KRAS inhibition activates an actionable CD24 'don't eat me' signal in pancreas cancer.

KRAS G12C inhibitor (G12Ci) has produced encouraging, albeit modest and transient, clinical benefit in pancreatic ductal adenocarcinoma (PDAC). Identifying and targeting resistance mechanisms to G12Ci treatment is therefore crucial. To better understand the tumor biology of the KRAS G12C allele and possible bypass mechanisms, we developed a novel autochthonous KRAS G12C -driven PDAC model. Compared to the classical KRAS G12D PDAC model, the G12C model exhibit slower tumor growth, yet similar histopathological and molecular features. Aligned with clinical experience, G12Ci treatment of KRAS G12C tumors produced modest impact despite stimulating a 'hot' tumor immune microenvironment. Immunoprofiling revealed that CD24, a 'do-not-eat-me' signal, is significantly upregulated on cancer cells upon G12Ci treatment. Blocking CD24 enhanced macrophage phagocytosis of cancer cells and significantly sensitized tumors to G12Ci treatment. Similar findings were observed in KRAS G12D -driven PDAC. Our study reveals common and distinct oncogenic KRAS allele-specific biology and identifies a clinically actionable adaptive mechanism that may improve the efficacy of oncogenic KRAS inhibitor therapy in PDAC. Significance: Lack of faithful preclinical models limits the exploration of resistance mechanisms to KRAS G12C inhibitor in PDAC. We generated an autochthonous KRAS G12C -driven PDAC model, which revealed allele-specific biology of the KRAS G12C during PDAC development. We identified CD24 as an actionable adaptive mechanisms in cancer cells induced upon KRAS G12C inhibition and blocking CD24 sensitizes PDAC to KRAS inhibitors in preclinical models.

Identification of biomarkers associated with diagnosis of postmenopausal osteoporosis patients based on bioinformatics and machine learning.

Background: Accumulating evidence suggests that postmenopausal osteoporosis (PMOP) is a common chronic systemic metabolic bone disease, but its specific molecular pathogenesis remains unclear. This study aimed to identify novel genetic diagnostic markers for PMOP. Methods: In this paper, we combined three GEO datasets to identify differentially expressed genes (DEGs) and performed functional enrichment analysis of PMOP-related differential genes. Key genes were analyzed using two machine learning algorithms, namely, LASSO and the Gaussian mixture model, and candidate biomarkers were found after taking the intersection. After further ceRNA network construction, methylation analysis, and immune infiltration analysis, ACACB and WWP1 were finally selected as diagnostic markers. Twenty-four clinical samples were collected, and the expression levels of biomarkers in PMOP were detected by qPCR. Results: We identified 34 differential genes in PMOP. DEG enrichment was mainly related to amino acid synthesis, inflammatory response, and apoptosis. The ceRNA network construction found that XIST-hsa-miR-15a-5p/hsa-miR-15b-5p/hsa-miR-497-5p and hsa-miR-195-5p-WWP1/ACACB may be RNA regulatory pathways regulating PMOP disease progression. ACACB and WWP1 were identified as diagnostic genes for PMOP, and validated in datasets and clinical sample experiments. In addition, these two genes were also significantly associated with immune cells, such as T, B, and NK cells. Conclusion: Overall, we identified two vital diagnostic genes responsible for PMOP. The results may help provide potential immunotherapeutic targets for PMOP.

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.

[Treatment of complex acetabular fractures with improved Stoppa combined with Kocher-Langenbeck approach assisted by 3D printing technology].

