Vectra 3.0 automated quantitative pathology imaging system

The tissue microarray (SMC-TMA) consisted of 120 tissue samples that were formalin fixed, paraffin embedded (FFPE), and sectioned (2-mm thickness): 6 normal samples, 35 low-grade gliomas, 1 IDH-mutant GBM, 3 IDH-status unknown GBMs, and 75 IDH-wild-type GBMs, including 14 SMC2 tumors. The FFPE tissues on slides were deparaffinized and rehydrated for multiplex immunohistochemistry staining. Epitope retrieval was performed using BOND Epitope Retrieval Solution 2 kits (Leica Biosystems, AR9640). Immunofluorescent signals were visualized using the OPAL 7-Color automation IHC kit (Akoya, NEL82100KT), TSA dyes 570 (PHGDH; Atlas Antibodies, RRID: AB_1855299, 1/1000), 690 (Nestin; Atlas Antibodies, RRID: AB_1854381, 1/700), and spectral DAPI. The stained slides were coverslipped using HIGHDEF® IHC fluoromount (Enzo, ADI-950-260-0025) and scanned using a Vectra® 3.0 Automated Quantitative Pathology Imaging System (PerkinElmer). Color separation, cell segmentation, and cell phenotyping were performed on inForm Advanced Image Analysis software (version 2.2, PerkinElmer) to extract image data. PHGDH and Nestin-positive cells were determined by thresholds of 0.6 (PHGDH) and 1.25 (Nestin), respectively.

The in vivo safety of macrocyclam derivatives was determined by histological and serological analyses. Five‐week‐old Balb/c mice were intravenously administered with at Gd@NrMC or Gd@ErMC at a Gd dose of 3.5 mg/kg. Briefly, NrMC (470 nmol) or ErMC (650 nmol) was mixed with gadolinium chloride in sodium acetate buffer (pH 5.0) and incubated for 2 h at 80°C. Unloaded Gd was removed using a PD‐SpinTrap™ G‐25 column. The samples including 70 μg of Gd were intravenously administered to mice. After 1 week, animals (n = 4 per group) were euthanized, and their organs (heart, lung, liver, spleen, and kidney) and sera were collected for histopathological and biochemical evaluation. Extracted organs were fixed in 10% formalin solution for 24 h and embedded in paraffin. For hematoxylin and eosin staining, each organ section was immersed in filtered Harris hematoxylin for 10 s and then in eosin for 30 s. The stained slides were visualized using a Vectra 3.0 Automated Quantitative Pathology Imaging System (PerkinElmer, Hopkinton, MA, USA), and the images were analyzed using InForm v2.4.11. software (PerkinElmer). The serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), aspartate aminotransferase (ALP), blood urea nitrogen (BUN), and total bilirubin (TBIL) were measured using an automatic chemistry analyzer (DRI‐CEHM 3500s; Fujifilm, Kanagawa, Japan).

Tissue sections were deparaffinized in xylene for 10 min, washed with 100% ethanol followed by 95, 80, 70, and 50% ethanol, and then rinsed in distilled water. Samples were processed for antigen retrieval, blocking and staining following the Opal Multiplex IHC protocol (PerkinElmer) as described elsewhere^{76 (link)}. Anti-CD19 (clone BT51E, NCL-L-CD19-163, Leica Biosystems), anti-CD31 (clone C31.3 + JC/70 A, ab199012, Abcam), anti-cytokeratin (clone PCK-26, ab6401, Abcam), and anti-CD138 (clone MI15, PA0088, Leica Biosystems) were used as primary antibodies. Opal 7-Color IHC Kit (PerkinElmer) was used as conjugated antibodies as described by the manufacturer’s instructions. Antibody dilutions are listed in Supplementary Table 6. Finally, after DAPI staining, slides were mounted with VECTASHIELD antifade mounting media (Vector Laboratories). Images were taken using Vectra 3.0 Automated Quantitative Pathology Imaging System (PerkinElmer) and InForm cell analysis software version 2.4.6 (PerkinElmer). Images were evaluated and validated by an experienced pathologist.

The antifibrotic effects of liposomes were tested in vivo using a hematological parameter assay and histological staining. BDL surgery was performed on 8-week-old female BALB/C mice (Raon Bio). On days 1 and 3 after BDL surgery, mice were intravenously administered liposomes at a phospholipid dose of 2.7 mg/mL. Four days after BDL surgery, blood samples were collected and assayed for ALT, AST, bile acid, and total bilirubin levels by the Neodin VET Diagnostics Institute (Seoul, Republic of Korea). For histological assessment of liver tissues, the liver was extracted 4 days after BDL surgery, fixed in 10% formalin for 48 h, and paraffin-embedded. The tissues sections were analyzed with H&E staining, and Masson’s trichrome staining. For H&E staining, tissue sections were immersed in filtered Harris hematoxylin for 10 s and then in eosin for 30 s. The percentage of connective tissue areas in fibrotic regions on stained slides was calculated using a Vectra 3.0 Automated Quantitative Pathology Imaging System (PerkinElmer, Hopkinton, MA, USA). Images were analyzed using the InForm v2.4.11. software (Perkin-Elmer).

Twenty-four-well Transwell membrane inserts (diameter: 6.5 mm, pore size: 8 μm; Corning, Tewksbury, MA, USA) were each coated with 10 μL of type I collagen (0.5 mg/mL, BD Biosciences, San Diego, CA, USA) and 20 μL of a 1:20 mixture of Matrigel (BD Biosciences) in PBS. After treatment with the indicated compounds for 24 h, MDA-MB-231 human TNBC cells were harvested, resuspended in serum-free medium, and plated (2 × 10⁵ cells/chamber) in the upper chambers of the Matrigel-coated Transwell inserts. Medium containing 30% FBS was used as a chemoattractant in the lower chambers. After a 24 h incubation, the cells that had migrated to the outer surfaces of the lower chambers were fixed and stained using the Diff-Quik Staining Kit (Sysmex, Kobe, Japan) and imaged using a Vectra 3.0 Automated Quantitative Pathology Imaging System (Perkin Elmer, Waltham, MA, USA). Representative images from three separate experiments are shown, and the numbers of invaded cells were counted in 5 randomly selected microscopic fields (200× magnification) [38 (link)].

Tissue slides were imaged at 20× magnification and, at least, three regions of interest containing cancer cells were analyzed per sample (1.34 mm²/region of interest) with the Vectra 3.0 Automated Quantitative Pathology Imaging System (Perkin Elmer). An analysis algorithm was trained for tissue and cell segmentation as well as immunophenotyping of cells. DAPI and keratin were employed for segmenting images into tumour, stroma, and ‘no tissue’ areas. Next, cellular segmentation was performed using a counterstain-based approach with DAPI to segment nuclei and membrane markers (CD8, CD3, CD163) to detect cell contours. The following phenotypes were identified: CD3⁺CD8⁻FOXP3⁻ T cells (which were mainly comprised of CD4⁺ T helper cells), CD3⁺CD8⁻FOXP3⁺ T cells (corresponding to Tregs) CD3⁺CD8⁺ T cells (corresponding to CTLs), and CD163⁺ myeloid cells to pinpoint tumor-associated macrophages. All images were visually inspected to confirm the correct attribution and quantification of phenotypes. For each case, cell counts were normalized by tissue area (number of cells/mm²) [37 (link)].