In this application note, we describe two HALO analysis tools that are used to quantify the distribution of immune cells relative to tumor cells in the tumor microenvironment, Proximity Analysis and Tumor Infiltration Analysis. The analysis tool used ultimately depends on the staining methods employed, and more importantly, on the research objectives and outputs required as described in more detail in the following sections.
A positive correlation between survival and T-cell density in the tumor core and invasive margin was first reported in colo-rectal cancer (Galon, J. et al., 2006). Independent investiga-tions have confirmed the predictive value of 'immune contex-ture' with respect to survival (as reviewed by Fridman, W.H. et al., 2011). Using HALOTM to analyze immunohistochemically (IHC)-labelled and immunofluorescently (IF)-labelled immune cell markers, Tumeh et al. reported that CD8+ cell density at the invasive margin could predict response to anti-PD1 thera-py in aggressive melanoma (2014). These studies highlight the importance of technologies which facilitate in situ, spatially-resolved analysis of immune markers to enable our under-standing of the complex interactions between the immune system and the tumor, and ultimately for predicting patient survival and selecting appropriate treatments.
Proximity analysis is used to quantify the spatial relationship between any two cell or object populations detected using HALO. In immuno-oncology, the primary applications for proximity analysis are to 1) count number of cells labelled with an immune cell marker (e.g. CD8, CD3, or CD4) within a certain distance of tumor cells (identified with tumor-specific marker) and 2) measure the distance between the two cell populations. Immune cells and tumor cells can be stained together on a single section using dual IHC, multiplex IHC, or multiplex IF or can be stained separately in parallel using serial sections.
An example of proximity analysis using serial sections from pancreatic tumor is shown in Figure 1. Here, the two serial section images probed individually for the tumor marker pancytokeratin (Pan-CK) and T-cell marker CD3 are aligned using whole-slide elastic registration. The Pan-CK-stained section is analyzed with membrane IHC and the CD3-stained section is analyzed with cytonuclear IHC to identify stained cells and map their x-y coordinates on each tissue section.
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