Cuproptosis, the novel type of oxidation-induced cell death in thoracic cancers: can it enhance the success of immunotherapy?

Copper is an important metal micronutrient, required for the balanced growth and normal physiological functions of human organism. Copper-related toxicity and dysbalanced metabolism were associated with the disruption of intracellular respiration and the development of various diseases, including cancer. Notably, copper-induced cell death was defined as cuproptosis which was also observed in malignant cells, representing an attractive anti-cancer instrument. Excess of intracellular copper leads to the aggregation of lipoylation proteins and toxic stress, ultimately resulting in the activation of cell death. Differential expression of cuproptosis-related genes was detected in normal and malignant tissues. Cuproptosis-related genes were also linked to the regulation of oxidative stress, immune cell responses, and composition of tumor microenvironment. Activation of cuproptosis was associated with increased expression of redox-metabolism-regulating genes, such as ferredoxin 1 (FDX1), lipoic acid synthetase (LIAS), lipoyltransferase 1 (LIPT1), dihydrolipoamide dehydrogenase (DLD), drolipoamide S-acetyltransferase (DLAT), pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1), and pyruvate dehydrogenase E1 subunit beta (PDHB)). Accordingly, copper-activated network was suggested as an attractive target in cancer therapy. Mechanisms of cuproptosis and regulation of cuproptosis-related genes in different cancers and tumor microenvironment are discussed in this study. The analysis of current findings indicates that therapeutic regulation of copper signaling, and activation of cuproptosis-related targets may provide an effective tool for the improvement of immunotherapy regimens.

Quantitative Proteomic Analysis of Cell Responses to Electroporation, a Classical Gene Delivery Approach.

Electroporation, as an established nonviral technology for breaching cell membrane, has been accepted for the delivery of nucleic acids. Despite satisfactory delivery efficiencies have been achieved on multiple cell kinds by simply exhausting all possible electrical parameters, electroporation is still inefficient, or even invalid, for various kinds of cells. This is largely due to the lack of comprehensive understanding of cell responses to electrical stimulation at biological aspect. Moreover, a systematically investigation of protein variation of electroporated cells is also required for biosafety evaluation before clinically applying electroporation. By employing quantitative proteomic analysis, the biological mechanism of electroporation is explored from the molecular level. The results reveal that electrical stimulations widely influence many biological processes including nucleic acid stabilization, protein synthesis, cytoskeleton dynamic, inflammation, and cell apoptosis. It is found that several antivirus-related processes appeared in the enrichment results. Moreover, SAMD9, a broad spectrum antiviral and antitumor factor, is dramatically downregulated on easy-to-transfect cells while electroporation can not alter SAMD9 expression on hard-to-transfect cells, hinting that electroporation, a pure physical treatment, can induce antivirus-like defensive responses and the altering of SAMD9 can be used to predict the effectiveness of electroporation on transfecting specific kinds of cells.

pH-Sensitive Nanomicelles for High-Efficiency siRNA Delivery in Vitro and in Vivo: An Insight into the Design of Polycations with Robust Cytosolic Release.

The extremely low efficient cytosolic release of the internalized siRNA has emerged recently as a central issue for siRNA delivery, while there is a lack of guidelines to facilitate the cytosolic release of internalized siRNA. To address these concerns, we studied the contribution of the pH-sensitive inner core on handling the cytosolic release of siRNA delivered by a series of PG-P(DPAx-co-DMAEMAy)-PCB amphiphilic polycation nanomicelles (GDDC-Ms) with extremely low internalization (<1/4 of lipofactamine 2000 (Lipo2000)). Significantly, just by varying the mole ratio of DPA and DMAEMA to adjust the initial disassembly pH (pHdis) of the core near to 6.8, GDDC4-Ms/siRNA could get nearly 98.8% silencing efficiency at w/w = 12 with 50 nM siRNA and ∼78% silencing efficiency at w/w = 30 with a very low dose of 5 nM siRNA in HepG-2 cell lines, while Lipo2000 only got 65.7% with 50 nM siRNA. Furthermore, ∼98.4% silencing efficiency was also realized in the hard-to-transfect human acute monoblastic leukemia cell line U937 by GDDC4-Ms/siRNA (at w/w = 15, 50 nM siRNA), in the inefficient case for Lipo2000. Additionally, the high silencing efficiency (∼80%) in skin tissue in vivo was discovered. Undoubtedly, the robust potential of GDDC4-Ms in handling the cytosolic release paves a simple but efficient new way for the design of the nonviral siRNA vector.

A circulating miRNA signature as a diagnostic biomarker for non-invasive early detection of breast cancer.

Novel, non-invasive biomarkers to diagnose breast cancer with high sensitivity and specificity are greatly desired. Circulating microRNAs (miRNAs) show potential for breast cancer detection, but the existing results appear to be mixed. Using microscale serum, we established a novel serum-direct multiplex detection assay based on RT-PCR (SdM-RT-PCR). Ninety-three miRNAs dysregulated or with functions in breast cancer were selected as candidates, and additional 3 miRNAs were chosen as endogenous controls. We first conducted miRNA profiling of these 96 miRNAs by SdM-RT-PCR using the sera of 25 breast cancer patients at diagnosis prior to treatment and 20 age-matched healthy controls. miRNAs showing significantly different expression levels between patients and controls were further analyzed using a logistic regression model. A miRNA signature was validated in an independent set of 128 serum samples composed of 76 breast cancer patients and 52 healthy controls. In the discovery stage, we identified 23 miRNAs as significantly dysregulated in breast cancer patients compared with healthy controls. Of these, 10 miRNAs were previously identified as dysregulated in breast cancer; 14 miRNAs remained significant after P-values were adjusted by both correction methods. Principal component analysis and hierarchical clustering of these miRNAs separated patients from controls. Furthermore, the 3-miRNA signature (miR-199a, miR-29c, and miR-424) with the highest diagnostic accuracy for distinguishing breast cancer patients from controls by ROC curve analysis (AUC = 0.888) was successfully confirmed in the validation set (AUC = 0.901). Our data demonstrate that the SdM-RT-PCR assay is an effective breast cancer profiling method that utilizes very small volumes and is compatible with Biobank. Furthermore, the identified 3-miRNA signature is a promising circulating biomarker for breast cancer diagnosis.

Flow-through cell electroporation microchip integrating dielectrophoretic viable cell sorting.

Microfluidics based continuous cell electroporation is an appealing approach for high-throughput cell transfection, but cell viability of existing methods is usually compromised by adverse electrical or hydrodynamic effects. Here we present the validation of a flow-through cell electroporation microchip, in which dielectrophoretic force was employed to sort viable cells. By integrating parallel electroporation electrodes and dielectrophoresis sorting electrodes together in a simple straight microfluidic channel, sufficient electrical pulses were applied for efficient electroporation, and a proper sinusoidal electrical field was subsequently utilized to exclude damaged cells by dielectrophoresis. Thus, the difficulties for seeking the fine balance between electrotransfection efficiency and cell viability were steered clear. After careful investigation and optimization of the DEP behaviors of electroporated cells, efficient electrotransfection of plasmid DNA was demonstrated in vulnerable neuron cells and several hard-to-transfect primary cell types with excellent cell viability. This microchip constitutes a novel way of continuous cell transfection to significantly improve the cell viability of existing methodologies.

Method for electric parametric characterization and optimization of electroporation on a chip.

We have developed a rapid method to optimize the electric parameters of cell electroporation. In our design, a pair of ring-dot formatted electrodes was used to generate a radial distribution of electric field from the center to the periphery. Varied electric field intensity was acquired in different annulus when an electric pulse was applied. Cells were cultured on the microchips for adherent cell electroporation and in situ observation. The electroporation parameters of electric field intensity were explored and evaluated in terms of cell viability and transfection efficiency. The optimization was performed in consideration of both cell viability, which was investigated to decrease as electric field increases, and the transfection rate, which normally increases at stronger electric field. The electroporation characteristics HEK-293A and Hela cells were investigated, and the optimum parameters were obtained. Verified by a commercial electroporation system as well as self-made microchips endowed the optimization with wider meaning. At last, as applications, we acquired the optimal electroporation pulse intensity of Neuro-2A cells and a type of primary cell (human umbilical vein endothelial cell, HUVEC) by one time electroporation using the proposed method.

Dual effects of radiotherapy on tumor microenvironment and its contribution towards the development of resistance to immunotherapy in gastrointestinal and thoracic cancers.

