Signaling crosstalk between tumor endothelial cells and immune cells in the microenvironment of solid tumors.

Tumor-associated endothelial cells (TECs) are crucial mediators of immune surveillance and immune escape in the tumor microenvironment (TME). TECs driven by angiogenic growth factors form an abnormal vasculature which deploys molecular machinery to selectively promote the function and recruitment of immunosuppressive cells while simultaneously blocking the entry and function of anti-tumor immune cells. TECs also utilize a similar set of signaling regulators to promote the metastasis of tumor cells. Meanwhile, the tumor-infiltrating immune cells further induce the TEC anergy by secreting pro-angiogenic factors and prevents further immune cell penetration into the TME. Understanding the complex interactions between TECs and immune cells will be needed to successfully treat cancer patients with combined therapy to achieve vasculature normalization while augmenting antitumor immunity. In this review, we will discuss what is known about the signaling crosstalk between TECs and tumor-infiltrating immune cells to reveal insights and strategies for therapeutic targeting.

Geographic EBV variants confound disease-specific variant interpretation and predict variable immune therapy responses.

Epstein-Barr virus (EBV) is a potent carcinogen linked to hematologic and solid malignancies, causing significant global morbidity and mortality. Therapy using allogeneic EBV-specific lymphocytes shows promise in certain populations, but the impact of EBV genome variation on these strategies remains unexplored. To address this, we sequenced 217 EBV genomes, including hematologic malignancies from Guatemala, Peru, Malawi, and Taiwan, and analyzed them alongside 1,307 publicly available EBV genomes from cancer, non-malignant diseases, and healthy individuals across Africa, Asia, Europe, North America, and South America. These included the first NK/T-cell lymphoma (NKTCL) EBV genomes reported outside East Asia. Our findings indicate that previously proposed EBV genome variants specific to certain cancer types are more closely tied to geographic origin than cancer histology. This included variants previously reported to be specific to NKTCL but were prevalent in EBV genomes from other cancer types and healthy individuals in East Asia. After controlling for geographic region, we did identify multiple NKTCL-specific variants associated with a 7.8- to 21.9- fold increased risk. We also observed frequent variations in EBV genomes affecting peptide sequences previously reported to bind common MHC alleles. Finally, we found several non-synonymous variants spanning the coding sequences of current vaccine targets BALF4, BKRF2, BLLF1, BXLF2, BZLF1, and BZLF2. These results highlight the need to consider geographic variation in EBV genomes when devising strategies for exploiting adaptive immune responses against EBV-related cancers, ensuring greater global effectiveness and equity in prevention and treatment.

Therapeutic targeting of tumor spheroids in a 3D microphysiological renal cell carcinoma-on-a-chip system.

Metastatic renal cell carcinoma (RCC) remains an incurable disease for most patients highlighting an urgent need for new treatments. However, the preclinical investigation of new therapies is limited by traditional two-dimensional (2D) cultures which do not recapitulate the properties of tumor cells within a collagen extracellular matrix (ECM), while human tumor xenografts are time-consuming, expensive and lack adaptive immune cells. We report a rapid and economical human microphysiological system ("RCC-on-a-chip") to investigate therapies targeting RCC spheroids in a 3D collagen ECM. We first demonstrate that culture of RCC cell lines A498 and RCC4 in a 3D collagen ECM more faithfully reproduces the gene expression program of primary RCC tumors compared to 2D culture. We next used bortezomib as a cytotoxin to develop automated quantification of dose-dependent tumor spheroid killing. We observed that viable RCC spheroids exhibited collective migration within the ECM and demonstrated that our 3D system can be used to identify compounds that inhibit spheroid collective migration without inducing cell death. Finally, we demonstrate the RCC-on-a-chip as a platform to model the trafficking of tumor-reactive T cells into the ECM and observed antigen-specific A498 spheroid killing by engineered human CD8+ T cells expressing an ROR1-specific chimeric antigen receptor. In summary, the phenotypic differences between the 3D versus 2D environments, rapid imaging-based readout, and the ability to carefully study the impact of individual variables with quantitative rigor will encourage adoption of the RCC-on-a-chip system for testing a wide range of emerging therapies for RCC.

Single cell atlas of kidney cancer endothelial cells reveals distinct expression profiles and phenotypes.

