DNA methylation (5-methylcytosine, 5-mC) is the best characterized epigenetic mark that has regulatory roles in diverse biological processes. Recent investigation of RNA modifications also raises the ...possible functions of RNA adenine and cytosine methylations on gene regulation in the form of “RNA epigenetics.” Previous studies demonstrated global DNA hypomethylation in tumor tissues compared to healthy controls. However, DNA and RNA methylation in circulating tumor cells (CTCs) that are derived from tumors are still a mystery due to the lack of proper analytical methods. In this respect, here we established an effective CTCs capture system conjugated with a combined strategy of sample preparation for the captured CTCs lysis, nucleic acids digestion, and nucleosides extraction in one tube. The resulting nucleosides were then further analyzed by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS). With the developed method, we are able to detect DNA and RNA methylation (5-methyl-2′-deoxycytidine, 5-methylcytidine, and N 6-methyladenosine) in a single cell. We then further successfully determined DNA and RNA methylation in CTCs from lung cancer patients. Our results demonstrated, for the first time, a significant decrease of DNA methylation (5-methyl-2′-deoxycytidine) and increase of RNA adenine and cytosine methylations (N 6-methyladenosine and 5-methylcytidine) in CTCs compared with whole blood cells. The discovery of DNA hypomethylation and RNA hypermethylation in CTCs in the current study together with previous reports of global DNA hypomethylation in tumor tissues suggest that nucleic acid modifications play important roles in the formation and development of cancer cells. This work constitutes the first step for the investigation of DNA and RNA methylation in CTCs, which may facilitate uncovering the metastasis mechanism of cancers in the future.
Comprehensive phenotypic profiling of heterogeneous circulating tumor cells (CTCs) at single‐cell resolution has great importance for cancer management. Herein, a novel spectrally combined encoding ...(SCE) strategy was proposed for multiplex biomarker profiling of single CTCs using a multifunctional nanosphere‐mediated microfluidic platform. Different cellular biomarkers uniquely labeled by multifunctional nanosphere barcodes, possessing identical magnetic tags and distinct optical signatures, enabled isolation of heterogeneous CTCs with over 91.6 % efficiency and in situ SCE of phenotypes. By further trapping individual CTCs in ordered microstructures on chip, composite single‐cell spectral signatures were conveniently and efficiently obtained, allowing reliable spectral‐readout for multiplex biomarker profiling. This SCE strategy exhibited great potential in multiplex profiling of heterogeneous CTC phenotypes, offering new avenues for cancer study and precise medicine.
A spectrally combined encoding strategy was proposed for multiplex biomarker profiling of heterogeneous circulating tumor cells (CTCs) using a multifunctional nanosphere‐mediated microfluidic platform. Different cellular biomarkers simultaneously encoded with both magnetic tags and distinct optical signatures, enabled efficient isolation and in situ on‐chip spectrally combined encoding of heterogeneous CTCs at single‐cell resolution.
Similar to the reversible epigenetic modifications on DNA, dynamic RNA modifications were recently considered to constitute another realm for biological regulation in the form of "RNA epigenetics". ...5-Methylcytosine (5-mC) has long been known to be present in RNA from all three kingdoms of life. However, the functions of 5-mC in RNA have not been fully understood, especially for the RNA demethylation mechanism. The discovery of 5-hydroxymethylcytosine (5-hmC) in RNA together with our recently reported 5-formylcytosine (5-foC) in RNA indicated that 5-mC in RNA may undergo the same cytosine oxidation demethylation pathway with generating intermediates 5-hmC, 5-foC, and 5-carboxylcytosine (5-caC) by ten-eleven translocation (Tet) proteins as that in DNA. However, endogenous 5-caC in RNA has not been observed so far. In the current study, we established a method using chemical labeling coupled with liquid chromatography-mass spectrometry analysis for the sensitive and simultaneous determination of the oxidative products of 5-mC. Our results demonstrated that the detection sensitivities of 5-mC, 5-hmC, 5-foC and 5-caC in RNA increased by 70-313 folds upon 2-bromo-1-(4-diethylaminophenyl)-ethanone (BDEPE) labeling. Using this method, we discovered the existence of 5-caC in the RNA of mammals. In addition, we found the 5-mC occurs in all RNA species including mRNA, 28S rRNA, 18S rRNA and small RNA (<200 nt). However, 5-hmC, 5-foC and 5-caC mainly occur in mRNA, and barely detected in other types of RNA. Furthermore, we found that the content of 5-hmC in the RNA of human colorectal carcinoma (CRC) and hepatocellular carcinoma (HCC) tissues significantly decreased compared to tumor adjacent normal tissues, suggesting that 5-hmC in RNA may play certain functional roles in the regulation of cancer development and formation.
