Nano Pom-Pom: A New Method for Detecting Cancer Biomarkers

Nano-sized extracellular vesicle (EV) exosomes serve as a reliable source of biomarkers. However, identification of cancer biomarkers through mapping of exosomal multi-omic molecular information is challenging due to the heterogeneous population of exosomes derived from diverse cell types.

Study: Nano pom-pom prepared exosomes allow detection of highly specific cancer biomarkers. Image Credit: Meletios Verras/Shutterstock.com

In an article published in the journal Communication Biology, the researchers introduced new three-dimensional (3D) nanographene immunomagnetic particles for specific capture and release (defined by markers) from intact exosomes. The newly designed 3D structured nanographene particles have a unique pom-pom flower morphology.

In addition, the capture and release of intact exosomes is carried out via photo-click chemistry. This exosome isolation approach allows the identification of cancer biomarkers with enhanced sensitivity and specificity. Multi-omic exosome analysis for the identification of cancer biomarkers was performed using tissue fluid obtained from bladder cancer patients.

Exosome-prepared nanographene immunomagnetic particles show differences in vivo biodistribution, exhibiting integral qualities with high viability. This nanographene immunomagnetic particle-based technique is an easy-to-apply approach to a wide range of biological fluids. Changing the developed method facilitates the enhancement, enrichment, and isolation of high-throughput exosomes.

What are Cancer Biomarkers?

Despite continuous efforts to develop cancer biomarkers, only a few are clinically approved by the United States Food and Drug Administration (USFDA), including the progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2/neu), cancer antigen 125 (CA-125), and prostate-specific antigen (PSA).

EV is a new source of biomarkers being explored for finding various cancer biomarkers for achieving cancer diagnosis, drug targeting and delivery, and immunotherapy. Exosome-type small EVs and their molecular components are associated with various physiological functions and disease pathologies. Exosomes are secreted from tumor cells and enriched with tumor markers. As a result, their presence is increased in the plasma and ascites of patients diagnosed with cancer.

Body fluids contain different types of EV. Therefore, a cellular-specific homogeneous exosome population could not be achieved. Cell-secreted membrane vesicles (EVs) are of heterogeneous origin, present in several subpopulations, in a wide size range of 30 to 1000 nanometers. Thus, due to the heterogeneity within the vesicles and their overlapping size ranges, the exact cellular origin of the exosomes could not be identified to understand disease pathogenesis.

Existing purification methods used to recover extracellular material (EMs) with vesicular or non-vesicular molecules include ultracentrifugation (UC)-based precipitation and precipitation polymer kits. However, this approach is not scalable, and populations of exosomes derived from different cell types or EV subtypes are indistinguishable. Studying cancer biomarkers from exosomes derived from cancer cells is therefore challenging.

Schematic illustration of Nano pom pom fabrication. b TEM and SEM images show unique 3D nanoscale flower pom-pom morphology compared to commercial immunomagnetic beads. c TEM imaging of immune gold nanoparticles staining of the captured exosomes completely covering the surface of the Nano pom-poms. The captured EVs were confirmed by antiCD63 gold nanoparticles. The insert shows a single exosome captured in the ~100 nm size range with three bound gold nanoparticles (~10 nm). d Nanoparticle tracking analysis of NanoPoms isolated exosomes with a much narrower size distribution compared to UC isolated EVs. e Tracing analysis of nanoparticles of exosome sizes isolated by NanoPoms (n = 4 independent experiments, mean ± SD), compared with that of ExoEasy isolates (n = 4 independent experiments, mean ± SD), indicating a reproducible exosome size and smaller than the NanoPoms preparation. f SEM image showing dense exosomes captured covering the surface of the Nano pom-pom, and can be completely released via photo cleavage on demand. Upon release, intact exosomes can be harvested for downstream multi-omic analysis including DNA, RNA sequencing, western blotting and next-generation proteomic analysis, as well as in vivo studies. © He, N., Thippabhotla, S., Zhong, C., Greenberg, Z., Xu, L., Pessetto, Z., Godwin, AK. et al. (2022)

Nanographene Exosomes Towards Detection Of Specific Cancer Biomarkers

This study demonstrates a novel approach for the specific capture and release of intact exosomes using 3D structured nanographene immunomagnetic particles. These nanographene immunomagnetic particles have a flower pom pom-like morphology, and the exosome capture and release mechanism is based on photo-click chemistry.

Whole exosomes for this study were isolated from different biological fluids, including human urine and blood, cow’s milk, and cell culture media. Furthermore, isolation of exosomes via nanographene immunomagnetic particles facilitated the identification of effective cancer biomarkers with high sensitivity and specificity compared to those isolated via immunomagnetic beads.

It in vivo biodistribution was assayed for exosomes released from nanographene immunomagnetic particles. The results show distinct biodistribution patterns with insignificant changes in the surface properties of exosomes, implying the potential of nanographene immunomagnetic particles in therapeutic development.

Exosomes derived from bladder cancer tissue fluid (urine and plasma) were compared with those derived from tumor tissue by next-generation sequencing (NGS) miRNA, global proteome, and somatic DNA mutations to reach an invasive and ultra-sensitive bladder diagnosis. cancer.

The results revealed that compared to the ultracentrifugation or bead isolation approaches, exosomes isolated from nanographene immunomagnetic particles showed increased sensitivity and specificity for the detection of urological tumor biomarkers.

The impact of external stimuli such as light release was observed on exosome isolation via a nanographene immunomagnetic particle-based approach in which miRNA profiles were compared in the presence and absence of light release processes. The light-releasing process exhibits the specificity of releasing only the captured exosomes. These observations confirmed the quality and integrity of exosomes prepared by nanographene immunomagnetic particles as a robust and easy method.

What Did the Study Find?

To summarize, the isolation of exosomes via nanographene-based immunomagnetic particle-based methods was applied to a variety of biological fluids, including human urine and blood, cell culture media, and cow’s milk. This isolation method is simple and does not require an additional ultracentrifugation process.

The 3D structure of the nanographene immunomagnetic particles and the release-capture process regulated by specific markers help prepare homogeneous exosomal subpopulations that can enrich cancer biomarkers.

NGS and droplet digital polymerase chain reaction (ddPCR) demonstrated that DNA isolation from exosomes prepared by nanographene immunomagnetic particles could be enriched for DNA mutations in tumors relevant to bladder cancer and aid in the identification of cancer biomarkers.

Reference

He, N., Thippabhotla, S., Zhong, C., Greenberg, Z., Xu, L., Pessetto, Z., Godwin, AK. et al. (2022) Nano pom-pom prepared exosomes allow the detection of highly specific cancer biomarkers. Communication Biology. https://doi.org/10.1038/s42003-022-03598-0

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