Background: Tumor endothelial cells (TECs) represent the primary interface between the tumor microenvironment and circulating immune cells, however their phenotypes are incompletely understood in highly vascularized clear cell renal cell carcinoma (ccRCC). Methods: We purified tumor and matched normal endothelial cells (NECs) from ccRCC specimens and performed single-cell RNA-sequencing to create a reference-quality atlas available as a searchable web resource for gene expression patterns. We established paired primary TECs and NECs cultures for ex vivo functional testing. Results: TECs from multiple donors shared a common phenotype with increased expression of pathways related to extracellular matrix regulation, cell-cell communication, and insulin-like growth factor signaling that was conserved in comparison to hepatocellular carcinoma associated TECs, suggesting convergent TEC phenotypes between unrelated tumors. Cultured TECs stably maintained a core program of differentially regulated genes, were inherently resistant to apoptosis after vascular endothelial growth factor removal and displayed increased adhesiveness to subsets of immune cells including regulatory T-cells. Conclusions: Our studies delineate unique functional and phenotypic properties of TECs, which may provide insights into their interactions with available and emerging therapies. Functional phenotypes of cultured TECs suggest potential mechanisms of resistance to both antiangiogenic and immune-based therapies.

Immunophenotypic correlates of sustained MRD negativity in patients with multiple myeloma.

The role of the immune microenvironment in maintaining disease remission in patients with multiple myeloma (MM) is not well understood. In this study, we comprehensively profile the immune system in patients with newly diagnosed MM receiving continuous lenalidomide maintenance therapy with the aim of discovering correlates of long-term treatment response. Leveraging single-cell RNA sequencing and T cell receptor ß sequencing of the peripheral blood and CyTOF mass cytometry of the bone marrow, we longitudinally characterize the immune landscape in 23 patients before and one year after lenalidomide exposure. We compare patients achieving sustained minimal residual disease (MRD) negativity to patients who never achieved or were unable to maintain MRD negativity. We observe that the composition of the immune microenvironment in both the blood and the marrow varied substantially according to both MRD negative status and history of autologous stem cell transplant, supporting the hypothesis that the immune microenvironment influences the depth and duration of treatment response.

Immunohistochemical Detection of 5T4 in Renal Cell Carcinoma.

5T4 (trophoblast glycoprotein encoded by TPBG ) is a cancer/testis antigen highly expressed in renal cell carcinoma (RCC) and many other cancers but rarely in normal tissues. Interest in developing 5T4 as a prognostic biomarker and direct targeting of 5T4 by emerging receptor-engineered cellular immunotherapies has been hampered by the lack of validated 5T4-specific reagents for immunohistochemistry (IHC). We tested 4 commercially available monoclonal antibodies (mAbs) for the detection of 5T4 in formalin-fixed, paraffin-embedded RCC and normal tissues. Using parental and TPBG -edited A498 cells, 3 mAbs showed 5T4 specificity. Further analyses focused on 2 mAbs with the most robust staining (MBS1750093, Ab134162). IHC on tissue microarrays incorporating 263 renal tumors showed high staining concordance of these 2 mAbs ranging from 0.80 in chromophobe RCC to 0.89 in advanced clear cell RCC (ccRCC). MBS1750093, the most sensitive, exhibited 2+/3+ staining in papillary RCC (92.2%) > advanced ccRCC (60.0%) > chromophobe RCC (43.6%) > localized ccRCC (39.6%) > oncocytoma (22.7%). RNA in situ hybridization also revealed high levels of TPBG RNA were present most frequently in papillary and advanced ccRCC. In advanced ccRCC, there was a trend towards higher 5T4 expression and regional or distant metastases. Normal organ controls showed no or weak staining with the exception of focal moderate staining in kidney glomeruli and distal tubules by IHC. These data identify mAbs suitable for detecting 5T4 in formalin-fixed, paraffin-embedded tissues and demonstrate both interpatient and histologic subtype heterogeneity. Our validated 5T4 IHC protocol will facilitate biomarker studies and support the therapeutic targeting of 5T4.

Integrated TCR repertoire analysis and single-cell transcriptomic profiling of tumor-infiltrating T cells in renal cell carcinoma identifies shared and tumor-restricted expanded clones with unique phenotypes.

