Validated Biomarkers
QF-Pro® is currently validated for various applications and biomarkers, including:
Description
Violet 3.0 is the all-in-one microscopy platform for QF-Pro® technology, bringing Fluorescence Lifetime Imaging Microscopy (FLIM) and Förster Resonance Energy Transfer (FRET) to any lab. Our comprehensive benchtop solution features a high-end modulated laser, sensitive image sensor, and also a multi-channel LED for immunofluorescence. Violet 3.0 includes two stage inserts: one for up to 4 microscopy slides and another for a standard multi-well plate, accommodating various sample types. Its ease of use supports both experienced scientists and beginners in exploring spatial biology with confidence. Experience research as never before and pave the way for groundbreaking advancements in spatial biology.
Violet 3.0 – where simplicity meets innovation, opening whole new avenues of research.
Features:
Works with plates or slides
Violet 3.0 has an easy to switch stage insert, allowing for the analysis of microwell plates or up to four standard microscope slides at a time. It is equipped with 10X, 20X and 40X dry objectives.
Easy to use software
Violet 3.0 is accompanied with one desktop licence of our QF-Pro® software allowing users of all expertise to easily run the device and generate groundbreaking data.
Includes 4 LED channels
Alongside the modulated laser required for QF-Pro® analysis a secondary multichannel LED (365nm, 488nm, 585nm and 635nm) is included for the illumination of secondary biomarkers of interest.
QF-Pro® Software
What makes Violet 3.0 even more accessible and easy to use is the accompanying QF-Pro® software suite. This guides users of all experience through the process of loading their samples and creating a map of Regions of Interest (ROIs) to be acquired. The platform then sequentially acquires all mapped ROIs and calculates a QF-Pro® score and image per region. Post-acquisition analyses, such as the application of a threshold or change in Lookup Table (LUT) is automated, with the option of fine-tuning for more experienced users. All data can then be seamlessly exported from the QF-Pro® software into Microsoft Excel (data) or PowerPoint/PDF (images). The combination of Violet 3.0 and QF-Pro® software allows any laboratory or researcher to access to benefits of QF-Pro®.
What is QF-Pro®?
The spatial biology technology with proven clinical value.
Quantifying Functions in Proteins (QF-Pro®) is a state-of-the-art bio-imaging platform able to spatially quantify functional proteomic events within fixed patient samples (FFPE, cell lines, PBMC, etc.), such as protein-protein interactions and protein post-translational modifications, at an unrivalled resolution and dynamic range. It does this by using an adaptation of Förster Resonance Energy transfer (FRET). This patented adaptation allows FRET to work reliably in patient tissue samples, with a high signal to noise ratio, for the first time.
Unlike competing technologies, such as the proximity ligation assay (PLA) or co-localisation experiments, QF-Pro® precisely measures these events at a ≤10nm resolution. Quantifying protein functions at this resolution yields mechanistic insights into signalling pathways and drug targets directly within samples.
How does QF-Pro® work?
QF-Pro® Assay Overview
QF-Pro® has modified established technologies (FRET/FLIM) to create a patented assay which is able to spatially quantify protein functions in situ within FPPE tissue samples for the first time at an unprecedented resolution. This is a feat that is unachievable with existing technologies.
Utilisation of Advanced Technologies
QF-Pro® utilises a two-site immunofluorescence-styled assay in conjunction with amplified Förster Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FLIM). Essentially operating as a “biochemical ruler”, QF-Pro® makes 1-10nm measurements within samples to quantify protein functional states. Our patented amplification step has allowed these readouts to perform in patient tissue samples, a feat that has been unachievable with previous FRET-based technologies.
Antibody Labeling Strategy
Two epitopes are labelled simultaneously using species-distinct antibodies. These are then detected with QF-Pro® secondary labelling reagents, one combined with a green chromophore (the donor) and the other to an amplified red chromophore (the acceptor).
Detection
Violet 3.0 and our QF-Pro® software then automatically measures the distance between the donor and acceptor chromophore, based on the amount of energy given from the donor to the acceptor (this is FRET). This allows us to quantify the interaction or post-translational modification of a protein and spatially measure these events across a sample. Violet 3.0 makes FRET imaging easier and more accessible than ever before.
Applications
Discover how QF-Pro® is transforming clinical biomarker research.
Understanding the intricate interactions of proteins is crucial for deciphering biological processes. Proteins govern a plethora of functions and serve as pivotal targets in health and disease. QF-Pro® not only enhances our understanding of biological pathways and protein functional states but also facilitates the identification of new biomarkers at single-cell resolution. Moreover, it enables deep phenotypic profiling in both cell and tissue models, providing early insights into drug mechanism of action and facilitating precise drug target engagement in tissues.
