An official website of the United States government
Membrane antigens control cell function by regulating biochemical interactions and hence are routinely used as diagnostic and prognostic targets in biomedicine. Fluorescent labeling and subsequent optical interrogation of cell membrane antigens, while highly effective, limit expression profiling to centralized facilities that can afford and operate complex instrumentation. Here, we introduce a cytometry technique that computes surface expression of immunomagnetically labeled cells by electrically tracking their trajectory under a magnetic field gradient on a microfluidic chip with a throughput of >500 cells per min. In addition to enabling the creation of a frugal cytometry platform, this immunomagnetic cell manipulation-based measurement approach allows direct expression profiling of target subpopulations from non-purified samples. We applied our technology to measure epithelial cell adhesion molecule expression on human breast cancer cells. Once calibrated, surface expression and size measurements match remarkably well with fluorescence-based measurements from a commercial flow cytometer. Quantitative measurements of biochemical and biophysical cell characteristics with a disposable cytometer have the potential to impact point of care testing of clinical samples particularly in resource limited settings.
more »
« less
Electronic measurement of cell antigen expression in whole blood
Membrane antigens are phenotypic signatures of cells used for distinguishing various subpopulations and, therefore, are of great interest for diagnosis of diseases and monitoring of patients in hematology and oncology. Existing methods to measure antigen expression of a target subpopulation in blood samples require labor-intensive lysis of contaminating cells and subsequent analysis with complex and bulky instruments in specialized laboratories. To address this long-standing limitation in clinical cytometry, we introduce a microchip-based technique that can directly measure surface expression of target cells in hematological samples. Our microchip isolates an immunomagnetically-labeled target cell population from the contaminating background in whole blood and then utilizes the differential responses of target cells to on-chip magnetic manipulation to estimate their antigen expression. Moreover, manipulating cells with chip-sized permanent magnets and performing quantitative measurements via an on-chip electrical sensor network allows the assay to be performed in a portable platform with no reliance on laboratory infrastructure. Using our technique, we could successfully measure expressions of the CD45 antigen that is commonly expressed by white blood cells, as well as CD34 that is expressed by scarce hematopoietic progenitor cells, which constitutes only ∼0.0001% of all blood cells, directly from whole blood. With our technology, flow cytometry can potentially become a rapid bedside or at-home testing method that is available around the clock in environments where this invaluable assay with proven clinical utility is currently either outsourced or not even accessible.
more »
« less
- Award ID(s):
- 1752170
- PAR ID:
- 10407951
- Date Published:
- Journal Name:
- Lab on a Chip
- Volume:
- 22
- Issue:
- 2
- ISSN:
- 1473-0197
- Page Range / eLocation ID:
- 296 to 312
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Small-molecule inhibitors of PD-L1 are postulated to control immune evasion in tumors similar to antibodies that target the PD-L1/PD-1 immune checkpoint axis. However, the identity of targetable PD-L1 inducers is required to develop small-molecule PD-L1 inhibitors. In this study, using chromatin immunoprecipitation (ChIP) assay and siRNA, we demonstrate that vitamin D/VDR regulates PD-L1 expression in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) cells. We have examined whether a VDR antagonist, MeTC7, can inhibit PD-L1. To ensure that MeTC7 inhibits VDR/PD-L1 without off-target effects, we examined competitive inhibition of VDR by MeTC7, utilizing ligand-dependent dimerization of VDR-RXR, RXR-RXR, and VDR-coactivators in a mammalian 2-hybrid (M2H) assay. MeTC7 inhibits VDR selectively, suppresses PD-L1 expression sparing PD-L2, and inhibits the cell viability, clonogenicity, and xenograft growth of AML cells. MeTC7 blocks AML/mesenchymal stem cells (MSCs) adhesion and increases the efferocytotic efficiency of THP-1 AML cells. Additionally, utilizing a syngeneic colorectal cancer model in which VDR/PD-L1 co-upregulation occurs in vivo under radiation therapy (RT), MeTC7 inhibits PD-L1 and enhances intra-tumoral CD8+T cells expressing lymphoid activation antigen-CD69. Taken together, MeTC7 is a promising small-molecule inhibitor of PD-L1 with clinical potential.more » « less
-
null (Ed.)We demonstrate a label free and high-throughput microbubble-based acoustic microstreaming technique to isolate rare circulating cells such as circulating cancer associated fibroblasts (cCAFs) in addition to circulating tumor cells (CTCs) and immune cells ( i.e. leukocytes) from clinically diagnosed patients with a capture efficiency of 94% while preserving cell functional integrity within 8 minutes. The microfluidic device is self-pumping and was optimized to increase flow rate and achieve near perfect capturing of rare cells enabled by having a trapping capacity above the acoustic vortex saturation concentration threshold. Our approach enables rapid isolation of CTCs, cCAFs and their associated clusters from blood samples of cancer patients at different stages. By examining the combined role of cCAFs and CTCs in early cancer onset and metastasis progression, the device accurately diagnoses both cancer and the metastatic propensity of breast cancer patients. This was confirmed by flow cytometry where we observed that metastatic breast cancer blood samples had significantly higher percentage of exhausted CD8 + T cells expressing programmed cell death protein 1 (PD1), higher number of CD4 + T regulatory cells and T helper cells. We show for the first time that our lateral cavity acoustic transducers (LCATs)-based approach can thus be developed into a metastatic propensity assay for clinical usage by elucidating cancer immunological responses and the complex relationships between CTCs and its companion tumor microenvironment.more » « less
-
Due to the vast difference in surface expression levels among cell populations, flow cytometers must possess a dynamic range sufficiently high to accommodate such variations. We recently introduced a microchip-based flow cytometer that combines magnetophoresis and distributed Coulter sensing. Inspired from digital photography techniques, we implemented exposure bracketing in magnetophoretic cell sorting to enhance the dynamic range of cell surface expression measurements with our electronic cytometry chip.more » « less
-
ObjectiveTo obtain the comprehensive transcriptome profile of human citrulline‐specific B cells from patients with rheumatoid arthritis (RA). MethodsCitrulline‐ and hemagglutinin‐specific B cells were sorted by flow cytometry using peptide–streptavidin conjugates from the peripheral blood ofRApatients and healthy individuals. The transcriptome profile of the sorted cells was obtained byRNA‐sequencing, and expression of key protein molecules was evaluated by aptamer‐basedSOMAscan assay and flow cytometry. The ability of these proteins to effect differentiation of osteoclasts and proliferation and migration of synoviocytes was examined by in vitro functional assays. ResultsCitrulline‐specific B cells, in comparison to citrulline‐negative B cells, from patients withRAdifferentially expressed the interleukin‐15 receptor α (IL‐15Rα) gene as well as genes related to protein citrullination and cyclicAMPsignaling. In analyses of an independent cohort of cyclic citrullinated peptide–seropositiveRApatients, the expression ofIL‐15Rα protein was enriched in citrulline‐specific B cells from the patients’ peripheral blood, and surprisingly, all B cells fromRApatients were capable of producing the epidermal growth factor ligand amphiregulin (AREG). Production ofAREGdirectly led to increased migration and proliferation of fibroblast‐like synoviocytes, and, in combination with anti–citrullinated protein antibodies, led to the increased differentiation of osteoclasts. ConclusionTo the best of our knowledge, this is the first study to document the whole transcriptome profile of autoreactive B cells in any autoimmune disease. These data identify several genes and pathways that may be targeted by repurposing severalUSFood and Drug Administration–approved drugs, and could serve as the foundation for the comparative assessment of B cell profiles in other autoimmune diseases.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/d1lc00889g
