Liposomal STAT3-Degrading PROTAC Prodrugs Promote Anti-Hepatocellular Carcinoma Immunity via Chemically Reprogramming Cancer Stem Cells.

Cancer stem cells (CSCs) with hyperactivated signal transducer and activator of transcription 3 (STAT3) are a major driver of hepatocellular carcinoma (HCC). Herein, we report a nanointegrative proteolysis-targeting chimera (PROTAC)-based STAT3 degradation strategy that enables efficient chemical reprogramming of HCC-associated CSCs, which potently inhibits CSC growth while evoking anti-HCC immune responses. The PROTAC prodrug was synthesized by conjugating the STAT3 binding domain (inS3) with a thioketal-caged E3 ligase ligand (VL-TK) via an oligo(ethylene glycol) linker (OEG) with tuned length and flexibility and encapsulating it in cRGD-modified cationic liposomes for CSC-targeted delivery while facilitating their lysosomal escape. The PROTAC prodrugs were activated by the upregulated ROS levels in CSCs and efficiently degraded STAT3 for chemical reprogramming, which would not only impair their stemness features but also remodel the immunosuppressive TME into an immunosupportive state to boost anti-HCC immunity. This strategy provides an approach for improving HCC treatment in clinics.

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.

Nanointegrative Glycoengineering-Activated Necroptosis of Triple Negative Breast Cancer Stem Cells Enables Self-Amplifiable Immunotherapy for Systemic Tumor Rejection.

Triple-negative breast cancer stem cells (TCSCs) are considered as the origin of recurrence and relapse. It is difficult to kill not only for its resistance, but also the lacking of targetable molecules on membrane. Here, it is confirmed that ST6 ß-galactoside alpha-2,6-sialyltransferase 1 (ST6Gal-1) is highly expressed in TCSCs that may be the key enzyme involved in glycoengineering via sialic acid (SA) metabolism. SA co-localizes with a microdomain on cell membrane termed as lipid rafts that enrich CSCs marker and necroptosis proteins mixed lineage kinase domain-like protein (MLKL), suggesting that TCSCs may be sensitive to necroptosis. Thus, the triacetylated N-azidoacetyl-d-mannosamine (Ac3ManNAz) is synthesized as the glycoengineering substrate and applied to introduce artificial azido receptors, dibenzocyclooctyne (DBCO)-modified liposome is used to deliver Compound 6i (C6), a receptor-interacting serine/threonine protein kinase 1(RIPL1)-RIP3K-mixed lineage kinase domain-like protein(MLKL) activator, to induce necroptosis. The pro-necroptosis effect is aggravated by nitric oxide (NO), which is released from NO-depot of cholesterol-NO integrated in DBCO-PEG-liposome@NO/C6 (DLip@NO/C6). Together with the immunogenicity of necroptosis that releases high mobility group box 1(HMGB1) of damage-associated molecular patterns, TCSCs are significantly killed in vitro and in vivo. The results suggest a promising strategy to improve the therapeutic effect on the non-targetable TCSCs with high expression of ST6Gal-1 via combination of glycoengineering and necroptosis induction.

Perspectives for Using CO2 as a Feedstock for Biomanufacturing of Fuels and Chemicals.

