We thank Dr. Carla Cal&#231;ada (Postdoctoral Fellow, Princess Margaret Cancer Centre) and Dr. Timothy Samuel (Ph.D. Student, Princess Margaret Cancer Centre) for help with tumor cell culturing and inoculation protocol development. Dr. Kathleen Ma, Dr. Anna Pietraszek, and Dr. Alyssa Goldstein (Animal Research Centre, Princess Margaret Cancer Centre) helped with surgery protocol development. Jacob Broske (Medical Engineering Technologist, Princess Margaret Cancer Centre) and Wayne Keller (Hardware Client Executive, Javelin Technologies &#8211; A TriMech Group Company) 3D printed the window chambers. James Jonkman (Advanced Optical Microscopy Facility, University Health Network) provided valuable guidance for brightfield and fluorescence microscopy image acquisition.

Guided Tumor Inoculation and Multimodal Imaging in Mice

<ol>
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The authors have no conflicts of interest to disclose.

In this work, we have developed a workflow to perform both intravital microscopy and clinically applicable imaging (CT, MRI, and PET) in the same animal. This was done with the goal of translating preclinical microscopy findings to the clinic by direct correlation of intravital microscopy with clinical imaging modalities such as MRI. Although conventional DSFC designs are made of metal2,3, we have adapted the DSFC to be MR-compatible by using 3D-printed window chambers made of a biocompatible plastic resin. The registration of the modalities was accomplished by the use of fiducial markers for the clinical imaging modalities and by obtaining a widefield microscopy image to match the fiducial marker locations on the DSFC with the vascular landmarks that can be seen on the widefield microcopy image as well as the high-resolution intravital microscopy microvascular images. A MATLAB code to co-register the modalities was developed and is included in Supplementary File 2.
DSFCs are not new in preclinical research2,3,27,29,30,31,32 with some studies developing MR-compatible DSFCs out of biocompatible plastics for intravital microscopy and clinically applicable imaging27,29,30,31,32. However, these studies perform whole-tumor level correlations with MRI and intravital microscopy findings. The novelty of this work is our ability to directly co-register intravital microcopy images to clinically applicable imaging modalities using a fiducial marker attachment to the DSFC enabling direct spatial correlation analysis. Gaustad et al. have also performed spatial correlation analysis between MRI and intravital microscopy; however, specific detailed steps on the co-registration process between modalities are not included29. This work provides a protocol for spatially correlating multiple modalities with each other using the fiducial marker ring attachment and includes a MATLAB pipeline.
There are several details of the DSFC design and procedure that offer improved imaging and tumor growth characteristics compared to other similar studies. First, most window chamber methods involve inoculation of the tumor after&#160;window chamber surgery. This is typically done since it is easier to center the tumor in the window chamber FOV; however, previous work has shown that this is suboptimal, leading to an &#39;unrealistic&#39; mono-layer tumor growth with spontaneous regression in many instances33. Instead, we allow the tumor to grow unrestricted in the skin for several weeks before DSFC implantation, which allows for a more realistic 3D tumor with improved growth characteristics33.
This DSFC design is also extremely lightweight at 0.83 g with a large imaging window FOV (1.2 cm diameter glass coverslip). A unique characteristic of our DSFC design is that it does not include a snap ring to secure the glass coverslip to the front frame. Instead, the glass coverslip is permanently affixed using UV-cured glue allowing the bottom portion of the front frame to be completely flat. Furthermore, the front frame is secured underneath the skin of the mouse. This was done to minimize the chance of any air gap between the tumor-glass interface. It has been noted in previous designs that if there is a gap at the tumor-glass interface there is a high probability of fluid buildup in this region. This significantly impacts results by attenuating the intravital microscopy signal thus reducing image quality. Additionally, it makes it more difficult to target the MRI slices to the tumor since this fluid buildup will appear bright on the T2w localization scans. This makes it challenging to identify which slices are imaging the tissue and which slices are imaging the fluid buildup in the DSFC. One drawback to this design is that if the glass coverslip breaks, it cannot be easily replaced. We have also designed a front frame that is made entirely out of an optically clear plastic thus eliminating the glass breakage issue34.
Throughout this procedure, several crucial steps are more technically challenging than others. One step that may cause difficulties is during the DSFC surgery when suturing the front frame to the skin (protocol steps 3.18-3.24). It is important to keep some extra loose skin along the top of the front frame of the DSFC. If this is not done, then the skin in this region will lose blood supply and become necrotic, thus ruining the preparation. Another technically challenging task is the targeting of the MRI slice package to include the most superficial part of tissue underneath the glass. If this slice is not targeted precisely to the region, then partial volume effects with the glass may occur, thus leading to erroneous MRI results. Uneven surfaces of the tissue underneath the glass of the DSFC may make this even more challenging. It is important to ensure that the skin is taut inside the DSFC to keep the tissue surface as flat and uniform as possible to reduce this source of error.
Here we have demonstrated the use of this DSFC workflow using svOCT, which has a large imaging FOV of 6 x 6 mm2. This large FOV is not common in other types of intravital microscopy with many imaging methods having FOVs of 1 mm2 or smaller13. This may cause some challenges when co-registering the widefield microscopy image to the high-resolution intravital microscopy images since microvascular landmarks may be difficult to identify. To overcome this, another widefield microscopy image may be required with a smaller FOV (~6 x 6 mm2) to act as an additional intermediate co-registration step between the imaging modalities to further refine the region of interest to the one imaged by intravital microscopy.
In this work, we have focused on imaging the tumor microvasculature, but other tumor microenvironmental components may be imaged using this model as well. For example, intravital microscopy has been shown to successfully image the tumor stroma35, and diffusion-weighted MRI methods such as diffusion tensor imaging or diffusion kurtosis imaging may have the ability to provide information on the structure of the tumor stroma36,37. This workflow may provide exciting opportunities to translate stromal tumor information into the clinic by directly co-registering high-resolution intravital microscopy images of the tumor stroma with MRI-based measurements of the stroma. However, it should be noted that because this method uses vascular landmarks for co-registration, those should also be present in intravital microscopy stromal images.
The presented workflow may prove to be an important step in bridging the gap between preclinical intravital microscopy and clinically applicable imaging modalities. This model can be used to validate clinical imaging methods with ground truth intravital microscopy images, develop new clinically applicable imaging methods through comparison with intravital microscopy, and longitudinally monitor the response of treatments using multiple imaging modalities.

