Shikonin reduces M2 macrophage population in ovarian cancer by repressing exosome production and the exosomal galectin 3-mediated ß-catenin activation.

BACKGROUND: Shikonin (SK), a naphthoquinone with anti-tumor effects, has been found to decrease production of tumor-associated exosomes (exo). This study aims to verify the treatment effect of SK on ovarian cancer (OC) cells, especially on the production of exo and their subsequent effect on macrophage polarization. METHODS: OC cells SKOV3 and A2780 were treated with SK. The exo were isolated from OC cells with or without SK treatment, termed OC exo and SK OC exo, respectively. These exo were used to treat PMA-induced THP-1 cells (M0 macrophages). M2 polarization of macrophages was determined by measuring the M2 specific cell surface markers CD163 and CD206 as well as the secretion of M2 cytokine IL-10. The functions of galectin 3 (LGALS3/GAL3) and ß-catenin in macrophage polarization were determined by gain- or loss-of-function assays. CB-17 SCID mice were subcutaneously injected with SKOV3 cells to generate xenograft tumors, followed by OC exo or SK OC exo treatment for in vivo experiments. RESULTS: SK suppressed viability, migration and invasion, and apoptosis resistance of OC cells in vitro. Compared to OC exo, SK OC exo reduced the M2 polarization of macrophages. Regarding the mechanism, SK reduced exo production in cancer cells, and it decreased the protein level of GAL3 in exo and recipient macrophages, leading to decreased ß-catenin activation. M2 polarization of macrophages was restored by LGALS3 overexpression but decreased again by the ß-catenin inhibitor FH535. Compared to OC exo, the SK OC exo treatment reduced the xenograft tumor growth in mice, and it decreased the M2 macrophage infiltration within tumor tissues. CONCLUSION: This study suggests that SK reduces M2 macrophage population in OC by repressing exo production and blocking exosomal GAL3-mediated ß-catenin activation.

Spatial distribution of tumor-associated macrophages in an orthotopic prostate cancer mouse model.

Mounting evidence suggests that the immune landscape within prostate tumors influences progression, metastasis, treatment response, and patient outcomes. In this study, we investigated the spatial density of innate immune cell populations within NOD.SCID orthotopic prostate cancer xenografts following microinjection of human DU145 prostate cancer cells. Our laboratory has previously developed nanoscale liposomes that attach to leukocytes via conjugated E-selectin (ES) and kill cancer cells via TNF-related apoptosis inducing ligand (TRAIL). Immunohistochemistry (IHC) staining was performed on tumor samples to identify and quantify leukocyte infiltration for different periods of tumor growth and E-selectin/TRAIL (EST) liposome treatments. We examined the spatial-temporal dynamics of three different immune cell types infiltrating tumors using QuPath image analysis software. IHC staining revealed that F4/80+ tumor-associated macrophages (TAMs) were the most abundant immune cells in all groups, irrespective of time or treatment. The density of TAMs decreased over the course of tumor growth and decreased in response to EST liposome treatments. Intratumoral versus marginal analysis showed a greater presence of TAMs in the marginal regions at 3 weeks of tumor growth which became more evenly distributed over time and in tumors treated with EST liposomes. TUNEL staining indicated that EST liposomes significantly increased cell apoptosis in treated tumors. Additionally, confocal microscopy identified liposome-coated TAMs in both the core and periphery of tumors, highlighting the ability of liposomes to infiltrate tumors by "piggybacking" on macrophages. The results of this study indicate that TAMs represent the majority of innate immune cells within NOD.SCID orthotopic prostate tumors, and spatial density varies widely as a function of tumor size, duration of tumor growth, and treatment of EST liposomes.

Tumor Cell-Associated IL-1α Affects Breast Cancer Progression and Metastasis in Mice through Manipulation of the Tumor Immune Microenvironment.

