Ex-Vivo and Pathology Services

Invicro’s Advanced Pathology Services (APS) team is focused on supporting our sponsors’ discovery and development efforts by providing a combination of unique and high-quality tissue imaging and measurement services. We also work in conjunction with Konica Minolta’s broad precision medicine service portfolio to support a comprehensive biomarker discovery and validation strategy. APS provides a diverse range of services that enables visualization and quantitation of morphological features and protein expression in tissues. Our consultative approach allows us to collaborate with investigators to recommend the best methods to meet the goals of the project and play an integral role in protocol development and validation.

Core Capabilities Expermimental Techniques Available
Routine and specialized histochemical stains 2D and 3D quantitative autoradiography
Immunohistochemistry (IHC) Whole-body autoradiography
Immunofluorescence (IF) IHC Micro-autoradiography (MARG)
High sensitivity quantitative IHC using Konica Minolta's phosphor integrated dots (PIDs) technology Cryo-fluorescence tomography (CFT)
Pathologist consultation and staining interpretation for our IHC services Fluorescence Microscopy
Receptor binding assays
Image Analysis: Custom analysis tools and segmentation available
Machine Learning

Our experienced team of research scientists is trained and prepared to support projects at any drug development phase, from pre-clinical to late-phase clinical trials, supporting all therapeutic areas including; oncology, neuroscience, immunology, and organ system pathology (i.e. bone and joint, ocular, renal, and CNS).

We provide a comprehensive panel of markers for evaluation of pathophysiological events (i.e. immuno-oncology, proliferation, apoptosis, vascularization, inflammation), and validation services for new markers for both paraffin embedded and frozen samples.  Our skilled scientists have experience working with a broad range of organisms, including human, non-human primates and rodents, which we continue to expand for antibody optimization.

Invicro’s unique pathology specialties:

  • Support discovery research projects by performing pilot optimization work on novel and challenging techniques.
  • A multimodal approach of co-registering ex vivo 3D images with in vivo nuclear scans generate temporal and spatial information from low to high resolution in the same sample.
  • Offer routine and custom image analysis:
    • Standard bright field and epifluorescence microscopy of thin sections,
    • Whole slide scanning
    • Signal co-localization
    • Signal quantification
    • Segmentation and classification
  • Automated workflow for better inter- and intra-observer reproducibility.

Through our sister company, Ambry Genetics, our histology lab is College of American Pathologists (CAP)-accredited and Clinical Laboratory Improvement Amendments (CLIA)-certified.

Dr. Kenneth Bloom, Chief Medical Officer, APS and Advanced Genomics Services (AGS), Invicro and Ambry Genetics is a recognized leader and innovator in the industry, and is actively involved in developing collaborations that best align our pathology services with the needs of our pharmaceutical sponsors.

Contact us for more information.

  • Quantitative Immunohistochemistry

    Quantitative IHC assays developed by Konica Minolta’s precision medicine group utilizes a novel fluorescent phosphor-integrated dots (PIDs) technology that provides superior sensitivity and uniformity compared to conventional immunofluorescent imaging and immunohistochemical methods; thus allowing quantitative assessment of target proteins. PIDs are organic fluorophore assembly-conjugated nanoparticles with unique properties including: uniform size, high intensity fluorescent signal (30,000X brighter than conventional fluorescent dyes), high photostability (densely packed fluorophores networked via chemical binding), high sensitivity, and broad dynamic range (10-6 to 10-1 mM).

    Used extensively in Japan, and now available through Invicro’s advanced pathology laboratory in Boston, primary applications to which PIDs can be applied towards include:

    1. Direct detection of a therapeutic agent (i.e. peptides, antibodies, ADCs) in tissue sections following intravital administration.
    2. Quantitative scoring of protein expression level in tissue sections.
    3. Detection of proteins with low cellular expression.

    As part of Invicro’s quantitative biomarker tissue services, dedicated image-processing algorithms are utilized that provides readouts (i.e. PID score/cell, PID score/ROI, number of drugs bound/cell) for expression levels of target proteins on tissues.

    Examples:

    • Wide selection of verified antibody clones for clinical markers. A suite of antibody clones for clinical markers (i.e. HER2, HER3, PD-L1, PD-1, EGFR, ki67, ER, PR, CSF1R and many more) are under development. Screening and validation of new markers, by performing rigorous evaluation mimicking the intended diagnostic situation, is available.
    • Multiplex platform. For a more comprehensive coverage of tumor immune contexture, the assay is amenable for multiplexing with standard IHC for markers i.e. CTL (CD8), TAMs (CD68, CD163, CSF1R, Tregs (FoxP3, CD25), CAFs (smooth muscle actin, vimentin).
    • Quantitative analysis of ADCs. Whole tumor and intra-tumor PK analysis provides quantitative results on trastuzumab distribution.
    • High-resolution imaging of cellular receptors. In clinical breast cancer samples (hormone receptor +ve and Her 2 -ve), non-nuclear ER expression showed superior prognostic value compared to the total ER. (Matsukane et al. (2017) AACR).
    • Improved patient stratification with prognostic value. PID-IHC provides distinction between pCR and non-pCR cohorts when compared to other methods of detection of HER2 expression (DAB-score, DAB-IHC, or FISH score) (Gonda et al. (2017)). Similarly, PD-L1 staining of PDAC samples showed negative correlation between PID-IHC and overall survival; TIC score, determined by DAB-IHC duplexing was found to be an independent prognostic factor (Yamaki et al. (2017) Int J. Clin. Oncol. 22, 726).
    • Integration of PID-IHC with clinically used diagnostic method improves patient stratification. PID score was determined in breast cancer samples and a subgroup was identified that was FISH positive (polysomy “classical” amplified cases) but with low PID-IHC score. Speculatively, these patients may not respond to HER2 targeted therapy. (Shen et al. (2018) USCAP).
  • Cryofluorescence tomography (CFT)

    CFT is an ex vivo multimodality imaging technique that provides white light anatomical and molecular fluorescence 3D images with micron scale resolution. CFT bridges the gap between fluorescence histology and bulk fluorescence imaging by providing high resolution imaging of multiple fluorophores simultaneously in a range of sample sizes (i.e. rodent organ, whole rodent body, dog/monkey head). In this technique, block-face 2D epi-fluorescence images are captured using sequential section-image cycles and corrected for sub-surface fluorescence. A high-throughput imaging workflow can be established by embedding multiple samples per block to be imaged. Histological sections can be selected during imaging based on real-time molecular information, eliminating guesswork in collection. The 3D mapping of the fluorescent signal is done with anatomical white light reference (endogenous or contrast enhanced) using the Invicro’s software platform. Images can be viewed by maximum intensity projection (MIP) display, slice-by-slice flythrough of the white light and fluorescence images, or via 3D multiplane rendering. We use a fluorescence imaging system customized with superior NIR and visible imaging capabilities supported by Emit Imaging http://emit-imaging.com and Curadel.

    Fig 1. A general volumetric resolution for fluorescent imaging technologies as a function of interrogation scale (subcellular to whole-body). The cluster of technologies in the upper right-hand corner correspond to in vivo “macro” imaging while ex vivo fluorescent microscopy technologies are shown in the lower left-hand corner. Adapted and modified from Hargreaves et al (2015). Optimizing Central Nervous System Drug Development Using Molecular Imaging. Clin. Pharmacol. Ther. 98(1):47-60.

    Image analysis, Access and Storage Using VivoQuant®
    VivoQuant® image processing software enables co-registration of cryoimaging to other imaging modalities (e.g. MRI, CT), while seamlessly integrating with the iPACS database system for easy access Each subject is associated with metainformation including unique identification numbers for the subject, series, and study in accordance with DICOM standards.

    Features and Applications
    Few unique applications of CFT technique include –

    1. Capability for multispectral fluorescence imaging click here for visuals.
    2. Assessment of temporal drug PK kinetics in the organ of interest.CFT technique enables PK/PD analysis of the fluorescence tracer at the organ level (i.e. brain).  After necropsy, the specimen is immediately cryopreserved (OCT or dry ice) to capture real time in vivo drug distribution. Notably, white light images can be used, with established reference tools, to perform anatomical segmentation.
    3. White light images can be used for anatomical segmentation. Endogenous or contrast enhanced (i.e. India ink, Evans blue dye) can be used for anatomical referencing. Wolf et al (2016). Dynamic dual-isotope molecular imaging elucidates principles for optimizing intrathecal drug delivery (view distribution maps of probes in the CSF space).
    4. Imaging of micro-metastasis in cancer. CFT provides superior resolution with improved sensitivity compared to other imaging modalities like epi-fluorescence imaging, and bioluminescence imaging.
    5. Multimodal imaging capability. CFT images integrate with other modalities (i.e. MRI, CT).
    6. Biodistribution analysis of tracers in whole animal across multiple organ systems. Potential applications include imaging gene transduction, adoptively transferred stem cells or immune cells.

    #1:

    In this study, multispectral CFT was performed on a rat kidney to simultaneously visualize the renal clearance of PEG909-ZW800-1 and PEG909-ZW700-1 tracers. Spectral filtration was used to gather fluorescence images of both tracers simultaneously. White light images were concurrently captured to provide anatomical context. Images were aligned and built into a 3D fluorescence distribution. A correction algorithm was applied to the fluorescence images to mitigate the effect of optical scattering and absorption.  Reconstructed images for both tracers are shown below.

