Secrets of the tumour microenvironment revealed with multiplex imaging

“The immune response is really important for cancer control, but it was neglected for decades,” says Anna Wilkins, urological cancer consultant at The Royal Marsden and clinician scientist at The Institute of Cancer Research in London.

Not anymore. Over the past couple of decades, researchers have been paying more attention to the co-evolution of tumours and the immune system. “We now know that the degree of immune-cell infiltration into a tumour influences its growth and how it can be treated,” Wilkins adds.

As immunotherapies become a first-line treatment for several cancers, oncologists acknowledge that a deeper understanding of the location and interaction of immune-cell subpopulations within the tumour microenvironment (TME) is crucial to guide the development of new therapies and the use of existing ones.

Despite solid evidence that it takes multiple biomarkers to indicate whether cancer patients are going to respond to immunotherapy, most treatment decisions are based on the detection of just one or two markers per tissue section using conventional immunohistochemistry.

Multiplex-imaging techniques involve simultaneous detection of multiple markers on a single tissue section. This approach massively enhances the spatial analysis of the cells and structures immediately surrounding a tumour.

“Multiplexing provides an overview of the battle between cancer cells and the immune system, which is impossible when staining for just one or two markers,” says Carlo Bifulco, medical director of oncological molecular pathology and pathology informatics at the Providence Oregon regional laboratory.

Advances in multiplex immunohistochemistry/immunofluorescence (m-IHC/IF) methods, including new dyes and cyclic staining, alongside helpful guidance on best practice, are allowing scientists to detect an increasing number of markers with high precision and accuracy.

“We are choosing therapies based on a very small amount of biological information when there is so much more data we could be using,” says Wilkins. “There is a huge potential to do better.”

Multiplex-imaging approaches

Antibodies that specifically and selectively bind to the target of interest are crucial to the detection of proteins in situ. Multiplexed antibody-based imaging methods are classified according to the type of antibody conjugate and the detection modality. m-IHC/IF imaging of antibodies conjugated with fluorophores, oligonucleotides and/or reporter enzymes, using a light microscope, is the most established method, and has a wide range of available reagents and imaging systems.

Pathologist Giorgio Cattoretti at The University of Milano-Bicocca, Italy, and colleagues in Leuven have developed a Multiple Iterative Labeling by Antibody Neodeposition (MILAN) method to sequentially immunostain formalin-fixed, paraffin-embedded (FFPE) tissue samples. “The MILAN method is very versatile and convenient for studying the TME,” he says.

MILAN relies on unconjugated and extensively validated primary antibodies and fluorochrome-conjugated secondary antibodies¹. “We found experimentally that Jackson ImmunoResearch have the best reagents available (and we tested most companies),” he notes.

The method involves sequential rounds of staining, imaging and removal of antibodies. A combination of the reducing agent beta-mercaptoethanol and the detergent sodium dodecyl sulphate (SDS) disassembles and removes the bound antibodies without damaging the tissue.

“We’ve shown that antigenic epitopes are preserved after more than 30 cycles of staining,” Cattoretti says. “Moreover, we can reproduce the results in the same sample six months later with less than 10% variation in signal intensity.”

With cyclic-staining methods, new dyes designed to fit in existing spectral gaps, and better detection equipment, researchers can easily image dozens of fluorophores in a single tissue section. However, they also need to be mindful of both the investment needed to process multiplexed samples and, ultimately, their clinical utility. “We need to weigh up the benefits of scanning large areas quickly and affordably for a small number of targets against very high-plex platforms that detect over 100 targets and generate fantastic amounts of biological data but from small areas of tissue,” says Wilkins.

Cooperative clinical research for multi-site multiplexing

Improving the intra- and inter-site reproducibility of multiplex data is essential if they are to be used in the clinic. Adequate antibody selection, antibody validation, panel design, staining optimization and validation, and correct data gathering and analysis strategies are key to minimizing variability between laboratories. Even then, there are some aspects of the process which make it difficult to standardize m-IHC/IF imaging, such as the use of different tissue-processing procedures and interobserver variability.

Several collaborative projects are aiming to harmonize multiplex imaging by developing workflows that can deliver robust and comparable m-IHC/IF data using similar tissue specimens. Results from the Cancer Immune Monitoring and Analysis Centers and Cancer Immunologic Data Commons (CIMAC-CIDC) network support aligning methodologies across multiple institutions, not by enforcing identical protocols, instruments and reagents, but rather by ensuring best practice and concordant quantification and interpretation of data².

Further tools are the community-validated organ-mapping antibody panels — combinations of antibodies that define cell populations and anatomical structures reproducibly in diverse tissues of human origin. These panels are not only enhancing standardization, but also save researchers time and money when carrying out multiplex imaging³. “Optimizing antibody-panel selection is crucial to appreciate the biological complexity of the TME and accelerate biological insights gained from multiplexed tissue imaging,” Cattoretti adds.

Several global initiatives aim to derive consensus-based checklists for m-IHC/IF data validation and reporting. Bifulco helps organize the Society for Immunotherapy of Cancer (SITC)’s multiplex immunofluorescence special interest group (MxIF-SIG). He highlights the importance of community efforts to translate the advances in multiplex imaging of the TME into better cancer outcomes. He puts the improvements in robustness and reproducibility of m-IF down to innovation at every step of the process — from staining platforms and protocols to image acquisition and analysis platforms.

The SITC has already published a statement on best practices for m-IHC/IF staining and validation⁴,and will soon be releasing another on best practices for reporting the obtained data. “The field is booming right now,” says Bifulco. “The aim of these guidance documents is to improve the quality of the papers that are submitted and published.”

Wilkins is excited about the developments in the field, particularly around how machine-learning algorithms for images will improve understanding of clinically relevant TME features⁵. “As m-IF becomes integrated with spatial omics and advanced data analysis,” she says, “we’ll be able to realise its full potential, improve patient selection and direct personalized therapy choices.”