Discovery of G-quadruplex-forming sequences in SARS-CoV-2.

The outbreak caused by the novel coronavirus SARS-CoV-2 has been declared a global health emergency. G-quadruplex structures in genomes have long been considered essential for regulating a number of biological processes in a plethora of organisms. We have analyzed and identified 25 four contiguous GG runs (G2NxG2NyG2NzG2) in the SARS-CoV-2 RNA genome, suggesting putative G-quadruplex-forming sequences (PQSs). Detailed analysis of SARS-CoV-2 PQSs revealed their locations in the open reading frames of ORF1 ab, spike (S), ORF3a, membrane (M) and nucleocapsid (N) genes. Identical PQSs were also found in the other members of the Coronaviridae family. The top-ranked PQSs at positions 13385 and 24268 were confirmed to form RNA G-quadruplex structures in vitro by multiple spectroscopic assays. Furthermore, their direct interactions with viral helicase (nsp13) were determined by microscale thermophoresis. Molecular docking model suggests that nsp13 distorts the G-quadruplex structure by allowing the guanine bases to be flipped away from the guanine quartet planes. Targeting viral helicase and G-quadruplex structure represents an attractive approach for potentially inhibiting the SARS-CoV-2 virus.

Nucleic acid therapy in pediatric cancer.

The overall survival, progress free survival, and life quality of cancer patients have improved due to the advance in minimally invasive surgery, precision radiotherapy, and various combined chemotherapy in the last decade. Furthermore, the discovery of new types of therapeutics, such as immune checkpoint inhibitors and immune cell therapies have facilitated both patients and doctors to fight with cancers. Moreover, in the context of the development in biocompatible and cell type targeting nano-carriers as well as nucleic acid-based drugs for initiating and enhancing the anti-tumor response have come to the age. The treatment paradigms utilization of nucleic acids, including short interfering RNA (siRNA), antisense oligonucleotides (ASO), and messenger RNA (mRNA), can target specific protein expression to achieve the therapeutic effects. Over ten nucleic acid therapeutics have been approved by the FDA and EMA in rare diseases and genetic diseases as well as dozens of registered clinical trails for varies cancers. Though generally less dangerous of pediatric cancers than adult cancers was observed during the past decades, yet pediatric cancers accounted for a significant proportion of child deaths which hurt those family very deeply. Therefore, it is necessary to pay more attention for improving the treatment of pediatric cancer and discovering new nucleic acid therapeutics which may help to improve the therapeutic effect and prognoses in turns to ameliorate the survival period and quality of life for children patient. In this review, we focus on the nucleic acid therapy in pediatric cancers.

A PD-L1 Aptamer Selected by Loss-Gain Cell-SELEX Conjugated with Paclitaxel for Treating Triple-Negative Breast Cancer.

BACKGROUND The clinical challenges of triple-negative breast cancer (TNBC) includes the lack of targeted therapy and chemoresistance. TNBC has relatively high PD-L1 expression, and PD-L1 antibody in combination with nab-paclitaxel has been approved by FDA for TNBC treatment. Aptamers, also termed chemical antibody, are widely used in targeted drug delivery. The present study aimed to select a DNA aptamer that could specifically bind and deliver drugs to TNBC cells. MATERIAL AND METHODS An innovative loss-gain cell-SELEX strategy was used to select DNA aptamer for PD-L1 protein. Construction of PD-L1 knock-out and over-expression MDA-MB-231 cell lines were conducted through transfection and confirmed by western blot and flow cytometry. Confocal microscopy and flow cytometry were used to analyze the binding ability of aptamer with TNBC cells. The cytotoxicity of aptamer-paclitaxel complex against TNBC cells was evaluated by Cell Counting Kit-8 assay. The reactivation of the T cell function by aptamer was measured by IL-2 enzyme-linked immunosorbent assay after T cells co-cultured with tumor cells. RESULTS In this work, using an innovative loss-gain cell-SELEX strategy, we screened a PD-L1-targeting aptamer. PD-L1 aptamer selectively bound to PD-L1 over-expressed TNBC cells with a dissociation constant in the nanomolar range. PD-L1 aptamer could also inhibit PD-1/PD-L1 interaction and restore the function of T cells. Moreover, we developed a PD-L1 aptamer-paclitaxel conjugate which showed improved cellular uptake and anti-proliferation efficacy in PD-L1 over-expressed TNBC cells. CONCLUSIONS In summary, these findings suggest that the selected PD-L1 aptamer might have potential implication in immune modulation and targeted therapy against TNBC.

