Targeting ALK averts ribonuclease 1-induced immunosuppression and enhances antitumor immunity in hepatocellular carcinoma.

Tumor-secreted factors contribute to the development of a microenvironment that facilitates the escape of cancer cells from immunotherapy. In this study, we conduct a retrospective comparison of the proteins secreted by hepatocellular carcinoma (HCC) cells in responders and non-responders among a cohort of ten patients who received Nivolumab (anti-PD-1 antibody). Our findings indicate that non-responders have a high abundance of secreted RNase1, which is associated with a poor prognosis in various cancer types. Furthermore, mice implanted with HCC cells that overexpress RNase1 exhibit immunosuppressive tumor microenvironments and diminished response to anti-PD-1 therapy. RNase1 induces the polarization of macrophages towards a tumor growth-promoting phenotype through activation of the anaplastic lymphoma kinase (ALK) signaling pathway. Targeting the RNase1/ALK axis reprograms the macrophage polarization, with increased CD8+ T- and Th1- cell recruitment. Moreover, simultaneous targeting of the checkpoint protein PD-1 unleashes cytotoxic CD8+ T-cell responses. Treatment utilizing both an ALK inhibitor and an anti-PD-1 antibody exhibits enhanced tumor regression and facilitates long-term immunity. Our study elucidates the role of RNase1 in mediating tumor resistance to immunotherapy and reveals an RNase1-mediated immunosuppressive tumor microenvironment, highlighting the potential of targeting RNase1 as a promising strategy for cancer immunotherapy in HCC.

Coffee as a dietary strategy to prevent SARS-CoV-2 infection.

BACKGROUND: To date, most countries lifted the restriction requirement and coexisted with SARS-CoV-2. Thus, dietary behavior for preventing SARS-CoV-2 infection becomes an interesting issue on a daily basis. Coffee consumption is connected with reduced COVID-19 risk and correlated to COVID-19 severity. However, the mechanisms of coffee for the reduction of COVID-19 risk are still unclear. RESULTS: Here, we identified that coffee can inhibit multiple variants of the SARS-CoV-2 infection by restraining the binding of the SARS-CoV-2 spike protein to human angiotensin-converting enzyme 2 (ACE2), and reducing transmembrane serine protease 2 (TMPRSS2) and cathepsin L (CTSL) activity. Then, we used the method of "Here" (HRMS-exploring-recombination-examining) and found that isochlorogenic acid A, B, and C of coffee ingredients showed their potential to inhibit SARS-CoV-2 infection (inhibitory efficiency 43-54%). In addition, decaffeinated coffee still preserves inhibitory activity against SARS-CoV-2. Finally, in a human trial of 64 subjects, we identified that coffee consumption (approximately 1-2 cups/day) is sufficient to inhibit infection of multiple variants of SARS-CoV-2 entry, suggesting coffee could be a dietary strategy to prevent SARS-CoV2 infection. CONCLUSIONS: This study verified moderate coffee consumption, including decaffeination, can provide a new guideline for the prevention of SARS-CoV-2. Based on the results, we also suggest a coffee-drinking plan for people to prevent infection in the post-COVID-19 era.

Continuous monitoring of a monoclonal antibody by size exclusion chromatography reveals a correlation between system suitability parameters and column aging.

Size exclusion chromatography (SEC) is a foundational analytical method to assess product purity of biological molecules. To ensure accurate and reproducible data that meet regulatory agency standards, it is critical to monitor the chromatographic column with efficient and continuous approaches. In this study, 19 SEC columns (Waters Acquity BEH200) were evaluated using an in-house monoclonal antibody made at Regeneron. System suitability parameters (SSPs) were used to monitor the performance of the SEC assay, including USP resolution, USP plate count, USP tailing factor, asymmetry factor, elution time, peak width, and peak height. A general linear model was built and revealed that elution time, peak width, asymmetry factor, and tailing factor increased with injection number, while peak height, resolution, and plate count decreased. After 1000 injections, tailing factor and peak width increased by more than 10%, while resolution and plate count decreased by more than 10% from their respective starting values.