Protein-Protein Interactions (PPIs)
QF-Pro® can be used in order to quantify a range of protein-protein interactions (PPIs) within tissue samples.
This includes intracellular PPIs such as HER2/HER3 dimerisation or Akt/PDK1 interaction states. QF-Pro® can also quantify intercellular interaction states such as those of immune checkpoints, i.e., PD-1/PD-L1, CTLA-4/CD80, TIGIT/CD155.
These intercellular PPIs are quantified in-situ without the need for cell permeabilisation. Other interaction states that can be quantified include DNA-protein interactions.
Post-Translational Modifications (PTMs)
Our intracellular PTM assays quantify events such as the phosphorylation or methylation states of intracellular proteins. The spatial mapping of these PTMs allows the user to quantify the functional states (e.g. activation) of proteins within samples. PTMs that can be quantified by QF-Pro® also include glycosylation and acetylation events. These readouts are particularly useful for gaining insights on intracellular signalling pathway dynamics and designing small molecule therapeutics.
Optimised biomarkers include Akt activation state, PD1 activation state and STAT3 (Y705 or S727) activation state quantification.
Immuno-Oncology
Our technology has demonstrated a direct clinical value in the domain of immuno-oncology. Immune checkpoints, such as PD-1/PD-L1, provide a key mechanism to target cancer, by re-enabling the hosts immune system to fight and destroy malignant cells. However, only 12-20% of solid tumour patients respond to these therapies and the lack of a robust biomarker to indicate which patients will or will not respond to these immunotherapies only contributes to worsen this situation.
Published in the Journal of Clinical Oncology in 2023, we have validated a QF-Pro® assay for the determination of PD-1/PD-L1 interaction state (functional checkpoint engagement) in non-small cell lung cancer (NSCLC). In this study of 188 patients, PD-1/PD-L1 interaction state measured by QF-Pro®, but not PD-L1 expression TPS, was highly predictive of patient outcome and response to immune checkpoint blockade treatments.
QF-Pro® identified that patients with a high PD-1/PD-L1 interaction state in their biopsy samples (as quantified by QF-Pro®) responded positively to immune checkpoint blockade, irrespective of their PD-L1 expression. This accounts for almost 25% of all NSCLC patients who would therefore not routinely be selected for therapy despite responding.
Our QF-Pro® analyses were compared to classical PD-L1 expression scores (TPS) determined by IHC. PD-L1 TPS alone did not predict response to immune checkpoint inhibitors. Moreover, in this study, 22.5% of NSCLC patients with high PD-L1 expression but low PD-1/PD-L1 interaction state QF-Pro® read-outsfailed to respond to therapies. Thus, if QF-Pro® would be used to stratify NSCLC patients to IO treatments, it could significantly boost patient response rates to these treatments.
As a versatile platform technology, QF-Pro® can be tailored to any functional protein target of interest. We work closely with clients to design and create novel functional biomarkers in a range of indications. We are constantly working to increase our off-the-shelf repertoire of biomarkers, of which you can find some examples below:
Immune Checkpoints
- PD1/PD-L1 interaction state – validated in cell models, ccRCC, NSCLC, melanoma, CRC and tonsil
- CTLA4/CD80 interaction state – validated in cell models, NSCLC, CRC-derived lung metastases and tonsil
- TIGIT/CD155 interaction state – validated in cell models, NSCLC, melanoma and tonsil
- LAG3/MHC II interaction state – validated in NSCLC, lymph nodes, thymus and appendix
- TIM-3/Gal9 interaction state – validated in NSCLC and tonsil
Example 1: Interaction of the immune checkpoint TIGIT/CD155 between Jurkat T cells and CHO-K1 cells.
Example 2: Activation state of Akt in clear cell renal cell carcinoma tissue.
Protein Activation States
- Akt/PKB activation state - validated in HNSCC, ccRCC, breast cancer, prostate cancer, sarcoma and cell models
- STAT3 activation state – validated in ccRCC and cell models
Protein interaction states
- HER2/HER3 interaction state – validated in cell models and FFPE tissue samples
- PKB/PDK1 interaction state – validated in cell models
- Beta-catenin/e-cadherin interaction state - validated in cell models, NSCLC and breast cancer
Example 3: Interaction state of HER2/HER3 in clear cell renal cell carcinoma tissue.
In addition to our validated biomarkers - please contact us to see how we can create a new QF-Pro® assay for you.