Tumor microvasculature is an important component of the tumor microenvironment that can be a target for therapy and a determinant of treatment response. In the preclinical setting, the microvasculature is typically studied using intravital microscopy in orthotopic or heterotopic window chamber animal models1,2. This has several advantages over histological studies since the imaging is done in live tissues and the tumor can be monitored longitudinally over several weeks or even months2,3. These studies can leverage the high-resolution imaging capabilities of intravital microscopy to study the delivery of therapeutics to the tumor4,5, the causes of treatment resistance6, and the response of the micro vessels to therapies such as antiangiogenic treatment7,8 and radiotherapy2,9.
Intravital microscopy clearly plays an important role in preclinical cancer research; however, how can tumor microenvironmental features be measured in the clinic? Microvascular information would be useful in the clinic for measuring blood supply and tumor cell hypoxia, which is important for determining treatment resistance in radiotherapy10, as well as the ability of the microvasculature to deliver chemotherapeutic agents to the surrounding tumor cells11. For example, in radiotherapy, spatial information on the structure and function of the tumor microvasculature may help personalize a patient&#39;s treatment plan by adjusting the fractionation schedule or by preferentially boosting the dose to avascular and likely hypoxic regions12.
Intravital microscopy can measure these important microvascular features since it has a very high resolution (&#956;m scale); however, its depth penetration into tissue is limited to several hundred microns or a few millimeters, at most making clinical implementation challenging. Indeed, there are some novel applications of intravital microscopy in the clinic13; however, these are still limited to examinations of near-surface level tissue such as the skin14 or mucosal/endothelial linings of various body cavities via flexible catheters/endoscopes15,16.
More commonly, the microvasculature is studied using imaging modalities such as CT17 or MRI18. These clinical imaging modalities can image to any depth within the body, but they have a much lower spatial resolution (mm scale). Thus, there is a need to bridge this resolution gap between preclinical intravital microscopy and clinical imaging modalities to bring high-resolution and detailed microvascular information into the clinic19. Several functional imaging methods have been developed to improve the microvascular imaging capabilities of clinical imaging modalities such as dynamic contrast-enhanced (DCE) MRI and CT20, and Intravoxel incoherent motion (IVIM) MRI21. However, these are model-based methods that provide indirect measurements of the microvasculature and thus, must be validated with appropriate &#34;ground truth&#34; measurements of the microvasculature19,22.
We have developed a dorsal skinfold window chamber (DSFC) tumor mouse model to bridge this gap between preclinical intravital microscopy and clinically applicable imaging modalities such as CT and MRI. The DSFC provides direct access to the tumor for high-resolution, intravital microscopy imaging through a glass window but also clinically applicable imaging such as MRI as it is made of MR-compatible materials (plastic and glass). Furthermore, an included MATLAB code performs multimodality 3D co-registration for direct spatial correlations between preclinical intravital microscopy and clinically applicable imaging modalities. Here we will describe the design and surgery to install the DSFC as well as the procedure to co-register intravital microscopy and clinically applicable imaging modalities.

