Detection of circulating tumor cells
Approximately 90% of deaths from solid tumors are attributed to metastasis [
47]. In the course of metastatic dissemination, a cancer cell from the primary tumor first invades the surrounding tissue and then enters the microvasculature of the blood (intravasation) and lymph systems, followed by survival and translocation through the bloodstream to microvessels in distant tissues, subsequent exit from the bloodstream (extravasation) and survival in the microenvironment of distant tissues, which present a suitable foreign microenvironment for development of a macroscopic secondary tumor [
48]. Early detection of metastatic cancer cells in the bloodstream, also known as circulating tumor cells (CTCs), can potentially affect cancer prognosis and diagnosis.
As a portion of a liquid biopsy, CTCs have been studied broadly due to their potential applications. CTC detection can help us understand the molecular organization of a tumor in a minimally invasive manner. Nevertheless, CTCs exhibit relatively low abundance and heterogeneity, which presents technical challenges for CTC isolation and characterization. In recent years, researchers have focused on the application of nanotechnologies for sensitive detection of CTCs; these technologies can help characterize cells and molecules, thereby enjoying broad clinical applications, such as disease detection at an early stage and evaluation of the treatment response and disease development.
As demonstrated in many studies, it is possible for cell pseudopodia to form on surfaces with nanostructure, thereby enhancing the local topographical interactions between cancer cells and nanostructured substrates, which is beneficial to CTC enrichment. For CTC detection, nanomaterials have an essential advantage in their large surface-to-volume ratio, which enables adsorption of high-efficiency targeting ligands with the ability to recognize specific molecules on cancer cells; therefore, CTC isolation shows high specificity and recovery, and the detection sensitivity is enhanced.
People have reported different types of nanomaterials, such as magnetic nanoparticles (MNPs), gold nanoparticles (AuNPs), quantum dots (QDs), nanowires, nanopillars, silicon nanopillars, carbon nanotubes, dendrimers, graphene oxide, and polymers, for CTC detection (Table
1) [
61]. It has been demonstrated that these nanomaterials can improve the sensitivity and specificity of CTC capture devices and have the potential to facilitate cancer diagnosis and prognosis.
Table 1
Nanotechnology for detection of cancer cells
Magnetic nanoparticles | Antibody | EpCAM | Colon/liver /lung/breast | |
Quantum dots | Aptamer | PTK7 | Leukemia | |
Polymer dots | Antibody | EpCAM | Breast | |
Gold nanoparticles | Aptamer Antibody | Her2 Cd2/cd3 | Breast Leukemia | |
Carbon nanotubes | Antibody | EpCAM | Liver | |
Upconversion NPs | Antibody | Her2 | Breast | |
Nanorod arrays | DNA aptamer | EpCAM | Breast | |
Nanoparticle-coated silicon bead | Antibody | EpCAM/CD146 | Breast Colorectal | |
Nanofibers | Antibody | EpCAM | Breast | |
In the field of nanobiotechnology, MNPs are mature nanomaterials that can bind to cells and have long been used for in vitro separation with the help of an external magnetic field [
62]. Antibody-functionalized MNPs, namely, immunomagnetic nanoparticles, are frequently applied in the biomedical field. For CTC detection, immunomagnetic technologies usually specifically target EpCAM-expressing CTCs with anti-EpCAM functionalized MNPs.
To perform single-cell transcriptional profiling of CTCs purified from breast cancer patients, Powell et al .[
63] used MagSweeper, which is an immunomagnetic enrichment device that can isolate tumor cells from unfractionated blood. MagSweeper serves as a magnetic cell sorting system that uses magnetic rods covered by a sheath to sweep across capture wells and attract target cells labeled with magnetic nanoparticles [
64]. It can be used to acquire high-purity CTCs from patient blood, while preserving their capacity to initiate tumors and metastasize, facilitating robust analysis of single CTCs. Using the system, the authors successfully purified CTCs from 70% of patients with primary and metastatic breast cancer and performed direct measurement of the gene expression in individual CTCs.
QDs are characterized by special optical properties, which enhance their usefulness in cancer cell detection [
65]. Due to their high quantum yields, QDs are helpful in the detection of materials with low abundance. However, to enhance QD electrical characteristics, Pang et al. [
52] hybridized ZnO NDs and g-C3N4 QDs to afford higher photoelectron transfer and separation efficiency. Due to the outstanding advantages, ZnO NDs and g-C3N4 QDs enjoyed the extended application, and a photocatalyzed renewable self-powered cytosensing device was presented on the basis of ZnO NDs@g-C3N4 QDs. Through conjugation to the membrane PTK7-specific aptamer Sgc8c, the device was used to detect CCRF-CEM cells (human acute lymphoblastic leukemia cells), which express PTK7. The results showed that the device offers better performance in terms of detection range, detection limit, selectivity, and reproducibility. It captured only CCRF-CEM cells (500 cell/mL) and no other cell types, such as HL-60, K562, and HeLa cells. The authors think that the device could be an effective platform for monitoring the progression of leukemia and shows great promise.
