Fish oil-based microemulsion can efficiently deliver oral peptide blocking PD-1/PD-L1 and simultaneously induce ferroptosis for cancer immunotherapy.

Peptide immune checkpoint inhibitors in cancer immunotherapy have attracted great attention recently, but oral delivery of these peptides remains a huge challenge due to the harsh gastrointestinal environment, large molecular size, high hydrophilic, and poor transmembrane permeability. Here, for the first time, a fish oil-based microemulsion was developed for oral delivery of programmed death-1/programmed cell death-ligand 1 (PD-1/PD-L1) blocking model peptide, OPBP-1. The delivery system was characterized, in vitro and in vivo studies were conducted to evaluate its overall implication. As a result, this nutraceutical microemulsion was easily formed without the need of co-surfactants, and it appeared light yellow, transparent, good flowability with a particle size of 152 ± 0.73 nm, with a sustained drug release manner of 56.45 ± 0.36% over 24 h and a great stability within the harsh intestinal environment. It enhanced intestinal drug uptake and transportation over human intestinal epithelial Caco-2 cells, and drastically elevated the oral peptide bioavailability of 4.1-fold higher than that of OPBP-1 solution. Meanwhile, the mechanism of these dietary droplets permeated over the intestinal enterocytic membrane was found via clathrin and caveolae-mediated endocytic pathways. From the in vivo studies, the microemulsion facilitated the infiltration of CD8+ T lymphocytes in tumors, with increased interferon-γ (IFN-γ) secretion. Thus, it manifested a promising immune anti-tumor effect and significantly inhibited the growth of murine colonic carcinoma (CT26). Furthermore, it was found that the fish oil could induce ferroptosis in tumor cells and exhibited synergistic effect with OPBP-1 for cancer immunotherapy. In conclusion, this fish oil-based formulation demonstrated great potential for oral delivery of peptides with its natural property in reactive oxygen species (ROS)-related ferroptosis of tumor cells, which provides a great platform for functional green oral delivery system in cancer immunotherapy.

Immune and oxidative stress disorder in ovulation-dysfunction women revealed by single-cell transcriptome.

Introduction: Ovulation dysfunction is now a widespread cause of infertility around the world. Although the impact of immune cells in human reproduction has been widely investigated, systematic understanding of the changes of the immune atlas under female ovulation remain less understood. Methods: Here, we generated single cell transcriptomic profiles of 80,689 PBMCs in three representative statuses of ovulation dysfunction, i.e., polycystic ovary syndrome (PCOS), primary ovarian insufficiency (POI) and menopause (MENO), and identified totally 7 major cell types and 25 subsets of cells. Results: Our study revealed distinct cluster distributions of immune cells among individuals of ovulation disorders and health. In patients with ovulation dysfunction, we observed a significant reduction in populations of naïve CD8 T cells and effector memory CD4 T cells, whereas circulating NK cells and regulatory NK cells increased. Discussion: Our results highlight the significant contribution of cDC-mediated signaling pathways to the overall inflammatory response within ovulation disorders. Furthermore, our data demonstrated a significant upregulation of oxidative stress in patients with ovulation disorder. Overall, our study gave a deeper insight into the mechanism of PCOS, POI, and menopause, which may contribute to the better diagnosis and treatments of these ovulatory disorder.

Development of a novel peptide targeting GPR81 to suppress adipocyte-mediated tumor progression.

GPR81, initially discovered in adipocytes, has been found to suppress lipolysis when activated. However, the current small molecules that target GPR81 carry the risk of off-target effects, and their impact on tumor progression remains uncertain. Here, we utilized phage display technology to screen a GPR81-targeting peptide named 7w-2 and proceeded to demonstrate its bioactivity. Although 7w-2 did not affect the proliferation of tumor cells, it effectively reduced adipocyte catabolism in vitro, consequently restraining the proliferation of co-cultured tumor cells. Furthermore, our findings revealed that 7w-2 could inhibit lipolysis in vivo, leading to a significant impediment in tumor growth and metastasis in the 4T1 murine tumor model. Additionally, 7w-2 exhibited the ability to significantly elevate the proportion and functionality of CD8+ T cells. Our study introduces 7w-2 as the first peptide targeting GPR81, shedding light on its potential role in adipocytes in suppressing tumor progression.