<table><tbody><tr><td>&lt;strong&gt;Cell Culture Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BxPC-3 Human Pancreatic Cancer Cells</td><td>ATCC (American Type Culture Collection)</td><td>CRL-1687</td><td></td></tr><tr><td>Corning Matrigel Basement Membrane Matrix, LDEV-free, 10 mL</td><td>Corning</td><td>354234</td><td></td></tr><tr><td>Corning Stripettor Ultra Pipet Controller</td><td>Corning</td><td>07-202-350</td><td></td></tr><tr><td>Dulbecco Phospphate buffered saline without Calcium, Magnesium, or phenol red, 500 mL</td><td>Gibco</td><td>14190144</td><td></td></tr><tr><td>Fetal Bovine Serum (Canada), 500 mL</td><td>Sigma-Aldrich</td><td>F1051-500ML</td><td></td></tr><tr><td>Penicillin-Streptomycin 100x (liquid,stabilized, sterile-filtered, cell culture tested)</td><td>Sigma-Aldrich</td><td>P4333-100ML</td><td></td></tr><tr><td>RPMI Medium 1640 (1x), liquid; with L-Glutamine, 500 mL</td><td>Gibco</td><td>11875093</td><td></td></tr><tr><td>TrypLE Express Enzyme, 500 mL</td><td>Gibco</td><td>12605028</td><td></td></tr><tr><td>&lt;strong&gt;Window Chamber Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>12 mm Glass Coverslip</td><td>Harvard Apparatus&amp;nbsp;&amp;nbsp;</td><td>CS-12R No. 1.5</td><td></td></tr><tr><td>Connex 500 3D Printer</td><td>Stratasys</td><td>N/A</td><td></td></tr><tr><td>Biocompatible clear MED610 resin</td><td>Stratasys</td><td>&amp;nbsp;RGD810</td><td></td></tr><tr><td>Loctite AA 3105 UV curable glue</td><td>Loctite</td><td>LCT1214249</td><td></td></tr><tr><td>Window chamber back frame</td><td>Trimech Inc</td><td>N/A</td><td></td></tr><tr><td>Window chamber fiducial marker</td><td>Trimech Inc</td><td>N/A</td><td></td></tr><tr><td>Window Chamber front frame</td><td>Trimech Inc</td><td>N/A</td><td></td></tr><tr><td>Window chamber support clip</td><td>Trimech Inc</td><td>N/A</td><td></td></tr><tr><td>&lt;strong&gt;inoculation and Surgery Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>BD SafetyGlide Insulin Syringes with Permanently Attached Needles, 0.5 mL, 29 G x 1/2&quot;</td><td>BD</td><td>CABD305932</td><td></td></tr><tr><td>Betadine Solution</td><td>Betadine</td><td>AP-B002C2R98U</td><td></td></tr><tr><td>Cidex OPA 14 Day Solution 3.8 L</td><td>ASP</td><td>JOH20394</td><td></td></tr><tr><td>Disposable Surgical Underpads 23 inch x 24 inch</td><td>Kendall</td><td>7134</td><td></td></tr><tr><td>Eye lubricant&amp;nbsp;&amp;nbsp;</td><td>Optixcare</td><td>50-218-8442</td><td></td></tr><tr><td>Hair removal cream</td><td>Nair</td><td>&amp;lrm;061700222611</td><td></td></tr><tr><td>Halstead Hemostatic Forceps</td><td>Almedic</td><td>7742-A12-150</td><td></td></tr><tr><td>Heating pad</td><td>Sunbeam&amp;nbsp;</td><td>B086MCN59R</td><td></td></tr><tr><td>Iris Scissors</td><td>Almedic</td><td>7601-A8-690</td><td></td></tr><tr><td>Isoflurane</td><td>Sigma</td><td>792632</td><td></td></tr><tr><td>Metacam</td><td>&amp;nbsp;Boehringer Ingelheim Animal Health USA Inc</td><td>NDC 0010-6015-03</td><td></td></tr><tr><td>NOD.Cg-Rag1tm1Mom&amp;nbsp;Il2rgtm1Wjl/SzJ mouse</td><td>the Jackson laboratory</td><td>7799</td><td></td></tr><tr><td>Peanut Clipper &amp;amp; Trimmer</td><td>Wahl</td><td>8655-200</td><td></td></tr><tr><td>&amp;nbsp;SOFSILK Nonabsorbable Surgical Suture #5-0 with 3/8&quot; Taper point needle (17 mm) (Wax Coated,Braided Black Silk, Sterile)&amp;nbsp;</td><td>&amp;nbsp;Syneture&amp;nbsp;</td><td>&amp;nbsp;VS880</td><td></td></tr><tr><td>Splinter Forceps</td><td>Almedic</td><td>7725-A10-634</td><td></td></tr><tr><td>&lt;strong&gt;MR Imaging&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>3D printed window chamber immobilization device.</td><td></td><td></td><td>custom 3D printed, refer to figure 3 for details.</td></tr><tr><td>Convection heating device</td><td>3M Bair Hugger</td><td>70200791401</td><td></td></tr><tr><td>Drug injection system</td><td>Harvard Apparatus&amp;nbsp;&amp;nbsp;</td><td>PY2 70-2131</td><td>PHD 22/2200 MRI compatible Syringe Pump</td></tr><tr><td>Gadovist 1.0</td><td>Bayer</td><td>2241089</td><td></td></tr><tr><td>Respiratory monitoring system</td><td>SAII</td><td>Model 1030</td><td>MR-compatible monitoring and gating system for small animals.</td></tr><tr><td>Tail vein catheter (27 G 0.5&quot; )</td><td>Terumo Medical Corp</td><td>15253</td><td></td></tr><tr><td>&lt;strong&gt;Optical Imaging&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>3D printed imaging stage</td><td></td><td></td><td>Custom 3D printed, refer to supplementary figure 3 for details.</td></tr><tr><td>12 V 7 W Flexible Polyimide Heater Plate Thin Adhesive PI Heating Film 25 mm x 50 mm</td><td>BANRIA&amp;nbsp;</td><td>B09X16XCVS</td><td>Heating element used for mouse body temeprature regulation.</td></tr><tr><td>DC power supply</td><td>BK Precission</td><td>1761</td><td>Used to power the heating element.</td></tr><tr><td>Leica MZ FLIII</td><td>Leica Microsystems</td><td>15209</td><td></td></tr><tr><td>svOCT imaging system</td><td></td><td></td><td>In-house made imaging system. Details can be found in reference 23.</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MATLAB Software</td><td>MathWorks</td><td>R2020A</td><td></td></tr></tbody></table>

