Deep learning-assisted monitoring of trastuzumab efficacy in HER2-Overexpressing breast cancer via SERS immunoassays of tumor-derived urinary exosomal biomarkers.

Monitoring drug efficacy is significant in the current concept of companion diagnostics in metastatic breast cancer. Trastuzumab, a drug targeting human epidermal growth factor receptor 2 (HER2), is an effective treatment for metastatic breast cancer. However, some patients develop resistance to this therapy; therefore, monitoring its efficacy is essential. Here, we describe a deep learning-assisted monitoring of trastuzumab efficacy based on a surface-enhanced Raman spectroscopy (SERS) immunoassay against HER2-overexpressing mouse urinary exosomes. Individual Raman reporters bearing the desired SERS tag and exosome capture substrate were prepared for the SERS immunoassay; SERS tag signals were collected to prepare deep learning training data. Using this deep learning algorithm, various complicated mixtures of SERS tags were successfully quantified and classified. Exosomal antigen levels of five types of cell-derived exosomes were determined using SERS-deep learning analysis and compared with those obtained via quantitative reverse transcription polymerase chain reaction and western blot analysis. Finally, drug efficacy was monitored via SERS-deep learning analysis using urinary exosomes from trastuzumab-treated mice. Use of this monitoring system should allow proactive responses to any treatment-resistant issues.

Colorimetric Detection of HER2-Overexpressing-Cancer-Derived Exosomes in Mouse Urine Using Magnetic-Polydiacetylene Nanoparticles.

Breast cancer (BC) is a major global health problem, with ≈20-25% of patients overexpressing human epidermal growth factor receptor 2 (HER2), an aggressive marker, yet access to early detection and treatment varies across countries. A low-cost, equipment-free, and easy-to-use polydiacetylene (PDA)-based colorimetric sensor is developed for HER2-overexpressing cancer detection, designed for use in low- and middle-income countries (LMICs). PDA nanoparticles are first prepared through thin-film hydration. Subsequently, hydrophilic magnetic nanoparticles and HER2 antibodies are sequentially conjugated to them. The synthesized HER2-MPDA can be concentrated and separated by a magnetic field while inheriting the optical characteristics of PDA. The specific binding of HER2 antibody in HER2-MPDA to HER2 receptor in HER2-overexpressing exosomes causes a blue-to-red color change by altering the molecular structure of the PDA backbone. This colorimetric sensor can simultaneously separate and detect HER2-overexpressing exosomes. HER2-MPDA can detect HER2-overexpressing exosomes in the culture medium of HER2-overexpressing BC cells and in mouse urine samples from a HER2-overexpressing BC mouse model. It can selectively isolate and detect only HER2-overexpressing exosomes through magnetic separation, and its detection limit is found to be 8.5 × 108 particles mL-1. This colorimetric sensor can be used for point-of-care diagnosis of HER2-overexpressing BC in LMICs.

Background free in vivo29Si MR imaging with hyperpolarized PEGylated silicon nanoparticles.

This study demonstrated the potential of 50 nm PEGylated Si NPs for high-resolution in vivo29Si MR imaging, emphasizing their biocompatibility and water dispersibility. The acquisition of in vivo Si MR images using the lowest reported dose after subcutaneous and intraperitoneal administration opens new avenues for future 29Si MR studies.

The lipoprotein-associated phospholipase A2 inhibitor Darapladib sensitises cancer cells to ferroptosis by remodelling lipid metabolism.

Arachidonic and adrenic acids in the membrane play key roles in ferroptosis. Here, we reveal that lipoprotein-associated phospholipase A2 (Lp-PLA2) controls intracellular phospholipid metabolism and contributes to ferroptosis resistance. A metabolic drug screen reveals that darapladib, an inhibitor of Lp-PLA2, synergistically induces ferroptosis in the presence of GPX4 inhibitors. We show that darapladib is able to enhance ferroptosis under lipoprotein-deficient or serum-free conditions. Furthermore, we find that Lp-PLA2 is located in the membrane and cytoplasm and suppresses ferroptosis, suggesting a critical role for intracellular Lp-PLA2. Lipidomic analyses show that darapladib treatment or deletion of PLA2G7, which encodes Lp-PLA2, generally enriches phosphatidylethanolamine species and reduces lysophosphatidylethanolamine species. Moreover, combination treatment of darapladib with the GPX4 inhibitor PACMA31 efficiently inhibits tumour growth in a xenograft model. Our study suggests that inhibition of Lp-PLA2 is a potential therapeutic strategy to enhance ferroptosis in cancer treatment.

