One protocol across four institutes
The aim was to test whether the same 3D cancer imaging experiment could be carried out independently at different research institutes and still produce consistent biological results.
It is rare for the same 3D imaging experiment to be performed independently at different research institutes within a single study. We developed one protocol that could be run across four centres in the UK and Spain, allowing us to ask how much experiments could vary between laboratories while still recovering the same response to treatment.
As a way to study this, we modified a mouse melanoma cell line, where NRAS activation drives MAP kinase signalling.[1] The cells grow as 3D spheroids in a gel in ordinary 96-well plates, and we added an ERK sensor so we could measure signalling responses to treatment throughout them. The ERK sensor has been widely used by other labs, and was invented during a period of development in fluorescent signalling reporters,[2] when researchers were finding new ways to turn signalling activity into visible changes inside living cells.
The NRAS activation increases the activity of the MAP kinase pathway, a signalling cascade from RAS or RAF, through MEK, to ERK. This pathway controls processes including cell proliferation. In 2002, Mike Stratton and colleagues at the Wellcome Trust Sanger Institute discovered that BRAF is frequently mutated in melanoma, in work that also involved researchers at the Institute of Cancer Research.[3] This helped lead to treatments that target MAP kinase signalling in melanoma. Binimetinib is used clinically as part of a treatment for melanomas with BRAF mutations. Our model is driven by NRAS, which switches on the same pathway.
Fast multiwell light-sheet imaging
We started developing the protocol for these experiments around 2022 using dOPM, developed in the Photonics Group at Imperial.[4] We worked out the practicalities of imaging and adding treatments, and developed a protocol that researchers at four centres in the UK and Spain could use independently. The first microscope for the project was implemented at the Francis Crick Institute, and the design was then installed at the other experimental sites.
Dual-view oblique plane microscopy (dOPM) is an extension of oblique plane microscopy (OPM) that images the sample from two different angles. It allows rapid 3D imaging from two directions using a single objective.[4]
Our ERK sensor changes its location within the cell. When ERK signalling is turned down, the sensor moves into the nucleus, allowing us to measure signalling throughout the 3D spheroids.
Robust therapeutic responses across sites
Despite differences between sites, all four centres recovered the same major biological response: binimetinib reduced ERK signalling, while TPA increased it. There was some variation between the sites, including differences we didn't control, such as laser power and reagent batches.
I hope we can expand on this approach with models of a wider range of cancers, more targeted therapies and therapeutic combinations, and extended live imaging to see how responses to treatment progress over hours and days of treatment.
Groups involved
This work was carried out as part of the MACH3CANCER consortium, bringing together research groups across four experimental centres with the team that developed the dOPM technology:
- Tumour Cell Biology Laboratory — Erik Sahai · Francis Crick Institute
- Edinburgh Drug Discovery — Neil Carragher · Institute of Genetics and Cancer, University of Edinburgh
- Dynamical Cell Systems — Chris Bakal · Institute of Cancer Research
- Colorectal Cancer Laboratory — Eduard Batlle · IRB Barcelona
- Biophotonics Group — Imperial College London · dOPM development
Sharing the data
The raw data we generated is openly shared. This kind of 3D imaging data is still relatively rare, and it can be used by others to train image analysis algorithms, compare with other datasets, or reuse to ask new questions.
You can explore and download the dataset from the BioImage Archive.
References
- Pedersen M, et al. Primary melanoma of the CNS in children is driven by congenital expression of oncogenic NRAS in melanocytes. Cancer Discovery (2013). doi:10.1158/2159-8290.CD-12-0464
- Regot S, et al. High-sensitivity measurements of multiple kinase activities in live single cells. Cell (2014). doi:10.1016/j.cell.2014.04.039 · ERK-KTR-mRuby2 on Addgene
- Davies H, et al. Mutations of the BRAF gene in human cancer. Nature (2002). doi:10.1038/nature00766
- Sparks H, Dent L, Bakal C, Behrens A, Salbreux G, Dunsby C. Dual-view oblique plane microscopy (dOPM). Biomedical Optics Express (2020). doi:10.1364/BOE.409781
You can read our report about the study here (it's a preprint, so it hasn't been peer reviewed yet).