Potential Diagnostic and Therapeutic Applications of Oligonucleotide Aptamers in Breast Cancer.

Breast cancer is one of the most common causes of cancer related deaths in women. Currently, with the development of early detection, increased social awareness and kinds of treatment options, survival rate has improved in nearly every type of breast cancer patients. However, about one third patients still have increased chances of recurrence within five years and the five-year relative survival rate in patients with metastasis is less than 30%. Breast cancer contains multiple subtypes. Each subtype could cause distinct clinical outcomes and systemic interventions. Thereby, new targeted therapies are of particular importance to solve this major clinical problem. Aptamers, often termed "chemical antibodies", are functionally similar to antibodies and have demonstrated their superiority of recognizing target with high selectivity, affinity and stability. With these intrinsic properties, aptamers have been widely studied in cancer biology and some are in clinical trials. In this review, we will firstly discuss about the global impacts and mechanisms of breast cancer, then briefly highlight applications of aptamers that have been developed for breast cancer and finally summarize various challenges in clinical translation of aptamers.

Development of Cell-SELEX Technology and Its Application in Cancer Diagnosis and Therapy.

SELEX (systematic evolution of ligands by exponential enrichment) is a process involving the progressive isolation of high selective ssDNA/RNA from a combinatorial single-stranded oligonucleotide library through repeated rounds of binding, partitioning and amplification. SELEX-derived single-stranded DNA/RNA molecules, called aptamers, are selected against a wide range of targets, including purified proteins, live cells, tissues, microorganisms, small molecules and so on. With the development of SELEX technology over the last two decades, various modified SELEX processes have been arisen. A majority of aptamers are selected against purified proteins through traditional SELEX. Unfortunately, more and more evidence showed aptamers selected against purified membrane proteins failed to recognize their targets in live cells. Cell-SELEX could develop aptamers against a particular target cell line to discriminate this cell line from others. Therefore, cell-SELEX has been widely used to select aptamers for the application of both diagnosis and therapy of various diseases, especially for cancer. In this review, the advantages and limitations of cell-SELEX and SELEX against purified protein will be compared. Various modified cell-SELEX techniques will be summarized, and application of cell-SELEX in cancer diagnosis and therapy will be discussed.

A Mini-Review for Cancer Immunotherapy: Molecular Understanding of PD-1/PD-L1 Pathway & Translational Blockade of Immune Checkpoints.

Interference of the binding of programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) has become a new inspiring immunotherapy for resisting cancers. To date, the FDA has approved two PD-1 monoclonal antibody drugs against cancer as well as a monoclonal antibody for PD-L1. More PD-1 and PD-L1 monoclonal antibody drugs are on their way in clinical trials. In this review, we focused on the mechanism of the PD-1/PD-L1 signaling pathway and the monoclonal antibodies (mAbs) against PD-1 and PD-L1, which were approved by the FDA or are still in clinical trials. And also presented is the prospect of the PD-1/PD-L1 immune checkpoint blockade in the next generation of immunotherapy.

Aptamers for nanobodies: A nontoxic alternative to toxic ochratoxin A in immunoassays.

To measure toxins using immunoassays, hazardous toxin standards need to be added for quantification. To solve this problem, we propose to use aptamers as competitors to replace toxin standards. In this work, aptamers specific for ochratoxin A (OTA) nanobodies were selected using a DNA library containing a 36 nucleotide random region. The obtained sequences were highly aligned and the best competitor was identified to be a sequence named apt2-OT based on an indirect competitive enzyme-linked immunosorbent assay (ELISA). The Kd of apt2-OT was measured to be 2.86 µM using local surface plasmon resonance spectroscopy. The optimal apt2-OT was identified to substitute the OTA standard with a concentration needed for 50% inhibition of binding (IC50) of 3.26 µM based on a nontoxic direct competitive ELISA. The equivalence relationship between the aptamer and OTA was established in a flour sample, and a recovery experiment was performed. The detection limit for this method was 0.23 ng/mL, with a linear range from 0.25 to 10.50 ng/mL. The recovery rate was 97.5%-115.5%. This study provides a low-cost, rapid and environmentally friendly alternative to the development of immunoassays for toxins.

