Quantification of Dual LAG-3 and CD3 IHC-stained T Cells in Clinical Tumor Tissue using Image Analysis
8 August 2017
By Hannah Thomson

The characterisation of immune cells within the tumor microenvironment and their potential impact on cancer treatment and prognosis has been the subject of intense investigation over recent years. Non-small cell lung cancer (NSCLC) patients have impaired immune responses, where the expression of inhibitory checkpoint molecules on T cells may lead to reduced immune cell activity.

One implication has been the engagement of LAG-3 inhibiting T cell proliferation, resulting in T cell suppression and poor prognosis. Consequently, LAG-3 immune checkpoint molecules are being investigated as a potential oncology target for modulating T cell responses in NSCLC disease.

One of the challenges in characterising LAG-3 within the tumour microenvironment is identifying the population of T cells expressing LAG-3 over other LAG-3 positive cells including B cells, NK cells, macrophages and dendritic cells.

OracleBio has been collaborating with one of our histology CRO partners, Indivumed GmbH, to develop a dual IHC staining assay which, when combined with quantitative analysis for specific identification and evaluation of LAG-3 positive T cells within clinical tumor samples.

Figure 1. (A) Magnified tissue section with IHC staining showing CD3 single positive cells (purple), LAG-3 single positive cells (brown), LAG-3/CD3 dual positive (purple/brown) and nuclear counterstain (blue). (B) Deconvoluted image showing CD3 positive staining. (C) Deconvoluted image showing LAG3 positive staining. (D) Image analysis overlay, where cytoplasmic borders for CD3 positive cell (light purple), LAG-3 positive (brown), LAG-3+/CD3+ dual positive (purple/brown) and nuclei (blue).

To exemplify the utilisation of this approach, a clinical sample of NSCLC tissue was dual IHC stained for LAG-3 and CD3 and analysed to quantify the number of LAG-3 positive T cells and associations with surrounding tumour microenvironment.

The approach described above highlights the application of high quality dual IHC staining with whole slide image analysis to characterise exemplar immune cells, or any specified dual stained immune cell combinations, in order to evaluate cell relationships within the tumour microenvironment.

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