Three-Dimensional Imaging of Tumor-Bearing Tissue Using the Iterative Bleaching Extends Multiplexity Approach

This research focuses on identifying targets for immunomodulatory drugs and complex solid gastrointestinal tumors. Analyzing the tumor microenvironment cellular landscape is absolutely crucial for understanding susceptibility to treatments like immunotherapy. Single-cell sequencing and flow cytometry help characterize the tumor microenvironment, but lacks spatial context. Recently, multiplex imaging and transcriptomic platforms have emerged to study the tumor microenvironment in its in-vivo context.

Available multiplex technologies for analyzing the tumor microenvironment, whether iterative or all in one, are restricted to two dimensions. This protocol extends characterization into the depth dimension, providing insights beyond single FFPE or frozen sections.

[Narrator] To begin, transfer the tissue sheets to a sample cup containing warm harvest medium and place it on a heating plate at the preparation table. Mount the tissue on platforms in a 15-centimeter plate containing harvest medium. Carefully drape the tumor-bearing tissue with the mesothelial side facing up over the small orifice side of the platform and secure it with a 2-0 silk suture. Place the prepared tissue platform inversely fully submerged in a 24-well plate containing harvest medium. For fixation, add 10 milliliters of fixative stock to a 50-milliliter conical tube containing 30 milliliters of cold PBS to prepare 40 milliliters of fixation buffer. Using forceps, transfer the tissue mounted on platforms to the fixation buffer. And incubate for 24 hours at 4 degrees Celsius. Decant the fixative. Replace it with 40 milliliters of cold PBS. And incubate for 10 minutes at 4 degrees Celsius. For staining, add 1 milliliter of blocking buffer into a well and insert the tissue platform. Block for at least two hours at room temperature on a rocker set to a low speed of 30 RPM. Prepare 0.8 milliliters of antibody dye solution in a microcentrifuge tube and centrifuge at 10,000 g for two minutes at room temperature. Transfer 0.75 milliliters of the supernatant to a clean well of a 9-well incubation plate. And insert a washed platform. Wrap the plate in aluminum foil. And incubate for at least two hours at room temperature on a rocker set to a low speed of 30 RPM. Wash the platform in a 50-milliliter conical tube containing 40 milliliters of PBS for 10 minutes at 4 degrees Celsius. Add 0.1 milliliters of PBS to the center of the glass bottom of a 3.5-centimeter imaging dish, and set it aside. Screw the height adjustment ring loosely in a clockwise direction into the outer lid. And mount the platform in the inner plastic holder, ensuring notch groove alignment. Secure the platform with the slider. Place the assembled imaging adapter on the glass bottom dish and carefully lower the height adjustment ring until the tissue touches the buffer. Once the optimal distance from the glass is reached, add 0.2 milliliters of PBS onto the tissue with the bottom side up to prevent tissue dehydration during imaging. Start the image acquisition software. Select the appropriate objective, such as 20X or 40X, and add the corresponding immersion medium. Choose the image acquisition parameters, such as a scan speed of 600 hertz, an XY resolution of 1024 by 1024 pixels, three-line averages, and a bidirectional scan. Select the appropriate laser lines or adjust the white light laser to match the excitation and emission spectra of the fluorophores used for staining. Place the assembled imaging dish into the sample holder on the microscope stage. Center the specimen using the XY control, bring the objective up to the cover glass, and start image acquisition. After each imaging round, perform a bleaching step. Prepare 5 milliliters of 1.5 milligrams per milliliter lithium borohydride solution in distilled water, and incubate for 30 minutes at room temperature. Transfer 1 milliliter of the lithium borohydride solution to a clean well of the 9-well incubation plate and insert the washed platform for 60 minutes at room temperature. Briefly wash the platform in 20 milliliters of PBS inside a conical tube. Check for the remaining fluorescent signal using the highest laser setting for Z compensation. A tumor-bearing peritoneal sample stained with the first round of iterative bleaching extends multiplexity, or IBEX 1, panel for visualization is shown here. Tumor lesions were identified as rosette-like structures that were double-positive for CD44 and podoplanin with nuclear arrangements characteristic of papillary mesothelioma. High resolution immunofluorescence images of selected regions from the tumor-bearing peritoneal sample are shown here. These images reveal tumor regions and tumor tertiary lymphoid structure interfaces, highlighting consistent immune cell infiltration with a high density of CD45-positive cells. This figure presents the iterative immunofluorescence staining across IBEX cycles one to six, illustrating the spatial distribution of immune and structural markers within the tumor lesion and tumor tertiary lymphoid structure interface. The maximum projections of different optical sections are presented here. This 3D imaging confirmed that tumor lesions and tertiary lymphoid structures reside at different Z depths, demonstrating the limitations of 2D imaging in capturing complex tissue architecture.