Microfluidic-based preparation of artificial antigen-presenting gel droplets for integrated and minimalistic adoptive cell therapy strategies.

Adoptive T-cell transfer for cancer therapy is limited by the inefficiency ofin vitroT-cell expansion and the ability ofin vivoT-cells to infiltrate tumors. The construction of multifunctional artificial antigen-presenting cells is a promising but challenging approach to achieve this goal. In this study, a multifunctional artificial antigen-presenting gel droplet (AAPGD) was designed. Its surface provides regulated T-cell receptor (TCR) stimulation and co-stimulation signals and is capable of slow release of mitogenic cytokines and collagen mimetic peptide. The highly uniform AAPGD are generated by a facile method based on standard droplet microfluidic devices. The results of the study indicate that, T-cell proliferatedin vitroutilizing AAPGD have a fast rate and high activity. AAPGD increased the proportion ofin vitroproliferating T cells low differentiation and specificity. The starting number of AAPGDs and the quality ratio of TCR-stimulated and co-stimulated signals on the surface have a large impact on the rapid proliferation of low-differentiated T cellsin vitro. During reinfusion therapy, AAPGD also enhanced T-cell infiltration into the tumor site. In experiments using AAPGD for adoptive T cell therapy in melanoma mice, tumor growth was inhibited, eliciting a potent cytotoxic T-lymphocyte immune response and improving mouse survival. In conclusion, AAPGD promotes rapid low-differentiation proliferation of T cellsin vitroand enhances T cell infiltration of tumorsin vivo. It simplifies the preparation steps of adoptive cell therapy, improves the therapeutic effect, and provides a new pathway for overdosing T cells to treat solid tumors.

A review for the impacts of circadian disturbance on urological cancers.

Circadian rhythm is an internal timing system and harmonizes a variety of cellular, behavioral, and physiological processes to daily environment. Circadian disturbance caused by altered life style or disrupted sleep patterns inevitably contributes to various disorders. As the rapidly increased cancer occurrences and subsequent tremendous financial burdens, more researches focus on reducing the morbidity rather than treating it. Recently, many epidemiologic studies demonstrated that circadian disturbance was tightly related to the occurrence and development of cancers. For urinary system, numerous clinical researches observed the incidence and progress of prostate cancer were influenced by nightshift work, sleep duration, chronotypes, light exposure, and meal timing, this was also proved by many genetic and fundamental findings. Although the epidemiological studies regarding the relationship between circadian disturbance and kidney/bladder cancers were relative limited, some basic researches still claimed circadian disruption was closely correlated to these two cancers. The role of circadian chemotherapy on cancers of prostate, kidney, and bladder were also explored, however, it has not been regularly recommended considering the limited evidence and poor standard protocols. Finally, the researches for the impacts of circadian disturbance on cancers of adrenal gland, penis, testis were not found at present. In general, a better understanding the relationship between circadian disturbance and urological cancers might help to provide more scientific work schedules and rational lifestyles which finally saving health resource by reducing urological tumorigenesis, however, the underlying mechanisms are complex which need further exploration.

Circadian disturbance induces erectile dysfunction by impairing endothelial function.

ABSTRACT: In order to explore the impact of circadian disturbance on erectile function, we randomly divided 24 adult male rats into groups of control (light on at 8:00 a.m. and off at 8:00 p.m.), dark/dark (DD; constant dark), light/light (LL; constant light), and shift dark/light (DL; light off at 8:00 a.m. and on at 8:00 p.m.). Four weeks later, erectile function was measured and corpora cavernosa were harvested for analysis. The maximum intracavernous pressure (mICP) and mICP/mean arterial pressure (MAP) ratio in the DD, LL, and DL groups were significantly lower than that in the control group. The LL and DL groups showed significantly attenuated endothelial nitric oxide synthase (eNOS), while DD, LL, and DL showed reduced neuronal nitric oxide synthase (nNOS) at both mRNA and protein levels. The production of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) was inhibited by altered light/dark cycles to varying degrees. Circadian disturbance impaired endothelial function and contributed to erectile dysfunction. For the core circadian elements, mRNA expression of circadian locomotor output cycles kaput (Clock) and brain/muscle aryl-hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) was elevated in the DL group, but their protein expression was not significantly changed. DD, LL, and DL increased period 1 (Per1) and Per3 levels, while LL and DL increased PER1 levels. No significant difference was found for Per2 levels, and PER2 and PER3 concentrations were not significantly changed. Moreover, LL and DL significantly increased cryptochrome-1 (CRY1) and CRY2 at both mRNA and protein levels. The altered light/dark rat model showed that circadian disturbance contributed to erectile dysfunction probably by impairing endothelial function. Meanwhile, the core circadian elements were detected in the corpora cavernosa, but these were disrupted. However, which circadian element regulates erectile function and how it works need further analysis.

