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 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, our contract flow cytometry service, provides you with an advanced, state-of-the-art analytical flow cytometry resource to support all aspects of your drug development needs. Run your sample generation studies with us or overnight-ship us your preclinical or non-CLIA regulated clinical samples and we’ll take care of the flow cytometry for you.
- Our in-house services for sample processing and data acquisition begin immediately after a study is taken down, eliminating the risk of sample degradation.
- Our instrumentation includes two Beckman Coulter CytoFLEX LX and two Invitrogen™ Attune™ flow cytometers, uniquely designed to handle complex tumor-derived samples and acquire data at rates greater than 10,000 cells per second.
- All instrument are fully upgraded enabling the detection of up to 23 parameters, and most commercially available flow cytometry fluorophores. This supports analysis using complex antibody panels required for in-depth immunophenotyping and functional characterization of heterogeneous samples.
- We can generate actionable data on rare cell populations such as regulatory T-cells that can comprise only 1-2 percent of total cells.
- Our instruments are configured with automated plate loaders allowing for high throughput capture rates.
Basic to Comprehensive Immune Profiling
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. Our list of standardized antibody panels (see below) have been validated in multiple in vivo cancer models and provide basic to comprehensive analysis of a wide range of lymphoid and myeloid lineage immune subsets to capture shifts that occur in the immune response triggered by in vivo test agent treatment. See a list of our panels below.
Comprehensive Leukocyte Quantitation
Comprehensive Service for Lymphoid and Myeloid Subset Analysis including Absolute Counts (MI-CompLeukocyte™)
The MI-CompLeukocyte™ package provides our portfolio’s most comprehensive profile capability by enabling the analysis of 11 immune cell subsets. The MI-CompLeukocyte™ package includes 2 immunophenotyping panels and 1 quantitation panel to enable absolute cell counts within tissues.
In the data shown, CT26 tumor-bearing mice were treated with an anti-mCTLA-4 checkpoint inhibitor or an isotype control. Tumors were harvested on day 18 to measure:
(A) Absolute counts of tumor-infiltrating cells
(B) CD69* and PD-1 on tumor-derived CD8+ T cells
(C) Ki-67 median fluorescence intensity (MFI) to examine CD8+ T cell proliferation
*CD69 can be substituted for other markers including LAG3, ICOS, or CTLA-4.
Lymphoid Lineage Panels (Mouse)
16-Color T Cell Analysis for CD4+/CD8+, Regulatory, and Effector/Memory Subsets including 7 Activation/Exhaustion Markers (MI-Expanded CompT™)
Analysis of T Cell Subsets in MC38 Tumors Using the MI-Expanded CompT™ Panel
The MI-Expanded CompT™ panel combines the utility of the MI-CompT™ and MI-Effector/Memory™ panels (A & B), and then adds four additional markers for T cell activation and exhaustion (C). ICOS, LAG-3, TIM-3, and granzyme B are intensively investigated biomarkers for T cell functionality, which can provide insight into the anti-tumor potential of CD8+ T cells. Evidence supports a co-stimulatory and anti-tumor role for ICOS receptor signaling, thus making ICOS an attractive therapeutic target. Granzyme B is often used as a biomarker for cytolytic activity and can correlate with CD8+ T cell anti-tumor responses. Conversely, PD-1, LAG-3 and TIM-3 are inhibitory receptors and the expression of these three receptors has been linked to T cell exhaustion. The data above demonstrates how the MI-Expanded CompT™ panel can be used to immunophenotype murine MC38 colon adenocarcinoma tumors.
Comprehensive T Cell Analysis for Regulatory and CD4+/CD8+ Subsets Including Activation/Exhaustion Marker Profiles (MI-CompT™)
CD4+, CD8+, and Regulatory T-Cell Analysis Combined with Immune Checkpoint/Activation Marker Interrogation Using the MI-CompT™ Panel
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.
Natural Killer/Natural Killer T Cell Analysis (MI-NK™)
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 Ly-6G immune cells.