OBJECTIVE: To explore the clinical effect of the modified Stoppa combined with Kocher-Langenbeck(K-L) approach and 3D printing technology in the treatment of complex acetabular fractures. METHODS: The surgical methods of 70 patients with complex acetabular fractures admitted from April 2013 to July 2019 were retrospectively analyzed. Among them, 25 cases were operated with 3D printing technology(3D printing group) and 45 cases were operated with conventional operation(conventional operation group). The operation time, intraoperative blood loss, intraoperative fluoroscopy times, complications and functional recovery of the hip joint of two groups were recorded. RESULTS: There were statistically significant differences between two groups in terms of operation time, intraoperative bleeding volume and intraoperative fluoroscopy times(P<0.01). All patients were followed up for (15.8±3.5) months, and all patients achieved bony healing. There was no significant difference in the results of Matta reduction between two groups(P>0.05). There was no significant difference in Harris score at 6 months after operation between two groups(P>0.05). CONCLUSION: With the aid of 3D printing technology, the improved Stoppa combined with K-L approach is used to treat complex acetabular fractures. It has certain advantages in helping clinicians to formulate a reasonable operation plan before operation, avoid unnecessary operation and trauma during operation, shorten the operation time and reduce the amount of bleeding during operation. It can improve the safety during the perioperative period, facilitate the accurate reduction of fractures during operation and facilitate the recovery of patients.

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.

Phosphorylation and Stabilization of PD-L1 by CK2 Suppresses Dendritic Cell Function.

Targeting immune checkpoints such as programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) has transformed cancer treatment, with durable clinical responses across a wide range of tumor types. However, a high percentage of patients fail to respond to anti-PD-1/PD-L1 treatment. A greater understanding of PD-L1 regulation is critical to improving the clinical response rate of PD-1/PD-L1 blockade. Here, we demonstrate that PD-L1 is phosphorylated and stabilized by casein kinase 2 (CK2) in cancer and dendritic cells (DC). Phosphorylation of PD-L1 at Thr285 and Thr290 by CK2 disrupted PD-L1 binding with speckle-type POZ protein, an adaptor protein of the cullin 3 (CUL3) ubiquitin E3 ligase complex, protecting PD-L1 from CUL3-mediated proteasomal degradation. Inhibition of CK2 decreased PD-L1 protein levels by promoting its degradation and resulted in the release of CD80 from DC to reactivate T-cell function. In a syngeneic mouse model, combined treatment with a CK2 inhibitor and an antibody against T-cell immunoglobulin mucin-3 (Tim-3) suppressed tumor growth and prolonged survival. These findings uncover a mechanism by which PD-L1 is regulated and suggest a potential antitumor treatment option to activate DC function by blocking the CK2-PD-L1 pathway and inhibiting Tim-3. SIGNIFICANCE: This work identifies a role for CK2 in immunosuppression by phosphorylation and stabilization of PD-L1, identifying CK2 inhibition as an immunotherapeutic approach for treating cancer.

An optimized protocol for PD-L1 pathological assessment with patient sample deglycosylation to improve correlation with therapeutic response.

Immunotherapy via PD-1/PD-L1 blockade is a promising strategy to eradicate cancer cells. However, the PD-L1 pathological level is inconsistent with the therapeutic response and is not a reliable biomarker to stratify patients for anti-PD-1/PD-L1 therapy. Here, we describe patient sample deglycosylation in an immunohistochemistry (IHC) assay to resolve this challenge. This protocol facilitates antigen retrieval by removing N-glycans from surface antigens on formalin-fixed paraffin-embedded (FFPE) tissue slides and can be applied in medical pathology for multiple cancer types. For complete details on the use and execution of this profile, please refer to Lee et al. (2019).

miR-424-5p shuttled by bone marrow stem cells-derived exosomes attenuates osteogenesis via regulating WIF1-mediated Wnt/ß-catenin axis.