Design of a novel chimeric peptide via dual blockade of CD47/SIRPα and PD-1/PD-L1 for cancer immunotherapy.

Although immune checkpoint inhibition has been shown to effectively activate antitumor immunity in various tumor types, only a small subset of patients can benefit from PD-1/PD-L1 blockade. CD47 expressed on tumor cells protects them from phagocytosis through interaction with SIRPα on macrophages, while PD-L1 dampens T cell-mediated tumor killing. Therefore, dual targeting PD-L1 and CD47 may improve the efficacy of cancer immunotherapy. A chimeric peptide Pal-DMPOP was designed by conjugating the double mutation of CD47/SIRPα blocking peptide (DMP) with the truncation of PD-1/PD-L1 blocking peptide OPBP-1(8-12) and was modified by a palmitic acid tail. Pal-DMPOP can significantly enhance macrophage-mediated phagocytosis of tumor cells and activate primary T cells to secret IFN-γ in vitro. Due to its superior hydrolysis-resistant activity as well as tumor tissue and lymph node targeting properties, Pal-DMPOP elicited stronger anti-tumor potency than Pal-DMP or OPBP-1(8-12) in immune-competent MC38 tumor-bearing mice. The in vivo anti-tumor activity was further validated in the colorectal CT26 tumor model. Furthermore, Pal-DMPOP mobilized macrophage and T-cell anti-tumor responses with minimal toxicity. Overall, the first bispecific CD47/SIRPα and PD-1/PD-L1 dual-blockade chimeric peptide was designed and exhibited synergistic anti-tumor efficacy via CD8+ T cell activation and macrophage-mediated immune response. The strategy could pave the way for designing effective therapeutic agents for cancer immunotherapy.

Early differentiation and gene expression characteristics of trophoblast lineages†.

With the development of the embryo, the totipotent blastomere undergoes the first lineage decision to the inner cell mass (ICM) and the trophectoderm (TE). The ICM forms the fetus while the TE forms the placenta, which is one of the unique organs in mammals serving as the interface between maternal and fetal bloodstreams. Proper trophoblast lineage differentiation is crucial for correct placental and fetal development, including the TE progenitor self-renewal and its differentiation toward mononuclear cytotrophoblast, which later either develops into invasive extravillous trophoblast, remodeling the uterine vascular, or fuses into multinuclear syncytiotrophoblast, secreting pregnancy-sustaining hormone. Aberrant differentiation and gene expression of trophoblast lineage is associated with severe pregnancy disorders and fetal growth restriction. This review focuses on the early differentiation and key regulatory factors of trophoblast lineage, which have been poorly elucidated. Meanwhile, the recent development of trophoblast stem cells, trophectoderm stem cells, and blastoids derived from pluripotent stem cells bring the accessible model to investigate the profound mystery of embryo implantation and placentation and were also summarized.

Dietary-Polysaccharide-Modified Fish-Oil-Based Double Emulsion as a Functional Colloidal Formulation for Oral Drug Delivery.

Oral delivery is the most convenient drug administration route. However, oral delivery of peptides is extremely challenging due to the physical and chemical barriers within the gastrointestinal tract. Polysaccharides are often utilized as polymeric biomaterials in drug delivery. Among these, dietary polysaccharides extracted from okra, yam, and spirulina have been reported to stimulate innate immunity with well-known nutritional benefits. In this study, we developed a dietary-polysaccharide-modified fish-oil-based emulsion for oral co-delivery of a hydrophilic PD-L1 blocking peptide and the hydrophobic small molecule simvastatin. The optimal emulsion was nano-sized and exhibited a negative surface charge, high drug encapsulation efficiency of over 97%, low viscosity, and sustained drug release manner. The formulation could significantly increase the uptake of peptides by intestinal Caco-2 cells, which demonstrated the great potential of the formulation for promoting the oral absorption of peptides. Additionally, these dietary polysaccharides could promote dendritic cell maturation and cytokine expression in macrophages, demonstrating that these nutraceutical polysaccharides had dual roles of functioning as promising colloidal delivery systems and as potential immune modulators or adjuvants. Thus, this food-based colloidal delivery system shows promise for the oral delivery of peptide drugs and lays a great platform for future applications in immunotherapy.

