Papers by Michel Nederlof
Scientific reports, Mar 28, 2024
Scientific Reports, Nov 28, 2017
We describe here a method, termed immunoFISH, for simultaneous in situ analysis of the compositio... more We describe here a method, termed immunoFISH, for simultaneous in situ analysis of the composition and distribution of proteins and individual RNA transcripts in single cells. Individual RNA molecules are labeled by hybridization and target proteins are concurrently stained using immunofluorescence. Multicolor fluorescence images are acquired and analyzed to determine the abundance, composition, and distribution of hybridized probes and immunofluorescence. We assessed the ability of immunoFISH to simultaneous quantify protein and transcript levels and distribution in cultured HER2 positive breast cancer cells and human breast tumor samples. We demonstrated the utility of this assay in several applications including demonstration of the existence of a layer of normal myoepithelial KRT14 expressing cells that separate HER2+ cancer cells from the stromal and immune microenvironment in HER2+ invasive breast cancer. The abundance, composition and spatial distribution of diverse RNA transcripts and proteins are fundamental determinants of the behavior of normal and cancer cells and numerous technologies have been developed to measure these cellular features. In the past decade, work has focused on the development of 'omic methods such as mass spectrometry and massively parallel sequencing that enable comprehensive and quantitative measures of protein and transcript profiles 1,2 . These methods have enabled discovery of important functional components of normal cells and have revealed changes in specific transcripts and proteins from normal that suggest mechanisms by which diseases arise, progress and respond to treatment. These analyses also have guided the development of new therapeutic approaches and diagnostic assays and stimulated the current interest in precision medicine. However, 'omic analysis methods provide little information about the rich and functionally important variations in transcriptional and proteomic features that exist between and within cells. In cancer, for example, variation between cells is caused by genomic and epigenomic instability and variability in signals from the diverse microenvironments in which the cancer cells reside 6-9 . These differences may translate into differences in biological behavior, metastatic potential 10 and therapeutic sensitivity 11 . Assessment of the locations of specific mRNA transcripts and proteins within cells allows study of a variety of cancer related processes; for example, nuclear processing of nascent pre-messenger RNA (pre-mRNA) transcripts, alternative splicing and receptor recycling following treatment with targeted therapeutics and the spatial organization of molecularly distinct cells within tissues. We show in this paper that simultaneous in situ measurement of transcripts location and composition and protein levels is possible in most circumstances by combining aspects of RNA fluorescence in situ hybridization (FISH) and immunofluorescence staining. In our approach, hereafter referred to as immunoFISH, we detect specific transcripts using synthetic oligonucleotide probes labeled with bright fluorophores that target multiple regions along the desired transcripts and specific proteins by immunofluorescent staining. Signals from fluorescently labeled proteins and individual transcripts are quantified by analysis of images acquired using high-resolution fluorescence microscopy. This approach builds on previous reports showing that coding and noncoding RNA transcripts can be quantified and spatially localized and even sequenced in situ . We demonstrate the utility of immunoFISH by assessing the location and abundance of HER2 introns and exons relative to the nuclear boundary defined by immunostaining for nuclear lamins and HER2 protein in
Scientific Reports, Mar 15, 2018
Multicolor microscopy tools necessary to localize and visualize the complexity of subcellular sys... more Multicolor microscopy tools necessary to localize and visualize the complexity of subcellular systems are limited by current fluorophore technology. While commercial fluorophores cover spectral space from the ultraviolet to the near infrared region and are optimized for conventional bandpass based fluorescence microscopy, they are not ideal for highly multiplexed fluorescence microscopy as they tend to have short Stokes shifts, restricting the number of fluorophores that can be detected in a single sample to four to five. Herein, we synthesized a library of 95 novel boron-dipyrromethene (BODIPY)based fluorophores and screened their photophysical, optical and spectral properties for their utility in multicolor microscopy. A subset of our BODIPY-based fluorophores yielded varied length Stokes shifts probes, which were used to create a five-color image using a single excitation with confocal laser scanning microscopy for the first time. Combining these novel fluorophores with conventional fluorophores could facilitate imaging in up to nine to ten colors using linear unmixing based microscopy approaches.
