tumor cells inoculation in mice through precise landmarking and injection techniques

マウスにおける腫瘍のガイド下接種とマルチモーダルイメージング

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.

著者スポットライト:高解像度生体内イメージングとMRIを統合して、定位放射線治療計画を強化する

トランスレーショナルがん研究における生体内顕微鏡検査と磁気共鳴画像法の併用のための背側Skinfold Window Chamber腫瘍マウスモデル

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.

精密なランドマークおよび注射技術によるマウスへの腫瘍細胞接種

マウスの毛を麻酔して取り除いた後、オートクレーブ手術マットで包まれた加熱パッドの上に置きます。体をやさしくまっすぐにし、背骨に沿って皮膚を持ち上げます。サージカルマーカーを使用して、テントを張った皮膚の中央にあるマウスの片側に1つの点を描きます。BXPCを増殖させた後、3つのヒト膵臓癌細胞株を2週間。インキュベーターからフラスコを取り出し、生物学的安全キャビネットに入れます。培地を吸引し、細胞を5ミリリットルのPBSで洗浄します。5ミリリットルの細胞解離剤をフラスコに加え、5%二酸化炭素と摂氏37度で6〜7分間インキュベートします。細胞懸濁液を15ミリリットルのチューブに移し、500Gで5分間遠心分離します。上清を除去した後、細胞を完全増殖培地に再懸濁します。ヘモサイトメーターを使用して、細胞濃度と総細胞数を決定します。マウスあたり250, 000個の細胞を含む1.5ミリリットルの懸濁液を遠心分離し、500 Gで5分間接種します。上清を取り除いた後、ペレットを接種するマウスあたり10マイクロリットルのPBSに再懸濁し、チューブを氷上に置きます。冷やしたピペットチップを使用して、可溶化した基底膜をチューブに加えます。冷やした29ゲージのインスリン針に20マイクロリットルの細胞懸濁液を入れ、シリンジを氷の上に置きます。針をマウスのランドマークポイントに約1センチメートルのコドルを挿入し、針の斜角側を上にして、針をランドマークに到達するまで皮膚の下に移動します。20マイクロリットルの細胞懸濁液を注入し、45秒間待ってからシリンジを取り出します。マウスを麻酔から外した後、マウスが歩行可能になるまで待ってから、ホームケージに戻ります。

tumor-cells-inoculation-mice-through-precise-landmarking-injection

Research

JoVE Journal

Cancer Research

Tumor Cells Inoculation in Mice Through Precise Landmarking and Injection Techniques

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.

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 ...