Identification of natural product 3, 5-diiodotyrosine as APOBEC3B inhibitor to prevent somatic mutation accumulation and cancer progression.

BACKGROUND: The development of cancer is largely dependent on the accumulation of somatic mutations, indicating the potential to develop cancer chemoprevention agents targeting mutation drivers. However, ideal cancer chemoprevention agents that can effectively inhibit the mutation drivers have not been identified yet. METHODS: The somatic mutation signatures and expression analyses of APOBEC3B were performed in patient with pan-cancer. The computer-aided screening and skeleton-based searching were performed to identify natural products that can inhibit the activity of APOBEC3B. 4-nitroquinoline-1-oxide (4-NQO)-induced spontaneous esophageal squamous cell carcinoma (ESCC) and azoxymethane/dextran sulfate sodium (AOM/DSS)-induced spontaneous colon cancer mouse models were conducted to investigate the influences of APOBEC3B inhibitor on the prevention of somatic mutation accumulation and cancer progression. RESULTS: Here, we discovered that the cytidine deaminase APOBEC3B correlated somatic mutations were widely observed in a variety of cancers, and its overexpression indicated poor survival. SMC247 (3, 5-diiodotyrosine), as a source of kelp iodine without side effects, could strongly bind APOBEC3B (KD=65 nM) and effectively inhibit its deaminase activity (IC50=1.69 µM). Interestingly, 3, 5-diiodotyrosine could significantly reduce the clusters of mutations, prevent the precancerous lesion progression, and prolong the survival in 4-NQO-induced spontaneous ESCC and AOM/DSS-induced spontaneous colon cancer mouse models. Furthermore, 3, 5-diiodotyrosine could reduce colitis, increase the proportion and function of T lymphocytes via IL-15 in tumor microenvironment. The synergistic cancer prevention effects were observed when 3, 5-diiodotyrosine combined with PD-1/PD-L1 blockade. CONCLUSIONS: This is the first prove-of-concept study to elucidate that the natural product 3, 5-diiodotyrosine could prevent somatic mutation accumulation and cancer progression through inhibiting the enzymatic activity of APOBEC3B. In addition, 3, 5-diiodotyrosine could reduce the colitis and increase the infiltration and function of T lymphocytes via IL-15 in tumor microenvironment. 3, 5-diiodotyrosine combined with PD-1/PD-L1 blockade could elicit synergistic cancer prevention effects, indicating a novel strategy for both prevent the somatic mutation accumulation and the immune-suppressive microenvironment exacerbation.

Oral delivery of protein and peptide drugs: from non-specific formulation approaches to intestinal cell targeting strategies.

The past few years has witnessed a booming market of protein and peptide drugs, owing to their superior efficiency and biocompatibility. Parenteral route is the most commonly employed method for protein and peptide drugs administration. However, short plasma half-life protein and peptide drugs requires repetitive injections and results in poor patient compliance. Oral delivery is a promising alternative but hindered by harsh gastrointestinal environment and defensive intestinal epithelial barriers. Therefore, designing suitable oral delivery systems for peptide and protein drugs has been a persistent challenge. This review summarizes the main challenges for oral protein and peptide drugs delivery and highlights the advanced formulation strategies to improve their oral bioavailability. More importantly, major intestinal cell types and available targeting receptors are introduced along with the potential strategies to target these cell types. We also described the multifunctional biomaterials which can be used to prepare oral carrier systems as well as to modulate the mucosal immune response. Understanding the emerging delivery strategies and challenges for protein and peptide drugs will surely inspire the production of promising oral delivery systems that serves therapeutic needs in clinical settings.

Dual Blockade of Lactate/GPR81 and PD-1/PD-L1 Pathways Enhances the Anti-Tumor Effects of Metformin.