Emerging evidence proves that exosomes contain specific microRNAs(miRNAs) contribute to osteogenic differentiation of bone marrow stem cells (BMSCs). However, the role and mechanism of bone marrow stem cells (BMSCs)-derived exosomes overexpressing miR-424-5p in osteoblasts remains unclear. Firstly, the BMSCs-derived exosomes were isolated, and identified by Western blot with the exosome surface markers CD9, CD81 and CD63. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect the level of miR-424-5p in exosomes, and western blot was implemented to verify the WIF1/Wnt/ß-catenin expression. The binding association between miR-424-5p and WIF1 was determined by the dual-luciferase reporter gene assay. Functional enhancement experiments were adopted to determine the role of exosome-carried miR-424-5p and WIF1/Wnt/ß-catenin in osteogenic differentiation. ALP staining was adopted, and levels of RUNX2, OCN, and OPN were monitored using qRT-PCR to determine osteogenic differentiation. As a result, In vivo experiments showed that RUNX2, OCN and OPN levels decreased and the ALP activity was dampened after miR-424-5p overexpression in exosomes. Besides, exosomes overexpressing miR-424-5p attenuated osteogenic development via WIF1/Wnt/ß-catenin. Our findings may bring evidence for miR-424-5p as a new biomarker for the treatment of osteoporosis.

Activated T cell-derived exosomal PD-1 attenuates PD-L1-induced immune dysfunction in triple-negative breast cancer.

Programmed cell death 1 (PD-1) is widely expressed in tumor-infiltrating lymphocytes (TILs) of triple-negative breast cancer (TNBC). As a dominant inhibitory immune checkpoint (ICP) receptor, cell surface PD-1 is well-known to transduce negative signaling of effector T cell activity during cell-cell contact. However, despite its well-documented inhibitory effects, higher PD-1 expression in TILs is significantly associated with longer survival in TNBC patients. This phenomenon raises an interesting question whether PD-1 harbors positive activity to enhance anti-tumor immunity. Here, we show that PD-1 is secreted in an exosomal form by activated T cells and can remotely interact with either cell surface or exosomal programmed death-ligand 1 (PD-L1), induce PD-L1 internalization via clathrin-mediated endocytosis, and thereby prevent subsequent cellular PD-L1: PD-1 interaction, restoring tumor surveillance through attenuating PD-L1-induced suppression of tumor-specific cytotoxic T cell activity. Our results, through revealing an anti-PD-L1 function of exosomal PD-1, provide a positive role to enhance cytotoxic T cell activity and a potential therapeutic strategy of modifying the exosome surface with membrane-bound inhibitory ICP receptors to attenuate the suppressive tumor immune microenvironment.

Human ribonuclease 1 serves as a secretory ligand of ephrin A4 receptor and induces breast tumor initiation.

Human ribonuclease 1 (hRNase 1) is critical to extracellular RNA clearance and innate immunity to achieve homeostasis and host defense; however, whether it plays a role in cancer remains elusive. Here, we demonstrate that hRNase 1, independently of its ribonucleolytic activity, enriches the stem-like cell population and enhances the tumor-initiating ability of breast cancer cells. Specifically, secretory hRNase 1 binds to and activates the tyrosine kinase receptor ephrin A4 (EphA4) signaling to promote breast tumor initiation in an autocrine/paracrine manner, which is distinct from the classical EphA4-ephrin juxtacrine signaling through contact-dependent cell-cell communication. In addition, analysis of human breast tumor tissue microarrays reveals a positive correlation between hRNase 1, EphA4 activation, and stem cell marker CD133. Notably, high hRNase 1 level in plasma samples is positively associated with EphA4 activation in tumor tissues from breast cancer patients, highlighting the pathological relevance of the hRNase 1-EphA4 axis in breast cancer. The discovery of hRNase 1 as a secretory ligand of EphA4 that enhances breast cancer stemness suggests a potential treatment strategy by inactivating the hRNase 1-EphA4 axis.

TYRO3 induces anti-PD-1/PD-L1 therapy resistance by limiting innate immunity and tumoral ferroptosis.