Objective responses of metastatic renal cell carcinoma (RCC) associated with systemic immunotherapies suggest the potential for T-cell-mediated tumor clearance. Recent analyses associate clonally expanded T cells present in the tumor at diagnosis with responses to immune checkpoint inhibitors (ICIs). To identify and further characterize tumor-associated, clonally expanded T cells, we characterized the density, spatial distribution, T-cell receptor (TCR) repertoire, and transcriptome of tumor-infiltrating T cells from 14 renal tumors at the time of resection and compared them with T cells in peripheral blood and normal adjacent kidney. Multiplex immunohistochemistry revealed that T-cell density was higher in clear cell RCC (ccRCC) than in other renal tumor histologies with spatially nonuniform T-cell hotspots and exclusion zones. TCR repertoire analysis also revealed increased clonal expansion in ccRCC tumors compared with non-clear cell histologies or normal tissues. Expanded T-cell clones were most frequently CD8+ with some detectable in peripheral blood or normal kidney and others found exclusively within the tumor. Divergent expression profiles for chemokine receptors and ligands and the Ki67 proliferation marker distinguished tumor-restricted T-cell clones from those also present in blood suggesting a distinct phenotype for subsets of clonally expanded T cells that also differed for upregulated markers of T-cell activation and exhaustion. Thus, our single-cell level stratification of clonally expanded tumor infiltrating T-cell subpopulations provides a framework for further analysis. Future studies will address the spatial orientation of these clonal subsets within tumors and their association with treatment outcomes for ICIs or other therapeutic modalities.

Case report: A persistently expanded T cell response in an exceptional responder to radiation and atezolizumab for metastatic non-small cell lung cancer.

Most patients with advanced non-small cell lung cancer (NSCLC) do not achieve a durable remission after treatment with immune checkpoint inhibitors. Here we report the clinical history of an exceptional responder to radiation and anti-program death-ligand 1 (PD-L1) monoclonal antibody, atezolizumab, for metastatic NSCLC who remains in a complete remission more than 8 years after treatment. Sequencing of the patient's T cell repertoire from a metastatic lesion and the blood before and after anti-PD-L1 treatment revealed oligoclonal T cell expansion. Characterization of the dominant T cell clone, which comprised 10% of all clones and increased 10-fold in the blood post-treatment, revealed an activated CD8+ phenotype and reactivity against 4 HLA-A2 restricted neopeptides but not viral or wild-type human peptides, suggesting tumor reactivity. We hypothesize that the patient's exceptional response to anti-PD-L1 therapy may have been achieved by increased tumor immunogenicity promoted by pre-treatment radiation therapy as well as long-term persistence of oligoclonal expanded circulating T cells.

Downregulation of MEG3 and upregulation of EZH2 cooperatively promote neuroblastoma progression.

Neuroblastoma (NB), an embryonic tumour originating from sympathetic crest cells, is the most common extracranial solid tumour type in children with poor overall prognosis. Accumulating evidence has demonstrated the involvement of long non-coding RNA (lncRNA) in numerous biological processes and their associations with embryonic development and multiple diseases. Ectopic lncRNA expression is linked to malignant tumours. Previous studies by our team indicate that MEG3 attenuates NB autophagy through inhibition of FOXO1 and epithelial-mesenchymal transition via the mTOR pathway in vitro. Moreover, MEG3 and EZH2 negatively regulate each other. In present study, we first collected 60 NB tissues and 20 adjacent tissues for Quantitative real-time polymerase chain reaction (Q-PCR) experiments and performed clinical correlation analysis of the results. At the same time, nude mice were used for subcutaneous tumour formation to detect the effect of MEG3 in vivo. Two NB cell lines, SK-N-AS and SK-N-BE(2)C, were overexpressed MEG3 and rescued with EZH2 and then were subjected to proliferation, migration, invasion, apoptosis and autophagy experiments. RNA-binding protein immunoprecipitation (RIP) and Co-Immunoprecipitation (Co-IP) experiments were performed to explore the molecular mechanism of MEG3 and EZH2 interaction. Q-PCR revealed that MEG3 expression was negatively correlated with INSS stage and risk grade of NB. Moreover, MEG3 overexpression was associated with inhibition of NB growth in vivo. MEG3 exerted an anti-cancer effect via stimulatory effects on EZH2 ubiquitination leading to its degradation. Conversely, EZH2 interacted with DNMT1 and HDAC1 to induce silencing of MEG3. The EZH2 inhibitor, DZNep, and HDAC inhibitor, SAHA, displayed synergistic activity against NB. Combined treatment with DZNep and SAHA inhibited proliferation, migration and invasion of NB through suppression of the PI3K/AKT/mTOR/FOXO1 pathway. In conclusion, downregulation of MEG3 and upregulation of EZH2 forms a feedback loop that concertedly promotes the development of NB. Combined blockage of EZH2 and HDAC1 with the appropriate inhibitors may therefore present an effective treatment strategy for NB cases with low MEG3 and high EZH2 expression.

