CLM Specimens were made available to us from tissues stored in the Victorian Cancer Biobank (VCB) after approval from the Austin Health Human Research Ethics Committee HREC reference HREC/54233/Austin-2019.
1. Heat the slides at 60 °C for 60 minutes in an oven
Bath and solvents for deparaffinization and rehydration of FFPE tissue:
2. Xylene for deparaffinization, immerse into 3 successive baths of xylene for 10 minutes each time. Note: Do not let slides dry out between steps.
3. Histological grade ethanol (100%) for rehydration, immerse into 2 successive baths for 5 minutes each time.
4. Histological grade ethanol (70%) for rinsing, immerse for 5 minutes.
5. After rehydration, rinse in running RO water for ~1 minute.
6. Fix in 10% NBF for 30 minutes.
7. After fixing, wash in running RO water for ~1 minute.
Proceed with antigen retrieval and microwave treatment step as specified for the primary antibody for targets of interest:
8. Place slides in the Tissue-tek vertical 24 slide rack
9. Fill slide container with antigen retrieval buffer diluted in 1:10 with peroxidase-free water as follows:
• Day 1- AR6 buffer (PerkinElmer/Akoya Biosciences) (for anti-α-SMA)
• Day 2- AR6 buffer (PerkinElmer/Akoya Biosciences) (for anti-E-cadherin)
• Day 3- AR6 buffer (PerkinElmer/Akoya Biosciences) (for anti-Foxp3)
• Day 4-Target retrieval Solution, pH9 buffer (Agilent Dako) (for anti-CD103)
• Day 5- AR6 buffer (PerkinElmer/Akoya Biosciences) (for anti-CD8)
• Day 6- Proteinase K (Millipore) (for anti-CD3).
Note: Proteinase K does not require microwave pre-treatment (steps 10-11), therefore it should be last antibody of the panel. Leave at room temperature (RT) for 30 minutes.
10. Heat slides in antigen retrieval buffer in a microwave oven as follows:
• 2 minutes at 100 % power or until boiling point is reached.
• Continue with 10 minutes at 20 % power.
11. Cool down the slides at RT for 15 minutes.
12. Wash the slides in distilled water (dH2O) for 1 minute and then rinse in TBST 3 times for 2 minutes each time.
13. Block endogenous peroxidase activity (Dako REAL Peroxidase-Blocking Solution; Agilent) for 30 minutes.
14. Block non-specific binding with Ab Diluent/ Block Buffer (PerkinElmer/Akoya Biosciences) for 30 minutes.
15. Add Primary Antibody diluted at the optimized ratio in Ab Diluent/ Block Buffer (PerkinElmer/Akoya Biosciences) for targets of interest in the following order:
• Day 1-α- SMA at 1:500
• Day 2- E-cadherin at 1:500
• Day 3- Foxp3 at 1:50
• Day 4-CD103 at 1:1000
• Day 5- CD8 at 1:100
• Day 6- CD3 at 1:100
16. Incubate with each antibody overnight at 4oC in a humidified chamber.
17. Wash the slides 3 times for 2 minutes each time with TBST Buffer at RT with agitation.
18. Add Opal Polymer HRP Ms + Rb (PerkinElmer/Akoya Biosciences) and incubate for 40 minutes at RT.
19. Wash the slides 3 times for 2 minutes each time with TBST Buffer at RT with agitation.
20. Add 50-150µl of TSA-Opal Working Solution per slide (1:50 dilution with 1 x Plus Amplification Diluent) depending on the size of the tissue and the specific conjugated dye to the target of interest:
• Day 1- To α- SMA add TSA-Opal690
• Day 2- To E-cadherin add TSA-Opal620
• Day 3- To Foxp3 add TSA-Opal520
• Day 4- To CD103 add TSA-Opal540
• Day 5- To CD8 add TSA-Opal570
• Day 6- To CD3 add TSA-Opal650
21. Incubate the slides for 10 minutes at RT
22. Wash the slides 3 time for 2 min each with TBST Buffer at RT with agitation
23. Repeat process from point 9, preparing the second day’s antigen-retrieval buffer and repeating the process until the final day’s procedure is complete.
Counterstaining and Mounting
24. After the last TSA-Opal color treatment, wash the slides in dH2O for 2 minutes and rinse in TBST for 2 minutes.
25. Add DAPI Solution (1:4000; ThermoFisher scientific) and incubate the slides for 2-5 min at RT.
26. Rinse the slides with TBST 3 times for 2 minutes
27. Rinse in running RO water for ~ 1 minutes
28. Cover the slide with Fluorescent Mounting Medium (Agilent Dako) and apply coverslip Menzel- Glazer 22x50 mm (Thermo scientific).
29. Leave the slides to set at room temperature overnight and protected from light.
30. Opal stained slides should be protected from light and maintained at 4oC in a humidified chamber for short/ long term image acquisition.
Image acquisition and quantification
31. Use the multispectral fluorescent microscope Vectra® 3.0 Automated Quantitative Pathology Imaging system (Perkin Elmer, MA) for whole tissue scan.
32. Use Phenochart® software (low power scanned image) to select high-powered fields (HPF) (equivalent fixed-size stamp 3x3= 2046 x 1530 µm; ×20 object lens, resolution= 0.5 µm equivalent to 1 pixel) across the tumor and adjacent liver area, selecting areas of tumor with the highest infiltrating lymphocyte density (Figure 1).
33. Use inForm® software v2.4.2Tissue finder algorithm for trainable pattern-recognition to reach over 91% accuracy for the following tissue segments: adjacent liver parenchyma depicted in red (LP), invasive tumor margin in blue (IM) and tumor core (TC) in green (Figure 2).
34. Use inForm® software v2.4.2 Cell segmentation and phenotyping algorithm to calculate cell count per HPF according to the trainable phenotypes of interest: CD3+ T Lymphocytes (CD3+ CD8-CD103-), CD8+ T cells (CD3+CD8+CD103-), TRM (CD3+CD8+CD103+), T regulatory cells (CD3+FoxP3+) , non T cells expressing CD8+ or CD103+ and cells expressing EMT markers such as α-SMA or E-cadherin. Every sample had background control for subtraction of autofluorescence (Figure 3).
35. Use Pixel-based colocalization analysis using inForm® for quantitation of fluorescent intensity for discovery of novel phenotypes not included in the previous algorithm.