Masha G. Savelieff, PhD
Flow cytometry has become a mainstay in the clinical laboratory as a reliable, rapid, and sensitive immunodiagnostic method. At present, its uses in the clinic have been limited to diagnostics. However, flow cytometry’s many strengths and advantages, which include single-cell resolution and multiplexed biomarker identification by a powerful instrument with a suite of analysis software, make the technique ideal for other potential clinical applications. Indeed, flow cytometry methods for therapeutic applications are emerging.
FACS, or fluorescence-activated cell sorting, is a specialized flow application that, in addition to analyzing the fluorophore content and biomarkers on single cells, can sort them based on the desired phenotype. One potential therapeutic application is for collected cells to be expanded ex vivo and reinfused into the patient as cell-based therapies.
The ability to perform multiplex biomarker analysis using flow cytometry is significant because it enables the identification and isolation of immune cells to a very specific phenotype. In addition, the ability to serially analyze single cells can lead to a high purity of sorted cells. This is important for cell-based therapies, which must be free of contaminating harmful cells, e.g., cancer cells, or therapeutically ineffective cells, e.g., over-differentiated T-cells. Despite its great potential for cell-based therapies, FACS is presently limited to preclinical and early-phase studies for such applications. However, intensive research aims to translate these findings to the clinic, particularly for sorting immune and stem cells for immunotherapy and regenerative medicine.
Isolating immune cells for immunotherapy
FACS can be employed to enrich hematopoietic stem cells (HSCs) from a patient’s blood for autologous reimplantation after high-dose chemotherapy, which ablates the process of hematopoiesis from the patient’s bone marrow. In a study of 22 patients with metastatic breast cancer enrolled in a phase I/II trial, FACS was employed to purify HSCs bearing a CD34+Thy-1+ signature for autologous mobilized peripheral blood (MPB) infusion, a process generally considered suboptimal due to contaminating circulating cancer cells. However, adding a FACS sorting step after enrichment of HSCs on an apheresis CD34+ column resulted in a 250,000-fold reduction in cancer cells. Moreover, patients receiving FACS-sorted MPB had longer progression-free survival and overall survival compared with patients who received regular MPB. Although limited in sample size, the study demonstrated the ability of FACS to purify HSCs to a high level for autologous MPB.
Autologous adoptive cell transfer is a process of purifying immune cells from a patient’s blood sample, expanding them ex vivo, and reinfusing them into the patient for therapeutic benefit. In cancer patients, this could involve reinfusion of cancer-specific antigen (Ag)-activated T-cells or tumor-infiltrating lymphocytes (TILs) to combat hematological or solid cancers. TILs exhibit a range of phenotypes, from the immature and least-differentiated stem cell memory T-cells (TSCMs) to the terminally differentiated effector T-cells. Studies have shown a correlation between clinical outcome and reinfusion of less-differentiated cells, suggesting that sorting TILs to enrich them in TSCMs or other less-differentiated forms prior to reinfusion could improve survival outcomes for patients. FACS could potentially accomplish this task, although the application is still in the preclinical phase.
The FDA has recently approved a novel therapy called chimeric antigen receptor-modified T-cells (CAR-Ts) to treat relapsed or refractory diffuse large B-cell lymphoma and B-cell precursor acute lymphoblastic leukemia. The treatment process involves obtaining T-cells from the patient and modifying them genetically to express a surface antibody specific to a cancer Ag fused to a protein domain that activates the T-cell. The CAR-Ts are then expanded and reinfused into the patient as an immunotherapy to target cancer cells. Various T-cell subsets differ in their efficacy and lasting effects in vivo as CAR-Ts; consequently, sorting T-cells by FACS prior to their genetic manipulation could boost their efficacy in patients, although this is still in the research stages. Additionally, flow cytometry can be used to characterize CAR-Ts and their expression of Ag receptors.
Selecting stem cells for regenerative medicine
Stem cells retain the ability to transform into a variety of different cell types and are therefore of potential use in regenerative medicine to treat degenerative diseases, congenital conditions, or tissue damage from injury. The list of potential applications for stem cells (embryonic, induced pluripotent, neural, placental, or mesenchymal) is staggering, with numerous ongoing clinical trials testing stem cell therapies for bone, cardiovascular, immune, and neurodegenerative diseases, to name a few. FACS is well-suited to sorting stem cells9 and is extensively employed in research, but its clinical applications are still in the nascent stages of development. Presently, flow cytometry can be used to verify the phenotype of cultured or isolated stem cells.
Limitations and potential solutions
FACS is an antibody-dependent technology and is therefore limited in its clinical applications by the availability of antibodies conforming to good manufacturing practices. Cells sorted by FACS can also suffer shear—or electrically induced damage. Newer formats of cell sorting, such as microelectromechanical systems in tandem with a flow cytometer, can mitigate these problems. A general issue facing all cell-based therapies is the labor intensity of culturing cells, which limits production volume. A potential solution would be to integrate existing automated cell culturing systems with sorting machines for a seamless transition from automated culturing to automated sorting. Overall, many of these limitations are technical in nature. Improvements could increase the applicability of FACS to cell-based therapies.