The most recent breakthroughs in cancer research and treatment have been in the field of immuno-oncology, using novel immunotherapies to bolster the host’s immune system to more effectively target and destroy tumor cells. Among the list of FDA-approved therapeutics with immunoregulatory activity are antibody and cytokine-based immunotherapies, small molecule drugs, and cell-based therapy. The success that these and other therapies have had at prolonging survival has placed emphasis on the development of new single agent and combination therapies with greater efficacy at treating cancer.
Productive preclinical development of immunotherapies requires high-throughput and robust quantitative analysis of the immune system in the tumor micro-environment, peripheral blood, and other host tissues.
To meet this need, MI Bioresearch offers state-of-the-art analytical flow cytometry services supporting all aspects of your preclinical drug development.
- Our in-house services for sample processing and data acquisition begin immediately after a study is taken down, eliminating the risk of sample degradation that can occur during overnight shipment.
- Our instrumentation includes two Invitrogen™ Attune™ flow cytometers uniquely designed to handle complex tumor-derived cell samples and acquire data at rates greater than 10,000 cells per second.
- We can generate actionable data on even rare cell populations such as regulatory T-cells that can comprise only 1-2 percent of total cells.
- Both of our Attune™ NxT’s are fully upgraded with four lasers and 16 detection parameters to support complex antibody panels required for in-depth cell immunophenotyping and functional characterization of heterogeneous cell samples.
- Our instruments are also configured with Attune™ Autosampler plate loaders to support both 96- and 384-well plates, which enable the analysis of hundreds of samples in only a few hours.
With over 50 years of combined expertise, our full-service analytical team can add an ex vivo flow cytometry arm to any in vivo study. Select from our list of standardized antibody panels (see below) that have been validated in multiple in vivo cancer models. Our panels provide basic to comprehensive analysis of a wide range of lymphoid and myeloid lineage immune subsets in order to capture any shifts that occur in the intra-tumor immune response that is triggered by in vivo test agent treatment.
Lymphoid Lineage Panels
Tumor-infiltrating CD4+ and CD8+ T-Cell Analysis in a Murine 4T1 Breast Carcinoma Model
Quantitative measurement of tumor-infiltrating T-cells can be crucial when evaluating the immune-modulating effects of new immunotherapies. The MI-BasicT™ provides T-cell subset measurement and has been validated in multiple tissue types. In the above study, T-cell subsets in a mammary fat pad implanted 4T1 mammary carcinoma were analyzed ex vivo. CD3+ T-cells were first identified by gating on CD45+ hematopoietic cells. T-cell populations were further analyzed based on CD4 and CD8 lineage marker expression.
Regulatory T-Cell Analysis in the Murine CT26 Colorectal Tumor Model
Regulatory T-cells suppress anti-tumor responses in several disease models and are an attractive target for new immunotherapies. The MI-Treg™ is an expansion of the MI-BasicT™ and can be used to evaluate the effects of new therapeutics that target regulatory T-cells. In the study above, regulatory T-cells were analyzed ex vivo in spleen from CT26 adenocarcinoma-bearing hosts. CD4+ T-cells were first identified by gating on CD45+CD3+ cells. Regulatory T-cells were further differentiated based on co-expression of CD25 and intracellular FoxP3.
T-Cell and B-Cell Subset Analysis in Murine Spleen
The MI-BasicT&B™ can be utilized to quantify CD4+ and CD8+ T-cells as well as B-cells fractions in heterogeneous samples. In the study above, CD3+ T-cells and CD19+ B-cells were analyzed in the CD45+ gate. T-cells were further subdivided into CD4+ and CD8+ populations. Only six fluorescence channels are occupied by this panel, which leaves several channels available for further cell characterization by dropping in additional antibodies.
Natural Killer (NK) Cell NKT Cell Subset Analysis in CT26 Colorectal Adenocarcinoma Model
NK cell subsets are well known for their anti-tumor activity and ability to control the growth of many tumors. The MI-NK™ can be used to identify theses subsets. The study above was performed to quantify percentages of NK cell and NKT cell subsets within tumor-derived Ly6G-Ly6C- immune cells.
Analysis of T-Cell Activation in the Murine A20 B-Cell Lymphoma Model
One challenge faced during the development of new immunotherapies is overcoming the loss of anti-tumor activity that occurs in T-cells within the tumor microenvironment. A state referred to as T-cell exhaustion can be identified based on the relative expression of different immune checkpoints and activation markers. The MI-T A&E™ is an expansion of the MI-BasicT™ and can be used to measure expression for select key biomarkers in different tissues. In the study above, expression of immune checkpoint proteins CTLA-4 and PD-1 as well as the surrogate proliferation marker Ki-67 were measured in T-cell subsets within A20 cell line-derived tumors.