Polymer nanoparticles derived from various conductive hydrophobic polymers have been applied to produce nanoparticles with high quantum yields, photostability and that are nontoxic. Therefore, PD is ideal for CTC detection. Wu et al. [
66] reported a strategy for semiconducting polymer dots (PDs) functionalization through entrapment of heterogeneous polymer chains into a single dot, which was facilitated by hydrophobic interactions, during nanoparticle formation. A few amphiphilic polymers with functional groups for subsequent covalent conjugation of biomolecules, such as streptavidin and immunoglobulin G (IgG), show co-condensation with most semiconducting polymers for modification and functionalization of a nanoparticle surface. The PDs bioconjugates were able to label cellular targets in an effective and specific manner, including a cell surface marker on human breast cancer cells, with no need to detect nonspecific binding. The authors demonstrated that the fluorescence exhibited by PD-labeled MCF-7 cells was 25 times higher than that of QD-labeled cells and 18 times higher than that of Alexa Fluor-labeled cells, according to flow cytometry analysis [
67]. Based on the results, the authors believe that these ultrabright nanoparticles were successfully bioconjugated. Thus, this strategy provides a new opportunity for applying versatile semiconducting polymers to different fluorescence measurement methods in biomedicine and modern biology.
Upconversion nanoparticles (UCNPs) are usually selected for fluorescent labeling considering the ability to excite UCNPs with near-infrared (NIR) light to infrared (IR) light for generation of fluorescence emission in the visible region of the spectrum, leading to minimal background noise. Furthermore, applying NIR light as the excitation source prevents damage to normal tissues on one hand and allows deep tissue penetration on the other hand [
68].
Shen et al. [
69] described a simple method to conjugate multifunctional nanoparticles (MFNPs) assembled by the formation of various layers with a monoclonal anti-HER2 antibody and confirmed that the MFNPs exhibited the specific detection of breast cancer BT474 cells (biomarker HER2 positive) with a high signal-to-noise ratio. The MFNPs have an obvious core-shell structure of UCNP@Fe3O4@Au coated with anti-HER2 antibody and poly(ethylene glycol) (PEG) and exhibited an outstanding dispersity in different aqueous solutions and a high signal-to-noise ratio. The authors revealed that the photothermal effect exhibited a new high-localization feature at the single-cell level based under a continuous-wave near-IR laser. Using these nanoparticles, the authors exerted a photothermal effect at the single-cell level.
For differentiating types of cells and cancer states, the authors used AuNPs capped with ligands of different hydrophobicity and coated with green fluorescent protein (GFP). Because the capping ligands used showed different chemical structures, each AuNP-GFP complex was related to cancer cells to a different degree, considering cell membrane composition differences [
70]. Magnetic biotargeting-multifunctional nanobioprobes (MBMNs) were used to detect and isolate a small subset of malignant cells from normal cells. CoFe2O4@BaTiO3 magnetoelectric nanoparticles distinguished different cancer cells from each other and from their normal counterparts through a magnetoelectric effect [
71].
Detection through cell surface protein recognition
The main method to detect cancer cells relies on binding of nanoparticle probes conjugated with moieties (protein, short peptides, antibodies, oligonucleotide aptamers) to surface markers on cancer cells and on those entering cells and detecting genetic content. For the detection of cancer cells, such as CTCs, capture or isolation is the first and most important stage. Although the cell physical properties, such as size, deformability, and density, are sometimes used, capture is primarily based on the affinity of cell surface molecules on CTCs detected with materials such as antibodies or aptamers. Unique surface proteins on CTCs are the primary targets.
Since many studies have demonstrated that EpCAM is highly expressed on CTCs from many human malignancies, EpCAM can be used as a cell surface biomarker. Hence, anti-EpCAM molecules are often applied to screening of CTCs. CTCs undergoing EMT would cause inefficient positive sorting on the basis of EpCAM expression. Therefore, another approach is to find supplemental or replacement markers for EpCAM. Many cell surface markers, such as vimentin, androgen receptor, glycan, major vault protein (MVP), and fibroblast activation protein α (FAPα), have been studied for the detection of CTCs. However, a majority of these markers are only specific to certain cells, and many markers do not exist after CTCs experienced EMT. More mesenchymal CTCs can be seen in the metastatic stages of cancer, and thus, seeking proper EMT markers to evaluate prognosis and metastasis in cancer patients is important. Here, we compile the recently identified cell surface protein markers for detection of CTCs in different cancer types (Table
2).
