CRISPR-clear imaging of melanin-rich B16-derived solid tumors
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.
Additional Information
© The Author(s) 2023. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. We acknowledge Gerard M. Coughlin & Alon Greenbaum (California Institute of Technology) for assistance with PACT clearing and vasculature staining protocols. We thank Manfred Kopf and Louis Weiner for providing the mouse strains and/or cell lines used in this study. We acknowledge the UZH Department of Biochemistry Workshop and the Center for Microscopy and Image Analysis (ZMB), and Flow Cytometry Facilities (FCF). This research is supported by the Burroughs Welcome Fund 88492 and NSF (to R.S), National Research Foundation (NRF) of Korea under grant numbers 2020R1I1A2075393, 2021M3A9I4024452 and 2022M3A9H1014157 (to J.K.H), SNF Sinergia grant CRSII5_170929 (to A.P.), and National Cancer Institute of the National Institutes of Health under Award number F32CA189372 (to S.N.S.). Contributions: The project was designed and coordinated by R.S., J.K.H., S.N.S. & A.P; J.H, T.G., and S.P. performed gene editing, RT-qPCR, western blot, and sequencing experiments and analysis; R.S. generated the B16-tdTomato cell line, performed tissue clearing, immunohistochemistry, and confocal microscopy with the assistance of B.S. and G.N.D; All imaging data experiments were performed, analyzed and interpreted by R.S.; B.S. & S.N.S. performed all animal experimentation; B.S. performed nanoparticle injection, tissue perfusion-fixation and sectioning and tumor morphology analysis, SDS-PAGE and western blot analysis; S.N.S performed cell characterization, hydroquinone experiments, western blots for tyrosinase, and flow cytometry and analysis. V.G. trained R.S. for PACT imaging; A.P. and J.K.H. supervised the project and acquired funding; R.S., J.K.H., S.N.S, B.S., A.P. and T.G. prepared the manuscript with feedback from all authors. The authors declare no competing interests. Data availability: The sequencing data are available at the NCBI Sequence Read Archive (SRA, http://www.ncbi.nih.gov/sra) under the accession number PRJNA910290. All other data are available from the corresponding author (or other sources, as applicable) on reasonable request. Associated raw data for Figs. 1b, 2f, S4a, S5e, S7b and S7d can be found as Supplementary Data, and uncropped western blots are shown in Fig. S11.Attached Files
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Additional details
- PMCID
- PMC10073193
- Eprint ID
- 121521
- Resolver ID
- CaltechAUTHORS:20230525-772152300.6
- Burroughs Wellcome Fund
- 88492
- NSF
- National Research Foundation of Korea
- 2020R1I1A2075393
- National Research Foundation of Korea
- 2021M3A9I4024452
- National Research Foundation of Korea
- 2022M3A9H1014157
- Swiss National Science Foundation (SNSF)
- CRSII5_170929
- NIH Postdoctoral Fellowship
- F32CA189372
- Created
-
2023-06-28Created from EPrint's datestamp field
- Updated
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2023-06-28Created from EPrint's last_modified field
- Caltech groups
- Division of Biology and Biological Engineering