Key elements of the human bocavirus type 1 (HBoV1) promoter and its trans-activation by NS1 protein.

BACKGROUND: Human bocavirus (HBoV), a parvovirus, is suspected to be an etiologic agent of respiratory disease and gastrointestinal disease in humans. All mRNAs of HBoV1 are transcribed from a single promoter. METHODS: In this study, we constructed EGFP and luciferase reporter gene vectors under the control of the HBoV1 full promoter (nt 1-252) and its mutated variants, respectively. Fluorescence microscopy was used to observe expression activities of the EGFP. Dual-luciferase reporter vectors were employed in order to evaluate critical promoter elements and the effect of NS1 protein on promoter activity. RESULTS: The HBoV1 promoter activity was about 2.2-fold and 1.9-fold higher than that of the CMV promoter in 293 T and HeLa cells, respectively. The putative transcription factor binding region of the promoter was identified to be located between nt 96 and nt 145. Mutations introduced in the CAAT box of the HBoV1 promoter reduced promoter activity by 34%, whereas nucleotide substitutions in the TATA box had no effect on promoter activity. The HBoV1 promoter activities in 293 T and HeLa cells, in the presence of NS1 protein, were 2- to 2.5-fold higher than those in the absence of NS1 protein. CONCLUSION: The HBoV1 promoter was highly active in 293 T and HeLa cell lines, and the sequence from nt 96 to nt 145 was critical for the activity of HBoV1 promoter. The CAAT box, in contrast to the TATA-box, was important for optimum promoter activity. In addition, the transcriptional activity of this promoter could be trans-activated by the viral nonstructural protein NS1 in these cells.

[Modulation of cellular transcriptional factors by human bocavirus 1 nonstructural protein NP1].

OBJECTIVE: We studied the regulating effect of human bocavirus 1 (HBoV1) nonstructural protein NP1 on the activity of cellular transcription factors and the expression of inflammatory cytokine TNF-alpha and IL-6. METHODS: The modulation of NP1 was measured by the Dual Luciferase Reporter Assay System and the expression of cytokines TNF-alpha and IL-6 was detected by ELISA and Real-time PCR. The luciferase based mammalian two-hybrid system was used to analyze whether the function of NP1 protein aroused from oligomerization. RESULTS: The transcription factors AP-1, STAT3 and STAT1 but not NF-kappaB were up-regulated by NP1, which was evidenced by approximately 2-3-fold increase of the luciferase activity compared to the control vector. Moreover, NP1 increased the TNF-alpha mRNA expression, but not contributed to cytokine IL-6 secretion. We also found that the self-interaction did not exist when NPI was solely expressed in 293T cells. CONCLUSION: This study demonstrates for the first time that NP1 may play important roles in activation of transcription factors and up-regulation of inflammatory cytokines expression, suggesting that NP1 be involved in HBoV1 pathogenesis.

Progress in building clinically relevant patient-derived tumor xenograft models for cancer research.

Patient-derived tumor xenograft (PDX) models, a method involving the surgical extraction of tumor tissues from cancer patients and subsequent transplantation into immunodeficient mice, have emerged as a pivotal approach in translational research, particularly in advancing precision medicine. As the first stage of PDX development, the patient-derived orthotopic xenograft (PDOX) models implant tumor tissue in mice in the corresponding anatomical locations of the patient. The PDOX models have several advantages, including high fidelity to the original tumor, heightened drug sensitivity, and an elevated rate of successful transplantation. However, the PDOX models present significant challenges, requiring advanced surgical techniques and resource-intensive imaging technologies, which limit its application. And then, the humanized mouse models, as well as the zebrafish models, were developed. Humanized mouse models contain a human immune environment resembling the tumor and immune system interplay. The humanized mouse models are a hot topic in PDX model research. Regarding zebrafish patient-derived tumor xenografts (zPDX) and patient-derived organoids (PDO) as promising models for studying cancer and drug discovery, zPDX models are used to transplant tumors into zebrafish as novel personalized medical animal models with the advantage of reducing patient waiting time. PDO models provide a cost-effective approach for drug testing that replicates the in vivo environment and preserves important tumor-related information for patients. The present review highlights the functional characteristics of each new phase of PDX and provides insights into the challenges and prospective developments in this rapidly evolving field.

