93 3D spatial quantification of CD8+ T-cells in the tumor microenvironment in intact FFPE cancer blocks using the 3D I/O Pro pipeline

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Background The complexity of the tumor microenvironment (TME) has been a major barrier in understanding the response to therapy in solid tumors. TME analysis has been traditionally based on thin 4 µm tissue sections, which represent less than 0.1% of a typical FFPE block. Recent literature suggests conventional histology techniques are insufficient to understanding the degree of immune infiltration in the TME. Here, we demonstrate how 3D imaging and analysis of whole tissue samples can be used to identify and quantitate cytotoxic (CD8+) T-cells in human FFPE cancer tissue samples. Methods Human colorectal cancer FFPE tissue was deparaffinized and processed before being stained with nuclear dye TO-PRO-3 and an anti-CD8 antibody. Samples were optically cleared using a modified iDISCO+ protocol. Entire samples were imaged at 2 µm/pixel resolution with a hybrid open-top light-sheet microscope; the 3Di™. CD8+ lymphocytes were quantified by 3D spatial analysis performed using 3Dai™ tools. We then evaluated virtual standard histological sections and compared the variability in individual sections to the whole enumeration provided by the 3D imaging and analysis in a single, representative FFPE block measuring approximately 6 mm x 4.3 mm x 4.2 mm. Results The number of CD8+ T-cells averaged 49 per 4 µm section with a range of 0-352. Over 50% of 4 µm sections had 0-5 lymphocytes. The section that contained the greatest number of CD8+ T-cells was located over 400 µm deep in the FFPE block, indicating that sections taken from the surface of the block were not representative of the area of highest CD8+ T-cell density in the cancer tissue. Conclusions We demonstrated that the 3D I/O Pro™ pipeline can quantify the total number of cytotoxic T-cells in whole tissue samples, providing a valuable ground truth for evaluation of immune infiltration in the tumor microenvironment versus conventional histology which analyzes only a portion of the tissue. The number of CD8+ T-cells varied throughout the depth of the tissue, a phenomenon that became most apparent at 400 µm deep in the FFPE block. In the future, we plan to correlate features, including total CD8+ T-cell quantification and spatial location versus tumor cells, and other features from the 3D TME with response to immunotherapy, to refine histologic definitions of immune exclusion.

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