HCG supplement did not accelerate tunica albuginea remodeling to facilitate penile growth.

Penile size is closely concerned and short penis contributes serious sexual dysfunction and tremendous psychological problems to couples. Androgen is essential for penile development and testosterone replacement is recommended to patients with micropenis. We previously proved that inhibiting activity of lysyl oxidase (Anti-lysyl oxidase, Anti-LOX) combined with vacuum erectile device (VED) lengthened penis by remodeling tunica albuginea. We thus explored whether HCG supplement could accelerate tunica albuginea remodeling (induced by Anti-LOX + VED) to promote penile growth. Forty-two SD male rats (4 weeks old) were purchased and divided into 7 groups: control, Anti-LOX, HCG, VED (with a negative aspirated pressure of - 300 mmHg), Anti-LOX + VED, HCG + VED, and Anti-LOX + HCG + VED. After an intervention for 4 weeks, all rats' penile length, exposed penile length, and erectile function were measured. Serum samples were collected to detect hormone levels and penile corpus cavernosum were harvested for histo-pathological analysis. All intervention groups showed significantly longer penis than controlled rats. Anti-LOX sharply increased penile length and exposed length by 15% and 9% respectively, this lengthening effect was more obvious in Anti-LOX + VED group (26% and 19%, respectively). Although HCG promoted penile length by 8%, this effect was slight for exposed length (3%). Moreover, Anti-LOX + HCG + VED dramatically increased penile length and exposed length by 22% and 18%, respectively, which was similar with that in Anti-LOX + VED (26% and 19%, respectively). HCG dramatically stimulated testosterone and dihydrotestosterone secretions than control group, whether with or without Anti-LOX and VED; while it induced more AR expression than other groups. Finally, all procedures did not improve or deteriorate normal erectile function. Although we verified that Anti-LOX + VED lengthened penis by inducing tunica albuginea remodeling, however, HCG supplement did not synergize with Anti-LOX + VED to accelerate albuginea remodeling to facilitate penile growth.

Microfluidic Chips: Emerging Technologies for Adoptive Cell Immunotherapy.

Adoptive cell therapy (ACT) is a personalized therapy that has shown great success in treating hematologic malignancies in clinic, and has also demonstrated potential applications for solid tumors. The process of ACT involves multiple steps, including the separation of desired cells from patient tissues, cell engineering by virus vector systems, and infusion back into patients after strict tests to guarantee the quality and safety of the products. ACT is an innovative medicine in development; however, the multi-step method is time-consuming and costly, and the preparation of the targeted adoptive cells remains a challenge. Microfluidic chips are a novel platform with the advantages of manipulating fluid in micro/nano scales, and have been developed for various biological research applications as well as ACT. The use of microfluidics to isolate, screen, and incubate cells in vitro has the advantages of high throughput, low cell damage, and fast amplification rates, which can greatly simplify ACT preparation steps and reduce costs. Moreover, the customizable microfluidic chips fit the personalized demands of ACT. In this mini-review, we describe the advantages and applications of microfluidic chips for cell sorting, cell screening, and cell culture in ACT compared to other existing methods. Finally, we discuss the challenges and potential outcomes of future microfluidics-related work in ACT.

Targeting the KLF5-EphA2 axis can restrain cancer stemness and overcome chemoresistance in basal-like breast cancer.

Ephrin type-A receptor 2 (EphA2) is a member of the tyrosine receptor kinases, a family of membrane proteins recognized as potential anticancer targets. EphA2 highly expressed in a variety of human cancers, playing roles in proliferation, migration, and invasion. However, whether and how EphA2 regulates basal-like breast cancer (BLBC) cell stemness and chemoresistance has not been revealed. Here, KLF5 was proven to be a direct transcription factor for EphA2 in BLBC cells, and its expression was positively correlated in clinical samples from breast cancer patients. The inflammatory factor TNF-α could promote BLBC cell stemness partially by activating the KLF5-EphA2 axis. Moreover, phosphorylation of EphA2 at S897 (EphA2 pS897) induced by TNF-α and PTX/DDP contributes to chemoresistance of BLBC. Furthermore, the EphA2 inhibitor ALW-II-41-27 could effectively reduce EphA2 pS897 and tumor cell stemness in vitro and significantly enhance the sensitivity of xenografts to the chemotherapeutic drugs PTX and DDP in vivo. Clinically, tumor samples from breast patients with less response to neoadjuvant chemotherapy showed a high level of EphA2 pS897 expression. In conclusion, KLF5-EphA2 promotes stemness and drug resistance in BLBC and could be a potential target for the treatment of BLBC.

RNF126-Mediated MRE11 Ubiquitination Activates the DNA Damage Response and Confers Resistance of Triple-Negative Breast Cancer to Radiotherapy.