The natural tannins oligomeric proanthocyanidins and punicalagin are potent inhibitors of infection by SARS-CoV-2.

The Coronavirus Disease 2019 (COVID-19) pandemic continues to infect people worldwide. While the vaccinated population has been increasing, the rising breakthrough infection persists in the vaccinated population. For living with the virus, the dietary guidelines to prevent virus infection are worthy of and timely to develop further. Tannic acid has been demonstrated to be an effective inhibitor of coronavirus and is under clinical trial. Here we found that two other members of the tannins family, oligomeric proanthocyanidins (OPCs) and punicalagin, are also potent inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection with different mechanisms. OPCs and punicalagin showed inhibitory activity against omicron variants of SARS-CoV-2 infection. The water extractant of the grape seed was rich in OPCs and also exhibited the strongest inhibitory activities for viral entry of wild-type and other variants in vitro. Moreover, we evaluated the inhibitory activity of grape seed extractants (GSE) supplementation against SARS-CoV-2 viral entry in vivo and observed that serum samples from the healthy human subjects had suppressive activity against different variants of SARS-CoV-2 Vpp infection after taking GSE capsules. Our results suggest that natural tannins acted as potent inhibitors against SARS-CoV-2 infection, and GSE supplementation could serve as healthy food for infection prevention.

Since it first surfaced in late 2019, the COVID-19 pandemic has had a significant impact on people's lives. While several vaccines have been created, infections have not disappeared. This is largely due to new variants of the virus responsible for the disease (SARS-CoV-2) emerging, which current vaccines do not work as well against. Indeed, several reports suggest that protection from the omicron variant wanes as shortly as four to six months after vaccination. Therefore, other strategies are needed to reduce the risk of SARS-CoV-2 infections. In 2022, researchers discovered that tannic acid blocked two proteins that SARS-CoV-2 needs to enter and replicate inside human cells. Tannic acid is part of the tannin family, which includes natural molecules found in plant-based meals and beverages. Here, Chen et al. ­ including some of the researchers involved in the 2022 studies ­ set out to find whether two other tannins found in nature (OPCs and punicalagin) could also inhibit SARS-CoV-2. Chen et al. administered tannic acid, OPCs and punicalagin to human cells cultured in a laboratory that had been infected with SARS-CoV-2. This revealed that all three tannins suppress the activity of the same proteins required for viral entry and replication, but to varying degrees suggesting that they block SARS-CoV-2 infections via different mechanisms. The compounds were also able to inhibit different variants of the virus, including omicron, from infecting the lab-grown cells. Further experiments revealed that water extracted from seeded grapes, which contains high levels of OPCs, could also block SARS-CoV-2 entry in the cell culture system. To test this further, Chen et al. gave 18 healthy individuals capsules containing different concentrations of grape seed extract and collected samples of their serum. The serum samples suppressed entry of different variants of SARS-CoV-2 in the cell culture system, with serums from subjects that received the higher dose having the greatest effect. These findings suggest that naturally occurring tannins can suppress multiple variants of SARS-CoV-2 from entering and replicating in cells. Consuming supplements of grape seed extract could potentially reduce the risk of SARS-CoV-2 infections. However, further experiments, including clinical trials, are needed to test this possibility.

Nuclear export signal mutation of epidermal growth factor receptor enhances malignant phenotypes of cancer cells.

