Background and Aims Recent development of multiple treatments for human hepatocellular carcinoma (HCC) has allowed for the selection of combination therapy to enhance the effectiveness of monotherapy. Optimal selection of therapies is based on both HCC and its microenvironment. Therefore, it is critical to develop and validate preclinical animal models for testing clinical therapeutic solutions. Approach and Results We established cell line–based or patient‐derived xenograft–based humanized‐immune‐system mouse models with subcutaneous and orthotopic HCC. Mice were injected with human‐specific antibodies (Abs) to deplete human immune cells. We analyzed the transcription profiles of HCC cells and human immune cells by using real‐time PCR and RNA sequencing. The protein level of HCC tumor cells/tissues or human immune cells was determined by using flow cytometry, western blotting, and immunohistochemistry. The HCC tumor size was measured after single, dual‐combination, and triple‐combination treatment using N‐(1ʹ,2‐Dihydroxy‐1,2ʹ‐binaphthalen‐4ʹ‐yl)‐4‐methoxybenzenesulfonamide (C188‐9), bevacizumab, and pembrolizumab. In this study, human immune cells in the tumor microenvironment were strongly selected and modulated by HCC, which promoted the activation of the IL‐6/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in tumor cells and led to augmented HCC proliferation and angiogenesis by releasing angiogenic cytokines in humanized‐immune‐system mice with HCC. In particular, intratumor human cluster of differentiation–positive (hCD14+) cells could produce IL‐33 through damage‐associated molecular pattern/Toll‐like receptor 4/activator protein 1, which up‐regulated IL‐6 in other intratumor immune cells and activated the JAK2/STAT3 pathway in HCC. Specific knockdown of the CD14 gene in human monocytes could impair IL‐33 production induced by cell lysates. Subsequently, we evaluated the in vivo anti‐HCC effect of C188‐9, bevacizumab, and pembrolizumab. The results showed that the anti‐HCC effect of triple‐combination therapy was superior to that of single or dual treatments. Conclusions Humanized‐immune‐system HCC mouse models are suitable for identifying targets from cancer and immune components and for testing combinational therapies.
Differentiation of endometrial fibroblasts into specialized decidual cells controls embryo implantation and transforms the cycling endometrium into a semi-permanent, immune-protective matrix that accommodates the placenta throughout pregnancy. This process starts during the midluteal phase of the menstrual cycle with decidual transformation of perivascular cells (PVC) surrounding the terminal spiral arterioles and endometrial stromal cells (EnSC) underlying the luminal epithelium. Decidualization involves extensive cellular reprogramming and acquisition of a secretory phenotype, essential for coordinated placental trophoblast invasion. Secreted metabolites are an emerging class of signaling molecules, collectively known as the exometabolome. Here, we used liquid chromatography-mass spectrometry to characterize and analyze time-resolved changes in metabolite secretion (exometabolome) of primary PVC and EnSC decidualized over 8 days. PVC were isolated using positive selection of the cell surface marker SUSD2. We identified 79 annotated metabolites differentially secreted upon decidualization, including prostaglandin, sphingolipid, and hyaluronic acid metabolites. Secreted metabolites encompassed 21 metabolic pathways, most prominently glycerolipid and pyrimidine metabolism. Although temporal exometabolome changes were comparable between decidualizing PVC and EnSC, 32 metabolites were differentially secreted across the decidualization time-course. Further, targeted metabolomics demonstrated significant differences in secretion of purine pathway metabolites between decidualized PVC and EnSC. Taken together, our findings indicate that the metabolic footprints generated by different decidual subpopulations encode spatiotemporal information that may be important for optimal embryo implantation.
In recent decades, chimeric antigen receptor (CAR)–engineered immune effector cells have demonstrated promising antileukemic activity. Nevertheless, their efficacy remains unsatisfactory on solid cancers, plausibly due to the influence of tumor microenvironments (TME). In a novel mouse cancer model with a humanized immune system, tumor-infiltrating immunosuppressive leukocytes and exhausted programmed death protein-1 (PD-1) high T cells were found, which better mimic patient TME, allowing the screening and assessment of immune therapeutics. Particularly, membrane-bound programmed death ligand 1 (PD-L1) level was elevated on a tumor cell surface, which serves as an attractive target for natural killer (NK) cell–mediated therapy. Hematopoietic stem cell–derived CAR-NK (CAR pNK) cells targeting the PD-L1 showed enhanced in vitro and in vivo anti-solid tumor function. The CAR pNK cells and nivolumab resulted in a synergistic anti-solid tumor response. Together, our study highlights a robust platform to develop and evaluate the antitumor efficacy and safety of previously unexplored therapeutic regimens.
