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other·preclinical research, biomedical research, research methods, oncology·PMC10073193

CRISPR-clear imaging of melanin-rich B16-derived solid tumors

Communications Biology · 9 authors, 9 centres

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TL;DR

The authors developed CRISPR-Clear, a CRISPR-based gene editing approach that eliminates melanin production in B16 mouse melanoma cells to enable deep optical imaging within tissue-cleared melanin-rich tumors. Tyrosinase knockout tumors showed no significant differences from parental B16 cells at the morphological, cancer pathway, or transcriptomic level.

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CRISPR-Clear is a CRISPR-based method integrated into the PACT tissue-clearing workflow to remove melanin, which normally causes strong light absorption and scattering that impairs optical imaging. The authors used CRISPR to knockout tyrosinase in B16 melanoma cells, generating a melanin(-) clone. Transcriptome analysis by RNA sequencing showed that gene expression profiles of knockout and parental B16 cells were overall very similar, with only four genes showing differences of potential statistical significance, none obviously related to cancer or melanin synthesis. Flow cytometry confirmed no measurable differences in cell morphology, and Western blots and IHC showed similar HER2 and tdTomato expression between edited and parental cells. The method was demonstrated on B16-derived solid tumors, including a HER2-transgenic variant, enabling high-resolution deep tissue imaging of antibodies, nanoparticles, fluorescent proteins, and vasculature.

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other

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preclinical research

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ORIGINAL PAPER

PMC10073193

CRISPR-clear imaging of melanin-rich B16-derived solid tumors

Communications Biology

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ABSTRACT

Tissue clearing combined with deep imaging has emerged as a powerful technology to expand classical histological techniques. Current techniques have been optimized for imaging sparsely pigmented organs such as the mammalian brain. In contrast, melanin-rich pigmented tissue, of great interest in the investigation of melanomas, remains challenging. To address this challenge, we have developed a CRISPR-based gene editing approach that is easily incorporated into existing tissue-clearing workflows such the PACT clearing method. We term this method CRISPR-Clear. We demonstrate its applicability to highly melanin-rich B16-derived solid tumors, including one made transgenic for HER2, constituting one of very few syngeneic mouse tumors that can be used in immunocompetent models. We demonstrate the utility in detailed tumor characterization by staining for targeting antibodies and nanoparticles, as well as expressed fluorescent proteins. With CRISPR-Clear we have unprecedented access to optical interrogation in considerable portions of intact melanoma tissue for stained surface markers, expressed fluorescent proteins, of subcellular compartments, and of the vasculature.

CITATION EXPORT

Rajib Schubert, Taegeun Bae, Branko Simic et al.. CRISPR-clear imaging of melanin-rich B16-derived solid tumors. Communications Biology. (2023). https://doi.org/10.1038/s42003-023-04614-7

AUTHORS

9 · 9 CENTRES

Rajib Schubert, Taegeun Bae, Branko Simic, Sheena N. Smith, Seong-Ho Park, Gabriela Nagy-Davidescu, Viviana Gradinaru, Andreas Plückthun, Junho K. Hur

1grid.7400.30000 0004 1937 0650Department of Biochemistry, University of Zürich, Zürich, Switzerland
2grid.20861.3d0000000107068890Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA USA
3grid.289247.20000 0001 2171 7818Department of Medicine, Graduate School, Kyung Hee University, Seoul, South Korea
4grid.49606.3d0000 0001 1364 9317Department of Medicine, Major in Medical Genetics, Graduate School, Hanyang University, Seoul, South Korea
5grid.49606.3d0000 0001 1364 9317Department of Genetics, College of Medicine, Hanyang University, Seoul, South Korea
6grid.289247.20000 0001 2171 7818Department of Pathology, College of Medicine, Kyung Hee University, Seoul, South Korea
7grid.418158.10000 0004 0534 4718Present Address: Research and early development, Roche Sequencing Solutions, Pleasanton, CA USA
8grid.411947.e0000 0004 0470 4224Present Address: College of Pharmacy, The Catholic University of Korea, Gyeonggi-do, South Korea
9Present Address: Vector BioPharma AG, Basel, Switzerland