FFPE Sections (4-μm thick) were mounted and routinely stained with H&E for histopathological examination (Supplementary Fig. S1a ). Multiplex immunofluorescence was applied to identify the expression patterns of key transcriptomic regulators of SCLC, including ASCL1 (Abcam, ab211327), NEUROD1 (Abcam, ab60704) and POU2F3 (Novus Biologicals, NBP1–83966). Multiplex immunofluorescence staining was performed using a PANO 7-plex IHC kit (Panovue, Cat# 0004100100), as previously described.65 In brief, the FFPE sections were subjected to deparaffinization, rehydration, and antigen retrieval according to the protocol supplied by the manufacturer. After blocking, the sections were incubated with a primary antibody and then a secondary antibody (polymer HRP-anti-mouse/Rabbit IgG). Other primary antibodies were sequentially applied by repeating the previous procedures. Nuclei were stained with DAPI (Sigma-Aldrich, D9542) after all the human antigens had been labeled. Multispectral images were obtained by scanning the stained slides with the Mantra System (PerkinElmer, Waltham, Massachusetts, US) and analyzed using inForm image analysis software (PerkinElmer, Waltham, Massachusetts, US) (Fig. 4c ).
Inform image analysis software
Manufactured by PerkinElmer
Sourced in United States
InForm image analysis software is a digital pathology platform designed for quantitative analysis of tissue samples. The software enables users to visualize, analyze, and quantify biomarkers within tissue sections.
Automatically generated - may contain errors
Lab products found in correlation
Mantra system, by PerkinElmer (18 mentions)
Pano 7 plex ihc kit, by Panovue (15 mentions)
Mantra quantitative pathology workstation, by PerkinElmer (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (4 mentions)
Vectra 3.0 automated quantitative pathology imaging system, by PerkinElmer (3 mentions)
D9542, by Merck Group (2 mentions)
Anti panck, by Cell Signaling Technology (2 mentions)
Mantra quantitative pathology imaging system, by PerkinElmer (2 mentions)
Ab92511, by Abcam (2 mentions)
Anti cd8, by Cell Signaling Technology (2 mentions)
Anti cd56, by Abcam (2 mentions)
Lsm 880, by Zeiss (2 mentions)
Ab211327, by Abcam (1 mentions)
Nbp1 83966, by Novus Biologicals (1 mentions)
X0909, by Agilent Technologies (1 mentions)
Cd163, by Cell Signaling Technology (1 mentions)
Pd l1 ihc 22c3 pharmdx assay, by Agilent Technologies (1 mentions)
Opal 7 colour manual ihc kit, by PerkinElmer (1 mentions)
Antibody diluent blocking buffer, by PerkinElmer (1 mentions)
Mab8214, by R&D Systems (1 mentions)
50 protocols using inform image analysis software
1
Multiplex Immunofluorescence Analysis of SCLC Markers
2
Multiplex Immunofluorescence Staining Protocol
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As previously described, mIF staining was conducted using a PANO 7-plex IHC kit (Panovue, Beijing, China) (31 (link)). Antibodies used included anti-panCK (CST4545, Cell Signaling Technology, Danvers, MA, USA), anti-CD8 (CST70306), anti-CD56 (CST3576), anti-CD68 (BX50031, Biolynx, China), and anti-HLA-DR (ab92511, Abcam, UK). The stained slides were scanned, and a single stacked image was constructed using the Mantra System (PerkinElmer, Waltham, MA, US). Furthermore, images of the sections were reconstructed based on a spectral library of multispectral unmixing. Finally, various cells were counted using the InForm image analysis software (PerkinElmer).
3
Multiplex IHC analysis of CRC biomarkers
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A TMA containing 56 CRC patient tissue samples was obtained. mIHC was performed to visualize the expression of HOXB13, DNMT3B and C-myc in the TMA according to the Opal immunostaining protocol. Briefly, the slides were deparaffined with xylene and ethanol, and heat-induced antigen retrieval was performed using microwave incubation. The sections were blocked in blocking buffer (Dako, X0909) for 10 min at room temperature. Then, the sections were incubated with primary antibody, HRP-conjugated secondary antibody and Opal working solution. Next, the sections were counterstained with DAPI. All the slides were scanned, and images were analyzed using inForm image analysis software (PerkinElmer).
4
Multispectral Imaging for Tissue Autofluorescence Removal
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The obtain multispectral images, the stained slides were scanned using the Mantra system (Perkin Elmer), which captures the fluorescent spectra at 20-nm wavelength intervals from 420 to 720 nm with identical exposure time. The scans were combined to build a single stack image. Images of unstained and single-stained sections were used to extract the spectrum of autofluorescence of tissues and each fluorescein, respectively. The extracted images were further used to establish a spectral library required for multispectral unmixing by InForm image analysis software (PerkinElmer). Using this spectral library, we obtained reconstructed images of sections with the autofluorescence removed.