Simultaneous Detection of RNAs and Proteins with Subcellular Resolution
Methods in molecular biology, 2020
We describe the detailed methods of "immunoFISH" to analyze the expression level and th... more We describe the detailed methods of "immunoFISH" to analyze the expression level and the spatial localization of RNA transcripts and proteins on cultured cells and formal-fixed, paraffin-embedded (FFPE) tissue sections. On cultured cells, we detect specific transcripts using the Stellaris fluorescence in situ hybridization (FISH) probes labeled with fluorophores that target multiple regions along the desired transcripts and proteins combining the immunofluorescent staining. On FFPE tissue sections, we use the RNAscope FISH probes, modified branched DNA (bDNA) probes to amplify the RNA signals, followed by immunofluorescent staining for protein detection. The abundance, composition, and spatial distribution are determined by signals from fluorescently labeled proteins and individual transcripts of images acquired using high-resolution fluorescence microscopy.
Goettingen image processing system gips
Cell systems, Mar 1, 2018
Extrinsic signals are implicated in breast cancer resistance to HER2-targeted tyrosine kinase inh... more Extrinsic signals are implicated in breast cancer resistance to HER2-targeted tyrosine kinase inhibitors (TKIs). To examine how microenvironmental signals influence resistance, we monitored TKI-treated breast cancer cell lines grown on microenvironment microarrays composed of printed extracellular matrix proteins supplemented with soluble proteins. We tested ~2,500 combinations of 56 soluble and 46 matrix microenvironmental proteins on basal-like HER2+ (HER2E) or luminal-like HER2+ (L-HER2+) cells treated with the TKIs lapatinib or neratinib. In HER2E cells, hepatocyte growth factor, a ligand for MET, induced resistance that could be reversed with crizotinib, an inhibitor of MET. In L-HER2+ cells, neuregulin1-β1 (NRG1β), a ligand for HER3,
Journal of Visualized Experiments, May 21, 2019
Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem... more Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth factors and matrix-associated proteins in the microenvironment in vivo. Furthermore, readily available reagents for the modeling of microenvironments in vitro typically utilize complex mixtures of proteins that are incompletely defined and suffer from batch to batch variability. The microenvironment microarray (MEMA) platform allows for the assessment of thousands of simple combinations of microenvironment proteins for their impact on cellular phenotypes in a single assay. The MEMAs are prepared in well plates, which allows the addition of individual ligands to separate wells containing arrayed extracellular matrix (ECM) proteins. The combination of the soluble ligand with each printed ECM forms a unique combination. A typical MEMA assay contains greater than 2,500 unique combinatorial microenvironments that cells are exposed to in a single assay. As a test case, the breast cancer cell line MCF7 was plated on the MEMA platform. Analysis of this assay identified factors that both enhance and inhibit the growth and proliferation of these cells. The MEMA platform is highly flexible and can be extended for use with other biological questions beyond cancer research.
<title>Knowledge-driven image analysis of cell structures</title>
Proceedings of SPIE, May 1, 1991
ABSTRACT Digital Imaging Light Microscopy technology has made major advancements over the past ye... more ABSTRACT Digital Imaging Light Microscopy technology has made major advancements over the past years. Changes have included significant improvements in sensitivity and image quality, but also the advent of innovative methods that have greatly increased the versatility of instruments. Methods to yield contrast in the specimen have made leaps in specificity and signal-to-noise ratio. The combination of these advanced technologies now permits the acquisition of 2 and dimensional image data, of up to five independently labeled parameters, in fixed specimens, or - in time lapse series - of living cells. Given the vast amount of data that can be generated with these instruments, there is a growing need for image analysis software that can yield quantitative measurements from these complex images. The authors are developing a knowledge driven image analysis system that is capable of handling the described image data. The proposed architecture tries to optimize robustness by taking advantage of knowledge that is available both from the expert user as well as from the multiple image sources. It therefore offers significant potential for improvements in performance over linear and mathematical morphology based systems. The authors applied this system to trace actin-based fiber bundles using knowledge constraints, and analyzed their distribution, shape, sizes and orientation in fixed cells by immunofluorescence with good results.
