48 Advances in multiplexed ion beam imaging (MIBI) for immune profiling of the tumor microenvironment

Abstract

BackgroundMultiplexed ion beam imaging (MIBI) combines time-of-flight secondary ion mass spectrometry (ToF-SIMS) with metal labeled antibodies to image 40+ proteins in a single scan at subcellular spatial resolution. Here, we show that the recently released MIBIscope provides improved sensitivity for detecting immune checkpoint markers and offers greater throughput at higher resolution than the alpha instrument.MethodsSerial sections from three FFPE NSCLC samples, in addition to a control slide consisting of various unremarkable tissues, were stained with a panel of 25 metal labeled antibodies. The tissue was imaged at subcellular resolution using the MIBIscope and the alpha instrument. Masses of detected species were assigned to target biomolecules given the unique label of each antibody and multi-step processing was used to create images. Cell classification was performed using two complementary methods that differed in the need for cell segmentation to phenotypically characterize the tissue environments and quantify marker expression.ResultsReplicate regions of interest (ROIs) were collected on both instruments with similarly sized ROIs acquired in 17 minutes with the MIBIscope compared to 280 minutes with the alpha instrument. Fourier Ring Correlation (FRC) showed the resolution to be greater on the MIBIscope as compared to the alpha instrument with FRC also demonstrating uniform resolution across an ROI 2.5X greater in size. Even with the 16X greater speed of the MIBIscope, the signal of the 25 markers across replicate ROIs was increased (y=x^1.07) and showed similar expression patterns to those observed on the alpha instrument (figure 1). This resulted in greater sensitivity to markers with low expression, such as checkpoint markers. Eleven cell populations were classified across the ROIs utilizing two methods, with both methods showing a similar frequency of tumor cells and B, T, and myeloid cell subsets between instruments. Segmentation enabled the number of cells within a population to be calculated but defining boundaries is laborious and signal from neighboring cells can result in misclassification. Performing classification at the pixel level, without segmentation, enabled the fraction of the tissue that is tumor or any other cell type to be rapidly determined.Abstract 48 Figure 1Comparison of images acquired between instrumentsThe signal intensity is greater on the MIBIscope and shows a similar staining pattern as achieved by the alpha instrument. Shown are 3 overlays from a single scan from replicate ROIs of an NSCLC sample displayed with the same contrast settings.ConclusionsThe MIBIscope enables the phenotypic characterization of tumor and non-tumor microenvironments. Co-expression of markers can be used to classify tumor and immune populations and to quantify the expression of markers associated with immune suppression. The increased sensitivity and throughput of the MIBIscope, in combination with the 40-parameter capability and subcellular resolution, provides a platform uniquely suited to understanding the complex tumor immune landscape.

The Pandora’s box of novel technologies that may revolutionize lung cancer

“…Multiplexed ion beam imaging (MIBI) Technology is developed on the basis of secondary ion mass spectrometry (SIMS) [86]. SIMS works by rastering a primary ion beam across the surface of a sample and releasing the reporting ions, which are then recorded pixel by pixel using TOF detection.…”

“…SIMS works by rastering a primary ion beam across the surface of a sample and releasing the reporting ions, which are then recorded pixel by pixel using TOF detection. In comparison to a laser, an ion beam allows for resolution adjustment over a wide range, such as 280 nm to 1 µm in the case of the MIBIScope [86]. Once released, the reporter ions travel at supersonic speeds from the sample to the detector, resulting in rapid acquisition and exceptional sensitivity [87].…”

“…In comparison to a laser, an ion beam allows for resolution adjustment over a wide range, such as 280 nm to 1 µm in the case of the MIBIScope [86]. Once released, the reporter ions travel at supersonic speeds from the sample to the detector, resulting in rapid acquisition and exceptional sensitivity [87]. This technology is therefore capable of quantifying protein expression, phenotyping immune infiltrates, and profiling tissue architecture.…”

“…This technology is therefore capable of quantifying protein expression, phenotyping immune infiltrates, and profiling tissue architecture. MIBI can also be applied to all common sample types, for example, FFPE and fresh-frozen tissue [87]. Using a standard IHC staining protocol, a tissue sample is stained with metal-tagged antibodies [88].…”

Direct detection of 5-MeV protons by flexible organic thin-film devices

“…For this reason, there is an increasing demand of systems optimized for the accurate recording and mapping of the dose delivered during a treatment plan (3). Besides this aspect, the next frontier for medical applications of proton beams is proton tomography, where high-energy (230 to 250 MeV) proton beams are used through the patient (4,5).…”