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.

All animal procedures were performed in accordance with the Guide to the Care and Use of Experimental Animals which is set forth by the Canadian Council on Animal Care. Experiments were performed according to a protocol approved by the University Health Network Institutional Animal Care and Use Committee in Toronto, Canada.
1. Tumor inoculation landmarking
NOTE: &#34;Landmarking&#34; refers to the process of marking the skin of the mouse to indicate w...

Guided Tumor Inoculation and Multimodal Imaging in Mice

<ol>
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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+vascular+permeability,+accumulation,+and+penetration+of+macromolecular+drug+carriers.">Tumor vascular permeability, accumulation, and penetration of macromolecular drug carriers.</a> J Natl Cancer Inst. 98 (5), 335-344 (2006).</li><li>Momiyama, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+real-time+imaging+of+the+efficacy+of+temozolomide+on+cancer+cells+in+the+brain+of+live+mice.">Subcellular real-time imaging of the efficacy of temozolomide on cancer cells in the brain of live mice.</a> Anticancer Res. 33 (1), 103-106 (2013).</li><li>Dadgar, S., Rajaram, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+approaches+to+investigating+radiation+resistance.">Optical imaging approaches to investigating radiation resistance.</a> Front Oncol. 9, 1152 (2019).</li><li>Fukumura, D., Jain, R. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-angiogenesis+therapy+can+overcome+endothelial+cell+anergy+and+promote+leukocyte-endothelium+interactions+and+infiltration+in+tumors.">Anti-angiogenesis therapy can overcome endothelial cell anergy and promote leukocyte-endothelium interactions and infiltration in tumors.</a> FASEB J. 20 (6), 621-630 (2006).</li><li>Allam, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Longitudinal+in-vivo+quantification+of+tumour+microvascular+heterogeneity+by+optical+coherence+angiography+in+pre-clinical+radiation+therapy.">Longitudinal in-vivo quantification of tumour microvascular heterogeneity by optical coherence angiography in pre-clinical radiation therapy.</a> Sci Rep. 12, 6140 (2022).</li><li>Stadlbauer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+hypoxia+and+alterations+in+microvascular+architecture+predict+glioblastoma+recurrence+in+humans.">Tissue hypoxia and alterations in microvascular architecture predict glioblastoma recurrence in humans.</a> Clin Cancer Res. 27 (6), 1641-1649 (2021).</li><li>Danquah, M. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+dorsal+skinfold+window+chamber+model+and+multi-parametric+characterization+of+tumor-associated+vasculature.">Optimization of the dorsal skinfold window chamber model and multi-parametric characterization of tumor-associated vasculature.</a> Intravital. 3 (1), e27935 (2014).</li><li>Allam, N., Taylor, E., Vitkin, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+3D-printed+tools+towards+robust+longitudinal+multi-modal+pre-clinical+imaging.">Low-cost 3D-printed tools towards robust longitudinal multi-modal pre-clinical imaging.</a> bioRxiv. , (2023).</li><li>Alexander, S., Weigelin, B., Winkler, F., Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+intravital+microscopy+of+the+tumour-stroma+interface:+invasion,+metastasis,+and+therapy+response.">Preclinical intravital microscopy of the tumour-stroma interface: invasion, metastasis, and therapy response.</a> Curr Opin Cell Biol. 25 (5), 659-671 (2013).</li><li>Steven, A. J., Zhuo, J., Melhem, E. 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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 ch...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cell Culture Materials</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>Window Chamber Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm Glass Coverslip</td>
			<td>Harvard Apparatus&#160;&#160;</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>&#160;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>inoculation and Surgery Materials</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&#34;</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&#160;&#160;</td>
			<td>Optixcare</td>
			<td>50-218-8442</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair removal cream</td>
			<td>Nair</td>
			<td>&#8206;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&#160;</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>&#160;Boehringer Ingelheim Animal Health USA Inc</td>
			<td>NDC 0010-6015-03</td>
			<td></td>
		</tr>
		<tr>
			<td>NOD.Cg-Rag1tm1Mom&#160;Il2rgtm1Wjl/SzJ mouse</td>
			<td>the Jackson laboratory</td>
			<td>7799</td>
			<td></td>
		</tr>
		<tr>
			<td>Peanut Clipper &#38; Trimmer</td>
			<td>Wahl</td>
			<td>8655-200</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;SOFSILK Nonabsorbable Surgical Suture #5-0 with 3/8&#34; Taper point needle (17 mm) (Wax Coated,Braided Black Silk, Sterile)&#160;</td>
			<td>&#160;Syneture&#160;</td>
			<td>&#160;VS880</td>
			<td></td>
		</tr>
		<tr>
			<td>Splinter Forceps</td>
			<td>Almedic</td>
			<td>7725-A10-634</td>
			<td></td>
		</tr>
		<tr>
			<td>MR Imaging</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&#160;&#160;</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&#34; )</td>
			<td>Terumo Medical Corp</td>
			<td>15253</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Imaging</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&#160;</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>Software</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.

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

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

Preclinical intravital imaging has very high resolution, but limited depth penetration in the tissue, which makes it very good for preclinical imaging studies. On the other hand, MRI is much more clinically applicable and has a higher depth penetration, but very low spatial resolution. The goal of this study is to correlate these two modalities with each other in order to better translate our findings from preclinical intravital microscopy into the clinic via magnetic resonance imaging.Perfusion-sensitive imaging methods in MRI and biophotonics, including our optical coherence tomography and geography system, are revealing how the microvasculature impacts tumor response to hypofractionated radiotherapy. 3D printing is also aiding through cost-effective custom tool manufacturing, facilitating longitudinal studies. Finally, AI image analysis is facilitating the identification of novel radiotherapy predictive biomarkers.We've made several findings concerning the tumor response to hypofractionated radiotherapy, including more recently, the temporal kinetics of both radiobiologically significant and abstracted AI-derived tissue response metrics. Additionally, we have demonstrated the promise for multimodal imaging being performed more accurately, correlating both MRI, macroscopic and optical coherence tomography, microscopic, angiography-derived metrics. By extending the effective length of longitudinal studies, we may better identify predictive biomarkers for long-term tumor treatment response.Additionally, by improving robustness of co-registration between macroscopic clinically available imaging modalities and microscopic preclinical imaging modalities, we may enhance the translatability of our findings from benchtop to bedside. During a stereotactic body radiation therapy, high doses of radiation are delivering a reduced number of fractions. One potential radiobiological target associated with increased cell death in SVRT is the tumor microvasculature.By combining high-resolution optical angiography and MRI, we want to enable functional MRI microvascular imaging for patient-specific SVRT treatment planning and improved outcomes.

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 sp...

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

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.

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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