Successful clinical methods for tumor elimination include a combination of surgical resection, radiotherapy, and chemotherapy. Radiotherapy is one of the crucial components of the cancer treatment regimens which allow to extend patient life expectancy. Current cutting-edge radiotherapy research is focused on the identification of methods that should increase cancer cell sensitivity to radiation and activate anti-cancer immunity mechanisms. Radiation treatment activates various cells of the tumor microenvironment (TME) and impacts tumor growth, angiogenesis, and anti-cancer immunity. Radiotherapy was shown to regulate signaling and anti-cancer functions of various TME immune and vasculature cell components, including tumor-associated macrophages, dendritic cells, endothelial cells, cancer-associated fibroblasts (CAFs), natural killers, and other T cell subsets. Dual effects of radiation, including metastasis-promoting effects and activation of oxidative stress, have been detected, suggesting that radiotherapy triggers heterogeneous targets. In this review, we critically discuss the activation of TME and angiogenesis during radiotherapy which is used to strengthen the effects of novel immunotherapy. Intracellular, genetic, and epigenetic mechanisms of signaling and clinical manipulations of immune responses and oxidative stress by radiotherapy are accented. Current findings indicate that radiotherapy should be considered as a supporting instrument for immunotherapy to limit the cancer-promoting effects of TME. To increase cancer-free survival rates, it is recommended to combine personalized radiation therapy methods with TME-targeting drugs, including immune checkpoint inhibitors.

AIFM2 promotes hepatocellular carcinoma metastasis by enhancing mitochondrial biogenesis through activation of SIRT1/PGC-1α signaling.

AIFM2 is a crucial NADH oxidase involved in the regulation of cytosolic NAD+. However, the role of AIFM2 in the progression of human cancers remains largely unexplored. Here, we elucidated the clinical implications, biological functions, and molecular mechanisms of AIFM2 in hepatocellular carcinoma (HCC). We found that AIFM2 is significantly upregulated in HCC, which is most probably caused by DNA hypomethylation and downregulation of miR-150-5p. High expression of AIFM2 is markedly associated with poor survival in patients with HCC. Knockdown of AIFM2 significantly impaired, while forced expression of AIFM2 enhanced the metastasis of HCC both in vitro and in vivo. Mechanistically, increased mitochondrial biogenesis and oxidative phosphorylation by activation of SIRT1/PGC-1α signaling contributed to the promotion of metastasis by AIFM2 in HCC. In conclusion, AIFM2 upregulation plays a crucial role in the promotion of HCC metastasis by activating SIRT1/PGC-1α signaling, which strongly suggests that AIFM2 could be targeted for the treatment of HCC.

CircATIC inhibits esophageal carcinoma progression and promotes radiosensitivity by elevating RHCG through sponging miR-10-3p.

BACKGROUND: Circular RNAs (circRNAs) are implicated in the progression and radiosensitivity of human cancers, including esophageal carcinoma (ESCA). In this study, we aimed to explore the functions of circRNA 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (circATIC) in ESCA progression. METHODS: CircATIC expression, miR-10b-3p and Rh family C glycoprotein (RHCG) were examined via quantitative real-time polymerase chain reaction (qRT-PCR), western blot assay or immunohistochemistry (IHC) assay. 5'-ethynyl-2'-deoxyuridine (EdU), wound-healing, transwell, and cell colony formation assays and flow cytometry analysis were conducted to evaluate cell proliferation, migration, invasion, radiosensitivity and apoptosis, respectively. Dual-luciferase reporter assay and RNA pulldown assay were conducted to analyze the relationships among circATIC, miR-10b-3p and RHCG. A murine xenograft model assay was performed to explore the functions of circATIC in tumor formation and radiosensitivity in vivo. RESULTS: CircATIC was decreased in ESCA. CircATIC overexpression suppressed cell proliferation, migration and invasion and promoted radiosensitivity and apoptosis in ESCA cells in vitro and repressed tumor formation and radioresistance in vivo. Functionally, circATIC served as the sponge for miR-10b-3p, which directly targeted RHCG. MiR-10b-3p elevation reversed circATIC-mediated effect on ESCA cell progression. Moreover, miR-10b-3p inhibition suppressed cell growth and metastasis and enhanced radiosensitivity in ESCA cells by targeting RHCG. CONCLUSIONS: Overexpression of circATIC hampered ESCA progression and promoted radiosensitivity depending on the regulation of miR-10b-3p and RHCG.