Microbial cell factories offer an eco-friendly alternative for transforming raw materials into commercially valuable products because of their reduced carbon impact compared to conventional industrial procedures. These systems often depend on lignocellulosic feedstocks, mainly pentose and hexose sugars. One major hurdle when utilizing these sugars, especially glucose, is balancing carbon allocation to satisfy energy, cofactor, and other essential component needs for cellular proliferation while maintaining a robust yield. Nearly half or more of this carbon is inevitably lost as CO2 during the biosynthesis of regular metabolic necessities. This loss lowers the production yield and compromises the benefit of reducing greenhouse gas emissions-a fundamental advantage of biomanufacturing. This review paper posits the perspectives of using CO2 from the atmosphere, industrial wastes, or the exhausted gases generated in microbial fermentation as a feedstock for biomanufacturing. Achieving the carbon-neutral or -negative goals is addressed under two main strategies. The one-step strategy uses novel metabolic pathway design and engineering approaches to directly fix the CO2 toward the synthesis of the desired products. Due to the limitation of the yield and efficiency in one-step fixation, the two-step strategy aims to integrate firstly the electrochemical conversion of the exhausted CO2 into C1/C2 products such as formate, methanol, acetate, and ethanol, and a second fermentation process to utilize the CO2-derived C1/C2 chemicals or co-utilize C5/C6 sugars and C1/C2 chemicals for product formation. The potential and challenges of using CO2 as a feedstock for future biomanufacturing of fuels and chemicals are also discussed.

High Sensitivity Singlet Oxygen Luminescence Sensor Using Computational Spectroscopy and Solid-State Detector.

This paper presents a technique for high sensitivity measurement of singlet oxygen luminescence generated during photodynamic therapy (PDT) and ultraviolet (UV) irradiation on skin. The high measurement sensitivity is achieved by using a computational spectroscopy (CS) approach that provides improved photon detection efficiency compared to spectral filtering methodology. A solid-state InGaAs photodiode is used as the CS detector, which significantly reduces system cost and improves robustness compared to photomultiplier tubes. The spectral resolution enables high-accuracy determination and subtraction of photosensitizer fluorescence baseline without the need for time-gating. This allows for high sensitivity detection of singlet oxygen luminescence emission generated by continuous wave light sources, such as solar simulator sources and those commonly used in PDT clinics. The value of the technology is demonstrated during in vivo and ex vivo experiments that show the correlation of measured singlet oxygen with PDT treatment efficacy and the illumination intensity on the skin. These results demonstrate the potential use of the technology as a dosimeter to guide PDT treatment and as an analytical tool supporting the development of improved sunscreen products for skin cancer prevention.

Mechanotransduction in response to ECM stiffening impairs cGAS immune signaling in tumor cells.

The tumor microenvironment (TME) plays decisive roles in disabling T cell-mediated antitumor immunity, but the immunoregulatory functions of its biophysical properties remain elusive. Extracellular matrix (ECM) stiffening is a hallmark of solid tumors. Here, we report that the stiffened ECM contributes to the immunosuppression in TME via activating the Rho-associated coiled-coil-containing protein kinase (ROCK)-myosin IIA-filamentous actin (F-actin) mechanosignaling pathway in tumor cells to promote the generation of TRIM14-scavenging nonmuscle myosin heavy chain IIA (NMHC-IIA)-F-actin stress fibers, thus accelerating the autophagic degradation of cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) to deprive tumor cyclic GMP-AMP (cGAMP) and further attenuating tumor immunogenicity. Pharmacological inhibition of myosin IIA effector molecules with blebbistatin (BLEB) or the RhoA upstream regulator of this pathway with simvastatin (SIM) restored tumor-intrinsic cGAS-mediated cGAMP production and enhanced antitumor immunity. Our work identifies that ECM stiffness is an important biophysical cue to regulate tumor immunogenicity via the ROCK-myosin IIA-F-actin axis and that inhibiting this mechanosignaling pathway could boost immunotherapeutic efficacy for effective solid tumor treatment.

Coordination-driven FBXW7 DNAzyme-Fe nanoassembly enables a binary switch of breast cancer cell cycle checkpoint responses for enhanced ferroptosis-radiotherapy.