5
Multiplex Immunohistochemistry Analysis of Lymph Nodes
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Six pairs of lymph nodes were randomly selected, formalin-fixed, paraffin-embedded, and subjected to multiplex immunohistochemistry (mIHC). The mIHC was performed by using a PANO 7-plex IHC kit (Panovue, China) following the standard protocol [16 (link)]. Immune cell panels included the following antibodies: CD3 (1 : 200, Abcam, ab16669), CD8A (1 : 300, Cell Signaling Technology, 70306), Foxp3 (1 : 500, Abcam, 20034), PD1 (1 : 50, Cell Signaling Technology, 43248), and CD163 (1 : 100, Cell Signaling Technology, 93498). The slides were incubated with the primary antibodies, followed by 0.3% hydrogen peroxide solution for blocking endogenous peroxidase. DAPI (Sigma-Aldrich) was used for nuclear counterstaining. Images were acquired and analyzed by using a Mantra System (PerkinElmer) and inForm image analysis software (PerkinElmer), respectively.
6
Multiplex Immunofluorescence Analysis of PD-L1, Immune Cells, and Tumor Microenvironment
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PD-L1 expression was detected using the PD-L1 IHC 22C3 pharmDx assay (Agilent Technologies, Santa Clara, CA, USA) and was assessed by combined positive score (CPS), where CPS ≥1 was considered as positive. The mIF staining was performed using PANO 7-plex IHC kit (Panovue, Beijing, China), according to the manufacturer’s instructions as described previously (17 (link)). Briefly, CD8 marker was used to identify T cells. The natural killer (NK) cells were divided into CD56dim (weak staining) and CD56bright (strong staining) according to the intensity of membrane staining by CD56 antibody. Tumor-associated macrophages (TAMs) were identified by CD68 and HLA-DR and were divided into TAM1 (CD68+ and HLA-DR+) and TAM2 (CD68+ and HLA-DR−). S100 staining was used to define the tumor center and the invasive margin. The stained slides were scanned and built a single stack image subsequently by the Mantra System (PerkinElmer, Waltham, MA, USA). The reconstruction of images was performed using inForm image analysis software (PerkinElmer) for multispectral unmixing to remove autofluorescence.
7
Quantitative Analysis of Tumor-Infiltrating Treg Cells
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The abundance of TITreg cells was analyzed utilizing Opal 7-Colour Manual IHC Kit (PerkinElmer NEL811001KT) according to the manufacture’s protocol (14 (link)). In brief, the slides were incubated with Antibody Diluent blocking buffer (PerkinElmer) at room temperature (RT) for 10 min. The primary antibody for CD4 (Abcam, ab133616, 1:500) and FoxP3 (R&D, MAB8214, 1:400) were incubated at RT for 1 h. Then, a secondary HRP antibody were incubated at RT for 10 min. Signal amplification was performed using Opal 520 TSA (PerkinElmer) and incubated at RT for 10 min. Visualization of the slides was done using the Mantra Quantitative Pathology Imaging System (PerkinElmer) and analyzed using InForm Image Analysis software (PerkinElmer, version 2.1). The TITreg cell fraction in CD4+ cells were calculated and data were presented as mean ± SD.
8
Statistical Analysis of Pathologic Response
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We performed statistical analyses using InForm image analysis software (version 2.4, Perkin Elmer) and SPSS statistics version 21 (IBM Armonk). An exact 95% confidence interval (CI) was calculated for the pathologic response rate and the overall ORR using the Clopper–Pearson formula. The chi‐squared test was used to test for the significance of proportional differences in gene mutations among different pathologic responses, and Fisher's exact test was used for the pathologic responses in patients with gene sequence mutations and without mutations. For correlative analyses, Wilcoxon's rank‐sum test or the Mann–Whitney U‐test was used to compare continuous variables between two independent groups. Wilcoxon's signed‐rank test was used for the comparison of paired data. Spearman's correlation coefficient was used to assess the correlation between two continuous variables. We calculated hazard ratio (HR) and 95% CI using Cox's proportional hazards model; p‐values <0.05 were considered statistically significant.
9
Lung Imaging and Segmentation Protocol
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Lungs were inflated and fixed with 4% PFA, dried with hexamethyldisilazane (Alfa Aesar, Ward Hill, MA, USA) and CT-scanned using a SkyScan 1172 (Bruker-MicroCT, Kontich, Belgium) with 12.6 μm pixel size, as described previously [32 (link)]. Reconstructed images were segmented with InForm image analysis software (Perkin-Elmer, Waltham, MA, USA) to exclude background and to segment lungs into normal (green), fibrotic (blue) and airway regions. Data are either represented as percentage volume of fibrosis or sum pixel intensity, as a measure of lung density.
10
Immunofluorescent Image Acquisition and Analysis
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