Immune checkpoint blockade therapy has demonstrated promising clinical outcomes for multiple cancer types. However, the emergence of resistance as well as inadequate biomarkers for patient stratification have largely limited the clinical benefits. Here, we showed that tumors with high TYRO3 expression exhibited anti-programmed cell death protein 1/programmed death ligand 1 (anti-PD-1/PD-L1) resistance in a syngeneic mouse model and in patients who received anti-PD-1/PD-L1 therapy. Mechanistically, TYRO3 inhibited tumor cell ferroptosis triggered by anti-PD-1/PD-L1 and facilitated the development of a protumor microenvironment by reducing the M1/M2 macrophage ratio, resulting in resistance to anti-PD-1/PD-L1 therapy. Inhibition of TYRO3 promoted tumor ferroptosis and sensitized resistant tumors to anti-PD-1 therapy. Collectively, our findings suggest that TYRO3 could serve as a predictive biomarker for patient selection and a promising therapeutic target to overcome anti-PD-1/PD-L1 resistance.

[Relationship between fabella and posterolateral knee pain and common peroneal nerve injury].

OBJECTIVE: To analyze the incidence and characteristics of fabella in the Chinese population and its correlation with pain in the posterolateral region of the knee joint and common peroneal nerve palsy. METHODS: Total 732 patients including 405 males(450 knees) and 327 females(383 knees) who underwent unilateral or bilateral knee MR imaging from September 2015 to July 2019 were retrospectively evaluated. The basic information of all patients was extracted from the hospital's his system. The patient's medical records were checked by telephone follow-up or his system, and the number of patients with posterolateral knee pain and common peroneal nerve paralysis were recorded. RESULTS: The overall prevalence of fabella was 48.38%, 23.53% in men and 24.85% in women, there was no significant difference between them (P>0.05). All the subjects were divided into five age groups. The prevalence of fabella was significantly different among different age groups:6.6% in 20 year-old group, 33.8% in 21 to 34-year-old group, 53.5% in 35 to 44-year-old group, 57.5% in 45 to 59-year-old group and 73.9% in ≥ 60-year-old group, the difference was statistically significant (P<0.001). There was a significant correlation between the prevalence of fabella and the age of patients. With the increase of patients' age, the prevalence of fabella in knee joint also showed an obvious upward trend (P<0.001). According to the presence of fabella in the knee joint, 232 cases of knee joint pain were found, accounting for 57.57% of the patients with fabella, accounting for 27.85% of the total data(P<0.01). The correlation score analysis was R=1.546, P<0.01;when the presence of fabella in the knee joint, a total of 44 cases of common peroneal nerve paralysis occurred in the knee joint, accounting for all knees 28% of the total knee joint, and 29 cases of common peroneal nerve palsy (3.48% of the total knee joint) were found when there was no fabella in the knee joint(P<0.05). The correlation score analysis was performed with R=1.695, P<0.05. CONCLUSION: The prevalence of fabella us in Chinese population is 48.38%. There is no relationship between the incidence of gastrocnemius and gender, but the incidence of fabella is positively correlated with age, pain in the posterolateral region of the knee joint and the occurrence of common peroneal nerve symptoms.

Inhibition of CDK2 reduces EZH2 phosphorylation and reactivates ERα expression in high-grade serous ovarian carcinoma.

The cyclin-dependent kinase 2 (CDK2) inhibitor dinaciclib, a potential anti-cancer drug, has been tested in clinical trials and reported to suppress tumor initiating cells. Our recent study demonstrated that pharmacological inhibition of CDK2 or enhancer of zeste homolog 2 (EZH2) allows re-expression of ERα and converts triple-negative breast cancers (TNBC) to luminal ERα-positive, rendering TNBC cells targetable by tamoxifen. Like TNBC, EZH2 is also commonly overexpressed in ovarian cancers, and overexpression of cyclin E1 gene (CCNE1) and/or amplification of its associated kinase CDK2 gene is present in ovarian tumor specimens, both of which are associated with primary treatment resistance and poor outcome in high-grade serous ovarian cancer (HGSC). We determined whether inhibition of CDK2-mediated phosphorylation of EZH2 activates ERα expression in ERα-negative HGSOC cells, rendering them targetable by hormonal therapy. The specific CDK2 inhibitor repressed phosphorylation of EZH2 at T416, and in turn activated the expression of its downstream target ERα gene (ESR1). We tested the efficacy of the combination of CDK2 inhibitor and tamoxifen and found significant synergistic inhibition. We further demonstrated that CDK2 inhibitor is a more promising agent than EZH2 inhibitor in repressing TNBC and HGSOC due to a feedback increase in CDK2 activity by EZH2 inhibitor. Our results indicated that the combination treatment of CDK2 inhibitor and tamoxifen has the potential to benefit patients with ERα-negative HGSOC.

