Delving deeper into CAR T cell evaluation

Profound analysis of tumor physiology and distribution of anti-tumor CAR T cells in the tumor microenvironment (TME) requires comprehensive 3D imaging data. Common 2D histological and IHC microscopy techniques are not sufficient for this purpose as they are usually applied to a limited number of thin sections, which leads to the potential risk of missing relevant information.

Tissue clearing, combined with light sheet microscopy, has the potential to bridge this technological gap and enable single-cell resolution imaging of whole large samples, including solid tumors or even entire organisms. However, technically challenging instruments and the use of homemade, difficult-to-reproduce immunostaining and clearing protocols currently pose significant obstacles to the establishment of this workflow in the context of pre-clinical studies.

In this article, we showcase the efficacy of 3D fluorescence microscopy using the UltraMicroscope Blaze™ for analyzing CAR T cells in solid tumors, employing pancreatic carcinoma xenografts as a model. By using automated light sheet microscopy along with validated antibodies, straightforward tissue clearing, and immunolabeling protocols, we can visualize and quantify multiple tumor parameters and the infiltration of therapeutic CAR T cells at a cellular level. Hence, this workflow facilitates the unbiased evaluation of cellular therapies in large, heterogeneous samples like solid tumors and the TME.

In light sheet microscopy, the optical pathways for fluorescence excitation (horizontal) and detection (vertical) are decoupled. A laser light sheet is directed perpendicular to the observation axis, selectively exciting a single plane of the labeled sample. Illuminating only a thin layer along the z-axis produces high-resolution images with minimal and strictly localized photodamage and bleaching effects, unlike traditional epifluorescence microscopy techniques. By moving the sample along the z-axis through the light sheet, a series of images can be generated, forming a stack that enables the visualization of large biological samples in 3D with single-cell resolution. Imaged samples can range from small organoids and rodent organs to larger entities like whole mouse models or human kidneys.

3D light sheet imaging offers a valuable alternative to traditional histology and microscopy techniques in preclinical screenings, addressing the challenges associated with evaluating complex 3D samples such as solid tumors and their microenvironment.

• Implementing the 3D imaging workflow in CAR T cell evaluation results in cost and time savings by providing researchers an unbiased, reliable tool to identify the best therapeutic CAR T cell candidates against solid tumors and discard less effective cell lines as early as possible.
• Light sheet microscopy is a versatile imaging technique applicable to various cell types and research areas, from CAR T cells to blood vessels, from immuno-oncology to neuroscience.
• Our user-friendly 3D imaging workflow and fully automated UltraMicroscope Blaze overcomes concerns about laborious protocols and complex instruments, making 3D light sheet imaging accessible and reproducible to everyone.
   

To read the full article, visit https://tinyurl.com/Miltenyi-Biotec or scan the QR code with your mobile phone.

Top image caption: Pancreatic carcinoma, imaged using a light sheet microscope.