CD4 and CD8 co-receptors modulate functional avidity of CD1b-restricted T cells.

T cells recognize mycobacterial glycolipid (mycolipid) antigens presented by CD1b molecules, but the role of CD4 and CD8 co-receptors in mycolipid recognition is unknown. Here we show CD1b-mycolipid tetramers reveal a hierarchy in which circulating T cells expressing CD4 or CD8 co-receptor stain with a higher tetramer mean fluorescence intensity than CD4-CD8- T cells. CD4+ primary T cells transduced with mycolipid-specific T cell receptors bind CD1b-mycolipid tetramer with a higher fluorescence intensity than CD8+ primary T cells. The presence of either CD4 or CD8 also decreases the threshold for interferon-γ secretion. Co-receptor expression increases surface expression of CD3ε, suggesting a mechanism for increased tetramer binding and activation. Targeted transcriptional profiling of mycolipid-specific T cells from individuals with active tuberculosis reveals canonical markers associated with cytotoxicity among CD8+ compared to CD4+ T cells. Thus, expression of co-receptors modulates T cell receptor avidity for mycobacterial lipids, leading to in vivo functional diversity during tuberculosis disease.

STARD3NL inhibits the osteogenic differentiation by inactivating the Wnt/ß-catenin pathway via binding to Annexin A2 in osteoporosis.

Osteoporosis is one of the leading forms of systemic diseases related to bone metabolism in the world. STARD3 N-terminal like (STARD3NL) showed robust association with osteoporosis-related traits. Yet, the molecular functional mechanisms of STARD3NL in osteoblasts is still obscure. In this study, we demonstrated a high level of STARD3NL expression in the bone tissues from the patients with low bone mass and ovariectomized (OVX)-induced osteoporotic mice. We identified Stard3nl as a potent factor that negatively and positively regulates osteoblast differentiation and cell proliferation, respectively. Furthermore, inhibition of Stard3nl induced ß-catenin gene expression and the nuclear translocation of ß-catenin, as well as Wnt signalling activities, contributing to the activation of Wnt/ß-catenin signalling. Mechanistic studies revealed that Stard3nl bound with Annexin A2 (Anxa2) to suppress ß-catenin expression, resulting into the suppression of Wnt signalling and downstream osteogenic differentiation. Moreover, adeno-associated virus 9 (AAV9)-mediated silencing of Stard3nl reversed bone loss in OVX-induced osteoporotic mice by the injection into the knee joints. Collectively, our study revealed that Stard3nl suppressed osteogenesis via binding with Anxa2, resulting into the inactivation of Wnt signalling. It also highlights the preventive and therapeutic potential of STARD3NL as a specific and novel target for osteoporotic patients.

MiR-664-3p suppresses osteoblast differentiation and impairs bone formation via targeting Smad4 and Osterix.

Osteoporosis is a metabolic disorder characterized by low bone mass and deteriorated microarchitecture, with an increased risk of fracture. Some miRNAs have been confirmed as potential modulators of osteoblast differentiation to maintain bone mass. Our miRNA sequencing results showed that miR-664-3p was significantly down-regulated during the osteogenic differentiation of the preosteoblast MC3T3-E1 cells. However, whether miR-664-3p has an impact on bone homeostasis remains unknown. In this study, we identified overexpression of miR-664-3p inhibited the osteoblast activity and matrix mineralization in vitro. Osteoblastic miR-664-3p transgenic mice exhibited reduced bone mass due to suppressed osteoblast function. Target prediction analysis and experimental validation confirmed Smad4 and Osterix (Osx) are the direct targets of miR-664-3p. Furthermore, specific inhibition of miR-664-3p by subperiosteal injection with miR-664-3p antagomir protected against ovariectomy-induced bone loss. In addition, miR-664-3p expression was markedly higher in the serum from patients with osteoporosis compared to that from normal subjects. Taken together, this study revealed that miR-664-3p suppressed osteogenesis and bone formation via targeting Smad4 and Osx. It also highlights the potential of miR-664-3p as a novel diagnostic and therapeutic target for osteoporotic patients.