Radiotherapy is a mainstream modality for breast cancer treatment that employs ionizing radiation (IR) to damage tumor cell DNA and elevate ROS stress, which demonstrates multiple clinically-favorable advantages including localized treatment and low invasiveness. However, breast cancer cells may activate the p53-mediated cell cycle regulation in response to radiotherapy to repair IR-induced cellular damage and facilitate post-treatment survival. F-Box and WD Repeat Domain Containing 7 (FBXW7) is a promoter of p53 degradation and critical nexus of cell proliferation and survival events. Herein, we engineered a cooperative radio-ferroptosis-stimulatory nanomedicine through coordination-driven self-assembly between ferroptosis-inducing Fe2+ ions and FBXW7-inhibiting DNAzymes and further modification of tumor-targeting dopamine-modified hyaluronic acid (HA). The nanoassembly could be selectively internalized by breast cancer cells and disintegrated in lysosomes to release the therapeutic payload. DNAzyme readily abolishes FBXW7 expression and stabilizes phosphorylated p53 to cause irreversible G2 phase arrest for amplifying post-IR tumor cell apoptosis. Meanwhile, the p53 stabilization also inhibits the SLC7A11-cystine-GSH axis, which combines with the IR-upregulated ROS levels to amplify Fe2+-mediated ferroptotic damage. The DNAzyme-Fe-HA nanoassembly could thus systematically boost the tumor cell damaging effects of IR, presenting a simple and effective approach to augment the response of breast cancer to radiotherapy. STATEMENT OF SIGNIFICANCE: To overcome the intrinsic radioresistance in breast cancer, we prepared co-assembly of Fe2+ and FBXW7-targeted DNAzymes and modified surface with dopamine conjugated hyaluronic acid (HA), which enabled tumor-specific FBXW7-targeted gene therapy and ferroptosis therapy in IR-treated breast cancers. The nanoassembly could be activated in acidic condition to release the therapeutic contents. Specifically, the DNAzymes could selectively degrade FBXW7 mRNA in breast cancer cells to simultaneously induce accumulation of p53 and retardation of NHEJ repair, eventually inducing irreversible cell cycle arrest to promote apoptosis. The p53 stabilization would also inhibit the SLC7A11/GSH/GPX4 axis to enhance Fe2+ mediated ferroptosis. These merits could act in a cooperative manner to induce pronounced tumor inhibitory effect, offering new approaches for tumor radiosensitization in the clinics.

YTHDF1 upregulation mediates hypoxia-dependent breast cancer growth and metastasis through regulating PKM2 to affect glycolysis.

N6-methyladenosine modification is the most common RNA modification mechanism in mammals. YTHDF1, a m6A reader, can recognize the m6A of mRNAs to facilitate the interaction with the mRNA ribosome assembly and recruitment of translation initiators to promote translation. From a clinical perspective, YTHDF1 upregulation is frequently observed in breast cancer, but its involvement in those cancer-related events is still unclear. Here we report that YTHDF1 is a cancer driver capable of facilitating the proliferation and invasion of breast cancer cells as well as enhancing tumorigenicity and metastasis through promoting glycolysis. We found that tumor hypoxia can transcriptionally induce HIF1α and post-transcriptionally inhibit the expression of miR-16-5p to promote YTHDF1 expression, which could sequentially enhance tumor glycolysis by upregulating PKM2 and eventually increase the tumorigenesis and metastasis potential of breast cancer cells. Inhibiting YTHDF1 via gene knockdown or miR-16-5p would significantly abolish YTHDF1-dependent tumor growth and metastasis. In summary, we identified the role of the YTHDF1-PKM2 signal axis in the occurrence and development of breast cancer, which can be used as a potential target for breast cancer treatment.

An ROS-Activatable Nanoassembly Remodulates Tumor Cell Metabolism for Enhanced Ferroptosis Therapy.