An immuno-magnetophoresis-based microfluidic chip to isolate and detect HER2-Positive cancer-derived exosomes via multiple separation.

Exosomes are useful for cancer diagnosis and monitoring. However, clinical samples contain impurities that complicate direct analyses of cancer-derived exosomes. Therefore, a microfluidic chip-based magnetically labeled exosome isolation system (MEIS-chip) was developed as a lab-on-a-chip platform for human epidermal growth factor receptor 2 (HER2)-positive cancer diagnosis and monitoring. Various magnetic nanoclusters (MNCs) were synthesized with different degrees of magnetization, and antibodies were introduced to capture HER2-overexpressing and common exosomes using immunoaffinity. MNC-bonded exosomes were separated into different exits according to their magnetization degrees. The MEIS-chip efficiently separated HER2-overexpressing exosomes from common exosomes that did not contain disease-related information. The simultaneous separation of HER2-and non-HER2-overexpressing exosomes provided a means of analyzing high-purity HER2-overexpressing exosomes while minimizing the contribution of non-target exosomes, reducing misdiagnosis risk. Notably, common exosomes served as a negative control for monitoring real-time changes in HER2 expression. These findings support the application of MEIS-chip for cancer diagnosis and treatment monitoring via effective exosome isolation.

Clinical molecular subtyping reveals intrinsic mesenchymal reprogramming in gastric cancer cells.

The mesenchymal cancer phenotype is known to be clinically related to treatment resistance and a poor prognosis. We identified gene signature-based molecular subtypes of gastric cancer (GC, n = 547) based on transcriptome data and validated their prognostic and predictive utility in multiple external cohorts. We subsequently examined their associations with tumor microenvironment (TME) features by employing cellular deconvolution methods and sequencing isolated GC populations. We further performed spatial transcriptomics analysis and immunohistochemistry, demonstrating the presence of GC cells in a partial epithelial-mesenchymal transition state. We performed network and pharmacogenomic database analyses to identify TGF-ß signaling as a driver pathway and, thus, a therapeutic target. We further validated its expression in tumor cells in preclinical models and a single-cell dataset. Finally, we demonstrated that inhibition of TGF-ß signaling negated mesenchymal/stem-like behavior and therapy resistance in GC cell lines and mouse xenograft models. In summary, we show that the mesenchymal GC phenotype could be driven by epithelial cancer cell-intrinsic TGF-ß signaling and propose therapeutic strategies based on targeting the tumor-intrinsic mesenchymal reprogramming of medically intractable GC.

Elution-free DNA detection using CRISPR/Cas9-mediated light-up aptamer transcription: Toward all-in-one DNA purification and detection tube.

Accurate and efficient detection of DNA is crucial for disease diagnosis and health monitoring. The traditional methods for DNA analysis involve multiple steps, including sample preparation, lysis, extraction, amplification, and detection. In this study, we present a one-step elution-free DNA analysis method based on the combination of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated light-up aptamer transcription (CLAT) assay and a DNA-capturing poly(2-dimethylaminomethyl styrene) (pDMAMS)-coated tube. The sample solution and lysis buffer are added to the pDMAMS-coated tube, and the DNA is efficiently captured on the surface via electrostatic interaction and directly detected by CLAT assay. The ability of the CRISPR/Cas9 system to specifically recognize DNA enables direct detection of DNA captured on the pDMAMS-coated tube. The combination of CLAT assay and pDMAMS-coated tube simplifies DNA detection in a single tube without the need for complicated extraction steps, improving sensitivity. Our platform demonstrated attomolar sensitivity in the detection of target DNA in cell lysate (0.92 aM), urine (7.7 aM), and plasma (94.6 aM) samples within 1 h. The practical applicability of this method was further demonstrated in experiments with tumor-bearing mice. We believe that this approach brings us closer to an all-in-one DNA purification and detection tube system and has potential applications in tissue and liquid biopsies, as well as various other DNA sensing applications.