Proteomics reveals that cell density could affect the efficacy of drug treatment.

In vitro cell biology study plays a fundamental role in biological and drug development research, but the repeatability and accuracy of cell studies remain to be low. Various uncertainties during the cell culture process could introduce bias into drug research. In this study, we evaluate the potential effects and underlying mechanisms induced by cell number differences in the cell seeding process. Normally, drug experiments are initiated 24 h after cell seeding, and the difference in the cell number at the time of inoculation leads to the difference in cell confluence (cell density) when drug research is conducted. While cell confluence is closely related to intercellular communication, surface protein interaction, cell autocrine as well as paracrine protein expression of cells, it might have a potential impact on the effect of biological studies such as drug treatment. This study used proteomics technology to comprehensively explore the different protein expression patterns between cells with different confluences. Due to the high sensitivity and high throughput of liquid chromatography-mass spectrometry (LC-MS/MS) detection, it was hired to evaluate the protein expression differences of Hep3B cells with 3 different confluences (30%, 50%, and 70%). The differential expressed proteins were analyzed by the Reactome pathway and the Gene Ontology (GO) pathway. Significant differences were identified across three confluences in terms of the number of proteins identified, the protein expression pattern, and the expression level of certain KEGG pathways. We found that those proteins involved in the cell cycle pathway were differently expressed: the higher the cell confluence, the higher these proteins expressed. A cell cycle inhibitor palbociclib was selected to further verify this observation. Palbociclib in the same dose was applied to cells with different confluence, the results indicated that the growth inhibition effect of palbociclib increases along with the increasing trend of cell cycle protein expression. The result indicated that cell density did influence the effect of drug treatment. Furthermore, three other drugs, cisplatin, paclitaxel, and imatinib, were used to treat the three liver cancer cell lines Hep3B, SUN387, and MHCC97, and a similar observation was obtained that drug effect would be different when the cell confluences were different. Therefore, selecting an appropriate number of cells for plating is vitally important at the beginning of a drug study.

Investigation on acquired palbociclib resistance by LC-MS based multi-omics analysis.

Palbociclib is a specific CDK4/6 inhibitor that has been widely applied in multiple types of tumors. Different from cytotoxic drugs, the anticancer mechanism of palbociclib mainly depends on cell cycle inhibition. Therefore, the resistance mechanism is different. For clinical cancer patients, drug resistance is inevitable for almost all cancer therapies including palbociclib. We have trained palbociclib resistant cells in vitro to simulate the clinical situation and applied LC-MS multi-omics analysis methods including proteomic, metabolomic, and glycoproteomic techniques, to deeply understand the underly mechanism behind the resistance. As a result of proteomic analysis, the resistant cells were found to rely on altered metabolic pathways to keep proliferation. Metabolic processes related to carbohydrates, lipids, DNA, cellular proteins, glucose, and amino acids were observed to be upregulated. Most dramatically, the protein expressions of COX-1 and NDUFB8 have been detected to be significantly overexpressed by proteomic analysis. When a COX-1 inhibitor was hired to combine with palbociclib, a synergistic effect could be obtained, suggesting the altered COX-1 involved metabolic pathway is an important reason for the acquired palbociclib resistance. The KEGG pathway of N-glycan biosynthesis was identified through metabolomics analysis. N-glycoproteomic analysis was therefore included and the global glycosylation was found to be elevated in the palbociclib-resistant cells. Moreover, integration analysis of glycoproteomic data allowed us to detect a lot more proteins that have been glycosylated with low abundances, these proteins were considered to be overwhelmed by those highly abundant proteins during regular proteomic LC-MS detection. These low-abundant proteins are mainly involved in the cellular biology processes of cell migration, the regulation of chemotaxis, as well as the glycoprotein metabolic process which offered us great more details on the roles played by N-glycosylation in drug resistance. Our result also verified that N-glycosylation inhibitors could enhance the cell growth inhibition of palbociclib in resistant cells. The high efficiency of the integrated multi-omics analysis workflow in discovering drug resistance mechanisms paves a new way for drug development. With a clear understanding of the resistance mechanism, new drug targets and drug combinations could be designed to resensitize the resistant tumors.

