A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research
Summary
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.
Abstract
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.
Introduction
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'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 (μm 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 "ground truth" 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.
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Dr. Carla Calçada (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 – 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.
Materials
| Cell Culture Materials | |||
| BxPC-3 Human Pancreatic Cancer Cells | ATCC (American Type Culture Collection) | CRL-1687 | |
| Corning Matrigel Basement Membrane Matrix, LDEV-free, 10 mL | Corning | 354234 | |
| Corning Stripettor Ultra Pipet Controller | Corning | 07-202-350 | |
| Dulbecco Phospphate buffered saline without Calcium, Magnesium, or phenol red, 500 mL | Gibco | 14190144 | |
| Fetal Bovine Serum (Canada), 500 mL | Sigma-Aldrich | F1051-500ML | |
| Penicillin-Streptomycin 100x (liquid,stabilized, sterile-filtered, cell culture tested) | Sigma-Aldrich | P4333-100ML | |
| RPMI Medium 1640 (1x), liquid; with L-Glutamine, 500 mL | Gibco | 11875093 | |
| TrypLE Express Enzyme, 500 mL | Gibco | 12605028 | |
| Window Chamber Materials | |||
| 12 mm Glass Coverslip | Harvard Apparatus | CS-12R No. 1.5 | |
| Connex 500 3D Printer | Stratasys | N/A | |
| Biocompatible clear MED610 resin | Stratasys | RGD810 | |
| Loctite AA 3105 UV curable glue | Loctite | LCT1214249 | |
| Window chamber back frame | Trimech Inc | N/A | |
| Window chamber fiducial marker | Trimech Inc | N/A | |
| Window Chamber front frame | Trimech Inc | N/A | |
| Window chamber support clip | Trimech Inc | N/A | |
| inoculation and Surgery Materials | |||
| BD SafetyGlide Insulin Syringes with Permanently Attached Needles, 0.5 mL, 29 G x 1/2" | BD | CABD305932 | |
| Betadine Solution | Betadine | AP-B002C2R98U | |
| Cidex OPA 14 Day Solution 3.8 L | ASP | JOH20394 | |
| Disposable Surgical Underpads 23 inch x 24 inch | Kendall | 7134 | |
| Eye lubricant | Optixcare | 50-218-8442 | |
| Hair removal cream | Nair | 061700222611 | |
| Halstead Hemostatic Forceps | Almedic | 7742-A12-150 | |
| Heating pad | Sunbeam | B086MCN59R | |
| Iris Scissors | Almedic | 7601-A8-690 | |
| Isoflurane | Sigma | 792632 | |
| Metacam | Boehringer Ingelheim Animal Health USA Inc | NDC 0010-6015-03 | |
| NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ mouse | the Jackson laboratory | 7799 | |
| Peanut Clipper & Trimmer | Wahl | 8655-200 | |
| SOFSILK Nonabsorbable Surgical Suture #5-0 with 3/8" Taper point needle (17 mm) (Wax Coated,Braided Black Silk, Sterile) | Syneture | VS880 | |
| Splinter Forceps | Almedic | 7725-A10-634 | |
| MR Imaging | |||
| 3D printed window chamber immobilization device. | custom 3D printed, refer to figure 3 for details. | ||
| Convection heating device | 3M Bair Hugger | 70200791401 | |
| Drug injection system | Harvard Apparatus | PY2 70-2131 | PHD 22/2200 MRI compatible Syringe Pump |
| Gadovist 1.0 | Bayer | 2241089 | |
| Respiratory monitoring system | SAII | Model 1030 | MR-compatible monitoring and gating system for small animals. |
| Tail vein catheter (27 G 0.5" ) | Terumo Medical Corp | 15253 | |
| Optical Imaging | |||
| 3D printed imaging stage | Custom 3D printed, refer to supplementary figure 3 for details. | ||
| 12 V 7 W Flexible Polyimide Heater Plate Thin Adhesive PI Heating Film 25 mm x 50 mm | BANRIA | B09X16XCVS | Heating element used for mouse body temeprature regulation. |
| DC power supply | BK Precission | 1761 | Used to power the heating element. |
| Leica MZ FLIII | Leica Microsystems | 15209 | |
| svOCT imaging system | In-house made imaging system. Details can be found in reference 23. | ||
| Software | |||
| MATLAB Software | MathWorks | R2020A |
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