Nuclear epidermal growth factor receptor (EGFR) has been shown to be correlated with drug resistance and a poor prognosis in patients with cancer. Previously, we have identified a tripartite nuclear localization signal (NLS) within EGFR. To comprehensively determine the functions and underlying mechanism of nuclear EGFR and its clinical implications, we aimed to explore the nuclear export signal (NES) sequence of EGFR that is responsible for interacting with the exportins. We combined in silico prediction with site-directed mutagenesis approaches and identified a putative NES motif of EGFR, which is located in amino acid residues 736-749. Mutation at leucine 747 (L747) in the EGFR NES led to increased nuclear accumulation of the protein via a less efficient release of the exportin CRM1. Interestingly, L747 with serine (L747S) and with proline (L747P) mutations were found in both tyrosine kinase inhibitor (TKI)-treated and -naïve patients with lung cancer who had acquired or de novo TKI resistance and a poor outcome. Reconstituted expression of the single NES mutant EGFRL747P or EGFRL747S, but not the dual mutant along with the internalization-defective or NLS mutation, in lung cancer cells promoted malignant phenotypes, including cell migration, invasiveness, TKI resistance, and tumor initiation, supporting an oncogenic role of nuclear EGFR. Intriguingly, cells with germline expression of the NES L747 mutant developed into B cell lymphoma. Mechanistically, nuclear EGFR signaling is required for sustaining nuclear activated STAT3, but not for Erk. These findings suggest that EGFR functions are compartmentalized and that nuclear EGFR signaling plays a crucial role in tumor malignant phenotypes, leading to tumorigenesis in human cancer.

An interlaboratory capillary zone electrophoresis-UV study of various monoclonal antibodies, instruments, and ε-aminocaproic acid lots.

Capillary zone electrophoresis ultraviolet (CZE-UV) has become increasingly popular for the charge heterogeneity determination of mAbs and vaccines. The ε-aminocaproic acid (eACA) CZE-UV method has been used as a rapid platform method. However, in the last years, several issues have been observed, for example, loss in electrophoretic resolution or baseline drifts. Evaluating the role of eACA on the reported issues, various laboratories were requested to provide their routinely used eACA CZE-UV methods, and background electrolyte compositions. Although every laboratory claimed to use the He et al. eACA CZE-UV method, most methods actually deviate from He's. Subsequently, a detailed interlaboratory study was designed wherein two commercially available mAbs (Waters' Mass Check Standard mAb [pI 7] and NISTmAb [pI 9]) were provided to each laboratory, along with two detailed eACA CZE-UV protocols for a short-end, high-speed, and a long-end, high-resolution method. Ten laboratories participated each using their own instruments, and commodities, showing excellence method performance (relative standard deviations [RSDs] of percent time-corrected main peak areas from 0.2% to 1.9%, and RSDs of migration times from 0.7% to 1.8% [n = 50 per laboratory], analysis times in some cases as short as 2.5 min). This study clarified that eACA is not the main reason for the abovementioned variations.

Interdisciplinary treatment with implant-supported two-unit cantilever prosthesis for a patient with hypodontia: A clinical report.

A 21-year-old woman with multiple congenitally missing maxillary anterior teeth received interdisciplinary treatment to restore function and esthetics. The treatment was initiated with orthodontic treatment, followed by implant placement, bone and soft-tissue augmentation, and prosthetic treatment including a screw-retained implant-supported 2-unit cantilever fixed dental prosthesis.

Validation of a shape-optimized 2-unit cantilevered inlay-retained fiber-reinforced resin-bonded dental prosthesis.

