The tumor microenvironment (TME) is becoming a battlefield where cancer cells vie with cancer researchers for survival—the former for their own, the latter for cancer patients. Composed of tumor and immune cells, stroma, and extracellular matrix, the TME is a security buffer zone that a tumor creates to protect itself from immune system attacks and to help it thrive. The TME has profound effects on a tumor’s ability to metastasize, go dormant or die, or evade anti-cancer therapies. Therefore the nature of the TME is thought to contain clues to more effective immunotherapies.
Immunotherapies, such as checkpoint inhibitor or blockade therapy, harness the power of a patient’s own immune system to fight off cancer—when they work. Unfortunately, a disappointingly small proportion of patients respond to them. New technologies for profiling the spatial expression of biomarkers for immune and cancer cells in the TME indicate avenues to improve the proportion of immunotherapy responders. This article focuses on how spatial biomarker profiling of tumor samples is teaching researchers more about the TME, which may improve the effectiveness of cancer immunotherapy for more patients.
Multiplexed immunofluorescence
Tagging proteins with fluorescently labeled antibodies is a tried-and-true method whose power increases with greater multiplexing. Leica Microsystems’ Cell DIVE, a multiplexing imaging platform for research use only, maps expression of over 60 biomarkers in a tissue section with iterative staining, using a patented dye inactivation process. “This approach expands the number of biomarkers that can be examined for a given tissue sample, offering a more comprehensive view of the tumor microenvironment than what has been available with traditional chromogenic or fluorescence-based imaging techniques,” says Katie White, Cell DIVE product manager. The iterative staining rounds are gentle on tissues, which can be used in subsequent downstream applications.
Cell DIVE allows researchers to probe the spatial expression of multiple biomarkers in individual cells across the TME. A group headed by Michael Berens at the Translational Genomics Research Institute used Cell DIVE to compare glioma tumor samples from patients with a mutation in the IDH1 gene, compared to wild-type. Patients with mutant IDH1 have better prognoses compared to those with the wild-type gene, but the reason for this is unknown. Analysis with Cell DIVE revealed greater spatial heterogeneity in mutant tumors, which correlates with magnetic resonance imaging findings. Biomarker expression also showed patterns of cell functional states in wild-type tumors that are consistent with enhanced angiogenesis. “Additional studies further defining this heterogeneity may illuminate potential future therapeutic options for glioma patients,” says Melinda Angus-Hill, Cell DIVE global applications manager. “For example, given the interest in immunotherapy, Cell DIVE can define the landscape of immune cell infiltration in IDH1 mutant and WT gliomas.”
Akoya Biosciences also offers systems for spatially resolved, multiplex immunofluorescence in tissues: the CODEX platform for biomarker discovery, and the Phenoptics platform for translational and clinical biomarker research. A research group led by Garry Nolan at Stanford University recently used CODEX to study the TME in colorectal cancer, profiling 56 biomarkers at single-cell resolution. They found that the TME was organized into nine distinct “neighborhoods,” which interacted in ways that correlated with disease progression and patient prognosis. Harnessing such organized spatial interactions between groups of cells in the TME may open the door to new immunotherapies that benefit more patients.
Akoya’s Phenoptics platform plays a central role in a new collaboration with the Bloomberg-Kimmel Institute for Cancer Immunotherapy, and the Bloomberg Center for Physics and Astronomy, at the Johns Hopkins University School of Medicine. This collaboration aims to find predictive biomarker signatures in immuno-oncology by joining the spatial phenotyping of Phenoptics with Johns Hopkins’ AstroPath program. The Phenoptics platform generates spatial phenotypic signatures of tumor biopsy samples. AstroPath then tackles the challenge of analyzing multiplex immunofluorescence signals from cells in the tumor microenvironment, by applying an astronomy model for analyzing images of galaxies using celestial object–mapping algorithms. Drawing from expertise in immunology, pathology, computer science, and astronomy, AstroPath’s goal is to help identify the best treatment options based on tumor biopsy analyses.
Multiplexed ion beam imaging
For greater spatial information, Ionpath uses multiplexed ion beam imaging (MIBI™) technology for high-definition spatial proteomics. Tissues are labeled with antibodies tagged with elemental isotopes, and imaged using ion beams and time-of-flight mass spectrometry, to perform high-definition analysis of TME biology. “Our highly multiplexed approach was developed specifically for phenotypic analysis of FFPE and frozen tissue samples, both common sample types in preclinical and translational oncology studies,” says Brad Nelson, senior VP of marketing and corporate strategy at Ionpath. “Our platform was designed to fit a standard pathology workflow with FFPE tissue generating images with sub-cellular resolution, at the sensitivity required to detect and quantify proteins present at low or high abundance with clinical-grade reproducibility.”
Scientists use MIBI to learn more about the tumor microenvironments of patients who do and do not respond to treatment. When it comes to cancer immunotherapy, “we believe the tumor microenvironment is the most reliable readout of disease state and patient response,” says Nelson. “The Ionpath research services team can deliver a MIBI Spatial Signature that can predict response to treatment based upon spatial characteristics of tissue samples, such as cell abundance, protein expression, and spatial organization.” Recently a group headed by Michael Angelo in the pathology department at Stanford University used MIBI Spatial Signatures to predict disease progression in breast cancer patients with ductal carcinoma in situ —often a precursor to
Spatial profiling with barcodes
The GeoMx Digital Spatial Profiler (DSP) from NanoString Technologies uses barcodes for high-throughput studies of the spatial context of biomarkers in the TME. Tissue sections are labeled with antibodies tagged by fluorophores, and antibodies tagged by photocleavable oligonucleotides that will act as barcodes. Using the fluorescently labeled antibodies as visual guides, researchers zero in on a region of interest; then ultraviolet light shone on the region releases the barcodes from the oligo-tagged antibodies in that region. The freed oligo barcodes are collected and read by next-generation sequencing, or an nCounter gene expression system.
“GeoMx DSP is the only platform that enables segmentation of a region of interest into a pure tumor and pure microenvironment,” says Doug Farrell, VP of corporate communications at NanoString Technologies. “GeoMx allows researchers to use standard immunohistochemistry markers to visualize the tumor cells, and then uses micromirror technology to precisely shine light on those cells to release their barcodes and collect discrete cell populations.” The GeoMx DSP can assay hundreds of proteins in a sample, and can also perform whole transcriptome analysis as well as targeted RNA panels.
Last year, David Rimm’s lab at Yale University used the GeoMx DSP to examine the spatial context of biomarkers for PD-1 checkpoint blockade in samples from non-small cell lung cancer (NSCLC) patients. Though PD-1 checkpoint blockade therapy can save lives, only a minority of NSCLC patients benefit from it. Rimm’s group measured 39 immune parameters in four tissue compartments. They found that high levels of CD56 and CD4 markers measured in the leucocyte (CD45+) tissue compartment were predictive for all clinical outcomes, including progression-free and overall survival.
Our ability to identify spatially informed biomarkers in tissues is only just beginning to unlock the secrets of the TME and its constituents. It will be exciting to watch as researchers wield this powerful technology against cancer, and resistance to cancer therapies.