Vectra multispectral imaging system version 2

Multiplex stained slides were imaged using the Vectra Multispectral Imaging System version 2 (PerkinElmer). Regions of interest were selected by 2 × 2 (1338 μm × 1000 μm) stamp and divided into tumor core and tumor edge. The tumor edge was defined as within 1mm interface between tumor and non‐malignant tissue. The tumor core area was defined as the proximal tumor area within the tumor edge. High magnification (20×) multispectral images were acquired to encompass all the regions for tumor core and tumor edge with minimum overlaps between images.

IHC and multiplex-stained slides were imaged with use of the Vectra Multispectral Imaging system version 2 (PerkinElmer). All samples were scanned at 20× magnification for TMA annotation and larger tumor region selection. Low-powered images were then used to extract one 40× image of each TMA core and 100 to 300 images of the larger patient samples, depending on the size of the samples, with the use of a Phenochart slide viewer (see Supplementary Fig. S2). Filter cubes used for multispectral imaging were DAPI (440–680 nm), FITC (520–680 nm), Cy3 (570–690 nm), Texas Red (580–700 nm), and Cy5 (670–720 nm). The signal intensities for each marker were normalized, and spectral unmixing was performed with PerkinElmer inForm Analysis software (2.4.1.). An image encompassing the entire slide through the full emission spectrum of each filter (DAPI, fluorescein isothiocyante [FITC]), Cy3, Texas Red [TR], and Cy5) was captured. A spectral signature for each fluorophore was obtained by using the same multispectral imaging protocol of single-stained slides, as well as an unstained slide to obtain the auto-fluorescence signature of the tissue. Spectral unmixing was then used to separate these spectral signatures into individual signals (see Supplementary Fig. S3).

FFPE tumors from s.c. experiments underwent IHC analysis following staining with Ab against CD3 (catalog A0452, Dako). PerkinElmer’s Vectra multispectral slide analysis system was used to capture 40× magnification of images of tumors (10 images/tumor). inForm software quantified CD3-positive cells (Fast Red chromogen) within each image. Additional slices were stained for CD8 and α-SMA and scanned with an Olympus Nanozoomer whole slide scanner and analyzed using Qupath (CD8) or FIJI (NIH) for α-SMA. Orthotopic tumors were also FFPE. Dual stains for DAPI (Perkin Elmer) with CD4 and FOXP3 were performed using a Roche autostainer and detected with Opal 520 and Opal 630-conjugated secondary (Perkin Elmer), respectively. Slides were imaged using the Vectra Multispectral Imaging System version 2 (Perkin Elmer). Filter cubes for imaging were DAPI (440–680 nm), FITC (520–680 nm), Cy3 (570–690 nm), Texas Red (580–700 nm), and Cy5 (670–720 nm). Multispectral images were analyzed with Qupath (50 (link)).

Multiplex stained TMA slides were imaged using the Vectra Multispectral Imaging System version 2 (Perkin Elmer), where one raw image comprising four stitched 200 × multispectral image cubes was obtained for each TMA core. Each 200 × multispectral image cube was created by combining images obtained every 10 nm of the emission light spectrum across the range of each emission filter cube. Filter cubes used for multispectral imaging were DAPI (440–680 nm), FITC (520 nm-680 nm), Cy3 (570–690 nm), Texas Red (580–700 nm) and Cy5 (670–720 nm) (Supplementary Fig. 1B).