Multiplex Immunofluorescence Combined with Spatial Image Analysis for the Clinical and Biological Assessment of the Tumor Microenvironment

In our lab, we are studying human colorectal cancer, its progression to metastatic disease, and its response to treatment, including immunotherapy. We are trying to find new predictive and prognostic biomarkers by investigating the tumor immune microenvironment and the intratumoral microbiome in primary and metastatic disease. In our project, we are currently implementing several technologies such as RNA sequencing, whole exome sequencing, TCR sequencing, circulating cell-free DNA analysis, immunofluorescence and fluorescence in situ hybridization.

At this moment, our major challenge is to combine fluorescence in situ hybridization staining with tyramide signal amplification multiplex immunofluorescence to study the interaction between the microbiome and the immune cells. Our method is robust, reproducible, easy to use, and cost effective. It can be set up in any lab possessing a fluorescent slide scanner, plus, the method can be used with any commercially available IHC antibody in opposition to commercially available kits that are usually optimized for restricted panel of antigens.

Prior to multiplex immunofluorescent staining, fix the tissue and perform deparaffinization and endogenous peroxidase inhibition. Then for multiplex immunofluorescent staining, submerge the slides in a 300-milliliter staining jar containing 10 millimolar citrate buffer complemented with 0.1%Triton X-100. Place the staining jar with the lid closed in a microwave for three to five minutes at maximum power until the buffer starts to boil.

After boiling, leave the buffer at near-boiling temperature by putting the microwave on low power for 15 minutes. Again, heat the buffer by putting the microwave at maximum power for 90 seconds. Remove the jar from the microwave, and allow the buffer to cool down for 15 minutes at room temperature.

Rinse the slides three times for five minutes each in distilled water, followed by five minutes of rinsing and tris-buffered saline containing 0.1%Tween 20 or TBS Tween. After the last wash, remove the TBS Tween by blotting the slides on a paper towel. Next, place the slides on a microscope slide box, and encircle the tissue with a hydrophobic pen.

Block the non-specific binding sites by covering the tissue with 5%BSA dissolved in TBS Tween. After 30 minutes, remove the blocking buffer by blotting the slides on a paper towel. Next, incubate the tissue for 60 minutes with 300 microliters of primary antibody diluted in 1%BSA TBS Tween.

After incubation, rinse the slides three times for three minutes each with TBS Tween. After washing, incubate the tissue for 40 minutes with 300 microliters of poly-HRP secondary antibody. After incubation, rinse the slides three times for three minutes each with TBS Tween.

Next, incubate the tissue for 10 minutes with 300 microliters of fluorochrome tyramide reagent diluted 200-fold in borate buffer extemporaneously supplemented with 0.003%hydrogen peroxide. Then rinse the slides three times with TBS Tween, and incubate the tissue overnight at 4 degrees Celsius with human pan-cytokeratin antibody diluted in 1%BSA TBS Tween. The next day, rinse the tissue with TBS Tween and incubate for 120 minutes with the secondary antibody directly coupled with fluorochrome diluted 200-fold and 1%BSA TBS Tween.

After incubation, rinse the tissue with TBS Tween before staining the nuclei with bisbenzimide diluted 1000-fold in 10%BSA TBS Tween for five minutes. Rinse the tissue three times for three minutes each in distilled water before mounting it on a slide using a fluorescence mounting medium and borosilicate cover glass. For image analysis, import the scans into an image analysis software.

Then go to the Analysis tab and select the high-plex fluorescence algorithm by clicking Settings, Actions, and then Load. Select the Dye Selection tab and the dye of interest. In the Nuclear Detection tab, go to Nuclear Segmentation Type and select AI Custom.

Then in the Nuclear Segmentation classifier, select the saved trained AI.In the Membrane and Cytoplasm Detection tab, choose the maximum cytoplasm radius and the number of membrane dyes. For each dye, select the nucleus positive threshold, the cytoplasm positive threshold, and the membrane positive threshold. For each dye, select the nucleus membrane and cytoplasm percentage of completeness values.

Save the algorithm by selecting Settings, Actions, and Save. Analyze the region of interests by clicking Analyze and selecting Annotation Layer. Then go to the Results tab, and select all data in Object Data.

Export the data in CSV format by right-clicking Export and selecting Object Data CSV. A representative result of optimal staining by multiplex immunofluorescence is shown. Choosing the correct dilution was essential for verifying the antibody specificity and optimizing the signal-to-noise ratio of the staining.