Immunology and Immuno-Oncology (IO) drug discovery requires a multi-staged approach: preclinical in vitro assays, in vivo studies and histological analysis are key to determining the best route to clinic. Aquila demonstrate the power of incorporating a specialist histology approach in paraffin embedded tissue samples early in the drug discovery process. We can also demonstrate the power of complementing in vitro assay readouts with specialist histology tools, thus providing high quality data to inform on clinical trial strategy.
Simultaneously imaging multiple IO and immunology markers on tissue sections provides morphological, pathological and spatial information of the juxtaposition of different immune cell types (quantified by image analysis).
Aquila have multiple optimised markers and multiple detection strategies to assist with biomarker research in both human and mouse tissue. Aquila offer optimisation of novel markers, which allows us to tailor our services to the client’s study design and tissue of interest. The case study below illustrates some of Aquila’s capabilities.
The client required the most time efficient method of identifying specific cancers of interest for their research program. The screening process involved examining a wide variety of cancer patient samples, with a specific treatment regime, as specified by the client. Aquila sourced formalin fixed paraffin embedded (FFPE) tissue samples, accompanied by patient data, from an ethically approved bioresource and, together with the client, decided on which marker combinations would be most appropriate for the first stage of the study. The second phase of the study would involve the analysis of the expression of a more extended panel of markers on the chosen cancer types from phase 1.
Phase 1- Cancer Type Screening
We investigated the expression of five biomarkers in total in human FFPE samples, stained in two triple marker panels (Panel 1: markers A, B, C + DAPI. Panel 2: markers C, D, E + DAPI). Each panel consisted of a novel client biomarker and two of Aquila’s in-house optimised biomarkers. Staining for two triple panels allows for a relatively fast screening of multiple samples, and can provide a whole slide scan image per sample. Aquila optimised and imaged both novel triple panels. Each scan was analysed to provide data on the co-expression of the markers of interest. This informed the selection of cancers of interest for the client.
Figure 1. Optimised triple panel stain successfully applied to a human colon cancer. 1A. Overview triple IF scan of panel. 1B. x20 snapshot of selected ROI from figure 1A.
Phase 2 – Application of Complex Panel Staining on Selected Human Cancer Types
The next step in this study required the development of a novel 7-plex panel, combining the four previously optimised markers used in the triple panels of phase 1, with the client’s marker of interest and an additional new marker. This complex panel required more time for optimal development. However, a greater multiplex panel allows investigation of the relationship of multiple markers in one slide, and the data generated is far more complex and informative compared to two triple panels, such as those used in phase 1. The main challenge of this study was overcoming the complexity of the higher level of multiplexing (staining optimisation and spectral un-mixing of images). When staining more than four colours, complex and extensive validation is required to ensure staining specificity and quality. Therefore, a program of work can be tailored to suit budget and timescales, while generating a strong data package from good experimental design.
When working on a multiplex panel, in addition to the staining optimisation and validation, the imaging process is more challenging than dual or triple panels. A set of control slides (spectral reference slides) is required to separate the wavelength of each stained marker in such a way that the final spectrally unmixed image shows the true location of each marker on the client sample. Aquila now have the Vectra® Polaris™ from PerkinElmer that allows imaging of multiplex panels such as the one in this case study.
We provided an accurate and powerful data package for the client, while conserving precious clinical tissue, and using ethically approved human tissue sourced by Aquila. This case study demonstrates Aquila’s ability to optimise biomarkers and tailor them to your staining and tissue requirements, whether it be from Aquila’s range of in-house optimised biomarkers, or a novel biomarker.
Figure 2. CD8/CD4/CD163/PD-L1/CD68/Pan CK protein expression in colorectal carcinoma (CRC) stained by IF (with Opal™ Multiplex Detection Reagents using the Vectra® Polaris™ from PerkinElmer).