Immunohistochemistry (IHC) staining of in-vitro cancer cell-generated tumoroids.

Targeting different pathways in combinational therapy may lead to synergistic effects with higher drug efficiency. Due to a large number of candidate drugs and the variability in the genomic landscape of the disease, conventional cell culture models have limited success. Three-dimensional (3D) cell culture platforms such as tumoroids not only provide a pathophysiological relevant condition but also allow for low-cost and high-throughput drug screening strategies. Immunostaining of targeted proteins within a tumoroid is challenging as the interior cells are difficult to access via a non-destructive method. Immunohistochemistry (IHC) is an important technique in clinical research to explore the expression of various biomarkers. IHC staining of tumoroids allows non-destructive detection of unstable proteins by direct fixation of cells at the state of tumor microenvironment (TME) context, providing two main advantages. First, the target protein can be fixed without dissociating cells and disintegration of tumoroids into a single-cell suspension. Second, staining the preserved structure of tumoroids helps identify the location of the target proteins as well as the spatial distribution throughout the tumoroid geometry. In this protocol, we describe the detailed methodology of a non-destructive IHC staining of cancer biomarkers which minimizes the manipulation of tumoroids prior to fixation by eliminating multiple centrifugations and shaking steps typically required for removing excess hydrogel and collecting tumoroids. The protocol can be used in studies involving prognostic and predictive biomarker investigations in new anti-tumor drug development strategies.