Highly Selective FRET-Aided Single-Molecule Counting of MicroRNAs Labeled by Splinted Ligation.

MicroRNAs (miRNAs) are short non-coding RNAs that play an important role in regulating gene expression. Since miRNAs are abnormally expressed in various cancers, they are considered to be promising biomarkers for early cancer diagnosis. However, the short length and strong sequence similarity among miRNAs make their reliable quantification very challenging. We developed a highly selective amplification-free miRNA detection method based on Förster resonance energy transfer (FRET)-aided single-molecule counting. miRNAs were selectively labeled with FRET probes using splinted ligation. When imaged with a single-molecule FRET setup, the miRNA molecules were accurately identified by the probe's FRET. miRNA concentrations were estimated from the count of molecules. The high sensitivity of the method in finding sparse molecules enabled us to achieve a limit of detection of 31-56 amol for miR-125b, miR-100, and miR-99a. Single nucleotide mismatch could be discriminated with a very high target-to-mismatch ratio. The method accurately measured the high expression of miR-125b in gastric cancer cells, which agreed well with previous reports. The high sensitivity and accuracy of this technique demonstrated its clinical potential as a robust miRNA detection method.

MRI measurement of alanine uptake in a mouse xenograft model of U-87 MG glioblastoma.

The potential use of alanine as an MRI contrast agent was investigated. The relaxation properties of alanine solutions were measured at 9.4 T. The T2 relaxivity caused by the chemical exchange (R2ex) between amine protons and water protons was 0.10 mM-1 s-1 at 37 °C. As a demonstration, alanine uptake in a mouse xenograft model of U-87 MG glioblastoma was measured using MRI, and was compared with immunohistochemistry staining of ASCT2, a transporter that imports amino acids into cancer cells. Statistically significant (p = 0.0079) differences in ASCT2 distribution were found between regions that show strong and weak alanine uptake in MRI. To better understand the influence of perfusion, the effect of ASCT2 inhibition on the alanine uptake in MRI was investigated, and dynamic contrast enhanced MRI was compared with alanine MRI.

Cationic poly(amino acid) surface functionalized manganese nanoparticles for nitric oxide-based immunotherapy and magnetic resonance imaging.

The low therapeutic efficacy of conventional cancer chemotherapy has been associated with an immunosuppressive tumor microenvironment (TME). Tumor-associated macrophages (TAMs), which display an M2-like phenotype, are abundant in many tumors and facilitate tumor growth and resistance to therapy. Here, we show that poly(L-arginine) (PLR), a cationic poly(amino acid) can induce the polarization of macrophages into the tumor-suppressive M1 phenotype, in vitro. Further, we demonstrate that hyaluronic acid (HA) and PLR-coated manganese dioxide (MnO2) nanoparticles (hpMNPs) display efficient anti-cancer effects by upregulating nitric oxide (NO) production. Surface modification with biocompatible HA reduced the cytotoxicity of the cationic PLR. Additionally, manganese ions released from these nanoparticles by the high concentrations of glutathione (GSH) in the TME increased iNOS expression level in macrophages and enhanced the performance of T1 weighted magnetic resonance imaging. Particularly, our results illustrate the therapeutic effects, such as growth inhibition and apoptosis of tumor cells, of hpMNP treated macrophages. Therefore, the newly designed multifunctional PLR-assisted MNPs may facilitate the polarization of M2 macrophages into the M1 phenotype, which can mediate NO-dependent anticancer immunotherapy.

Ligation-free isothermal nucleic acid amplification.

