tumor cells inoculation in mice through precise landmarking and injection techniques

Guided Tumor Inoculation and Multimodal Imaging in Mice

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.

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

Инокуляция опухолевых клеток у мышей с помощью точных методов ориентировки и инъекций

После обезболивания и удаления шерсти мыши положите ее на грелку, завернутую в автоклавный хирургический коврик. Аккуратно выпрямите тело и подтяните кожу вдоль позвоночника. С помощью хирургического маркера нарисуйте одну точку на одной стороне мыши в центре тентовой кожи.После выращивания BXPC, трех клеточных линий рака поджелудочной железы человека в течение двух недель. Выньте колбу из инкубатора и поместите ее в шкаф биологической безопасности. Отсадите среду и промойте клетки пятью миллилитрами PBS.Добавьте в колбу пять миллилитров агента для диссоциации клеток и инкубируйте при температуре 37 градусов Цельсия с 5% углекислым газом в течение шести-семи минут. Перелейте клеточную суспензию в пробирку объемом 15 миллилитров и центрифугируйте при 500 G в течение пяти минут. После удаления надосадочной жидкости повторно суспендируйте клетки в полноценных питательных средах.С помощью гемоцитометра определяют концентрацию клеток и общее количество клеток. Центрифугируйте 1,5-миллилитровую суспензию, содержащую 250 000 клеток на каждую мышь, для инокулирования при 500 G в течение пяти минут. После удаления надосадочной жидкости повторно суспендируйте гранулу в 10 микролитрах PBS на каждую инокулируемую мышь и поместите трубку на лед.С помощью охлажденных наконечников для пипеток добавьте в пробирку растворимую базальную мембрану. Загрузите охлажденную иглу инсулина 29 калибра 20 микролитрами клеточной суспензии и положите шприц на лед. Введите иглу примерно на один сантиметр с няшки к ориентирующему пункту мыши и перемещайте иглу под кожу до тех пор, пока она не окажется на ориентире, скошенной стороной иглы вверх.Введите 20 микролитров клеточной суспензии и подождите 45 секунд, прежде чем извлечь шприц. После извлечения мыши из наркоза подождите, пока она начнет передвигаться, прежде чем вернуться в свою домашнюю клетку.

Research

JoVE Journal

Cancer Research

Tumor Cells Inoculation in Mice Through Precise Landmarking and Injection Techniques

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

Window Chamber Surgery in Tumor Cell Injected Mice for Tumor Growth Visualization and Imaging

Хирургия оконной камеры у мышей, которым вводили опухолевые клетки, для визуализации и визуализации роста опухоли

Integrative Imaging Techniques for Tumor Analysis in Mice

Методы интегративной визуализации для анализа опухолей у мышей