Conscription of Immune Cells by Light-Activatable Silencing NK-Derived Exosome (LASNEO) for Synergetic Tumor Eradication.

Exosomes derived from natural killer (NK) cells (NEO) constitute promising antineoplastic nano-biologics because of their versatile functions in immune regulation. However, a significant augment of their immunomodulatory capability is an essential need to achieve clinically meaningful treatment outcomes. Light-activatable silencing NK-derived exosomes (LASNEO) are orchestrated by engineering the NEO with hydrophilic small interfering RNA (siRNA) and hydrophobic photosensitizer Ce6. Profiling of genes involved in apoptosis pathway with Western blot and RNA-seq in cells receiving NEO treatment reveals that NEO elicits effective NK cell-like cytotoxicity toward tumor cells. Meanwhile, reactive oxygen species (ROS) generation upon laser irradiation not only triggers substantial photodynamic therapy effect but also boosts M1 tumor-associated macrophages polarization and DC maturation in the tumor microenvironment (TME). In addition, ROS also accelerates the cellular entry and endosomal escape of siRNA in TME. Finally, siRNAs targeting PLK1 or PD-L1 induce robust gene silencing in cancer cells, and downregulation of PD-L1 restores the immunological surveillance of T cells in TME. Therefore, the proposed LASNEO exhibit excellent antitumor effects by conscripting multiple types of immune cells. Considering that its manufacture is quite simple and controllable, LASNEO show compelling potential for clinical translational application.

Ionizable lipid-assisted efficient hepatic delivery of gene editing elements for oncotherapy.

CRISPR/Cas9-based gene editing has emerged as a powerful biotechnological tool, that relies on Cas9 protein and single guided RNA (sgRNA) to edit target DNA. However, the lack of safe and efficient delivery carrier is one of the crucial factors restricting its clinical transformation. Here, we report an ionizable lipid nanoparticle (iLP181, pKa = 6.43) based on iLY1809 lipid enabling robust gene editing in vitro and in vivo. The iLP181 effectively encapsulate psgPLK1, the best-performing plasmid expressing for both Cas9 protein and sgRNA targeting Polo-like kinase 1 (PLK1). The iLP181/psgPLK1 nanoformulation showed uniformity in size, regular nanostructure and nearly neutral zeta potential at pH 7.4. The nanoformulation effectively triggered editing of PLK1 gene with more than 30% efficiency in HepG2-Luc cells. iLP181/psgPLK1 significantly accumulated in the tumor for more than 5 days after a single intravenous injection. In addition, it also achieved excellent tumor growth suppression compared to other nucleic acid modalities such as siRNA, without inducing adverse effects to the main organs including the liver and kidneys. This study not only provides a clinically-applicable lipid nanocarrier for delivering CRISPR/Cas system (even other bioactive molecules), but also constitutes a potential cancer treatment regimen base on DNA editing of oncogenes.

A laminar flow electroporation system for efficient DNA and siRNA delivery.

By introducing a hydrodynamic mechanism into a microfluidics-based electroporation system, we developed a novel laminar flow electroporation system with high performance. The laminar buffer flow implemented in the system separated the cell suspension flow from the electrodes, thereby excluding many unfavorable effects due to electrode reaction during electroporation, such as hydrolysis, bubble formation, pH change, and heating. Compared to conventional microfluidic electroporation systems, these improvements significantly enhanced transfection efficiency and cell viability. Furthermore, successful electrotransfection of plasmid DNA and, more importantly, synthetic siRNA, was demonstrated in several hard-to-transfect cell types using this system.

Profiling of mismatch discrimination in RNAi enabled rational design of allele-specific siRNAs.