Metformin is a widely used antidiabetic drug for cancer prevention and treatment. However, the overproduction of lactic acid and its inefficiency in cancer therapy limit its application. Here, we demonstrate the synergistic effects of the lactate/GPR81 blockade (3-hydroxy-butyrate, 3-OBA) and metformin on inhibiting cancer cells growth in vitro. Simultaneously, this combination could inhibit glycolysis and OXPHOS metabolism, as well as inhibiting tumor growth and reducing serum lactate levels in tumor-bearing mice. Interestingly, we observed that this combination could enhance the functions of Jurkat cells in vitro and CD8+ T cells in vivo. In addition, considering that 3-OBA could recover the inhibitory effects of metformin on PD-1 expression, we further determined the dual blockade effects of PD-1/PD-L1 and lactate/GPR81 on the antitumor activity of metformin. Our results suggested that this dual blockade strategy could remarkably enhance the anti-tumor effects of metformin, or even lead to tumor regression. In conclusion, our study has proposed a novel and robust strategy for a future application of metformin in cancer treatment.

An orally available PD-1/PD-L1 blocking peptide OPBP-1-loaded trimethyl chitosan hydrogel for cancer immunotherapy.

Blockade of the immune checkpoint PD-1/PD-L1 with monoclonal antibodies demonstrated unprecedented clinical efficacies in many cancers. But the orally available low molecular weight inhibitors remain infancy. Compared to small molecules, peptide exhibits better selectivity and fewer side effects, but poor half-life and a big challenge to be orally administrated. Here, we developed a proteolysis-resistant D peptide OPBP-1 (Oral PD-L1 Binding Peptide 1) which could selectively bind PD-L1, significantly block PD-1/PD-L1 interaction and enhance IFN-γ (interferon γ) secretion from CD8+ T cells in human PBMCs (Peripheral blood mononuclear cells). OPBP-1 could significantly inhibit tumor growth in murine colorectal CT26 and melanoma B16-OVA models at a relatively low dose of 0.5 mg/kg, with enhancing the infiltration and function of CD8+ T cells. More interestingly, oral delivery of OPBP-1 loaded TMC (N, N, N-trimethyl chitosan) hydrogel (OPBP-1@TMC) showed promising OPBP-1 oral bioavailability (52.8%) and prolonged half-life (14.55 h) in rats, and also significantly inhibited tumor growth in CT26 model. In conclusion, we discovered and optimized a PD-1/PD-L1 blocking peptide OPBP-1, and subsequently loaded into a TMC based hydrogel oral delivery system, in order to maximally elevate the oral bioavailability of the peptide drug and effectively inhibit tumor growth. These results opened up a new prospect for oral drug development in cancer immunotherapy.

Autopsy and statistical evidence of disturbed hemostasis progress in COVID-19: medical records from 407 patients.

BACKGROUND: The progression of coagulation in COVID-19 patients with confirmed discharge status and the combination of autopsy with complete hemostasis parameters have not been well studied. OBJECTIVE: To clarify the thrombotic phenomena and hemostasis state in COVID-19 patients based on epidemiological statistics combining autopsy and statistical analysis. METHODS: Using autopsy results from 9 patients with COVID-19 pneumonia and the medical records of 407 patients, including 39 deceased patients whose discharge status was certain, time-sequential changes in 11 relevant indices within mild, severe and critical infection throughout hospitalization according to the Chinese National Health Commission (NHC) guidelines were evaluated. Statistical tools were applied to calculate the importance of 11 indices and the correlation between those indices and the severity of COVID-19. RESULTS: At the beginning of hospitalization, platelet (PLT) counts were significantly reduced in critically ill patients compared with severely or mildly ill patients. Blood glucose (GLU), prothrombin time (PT), activated partial thromboplastin time (APTT), and D-dimer levels in critical patients were increased compared with mild and severe patients during the entire admission period. The International Society on Thrombosis and Haemostasis (ISTH) disseminated intravascular coagulation (DIC) score was also high in critical patients. In the relatively late stage of nonsurvivors, the temporal changes in PLT count, PT, and D-dimer levels were significantly different from those in survivors. A random forest model indicated that the most important feature was PT followed by D-dimer, indicating their positive associations with disease severity. Autopsy of deceased patients fulfilling diagnostic criteria for DIC revealed microthromboses in multiple organs. CONCLUSIONS: Combining autopsy data, time-sequential changes and statistical methods to explore hemostasis-relevant indices among the different severities of the disease helps guide therapy and detect prognosis in COVID-19 infection.

Computer-aided design of PVR mutants with enhanced binding affinity to TIGIT.