STATEMENT OF PROBLEM: Current designs of fiber-reinforced composite (FRC) resin-bonded fixed dental prostheses (RBFDPs) have a limited lifespan, failing mainly through veneer-fiber delamination, debonding, and fracture. PURPOSE: The purpose of this in vitro study was to validate a new inlay-retained 2-unit cantilevered RBFDP with an optimized cavity and fiber layout proposed in a previous study by using simulated occlusal loading. MATERIAL AND METHODS: Two groups of specimens (n=20), 1 with and 1 without glass fibers, were used to test the influence of the cavity design and that of the fiber layout on their load capacity, respectively. The specimens without fibers were directly cut from a resin-ceramic block by using a computer-aided manufacturing system, while those with fibers were manually fabricated with unidirectional glass fibers and composite resin in a silicone mold. The specimens with and without fibers were attached to abutments made of the same resin-ceramic with a cyanoacrylate-based adhesive and a resin-based dental cement, respectively. An increasing compressive load was applied on the mesial fossa of the premolar pontic until failure. Cracking in the specimens during loading was monitored with a 2-channel acoustic emission (AE) system. RESULTS: All the specimens without fiber reinforcement debonded from the abutments. Those using the optimized shovel-shaped cavity design had a mean ±standard deviation failure load (50.0 ±17.3 N) that was 193% higher than that of those with the conventional step-box design (17.1 ±6.2 N; P<.001). No significant difference was found between the groups for the mean number of AE events per specimen (step-box: 49 ±34 versus shovel-shaped: 63 ±34; P=.427), the mean amplitude of each event (58.4 ±1.3 dB versus 59.5 ±2.4 dB; P=.299), or the mean time to failure (283.2 ±122.3 seconds versus 297.5 ±66.7 seconds; P=.798). Between the groups of specimens with reinforcing fibers, the mean failure load of the conventional design was approximately half that of the optimized one. Again, no significant difference was found for the mean number of AE events per specimen (conventional: 28 ±18 versus optimized: 52 ±53; P=.248) or the mean amplitude for each AE event (64.9 ±4.2 dB versus 61.7 ±5.2 dB; P=.187). The connectors of 8 fiber-reinforced specimens with the conventional design fractured; the other 2 debonded from the abutments. Half of the shape-optimized fiber-reinforced specimens had fractured abutments, but the cantilevers remained intact, 4 specimens fractured at the connector, and only 1 debonded from its abutment. CONCLUSIONS: The shape-optimized 2-unit cantilevered FRC RBFDP had a higher load capacity than the conventional design.

Targeting the ALK-CDK9-Tyr19 kinase cascade sensitizes ovarian and breast tumors to PARP inhibition via destabilization of the P-TEFb complex.

Poly(ADP-ribose) polymerase (PARP) inhibitors have demonstrated promising clinical activity in multiple cancers. However, resistance to PARP inhibitors remains a substantial clinical challenge. In the present study, we report that anaplastic lymphoma kinase (ALK) directly phosphorylates CDK9 at tyrosine-19 to promote homologous recombination (HR) repair and PARP inhibitor resistance. Phospho-CDK9-Tyr19 increases its kinase activity and nuclear localization to stabilize positive transcriptional elongation factor b and activate polymerase II-dependent transcription of HR-repair genes. Conversely, ALK inhibition increases ubiquitination and degradation of CDK9 by Skp2, an E3 ligase. Notably, combination of US Food and Drug Administration-approved ALK and PARP inhibitors markedly reduce tumor growth and improve survival of mice in PARP inhibitor-/platinum-resistant tumor xenograft models. Using human tumor biospecimens, we further demonstrate that phosphorylated ALK (p-ALK) expression is associated with resistance to PARP inhibitors and positively correlated with p-Tyr19-CDK9 expression. Together, our findings support a biomarker-driven, combinatorial treatment strategy involving ALK and PARP inhibitors to induce synthetic lethality in PARP inhibitor-/platinum-resistant tumors with high p-ALK-p-Tyr19-CDK9 expression.

Patient-derived tumor organoids as a platform of precision treatment for malignant brain tumors.

Malignant brain tumors consist of malignancies originated primarily within the brain and the metastatic lesions disseminated from other organs. In spite of intensive studies, malignant brain tumors remain to be a medical challenge. Patient-derived organoid (PDO) can recapitulate the biological features of the primary tumor it was derived from and has emerged as a promising drug-screening model for precision therapy. Here we show a proof-of-concept based on early clinical study entailing the organoids derived from the surgically resected tumors of 26 patients with advanced malignant brain tumors enrolled during December 2020 to October 2021. The tumors included nine glioma patients, one malignant meningioma, one primary lymphoma patient, and 15 brain metastases. The primary tumor sites of the metastases included five from the lungs, three from the breasts, two from the ovaries, two from the colon, one from the testis, one of melanoma origin, and one of chondrosarcoma. Out of the 26 tissues, 13 (50%) organoids were successfully generated with a culture time of about 2 weeks. Among these patients, three were further pursued to have the organoids derived from their tumor tissues tested for the sensitivity to different therapeutic drugs in parallel to their clinical care. Our results showed that the therapeutic effects observed by the organoid models were consistent to the responses of these patients to their treatments. Our study suggests that PDO can recapitulate patient responses in the clinic with high potential of implementation in personalized medicine of malignant brain tumors.