a dorsal skinfold window chamber tumor mouse model for combined intravital microscopy and magnetic resonance imaging in translational cancer research

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor microenvironment and its response to therapy. These imaging modalities have micron-scale resolution but have limited use in the clinic due to their shallow penetration depth into tissue. More clinically applicable imaging modalities such as CT, MRI, and PET have much greater penetration depth but have comparatively lower spatial resolution (mm scale). 
To translate preclinical intravital imaging findings into the clinic, new methods must be developed to bridge this micro-to-macro resolution gap. Here we describe a dorsal skinfold window chamber tumor mouse model designed to enable preclinical intravital and clinically applicable (CT and MR) imaging in the same animal, and the image analysis platform that links these two disparate visualization methods. Importantly, the described window chamber approach enables the different imaging modalities to be co-registered in 3D using fiducial markers on the window chamber for direct spatial concordance. This model can be used for validation of existing clinical imaging methods, as well as for the development of new ones through direct correlation with "ground truth" high-resolution intravital findings. 
Finally, the tumor response to various treatments-chemotherapy, radiotherapy, photodynamic therapy-can be monitored longitudinally with this methodology using preclinical and clinically applicable imaging modalities. The dorsal skinfold window chamber tumor mouse model and imaging platforms described here can thus be used in a variety of cancer research studies, for example, in translating preclinical intravital microscopy findings to more clinically applicable imaging modalities such as CT or MRI.