Blocking immunosuppressive neutrophils deters pY696-EZH2-driven brain metastases.

The functions of immune cells in brain metastases are unclear because the brain has traditionally been considered "immune privileged." However, we found that a subgroup of immunosuppressive neutrophils is recruited into the brain, enabling brain metastasis development. In brain metastatic cells, enhancer of zeste homolog 2 (EZH2) is highly expressed and phosphorylated at tyrosine-696 (pY696)-EZH2 by nuclear-localized Src tyrosine kinase. Phosphorylation of EZH2 at Y696 changes its binding preference from histone H3 to RNA polymerase II, which consequently switches EZH2's function from a methyltransferase to a transcription factor that increases c-JUN expression. c-Jun up-regulates protumorigenic inflammatory cytokines, including granulocyte colony-stimulating factor (G-CSF), which recruits Arg1+- and PD-L1+ immunosuppressive neutrophils into the brain to drive metastasis outgrowth. G-CSF-blocking antibodies or immune checkpoint blockade therapies combined with Src inhibitors impeded brain metastasis in multiple mouse models. These findings indicate that pY696-EZH2 can function as a methyltransferase-independent transcription factor to facilitate the brain infiltration of immunosuppressive neutrophils, which could be clinically targeted for brain metastasis treatment.

The gluconeogenic enzyme PCK1 phosphorylates INSIG1/2 for lipogenesis.

Cancer cells increase lipogenesis for their proliferation and the activation of sterol regulatory element-binding proteins (SREBPs) has a central role in this process. SREBPs are inhibited by a complex composed of INSIG proteins, SREBP cleavage-activating protein (SCAP) and sterols in the endoplasmic reticulum. Regulation of the interaction between INSIG proteins and SCAP by sterol levels is critical for the dissociation of the SCAP-SREBP complex from the endoplasmic reticulum and the activation of SREBPs1,2. However, whether this protein interaction is regulated by a mechanism other than the abundance of sterol-and in particular, whether oncogenic signalling has a role-is unclear. Here we show that activated AKT in human hepatocellular carcinoma (HCC) cells phosphorylates cytosolic phosphoenolpyruvate carboxykinase 1 (PCK1), the rate-limiting enzyme in gluconeogenesis, at Ser90. Phosphorylated PCK1 translocates to the endoplasmic reticulum, where it uses GTP as a phosphate donor to phosphorylate INSIG1 at Ser207 and INSIG2 at Ser151. This phosphorylation reduces the binding of sterols to INSIG1 and INSIG2 and disrupts the interaction between INSIG proteins and SCAP, leading to the translocation of the SCAP-SREBP complex to the Golgi apparatus, the activation of SREBP proteins (SREBP1 or SREBP2) and the transcription of downstream lipogenesis-related genes, proliferation of tumour cells, and tumorigenesis in mice. In addition, phosphorylation of PCK1 at Ser90, INSIG1 at Ser207 and INSIG2 at Ser151 is not only positively correlated with the nuclear accumulation of SREBP1 in samples from patients with HCC, but also associated with poor HCC prognosis. Our findings highlight the importance of the protein kinase activity of PCK1 in the activation of SREBPs, lipogenesis and the development of HCC.

Blocking c-Met and EGFR reverses acquired resistance of PARP inhibitors in triple-negative breast cancer.