Ferroptosis is an emerging antitumor option and has demonstrated unique advantages against many tumor indications. However, its efficacy is potentially hindered by the endogenous lipid peroxide-scavenging mechanisms and the reliance on acidic pH. Herein, a nanointegrated strategy based on clinically-safe components to synergistically remodel glutathione and lactate metabolism in tumor cells for enhanced ferroptosis therapy is developed. First ferrocene is conjugated on PEGylated polyamidoamine dendrimers via reactive oxygen species (ROS)-cleavable thioketal linkage, which would further self-assemble with the glutathione (GSH)-depleting agent diethyl maleate (DEM) and monocarboxylate transporter 4-inhibiting siRNA (siMCT4) to afford biostable nanoassemblies (siMCT4-PAMAM-PEG-TK-Fc@DEM). The nanoassemblies can be activated by the elevated ROS levels in tumor intracellular environment and readily release the incorporated therapeutic contents, afterward DEM can directly conjugate to GSH to disrupt the glutathione peroxidase 4 (GPX4)-mediated antioxidant defense, while siMCT4 can block the MCT4-mediated efflux of lactic acid and acidify the intracellular milieu, both of which can improve the ferrocene-catalyzed lipid peroxidation and induce pronounced ferroptotic damage. The siMCT4-PAMAM-PEG-TK-Fc@DEM nanoplatform demonstrates high ferroptosis-based antitumor potency and good biocompatibility in vitro and in vivo, which may offer new avenues for the development of more advanced antitumor therapeutics with improved translatability.

Activatable Biomineralized Nanoplatform Remodels the Intracellular Environment of Multidrug-Resistant Tumors for Enhanced Ferroptosis/Apoptosis Therapy.

Ferroptosis is a new form of regulated cell death with significant therapeutic prospect, but its application against drug-resistant tumor cells is challenging due to their ability to effuse antitumor agents via p-glycoprotein (P-gp) and anti-lipid peroxidation alkaline intracellular environment. Herein, an amorphous calcium phosphate (ACP)-based nanoplatform is reported for the targeted combinational ferroptosis/apoptosis therapy of drug resistant tumor cells by blocking the MCT4-mediated efflux of lactic acid (LA). The nanoplatform is fabricated through the biomineralization of doxorubicin-Fe2+ (DOX-Fe2+ ) complex and MCT4-inhibiting siRNAs (siMCT4) and can release them to the tumor cytoplasm after the hydrolysis of ACP and dissociation of DOX-Fe2+ in the acidic lysosomes. siMCT4 can inhibit MCT4 expression and force the glycolysis-generated lactic acid (LA) to remain in cytoplasm for rapid acidification. The nanoplatform-induced remodeling of the tumor intracellular environment can not only interrupt the ATP supply required for P-gp-dependent DOX effusion to enhance H2 O2 production, but also increase the overall catalytic efficiency of Fe2+ for the initiation and propagation of lipid peroxidation. These features could act in concert to enhance the efficacy of the combinational ferroptosis/chemotherapy and prolong the survival of tumor-bearing mice. This study may provide new avenues for the treatment of multidrug-resistant tumors.

High optical-throughput spectroscopic singlet oxygen and photosensitizer luminescence dosimeter for monitoring of photodynamic therapy.

We report a high light-throughput spectroscopic dosimeter system that is able to noninvasively measure luminescence signals of singlet oxygen (1 O2 ) produced during photodynamic therapy (PDT) using a CW (continuous wave) light source. The system is based on a compact, fiber-coupled, high collection efficiency spectrometer (>50% transmittance) designed to maximize optical throughput but with sufficient spectral resolution (~7 nm). This is adequate to detect 1 O2 phosphorescence in the presence of strong luminescence background in vivo. This system provides simultaneous acquisition of multiple spectral data points, allowing for more accurate determination of luminescence baseline via spectral fitting and thus the extraction of 1 O2 phosphorescence signal based solely on spectroscopic decomposition, without the need for time-gating. Simultaneous collection of photons at different wavelengths improves the quantum efficiency of the system when compared to sequential spectral measurements such as filter-wheel or tunable-filter based systems. A prototype system was tested during in vivo PDT tumor regression experiments using benzoporphyrin derivative (BPD) photosensitizer. It was found that the treatment efficacy (tumor growth inhibition rate) correlated more strongly with 1 O2 phosphorescence than with PS fluorescence. These results indicate that this high photon-collection efficiency spectrometer instrument may offer a viable option for real-time 1 O2 dosimetry during PDT treatment using CW light.