In this study, we uncover a ligation-free DNA extension method in two adjacent fragmented probes, which are hybridized to target RNA, for developing a ligation-free nucleic acid amplification reaction. In this reaction, DNA elongation occurs from a forward probe to a phosphorothioated-hairpin probe in the presence of target RNA regardless of ligation. The second DNA elongation then occurs simultaneously at the nick site of the phosphorothioated probe and the self-priming region. Therefore, the binding site of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 12a is repeatedly amplified, inducing a fluorescence signal in the presence of CRISPR-Cas12a. This ligation-free isothermal gene amplification method enables the detection of target RNA with 49.2 fM sensitivity. Moreover, two types of mRNA detection are feasible, thus, demonstrating the potential of this method for cancer companion diagnostics. Notably, the proposed method also demonstrates efficacy when applied for the detection of mRNA extracted from human cells and tumor-bearing mouse tissue and urine samples. Hence, this newly developed ligation-free isothermal nucleic acid amplification system is expected to be widely used in a variety of gene detection platforms.

Simultaneous dual-targeted monitoring of breast cancer circulating miRNA via surface-enhanced Raman spectroscopy.

Breast cancer is one of the most common cancers globally. Because the 5-year survival rate of breast cancer greatly increases when treated in its initial stage, the importance of early detection has been increasing. Herein, one-spot multiple breast cancer circulating microRNA (miRNA) detection via surface-enhanced Raman spectroscopy (SERS) with seed-mediated grown Ag nanopillars (SMGAPs) is described. The electrochemical reduction on the pre-distributed 40 nm gold nanoparticle seeds (sGNP) acted as scaffolds for silver ion growth, and a nanopillar-shaped silver structure was successfully grown on the gold substrate surface. The synthesized structure showed uniform and remarkably increased signal enhancement for malachite green isothiocyanate. Based on this consistency, two circulating miRNA markers for breast cancer (miR-21 and miR-155) were used as the SERS diagnostic target. The limit of detection (LOD) of each labeled target was 451 zmol and 1.65 amol respectively. Moreover, miRNAs in four types of cancer cell extracts (HCC1143, HCC1954, MDA-MB-231, MCF-7) were sorted by miR-21 and miR-155 copies. Finally, quantitative analysis of miRNA in urine was successful compared to that in the healthy group.

Microfluidic device for one-step detection of breast cancer-derived exosomal mRNA in blood using signal-amplifiable 3D nanostructure.

Metastasis attributed to approximately 90% of cancer-related deaths; hence, the detection of metastatic tumor-derived components in the blood assists in determining cancer recurrence and patient survival. Microfluidic-based sensors facilitate analysis of small fluid volumes and represent an accurate, rapid, and user-friendly method of field diagnoses. In this study, we have developed a microfluidic chip-based exosomal mRNA sensor (exoNA-sensing chip) for the one-step detection of exosomal ERBB2 in the blood by integrating a microfluidic chip and 3D-nanostructured hydrogels. The exoNA-sensing chip is a vacuum-driven power-free microfluidic chip that can accurately control the flow of trace fluids (<100 µL). The sensing part of the exoNA-sensing chip includes 3D-nanostructured hydrogels capable of detecting ERBB2 and a reference gene by amplifying a fluorescent signal via an enzyme-free catalytic hairpin assembly reaction at room temperature. This hydrogel offers a detection limit of 58.3 fM with good selectivity for target sequences. The performance of the exoNA-sensing chip was evaluated by testing in vitro and in vivo samples and was proven to be effective for cancer diagnosis and liquid biopsies.

29Si Isotope-Enriched Silicon Nanoparticles for an Efficient Hyperpolarized Magnetic Resonance Imaging Probe.