Speckle variance optical coherence tomography (svOCT) was performed to obtain large field-of-view (FOV) 3D microvascular images (6 x 6 mm2 lateral x 1 mm depth). To obtain these images, a previously described swept source OCT system based on a quadrature interferometer was used23. OCT images were acquired by stitching together two laterally adjacent 3 x 6 mm2 FOV scans. Each B-scan consisted of 400 A-scans and was performed 24x per location (25 ms apart) to enable accurate speckle variance processing as described in our previous work24.
To co-register the MRI dataset to the microvascular svOCT dataset several steps were performed. The first step was done in step 5.12 and is illustrated in Figure 4. This step co-registers the two datasets along the depth direction (z-axis in Figure 4). To co-register the datasets along the lateral dimensions (xy-plane in Figure 4), the fiducial marker ring along with a series of two point-based affine registrations are used. First, the MRI dataset is co-registered to the brightfield microscopy image by matching the fiducial marker locations in the ring, with the corresponding divots around the perimeter of the glass window on the front frame (green points in Figure 5B,C). Next, vascular landmarks that are identifiable in both the svOCT microvascular dataset and the brightfield microscopy image are used to co-register the brightfield image (and corresponding MRI dataset) to the svOCT dataset (blue points in Figure 5A,B). Therefore, the brightfield microscopy image acts as an intermediate-resolution image that contains features from both the MRI (divot locations for fiducial markers) and svOCT microvascular image (microvascular landmarks) to enable multimodality co-registration across vastly different imaging resolutions.
Images of the successful co-registration between all three modalities (microvascular imaging, widefield microscopy, and MRI) for a window chamber preparation mouse with no tumor (healthy bare skin) and tumor are shown in Figure 6A-C and Figure 6D-F, respectively. Once all three modalities are co-registered, correlation analysis can be performed. To take advantage of the co-registered datasets, spatial correlations should be performed either on a voxel-by-voxel basis or by using appropriate averaging techniques over selected volumes of interest. Figure 7 shows some representative results of the correlation between svOCT-derived microvascular metrics and functional MRI-derived macrovascular metrics. These plots include the data from two tumor-bearing mice and one &#39;healthy&#39; bare skin mouse. These plots were generated using a 1 mm2 lateral x 500 &#956;m depth sliding window volume of interest (VOI) technique with a 500 &#956;m lateral step size with each point representing one position of the VOI. For the healthy mouse, vascular analysis was restricted to a 3 x 3 mm2 region of interest in the center of the FOV to match the 3D spatial extent of the examined tumors. A more detailed study involving svOCT to MRI correlations using this analysis method can be found in our recent work22.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66383/66383fig06.jpg" /> 
Figure 6: Co-registered healthy and tumor imaging datasets. The top row shows the (A) color depth-encoded svOCT, (B) co-registered brightfield microscopy image with analysis region shown by the blue dotted line, and (C) co-registered DCE-MRI parameter map for a healthy bare skin mouse. The bottom row shows the (D) color depth-encoded svOCT, (E) co-registered brightfield microscopy image with tumor contour shown by the blue dotted line, and (F) co-registered DCE-MRI parameter map for a tumor-bearing mouse. The black voxels in C and F are outside the analysis region and tumor, respectively, and are, therefore, not considered. Scale bars = 1 mm. Abbreviations: svOCT = speckle variance optical coherence tomography; DCE-MRI = dynamic contrast-enhanced magnetic resonance imaging.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66383/66383fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 7A shows MRI&#39;s ktrans parameter versus the svOCT-measured vascular volume fraction (VVF). VVF is the proportion of tissue that is occupied by vessels and&#160;ktrans is the rate constant for gadolinium moving from the intravascular space to the extravascular space. ktrans is obtained by fitting of the Toft&#39;s model to the gadolinium time concentration curves on a voxel-by-voxel basis25,26. VVF measurements from svOCT show that tumors have a smaller VVF than healthy tissue, potentially indicating the presence of poorly vascularized regions inside the tumor. As ktrans is dependent on blood flow, vascular permeability, and capillary surface area25,26, a positive correlation between ktrans and VVF is expected since a larger VVF implies a larger vascular surface area and blood flow. However, more studies such as Dextran extravasation experiments are needed to further elucidate the impact of tumor vessel permeability in this relationship27.
Figure 7B shows the correlation between MRI&#39;s time to peak contrast enhancement (TTP) versus svOCT&#39;s mean distance to the nearest vessel (<img alt="Equation 2" src="/files/ftp_upload/66383/66383eq02.jpg" style="margin: auto;" />). TTP is a semi-quantitative metric that measures the time required for the gadolinium time concentration curve to reach its maximum value and this is calculated on a voxel-by-voxel basis26. The <img alt="Equation 2" src="/files/ftp_upload/66383/66383eq02.jpg" style="margin: auto;" /> is a svOCT microvascular metric that measures the average distance to the nearest vessel within the analyzed volume22. In general, there is a smaller <img alt="Equation 2" src="/files/ftp_upload/66383/66383eq02.jpg" style="margin: auto;" /> in healthy tissue compared to tumor tissue, indicating that the healthy microvasculature is more densely packed leading to more uniformly oxygenated tissue, and the tumor tissue is less well vascularized28. The higher <img alt="Equation 2" src="/files/ftp_upload/66383/66383eq02.jpg" style="margin: auto;" /> values in tumor tissue may indicate the presence of avascular tissue regions. Figure 7B shows a positive correlation between TTP and <img alt="Equation 2" src="/files/ftp_upload/66383/66383eq02.jpg" style="margin: auto;" />. This demonstrates that healthy tissue is well vascularized with gadolinium washing into and out of the tissue efficiently and quickly. However, tumor microvasculature is more malformed with larger intervascular distances, which increases the time for gadolinium to reach the tissue thus resulting in an increase in TTP.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/66383/66383fig07.jpg" /> 
Figure 7: MRI to svOCT macro to micro-vascular spatial correlations. These plots contain the findings from two tumor-bearing mice and one healthy bare-skin mouse using a x 1 x 1 mm2 lateral &#215; 500 &#956;m depth, sliding window VOI with a 500 &#956;m step size, with each point representing the average values of the given metric for one position of the sliding window VOI. Abbreviations: svOCT = speckle variance optical coherence tomography; DCE-MRI = dynamic contrast-enhanced magnetic resonance imaging; VOI = volume of interest. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66383/66383fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: 3D STL files of window chamber components. A zip file containing the 4 components of the DSFC chamber (front frame, back frame, support clip, and fiducial marker ring), which was used to 3D print the parts. Abbreviation: DSFC = dorsal skinfold window chamber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66383/Supplementary file 1.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 2: MATLAB script for multimodal image co-registration. This MATLAB script (Multimodal_Image_Register.m) takes in the brightfield microscopy, microvascular image data, and structural and functional (DCE and/or IVIM parameter maps) MR images and outputs all datasets co-registered to the microvascular dataset. The MATLAB script is written to support the following file formats: MRI - .dcm, svOCT - .mat, and microscopy - .jpg, or .TIF. The MATLAB code is written to accommodate the file sizes/resolutions that are described in the protocol section: MRI - 32 x 32 mm field-of-view with 64 x 64 matrix for 0.5 x 0.5 mm in-plane resolution; 10 contiguous imaging slices, microscopy - 2D image with &#62;1.5 cm2 field of view (any resolution acceptable), and svOCT - 6 x 6 mm field of view 7.5 &#181;m resolution. The MATLAB code should be modified if different file formats, image resolution, or imaging fields of view are used. Abbreviations: DCE = dynamic contrast-enhanced; IVIM = intravoxel incoherent motion; MRI = magnetic resonance imaging; svOCT = speckle variance optical coherence tomography. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66383/Multimodal_Image_Register.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure S1: Temporary sutures to affix window chamber to the skin of the mouse. The suture starts at (1) and goes through the front side skin of the mouse and through the front frame of the window chamber. (2) The suture then goes through the back skin of the mouse and out the back frame of the window chamber. The suture is then threaded back through the back frame, (3) into the backside skin, through the front frame, and (4) back out the front skin. (5) The suture is then brought back through both DSFC frames and skin layers. (6) Finally, the suture is brought back through the top hole of the window chamber and all layers of the skin. The DSFC + skin assembly is then brought together by tightening and knotting the sutures at (1). Abbreviation: DSFC = dorsal skinfold window chamber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66383/Supplementary Figure 1.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure S2: Optimal placement of permanent sutures. The permanent sutures should be threaded through the front skin/frame out of the back skin/frame, back through the entire assembly, and knotted in the front. Each number represents one individual suture. The sutures should be placed in the order of the numbers to evenly apply pressure to the entire assembly as it is secured. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66383/Supplementary Figure 2.zip" target="_blank">Please click here to download this File.</a>
Supplementary Figure S3: 3D printed stage for intravital microscopy imaging. (A)&#160;A 3D printed stage with isoflurane gas anesthesia attachment is used for intravital microscopy imaging. (B) The window chamber is secured to the stage by three bolts to immobilize it and eliminate any breathing motion. (C) Two heating elements connected in series to a DC power source are glued to the underside of the stage to maintain the mouse&#39;s body temperature while anesthetized. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66383/Supplementary Figure 3.zip" target="_blank">Please click here to download this File.</a>

