Vectra polaris automated quantitative pathology imaging system

Formalin-fixed lymph nodes, salivary glands, pancreas, and lacrimal glands biopsies from IgG4-RD patients were paraffin-embedded. Slides were deparaffinized in xylene and antigens were retrieved. Then, the sections were stained with anti-CD4 (catalog 93518, Cell Signaling Technology), anti-CD8 (catalog 85336, Cell Signaling Technology), anti-GNLY (catalog ER61826, Huabio), and anti-γδTCR (catalog TCR1061, Thermo Fisher Scientific) according to the manufacturer’s protocol (Opal Polaris, Akoya Bioscience) and then were screened and analyzed with the Vectra Polaris Automated Quantitative Pathology Imaging system (PerkinElmer). For CCL5 and CX3CL1 staining, antibodies against CCL5 (catalog ET1705-70, Huabio) and CX3CL1 (catalog 60339-1-Ig, Proteintech) were used as primary antibodies, while Alexa Fluor 647– and 488–conjugated anti-IgG (catalog ab150079 and ab150113, Abcam) were used as secondary antibodies. Salivary gland biopsies from patients with mucocele as controls. The images were captured with a Leica microscope.

Formalin-fixed, paraffin-embedded biopsy specimens and parallel surgical excisions from patients with early stage breast cancer were subjected to immunohistochemistry (IHC) staining using anti-human Ki67 antibody (Dako, clone 30-9). Results were analyzed using the Vectra® Polaris™ Automated Quantitative Pathology Imaging System and InForm software (PerkinElmer) (1 × 1 field, 20× resolution).

Opal multiplex staining was performed according to the Opal 5-Color Manual IHC Kit (PerkinElmer). Opal DAPI, Opal 520, Opal 570, and Opal 650 from Abcam were used to generate different signals corresponding to Ku70, VAV2, or Ku80 antibody, respectively. The slides were counterstained with DAPI for nuclear visualization and subsequently coverslipped using VectaShield Hardset mounting media. The slides were imaged using a Vectra Polaris Automated Quantitative Pathology Imaging System (PerkinElmer). We used inForm software (PerkinElmer) to unmix and remove autofluorescence and to analyze the multispectral images.

Formalin-fixed paraffin-embedded (FFPE) sections of esophageal precursor lesions were collected from 30 patients between 2016 and 2018 in Linzhou Cancer Hospital, including INF (n = 5), HYP (n = 13), DYS (n = 8) and CIS (n = 3), to validate the results obtained in mice. The protein expression levels of the marker genes were detected by IHC staining for mice tissues and immunofluorescence for human specimens with antibodies (Abcam) shown in Supplementary Table 3. The samples were incubated with antibody against Ki67 (1:50 for IHC, ab16667), Top2a (1:8000 for IHC, 1:10,000 for IF, ab52934), Aldh3a1 (1:200 for IHC, 1:600 for IF, ab76976), Atf3 (1:200 for IHC, 1:600 for IF, ab216569), S100a8 (1:500 for IHC, 1:1500 for IF, ab92331), Mmp14 (1:2000 for IHC, 1:6000 for IF, ab51074), or Itga6 (1:250 for IHC, 1:750 for IF, ab181551). Opal multiplex staining was performed according to the Opal 5-Color Manual IHC Kit (Perkin Elmer). Opal DAPI, Opal 520, Opal 570, Opal 620, and Opal 690 were used to generate different signals. Slides were counterstained with DAPI (1:2000) for nuclei visualization, and subsequently coverslipped using a VectaShield Hardset mounting media. The slides were imaged using Vectra Polaris Automated Quantitative Pathology Imaging System (Perkin Elmer). We used inForm software (Perkin Elmer) to unmix and remove autofluorescence and to analyze the multispectral images.

The sections were deparaffinized, and rehydrated as described for the IHC procedure. Opal multiplex staining was performed according to the Opal 5-Color Manual IHC Kit (PANOVUE). The sections were incubated by antibodies against A3A (1:300, PA5-99584, Thermo Fisher), CD8 (1:800, ab93278, Abcam), Granzyme B (1:300, #17215, CST), PD-L1 (1:400, ab205921, Abcam), and FOSL1 (1:50, sc-28310, Santa Cruz) at 4°C overnight. Opal 520 corresponding to the A3A antibody, Opal 570 to Granzyme B (or PD-L1) antibodies and Opal 650 to CD8 (or FOSL1) antibodies were used to generate different immunofluorescent signals. Slides were counterstained with DAPI for nuclei visualization, and subsequently coverslipped using a VectaShield Hardset mounting media. The slides were imaged using Vectra Polaris Automated Quantitative Pathology Imaging System (Perkin Elmer). We used inForm software (Perkin Elmer) to unmix and remove autofluorescence and analyze the multispectral images.

Sections stained for T- and B-cell markers were scanned with a slide scanner (NanoZoomer Digital Pathology, Hamamatsu Photonics) at ×200 magnification. Cells per mm² were counted manually using the Hamamutsu NDP.view program. The selected regions ranged from 7.6 to 111 mm². To quantify multilabelled cells, fluorescent staining was scanned with the Vectra Polaris Automated Quantitative Pathology Imaging system from Perkin Elmer and quantified semi-automatically with Qupath. This software provides a machine learning tool for object classification. As a first step, the algorithm had to be trained. Therefore, the image was split in different channels, one for each marker. Cells were detected by nuclear staining (DAPI). Next, the object classifier was trained and checked manually for accuracy. Subsequently, the object classifier was saved, a region of interest (from 4 to 102 mm²) selected and the algorithm applied. To quantify multilabelled cells, the classifiers were applied at the same time. Finally, to obtain the numbers of cells per mm², the absolute numbers of quantified cells were divided by the analysed areas.