Silencing specificity is a critical issue in the therapeutic applications of siRNA, particularly in the treatment of single nucleotide polymorphism (SNP) diseases where discrimination against single nucleotide variation is demanded. However, no generally applicable guidelines are available for the design of such allele-specific siRNAs. In this paper, the issue was approached by using a reporter-based assay. With a panel of 20 siRNAs and 240 variously mismatched target reporters, we first demonstrated that the mismatches were discriminated in a position-dependent order, which was however independent of their sequence contexts using position 4th, 12th and 17th as examples. A general model was further built for mismatch discrimination at all positions using 230 additional reporter constructs specifically designed to contain mismatches distributed evenly along the target regions of different siRNAs. This model was successfully employed to design allele-specific siRNAs targeting disease-causing mutations of PIK3CA gene at two SNP sites. Furthermore, conformational distortion of siRNA-target duplex was observed to correlate with the compromise of gene silencing. In summary, these findings could dramatically simplify the design of allele-specific siRNAs and might also provide guide to increase the specificity of therapeutic siRNAs.

Development of A Decahedral Nanoenzyme Capable of Overcoming Hypoxia to Facilitate the Iodine-125 Radiosensitization of Esophageal Cancer.

Radioisotopes have long been leveraged for internal radiotherapy-mediated cancer treatment. However, such therapeutic approaches are associated with serious side effects, and their efficacy is limited by intratumoral hypoxia. Herein, we prepared a folic acid-decorated palladium decahedral platform capable of enhancing the radiotherapeutic efficacy of iodine-125 (125I) seed treatment. This decahedral nanoenzyme was able to target tumor regions and catalyze the conversion of intracellular H2O2 to O2, thereby alleviating hypoxia within the tumor microenvironment. In addition, palladium was hypoxia can be alleviated, on the other hand, palladium was able to enhance the radiotherapeutic energy deposition within tumor tissues. The results of this analysis indicated that synthesized decahedral constructs can efficiently target and modify the hypoxic tumor microenvironment while simultaneously enhancing radiation energy deposition therein. Relative to palladium nanodots, the prolonged in vivo circulation of these decahedral constructs better enabled them to facilitate sustained radiosensitization. Overall, the results of this study highlight a novel approach to improving the therapeutic utility of 125I seed interstitial implantation, thus underscoring an important direction for future clinical research.

A photo-triggerable aptamer nanoswitch for spatiotemporal controllable siRNA delivery.

A photo-triggerable aptamer nanoswitch was proposed for spatiotemporal regulation of siRNA delivery. Recognition between AS1411 and nucleolin was effectively blocked by a photo-labile complementary oligonucleotide, which could be reactivated with photo-irradiation, resulting in efficient tumor-targeted siRNA internalization and gene silencing in vitro and in vivo.

Efficient hepatic delivery and protein expression enabled by optimized mRNA and ionizable lipid nanoparticle.

mRNA is a novel class of therapeutic modality that holds great promise in vaccination, protein replacement therapy, cancer immunotherapy, immune cell engineering etc. However, optimization of mRNA molecules and efficient in vivo delivery are quite important but challenging for its broad application. Here we present an ionizable lipid nanoparticle (iLNP) based on iBL0713 lipid for in vitro and in vivo expression of desired proteins using codon-optimized mRNAs. mRNAs encoding luciferase or erythropoietin (EPO) were prepared by in vitro transcription and formulated with proposed iLNP, to form iLP171/mRNA formulations. It was revealed that both luciferase and EPO proteins were successfully expressed by human hepatocellular carcinoma cells and hepatocytes. The maximum amount of protein expression was found at 6 h post-administration. The expression efficiency of EPO with codon-optimized mRNA was significantly higher than that of unoptimized mRNA. Moreover, no toxicity or immunogenicity was observed for these mRNA formulations. Therefore, our study provides a useful and promising platform for mRNA therapeutic development.

Continuous Vector-free Gene Transfer with a Novel Microfluidic Chip and Nanoneedle Array.

BACKGROUND: Delivery of foreign cargoes into cells is of great value for bioengineering research and therapeutic applications. OBJECTIVE: In this study, we proposed and established a carrier-free gene delivery platform utilizing staggered herringbone channel and silicon nanoneedle array, to achieve high-throughput in vitro gene transfection. METHODS: With this microchip, fluidic micro vortices could be induced by the staggered-herringboneshaped grooves within the channel, which increased the contact frequency of the cells with the channel substrate. Transient disruptions on the cell membrane were well established by the nanoneedle array on the substrate. RESULT: Compared to the conventional nanoneedle-based delivery system, proposed microfluidic chip achieved flow-through treatment with high gene transfection efficiency (higher than 20%) and ideal cell viability (higher than 95%). CONCLUSION: It provides a continuous processing environment that can satisfy the transfection requirement of large amounts of biological molecules, showing high potential and promising prospect for both basic research and clinical application.