Growth-promoting function of the cGAS-STING pathway in triple-negative breast cancer cells.

The cGAS-STING axis is one of the key mechanisms guarding cells from pathogen invasion in the cytoplasmic compartment. Sensing of foreign DNA in the cytosol by the cGAS-STING axis triggers a stress cascade, culminating at stimulation of the protein kinase TBK1 and subsequently activation of inflammatory response. In cancer cells, aberrant metabolism of the genomic DNA induced by the hostile milieu of tumor microenvironment or stresses brought about by cancer therapeutics are the major causes of the presence of nuclear DNA in the cytosol, which subsequently triggers a stress response. However, how the advanced tumors perceive and tolerate the potentially detrimental effects of cytosolic DNA remains unclear. Here we show that growth limitation by serum starvation activated the cGAS-STING pathway in breast cancer cells, and inhibition of cGAS-STING resulted in cell death through an autophagy-dependent mechanism. These results suggest that, instead of being subject to growth inhibition, tumors exploit the cGAS-STING axis and turn it to a survival advantage in the stressful microenvironment, providing a new therapeutic opportunity against advanced cancer. Concomitant inhibition of the cGAS-STING axis and growth factor signaling mediated by the epidermal growth factor receptor (EGFR) synergistically suppressed the development of tumor organoids derived from primary tumor tissues of triple-negative breast cancer (TNBC). The current study unveils an unexpected function of the cGAS-STING axis in promoting cancer cell survival and the potential of developing the stress-responding pathway as a therapeutic target, meanwhile highlights the substantial concerns of enhancing the pathway's activity as a means of anti-cancer treatment.

Prospects of Coffee Leaf against SARS-CoV-2 Infection.

In the current climate, many countries are in dire need of effective preventive methods to curb the Severe Acute Respiratory Syndrome Coronavirus Type 2 (SARS-CoV-2) pandemic. The purpose of this research is to screen and explore natural plant extracts that have the potential to against SARS-CoV-2 and provide alternative options for SARS-CoV-2 prevention and hand sanitizer or spray-like disinfectants. We first used Spike-ACE2 ELISA and TMPRSS2 fluorescence resonance energy transfer (FRET) assays to screen extracts from agricultural by-products from Taiwan with the potential to impede SARS-CoV-2 infection. Next, the SARS-CoV-2 pseudo-particles (Vpp) infection assay was tested to validate the effectiveness. We identified an extract from coffee leaf (Coffea Arabica), a natural plant that effectively inhibited wild-type SARS-CoV-2, and five Variants of Concern (Alpha, Beta, Gamma, Delta, and Omicron strain) from entering host cells. In an attempt to apply coffee leaf extract for hand sanitizer or spray-like disinfectants, we designed a skin-like gelatin membrane experiment. We showed that the high concentration of coffee leaf extract on the skin surface could block SARS-CoV-2 into cells more potently than 75% Ethanol, a standard disinfectant to inactivate SARS-CoV-2. Finally, LC-HRMS analysis was used to identify compounds such as caffeine, chlorogenic acid (CGA), quinic acid, and mangiferin that are associated with an anti-SARS-CoV-2 activity. Our results demonstrated that coffee leaf extract, an agricultural by-product effectively inhibits SARS-CoV-2 Vpp infection through an ACE2-dependent mechanism and may be utilized to develop products against SARS-CoV-2 infection.

Natural tannins as anti-SARS-CoV-2 compounds.