Current status of antigen-specific T-cell immunotherapy for advanced renal-cell carcinoma.

In renal-cell carcinoma (RCC), tumor-reactive T-cell responses can occur spontaneously or in response to systemic immunotherapy with cytokines and immune checkpoint inhibitors. Cancer vaccines and engineered T-cell therapies are designed to selectively augment tumor antigen-specific CD8+ T-cell responses with the goal to elicit tumor regression and avoid toxicities associated with nonspecific immunotherapies. In this review, we provide an overview of the central role of T-cell immunity in the treatment of advanced RCC. Clinical outcomes for antigen-targeted vaccines or other T-cell-engaging therapies for RCC are summarized and evaluated, and emerging new strategies to enhance the effectiveness of antigen-specific therapy for RCC are discussed.

Application of dexmedetomidine-assisted intravertebral anesthesia in hip replacement and its influence on T-lymphocyte subsets.

Application of dexmedetomidine-assisted intravertebral anesthesia for elderly patients with hip replacement and its influences on T-lymphocyte subsets in peripheral blood were assessed. Eighty-six patients undergoing intravertebral anesthesia in hip replacement were treated as group A, and one hundred patients undergoing intravertebral anesthesia combined with dexmedetomidine were treated as group B. Hemodynamic changes in both groups were compared 5 min before anesthesia (T0), immediately after skin incision (T1) and after surgery (T2). General operation conditions of patients in both groups were recorded. T-lymphocyte subsets, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), visual analogue scale (VAS) pain scores and mini-mental state examination (MMSE) cognitive function changes before surgery and 24 h after surgery were compared between the groups, and the incidence of complications in both groups after 24 h was recorded. The recovery time of patients in group B was shorter than that of group A (P<0.05). Changes of systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate fluctuations in group B were lower than those in group A (P<0.05). At 24 h after surgery, VAS scores of group B were lower than those of group A (P<0.05); levels of IL-6 and TNF-α were lower than those of group A (P<0.05); CD3+ cells, CD4+ cells, CD8+ cells, and CD4/CD8 ratio were higher than those of group A (P<0.05), and MMSE score was higher than that of group A (P<0.05). The incidence of gastrointestinal reactions and postoperative cognitive dysfunction (POCD) in group B was lower than that in group A (P<0.05). In conclusion, administration of dexmedetomidine can effectively shorten the recovery time of patients, stabilize intraoperative hemodynamics of patients, protect immune function, and reduce postoperative pain and POCD occurrence during anesthesia of hip replacement.

Selective Vascular Endothelial Growth Factor Receptor Inhibitors Provide Limited Benefits for Metastatic Colorectal Cancer: A Meta-Analysis.

