Vectra 2

Detection of CTCs in breast and prostate patient samples was performed using an automated fluorescence multispectral microscopy-scanning platform (Vectra 2.0, Perkin Elmer) that employed a unique 5-color immunofluorescence assay panel for each cancer type. All clinical samples were imaged at empirically derived exposure times developed with spike cell samples as well as healthy donors and patients. Circulating tumor cells were identified and scored according to specific criteria (Supplementary Information Table 4) for each fluorescent marker against cellular/staining parameters developed with model systems using cancer cell lines positive for disease specific signals as well as blood cells. The criteria were embedded in an automated algorithm used by the Vectra platform to automatically identify and classify candidate CTC targets. All candidate CTCs were manually scored by two blinded human reviewers against the established criteria and subsequently tabulated to generate counts according to signal presence.

For quantitative staining analysis, Vectra 2.0 automated quantitative tissue imaging system with Nuance and inForm software 2.2.1 (PerkinElmer) was used as described previously^{39 (link)}. The multiplexed TMA was imaged with the Vectra slide scanner, using a scanning protocol that was created based on core size and layout, as well as an acquisition of spectral library. For each slide, an 8-bit image cube from each of the TMA tissue cores was acquired and the inForm advanced image analysis software was used to segment tissues (melanoma versus others) to analyze the protein levels. Then, the target signals were quantitated after unmixing the spectral curves with InForm software. Continuous signal intensity (mean optical density per pixel) was generated for each identified tissue/cell type. Data were exported for further analysis. Quantitation for PLK1, N-cadherin, and E-cadherin was performed in S100-positive cells in melanoma tissue.

Esophageal carcinoma tissue microarrays were purchased from Bioaitech Co., Ltd. (Xi’an, China; ethical license: 2005DKA21300), which contained a total of 148 specimens consisting of EAC (n = 50), ESCC (n = 78), and normal esophagus (NE) tissues (n = 20). The streptavidin–peroxidase method was used for immunohistochemistry analysis of the tissue microarrays. The slides were stained with the GPER1 antibody (PA5-28647; Invitrogen, Carlsbad, CA, USA) [12 (link),52 (link),53 (link)] and visualized with DAB after incubation with the secondary antibody.
The staining results were analyzed using a Vectra Multispectral Imaging System (Perkin Elmer, Waltham, MA, USA). Vectra 2.0.8 (Perkin Elmer) was used for scanning the slides. After importing the high-magnification images and spectral information in inForm 1.2 (Perkin Elmer), positive cells were identified in the target area and the positive rate was determined by setting the threshold intensity. The expression of GPER1 in the tissue microarray is shown with an automatically calculated H-score (=Σpi*i, where i means staining intensity, i = 0,1,2,3; pi is the percentage of the number of cells with corresponding staining intensity in the overall cells). Positive staining was defined as an H-score ≥ 100 (maximum = 300) in this study.

For image segmentation and quantification, regions of interest from individual images or image stacks containing normal breast ducts or multicellular cancer groups within the marginal adipose tissue were identified in IDC and ILC tissue sections. Image analysis from both spectrally unmixed epifluorescent images (Vectra 2.0.8, PerkinElmer Inc.) or maximum intensity projections from 3D confocal stacks were manually segmented, background corrected, and the mean gray values of vimentin and cytokeratin were obtained using ImageJ (ImageJ; 1.40v; National Institute of Health) from the following regions: luminal epithelium of normal cytokeratin-positive ducts in the tumor-free margin; multicellular epithelial cytokeratin-positive groups in the marginal adipose tissue; and cytokeratin-negative vimentin-positive stromal cells which were further identified by elongated morphology and spindle-shaped nuclei.

Tumor samples and PDOs pellets were fixed in 4% paraformaldegyde at 4 ℃ for 24 h and 30 min, respectively, and then followed by dehydration, paraffin embedding, sectioning and standard HE staining. IHC were performed for LGR5 (1:100, Affinity DF2816), E-cadherin (1:2000, proteintech 20874-1-AP), Ki-67 (1:10000, proteintech 27309-1-AP), and CEA (1:200, Bioss bs-0060R). Briefly, paraffin sections were deparaffinized in xylene and rehydrated through a graded ethanol series. After heat-mediated antigen retrieval with a sodium citrate buffer (10 mM, PH 6.0), the sections were blocked at room temperature for 30 min. The primary antibodies were diluted in 3% bull serum albumin (BSA), and staining was performed overnight at 4 ℃. Sections were then incubated with secondary antibodies at room temperature for 60 min. HE and IHC images were viewed and captured using an automatic multispectral imaging system (PerkinElmer Vectra II, USA).

Ten-μm-thick tissue sections (human and mice) were de-waxed and rehydrated through graded alcohols. Antigen retrieval was carried out using Dako PT link (Dako/Agilent Technologies, Santa Clara, CA). IHC staining of individual markers lactate dehydrogenase A (LDHA), monocarboxylate transporter 4 (MCT4), or proliferation marker (Ki67) was performed using EnVision™ G|2 Doublestain System, rabbit/mouse (DAB/Permanent Red) kit (Dako/Agilent Technologies, Santa Clara, CA), according to the manufacturer’s instructions. Slices were imaged by an automatic multispectral imaging system (Vectra II, PerkinElmer version 2.0.7.1).

After the completion of nanoagent administration, three mice were taken from each group for immunofluorescence section and then imaging. Subsequently, the corresponding quantitative analysis was acquired based on the representative images. The expression of uncoupling protein 2 (UCP 2) in mitochondrial membrane induced by the synergistic effect was also verified by immunohistochemistry. The images of immunohistochemistry (IHC) were visualized by using an automatic multispectral imaging system with associated software (Vectra II, PerkinElmer, version 2.0.7.1).