Tannins are polyphenols enriched in wood, bark, roots, leaves, seeds and fruits of a variety of plants. Over the last two decades, there has been an increasing interest in understanding the biological functions of tannins and their applications as antioxidants, anticancer drugs, and food additives. Since the outbreak of the COVID-19 pandemic, much effort has been devoted to finding an expedient cure. Tannins have been put forward as having possible anti-COVID-19 properties; however, owing to the profuse nature of the structurally diverse derivatives of tannins, the tannin species in the family associated with an indication of anti-COVID-19 have been poorly defined, compounded by frequent terminology misnomers. This article reviews the tannin family in fruits and the current knowledge about the activities of the compounds with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It will aid molecular and cellular biologists in developing natural anti-viral chemicals as means of overcoming the current and future pandemics.

Peimine inhibits variants of SARS-CoV-2 cell entry via blocking the interaction between viral spike protein and ACE2.

Coronavirus disease 2019 (COVID-19) is caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Several vaccines against SARS-CoV-2 have been approved; however, variants of concern (VOCs) can evade vaccine protection. Therefore, developing small compound drugs that directly block the interaction between the viral spike glycoprotein and ACE2 is urgently needed to provide a complementary or alternative treatment for COVID-19 patients. We developed a viral infection assay to screen a library of approximately 126 small molecules and showed that peimine inhibits VOCs viral infections. In addition, a fluorescence resonance energy transfer (FRET) assay showed that peimine suppresses the interaction of spike and ACE2. Molecular docking analysis revealed that peimine exhibits a higher binding affinity for variant spike proteins and is able to form hydrogen bonds with N501Y in the spike protein. These results suggest that peimine, a compound isolated from Fritillaria, may be a potent inhibitor of SARS-CoV-2 variant infection. PRACTICAL APPLICATIONS: In this study, we identified a naturally derived compound of peimine, a major bioactive alkaloid extracted from Fritillaria, that could inhibit SARS-CoV-2 variants of concern (VOCs) viral infection in 293T/ACE2 and Calu-3 lung cells. In addition, peimine blocks viral entry through interruption of spike and ACE2 interaction. Moreover, molecular docking analysis demonstrates that peimine has a higher binding affinity on N501Y in the spike protein. Furthermore, we found that Fritillaria significantly inhibits SARS-CoV-2 viral infection. These results suggested that peimine and Fritillaria could be a potential functional drug and food for COVID-19 patients.

The Functions of PCNA in Tumor Stemness and Invasion.

Invasion is the most prominent lethal feature of malignant cancer. However, how cell proliferation, another important feature of tumor development, is integrated with tumor invasion and the subsequent cell dissemination from primary tumors is not well understood. Proliferating cell nuclear antigen (PCNA) is essential for DNA replication in cancer cells. Loss of phosphorylation at tyrosine 211 (Y211) in PCNA (pY211-PCNA) mitigates PCNA function in proliferation, triggers replication fork arrest/collapse, which in turn sets off an anti-tumor inflammatory response, and suppresses distant metastasis. Here, we show that pY211-PCNA is important in stromal activation in tumor tissues. Loss of the phosphorylation resulted in reduced expression of mesenchymal proteins as well as tumor progenitor markers, and of the ability of invasion. Spontaneous mammary tumors that developed in mice lacking Y211 phosphorylation contained fewer tumor-initiating cells compared to tumors in wild-type mice. Our study demonstrates a novel function of PCNA as an essential factor for maintaining cancer stemness through Y211 phosphorylation.

Evaluation of Shear Capacity of Steel Fiber Reinforced Concrete Beams without Stirrups Using Artificial Intelligence Models.