Protein post-translational modifications: A key factor in colorectal cancer resistance mechanisms.

Colorectal cancer (CRC) is one of the leading causes of cancer-related death. Despite advances in treatment, drug resistance remains a critical impediment. Post-translational modifications (PTMs) regulate protein stability, localization, and activity, impacting vital cellular processes. Recent research has highlighted the essential role of PTMs in the development of CRC resistance. This review summarizes recent advancements in understanding PTMs' roles in CRC resistance, focusing on the latest discoveries. We discuss the functional impact of PTMs on signaling pathways and molecules involved in CRC resistance, progress in drug development, and potential therapeutic targets. We also summarize the primary enrichment methods for PTMs. Finally, we discuss current challenges and future directions, including the need for more comprehensive PTM analysis methods and PTM-targeted therapies. This review identifies potential therapeutic interventions for addressing medication resistance in CRC, proposes prospective therapeutic options, and gives an overview of the function of PTMs in CRC resistance.

The application of Aptamer in biomarker discovery.

Biomarkers are detectable molecules that can reflect specific physiological states of cells, organs, and organisms and therefore be regarded as indicators for specific diseases. And the discovery of biomarkers plays an essential role in cancer management from the initial diagnosis to the final treatment regime. Practically, reliable clinical biomarkers are still limited, restricted by the suboptimal methods in biomarker discovery. Nucleic acid aptamers nowadays could be used as a powerful tool in the discovery of protein biomarkers. Nucleic acid aptamers are single-strand oligonucleotides that can specifically bind to various targets with high affinity. As artificial ssDNA or RNA, aptamers possess unique advantages compared to conventional antibodies. They can be flexible in design, low immunogenicity, relative chemical/thermos stability, as well as modifying convenience. Several SELEX (Systematic Evolution of Ligands by Exponential Enrichment) based methods have been generated recently to construct aptamers for discovering new biomarkers in different cell locations. Secretome SELEX-based aptamers selection can facilitate the identification of secreted protein biomarkers. The aptamers developed by cell-SELEX can be used to unveil those biomarkers presented on the cell surface. The aptamers from tissue-SELEX could target intracellular biomarkers. And as a multiplexed protein biomarker detection technology, aptamer-based SOMAScan can analyze thousands of proteins in a single run. In this review, we will introduce the principle and workflow of variations of SELEX-based methods, including secretome SELEX, ADAPT, Cell-SELEX and tissue SELEX. Another powerful proteome analyzing tool, SOMAScan, will also be covered. In the second half of this review, how these methods accelerate biomarker discovery in various diseases, including cardiovascular diseases, cancer and neurodegenerative diseases, will be discussed.

Aptamer nucleotide analog drug conjugates in the targeting therapy of cancers.

Aptamers are short single-strand oligonucleotides that can form secondary and tertiary structures, fitting targets with high affinity and specificity. They are so-called "chemical antibodies" and can target specific biomarkers in both diagnostic and therapeutic applications. Systematic evolution of ligands by exponential enrichment (SELEX) is usually used for the enrichment and selection of aptamers, and the targets could be metal ions, small molecules, nucleotides, proteins, cells, or even tissues or organs. Due to the high specificity and distinctive binding affinity of aptamers, aptamer-drug conjugates (ApDCs) have demonstrated their potential role in drug delivery for cancer-targeting therapies. Compared with antibodies which are produced by a cell-based bioreactor, aptamers are chemically synthesized molecules that can be easily conjugated to drugs and modified; however, the conventional ApDCs conjugate the aptamer with an active drug using a linker which may add more concerns to the stability of the ApDC, the drug-releasing efficiency, and the drug-loading capacity. The function of aptamer in conventional ApDC is just as a targeting moiety which could not fully perform the advantages of aptamers. To address these drawbacks, scientists have started using active nucleotide analogs as the cargoes of ApDCs, such as clofarabine, ara-guanosine, gemcitabine, and floxuridine, to replace all or part of the natural nucleotides in aptamer sequences. In turn, these new types of ApDCs, aptamer nucleotide analog drug conjugates, show the strength for targeting efficacy but avoid the complex drug linker designation and improve the synthetic efficiency. More importantly, these classic nucleotide analog drugs have been used for many years, and aptamer nucleotide analog drug conjugates would not increase any unknown druggability risk but improve the target tumor accumulation. In this review, we mainly summarized aptamer-conjugated nucleotide analog drugs in cancer-targeting therapies.

