Spatial Analysis of CD4+ Cells in Colorectal Cancer
In Europe colorectal cancer was estimated as the second most common malignancy among new cancer cases, making it an important object of scientific immune-oncology research. Various immune cell populations contribute to the tumor microenvironment and thereby have a strong impact on the development and progression of the tumor. Therefore, investigating and deciphering the tumor-triggered immune response assists for prognostic and predictive assessments in clinics. The various immune cell populations are often investigated in a spatial context, meaning that not only the immune cell phenotypes are of interest, but also their spatial distribution and interaction in the affected tissue. A very powerful software that is able to address these complex questions is the contextual image analysis software StrataQuest.
The sample below shows a virtual slide of colorectal cancer. The image was acquired with 20x objective and consists of 20 individual fields of view which are seamlessly stitched together. The tissue was stained for five different immunofluorescent markers: DAPI (nuclei in dark blue), cytokeratin (epithelial cells, light blue), CD4 (cytotoxic T cells, red), FOXP3 (regulatory T cells, green), and Ki67 (proliferation marker, white).
Image courtesy Dr. Melanie McCoy, University of Western Australia, Perth.
The aim of this project was to identify single cells, in particular CD4+ cells, to stratify the tissue into tumor and stroma, and to analyze where CD4+ cells are located with respect to the tumor areas. For this, the StrataQuest App ‘IF Immune Status in situ’ was developed to fully automate the analysis process.
First, all nuclei are identified, using TissueGnostics‘ proprietary algorithms for nuclear segmentation (a).
Next, using cytokeratin as an epithelial marker, all epithelial areas (which are also tumorous) are recognized (b).
The nuclear mask can be combined with the epithelial mask (c) in order to address the question: ‘How many cells are CD4+, and where are they?’. CD4+ cells are identified by the intensity of the CD4+ staining within a detected cellular mask (see image (d) for visualized result).
Scattergrams are especially useful for this purpose: after selecting a scattergram of interest (for example, CD4+ means intensity vs nuclei size) and setting appropriate cut-off values, one can use the backward connection to see cells. In the following case, more than 42 thousand CD4+ cells have been detected and are outlined in red (d).
Apart from quantification in general, StrataQuest can also output data on distance measurements: for example, most CD4+ cells (outlined in red) were found to be very close to the tumorous epithelium – ranging from 1 to 25µm (e).
StrataQuest by TissueGnostics is a powerful tool for tissue imaging and tissue microenvironment analysis since it can detect tissue structures, cellular organelles and compartments, and quantify spatial relationships between cells. If you are interested in learning more about tissue microenvironment analysis, you can read our blog posts on this topic:
