Multiplex Panels2022-02-03T08:50:23-05:00

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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Validated Antibodies

Validated antibodies have been validated with the Visikol multiplex immunofluorescence approach. However, for all service projects, Visikol conducts a sample validation with positive and negative control samples prior to processing project samples.

Antibody

Target Species

Host

Alpha Smooth Actin (SMA)Hu, Mu, Rb, Rt, CaGoat
Caspase-3Hu, MuRabbit
Caspase-9Hu, Mu, Rt, Ca, MaRabbit
CD3Hu, MuRabbit
CD3Hu, MuRabbit
CD4MuRat
CD8aMuRabbit
CD8Hu, Rt, PgRabbit
CD8Hu, MuRat
CD8Hu, MuRabbit
CD11bHu, Mu, Rt, Bv, Ce, ChHa, Pm, RMRabbit
CD20Hu, PmMouse
CD25Hu, MuRabbit
CD31Hu, Mu, Rt, Rb, Ca, Pr, RMMouse
CD34Hu, Mu, Rt,Rabbit
CD45roHu, PmMouse
CD45Hu, Mk, Mu, Rt, CaGoat
CD68MuRat
CD68Hu, Mu, Rt, Pm, FeMouse
CD80Hu, MuHamster
CD86Hu, Mu, Rt, CaRabbit
CD117/c-kitHu, MuGoat
CD133HuRabbit
CD163Hu, Mu, RtRabbit
CD206 (MMR/Mannose Receptor)Hu, Mu, Pr, PoRabbit
Cytokeratin Pan Type I/II (PanCK)Hu, Mu, Rt, Pr, Sh, Ze, Ch, FiMouse
Pan CytokeratinHu, MuGuinea Pig
Cytokeratin 5Hu, Mu, Bo, Ca, Sh, Rt, Pg, Ha, BtMouse
Cytokeratin 10Hu, CaMouse
Cytokeratin 13Hu, Mu, Mo, Rt, Bo, Ca, Ha, Ho, PgRabbit
Cytokeratin 14Hu. Ca, PgMouse
Cytokeratin 14Hu, Mu, Rt, Fe, Ca, RM, Sh, HoMouse
Cytokeratin 18Hu, Mu, Rt, Po, Ca, Ch, Gp, Ha, Rb, ZeMouse
Cytokeratin 18Hu, Mu, Bo, Ca, Sh, Rt, Pg, HaMouse
F4/80Hu, MuRat
Fibroblast Activation Protein (FAP)Hu, Mu, RtRabbit
Granzyme bMuGoat
HIF1AHu, Mu, RtGoat
Ki67Hu, Mu, RtRabbit
Ki67Hu, Mu, Rt, Pm, Ca, CmRat
Ki67Hu, Mu, Rt, Pm, Ca, Bo, Pg, Sh, RMRabbit
LAMP1Hu, Mu, RtRabbit
Melan-AHuRabbit
MHC Class IIHuRabbit
NanogHuRabbit
NestinHu. Mu, Rt, PmChicken
Oct 3/4Hu. Mu, RtMouse
PD-1HuGoat
PD-L1MuGoat
S100A4 (FSP1)Hu. Mu, Rt, CaGoat
Sox2Hu. Mu, Bo, Ch, PmRat
Tyrosine HydroxylaseHu. Mu, RtRabbit
Tyrosine HydroxylaseHu. Mu, RtMouse
VEGF Receptor 2 (KDR)Hu. Mu, Rt, BoChicken
VimentinHu. Mu, Rt, Bo, Ch, Ho, PgChicken

Species Glossary

Bo = Bovine
Bt = Bat
Ca = Canine
Ce = C. Elegans
Ch = Chicken
ChHa = Chinese Hamster
Cm = Cynomolgus Monkey
Fe = Feline
Fi = Fish
Gp = Guinea Pig
Ha = Hamster
Ho = Horse
Hu = Human
Ma = Mammal
Mk = Monkey
Mu = Mouse
Pg = Pig
Po = Porcine
Pr = Primate
Rb = Rabbit
RM = Rhesus Macaque
Rt = Rat
Sh = Sheep
Ze = Zebrafish

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