Tumor-Targeted Disruption of Lactate Transport with Reactivity-Reversible Nanocatalysts to Amplify Oxidative Damage.

Nanocatalytic tumor therapy is an emerging antitumor option that employs catalytically-active inorganic nanostructures to produce tumor-damaging reactive oxygen species. However, initiation of nanocatalytic reactions in the tumor intracellular environment is a challenge due to the reliance on acidic pH. By exploiting the pH-selective multifaceted catalytic activities of Prussian blue-based nanomaterials (PBNM) as well as the hyperglycolysis characteristics of tumors, it is demonstrated that blocking the monocarboxylate transporter 4 (MCT4)-mediated lactate effusion in tumor cells can reverse the pH gradient across the tumor cell membrane and cause rapid intracellular acidification as well as neutralization of the extracellular compartment, thus creating vulnerabilities for PBNM-based nanocatalytic therapies in situ while suppressing tumor stemness/metastasis in vivo. For this purpose, MCT4-inhibiting siRNAs are incorporated into reactivity-switchable PBNM-based nanocatalysts to initiate hydroxyl radical production. Meanwhile, ß-lapachone, a clinically-approved drug with H2 O2 -generating capabilities, is also integrated to sustain the nanocatalytic process. In contrast, the nanocatalyst shows no apparent toxicity to normal cells due to its catalase-like activities under neutral pH. This treatment strategy can inhibit tumor growth in mice at optimal safety as well as to suppress the cancer cell stemness and lung metastasis, suggesting the clinical translational potential of the findings.

HCAR1/MCT1 Regulates Tumor Ferroptosis through the Lactate-Mediated AMPK-SCD1 Activity and Its Therapeutic Implications.

Ferroptosis is a recently discovered form of programed cell death caused by the metabolically regulated lipid peroxidation and holds promise for cancer treatment, but its regulatory mechanisms remain elusive. In this study, we observe that lactate-rich liver cancer cells exhibit enhanced resistance to the ferroptotic damage induced by common ferroptosis inducers such as Ras-selective lethal small molecule 3 (RSL3) and Erastin and that the monocarboxylate transporter 1 (MCT1)-mediated lactate uptake could promote ATP production in hepatocellular carcinoma (HCC) cells and deactivate the energy sensor AMP-activated protein kinase (AMPK), leading to the upregulation of sterol regulatory element-binding protein 1 (SREBP1) and the downstream stearoyl-coenzyme A (CoA) desaturase-1 (SCD1) to enhance the production of anti-ferroptosis monounsaturated fatty acids. Additionally, blocking the lactate uptake via hydroxycarboxylic acid receptor 1 (HCAR1)/MCT1 inhibition promotes ferroptosis by activating the AMPK to downregulate SCD1, which may synergize with its acyl-coenzyme A synthetase 4 (ACSL4)-promoting effect to amplify the ferroptotic susceptibility. In vitro and in vivo evidence confirms that lactate regulates the ferroptosis of HCC cells and highlights its translational potential as a therapeutic target for ferroptosis-based tumor treatment.

Multispectral singlet oxygen and photosensitizer luminescence dosimeter for continuous photodynamic therapy dose assessment during treatment.