Differentiation of endometrial fibroblasts into specialized decidual cells controls embryo implantation and transforms the cycling endometrium into a semi-permanent, immune-protective matrix that accommodates the placenta throughout pregnancy. This process starts during the midluteal phase of the menstrual cycle with decidual transformation of perivascular cells (PVC) surrounding the terminal spiral arterioles and endometrial stromal cells (EnSC) underlying the luminal epithelium. Decidualization involves extensive cellular reprogramming and acquisition of a secretory phenotype, essential for coordinated placental trophoblast invasion. Secreted metabolites are an emerging class of signalling molecules. Here, we used liquid chromatography-mass spectrometry to characterise the dynamic changes in metabolite secretion (exometabolome) of primary PVC and EnSC decidualized over 8 days. We identified 79 annotated metabolites differentially secreted upon decidualization, including prostaglandin, sphingolipid, and hyaluronic acid metabolites. Secreted metabolites encompassed 21 metabolic pathways, most prominently glycerolipid and pyrimidine metabolism. Although temporal exometabolome changes were comparable between decidualizing PVC and EnSC, 32 metabolites were differentially secreted across the decidualization time-course. Further, targeted metabolomics demonstrated a conspicuous difference in xanthine secretion between decidualized PVC and EnSC. Taken together, our findings indicate that the metabolic footprints generated by different decidual subpopulations encode spatiotemporal information that may be important for optimal embryo implantation.
Women with endometriosis have a profound association with autoimmunity. An excess of autoantigens in the peritoneal cavity resulting from retrograde menstruation could lead to inflammation and pathologic autoimmunity. Using a native-conformation protein array, proteome-wide analysis of autoantibodies (AAbs) against 1623 proteins were profiled in peritoneal fluids (PF) of 25 women with endometriosis and 25 endometriosis-negative women. 46% of endometriotic women have five or more AAbs. Diverse cognate autoantigens were identified and corresponding AAbs against proteins involved in implantation, B-cell activation/development, and aberrant migration and mitogenicity. AAbs recognizing tumour suppressor protein p53 were the most frequent at 35% and were targeted against native and citrullinated p53 forms. Further, unsupervised hierarchical clustering and integrative pathway analysis, we observed clusters of endometriosis-associated infertile women with 60% positive for two or more AAbs which are involved in PDGF, TGF-β, RAC1/PAK1/p38/MMP2 signaling, LAT2/NTAL/LAB-mediated calcium mobilisation and integrin-mediated cell adhesion. Together, our data identifies peritoneal autoimmunity in a significant subset of women with endometriosis, with diverse impact on infertility and disease pathophysiology.
Study question Are alterations in the transcript levels of genes from the methionine cycle associated with maternal associated miscarriage risk? Summary answer Lower expression of AHCY is associated with a greater number of prior pregnancy losses What is known already Around 15% of pregnancies end in miscarriage, and the risk of recurrence increases with each pregnancy loss. Aberrant differentiation (decidualization) of endometrial stromal cells into specialised decidual cells to accommodate implantation is a key maternal factor for miscarriage risk. Our previous work identified secretory changes in cysteine and methionine metabolites upon decidualization. The methionine cycle contributes to vital cellular functions, including producing methionine for proliferation, regulating cell differentiation, and S-adenosylmethionine (SAM) production. SAM is required for protein, RNA and DNA methylation, thereby influencing pathways at the metabolic, epigenetic, and proteomic levels. AHCY clears S-adenosylhomocysteine (SAH), reducing its inhibition of methylation. Study design, size, duration Endometrial biopsies (n = 250) were collected during the luteal phase (LH + 6-11). Patients were grouped based on their miscarriage history. Accordingly, expression of genes from the methionine cycle were quantified using RT-qPCR. Participants/materials, setting, methods Endometrial biopsies were obtained, with written informed consent, from women attending the Implantation Clinic at University Hospitals Coventry and Warwickshire NHS Trust, following transvaginal ultrasounds to exclude uterine pathology. Isolated RNA was converted into cDNA. Expression of AHCY, AMD1, BHMT2, CBS, MAT2A, MAT2B, and MTR were normalised to L19. Statistical analysis was performed in Graphpad Prism; with significance accepted at p < 0.05. AHCY was silenced in an endometrial cell line to determine its effect on decidualization. Main results and the role of chance This study reports a distinct reduction in expression of methionine cycle genes (MAT2A, AHCY, AMD1, MTR, BHMT2) in the late-luteal phase of the cycle consistent with a reduction in proliferation. By plotting percentile graphs based on the statistical distribution in gene expression for each day of the luteal phase, comparisons have been made between groups. AHCY expression is significantly reduced in patients with increasing number of prior miscarriages, particularly between 0-2 and 5+ previous miscarriages (p = 0.0334). Neither patient age nor BMI are a factor in this reduced expression. In contrast, there is a stromal specific increase in AHCY upon decidualization in vitro, suggesting it is required in the decidua. Silencing AHCY in an endometrial cell line significantly reduces PRL expression upon decidualization. Reduction in AHCY may lead to a decreased “methylation potential” as SAH cannot be cleared. SAH accumulation inhibits methylation, and limits SAM production, thus compounding its effect. Decreased methylation potential could prevent differentiation of the stromal compartment, resulting in lower levels of PRL, and altering decidual timing. Therefore, an embryo may implant into a tissue primed for disintegration, resulting a miscarriage. In summary, AHCY may contribute to aberrant decidualization augmenting the risk of miscarriage. Limitations, reasons for caution Results are based on endometrial biopsies from an implantation clinic, therefore studies into biopsies from women with normal reproductive histories should also be analysed. Further functional studies are needed to ascertain the mechanism of action of AHCY in miscarriage. Wider implications of the findings This study identified that a decrease in AHCY in whole endometrial tissue is associated with increased risk of miscarriage. Further, silencing AHCY perturbed decidual marker expression. Thus, AHCY may act as a biomarker for atypical decidualization, and the clearance of SAH may be a potential treatment. Trial registration number N/A
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