Cell Population Analysis as a Biomarker
Tuulia Huhtala, Christoph Eberle

Cell Population Analysis as a Biomarker

Flow cytometric technology has been around for 50 years. As we learn more about the immune system’s role in incurable diseases, flow cytometry is suddenly popular again.

Since the first instruments using fluorescence-based detection came to market flow cytometry has been widely utilized.  Nowadays its support reaches from clinical diagnostics to food safety testing to basic research and drug development.  Essentially, it is used to separate, count and characterize particles in a fluid – simultaneously and with high speed. For most applications these particles are variably heterogenous populations of cells. Any sample collected, for example from a patient or an animal model, is processed as single-cell suspension, and cell targets are typically identified by fluorochromes linked to primary antibodies directed against a preselected set of antigens. For analytical interrogation these tagged cells are forced into a line by hydrodynamic focusing (this can be done even more efficiently with the help of sound waves), so that one by one each cell passes the laser beam in a flow cytometer. Thereby the fluorophores conjugated to bound antibodies get excited and emit fluorescence, which is detected and ultimately converted to an electronic file.

Light-scatter properties reveal cell size and complexity, whereas molecular details can be determined based on the pattern, in which antigenic markers are expressed on the surface and intracellularly. Datasets generated with all this stored information can be enormous. A common analytical method  is to gate cell populations in two-dimensional dot plots displaying the fluorescence intensity of extracted subsets. As a result, disease-specific phenotypes of immune responses can be identified. The occurrence of such phenotypes with potential changes in immune cell composition and frequency can serve as a translational biomarker in drug development. 

How Immune Responses Harm Our CNS

As immunophenotyping may reveal not only inflammatory processes, but also the activation status of immune cell subsets, it can guide selecting individual immunomodulatory therapies. In central nervous system (CNS) animal models this provides insight in the dynamics of peripheral and resident cells. Understanding the innate and adaptive immune cell status within the CNS and in the periphery during disease progression can be applied to monitor biomarker expression profiles as well possible pharmacodynamic changes related to treatment.

Diseases affecting the CNS originate from various sources including tumors, degeneration, trauma, blood circulation interruptions, traumas, structural defects, infection and autoimmune disorders. In these pathogeneses the immune and nervous systems are closely related to either disease origin or progression of disease. Homeostasis and regulation of inflammatory markers is an active process in the brain, and if they malfunction it turns pathological. Both acute and chronic inflammation responses in CNS have been identified with regards to various diseases. Acute inflammation response starts and becomes severe rapidly. Symptoms are typically only present for a few days. In the CNS acute inflammation usually follows an injury, e.g. in traumatic brain injury, spinal cord injury or stroke. Contrary to acute symptoms, chronic inflammation can last from several months to years and is present in diseases such as Alzheimer’s, Parkinson’s, Huntington’s and multiple sclerosis. Increased T-cell numbers have been associated with the mentioned disorders contributing both to inflammation and neuronal dysfunction as well as to  deferring inflammatory responses leading to neurodegeneration.

How Immune Responses Fight Cancer

On the other hand, T-lymphocytes join the fight against cancer. They and other immune cells like macrophages, neutrophils or myeloid-derived suppressor cells (MDSC) accumulate in the microenvironment, which surrounds a tumor and nurtures it to promote or supress its growth. In general, this physiologically abnormal arrangement keeps an immune response down. Novel treatments explore how to put the tumor on the immune system’s radar and thus initiate a directed attack. Phenotyping of tumor-infiltrating lymphocytes (TIL) by flow cytometry is a common readout from animal models used in oncology discovery. Experimental studies assess whether and how immune cells within this disguising microenvironment may change under various dosing regimen. Measurable changes in their frequency, composition, distribution and function may help determining a possible therapeutic outcome. Ultimately, the same information can be of prognostic and predictive value down the drug development journey.   

Recruiting the body's own immune system as ally to fight cancer is the paradigm of a set of new immunotherapies.  This approach has been emerging as the "fourth pillar" of tumor treatment, most notably recognized by this year's Nobel Prize in Physiology or Medicine. Both defense lines, the innate and the adaptive, are utilized for creating an anti-tumor immune response. This form of immunization may be done either actively by targeting selected lymphoid cells (e.g. checkpoint inhibitors) or passively by stimulating the whole immune system (e.g. adoptive cell transfer), both of which depends on the actual therapeutic concept. 

Hopes remain high that among these new strategies one or more will be our decisive move in the battle against the “emperor of all maladies”. For now, this one has yet to meet his Waterloo.