SIGNIFICANCE: Photodynamic therapy (PDT) involves complex light-drug-pathophysiology interactions that can be affected by multiple parameters and often leads to large variations in treatment outcome from patient to patient. Direct PDT dosimetry technologies have been sought to optimize the control variables (e.g., light dose, drug administration, tissue oxygenation, and patient conditioning) for best patient outcomes. In comparison, singlet oxygen (O21) dosimetry has been tested in various forms to provide an accurate and perhaps comprehensive prediction of the treatment efficacy. AIM: We discuss an advanced version of this approach provided by a noninvasive, continuous wave dosimeter that can measure near-infrared spectrally resolved luminescence of both photosensitizer (PS) and O21 generated during PDT cancer treatment. APPROACH: This dosimetry technology uses an amplified, high quantum efficiency InGaAs detector with spectroscopic decomposition during the light treatment to continuously extract the maximum signal of O21 phosphorescence while suppressing the strong PS luminescence background by spectrally fitting the data points across nine narrow band wavelengths. O21 and PS luminescence signals were measured in vivo in FaDu xenograft tumors grown in mice during PDT treatment using Verteporfin as the PS and a continuous laser treatment at 690 nm wavelength. RESULTS: A cohort of 19 mice was used and observations indicate that the tumor growth rate inhibition showed a stronger correlation with O21 than with just the PS signal. CONCLUSIONS: These results suggest that O21 measurement may be a more direct dosimeter of PDT damage, and it has potential value as a definitive diagnostic for PDT treatment, especially with spectral separation of the background luminescence and online estimation of the PS concentration.

Two-photon microscope using a fiber-based approach for supercontinuum generation and light delivery to a small-footprint optical head.

In this Letter, we report a low-cost, portable, two-photon excitation fluorescence microscopy imager that uses a fiber-based approach for both femtosecond supercontinuum (SC) generation and light delivery to the optical head. The SC generation is based on a tapered polarization-maintaining photonic crystal fiber that uses pre-chirped femtosecond narrowband pulses to generate a coherent SC spectrum with a bandwidth of approximately 300 nm. Using this approach, high-power, near-transform-limited, wavelength-selectable SC pulses are generated and directly delivered to the imaging optical head. Preliminary testing of this imager on brain slices is presented, demonstrating a high signal-to-noise ratio and sub-cellular imaging capabilities to a depth of approximately 200 µm. These results demonstrate the suitability of the technology for ex vivo and potentially in vivo cellular-level biomedical imaging applications.

Targeted inhibition of MCT4 disrupts intracellular pH homeostasis and confers self-regulated apoptosis on hepatocellular carcinoma.

The high lactate production rate in hepatocellular carcinoma cells (HCC) have a profound impact on their malignant properties. In adaptation to the enhanced lactate stress, lactate-effusing monocarboxylate transporter 4(MCT4) is usually overexpressed in a broad range of HCC subtypes. In this study, the MCT4-mediated lactate efflux in HCC was blocked using microRNA-145(miR-145), which would force the endogenously generated lactate to accumulate within tumor cells in a self-regulated manner, resulting in the acidification of the cytoplasmic compartment as well as partial neutralization for pH in the tumor extracellular environment. Evaluations on multiple representative HCC subtypes (HepG2, Hep3B and HuH7) suggested that the disrupted pH homeostasis would amplify the lactate stress to initiate HCC apoptosis, while at the same time also suppressing their migration and invasion abilities. Moreover, safety tests on 7702 cells and living animals revealed that MCT4-blockade treatment has no cytotoxicity against healthy cells/tissues. The results indicate the MCT4-inhibition-induced disruption of tumor intracellular pH holds promise as a therapy against not only HCC, but a broader spectrum of MCT4-overexpressing hyperglycolytic tumors.

Visualizing the vasculature of the entire human eye posterior hemisphere without a contrast agent.

The platform described here combines the non-invasive measurement of the retina/choroid structure and ocular blood flow based on optical coherence tomography (OCT) and wide-field semi-quantitative global flow visualization using line-scanning Doppler flowmetry (LSDF). The combination of these two imaging modalities within the same platform enables comprehensive assessment of blood flow in the retina and choroid in animals and human subjects for diagnostic purposes. Ultra-widefield vasculature visualization is demonstrated here for the first time without injecting additional contrast agents and based only on the motion of particles within the vasculature.

Progesterone-induced miR-152 interferes with embryonic implantation by downregulating GLUT3 in endometrial epithelium.

