Biology subjects

Palmer, A. F.

Publications and source records attributed to Palmer, A. F..

3 recordsLinked to original sources

bioRxiv2022

Engineering a Single Extracellular Vesicle Protein and RNA Assay (siEVPRA) via In Situ Fluorescence Microscopy in a UV Micropatterned Array

The physical and molecular heterogeneity of extracellular vesicles (EVs) confounds bulk biomarker characterization, thus encouraging the development of novel assays capable of profiling EVs at a single-vesicle resolution. Here, we present a single EV (siEV) protein and RNA assay (siEVPRA) to simultaneously detect proteins, messenger RNAs (mRNAs), and microRNAs (miRNAs) in siEVs. The siEVPRA consists of an array of microdomains embedded on a polyethylene glycol (PEG)-coated glass surface produced via UV photopatterning, functionalized with antibodies to target siEV subpopulations. Fluorescently labeled antibodies and RNA-targeting molecular beacons (MBs) were used to generate signals for proteins, mRNAs, and miRNAs on siEVs detected by total internal reflection fluorescence microscopy (TIRFM), outperforming the sensitivities of ELISA and PCR by three orders of magnitude. Using the siEVPRA, we analyzed EVs harvested from glioblastoma (GBM) cell lines and demonstrated vesicular heterogeneity in protein, mRNA, and miRNA expression through colocalization analyses, and validated the results by bulk RNA sequencing. We further demonstrated the clinical utility of the siEVPRA by detecting different mRNAs and miRNAs associated with GBM in patient samples. Together, these results indicate that the siEVPRA provides an effective platform to investigate the heterogeneity of proteins and RNAs in subpopulations of EVs.

bioengineering↗

bioRxiv2021

An Immunogold Single Extracellular Vesicular RNA and Protein (AuSERP) Biochip to Predict Responses to Immunotherapy in Non-Small Cell Lung Cancer Patients

Conventional PD-L1 immunohistochemical tissue biopsies only predict 20~40% of non-small cell lung cancer (NSCLC) patients that will respond positively to anti-PD-1/PD-L1 immunotherapy. Herein, we present an immunogold biochip to quantify single extracellular vesicular RNA and protein (AuSERP) as a non-invasive alternative. With only 3 L of serum, PD-1/PD-L1 proteins on the surface of extracellular vesicles (EVs) and EV PD-1/PD-L1 messenger RNA (mRNA) cargo were detected at a single-vesicle resolution and exceeded the sensitivities of ELISA and qRT-PCR by 1000 times. By testing a cohort of 27 non-responding and 27 responding NSCLC patients, AuSERP indicated that the single-EV mRNA biomarkers surpass the single-EV protein biomarkers at predicting patient responses to immunotherapy. Dual single-EV PD-1/PD-L1 mRNA detection differentiated responders from non-responders with an accuracy of 72.2% and achieved an NSCLC diagnosis accuracy of 93.2%, suggesting the potential for AuSERP to provide enhanced immunotherapy predictions and cancer diagnoses within the clinical setting.

immunology↗

bioRxiv2020

Immunomagnetic Sequential Ultrafiltration (iSUF) Platform for Enrichment and Purification of Extracellular Vesicles from Biofluids

Extracellular vesicles (EVs) derived from tumor cells have the potential to provide a much-needed source of non-invasive molecular biomarkers for liquid biopsies. However, current methods for EV isolation have limited specificity towards tumor-derived EVs that limit their clinical use. Here, we present an approach called immunomagnetic sequential ultrafiltration (iSUF) that consists of sequential stages of purification and enrichment of EVs (nonspecifically and specifically) in approximately 2 h. In iSUF, EVs present in different volumes of biofluids (0.5 mL to 100 mL) can be significantly enriched (up to 1000 times), with up to 99 % removal of contaminating proteins (e.g., albumin). The EV recovery rate by iSUF for cell culture media (CCM), serum, and urine corresponded to 98.0% {+/-} 3.6%, 96.0% {+/-} 2.0% and 94.0% {+/-} 1.9%, respectively (p > 0.05). The final step of iSUF enables the separation of tumor-specific EVs by incorporating immunomagnetic beads specific to a target subpopulation of EVs. Serum from a small cohort of clinical samples from metastatic breast cancer (BC) patients and healthy donors were processed by the iSUF platform and the isolated EVs from patients showed significantly higher expression levels of BC biomarkers (i.e., HER2, CD24, and miR21).

bioengineering↗