BACKGROUND: Metastatic colorectal cancer (mCRC) is one of the most common and deadly cancers worldwide. For most patients diagnosed with mCRC and managed with 5-fluorouracil (5-FU)/leucovorin plus oxaliplatin (FOLFOX), the median survival time is still less than 2 years. Small molecule selective vascular endothelial growth factor receptor (VEGFR) inhibitors have been demonstrated to have strong anti-tumour activity in various cancer models. OBJECTIVE: To demonstrate the efficacy and safety of selective VEGFR inhibitors in the management of mCRC. METHODS: A comprehensive search in PubMed, EMBASE, Web of Science, Ovid MEDLINE, Google Scholar, Springer and Cochrane Central databases was performed for randomized controlled trials (RCTs) focusing on the effect of selective VEGFR inhibitors on mCRC. The primary outcome measures were progression-free survival (PFS) rates, overall survival (OS) rates, complete response (CR), partial response (PR), stable disease (SD), progressive disease (PD), objective response rates (ORRs), disease control rates (DCRs) and adverse effect (AE) rates. The dates of the included studies ranged from the inception of the database to January 15, 2020. RESULTS: Twenty-two RCTs were included. A total of 9362 patients met the inclusion criteria. Compared with placebo, selective VEGFR inhibitors significantly increased the PFS rate, SD, PR and DCR, reduced PD, caused more treatment-emergent adverse events (TEAEs), hypertension, hand-foot skin reaction, diarrhoea, fatigue, and thrombocytopaenia and increased aspartate aminotransferase(AST) concentration. There was no significant difference between selective VEGFR inhibitors and placebo regarding OS rate, CR, ORR, proteinuria, hyperbilirubinaemia or alkaline phosphatase(ALP) concentration. Additionally, compared with FOLFOX4+placebo, FOLFOX4+ selective VEGFR inhibitors, clearly reduced PD, and caused more 3-4 AEs, serious AEs, hypertension, hand-foot syndrome, diarrhoea, nausea, vomiting, decreased appetite, dehydration, fatigue, dizziness, neutropaenia and thrombocytopaenia. For PFS rate, OS rate, CR, PR, SD, ORR, abdominal pain, peripheral sensory neuropathy, asthaenia, anaemia and hypokalaemia rates, there was no significant difference between FOLFOX4+ selective VEGFR inhibitors and FOLFOX4+placebo. However, compared with FOLFOX4+bevacizumab, FOLFOX4+selective VEGFR inhibitors, led to increased hypertension, neutropaenia, fatigue, thrombocytopaenia and asthaenia. There is no clear difference between FOLFOX4+selective VEGFR inhibitors and FOLFOX4+ bevacizumab with regard to PFS rate, OS rate, CR, PR, SD, PD, ORR, diarrhoea, nausea, vomiting, peripheral neuropathy and abdominal pain rates. Selective VEGFR inhibitors+cetuximab increased PFS and PR and reduced PD compared to cetuximab, but there was no statistical difference between the two groups for OS and SD. CONCLUSION: Compared with placebo or cetuximab, selective VEGFR inhibitors alone or combined with cetuximab seemed to be more efficacious for mCRC respectively; however, the effects were not better than FOLFOX4 alone or when combined with bevacizumab for mCRC. Additionally, selective VEGFR inhibitors were not as safe as placebo or FOLFOX4 alone or in combination with bevacizumab in mCRC.

Icariin promotes osteogenic differentiation by suppressing Notch signaling.

Osteoporosis is a bone disease characterized by microarchitectural deterioration, low bone mass, and increased risk of fractures. Icariin (ICA), an active flavonoid glucoside isolated from Herba epimedii (HEF), is a potent stimulator of osteogenic differentiation and has potential applications for preventing bone loss in postmenopausal women. However, the molecular mechanism underlying the osteogenic effect of ICA has not yet been fully elucidated. In this study, we report that ICA treatment significantly elevated gene expression of osteogenic markers and increased alkaline phosphatase (ALP) activity in MC3T3-E1 and C3H10T1/2 cells. RNA sequencing revealed that the expression of several genes involved in the Notch pathway was decreased following ICA treatment. Real-time PCR further demonstrated that the mRNA levels of Notch ligands Jagged-1 (Jag1), lunatic fringe (Lfng), and Notch signaling downstream target gene Hey-1 were significantly decreased following ICA treatment. In addition, we found that constitutive activation of Notch signaling through overexpression of the intracellular domain of Notch (NICD) fully blocked ICA-induced osteoblast differentiation. Moreover, inhibiting Notch signaling with DAPT markedly enhanced osteogenic differentiation following ICA treatment. We found that the mRNA levels of Notch pathway molecules (Lfng, Notch1, Rbpjk and Nfatc1) were increased in ovariectomized (OVX) mice, and administration of ICA significantly decreased the expression of these genes. Our results suggest that ICA promotes osteogenic differentiation in vitro and alleviates osteoporosis in vivo through inhibition of the Notch signaling pathway.

Preclinical development of T-cell receptor-engineered T-cell therapy targeting the 5T4 tumor antigen on renal cell carcinoma.