Read this Scientific Article Protocol about Author Spotlight: Integrating High-Resolution Intravital Imaging and MRI to Enhance Stereotactic Body Radiation Therapy Planning at JoVE.com

Author Spotlight: Integrating High-Resolution Intravital Imaging and MRI to Enhance Stereotactic Body Radiation Therapy Planning

Translation of Intravital microscopy findings is challenged by its shallow depth penetration into tissue. Here we describe a dorsal window chamber mouse model that enables co-registration of intravital microscopy and clinically applicable imaging modalities (e.g., CT, MRI) for direct spatial correlation, potentially streamlining clinical translation of intravital microscopy findings.

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the ...

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Nanyang Technological University

Research

JoVE Journal

Cancer Research

1.2K Views.  University of Toronto. Preklinische intravitale beeldvorming heeft een zeer hoge resolutie, maar beperkte dieptepenetratie in het weefsel, waardoor het zeer goed is voor preklinische beeldvormingsstudies. Aan de andere kant is MRI veel meer klinisch toepasbaar en heeft het een hogere dieptepenetratie, maar een zeer lage ruimtelijke resolutie. Het doel van deze studie is om deze twee modaliteiten met elkaar te correleren om onze bevindingen van preklinische intravitale microscopie beter te vertalen naar de kliniek v...