Silicon particles have garnered attention as promising biomedical probes for hyperpolarized 29Si magnetic resonance imaging and spectroscopy. However, due to the limited levels of hyperpolarization for nanosized silicon particles, microscale silicon particles have primarily been the focus of dynamic nuclear polarization (DNP) applications, including in vivo magnetic resonance imaging (MRI). To address these current challenges, we developed a facile synthetic method for partially 29Si-enriched porous silicon nanoparticles (NPs) (160 nm) and examined their usability in hyperpolarized 29Si MRI agents with enhanced signals in spectroscopy and imaging. Hyperpolarization characteristics, such as the build-up constant, the depolarization time (T1), and the overall enhancement of the 29Si-enriched silicon NPs (10 and 15%), were thoroughly investigated and compared with those of a naturally abundant NP (4.7%). During optimal DNP conditions, the 15% enriched silicon NPs showed more than 16-fold higher enhancements─far beyond the enrichment ratio─than the naturally abundant sample, further improving the signal-to-noise ratio in in vivo 29Si MRI. The 29Si-enriched porous silicon NPs used in this work are potentially capable to serve as drug-delivery vehicles in addition to hyperpolarized 29Si in vivo, further enabling their potential future applicability as a theragnostic platform.

Immunomagnetic microfluidic integrated system for potency-based multiple separation of heterogeneous stem cells with high throughput capabilities.

Multipotent adult stem cells (MASCs) derived from Pluripotent stem cells (PSCs) have found widespread use in various applications, including regenerative therapy and drug screening. For these applications, highly pluripotent PSCs need to be selectively separated from those that show low pluripotency for reusage of PSCs, and MASCs need to be collected for further application. Herein, we developed immunomagnetic microfluidic integrated system (IM-MIS) for separation of stem cells depending on potency level. In this system, each stem cell was multiple-separated in microfluidics chip by magnetophoretic mobility of magnetic-activated cells based on the combination of two sizes of magnetic nanoparticles and two different antibodies. Magnetic particles had a difference in the degree of magnetization, and antibodies recognized potency-related surface markers. IM-MIS showed superior cell separation performance than FACS with high throughput (49.5%) in a short time (<15 min) isolate 1 × 107 cells, and higher purity (92.1%) than MACS. IM-MIS had a cell viability of 89.1%, suggesting that IM-MIS had no effect on cell viability during isolation. Furthermore, IM-MIS did not affect the key characteristics of stem cells including its differentiation potency, phenotype, genotype, and karyotype. IM-MIS may offer a new platform for the development of multi-separation systems for diverse stem cell applications.

Genetic changes and growth promotion of glioblastoma by magnetic nanoparticles and a magnetic field.

Aim: To confirm the biological effects of manganese ferrite magnetic nanoparticles (MFMNPs) and an external magnetic field on glioblastoma cells. Methods: U-87MG glioblastoma cells were prepared, into which the uptake of MFMNPs was high. The cells were then exposed to an external magnetic field using a neodymium magnet in vitro and in vivo. Results:LRP6 and TCF7 mRNA levels involved in the Wnt/ß-catenin signaling pathway were elevated by the influence of MFMNPs and the external magnetic field. MFMNPs and the external magnetic field also accelerated tumor growth by approximately 7 days and decreased survival rates in animal experiments. Conclusion: When MFMNPs and an external magnetic field are applied for a long time on glioblastoma cells, mRNA expression related to Wnt/ß-catenin signaling is increased and tumor growth is promoted.

In vivo monitoring platform of transplanted human stem cells using magnetic resonance imaging.

As stem cells show great promise in regenerative therapy, stem cell-mediated therapeutic efficacy must be demonstrated through the migration and transplantation of stem cells into target disease areas at the pre-clinical level. In this study, we developed manganese-based magnetic nanoparticles with hollow structures (MnOHo) and modified them with the anti-human integrin ß1 antibody (MnOHo-Ab) to enable the minimal-invasive monitoring of transplanted human stem cells at the pre-clinical level. Compared to common magnetic resonance imaging (MRI)-based stem cell monitoring systems that use pre-labeled stem cells with magnetic particles before stem cell injection, the MnOHo-Ab is a new technology that does not require stem cell modification to monitor the therapeutic capability of stem cells. Additionally, MnOHo-Ab provides improved T1 MRI owing to the hollow structure of the MnOHo. Particularly, the anti-integrin ß1 antibody (Ab) introduced in the MnOHo targets integrin ß1 expressed in the entire stem cell lineage, enabling targeted monitoring regardless of the differentiation stage of the stem cells. Furthermore, we verified that intravenously injected MnOHo-Ab specifically targeted human induced pluripotent stem cells (hiPSCs) that were transferred to mice testes and differentiated into various lineages. The new stem cell monitoring method using MnOHo-Ab demonstrates whether the injected human stem cells have migrated and transplanted themselves in the target area during long-term stem cell regenerative therapy.

