Immunohistochemistry and mass spectrometry for highly multiplexed cellular molecular imaging

Abstract

The role of immunohistochemistry (IHC) in the management of cancer has expanded to provide improved diagnostic classification, as well as guidance on disease prognosis, therapy, and relapse. These new tasks require evaluation of an increasing number of protein targets; however, conventional multiplexing, usually achieved using serial tissue sections stained for a single analyte per slide, can exhaust small biopsy specimens, complicate slide-to-slide protein expression correlation, and leave insufficient material for additional molecular assays. A new approach, mass spectrometry immunohistochemistry (MSIHC), compatible with high levels of target multiplexing and suitable for use on formalin-fixed, paraffin-embedded samples can circumvent many of these issues. The strategy employs antibodies that are labeled with elemental mass tags, such as isotopically pure lanthanides not typically found in biological specimens, rather than with typical fluorophores or chromogens. The metal-labeled antibodies are then detected in tissue using lasers or ion beams to liberate the tags for subsequent mass spectrometry detection. Within a given multiplexed IHC panel, the metal labels are selected so that their respective masses do not overlap. More than 30 antibodies have been imaged simultaneously, and up to 100 antibodies could potentially be detected at once if the full available mass spectrum is deployed. MSIHC has a number of advantages over conventional IHC techniques. Background due to autofluorescence is absent and the dynamic range is 10 5 , exceeding immunofluorescence and chromogenic IHC by 100-fold and 1000-fold, respectively. Detection of labeled primary antibodies improves assay linearity over both chromogenic and fluorescent IHC. Multiplexed mass-tagged antibodies incubated simultaneously with tissue do not appear to cross-interfere, and because the mass tags do not degrade, samples are stable indefinitely. The imaging resolution of multiplexed ion-beam imaging can be better than light microscopy. With appropriate instrumentation, MSIHC has the potential to transform research and clinical pathology practice.

Multiplexed immunofluorescence delineates proteomic cancer cell states associated with metabolism

“…These isotopes are not found in normal tissue, eliminating problems with tissue background signal and resulting in high sensitivity. However, this technology is limited by the availability of metal tags (only ~32) and validated antibody conjugates (34). Furthermore, the latter approach requires sample ablation for isotope release, rendering samples unsuitable for additional downstream analysis such as FISH.…”

Biologically Relevant Heterogeneity: Metrics and Practical Insights

“…Samples are ionized either with a laser or ion beam, the metal tags are quantified, and then the images are computationally reconstructed based on known raster positions of the laser or ion beams. 93, 102, 103 These approaches are still developing, but have already enabled quantification of >40 parameters at the single cell level, 103, 104105 and have been used to detect heterogeneity in breast cancer tissues 93, 106 …”

When machine vision meets histology: A comparative evaluation of model architecture for classification of histology sections

“…It is also worth to mention that alternative staining (e.g., fluorescence) and microscopy methods (multi-spectral imaging) have been proposed and studied in order to overcome the fundamental limitations/challenges in tissue histology (Stack et al, 2014; Levenson et al, 2015; Rimm, 2014; Huang et al, 2013; Ghaznavi et al, 2013); however, H&E stained tissue sections are still the gold standard for the assessment of tissue neoplasm. Furthermore, the efficient and effective representation and interpretation of H&E tissue histology sections in large cohorts (e.g., The Cancer Genome Atlas dataset) have the potential to provide predictive models of genomics and clinical outcome, and are therefore urgently required.…”