5T4 (trophoblast glycoprotein, TPBG) is a transmembrane tumor antigen expressed on more than 90% of primary renal cell carcinomas (RCC) and a wide range of human carcinomas but not on most somatic adult tissues. The favorable expression pattern has encouraged the development and clinical testing of 5T4-targeted antibody and vaccine therapies. 5T4 also represents a compelling and unexplored target for T-cell receptor (TCR)-engineered T-cell therapy. Our group has previously isolated high-avidity CD8+ T-cell clones specific for an HLA-A2-restricted 5T4 epitope (residues 17-25; 5T4p17). In this report, targeted single-cell RNA sequencing was performed on 5T4p17-specific T-cell clones to sequence the highly variable complementarity-determining region 3 (CDR3) of T-cell receptor α chain (TRA) and ß chain (TRB) genes. Full-length TRA and TRB sequences were cloned into lentiviral vectors and transduced into CD8+ T-cells from healthy donors. Redirected effector T-cell function against 5T4p17 was measured by cytotoxicity and cytokine release assays. Seven unique TRA-TRB pairs were identified. All seven TCRs exhibited high expression on CD8+ T-cells with transduction efficiencies from 59 to 89%. TCR-transduced CD8+ T-cells demonstrated redirected cytotoxicity and cytokine release in response to 5T4p17 on target-cells and killed 5T4+/HLA-A2+ kidney-, breast-, and colorectal-tumor cell lines as well as primary RCC tumor cells in vitro. TCR-transduced CD8+ T-cells also detected presentation of 5T4p17 in TAP1/2-deficient T2 target-cells. TCR-transduced T-cells redirected to recognize the 5T4p17 epitope from a broadly shared tumor antigen are of interest for future testing as a cellular immunotherapy strategy for HLA-A2+ subjects with 5T4+ tumors.

The Wnt-ß-catenin signaling regulated MRTF-A transcription to activate migration-related genes in human breast cancer cells.

MRTF-A is a transcriptional co-activator being critical for multiple processes including tissue fibrosis and cancer metastasis. The Rho-actin signaling stimulates the nuclear translocation and transcriptional activity of MRTF-A with little effect on the expression of MRTF-A gene. High expression of MRTF-A was observed in pancreatic cancer tissues and in TGF-ß treated breast cancer cells. However, the mechanism for the upregulation of MRTF-A gene remains unclear. In this study, we showed that the transcription of MRTF-A was regulated by the Wnt-ß-catenin signaling in breast cancer cells. LiCl treatment, Wnt3a treatment or ß-catenin overexpression enhanced the transcription of MRTF-A gene. In agreement, depletion of ß-catenin with siRNA diminished MRTF-A transcription. With ChIP assays, ß-catenin was identified to interact with the MRTF-A promoter whereby it increased histone H4 acetylation and RNA polymerase II association. Further, results of RT-qPCR and Western-blotting supported that the transcriptional co-activator activity of MRTF-A was controlled by both the Rho-actin and the Wnt-ß-catenin signaling pathways. MRTF-A was required for cell migration stimulated by the Wnt-ß-catenin signaling. Taken together, our results suggest that MRTF-A integrates the Rho-actin and the Wnt-ß-catenin signaling to regulate migration-related genes and consequently increases the mobility of breast cancer cells.

Oridonin induces G2/M cell cycle arrest and apoptosis via the PI3K/Akt signaling pathway in hormone-independent prostate cancer cells.

Oridonin is an active constituent isolated from the traditional Chinese herb Rabdosia rubescens, which exerts antitumor effects in experimental and clinical settings. However, its antitumor effects and underlying mechanisms on prostate cancer cells have not yet been clearly identified. In the present study, the androgen-independent prostate cancer PC3 and DU145 cell lines were used as models to investigate the effects and possible mechanisms of oridonin on cellular proliferation and apoptosis. Results demonstrated that oridonin inhibited cellular proliferation, and was able to significantly induce G2/M cell cycle arrest and apoptosis. Detailed signaling pathway analysis by western blotting demonstrated that the expression levels of p53 and p21 were upregulated, whereas the expression of cyclin-dependent kinase 1 was downregulated following oridonin treatment, which led to cell cycle arrest in the G2/M phase. Oridonin also upregulated the proteolytic cleaved forms of caspase-3, caspase-9 and poly (ADP-ribose) polymerase. Furthermore, the protein expression levels of B-cell lymphoma 2 were decreased and those of Bcl-2-associated X protein were increased following oridonin treatment. In addition, oridonin treatment significantly inhibited the expression of phosphoiniositide-3 kinase (PI3K) p85 subunit and the phosphorylation of Akt. The downstream gene murine double minute 2 was also downregulated, which may contribute to the elevated expression of p53 following oridonin treatment. In conclusion, the results of the present study suggested that oridonin is able to inactivate the PI3K/Akt pathway and activate p53 pathways in prostate cancer cells, resulting in the suppression of proliferation and the induction of caspase-mediated apoptosis.