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The current landscape of immuno-oncology biomarkers is evolving rapidly, bringing in a new era of immune-modulating therapeutic combinations for oncology studies. Flow Cytometry offers the potential for powerful biomarker screening paradigms and multiparameter cellular profiling that enable researchers to interrogate the immune system's mechanisms at work. Over the past decade, the FlowMetric team has supported a diverse range of immune system studies and delivered customized and qualified flow cytometry solutions to support the most complex immuno-oncology pre-clinical and clinical trials.

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Comprehensive Cytokine Profiling

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The Monitoring of Adaptive Cell Transfer

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Evaluation of Checkpoint Inhibitor Effects on Tumor Cells

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Functional Phenotyping of TILs

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Analysis of T-cell exhaustion

Analyzing Tumor Infiltrating Lymphocytes within the Tumor Microenvironment.

The ability to assess the tumor microenvironment and describe the phenotypic and metabolic adaptations of tumors is at the core of immuno-oncology. FlowMetric has developed robust and reproducible methods for efficient tumor disruption and recovery and immunophenotyping of Tumor Infiltrating Lymphocytes (TILs), performed with or without CD45+ enrichment. Fig. 1. Demonstrates flow cytometry based phenotypic subset distribution of immune cells and cytokine profiling of progressively growing tumors. By coupling this analysis with the ex vivo expansion of TILs, we are able to identify the immunomodulators (cytokines and growth factors) driving the T-cell function.

Immuno-profiling may be used to examine key checkpoints of cellular pathways that are crucial for in maintaining normal immune responses and protecting tissues from potential damage during an immune response. Tumor cells disrupt these pathways, resulting in immune resistance. One focus of immunotherapy is to ensure these checkpoints remain active to regulate active adaptive and innate immune responses to the tumor. Widely studied checkpoint regulators include CTLA-4, PD-1, PD-L1.

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Figure 1A.

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Figure 1B.

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Figure 1C.

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Figure 1. Immune Checkpoint Monitoring for Identification of Immune Cell Subsets.
PMBCs from normal healthy donors were stained with a 16-color T Cell Exhaustion Panel and analyzed by flow cytometry. (A) and (B) Assessment of expression levels of important checkpoint and activation molecules on CD4+ and CD8+ T cells. (C) T Reg cells were identified as CD25high CD127low and FoxP3+. Expression was induced by stimulating cells with anti-CD3, anti-CD28 coated beads.

Analyzing CAR-T by Flow Cytometry

Since CAR-T research focuses on various immune cell types and their behavior in cancer, flow cytometry represents a powerful and versatile technology for the interrogation of immune cell responses and their effects on cancer cells. An example of such a flow assay, involves the quantification and characterization of specific subsets of circulating T cells. This type of analysis provides a platform to better gauge the immunogenicity of the therapy and the ability of the immune cells to successfully identify and attack cancer cells.

The use of flow cytometry is at the forefront of this medical revolution in immuno-oncology, as an empowering and versatile technology enabling in-depth analysis of cellular behavior of CAR-T cells and their potential role in cancer biology and effective therapeutic intervention. FlowMetric provides a variety of assessment profiles to examine the stability, safety and efficacy of your CAR cells.

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Evaluation of CAR Expression on Cells and Phenotypic and Subset Composition

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In Vitro Cytotoxicity Testing.
Cell Mediated Cytotoxicity

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Verification of CAR Cell Immune Functions. Intracellular Cytokine Staining, Isoplexis Cytokine Profiling

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Tracking of T cell Activation, Expansion and Proliferation (MTT, MTS, CFSE)

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Therapeutic CAR-T activity leverages the expression of key receptors for the chemokines secreted by the tumor. Endogenous receptors include CXCR5 and CCR5, but increased activity can be achieved through the enhanced/engineered expression of receptors such as CCR2 and CCR4 for chemokines naturally secreted by tumors and this can improve tracking of the T-cells to the tumor. Antigen-activated CAR-T cells within the TME, upregulate expression of inhibitory receptors, resulting in T cell dysfunction. Drugs targeting these receptors can help overcome the inhibition of these checkpoints. The efficacy of immune checkpoint inhibitors is highly influenced by the TME. Responsive tumors tend to display increased levels of TILs, a higher T-effector to T-regulatory cell ratio, and increased cytokine secretions such as IFN-γ. In contrast, non-responsive tumors display higher levels of T-regulatory, and reduced levels of NK and activated lymphocytes.

The FlowMetric Advantage

Our scientific team has extensive experience in the development of flow cytometry panels to examine various aspects of immuno-oncology and immunotherapy. We provide advanced platforms that enable us to acquire and analyze large data sets, ensuring full support of your clinical reporting to regulatory agencies.