from the animal’s abdomen. After mouse placement, the mouse tail vein should be cannulated (Haney et al. 2009). This is a two-phase procedure; the first stage requires the insertion of the standard 24G i.v. catheter before being flushed with 0.2–0.3 mL of 1 % Heparin solution in saline. Afterward, stretched PE 10 Intramedic Polyethylene Tubing (Becton–Dickinson) should be inserted into the cannula. Finally, the double catheter is secured by tape and glue (VetBond®). Note that heparin flush must be repeated periodically and immediately after other i.v. injections to prevent clotting which may occlude the vessel.
The preparation for skin-flap surgery requires bringing the mouse to a deep plane of anesthesia (surgery plane). This can be achieved by maintaining the mouse at 2 % isofluorane for a sufficient amount of time (time for tail cannulation should suffice) and ensuring that there is no pain response. Next, the extremities of the mouse are fixed to the heated stage by applying copper tape to all four limbs and part of the tail close to the field avoiding the cannulated region of the tail. If desired, certain limbs can be taped together with copper tape (including at the nosecone) as appropriate without hindering the setup. Care should be taken to express the mouse bladder to avoid additional fluid in the RF field (bladder cannulation should be considered for long-term imaging protocols). At this point, the performance of skin-flap surgery may be carried out. This step requires: (1) creation of a midline incision; (2) gentle but firm usage of a cotton swab soaked with PBS to separate the fascia from the dermis; (3) suturing of skin flap in two or three locations (stretch sutures across the platform and over a small non-metallic, non-heating pad to support the tumor in the skin flap); (4) mounting the sutures to the side of the XYZ-controlled platform using surgical tape.
Setup of mouse in IVM system with retro-fitting of portable RF device
After mouse preparation, transfer the heated stage affixed with the mouse to the position under the objective lens of the confocal microscope, securing it to the holder connected to the XYZ-servo-controlled platform. Move the TX RF antenna within 2–3 mm of the side of the confocal microscope objective lens. Next, place a coverslip over the tumor, thoroughly wetting both the sides with PBS and bringing part of the tumor into focus. Next, switch to computer control of the microscope and select laser percent power and detection gain settings for excitation and emission.
Figure 3 shows the anesthetized mouse with open skin flap on the XYZ-servo-controlled platform in the RF-IVM setup. Copper tape is used both to ground the mouse to the platform and secure its nose to the isofluorane, Panel A. As shown in Panel B, the water-immersion lens is positioned over the coverslip, and coverslip over the tumor such that PBS creates a continuous optical path with these items. In Panel C, RX and TX antennas are brought as close to the platform as possible to maximize signal transmission.
Imaging procedure with in situ RF field exposure for tumor
After localization of fluorescence signal of tracer in tumor vessels, the position of infrared (IR) camera (FLIR SC 6000, FLIR Systems, Inc., Boston, MA) should be optimized, so tumor and normal tissue have temperature readings. Care should be taken to obtain IR photos every 30 s or run continuous IR video during the whole experimental time (prior, during, and post-RF). Afterward, the continuous IVM video can be started. The tracer-loaded syringe is attached to the cannula, and fluorescently-labeled tracer is slowly injected at ~5 μl/s. Next, the i.v. line is flushed with Heparin solution to remove reagents from the tubing. The NP solution loaded syringe is connected to the cannula, and injection is performed. Flushing with Heparin solution is then repeated. After the initiation of RF at 10 W, gradually reach 50–75 W over 5 min. When RF irradiation is complete, continue running video for 10–30 min or longer.
Image analysis
Movies were collected from the IVM system and were input into ImageJ software 1.49U (National Institutes of Health, USA). For intensity quantification, regions of interest (ROI) were selected from each image, and the mean gray value (MGV) was quantified at each time point. Surface intensity plots were also created from the same-segmented region to show the perfusion over time. For percent area fraction analysis, images were converted to binary images and subsequently inverted. The ratio of white pixels and black pixels were used to quantify the areas around the vasculature that were occupied by NP over time. In either intensity quantification or percent area fraction analysis, no thresholding was performed prior to image binarization, and background intensities were not subtracted.
The quantification of cell-sized objects in the blood stream involved tracking the X and Y co-ordinates over time of the object’s center of mass, as it interacts with the tunica intima. These objects were then used to create a vector that gave the total distance travelled by the object over the selected time frame. The speed of the object was then calculated from this information. During all image analysis techniques, frames that were out of focus due to mouse respiration or tissue expansion due to RF field hyperthermia were bypassed, and the nearest ‘in focus’ frame at the time point being considered was selected in its place.