Chiral carboxylic acids play important roles in energy metabolism and signal transduction in the human body. These enantiomers usually possess different bioactivities and are also associated with the ...development of some diseases. Therefore, simultaneous determination of multiple chiral carboxylic acids is vital for study of the pathogenesis of related diseases. However, it is still challenging to simultaneously detect the enantiomers of multiple chiral carboxylic acids in biological samples. Here, we developed a novel 4-plex chemical labeling strategy based on 4 analogues of cinchona alkaloid-derived primary amines (CAPAs) for simultaneous determination of 16 enantiomers of 8 chiral carboxylic acids by liquid chromatography-mass spectrometry (LC-MS). To achieve high-throughput analysis, one CAPA analogue was used to label chiral carboxylic acid standards and served as internal standards (ISs), while the other 3 CAPA analogues were used to label endogenous chiral carboxylic acids in 3 different biological samples. After CAPAs labeling, the 16 chiral carboxylic acid enantiomers could be detected by LC-MS, and their detection sensitivity was greatly enhanced by up to 3 orders of magnitude compared to intact analytes. Further, the developed method for the determination of 16 chiral carboxylic acid enantiomers was validated in human serums and mammalian cells. Finally, the proposed method was applied to the determination of chiral carboxylic acids in the serum samples from type 2 diabetes mellitus (T2DM) and colorectal cancer (CRC) patients. We found that 5 chiral carboxylic acid enantiomers in T2DM serum samples and 4 chiral carboxylic acid enantiomers in CRC serum samples exhibited significant change compared to the healthy control (HC).
Cytosine methylation (5-methylcytosine, 5-mC) in genomic DNA is an important epigenetic mark that has regulatory roles in diverse biological processes. 5-mC can be oxidized stepwise by the ten–eleven ...translocation (TET) proteins to form 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC), which constitutes the active DNA demethylation pathway in mammals. Owing to the extremely limited contents of endogenous 5-mC oxidation products, no reported method can directly determine all these cytosine modifications simultaneously. In the current study, we developed selective derivatization of cytosine moieties with 2-bromo-1-(4-dimethylamino-phenyl)-ethanone (BDAPE) coupled with liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) for the simultaneous determination of these cytosine modifications in genomic DNA. The chemical derivatization notably improved the liquid chromatography separation and dramatically increased detection sensitivities of these cytosine modifications. The limits of detection (LODs) of the derivatives of 5-mC, 5-hmC, 5-foC, and 5-caC were 0.10, 0.06, 0.11, and 0.23 fmol, respectively. Using this method, we successfully quantified 5-mC, 5-hmC, 5-foC, and 5-caC in genomic DNA from human colorectal carcinoma (CRC) tissues and tumor-adjacent normal tissues. The results demonstrated significant depletion of 5-hmC, 5-foC, and 5-caC in tumor tissues compared to tumor-adjacent normal tissues, and the depletion of 5-hmC, 5-foC, and 5-caC may be a general feature of CRC; these cytosine modifications could serve as potential biomarkers for the early detection and prognosis of CRC. Moreover, the marked depletion of 5-hmC, 5-foC, and 5-caC may also have profound effects on epigenetic regulation in the development and formation of CRC.
Profiling the heterogeneous phenotypes of individual circulating tumor cells (CTCs) from patients is a very challenging task, but it paves new ways for cancer management, especially personalized ...anticancer therapy. Herein, we propose a chip-assisted multifunctional-nanosphere system for efficient and reliable biomarker phenotype analysis of individual heterogeneous CTCs. Red fluorescent magnetic biotargeting multifunctional nanospheres and green fluorescent biotargeting nanospheres targeting to two kinds of CTC biomarkers are used for convenient dual-fluorescence labeling of CTCs along with magnetic tags. By integrating magnetic enrichment with a size-selective single-cell-trapping microfluidic chip (SCT-chip), over 90% of CTCs, even when the concentrations is as low as 10 CTCs per milliliter of blood, can be individually trapped at highly ordered micropillars, spatially separated from the minimal residual blood cells. Such single CTCs offer easy-readout fluorescence signals, facilitating efficient identification and reliable phenotype analysis in accordance with their biomarker expressions. Therefore, the phenotypes of breast tumor cells in terms of the expression level of human epidermal-growth-factor receptor 2, an important target of clinical anticancer drugs, are accurately assessed, and over 82% of them can be classified into corresponding cell subpopulations. Furthermore, this system demonstrates successful detection and subpopulation analysis of heterogeneous CTCs from seven breast cancer patients, which provides a promising new means for single-cell profiling of CTC-biomarker phenotypes and guiding of personalized anticancer therapy.
RNA molecules contain diverse modifications that display important functions in a variety of physiological and pathological processes. So far over 150 chemical modifications have been characterized ...to be present in various RNA species, such as in messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA). Previous studies revealed that certain RNA modifications were correlated to specific human diseases, indicating RNA modifications could serve as the potential indicator of human diseases. However, systemic investigation of the alteration of RNA modifications in different RNA species of carcinoma tissues are still lacked. Herein, we carried out the comprehensive profiling and evaluation of the alteration of RNA modifications in thyroid carcinoma by liquid chromatography-tandem mass spectrometry (LC-ESI-MS/MS) analysis. The developed method allowed us to simultaneously detect 48 different types of RNA modifications. Using this method, we detected 10, 15, 14, and 25 modifications in mRNA, 18S rRNA, 28S rRNA and small RNA (< 200 nt), respectively. Compared to the normal tissues, we revealed a total of 14 RNA modification exhibited significant increase and 2 RNA modifications showed significant decrease in thyroid carcinoma tissues. Our study provided the first comprehensive profile as well as the alteration of modifications in different RNA species in thyroid carcinoma and matched tumor-adjacent normal tissues. The altered pattern RNA modifications may serve as the indicator of thyroid carcinoma. Moreover, this study may promote the in-depth understanding of the regulatory roles of RNA modifications in thyroid carcinoma.