To investigate the role of progesterone-induced micro-RNA (miR)-152 in early embryonic development and implantation by regulating GLUT3 in endometrial epithelium, qRT-PCR was used to detect the expression of miR-152, GLUT1, and GLUT3 in the endometrial epithelial cells of female mice. GLUT1 and GLUT3 proteins were detected by immunohistochemical staining in the mouse endometrial epithelium. Bioinformatics prediction associated with a luciferase assay was performed to determine whether GLUT1 and GLUT3 are target genes of miR-152. Specific miR-152 mimics or inhibitors were transfected into the endometrial epithelial cells to, respectively, overexpress or downregulate miR-152. Next, the glucose concentration of uterine fluid was measured by conducting high-performance liquid chromatography in vivo, and the glucose uptake of the endometrial epithelial cells was observed using a fluorometric assay in vitro. Early embryonic development and implantation were also observed after the miR-152 mimics or inhibitors had been transfected. Embryo transfer was observed after the miR-152 mimic transfection. miR-152 was found to directly target and thereby downregulate GLUT3 expression. The expressions of both miR-152 and GLUT3 in the mouse endometrial epithelium had spatiotemporal characteristics on days 1-4 of pregnancy. miR-152 affected the glucose concentration of uterine fluid and the glucose uptake of endometrial epithelial cells. The transfection of specific miR-152 mimics led to impaired embryonic development and implantation. To conclude, in endometrial epithelial cells, progesterone-induced miR-152 downregulates GLUT3 at the posttranscriptional level to maintain a proper glucose concentration in the uterine fluid, which is necessary for early embryonic development and implantation.

Progesterone-Induced miR-145/miR-143 Inhibits the Proliferation of Endometrial Epithelial Cells.

Our previous study showed that progesterone (P4) can specifically regulate the expression of some microRNAs (miRNAs) in endometrial epithelium. In the present study, we verified the P4-dependent expression of miR-145/miR-143 in endometrial epithelial cells, explored the regulative mechanism of the P4 receptor (PR), and investigated their effects on the proliferation of endometrial epithelial cells. Our results showed that P4 can induce the expression of miR-145/143 in endometrial epithelial cells by acting on the PR A subtype. P4-induced miR-145/143 can inhibit the expression of cyclin D2 by binding to cyclin D2 mRNA 3'UTR. It can also inhibit cell proliferation in mouse endometrial epithelium by arresting the cell cycle during the G1-S checkpoint. Furthermore, miR-145 and miR-143 can inhibit the proliferation of human endometrial cancer cells. In conclusion, P4-induced miR-145/miR-143 is an important regulator in the proliferation of endometrial epithelial cells, and it can also inhibit the proliferation of human endometrial cancer cells. Our study indicates miRNAs are important mechanism of P4 in inhibiting the proliferation of endometrial epithelial cells. And these miRNAs are potential candidates for the diagnosis of endometrial cancer and therapeutic targets.

Slide-free virtual histochemistry (Part I): development via nonlinear optics.

Histochemistry is a microscopy-based technology widely used to visualize the molecular distribution in biological tissue. Recent developments in label-free optical imaging has demonstrated the potential to replace the conventional histochemical labels/markers (fluorescent antibodies, organic dyes, nucleic acid probes, and other contrast agents) with diverse optical interactions to generate histochemical contrasts, allowing "virtual" histochemistry in three spatial dimensions without preparing a microscope slide (i.e. labor-intensive sample preparation). However, the histochemical information in a label-free optical image has often been rather limited due to the difficulty in simultaneously generating multiple histochemical contrasts with strict spatial co-registration. Here, in the first part (Part I) of this two-part series study, we develop a technique of slide-free virtual histochemistry based on label-free multimodal multiphoton microscopy, and simultaneously generate up to four histochemical contrasts from in vivo animal and ex vivo human tissue. To enable this functionality, we construct and demonstrate a robust fiber-based laser source for clinical translation and phenotype a wide variety of vital cells in unperturbed mammary tissue.