BACKGROUND: TIGIT, as a novel immune checkpoint molecule involved in T cell and NK cell anergy, could induce the immune tolerance and escape through binding with its ligand PVR. Blockade of TIGIT/PVR is considered as a promising strategy in cancer immunotherapy. However, to facilitate the design of inhibitors targeting TIGIT/PVR, the structural characteristics and binding mechanism still need to be further studied. METHODS: In this study, molecular dynamics (MD) simulations and in silico mutagenesis were used to analyze the interaction between TIGIT and its ligand PVR. Then, PVR mutants were designed and their activities were determined by using TIGIT overexpressed Jurkat cells. RESULTS: The results suggested that the loops of PVR (CC' loop, C'C″ loop, and FG loop) underwent a large intra-molecular rearrangement, and more hydrogen bond crosslinking between PVR and TIGIT were formed during MD simulations. The potential residues for PVR to interact with TIGIT were identified and utilized to predict high affinity PVR mutants. Through the biological activity evaluation, four PVR mutants (PVRS72W, PVRS72R, PVRG131V and PVRS132Q) with enhanced affinity to TIGIT were discovered, which could elicit more potent inhibitory effects compared with the wild type PVR. CONCLUSIONS: The MD simulations analysis provided new insights into the TIGIT/PVR interaction model, and the identified PVR mutants (PVRS72W, PVRS72R, PVRG131V and PVRS132Q) could serve as new candidates for immunotherapy to block TIGIT/PVR. Video Abstract.

Effect of Sperm Cryopreservation on miRNA Expression and Early Embryonic Development.

Although sperm preservation is a common means of personal fertility preservation, its effects on embryonic development potential need further investigation. The purpose of this study was to identify key microRNA (miRNA) in cryopreserved sperm and determine the changes of these miRNAs and their target genes during embryonic development using cryopreserved sperm. Moreover, the embryonic development potential of cryopreserved sperm was estimated in assisted reproductive technology (ART), where key miRNAs and target genes were validated in sperm and subsequent embryos. Clinical data of embryonic development from cryopreserved sperm indicated a significant decrease in fertilization rate in both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cases, as well as a reduction in blastocyst formation rate in ICSI cases. Meanwhile there was a significant increase in blocked embryo ratio of Day1, Day2, and Day3.5 embryos when frozen-thawed mouse sperm was used, compared with fresh mouse sperm, suggesting a potential negative effect of sperm cryopreservation on embryonic development. From frozen-thawed and fresh sperm in humans and mice, respectively, 21 and 95 differentially expressed miRNAs (DEmiRs) were detected. miR-148b-3p were downregulated in both human and mouse frozen-thawed sperm and were also decreased in embryos after fertilization using cryopreserved sperm. Target genes of miR-148b-3p, Pten, was identified in mouse embryos using quantitative real-time PCR (qRT-PCR) and Western blot (WB). In addition, common characters of cryopreservation of mouse oocytes compared with sperm were also detected; downregulation of miR-148b-3p was also confirmed in cryopreserved oocytes. In summary, our study suggested that cryopreservation of sperm could change the expression of miRNAs, especially the miR-148b-3p across humans and mice, and may further affect fertilization and embryo development by increasing the expression of Pten. Moreover, downregulation of miR-148b-3p induced by cryopreservation was conserved in mouse gametes.

Blocking of the PD-1/PD-L1 interaction by a novel cyclic peptide inhibitor for cancer immunotherapy.

The interaction of PD-1/PD-L1 allows tumor cells to escape from immune surveillance. Clinical success of the antibody drugs has proven that blockade of PD-1/PD-L1 pathway is a promising strategy for cancer immunotherapy. Here, we developed a cyclic peptide C8 by using Ph.D.-C7C phage display technology. C8 showed high binding affinity with hPD-1 and could effectively interfere the interaction of PD-1/PD-L1. Furthermore, C8 could stimulate CD8+ T cell activation in human peripheral blood mononuclear cells (PBMCs). We also observed that C8 could suppress tumor growth in CT26 and B16-OVA, as well as anti-PD-1 antibody resistant B16 mouse model. CD8 T cells infiltration significantly increased in tumor microenvironment, and IFN-γ secretion by CD8+ T cells in draining lymph nodes also increased. Simultaneously, we exploited T cells depletion models and confirmed that C8 exerted anti-tumor effects via activating CD8+ T cells dependent manner. The interaction model of C8 with hPD-1 was simulated and confirmed by alanine scanning. In conclusion, C8 shows anti-tumor capability by blockade of PD-1/PD-L1 interaction, and C8 may provide an alternative candidate for cancer immunotherapy.