Engineering Alendronate-Composed Iron Nanochelator for Efficient Peritoneal Carcinomatosis Treatment.

Iron is an essential element for various cellular metabolism. Cancer cells also have high requirement of iron in their proliferation, invasion, and metastasis processes. Alendronate (ALN), a kind of FDA-approved bisphosphonates with metal-chelating capability, is initially certified to selectively bind to intracellular Fe3+ theoretically and experimentally in this study. Hence, CaALN iron nanochelator is rationally designed to kill cancer cells by synergism of Fe-depletion and calcium accumulation. In vitro experiments and RNA sequencing analysis indicate that CaALN nanomedicine inhibits the proliferation of cancer cells by depleting Fe, interfering with DNA replication, and triggering intracellular reactive oxygen species (ROS). Meanwhile, released Ca2+ and ROS mutually promote and induce damage of cellular macromolecules, which leads to mitochondrial apoptosis of cancer cells. In an intraperitoneal disseminated mouse model with the human ovarian cancer cells SKOV3, CaALN nanoparticles selectively accumulate in tumor tissues and result in significant retardation of tumor growth and ascites formation. The mean survival time of SKOV3-bearing mice in treatment group is prolonged from 33 to 90 d. These results indicate that the alendronate-originated iron chelator can serve as an efficient strategy for the treatment of peritoneal carcinomatosis.

Targeting loop3 of sclerostin preserves its cardiovascular protective action and promotes bone formation.

Sclerostin negatively regulates bone formation by antagonizing Wnt signalling. An antibody targeting sclerostin for the treatment of postmenopausal osteoporosis was approved by the U.S. Food and Drug Administration, with a boxed warning for cardiovascular risk. Here we demonstrate that sclerostin participates in protecting cardiovascular system and inhibiting bone formation via different loops. Loop3 deficiency by genetic truncation could maintain sclerostin's protective effect on the cardiovascular system while attenuating its inhibitory effect on bone formation. We identify an aptamer, named aptscl56, which specifically targets sclerostin loop3 and use a modified aptscl56 version, called Apc001PE, as specific in vivo pharmacologic tool to validate the above effect of loop3. Apc001PE has no effect on aortic aneurysm and atherosclerotic development in ApoE-/- mice and hSOSTki.ApoE-/- mice with angiotensin II infusion. Apc001PE can promote bone formation in hSOSTki mice and ovariectomy-induced osteoporotic rats. In summary, sclerostin loop3 cannot participate in protecting the cardiovascular system, but participates in inhibiting bone formation.

Nondestructive quantification of single-cell nuclear and cytoplasmic mechanical properties based on large whole-cell deformation.

The mechanical properties of cell nuclei have been recognized to reflect and modulate important cell behaviors such as migration and cancer cell malignant tendency. However, these nuclear properties are difficult to characterize accurately using conventional measurement methods, which are often based on probing or deforming local sites over a nuclear region. The corresponding results are sensitive to the measurement position, and they are not decoupled from the cytoplasmic properties. Microfluidics is widely recognized as a promising technique for bioassay and phenotyping. In this report, we develop a simple and nondestructive approach for the single-cell quantification of nuclear elasticity based on microfluidics by considering different deformation levels of a live cell captured along a confining microchannel. We apply two inlet pressure levels to drive the flow of human nasopharyngeal epithelial cells (NP460) and human nasopharyngeal cancerous cells (NPC43) into the microchannels. A model considering the essential intracellular components (cytoplasm and nucleus) for describing the mechanics of a cell deforming along the confining microchannel is used to back-calculate the cytoplasmic and nuclear properties. On the other hand, we also apply a widely used chemical nucleus extraction technique to examine its possible effects (e.g., reduced nuclear modulus and reduced lamin A/C expression). To determine if the decoupled nuclear properties are representative of cancer-related attributes, we classify the NP460 and NPC43 cells using the decoupled physical properties as classification factors, resulting in an accuracy of 79.1% and a cell-type specificity exceeding 74%. It should be mentioned that the cells can be recollected at the device outlet after the nondestructive measurement. Hence, the reported cell elasticity measurement can be combined with downstream genetic and biochemical assays for general cell research and cancer diagnostic applications.