The shear transfer mechanism of steel fiber reinforced concrete (SFRC) beams without stirrups is still not well understood. This is demonstrated herein by examining the accuracy of typical empirical formulas for 488 SFRC beam test records compiled from the literature. To steer clear of these cognitive limitations, this study turned to artificial intelligence (AI) models. A gray relational analysis (GRA) was first conducted to evaluate the importance of different parameters for the problem at hand. The outcomes indicate that the shear capacity depends heavily on the material properties of concrete, the amount of longitudinal reinforcement, the attributes of steel fibers, and the geometrical and loading characteristics of SFRC beams. After this, AI models, including back-propagation artificial neural network, random forest and multi-gene genetic programming, were developed to capture the shear strength of SFRC beams without stirrups. The findings unequivocally show that the AI models predict the shear strength more accurately than do the empirical formulas. A parametric analysis was performed using the established AI model to investigate the effects of the main influential factors (determined by GRA) on the shear capacity. Overall, this paper provides an accurate, instantaneous and meaningful approach for evaluating the shear capacity of SFRC beams containing no stirrups.

De-glycosylated membrane PD-L1 in tumor tissues as a biomarker for responsiveness to atezolizumab (Tecentriq) in advanced breast cancer patients.

The atezolizumab (Tecentriq), a humanized antibody against human programmed death ligand 1 (PD-L1), combined with nab-paclitaxel was granted with accelerated approval to treat unresectable locally advanced or metastatic triple-negative breast cancer (TNBC) due to the encouraging positive results of the phase 3 IMpassion130 trial using PD-L1 biomarker from immune cells to stratify patients. However, the post-market study IMpassion131 did not support the original observation, resulting in the voluntary withdrawal of atezolizumab from the indication in breast cancer by Genentech in 2021. Emerging evidence has revealed a high frequency of false negative result using the standard immunohistochemical (IHC) staining due to heavy glycosylation of PD-L1. The removal of glycosylation prevents from the false negative staining, enabling more accurate assessment of PD-L1 levels and improving prediction for response to immune checkpoint therapy. In the present study, the natural and de-glycosylated PD-L1 expression in tumor and immune cells from nine TNBC patients were analyzed by using clone 28-8 monoclonal antibody to correlate with treatment outcome. Our results demonstrate that: (1) Removal of the glycosylation indeed enhances the detection of PD-L1 by IHC staining, (2) The PD-L1 levels on tumor cell surface after removal of the glycosylation correlates well with clinical responses for atezolizumab treatment; (3) The criteria used in the IMpassion130 and IMpassion131 trials which scored the natural PD-L1 in the immune cells failed to correlate with the clinical response. Taken together, tumor cell surface staining of PD-L1 with de-glycosylation has a significant correlation with the clinical response for atezolizumab treatment, suggesting that treatment of atezolizumab may be worthy of further consideration with de-glycosylation procedure as a patient stratification strategy. A larger cohort to validate this important issue is warranted to ensure right patient population who could benefit from the existing FDA-approved drugs.

miR-4759 suppresses breast cancer through immune checkpoint blockade.

Programmed cell death protein 1 (PD-1)/ programmed cell death protein ligand 1 (PD-L1) is the key immune checkpoint governing evasion of advanced cancer from immune surveillance. Immuno-oncology (IO) therapy targeting PD-1/PD-L1 with traditional antibodies is a promising approach to multiple cancer types but to which the response rate remains moderate in breast cancer, calling for the need of exploring alternative IO targeting approaches. A miRNA-gene network was integrated by a bioinformatics approach and corroborated with The Cancer Genome Atlas (TCGA) to screen miRNAs regulating immune checkpoint genes and associated with patient survival. Here we show the identification of a novel microRNA miR-4759 which repressed RNA expression of the PD-L1 gene. miR-4759 targeted the PD-L1 gene through two binding motifs in the 3' untranslated region (3'-UTR) of PD-L1. Reconstitution of miR-4759 inhibited PD-L1 expression and sensitized breast cancer cells to killing by immune cells. Treatment with miR-4759 suppressed tumor growth of orthotopic xenografts and promoted tumor infiltration of CD8+ T lymphocytes in immunocompetent mice. In contrast, miR-4759 had no effect to tumor growth in immunodeficient mice. In patients with breast cancer, expression of miR-4759 was preferentially downregulated in tumors compared to normal tissues and was associated with poor overall survival. Together, our results demonstrated miR-4759 as a novel non-coding RNA which promotes anti-tumor immunity of breast cancer.