CD4+, CD8+, and Regulatory T-Cell Analysis Combined with Immune Checkpoint/Activation Marker Interrogation Using the MI-CompT™
The MI-CompT™ combines the utility of the MI-BasicT™, MI-Treg™, and MI-T A&E™ into one comprehensive analysis of the T-cell profile within tissues. The data above displays A) CD4+/CD8+ T-cell subset analysis in murine tumors using a 4T1 breast carcinoma model, B) regulatory T-cell analysis in spleens from CT26 colorectal adenocarcinoma bearing mice, and C) CTLA-4, PD-1, and Ki-67 expression levels in T-cell subsets from A20 (B cell lymphoma) cell line-derived tumors.
Myeloid Lineage Panels
Myeloid-Derived Suppressor Cell (MDSC) Analysis in a Murine CT26 Colorectal Oncology Model
MDSC subsets can influence several pro-tumor responses including the suppression of immune function and are an attractive target for immunotherapy. They are a heterogeneic cell population, the phenotype of which can be shaped by signals in the tumor microenvironment. The data above demonstrates how the MI-MDSC™ can be used for basic identification of granulocytic (G-) and monocytic (M-) MDSC subsets in CT26-derived tumors. CD11b+ myeloid cells were first identified in the CD45+ gate after CD3+CD19+ lymphocyte exclusion. G-MDSC and M-MDSC were further differentiated based on expression of Ly-6G and Ly-6C surface receptors.
Analysis of Classically (M1) and Alternatively (M2) Activated Tumor-associated Macrophages in a CT26 Colorectal Adenocarcinoma Model
M1 and M2 macrophages are the two major macrophage groups that reside within the tumor microenvironment. They have opposing functions with regard to cancer progression and are therefore targets for new immunotherapies. The study above demonstrates how the MI-TAM™ can be used to profile the ratios of these two groups. To this end, MDSC subsets were excluded so that CD11b+/F480+ macrophages could be identified. This fraction was analyzed for M2 macrophages (CD206+) and M1 macrophages (CD206-MHCII+).
Analysis of Myeloid-derived Suppressor Cell (MDSC), Natural Killer (NK) Cell, and Dendritic Cell (DC) Subsets in a Murine CT.26 Colorectal Tumor Model
The MI-ExpandedMyeloid™ is an expansion of the basic MI-MDSC™ to enable analysis of NK and DC subsets in addition to MDSC populations. In the data shown, these subsets were analyzed in CT26-derived tumors. Monocytic (M-) and granulocytic (G-) MDSCs express high levels of Ly-6C and Ly-6G protein respectively (Bright) and co-expressed the CD11b marker. NK and DCs were analyzed in the MDSC exclusion gate and further differentiated based on pan-NK markers (CD49b and CD335) vs DC-specific CD11c protein expression. Red and grey peaks represent CD11b stained and unstained cells respectively.
Myeloid Cell Analysis in Tumors Using MI-CompMyeloid™
The MI-CompMyeloid™ builds on the MI-MDSC™ for a more in-depth analysis of myeloid cell populations. It also enables the analysis of the immune inhibitory receptor PD-L1 on both tumor and immune cell subsets. The data above demonstrates how the MI-CompMyeloid™ can be used to A) measure PD-L1 expression on tumor and immune cells (4T1 breast carcinoma model), B) quantify macrophage and dendritic cell subsets (CT26 colorectal tumor model), and C) characterize MDSC subsets for expression of CD115, which is a receptor that has been shown to directly correlate with suppressive activity (CT26 colorectal tumor model). This panel can also be helpful in the analysis of neutrophils and monocyte populations (not shown). Red and grey peaks represent cells stained for target antigen and unstained cells respectively.
Consult with our experts to develop a custom antibody panel that meets your unique needs. We also offer a variety of in vitro cell-based functional assays to analyze biological responses following treatment. Take advantage of these services to gain insight into the mechanistic action of new drug candidates. Post-study support services and consultations with our experts are available to assist you in the interpretation of data and the outline of future study direction.
All MI Bioresearch, standard flow cytometry panels are copyrighted. Unauthorized disclosure is prohibited. Client shall not reverse engineer, perform any studies to determine the structure or chemical composition, or elucidate the physical properties in which any of our processes or panels have been created.