Video: Auteur in de schijnwerpers: integratie van intravitale beeldvorming met hoge resolutie en MRI om de planning van stereotactische lichaamsbestralingstherapie te verbeteren

Long-term High-Resolution Intravital Microscopy in the Lung with a Vacuum Stabilized Imaging Window

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung is the most difficult to assess using intravital optical imaging due to its enclosed position within the body, delicate nature and vital role in sustaining proper physiology. While clinical modalities (positron emission tomography (PET), magnetic resonance imaging (MRI) and computed tomography (CT)) are capable of providing noninvasive images of this tissue, they lack the resolution necessary to visualize the earliest seeding events, with a single pixel consisting of nearly a thousand cells. Current models of metastatic lung seeding postulate that events just after a tumor cell&#39;s arrival are deterministic for survival and subsequent growth. This means that real-time intravital imaging tools with single cell resolution 2 are required in order to define the phenotypes of the seeding cells and test these models. While high resolution optical imaging of the lung has been performed using various ex vivo preparations, these experiments are typically single time-point assays and are susceptible to artifacts and possible erroneous conclusions due to the dramatically altered environment (temperature, profusion, cytokines, etc.) resulting from removal from the chest cavity and circulatory system 3. Recent work has shown that time-lapse intravital optical imaging of the intact lung is possible using a vacuum stabilized imaging window 2,4,5 however, typical imaging times have been limited to approximately 6 hr. Here we describe a protocol for performing long-term intravital time-lapse imaging of the lung utilizing such a window over a period of 12 hr. The time-lapse image sequences obtained using this method enable visualization and quantitation of cell-cell interactions, membrane dynamics and vascular perfusion in the lung. We further describe an image processing technique that gives an unprecedentedly clear view of the lung microvasculature.

This protocol describes the use of multiphoton microscopy to perform long-term high-resolution, single cell imaging of the intact lung in real time using a vacuum stabilized imaging window.

Metastasis to secondary sites such as the lung, liver and bone is a traumatic event with a mortality rate of approximately 90% 1. Of these sites, the lung ...

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have emerged in conjunction with the state-of-the-art positron emission tomography with 18F-fluorodeoxyglucose ([18F]-FDG PET). 13C magnetic resonance spectroscopic imaging (13CMRSI) is a minimally invasive imaging method that enables the monitoring of metabolism in vivo and in real time. As with any other method based on 13C nuclear magnetic resonance (NMR), it faces the challenge of low thermal polarization and a subsequent low signal-to-noise ratio due to the relatively low gyromagnetic ratio of 13C and its low natural abundance in biological samples. By overcoming these limitations, dynamic nuclear polarization (DNP) with subsequent sample dissolution has recently enabled commonly used NMR and magnetic resonance imaging (MRI) systems to measure, study, and image key metabolic pathways in various biological systems. A particularly interesting and promising molecule used in 13CMRSI is [1-13C]pyruvate, which, in the last ten years, has been widely used for in vitro, preclinical, and, more recently, clinical studies to investigate the cellular energy metabolism in cancer and other diseases. In this article, we outline the technique of dissolution DNP using a 3.35 T preclinical DNP hyperpolarizer and demonstrate its usage in in vitro studies. A similar protocol for hyperpolarization may be applied for the most part in in vivo studies as well. To do so, we used lactate dehydrogenase (LDH) and catalyzed the metabolic reaction of [1-13C]pyruvate to [1-13C]lactate in a prostate carcinoma cell line, PC3, in vitro using 13CMRSI.

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging

Dynamic nuclear polarization with subsequent sample dissolution has enabled real-time studies of metabolism in biological systems. Hyperpolarized [1-13C]pyruvate was used to study lactate dehydrogenase activity in a prostate carcinoma cell line in vitro.

In the past decades, new methods for tumor staging, restaging, treatment response monitoring, and recurrence detection of a variety of cancers have ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Longitudinal Intravital Imaging of Brain Tumor Cell Behavior in Response to an Invasive Surgical Biopsy

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized treatment determination. However, perturbation of the tumor architecture by biopsy and other invasive procedures has been associated with undesired effects on tumor progression, which need to be studied in depth to further improve the clinical benefit of these procedures. Conventional static approaches, which only provide a snapshot of the tumor, are limited in their ability to reveal the impact of biopsy on tumor cell behavior such as migration, a process closely related to tumor malignancy. In particular, tumor cell migration is the key in highly aggressive brain tumors, where local tumor dissemination makes total tumor resection virtually impossible. The development of multiphoton imaging and chronic imaging windows allows scientists to study this dynamic process in living animals over time. Here, we describe a method for the high-resolution longitudinal imaging of brain tumor cells before and after a biopsy in the same living animal. This approach makes it possible to study the impact of this procedure on tumor cell behavior (migration, invasion, and proliferation). Furthermore, we discuss the advantages and limitations of this technique, as well as the ability of this methodology to study changes in the cancer cell behavior for other surgical interventions, including tumor resection or the implantation of chemotherapy wafers.