Therapeutic evaluation of palbociclib and its compatibility with other chemotherapies for primary and recurrent nasopharyngeal carcinoma.

BACKGROUND: Recent genomic analyses revealed that druggable molecule targets were only detectable in approximately 6% of patients with nasopharyngeal carcinoma (NPC). However, a dependency on dysregulated CDK4/6-cyclinD1 pathway signaling is an essential event in the pathogenesis of NPC. In this study, we aimed to evaluate the therapeutic efficacy of a specific CDK4/6 inhibitor, palbociclib, and its compatibility with other chemotherapeutic drugs for the treatment of NPC by using newly established xenograft models and cell lines derived from primary, recurrent, and metastatic NPC. METHODS: We evaluated the efficacies of palbociclib monotherapy and concurrent treatment with palbociclib and cisplatin or suberanilohydroxamic acid (SAHA) in NPC cell lines and xenograft models. RNA sequencing was then used to profile the drug response-related pathways. Palbociclib-resistant NPC cell lines were established to determine the potential use of cisplatin as a second-line treatment after the development of palbociclib resistance. We further examined the efficacy of palbociclib treatment against cisplatin-resistant NPC cells. RESULTS: In NPC cells, palbociclib monotherapy was confirmed to induce cell cycle arrest in the G1 phase in vitro. Palbociclib monotherapy also had significant inhibitory effects in all six tested NPC tumor models in vivo, as indicated by substantial reductions in the total tumor volumes and in Ki-67 proliferation marker expression. In NPC cells, concurrent palbociclib treatment mitigated the cytotoxic effect of cisplatin in vitro. Notably, concurrent treatment with palbociclib and SAHA synergistically promoted NPC cell death both in vitro and in vivo. This combination also further inhibited tumor growth by inducing autophagy-associated cell death. NPC cell lines with induced palbociclib or cisplatin resistance remained sensitive to treatment with cisplatin or palbociclib, respectively. CONCLUSIONS: Our study findings provide essential support for the use of palbociclib as an alternative therapy for NPC and increase awareness of the effective timing of palbociclib administration with other chemotherapeutic drugs. Our results provide a foundation for the design of first-in-human clinical trials of palbociclib regimens in patients with NPC.

Elasticity-Modulated Microbeads for Classification of Floating Normal and Cancer Cells Using Confining Microchannels.

Engineered microbeads have a wide range of applications in cancer research including identification, characterization, and sorting of cancer cells. In particular, the microbead-based cancer identification techniques are mainly based on the known genetic or biochemical biomarkers; and detection specificity is yet to be improved. On the other hand, it has been discovered that biomechanical properties of cancer cells such as cell-body elasticity can be considered as cancer biomarkers. Here, we report a straightforward microfluidic classification scheme for floating/dissociated normal and cancer epithelial cells using a confining microchannel device together with calcium-alginate hydrogel microbeads. The hydrogel microbeads are generated based on the microfluidic emulsion process, with characterization on the process parameters (e.g., liquid driving pressure and cross-linking duration) in order to specify the resultant bead diameter and elasticity. These engineered microbeads are first mixed with a cell mixture of dissociated human nasopharyngeal epithelial cells (NP460) and nasopharyngeal carcinoma cells (NPC43). The cell elasticity can then be reflected from the locations of captured cells in the device. Experiments further demonstrate that the cell classification has a success rate of >95%. Furthermore, we performed the microbead-based cell classification on a whole blood sample containing floating human breast epithelial cells (MCF-10A) and breast cancer epithelial cells (MDA-MB-231) with a success rate of >75%, revealing its directly applicability to identification of circulating tumor cells in human blood. Together, this research demonstrates a new application of engineered hydrogel microbeads for classification of cells based on their mechanical properties.