The New Technology of Light Microscopy
American Scientist, Jul 1, 1992
Quantitation of cytoskeletal fibers in fluorescence images: Stress fiber disassembly accompanies dephosphorylation of the regulatory light chains of myosin II
Bioimaging, Sep 1, 1993
ABSTRACT Stress fibers in serum-deprived fibroblasts provide an excellent system for studying the... more ABSTRACT Stress fibers in serum-deprived fibroblasts provide an excellent system for studying the assembly dynamics of the actin-based cytoskeleton. Previous studies suggested that fibers containing actin and myosin II are restructured via two alternative pathways: coupled solation-contraction and disassembly without contraction. The former process appears to be regulated by Ca++, but regulation of the latter process has not been extensively explored. To assess the possible role of protein phosphorylation, we examined the dynamic behavior of stress fibers in cells treated with inhibitors of protein kinases. Swiss 3T3 fibroblasts were microinjected with fluorescent analogs of actin and myosin II and fiber dynamics monitored using light-microscope imaging. Staurosporine and KT5926 caused reversible dispersal of stress fibers without contraction, along with dephosphorylation of the regulatory light chain of myosin II, LC20. In order to make direct comparisons between the dose responses of these biochemical and morphological effects, fiber disruption was quantitated using two independent measures: total edge strength in fluorescent images of actin and myosin II, and fiber length determined by automated object-identification. Loss of stress fibers was shown to parallel LC20 dephosphorylation. Quantitation of cytoskeletal organization provides a framework for testing relationships between structural events and potential biochemical regulatory signals.
Journal of pathology informatics, 2011
For many years pathologists have used Hematoxylin and Eosin (H&E), single marker immunohistochemi... more For many years pathologists have used Hematoxylin and Eosin (H&E), single marker immunohistochemistry (IHC) and in situ hybridization with manual analysis by microscopy or at best simple digital imaging. There is a growing trend to update pathology to a digital workflow to improve objectivity and productivity, as has been done in radiology. There is also a need for tissue-based multivariate biomarker assays to improve the accuracy of diagnostic, prognostic, and predictive testing. Multivariate tests are not compatible with the traditional single marker, manual analysis pathology methods but instead require a digital platform with brightfield and fluorescence imaging, quantitative image analysis, and informatics. Here we describe the use of the Hamamatsu NanoZoomer Digital Pathology slide scanner with HCImage software for combined brightfield and multiplexed fluorescence biomarker analysis and highlight its applications in biomarker research and pathology testing. This combined approach will be an important aid to pathologists in making critical diagnoses.
Scientific Reports
A number of highly multiplexed immunostaining and imaging methods have advanced spatial proteomic... more A number of highly multiplexed immunostaining and imaging methods have advanced spatial proteomics of cancer for improved treatment strategies. While a variety of methods have been developed, the most widely used methods are limited by harmful signal removal techniques, difficulties with reagent production and antigen sensitivity. Multiplexed immunostaining employing oligonucleotide (oligos)-barcoded antibodies is an alternative approach that is growing in popularity. However, challenges remain in consistent conjugation of oligos to antibodies with maintained antigenicity as well as non-destructive, robust and cost-effective signal removal methods. Herein, a variety of oligo conjugation and signal removal methods were evaluated in the development of a robust oligo conjugated antibody cyclic immunofluorescence (Ab-oligo cyCIF) methodology. Both non- and site-specific conjugation strategies were assessed to label antibodies, where site-specific conjugation resulted in higher retained ...
Fluorescent imaging technologies for in situ measurement of drug target engagement and cell signaling pathways
Successful cancer treatment continues to elude modern medicine and its arsenal of therapeutic str... more Successful cancer treatment continues to elude modern medicine and its arsenal of therapeutic strategies. Therapy resistance is driven by significant tumor heterogeneity, complex interactions between malignant, microenvironmental and immune cells and cross talk between signaling pathways. Advances in molecular characterization technologies such as next generation sequencing have helped unravel this network of interactions and identify druggable therapeutic targets. Tyrosine kinase inhibitors (TKI) are a class of drugs seeking to inhibit signaling pathways critical to sustaining proliferative signaling, resisting cell death, and the other hallmarks of cancer. While tumors may initially respond to TKI therapy, disease progression is near universal due to mechanisms of acquired resistance largely involving cellular signaling pathway reprogramming. With the ultimate goal of improved TKI therapeutic efficacy our group has developed intracellular paired agent imaging (iPAI) to quantify dr...
