Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

In the era of cancer immunotherapy, interest in elucidating tumor microenvironment dynamics has increased strikingly. This protocol details a mass spectrometry imaging technique with respect to its staining and imaging steps, which allow for highly multiplexed spatial analysis.

These protocols enable single cell resolution quantification, marker co-expression and a special analysis using a formalin-fixed and paraffin-embedded and fresh frozen tissue samples to elucidate the roles of immune cells in cancer. This technique is a powerful tool that facilitate the simultaneous characterization of a 40 marker on a single tissue section, widely preserve the structure of the tissue in the samples. The critical step is antibody optimization and validation.

Failure to complete the step, can result in low quality images that may compromise a retaining high quality reproducible data and results. Begin by spinning down all the antibody containing tubes at 10, 000 x G for five minutes, add individual antibodies to the blocking buffer and mix well. Do not disturb the bottom of the antibody tube when pipetting it.

Filter the antibody panel by pre-wetting a 0.1 micrometer centrifugal filter device with 100 microliters of blocking buffer. Spin the filter at 10, 000 x G for two minutes and remove the blocking buffer flow-through with a pipette. Then transfer the antibody panel into a spin column and spin the filter at 10, 000 x G for two minutes.

Discard the spin column and use the flow-through as the filtered antibody panel. For the antibody staining, remove the blocking buffer from the slides by tipping each slide on its side and gently tapping the edges against a task wiper. Place the slides in a moisture chamber and add 100 to 200 microliters of antibody master mix to the tissue.

Add positive and negative control slides to the staining batch and incubate the slides at 4 degrees Celsius overnight. To set up the instrument, login to the control software, and click on Wake Up, if the instrument is in sleep mode. Click on Exchange Sample to load a slide and then click on Continue to open the door.

Put the slide in the loading slot with a sample facing up and the label on the right, click on Continue to close the door. Select the project and then select the slide name for the loaded sample. Visualize the panoramic image on the slide optical image pane for the field of view or FOV selection, click on the Mode menu and select Secondary Electron Detector, then click on the Imaging Mode menu and pick QC 300 micrometer.

Click on Jog Stage to control the FOV location and then click on the arrows to navigate and select the position of the FOV. Click add FOV, set 400 micrometer by 400 micrometer as the field size and select Fine as the Imaging Mode and 1 millisecond of Dwell Time, click on Confirm. After creating an FOV list, proceed to the image focus and adjust the Stigmation by moving the beam to a tissue region that will not be imaged.

Adjust the parameters until a clear central image is obtained, click on Start Run. The acquired images will be automatically uploaded and stored in the web-based image management application. The matrix shown here describes the percent crosstalk derived from the probe purity and oxides.

Here blue boxes indicate greater than or equal to 0.5%crosstalk and clear boxes indicate less than 0.5%crosstalk. For the Mass tags, probes that contributed at least 0.5%crosstalk into no channels, one or two channels, or more than two channels are shown here. Mass channels receiving at least 0.5%crosstalk from no probes, one or two probes, or more than two probes are also shown.

The representative staining images in different tissues like lung adenocarcinoma, non-neoplastic kidney and tonsil are depicted here. An image of a tonsil tissue section stained using this technique and visualized using a third party digital image analysis software program before filtering and correction is presented here. The image of the same section under the same conditions after filtering and correction steps is shown here.

This method generate images that can be uploaded to a third party digital image analysis software, allowing comprehensive analysis such as tissue compartmentalization, cell segmentation, marker co-expression, as well as neighbor analysis and distant analysis. Highly multiplex imaging methods are available through research. By constructing different panels, researchers can investigate cancer, normal, paraneoplastic, inflammatory and infectious samples.