Table 2
Cell surface protein markers for CTC detection
EpCAM | CTC | Colorectal Breast Head and neck | CellSearch | |
EpCAM and FRα | CTC | Non-small cell lung cancer | CellSearch | |
Glycan | CTC | Breast | Flow cytometry | |
Vimentin | CTC | Gastrointestinal | Confocal microscopy | |
EMT CTC | Prostate | CellSearch | |
CTC | Sarcoma | Flow cytometry | |
Vimentin+PD-L1 | CTC | Colorectal, prostate cancer | Confocal microscopy | |
Synaptophysin | CTC | Castration resistant prostate cancer | CellSearch | |
Major vault protein | Mesenchymal and intermediate CTCs | Hepatocellular carcinomas | Flow cytometry | |
Androgen receptor | CTC | Metastatic breast cancer | Epic Sciences CTC platform | |
p75 Neurotrophin receptor+EpCAM | CTC | Esophageal squamous cell carcinoma | Flow cytometry | |
Carbonic anhydrase 9 and CD147 | CTC | Clear cell renal cell carcinoma | NanoVelcro | |
Excision repair cross-complementation group 1 | CTC | Platinum resistance ovarian cancer | Reverse-transcription PCR | |
Detection based on mRNA
In addition to the detection of extracellular nucleic acids, nanoparticles have also been developed as intracellular nucleic acid sensors. Seferos et al. [
85] demonstrated that it is possible to use novel gold nanoparticle probes modified by oligonucleotides hybridized to complements labeled with a fluorophore as transfection agents and cellular “nanoflares” to detect mRNA in living cells. Nanoflares overcome many challenges in the creation of effective and sensitive intracellular probes and show a large signal-to-noise ratio and sensitivity to changes in the number of RNA transcripts in cells. Nanoflares, which show high orientation, dense oligonucleotide coating and can enter cells without the need for cytotoxic transfection agent s[
86], are useful for detecting intracellular mRNA.
Meanwhile, researchers have developed nanoflares for simultaneous intracellular detection of various mRNA transcripts. In these multiplexed nanoflare studies, AuNPs functionalized with 2-3 DNA recognition strands and later hybridized with short complementary reporter strands were generated as nanoflares. For example, the use of multiplexed nanoflares to detect survivin in addition to actin has been investigated for normalizing nanoflare fluorescence differences in cellular uptake. Therefore, the technique is comparable with conventional qRT-PCR for quantification of intracellular mRNA but can be performed at the single live cell level.
In some cases, the nanoflare platform was expanded to quantify intracellular RNA and detect spatiotemporal localization in living cells [
87]. In this work, β-actin-targeting nanoflares were incubated with HeLa cells and presented an obviously different intracellular distribution, exhibiting strong colocalization with mitochondria, which has not been previously demonstrated. Further, Smart-Flares were utilized for studying melanoma tumor cell heterogeneit y[
88]. These Smart-Flares were able to quantify genomic expression at the single-cell level, thus expanding our knowledge of cancer and metastasis. Investigating the heterogeneity of cancer cells is crucial for identifying novel biomarkers for early cancer diagnosis.
Halo et al. [
89] reported nanoflares, which were applied to capture live circulating breast cancer cells. These nanoflares could detect target mRNA in model metastatic breast cancer cell (MBC) lines in human blood and exhibited high recovery and fidelity reaching 99%. They also used nanoflares together with later cultured mammospheres to reimplant the retrieved live recurrent breast cancer cells into whole human blood. Only 100 live cancer cells could be detected per mL of blood. Relying on the NanoFlare technology, it was possible to simultaneously isolate and characterize intracellular live cancer cells from whole blood. The authors demonstrated the ability of nanoflares to collect CTCs for future culture and study. In addition, nanoflares contribute to the technology of combining intracellular markers with cell-surface markers for dually identifying putative CTCs. The combined method is likely to enhance the function of more platforms to specifically identify CTCs and subpopulations of CTCs. The authors think that nanoflares provide the first gene-based approach to detect, isolate, and characterize live cancer cells in the blood and are likely to contribute to cancer diagnosis, prognosis, and prediction, as well as personalized treatment.
Lee et al. reported an approach based on an elegant plasmonic nanoparticle network structure, generating a plasmon-coupled dimer able to detect single mRNA variants [
90]. They applied the method to the detection and quantification of BRCA1 mRNA splice variants in vitro and in vivo. Two probes conjugated to nanoparticles were connected to the BRCA1 mRNA target in a sequence-specific manner, and as a result, the signal exhibited a spectral shift due to dimer formation. They demonstrated that their method is powerful and can successfully detect, quantify, and differentiate between different BRCA1 splice variants with single-copy sensitivity, thereby laying a foundation for quantitative, single-cell genetic profiling in the future.