Multiplex Panels2021-08-09T15:15:09-05:00

Since the discovery of immune checkpoint proteins and the first FDA approval of anti-CTLA 4 therapy (Ipilimumab) in 2011, there has been a rapid increase in the number of checkpoint inhibitors that are either approved or at various stages of clinical development. Despite their tremendous promise, the clinical benefits from checkpoint inhibition are limited due to the refractory nature of tumor microenvironments. As tumors grow, they recruit a variety of cells such as regulatory T and B cells, cancer associated fibroblast and myeloid cells that contribute to an immunosuppressive environment that favors tumor growth. In other instances, tumors negatively influence the expression of cell adhesion molecules that prevent the migration of cytotoxic T cells into the tumor, negatively impacting immunotherapy. To interrogate the complex tumor-immune environment, we at Visikol have developed four panels to label 16 different immune and cancer cell biomarkers.

T-cell Activation Panel

To study T cell activationtissue samples are labelled for CD3-a T cell receptor protein and granzyme b, a serine protease that is primarily secreted by cytotoxic CD8+ T cells and NK cells. Activated T cells are positive for both CD3 and granzyme b and tumor tissue is positively labelled for pan-cytokeratin. Ki67 marks regions of the tumor that are actively proliferating.

DAPI/Blue, PanCK/Yellow, CD3/Red, Granzyme b/Green, Ki67/CY5

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PD-L1 Checkpoint Inhibition Panel

Our Checkpoint inhibition panel labels PD-L1 expressed on tumor cells and macrophages. Cytotoxic T cells are labelled with CD8 and tumor associated macrophages are labelled with CD68. This panel also includes pan-cytokeratin that labels tumor cells. Binding of PD-L1 expressed on tumor cells to PD-1 expressed on T cells is known to dampen the immune response. This panel also detects the presence of immunosuppressive tumor associated macrophages that express both PD-L1 and CD68.

DAPI/Blue, CD68/Green, PanCK/Yellow (Very little expressed; normal for tonsil), CD8/Red, PD-L1/CY5

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Antigen Presenting Cell Panel

The Memory T cell panel labels CD45RO which marks memory T cells and exhausted T cells are labelled for PD-1. This panel also labels, CD3 a pan T cell marker and pan cytokeratin that labels tumor cells. The presence of double positive CD45RO and CD3 T cells in the tumor microenvironment would indicate a favorable adaptive immune response compared to the presence of double positive PD-1 and CD3 T cells that represents an immunosuppressed T cell population.

DAPI/Blue, CD68/Green, MHCII/Red, CD20/CY5, CD11b/Yellow

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Memory T-cell Panel

Finally, our Antigen presenting cell panel includes CD11c to label dendritic cell, CD68 that labels resident macrophages, CD20 that labels B cells and MHCII that labels professional antigen presenting cells. Dendritic cells are considered the most powerful antigen presenting cells and their presence in the tumor microenvironment is favorable to mounting an adaptive immune response against tumor cells. Dendritic cells bind tumor neoantigens and present them along with MHCII molecules to T cells.

DAPI/Blue, PD-1/Green, PanCK/Red, CD3/Yellow, CD45 RO/CY5

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