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 pre-established validated panels to label various immune and cancer cell biomarkers. Many of these panels can be adjusted with the addition or removal of markers to help reach your research goals, check out our collection of validated panels here.
Tumor Microenvironment Panel
To take an in-depth look into the complex and dynamic ecosystem of the tumor microenvironment, a 19-marker panel allows for a detailed analysis of various cell populations, activation status and characteristics of the surrounding microenvironment of tumor tissue. Macrophage polarization and T-cell phenotype markers allows for an overview of polarization status (M1, M2a, M2b, M2c and M2d) and T-cell populations (Helper, Cytotoxic and Regulatory). Activation status of fibroblasts status with FAP and Vimentin associated with common fibroblasts markers and profile markers for proliferation (Ki67), apoptosis (Caspase-3), angiogenesis (VEGF) and hypoxia (HIF1α) allow an assessment of tumor progression, severity, and changes within the extracellular matrix of a tumor.
DAPI/Blue, CD206/Yellow, CD8/Red, VEGF/Green, CD45/CY5
T-cell Activation Panel
To study T-cell activation, tissue 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
PD-L1 Checkpoint Inhibition Panel
Our checkpoint inhibition panel labels PD-L1 expressed on tumor cells and macrophages. Cytotoxic T-cells are labeled 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
Antigen Presenting Cell Panel
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, CD68/Green, MHCII/Red, CD20/CY5, CD11b/Yellow
Memory T-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-cell 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
Validated Antibodies
Validated antibodies have been validated with the Visikol multiplex immunofluorescence and standard immunohistochemistry chromogenic approaches to ensure positive labeling at an optimal concentration. However, for all service projects, Visikol conducts a sample validation with positive and negative control samples prior to processing project samples.