    3D rendering of the kidney was generated in VivoQuant®™ using the maximum intensity projection feature, enhancing the visualization of renal fluorescence distribution as well as structural mapping. The 3D distribution of both tracers in the kidney is shown in a maximum intensity projection.

    Click to animate:

    The Intravenously injected tracer.

    The Intravenously injected tracer.

    The Intrathecally injected tracer.

    The Intrathecally injected tracer.

    A slice-by-slice overlay of the white light and the fluorescence images were also created to better visualize the fluorescence distribution within the kidney. Additionally, the multiview layout in VivoQuant®™ was used to generate a slice-by-slice flythrough of the white light and fluorescence images. The fluorescence recovery and white light flythroughs are shown below.
  • Autoradiography Services

    The APS core offers autoradiography (ARG) services to assess radiotracer distribution in whole body (mice, rats) and in discrete organs, tissues, and cells. Whole body ARG enables benefits i.e. visual localization of drug pharmacokinetics, drug penetration into specific targets, besides also enabling applications like drug screening during the lead optimization process. A quantitative and 3D analysis of ARG signal can be performed using radioactive fiducials. We offer co-registration of ARG with high resolution optical modalities i.e. bright field images for anatomical referencing.

    At APS, we also perform microautoradiography (MARG, 0.3 µm image resolution) on frozen sections (soft tissues) enabling localization of radiotracer at the cellular and sub-cellular level. Other offerings include immersion ARG for analyzing radioligand binding parameters in tissues.

     

    Case studies:

      • Whole body 2D ARG can be used to infer tissue biodistribution of a radiotracer. ARG images can be aligned with a sister section images (i.e. optical, fluorescent, or histological stain) to gather superior spatial information of the in vivo distribution of a radiotracer.

     

      • Cryo-imaging quantitative ARG (CIQA). To evaluate the intratumoral distribution of a dual-labeled ADC in a preclinical setting, we combined CIQA with high-resolution white light images and 3D modeling to gain a simultaneous readout of both antibody and payload distribution in the tumor. Ohad et al (2018).
        CIQA technique can be applied to whole body imaging, simultaneous imaging of multiple organs, and is also amenable to a medium-throughput screening of several specimens (i.e. tumor, brains) embedded within a single block. A 3-D ARG reconstruction with volume rendering can be performed using the slice plane to gain a more detailed in vivo information on tracer distribution.

     

    • Tissue binding assays to determine specific binding parameters of a radioligand. In this study, rat brains were used for specific binding assay with 3H-Flumazenil (a GABA receptor antagonist). Parameters i.e. KD (ligand concentration that binds to half the receptor sites at equilibrium) and Bmax (maximum number of binding sites) were calculated.
  • A list of different proposed IHC antibodies (human, FFPE)

    Marker Clone Source
    PD-L1 22C3 & SP263 Dako (22C3) & Ventana (SP263)
    CD8 OKT8 Dako
    CD68 KP1 Dako
    CD4 OKT4 Cell Marque
    CD20 L26 Leica
    FOX3P 236a/E7 Dako
    Estrogen Receptor 6F11 OR 1D5 Dako
    Progesterone Receptor PR636 OR PR1294 Dako
    HER2 4B5 OR HERCEPTEST Ventana (4B5) & Dako (HercepTest)
    Ki-67 MIB-1 Dako
    PSMA DAKO 3E6 Dako
    Vimentin 3B4 Dako
    Cytokeratin OSCAR Signet
    CD45 & Leukocyte common antigen 2B11 + PD7/26 Dako
    S-100 proteins DAKO POLYCLONAL Dako
    MLH1 G168-15 Biocare
    MSH2 FE11 Biocare
    MSH6 EP49 Abcam
    PMS1 EP51 Abcam
    EGFR 3C6 Ventana
  • Routine Stain List
    Alcian Blue-PAS-hematoxylin stain Alkaline Phosphatase stain
    Aniline Blue stain Bielschowsky's silver stain
    Goldners Trichrome stain Gomori's Trichrome stain
    Gram stain H&E
    Luxol fast blue and PAS stain Masson's Trichrome stain
    Methylene blue/basic fuchsin stain Movat pentachrome stain
    Nissl stain PAS
    Periodic acid-methenamine-silver stain (PAM) Picrosirius red/van gieson picrofuchsins stain
    Safranin O/fast green stain Sirius red stain
    Toluidine blue Stain TRAP stain
    TUNEL stain Von kossa stain