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We carried out the comprehensive profiling and evaluation of the alteration of RNA modifications in thyroid carcinoma by LC-ESI-MS/MS analysis. The quantification results showed that a total of 14 RNA modification exhibited significant increase and 2 RNA modifications showed significant decrease in thyroid carcinoma tissues, which lays a foundation for diagnosis, monitoring disease evolution, and predicting response to treatment through specific RNA modification patterns.
•A fully automated in-tube SPME/LC-PCD-MS method was developed.•Satisfied linearity and recoveries were obtained for aldehydes analysis in urine.•HAHC was cheap, commercially available, and showed no ...contaminations to MS.•Advantages lay in rapidity, economy, reproducibility and simplicity.
A fully automated in-tube solid phase microextraction/liquid chromatography-post column derivatization-mass spectrometry (in-tube SPME/LC-PCD-MS) method was developed for the analysis of urinary hexanal and heptanal. Online in-tube SPME enabled effective enrichment of the low level aldehydes and elimination of matrix interferences. PCD could be simply realized by mixing the LC elute and hydroxylamine hydrochloride (HAHC) solution with just a tee. The peak broadening and loss in separation efficiency associated with post column dead-volume could be ignored and even completely eliminated by employing suitable PCD configuration. What’s more, HAHC is commercially available and quite cheap, and shows no contaminations to MS. The entire procedure, including the extraction of aldehydes by in-tube SPME, LC separation, post column derivatization and MS detection were integrated together and completely automated, offering competitive advantages in terms of rapidity, economy, reproducibility and simplicity. The developed protocol was then successfully performed to determine lung cancer biomarkers (hexanal, heptanal) levels in urine samples.
Detecting viable circulating tumor cells (CTCs) without disruption to their functions for in vitro culture and functional study could unravel the biology of metastasis and promote the development of ...personalized anti-tumor therapies. However, existing CTC detection approaches commonly include CTC isolation and subsequent destructive identification, which damages CTC viability and functions and generates substantial CTC loss. To address the challenge of efficiently detecting viable CTCs for functional study, we develop a nanosphere-based cell-friendly one-step strategy. Immunonanospheres with prominent magnetic/fluorescence properties and extraordinary stability in complex matrices enable simultaneous efficient magnetic capture and specific fluorescence labeling of tumor cells directly in whole blood. The collected cells with fluorescent tags can be reliably identified, free of the tedious and destructive manipulations from conventional CTC identification. Hence, as few as 5 tumor cells in ca. 1mL of whole blood can be efficiently detected via only 20min incubation, and this strategy also shows good reproducibility with the relative standard deviation (RSD) of 8.7%. Moreover, due to the time-saving and gentle processing and the minimum disruption of immunonanospheres to cells, 93.8±0.1% of detected tumor cells retain cell viability and proliferation ability with negligible changes of cell functions, capacitating functional study on cell migration, invasion and glucose uptake. Additionally, this strategy exhibits successful CTC detection in 10/10 peripheral blood samples of cancer patients. Therefore, this nanosphere-based cell-friendly one-step strategy enables viable CTC detection and further functional analyses, which will help to unravel tumor metastasis and guide treatment selection.
•CTCs and WBCs were efficiently and specifically labeled with different IFNs tags.•Simultaneous capture and identification of CTCs were realized by coupled IMNs and IFNs.•One-step strategy shortened time consumption and improved efficiency of CTC detection.•The strategy was cell-friendly to detect viable CTCs for culture and functional study.
The discovery of dynamic and reversible modifications in RNA expands their functional repertoires. Now, RNA modifications have been viewed as new regulators involved in a variety of biological ...processes. Among these modifications, thiolation is one kind of special modification in RNA. Several thiouridines have been identified to be present in RNA, and they are essential in the natural growth and metabolism of cells. However, detection of these thiouridines generally is challenging, and few studies could offer the quantitative levels of uridine modifications in RNA, which limits the in-depth elucidation of their functions. Herein, we developed a chemical derivatization in combination with mass spectrometry analysis for the sensitive and simultaneous determination of uridine thiolation and hydroxylation modifications in eukaryotic RNA. The chemical derivatization strategy enables the addition of easily ionizable groups to the uridine thiolation and hydroxylation modifications, leading up to a 339-fold increase in detection sensitivities of these modifications by mass spectrometry analysis. The limits of detection of these uridine modifications can be down to 17 amol. With the established method, we discovered and confirmed that a new modification of 5-hydroxyuridine (ho5U) was widely present in small RNAs of mammalian cells, expanding the diversity of RNA modifications. The developed method shows superior capability in determining low-abundance RNA modifications and may promote identifying new modifications in RNA, which should be valuable in uncovering the unknown functions of RNA modifications.
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