Triple-negative breast cancer (TNBC) has higher molecular heterogeneity and metastatic potential and the poorest prognosis. Because of limited therapeutics against TNBC, irradiation (IR) therapy is still a common treatment option for patients with lymph nodes or brain metastasis. Thus, it is urgent to develop strategies to enhance the sensitivity of TNBC tumors to low-dose IR. Here, the authors report that E3 ubiquitin ligase Ring finger protein 126 (RNF126) is important for IR-induced ATR-CHK1 pathway activation to enhance DNA damage repair (DDR). Mechanistically, RNF126 physically associates with the MRE11-RAD50-NBS1 (MRN) complex and ubiquitinates MRE11 at K339 and K480 to increase its DNA exonuclease activity, subsequent RPA binding, and ATR phosphorylation, promoting sustained DDR in a homologous recombination repair-prone manner. Accordingly, depletion of RNF126 leads to increased genomic instability and radiation sensitivity in both TNBC cells and mice. Furthermore, it is found that RNF126 expression is induced by IR activating the HER2-AKT-NF-κB pathway and targeting RNF126 expression with dihydroartemisinin significantly improves the sensitivity of TNBC tumors in the brain to IR treatment in vivo. Together, these results reveal that RNF126-mediated MRE11 ubiquitination is a critical regulator of the DDR, which provides a promising target for improving the sensitivity of TNBC to radiotherapy.

A feedforward circuit between KLF5 and lncRNA KPRT4 contributes to basal-like breast cancer.

Basal-like breast cancer (BLBC) is the most aggressive subtype of breast cancer with a poor prognosis. Long noncoding RNAs (lncRNAs) play critical roles in human cancers. Krüppel-like Factor 5 (KLF5) is a key oncogenic transcription factor in BLBC. However, the underlying mechanism of mutual regulation between KLF5 and lncRNA remains largely unknown. Here, we demonstrate that lncRNA KPRT4 promotes BLBC cell proliferation in vitro and in vivo. Mechanistically, KLF5 directly binds to the promoter of KPRT4 to promote KPRT4 transcription. Reciprocally, KPRT4 recruits the YB-1 transcription factor to the KLF5 promoter by interacting with YB-1 at its 5' domain and forming an RNA-DNA-DNA triplex structure at its 3' domain, resulting in enhanced transcription of KLF5 and ultimately establishing a feedforward circuit to promote cell proliferation. Moreover, the antisense oligonucleotide (ASO)-based therapy targeting KPRT4 substantially attenuated tumor growth in vivo. Clinically, the expression levels of YB-1, KLF5 and KPRT4 are positively correlated in clinical breast specimens. Together, our data suggest that KPRT4 is a major molecule for BLBC progression and that the feedforward circuit between KLF5 and KPRT4 may represent a potential therapeutic target in BLBC.

KLF5-induced lncRNA IGFL2-AS1 promotes basal-like breast cancer cell growth and survival by upregulating the expression of IGFL1.

Basal-like breast cancer (BLBC) is the most malignant subtype of breast cancer and has a poor prognosis. Kruppel-like factor 5 (KLF5) is an oncogenic transcription factor in BLBCs. The mechanism by which KLF5 promotes BLBC by regulating the transcription of lncRNAs has not been fully elucidated. In this study, we discovered that lncRNA IGFL2-AS1 is a downstream target gene of KLF5 and that IGFL2-AS1 mediates the pro-proliferation and pro-survival functions of KLF5. Additionally, we demonstrated that IGFL2-AS1 functions by upregulating the transcription of its neighboring gene IGFL1 via two independent mechanisms. On the one hand, nuclear IGFL2-AS1 promotes the formation of a KLF5/TEAD4 transcriptional complex at the IGFL1 gene enhancer. On the other hand, cytoplasmic IGFL2-AS1 inhibits the expression of miR4795-3p, which targets the IGFL1 gene. TNFα induces the expression of IGFL2-AS1 and IGFL1 through KLF5. Taken together, the results of this study indicate that IGFL2-AS1 and IGFL1 may serve as new therapeutic targets for BLBCs.

Arginine methyltransferase PRMT5 methylates and stabilizes KLF5 via decreasing its phosphorylation and ubiquitination to promote basal-like breast cancer.