Utilization of chromogenic enzyme substrates for signal amplification in multiplexed detection of biomolecules using surface mass spectrometry.

MicroRNAs (miRNAs) are important post-transcriptional gene regulators and can serve as potential biomarkers for many diseases. Most of the current miRNA detection techniques require purification from biological samples, amplification, labeling, or tagging, which makes quantitative analysis of clinically relevant samples challenging. Here we present a new strategy for the detection of miRNAs with uniformity over a large area based on signal amplification using enzymatic reactions and measurements using time-of-flight secondary ion mass spectrometry (ToF-SIMS), a sensitive surface analysis tool. This technique has high sequence specificity through hybridization with a hairpin DNA probe and allows the identification of single-base mismatches that are difficult to distinguish by conventional mass spectrometry. We successfully detected target miRNAs in biological samples without purification, amplification, or labeling of target molecules. In addition, by adopting a well-known chromogenic enzymatic reaction from the field of biotechnology, we extended the use of enzyme-amplified signal enhancement ToF (EASE-ToF) to protein detection. Our strategy has advantages with respect to scope, quantification, and throughput over the currently available methods, and is amenable to multiplexing based on the outstanding molecular specificity of mass spectrometry (MS). Therefore, our technique not only has the potential for use in clinical diagnosis, but also provides evidence that MS can serve as a useful readout for biosensing to perform multiplexed analysis extending beyond the limitations of existing technology.

Co-expression of cancer driver genes: IDH-wildtype glioblastoma-derived tumorspheres.

BACKGROUND: Driver genes of GBM may be crucial for the onset of isocitrate dehydrogenase (IDH)-wildtype (WT) glioblastoma (GBM). However, it is still unknown whether the genes are expressed in the identical cluster of cells. Here, we have examined the gene expression patterns of GBM tissues and patient-derived tumorspheres (TSs) and aimed to find a progression-related gene. METHODS: We retrospectively collected primary IDH-WT GBM tissue samples (n = 58) and tumor-free cortical tissue samples (control, n = 20). TSs are isolated from the IDH-WT GBM tissue with B27 neurobasal medium. Associations among the driver genes were explored in the bulk tissue, bulk cell, and a single cell RNAsequencing techniques (scRNAseq) considering the alteration status of TP53, PTEN, EGFR, and TERT promoter as well as MGMT promoter methylation. Transcriptomic perturbation by temozolomide (TMZ) was examined in the two TSs. RESULTS: We comprehensively compared the gene expression of the known driver genes as well as MGMT, PTPRZ1, or IDH1. Bulk RNAseq databases of the primary GBM tissue revealed a significant association between TERT and TP53 (p < 0.001, R = 0.28) and its association increased in the recurrent tumor (p < 0.001, R = 0.86). TSs reflected the tissue-level patterns of association between the two genes (p < 0.01, R = 0.59, n = 20). A scRNAseq data of a TS revealed the TERT and TP53 expressing cells are in a same single cell cluster. The driver-enriched cluster dominantly expressed the glioma-associated long noncoding RNAs. Most of the driver-associated genes were downregulated after TMZ except IGFBP5. CONCLUSIONS: GBM tissue level expression patterns of EGFR, TERT, PTEN, IDH1, PTPRZ1, and MGMT are observed in the GBM TSs. The driver gene-associated cluster of the GBM single cells were enriched with the glioma-associated long noncoding RNAs.