CD47/SIRPα blocking peptide identification and synergistic effect with irradiation for cancer immunotherapy.

BACKGROUND: Immunotherapy has achieved remarkable advances via a variety of strategies against tumor cells that evade immune surveillance. As important innate immune cells, macrophages play important roles in maintaining homeostasis, preventing pathogen invasion, resisting tumor cells and promoting adaptive immune response. CD47 is found to be overexpressed on tumor cells and act as a don't eat me' signal, which contributes to immune evasion. Macrophages mediated phagocytosis via blockade CD47/SIRPα (signal regulatory protein alpha) interaction was proved to induce effective antitumor immune response. METHODS: A novel peptide pep-20, specifically targeting CD47 and blocking CD47/SIRPα interaction, was identified via high-throughput phage display library bio-panning. The capability to enhance the macrophage-mediated phagocytosis activities and antitumor effects of pep-20 were investigated. The mechanism of pep-20 to induce T-cell response was explored by ex vivo analysis and confirmed via macrophage depleting strategy. The structure-activity relationship and D-amino acid substitution of pep-20 were also studied. The antitumor effects and mechanism of a proteolysis resistant D-amino acid derivate pep-20-D12 combined with irradiation (IR) were also investigated. RESULTS: Pep-20 showed remarkable enhancement of macrophage-mediated phagocytosis to both solid and hematologic tumor cells in vitro, and inhibited tumor growth in immune-competent tumor-bearing mice. Furthermore, pep-20 promoted macrophages to mobilize the antitumor T-cell response with minimal toxicity. Furthermore, systemic administration of the derivate pep-20-D12 showed robust synergistic antitumor efficacy in combination with IR. CONCLUSION: In summary, these results demonstrated that CD47/SIRPα blocking peptides, pep-20 and its derivate, could serve as promising candidates to promote macrophages-mediated phagocytosis and immune response in cancer immunotherapy.

A novel cyclic peptide targeting LAG-3 for cancer immunotherapy by activating antigen-specific CD8+ T cell responses.

PD-1 and CTLA-4 antibodies offer great hope for cancer immunotherapy. However, many patients are incapable of responding to PD-1 and CTLA-4 blockade and show low response rates due to insufficient immune activation. The combination of checkpoint blockers has been proposed to increase the response rates. Besides, antibody drugs have disadvantages such as inclined to cause immune-related adverse events and infiltration problems. In this study, we developed a cyclic peptide C25 by using Ph.D.-C7C phage display technology targeting LAG-3. As a result, C25 showed a relative high affinity with human LAG-3 protein and could effectively interfere the binding between LAG-3 and HLA-DR (MHC-II). Additionally, C25 could significantly stimulate CD8+ T cell activation in human PBMCs. The results also demonstrated that C25 could inhibit tumor growth of CT26, B16 and B16-OVA bearing mice, and the infiltration of CD8+ T cells was significantly increased while FOXP3+ Tregs significantly decreased in the tumor site. Furthermore, the secretion of IFN-γ by CD8+ T cells in spleen, draining lymph nodes and especially in the tumors was promoted. Simultaneously, we exploited T cells depletion models to study the anti-tumor mechanisms for C25 peptide, and the results combined with MTT assay confirmed that C25 exerted anti-tumor effects via CD8+ T cells but not direct killing. In conclusion, cyclic peptide C25 provides a rationale for targeting the immune checkpoint, by blockade of LAG-3/HLA-DR interaction in order to enhance anti-tumor immunity, and C25 may provide an alternative for cancer immunotherapy besides antibody drugs.