Here we describe a method for high-resolution time-lapse multiphoton imaging of brain tumor cells before and after invasive surgical intervention (e.g., biopsy) within the same living animal. This method allows studying the impact of these invasive surgical procedures on tumor cells&#39; migratory, invasive, and proliferative behavior at a single cell level.

Biopsies are standard of care for cancer treatment and are clinically beneficial as they allow solid tumor diagnosis, prognosis, and personalized ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro model in the study of cancer stem/initiating cells, preclinical cancer research, and drug screening. The 3D spheroid models are superior to conventional tumor cell culture and reproduce some important characters of real solid tumors. However, conventional 3D tumor spheroids are made up exclusively of tumor cells. They lack the participation of tumor stromal cells and have insufficient extracellular matrix (ECM) deposition, thus only partially mimicking the in vivo conditions of tumor tissues. We established a new multicellular 3D spheroid model composed of tumor cells and stromal fibroblasts that better mimics the in vivo heterogeneous tumor microenvironment and its native desmoplasia. The formation of spheroids is strictly regulated by the tumor stromal fibroblasts and is determined by the activity of certain crucial intracellular signaling pathways (e.g., Notch signaling) in stromal fibroblasts. In this article, we present the techniques for coculture of tumor cells-stromal fibroblasts, time-lapse imaging to visualize cell-cell interactions, and confocal microscopy to display the 3D architectural features of the spheroids. We also show two examples of the practical application of this 3D spheroid model. This novel multicellular 3D spheroid model offers a useful platform for studying tumor-stroma interaction, elucidating how stromal fibroblasts regulate cancer stem/initiating cells, which determine tumor progression and aggressiveness, and exploring involvement of stromal reaction in cancer drug sensitivity and resistance. This platform can also be a pertinent in vitro model for drug discovery.

A novel three-dimensional spheroid model based on the heterotypic interaction of tumor cells and stromal fibroblasts is established. Here, we present coculture of tumor cells and stromal fibroblasts, time-lapse imaging, and confocal microscopy to visualize the formation of spheroids. This three-dimensional model offers a pertinent platform to study tumor-stroma interactions and test cancer therapeutics.

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Medicine

Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an urgent need to understand the mechanisms behind metastasis development, and thus to propose efficient treatments for advanced cancer. Metastatic cancers are hard to treat, as biopsies are invasive and inaccessible. Recently, there has been considerable interest in liquid biopsies including both cell-free circulating deoxyribonucleic acid (DNA) and circulating tumor cells from peripheral blood and we have established several circulating tumor cell lines from metastatic colorectal cancer patients to participate in their characterization. Indeed, to functionally characterize these rare and poorly described cells, the crucial step is to expand them. Once established, circulating tumor cell (CTC) lines can then be cultured in suspension or adherent conditions. At the molecular level, CTC lines can be further used to assess the expression of specific markers of interest (such as differentiation, epithelial or cancer stem cells) by immunofluorescence or cytometry analysis. In addition, CTC lines can be used to assess drug sensitivity to gold-standard chemotherapies as well as to targeted therapies. The ability of CTC lines to initiate tumors can also be tested by subcutaneous injection of CTCs in immunodeficient mice.
Finally, it is possible to test the role of specific genes of interest that might be involved in cancer dissemination by editing CTC genes, by short hairpin ribonucleic acid (shRNA) or Crispr/Cas9. Modified CTCs can thus be injected into immunodeficient mouse spleens, to experimentally mimic part of the metastatic development process in vivo.
In conclusion, CTC lines are a precious tool for future research and for personalized medicine, where they will allow prediction of treatment efficiency using the very cells that are originally responsible for metastasis.

Culturing CTCs allows a deeper functional characterization of cancer, through assaying specific marker expression, and assessing drug resistance and the ability to colonize the liver among other possibilities. Overall, CTC culture could be a promising clinical tool for personalized medicine to improve patient outcome.

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static ...

Cranial Window-based Intravital Imaging in a Mouse Model: An Imaging Technique to Study the Behavior of Pre-injected Cancer Cells in the Brain of a Murine Model

Source: Alieva, M. and Rios, A. C. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/59278/longitudinal-intravital-imaging-brain-tumor-cell-behavior-response-to">Longitudinal intravital imaging of brain tumor cell behavior in response to an invasive surgical biopsy.</a>&#160;J. Vis. Exp.&#160;(2019)
In this video, we perform intravital imaging of pre-injected cancer cells in a mouse model via an implanted cranial window. This&#160;high-resolution time-lapse imaging technique enables the study of dynamic behavior of cancer cells in the live mouse.

Source: Alieva, M. and Rios, A. C. Longitudinal intravital imaging of brain tumor cell behavior in response to an invasive surgical biopsy.&#160;J. Vis. ...

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

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