The limited treatment options and therapeutic failure due to acquired resistance for patients with triple-negative breast cancer (TNBC) represent a significant challenge. Inhibitors against poly (ADP-ribose) polymerase (PARP), olaparib and talazoparib, were recently approved for the treatment of metastatic breast cancer (including TNBC) in patients with germline BRCA1/2 mutations. Despite impressive response rates of ~60%, the prolongation in median progression-free survival with a PARPi is modest, suggesting the emergence of resistance. Several studies have reported that receptor tyrosine kinases (RTKs), such as c-MET (also known as hepatocyte growth factor receptor), are involved in resistance to various anti-neoplastic agents, including PARPi. However, the mechanism by which c-MET contributes to acquired resistance to PARPi in TNBC is not fully understood. In this study, we show that hyperactivated c-Met is detected in TNBC cells with acquired resistance to PARPi, and the combination of talazoparib and crizotinib (a multi-kinase inhibitor that inhibits c-MET) synergistically inhibits proliferation in these cells. Unexpectedly, depleting c-MET had limited effect on talazoparib sensitivity in PARPi-resistant cells. Interestingly, we found evidence of epidermal growth factor receptor (EGFR) hyperactivation and interaction of EGFR/c-Met in these cells. Notably, combining EGFR and PARP inhibitors resulted in greater inhibition of proliferation in c-MET-depleted TNBC cells, and combined c-MET and EGFR inhibition increased sensitivity to talazoparib in TNBC cells with acquired resistance to PARPi. Our findings suggest that combined inhibition of c-MET and EGFR could potentially re-sensitize TNBC to the cytotoxic effects of PARPi.

Author Correction: CDK2-mediated site-specific phosphorylation of EZH2 drives and maintains triple-negative breast cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

CDK2-mediated site-specific phosphorylation of EZH2 drives and maintains triple-negative breast cancer.

Triple-negative breast cancer (TNBC), which lacks estrogen receptor α (ERα), progesterone receptor, and human epidermal growth factor receptor 2 (HER2) expression, is closely related to basal-like breast cancer. Previously, we and others report that cyclin E/cyclin-dependent kinase 2 (CDK2) phosphorylates enhancer of zeste homolog 2 (EZH2) at T416 (pT416-EZH2). Here, we show that transgenic expression of phospho-mimicking EZH2 mutant EZH2T416D in mammary glands leads to tumors with TNBC phenotype. Coexpression of EZH2T416D in mammary epithelia of HER2/Neu transgenic mice reprograms HER2-driven luminal tumors into basal-like tumors. Pharmacological inhibition of CDK2 or EZH2 allows re-expression of ERα and converts TNBC to luminal ERα-positive, rendering TNBC cells targetable by tamoxifen. Furthermore, the combination of either CDK2 or EZH2 inhibitor with tamoxifen effectively suppresses tumor growth and markedly improves the survival of the mice bearing TNBC tumors, suggesting that the mechanism-based combination therapy may be an alternative approach to treat TNBC.

Removal of N-Linked Glycosylation Enhances PD-L1 Detection and Predicts Anti-PD-1/PD-L1 Therapeutic Efficacy.

Reactivation of T cell immunity by PD-1/PD-L1 immune checkpoint blockade has been shown to be a promising cancer therapeutic strategy. However, PD-L1 immunohistochemical readout is inconsistent with patient response, which presents a clinical challenge to stratify patients. Because PD-L1 is heavily glycosylated, we developed a method to resolve this by removing the glycan moieties from cell surface antigens via enzymatic digestion, a process termed sample deglycosylation. Notably, deglycosylation significantly improves anti-PD-L1 antibody binding affinity and signal intensity, resulting in more accurate PD-L1 quantification and prediction of clinical outcome. This proposed method of PD-L1 antigen retrieval may provide a practical and timely approach to reduce false-negative patient stratification for guiding anti-PD-1/PD-L1 therapy.