Single-Cell Transcriptomic Analysis Reveals Mitochondrial Dynamics in Oocytes of Patients With Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS), characterized by polycystic ovarian morphology, ovarian follicular maturation arrest, and hormonal disorders, affects numerous women in the reproductive age worldwide. A recent study has found that mitochondria are likely to play an essential role in oocyte quality. However, it is still unclear whether oocyte development failure is associated with mitochondria in patients with PCOS. We analyzed the single-cell RNA sequencing data from the previous study, including data from 14 oocytes from 7 healthy fertile women and 20 oocytes from 9 patients with PCOS at the germinal vesicle (GV) stage, metaphase I (MI) stage, and metaphase II (MII) stage. We revealed the transcriptomic dynamics by weighted gene co-expression network analysis (WGCNA) and investigated the differences between stages using PCA and Deseq2 analyses to identify the differential expression genes (DEGs). Gene ontology (GO) was performed using clusterProfiler R package and Metascape. Our results indicated that specific gene modules were related to different stages of oocyte development using WGCNA. Functional enrichment analysis and gene co-expression network analysis found significant enrichment of the mitochondrial regulation genes at the GV stage. PCA (principal component analysis) and differential gene expression analysis suggested that GV was significantly different from the MI and MII stages between the two groups. Further analysis demonstrated that the upregulated differentially expressed genes at the GV stage of patients with PCOS mainly related to mitochondrial function, such as COX6B1, COX8A, COX4l1, and NDUFB9. Meanwhile, these genes tended to be activated at the MII stage in healthy cells, suggesting that some mitochondrial functions may be prematurely activated at the GV stage of PCOS oocytes, whereas this process occurs at the MII stage in healthy oocytes. Collectively, our study showed that aberrant mitochondrial function at the GV stage may contribute to a decline in oocyte quality of PCOS patients.

A Novel d-Peptide Identified by Mirror-Image Phage Display Blocks TIGIT/PVR for Cancer Immunotherapy.

The low response rate and adaptive resistance of PD-1/PD-L1 blockade demands the studies on novel therapeutic targets for cancer immunotherapy. We discovered that a novel immune checkpoint TIGIT expressed higher than PD-1 in many tumors especially anti-PD-1 resistant tumors. Here, mirror-image phage display bio-panning was performed using the d-enantiomer of TIGIT synthesized by hydrazide-based native chemical ligation. d-peptide D TBP-3 was identified, which could occupy the binding interface and effectively block the interaction of TIGIT with its ligand PVR. D TBP-3 showed proteolytic resistance, tumor tissue penetrating ability, and significant tumor suppressing effects in a CD8+ T cell dependent manner. More importantly, D TBP-3 could inhibit tumor growth and metastasis in anti-PD-1 resistant tumor model. This is the first d-peptide targeting TIGIT, which could serve as a potential candidate for cancer immunotherapy.

Intrinsic Expression of Immune Checkpoint Molecule TIGIT Could Help Tumor Growth in vivo by Suppressing the Function of NK and CD8+ T Cells.

TIGIT, an immune checkpoint molecule widely expressed on NK cells, activated T cells and Tregs, has been involved in delivering inhibitory signals through the interaction with PVR. The blockade of TIGIT/PVR interaction is a promising approach in cancer immunotherapy. Here, we unexpectedly discovered the expression of TIGIT in murine tumor cells. To elucidate the mechanism of such intrinsic expression, TIGIT knockout murine colorectal CT26 and MC38 cell lines were generated by using CRISPR/Cas9 system. Although TIGIT knockout showed no effects on proliferation and colony formation of tumor cells in vitro, the tumor growth in mice was considerably inhibited. TIGIT knockout led to the increase of IFN-γ secretion by NK and CD8+ T cells. Further, in BABL/c nude mice, CD8+ T cells depleting mice and NK cells depleting nude mice, the promotion of tumor growth was significantly diminished, suggesting that both NK cells and CD8+ T cells were involved in the tumor promoting process mediated by intrinsic TIGIT. In addition, blocking TIGIT/PVR interaction by the antibody or recombinant PVR protein could elicit anti-tumor effects by facilitating the tumor infiltration and restoring the function of CD8+ T cells, and the antibody-mediate TIGIT blockade could inhibit MC38 tumor growth through blocking TIGIT expressed on tumor cells. We therefore propose a novel TIGIT/PVR interaction mode that tumor intrinsic TIGIT delivers inhibitory signals to CD8+ T cells and NK cells by engaging with PVR.