Trends in Oncology

Non-human primates (NHPs) continue to be a valuable resource for preclinical research because of the similarities they share with humans. NHPs, especially rhesus macaques, are used in preclinical studies for evaluating new drugs or vaccines for safety and efficacy. Flow cytometry assays can be easily adapted to study cells from NHP. Consider these three factors if you are planning to adapt a flow cytometry assay for use with NHP samples.

1. Staining panel prep: Like human subjects, PBMCs can be collected from Non-human primates and evaluated by flow cytometry. Many immune cell subsets, especially B and T cells, share similar phenotypes with their human counterparts. Human antibodies are most often used to stain NHP cells because of similarities shared between surface markers. Consider which cells of interest you may want to study and how existing antibodies used in flow cytometry may be used in your panel.
2. Real-world variability: Many scientists are accustomed to working with an inbred mouse strain, which reduces background variability observed in experimental settings. In contrast, NHPs are outbred animals, and like humans, may display widely varying responses. As you develop your NHP protocols, consider how you will handle highly variable data, including flow cytometry data. These considerations will inform how many animals may be used in each group and how many cells are stained and evaluated in each sample.
3. Metabolism and toxicity: NHPs share many physiological similarities to humans and are a valuable model for pharmacokinetic and pharmacodynamic measurements of experimental drugs and biologics. Toxicity testing can also be done in NHPs and together these data are critical to evaluating candidates in preclinical pipelines.

Developing NHP protocols from inception to sample analysis requires working with experts who have experience writing protocols that are compliant with appropriate oversight committees, such as institutional animal care and use committee offices. Working with experts, such as contract research organizations, also assures that any NHP flow cytometry studies will satisfy any necessary regulatory compliance determinations related to drug and biological development. 

Hematologic malignancies include a wide array of lymphomas and leukemias that affect different immune cell subsets. Acute myelogenous leukemia (AML) is one of the most commonly occurring leukemias in adults and children. AML is a highly heterogeneous disease that can be caused by spontaneous gene mutations or chromosomal translocations, which result in the proliferation of dysfunctional myeloid cells. Cytogenetic and morphologic analyses have been the gold standard methods used in AML diagnosis. Still, flow cytometry-based protocols are becoming more widely used and validated as complementary diagnostic methods that can be coupled with these analyses to better guide treatment plans. Flow cytometry has also become an essential tool to understand AML progression and develop and evaluate novel therapeutics.

Consider these aspects of flow cytometry-based analysis of AML for exploratory or preclinical research:

Phenotype matters: Immunologists know a lot about what a normally developing myeloid cell should look like in terms of its immunophenotype. Changes in expression of different lineage markers correlate strongly with AML progression and are characterized by the presence of blasts (leukemic cells) in the bone marrow. Flow cytometry-based immunophenotyping offers researchers a rapid and sensitive method for detecting blasts at the onset of disease as well as monitoring changes in this population throughout an experimental therapy.

Rare cells from precious samples: A major consideration of following AML progression is the ability to detect relatively rare cells in bone marrow aspirates. This type of sample may be relatively small and is typically used fresh for morphologic evaluation, but even a small volume of remaining aspirate can be used for flow cytometry-based methods that are sensitive and robust enough to detect blasts.

Paired samples: Bone marrow cells can be analyzed along with peripheral blood using advanced flow cytometry methods that monitor the persistence of blasts and other leukemic subsets over time in these compartments. This type of analysis offers critical insight into the development of new therapeutics whether they are being evaluated in humanized mouse models or clinical trial participants.

Flow cytometry continues to advance immuno-oncology research, especially for diseases involving the detection of rare cell populations. Consider working with preclinical and clinical flow cytometry experts to develop protocols for future immuno-oncology studies.

 

 

In vivo models for numerous diseases and conditions have endpoints that have involved animals being gravely ill or dying. As researchers have sought to utilize animal models in more humane and practical ways, surrogate endpoints have been developed that prevent animals from suffering and provide critical research data. Flow cytometry has been instrumental to these advances. Consider these aspects of preclinical flow cytometry endpoint analysis as you develop new protocols.

 

1. What are the immune system features of your disease state? 

Flow cytometry provides the most useful data when the cell subsets of interest are well-defined and robust. You may need to analyze existing research literature or do pilot studies to define the immune cell subsets of interest for a particular disease state, be it changes in regulatory T cells in the tumor microenvironment, or the proliferation of plasmablasts in different leukemias. You must identify which profound changes in different cell populations are most closely correlated with morbidity and mortality in your animal model.

2. What is the desired treatment outcome?

Preclinical studies with surrogate endpoints are valuable for screening potential therapeutic candidates. These drugs or biologics may have undesirable off target effects as well. In designing a flow cytometry assay for alternative endpoints, it is critical to identify the changes in immune cell subsets that reflect therapeutic improvements or indicate potential toxicity or off target effects.

3. Can this be translated into a clinical flow cytometry protocol?

In some disease models, particularly models using humanized mice, flow cytometry endpoints can be used in both preclinical screens and to evaluate clinical trial specimens. This consideration is valuable as protocols are developed and cell phenotypes are identified as predictors of good or poor prognoses.

Flow cytometry endpoint analysis not only advances the humane use of animal models but can be translated into informative clinical protocols that are critical for the evaluation of potential therapies.