Effector/Memory T Cell Analysis (MI-T Effector/MemoryTM)
Analysis of Effector and Memory T Cell Subsets in CT26 Tumors Using the MI-T Effector/Memory™ Panel
Memory T cell formation following antigen challenge is essential for long lasting immunologic memory. In preclinical mouse models, the activity of memory T cells can determine whether successful rejection of tumor formation upon a tumor cell implant re-challenge is achieved. Memory T cells can be identified based on high levels of CD44 expression. Antigen-specific memory T cells can be further subdivided into CD62L– effector memory (Tem) and CD62L+ central memory (Tcm) subsets, which reside in peripheral and lymphoid tissues respectively. These cells can rapidly combat tumor formation by readily converting back into proliferating effector T cells (Teff) with robust cytokine producing potential. The data above demonstrates how the MI-T Effector/Memory™ panel can be used to identify naïve, effector, Tem, and Tcm subsets in both naïve spleen and CT26 tumor-derived cells. The splenic CD8+ T cell phenotype is predominantly naïve (CD44–CD62L+). In contrast, the tumor-infiltrating T cells are predominantly activated effector cells (CD44–CD62L–), and contain a minor subset with a Tem phenotype (CD44+CD62L–).
CD4+/CD8+ T Cell Analysis (MI-BasicT™)
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 and CD4+/CD8+ T Cell Analysis (MI-Treg™)
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.
CD4+/CD8+ T Cell and B Cell Analysis (MI-BasicT&B™)
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.
T Cell Activation, Exhaustion, and Proliferation Biomarker Analysis (MI-T A&E™)
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.
Lymphoid Lineage Panels (Human)
Human T Cell Analysis (Human BasicT™)
Analysis of T Cells Following Human PBMC Engraftment
Quantitative measurement of T cell engraftment and persistence useful for analysis of adoptive cell therapy and humanized in vivo models. The gating strategy above demonstrates analysis of human CD4+ (hCD4+) and hCD8+ T cells in human PBMC engrafted NSG mice (week 4 post‐PBMC transfer). This analysis has also been validated for use on tumors from xenograft-bearing CD34+ humanized mice.
Myeloid Lineage Panels
Comprehensive Myeloid Analysis for M1/M2 Tumor-Associated Macrophages, Myeloid-derived Suppressor Cell Subsets, and Dendritic Cells (MI-CompMyeloid™)
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-CompMyeloid™ can be used to profile the ratios of these two groups. To this end, M-MDSC/G-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+). Dendritic cells are also delineated using the MI-CompMyeloid™ panel, and are identified based on CD11c and MHC class II expression (not shown).
Comprehensive Dendritic Cell Analysis (MI-CompDC™)
Dendritic Cell Analysis in Tumors Using MI-CompDC™ Panel
Dendritic cells (DC) are professional antigen presenting cells that are instrumental in the initiation to T cell mediated anti-tumor responses. DCs can be activated to express numerous costimulatory ligands and can internalize antigen and present immunodominant peptides to both CD4+ and CD8+ T cells. The development of novel therapeutics that harness DC function is an area of intensive investigation. The data above demonstrates how the MI-CompDC™ panel is used to identify conventional DC subsets (cDC1 and cDC2) in B16-F10 tumor-derived cells. Additional markers in the panel enable delineation of CD8+ and XCR1+ DC subsets, as well as the measurement of maturation markers CD80 and CD86 (not shown).
In Depth Myeloid-derived Suppressor Cell Characterization Plus PD-L1 Analysis (MI-CompMDSC™)
The MI-CompMDSC™ panel builds on the MI-MDSC™ for a more in-depth analysis of the MDSC subsets. It also enables the analysis of the immune inhibitory receptor PD-L1 on both tumor and immune cells. The data above demonstrates how the MI-CompMDSC™ can be used to A) measure PD-L1 expression on tumor and immune cells (4T1 breast carcinoma model), and B) characterize MDSC subsets for expression of CD115, which is a receptor demonstrated to directly correlate with suppressive activity (CT26 colorectal tumor model). Red and grey peaks represent cells stained for target antigen and unstained cells respectively. F4/80 and CD11c expression on MDSCs can also be measured (not shown).
Myeloid-Derived Suppressor Cell Subset Analsysis (MI-MDSC™)
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.
We offer phospho-flow cytometry, which is a valuable tool for screening the effects of new drugs on the activation of cell signaling pathways in vitro; as well as having the ability to measure drug effects in vivo by multiplexing the technique with immunophenotyping cell surface markers to distinguish analysis in different cell subsets. This platform uses fluorescence-labeled antibodies that recognize proteins only when phosphorylated on specific amino acid residues that regulate their function. Phospho-flow data is highly consistent and reproducible, which in turn creates a unique platform for cell signaling analysis.
