OPM used by CRUK Edinburgh Centre to image 3D glioma mass
E57 Mesenchymal Glioma with Fucci reporter (Sartorius 4779) treated with 1μM PF05212384 (left) or DMSO control (right) imaged after 3 days treatment
Researchers at the Edinburgh CRUK centre have acquired their first image data using their new oblique plane microscope (OPM) system
A 3D mass of glioma (brain tumour) cells derived from a patient with a mesenchymal sub-type (the most aggressive glioma subtype) was treated with a drug that halts cell growth and division (left). This is measured using a reporter system that colours cell nuclei that are ready to divide and grow in green, and those that aren’t in red. By comparing to a control sample (right), we see that the drug (left) has greatly reduced the number of dividing cells. Studying cells in a 3D environment maximises cell-cell and cell-environment contacts as well as causes gradients of nutrients, oxygen and therapeutic drug to form across the 3D structure. This more closely resembles the behaviour of tumours in their native environment – compared to cell culture on in a monolayer of cells – providing a more accurate tool to help study cancer treatments
OPM implemented at the CRUK Edinburgh Centre
Hugh Sparks and Nils Gustafsson implemented a new dOPM instrument at the CRUK Edinburgh Centre in the phenotypic discovery facility at the University of Edinburgh, under the guidance of Chris Dunsby. This is being applied to image cancer organoids and other 3D cancer models by Jayne Culley and Martin Lee under the guidance of Neil Carragher.
Progress on OPM implemntation at IRB Barcelona
Nils Gustafsson implemented a new dOPM instrument at the IRB in Barcelona under the guidance of Chris Dunsby. This system is being integrated into the Advanced Digital Microscopy facility led by Julien Colombelli and will be exploited by the lab of Eduard Batlle.
OPM implemented at the Francis Crick Institute
The first CRUK Accelerator OPM instrument has been implemented and upgraded at the Francis Crick Institute
Nathan Curry and Nils Gustafsson implemented a new OPM instrument at the Francis Crick Institute under the guidance of Chris Dunsby. This was applied to image cancer organoids and has now been upgraded to a new design with higher resolution and signal to noise performance. This new OPM design will now be implemented for our other Accelerator partners at the Institute of Cancer Research, the University of Edinburgh and IRB Barcelona.
OPM image data– acquired at Imperial
Oblique Plane Microscopy (OPM) is a core technology for our Accelerator that we will implement at our partner sites. Here we present renderings of exemplar OPM image data of 3D cancer models from our consortium partners that have been imaewd on the first prototype OPM instrument at Imperial.
Sections of EGFR mutant mouse lung tissue treated with latest EGFR inhibitors- Sahai lab, Crick
This work aims to improve our understanding of residual disease and its microenvironment after treatment. The figure above shows a section of mouse lung tissue treated with an EGFR inhibitor imaged using the OPM system. Actin is labelled in green. The yellow-red-purple false-colour indicates the FRET ratio reported by the YPet/ECFP EKAR-EV FRET biosensor for ERK activity. The field of view shown is approximately 1.5×0.32×0.14 mm3.
Melanoma spheroids – Bakal lab, ICR
We are interested in how changes in cell size and shape drive melanoma metastasis in 3D. We aim to study this using melanoma spheroids expressing mutant NRAS (G12D) and also expressing membrane (CAAX-GFP) and nuclear (iRFP670-H2B) labels. The spheroids were cultured in reduced growth factor basement membrane extract (BME). Fifteen spheroids were imaged in the same experiment at 15 min intervals. The dOPM images above show a near-isotropic resolution in all three dimensions, which will aid the automated extraction of cell shape features for analysis.
Patient and mouse-derived colon cancer organoids – Battle lab, IRB
We aim to quantify the dynamics of cancer stem cell division and differentiation using engineered PDOs where specific marker genes are labelled to quantify the proportion of proliferating and non-proliferating stem cells. Readouts in this work include the specific markers for stem cells, LGR5-GFP and Ki67-RFP. We imaged 8 fields in a multi-well plate where the field of view was 2×0.32×0.14 mm3 and the image acquisition time was 3 minutes per field for 3 colours. The figure above shows representative data from patient-derived (well 2) and mouse-derived (well 3) organoids acquired using the OPM system .
Patient-derived glioma stem cell spheroid model – Carragher lab, CRUK Edinburgh
We are aiming to develop an automated 3D phenotypic screening assay to identify small therapeutic molecules targeting the quiescent glioma stem cell niche near the hypoxic core and not the proliferating glioma stem cells on the periphery of the spheroid. The figure above shows a fixed patient-derived glioma stem cell spheroid model expressing the FUCCI cell cycle reporter imaged using OPM.
GFP (green) nucleus – G1 phase cell cycle
Cherry (red) nucleus – S phase cell cycle
Red and green – G2/ML phase cell cycle