Parametric optimization of electric field strength for cancer electrochemotherapy on a chip-based model.

Electrochemotherapy (ECT), as one of the very few available treatments for cutaneous and subcutaneous tumors when surgery and radiotherapy are no longer available, requires applying a proper electric field to the tumor to realize electroporation-mediated cytotoxic drug delivery. It is impossible to exhaust all possible electrical parameters on patients to realize the optimal tradeoff between tumor suppression and adverse effects. To address this issue, this study provides a feasible solution by developing a four-leaf micro-electrode chip (F-MEC) in which the electric field was specially designed by linear distribution to cover all possible electric field strengths for ECT. Methods: We developed a F-MEC that provides a linearly varied electric field and a capacity for in situ observation of cell status. By culturing tumor cells on the F-MEC surface and in situ monitoring the cell responses to ECT drugs, the optimal electric field strength for any given cell type could be rapidly and accurately calculated in a few, or even only one, simple assay. Results: Using this chip, we monitored MCF-7 and A315 cell responses to ECT and determined the optimum ECT voltage. More importantly, we successfully verified that the in vitro determined voltage coincided with the optimal value for in vivo ECT in mice. Conclusion: In this proof-of-concept study, the in vivo tumor suppression assays proved that the optimal parameters acquired from in vitro F-MEC assay could be used for in vivo ECT.

Efficient delivery of nucleic acid molecules into skin by combined use of microneedle roller and flexible interdigitated electroporation array.

Rationale: Delivery of nucleic acid molecules into skin remains a main obstacle for various types of gene therapy or vaccine applications. Here we propose a novel electroporation approach via combined use of a microneedle roller and a flexible interdigitated electroporation array (FIEA) for efficient delivery of DNA and siRNA into mouse skin. Methods: Using micromachining technology, closely spaced gold electrodes were made on a pliable parylene substrate to form a patch-like electroporation array, which enabled close surface contact between the skin and electrodes. Pre-penetration of the skin with a microneedle roller resulted in the formation of microchannels in the skin, which played a role as liquid electrodes in the skin and provided a uniform and deep electric field in the tissue when pulse stimulation was applied by FIEA. Results: Using this proposed method, gene (RFP) expression and siRNA transfection were successfully achieved in normal mice skin. Anti-SCD1 siRNA electroporated via this method mediated significant gene silencing in the skin. Moreover, electroporation assisted by the microneedle roller showed significant advantages over treatment with FIEA alone. This allowed nucleic acid transportation at low voltage, with ideal safety outcomes. Principal conclusions: Hence, the proposed electroporation approach in this study constitutes a novel way for delivering siRNA and DNA, and even other nucleic acid molecules, to mouse skin in vivo, potentially supporting clinical application in the treatment of skin diseases or intradermal/subcutaneous vaccination.

The study of relationships between pKa value and siRNA delivery efficiency based on tri-block copolymers.

Tri-block copolymers have exhibited great potentials in small interfering RNA (siRNA) therapeutics. To reveal structure-activity relationships, we here synthesized a series of tri-block copolymers with different hydrophobic segments, PEG-PAMA-P(C6Ax-C7Ay-DPAz-DBAm) (EAAS) and PEG-PDAMAEMA-P(C6Ax-C7Ay-DPAz-DBAm) (EDAS), termed from EAASa to EAASh and EDASa to EDASh, with pKa ranging from 5.2 to 7.0. Our data showed that the better gene silencing efficiency was located in pKa of 5.8-6.2, which was contributed from higher endosomal escape observed with confocal images and hemolysis assay. EAASc, the leader polymer, showed excellent gene knockdown at w/w ratio of 14.5 on HepG2 (89.94%), MDA-MB-231 (92.45%), 293A (83.06%), and Hela cells (80.27%), all better than lipofectamine 2000. Besides, EAASc mediated effective gene silencing in tumor when performed peritumoral injection. This work found out that polymers with pKa ranging from 5.8 to 6.2 were efficient in siRNA delivery, which provided an optimization strategy for siRNA delivery systems, especially for tri-block copolymers.