Fluorescent Imaging for In Situ Measurement of Drug Target Engagement and Cell Signaling Pathways
Proceedings of SPIE--the International Society for Optical Engineering, 2020
Successful cancer treatment continues to elude modern medicine and its arsenal of therapeutic str... more Successful cancer treatment continues to elude modern medicine and its arsenal of therapeutic strategies. Therapy resistance is driven by significant tumor heterogeneity, complex interactions between malignant, microenvironmental and immune cells and cross talk between signaling pathways. Advances in molecular characterization technologies such as next generation sequencing have helped unravel this network of interactions and identify druggable therapeutic targets. Tyrosine kinase inhibitors (TKI) are a class of drugs seeking to inhibit signaling pathways critical to sustaining proliferative signaling, resisting cell death, and the other hallmarks of cancer. While tumors may initially respond to TKI therapy, disease progression is near universal due to mechanisms of acquired resistance largely involving cellular signaling pathway reprogramming. With the ultimate goal of improved TKI therapeutic efficacy our group has developed intracellular paired agent imaging (iPAI) to quantify dr...
Essai automatise servant a identifier des cellules individuelles pendant des essais cinetiques
L'invention concerne des procedes et un logiciel servant a reperer des cellules individuelles... more L'invention concerne des procedes et un logiciel servant a reperer des cellules individuelles pendant un essai de criblage de cellule cinetique.
Many critical advances in research utilize techniques that combine high-resolution with high-cont... more Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSimaTM Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at sub-cellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinityTM), release of antibodies (REAleaseTM) or their labels (REAdyeleaseTM). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 normal tissues. One potential target pair for chimeric antigen receptor (CAR) T-cell therapies identified for ovarian carcinoma is EPCAM/THY1. Using an adapter CAR T cell approach, we show selective killing of cells only in presence of both markers. MICS represents a ...
Identification of cells during kinetic test series
The New Technology of Light Microscopy
Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues XVII, Mar 4, 2019
Successful cancer treatment continues to elude modern medicine and its arsenal of therapeutic str... more Successful cancer treatment continues to elude modern medicine and its arsenal of therapeutic strategies. Therapy resistance is driven by significant tumor heterogeneity, complex interactions between malignant, microenvironmental and immune cells and cross talk between signaling pathways. Advances in molecular characterization technologies such as next generation sequencing have helped unravel this network of interactions and have vastly affected how cancer is diagnosed and treated. However, the translation of complex genomic analyses to pathological diagnosis remains challenging using conventional immunofluorescence (IF) staining, which is typically limited to 2–5 antigens. Numerous strategies to increase distinct antigen detection on a single sample have been investigated, but all have deleterious effects on the tissue limiting the maximum number of biomarkers that can be imaged on a single sample and none can be seamlessly integrated into routine clinical workflows. To facilitate ready integration into clinical histopathology, we have developed a novel cyclic IF (cycIF) technology based on antibody conjugated oligonucleotides (Ab-oligos). In situ hybridization of complementary oligonucleotides (oligos) facilitates biomarker labeling for imaging on any conventional fluorescent microscope. We have validated a variety of oligo configurations and their respective signal removal strategies capable of diminishing fluorescent signal to levels of autofluorescence before subsequent staining cycles. Robust signal removal is performed without the employment of harsh conditions or reagents, maintaining tissue integrity and antigenicity for higher dimensionality immunostaining of a single sample. Our platform Ab-oligo cycIF technology uses conventional fluorophores and microscopes, allowing for dissemination to a broad audience and congruent integration into clinical histopathology workflows.
Abstract PD5-01: Microenvironment microarrays show that microenvironment mediated resistance mechanisms to lapatinib differ between basal and luminal HER2+ cells
Cancer Research
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Papers by Michel Nederlof