Krüppel-like factor 5 (KLF5) is an oncogenic factor that is highly expressed in basal-like breast cancer (BLBC) and promotes cell proliferation, survival, migration, stemness, and tumor growth; however, its posttranslational modifications are poorly defined. Protein arginine methyltransferase 5 (PRMT5) is also an oncogene implicated in various carcinomas, including breast cancer. In this study, we found that PRMT5 interacts with KLF5 and catalyzes the di-methylation of KLF5 at Arginine 57 (R57) in a methyltransferase activity-dependent manner in BLBC cells. Depletion or pharmaceutical inhibition (using PJ-68) of PRMT5 decreased the expression of KLF5 and its downstream target genes in vitro and in vivo. PRMT5-induced KLF5R57me2 antagonizes GSK3ß-mediated KLF5 phosphorylation and subsequently Fbw7-mediated KLF5 ubiquitination and coupled degradation. Functionally, PRMT5 promotes breast cancer stem cell maintenance and proliferation, at least partially, by stabilizing KLF5. PRMT5 and KLF5 protein levels were positively correlated in clinical BLBCs. Taken together, PRMT5 methylates KLF5 to prevent its phosphorylation, ubiquitination, and degradation, and thus promotes breast cancer stem cell maintenance and proliferation. These findings suggest that PRMT5 is a potential therapeutic target for BLBC.

HDAC inhibitors induce proline dehydrogenase (POX) transcription and anti-apoptotic autophagy in triple negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with poor clinical outcomes and without effective targeted therapies. Numerous studies have suggested that HDAC inhibitors (TSA/SAHA) may be effective in TNBCs. Proline oxidase, also known as proline dehydrogenase (POX/PRODH), is a key enzyme in the proline metabolism pathway and plays a vital role in tumorigenesis. In this study, we found that HDAC inhibitors (TSA/SAHA) significantly increased POX expression and autophagy through activating AMPK. Depletion of POX decreased autophagy and increased apoptosis induced by HDAC inhibitors in TNBC cells. These results suggest that POX contributes to cell survival under chemotherapeutic stresses and might serve as a potential target for treatment of TNBC.

Multiple antiviral approaches of (-)-epigallocatechin-3-gallate (EGCG) against porcine reproductive and respiratory syndrome virus infection in vitro.

Porcine reproductive and respiratory syndrome virus (PRRSV) remains an economically important pathogen in the global pig industry, effective measures to control the virus are still lacking. (-)-Epigallocatechin-3-gallate (EGCG), the most abundant and bioactive catechin in green tea, has been reported to have antiviral effect against the diverse groups of viruses. In this study, the comprehensive anti-PRRSV activity of EGCG was investigated using various in vitro assays. EGCG effectively inhibited PRRSV infection and replication in porcine alveolar macrophages (PAMs), regardless of whether it was administrated pre- or post-infection, and the cytotoxicity to PAMs was low. Next, anti-PRRSV approaches of EGCG were characterized in MARC-145 cells. EGCG was demonstrated to be able to significantly prevent PRRSV from infecting MARC-145 cells either through blocking of EGCG-treated viruses docking to susceptible cells involving a direct virus-EGCG interaction or by blocking of the infective virus binding to EGCG pre-treated cells via triggering down-regulation of viral receptors and/or related proteins required for infection. In addition, PRRSV replication was suppressed in MARC-145 cells treated with EGCG post-infection, likely because of down-regulation of pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-8. Taken together, these data showed that treatment of primary PAMs with EGCG can inhibit PRRSV infection and revealed that multiple antiviral approaches of EGCG operate in PRRSV-susceptible MARC-145 cells.

Non-coding Transcripts from Enhancers: New Insights into Enhancer Activity and Gene Expression Regulation.

Long non-coding RNAs (lncRNAs) have gained widespread interest in the past decade owing to their enormous amount and surprising functions implicated in a variety of biological processes. Some lncRNAs exert function as enhancers, i.e., activating gene transcription by serving as the cis-regulatory molecules. Furthermore, recent studies have demonstrated that many enhancer elements can be transcribed and produce RNA molecules, which are termed as enhancer RNAs (eRNAs). The eRNAs are not merely the by-product of the enhancer transcription. In fact, many of them directly exert or regulate enhancer activity in gene activation through diverse mechanisms. Here, we provide an overview of enhancer activity, transcription of enhancer itself, characteristics of eRNAs, as well as their roles in regulating enhancer activity and gene expression.

Human eukaryotic elongation factor 1A forms oligomers through specific cysteine residues.

Eukaryotic elongation factor 1A (eEF1A) is a multifunctional protein involved in bundling actin, severing microtubule, activating the phosphoinositol-4 kinase, and recruiting aminoacyl-tRNAs to ribosomes during protein biosynthesis. Although evidence has shown the presence of the isoform eEF1A1 oligomers, the substantial mechanism of the self-association remains unclear. Herein, we found that human eEF1A1 could spontaneously form oligomers. Specifically, mutagenesis screen on cysteine residues demonstrated that Cys(234) was essential for eEF1A1 oligomerization. In addition, we also found that hydrogen peroxide treatment could induce the formation of eEF1A oligomers in cells. By cysteine replacement, eEF1A2 isoform displayed the ability to oligomerize in cells under the oxidative environment. In summary, in this study we characterized eEF1A1 oligomerization and demonstrated that specific cysteine residues are required for this oligomerization activity.