In Vitro Phospho-Flow Analysis of PI3K/AKT Signaling
Hyper-phosphorylation of AKT promotes tumor survival in various malignancies and is a target for cancer treatment. The data above demonstrates how the phospho-flow platform can be used to measure phosphorylation of AKT and the downstream target S6 in cultured MV-4-11 cells treated with the MK-2206. After 2 hours of treatment, cells were and stained simultaneously with fluorescent phospho-specific antibodies and analyzed by flow cytometry. Treatment-induced inhibition of AKT and S6 phosphorylation was measured by comparing the MFI from treated and untreated cells. Samples were treated and acquired in duplicate and the data is representative of three independent experiments.
In Vitro Phospho-Flow Analysis of MAPK Signaling
30% of all cancers have mutations in the KRAS gene triggering hyper-phosphorylation of MEK and ERK kinases. Therefore the development of new inhibitors that target this signaling pathway are a focus of pre-clinical research. The data above demonstrates how the phospho-flow platform can be used to quantify the inhibition of MEK and ERK phosphorylation induced by sorafenib treatment in cultured MV-4-11 cells. Cells were treated under the conditions shown for 15 minutes, stained simultaneously with fluorescent phospho-specific antibodies, and acquired by flow cytometry. Three independent experiments were combined and the MFI was used to measure phosphorylation states. Statistical analysis was performed using a Student’s T-test (* p<0.05).
Phospho-Flow Analysis of the JAK/STAT Signaling in T Cells
JAK/STAT pathways couple cytokine receptor signaling with de novo gene transcription and are important for both tumor cell survival and T cell activation. The data shown demonstrates how the phospho-flow platform can be used to measure STAT5 phosphorylation in cytokine treated CD4+ T cells in vitro. Purified murine CD4+ T cells were treated with IL-2, IL-7, or both for 15 minutes and stained with a fluorescent phospho-specific STAT5 antibody. MFI was used to measure phosphorylated STAT5 and data is presented in a histogram overlay and bar graphs. CD4+ T cells were purified from spleen by negative selection using antibody labeled magnetic beads. Data shown is representative of three separate experiments.
Phospho-Flow Analysis in a Disseminated AML Model for PI3K/AKT Signaling
The unique power of phospho-flow is demonstrated by the ability to detect phospho-proteins at a single cell level in heterogeneous samples. The data above combines phospho-flow with immunophenotyping techniques to enable detection of phosphorylated AKT and S6 in MV-4-11 tumor cells that have homed to the bone marrow. After intravenous implantation with MV-4-11 cells, mice (n=5/group) were dosed with an AKT inhibitor (MK-2206). Bone marrow was sampled and tumor cells (A) and host hematopoietic cells (B) were gated by flow cytometry using anti-human and anti-mouse CD45 antibodies. AKT and S6 phosphorylation was detected by fluorescent phospho-specific antibodies and MFI was used to measure inhibition triggered by the AKT inhibitor MK-2206. Statistical analysis was performed using a Student’s T-test (* p<0.05).
Solid Tumor Phospho-Flow Analysis of STAT5 and MAPK Signaling
We now offer phospho-flow services for solid tumor analysis. This platform can examine a test agent’s effect on cell signaling in both tumor and immune cells simultaneously. The data above shows inhibition of MEK and STAT5 phosphorylation in tumors from mice dosed with the FLT3 inhibitor cabozantinib. Analysis was performed ex vivo in cells isolated from subcutaneously implanted MV-4-11 tumors. Immunophenotyping for human CD45 expression enabled targeted analysis of tumor cell signaling (hCD45+) and the exclusion of the host immune cell infiltrate. Statistical analysis was performed using a Student’s T-test (** p<0.01).
For more information, read our blog related to this topic: “Phospho-Flow Cytometry for the Screening of Intracellular Signaling Events in Cancer Cells and Immune Cells” or view our webinar on this subject matter.
Flow Cytometry Functional Assays
We also offer a variety of in vitro cell-based functional assays to analyze biological responses following treatment. Flow-cytometry-based functional assays can be essential for elucidating the mechanism of a drug. While standard flow cytometry can identify immune subsets and their relative abundance within a sample, functional assays allow for the characterization of the biological significance of these populations including suppressive capability or activation status of a target population. Take advantage of the variety of functional assays we offer to gain insight into the mechanistic action of new drug candidates.
Custom Antibody Panels and Consultation
We understand that the complexity of today’s drug development, along with the complexity of the immune response creates a need for evaluation of additional immune cell types that are not part of our standard antibody panels. Our experts lend their scientific acumen to consult with our customers to develop unique antibody panels that meet individual needs. Pre- and post-study support services and consultations with our experts are available to assist you in the design and/or interpretation of data along with guidance toward future study direction.
Consult with our scientific team about your flow cytometry needs.
All 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.