IL-1α is a dual function cytokine that affects inflammatory and immune responses and plays a pivotal role in cancer. The effects of intracellular IL-1α on the development of triple negative breast cancer (TNBC) in mice were assessed using the CRISPR/Cas9 system to suppress IL-1α expression in 4T1 breast cancer cells. Knockout of IL-1α in 4T1 cells modified expression of multiple genes, including downregulation of cytokines and chemokines involved in the recruitment of tumor-associated pro-inflammatory cells. Orthotopical injection of IL-1α knockout (KO) 4T1 cells into BALB/c mice led to a significant decrease in local tumor growth and lung metastases, compared to injection of wild-type 4T1 (4T1/WT) cells. Neutrophils and myeloid-derived suppressor cells were abundant in tumors developing after injection of 4T1/WT cells, whereas more antigen-presenting cells were observed in the tumor microenvironment after injection of IL-1α KO 4T1 cells. This switch correlated with increased infiltration of CD3+CD8+ and NKp46+cells. Engraftment of IL-1α knockout 4T1 cells into immunodeficient NOD.SCID mice resulted in more rapid tumor growth, with increased lung metastasis in comparison to engraftment of 4T1/WT cells. Our results suggest that tumor-associated IL-1α is involved in TNBC progression in mice by modulating the interplay between immunosuppressive pro-inflammatory cells vs. antigen-presenting and cytotoxic cells.

Epstein-barr virus infections induce aberrant osteoclastogenesis in immune system-humanized NOD/Shi-scid/IL-2RγCnull mice.

Epstein-Barr virus (EBV) and other viral infections are possible triggers of autoimmune diseases, such as rheumatoid arthritis (RA). To analyze the causative relationship between EBV infections and RA development, we performed experiment on humanized NOD/Shi-scid/IL-2RγCnull (hu-NOG) mice reconstituted human immune system components and infected with EBV. In EBV-infected hu-NOG mice, breakdown of knee joint bones was found to be accompanied by the accumulation of receptor activator of nuclear factor-κB (NF-κB) (RANK) ligand (RANKL), a key factor in osteoclastogenesis, human CD19 and EBV-encoded small RNA (EBER)-bearing cells. Accumulation of these cells expanded in the bone marrow adjacent to the bone breakage, showing a histological feature like to that in bone marrow edema. On the other hand, human RANK/human matrix metalloprotease-9 (MMP-9) positive, osteoclast-like cells were found at broken bone portion of EBV-infected mouse knee joint. In addition, human macrophage-colony stimulating factor (M-CSF), an essential factor in development of osteoclasts, evidently expressed in spleen and bone marrow of EBV-infected humanized mice. Furthermore, RANKL and M-CSF were identified at certain period of EBV-transformed B lymphoblastoid cells (BLBCs) derived from umbilical cord blood lymphocytes. Co-culturing bone marrow cells of hu-NOG mice with EBV-transformed BLBCs resulted in the induction of a multinucleated cell population positive for tartrate-resistant acid phosphatase and human MMP-9 which indicating human osteoclast-like cells. These findings suggest that EBV-infected BLBCs induce human aberrant osteoclastogenesis, which cause erosive arthritis in the joints.

Bortezomib suppresses acute myelogenous leukaemia stem-like KG-1a cells via NF-κB inhibition and the induction of oxidative stress.

Acute myelogenous leukaemia (AML) originates and is maintained by leukaemic stem cells (LSCs) that are inherently resistant to antiproliferative therapies, indicating that a critical strategy for overcoming chemoresistance in AML therapy is to eradicate LSCs. In this work, we investigated the anti-AML activity of bortezomib (BTZ), emphasizing its anti-LSC potential, using KG-1a cells, an AML cell line with stem-like properties. BTZ presented potent cytotoxicity to both solid and haematological malignancy cells and reduced the stem-like features of KG-1a cells, as observed by the reduction in CD34- and CD123-positive cells. A reduction in NF-κB p65 nuclear staining was observed in BTZ-treated KG-1a cells, in addition to upregulation of the NF-κB inhibitor gene NFΚBIB. BTZ-induced DNA fragmentation, nuclear condensation, cell shrinkage and loss of transmembrane mitochondrial potential along with an increase in active caspase-3 and cleaved PARP-(Asp 214) level in KG-1a cells. Furthermore, BTZ-induced cell death was partially prevented by pretreatment with the pancaspase inhibitor Z-VAD-(OMe)-FMK, indicating that BTZ induces caspase-mediated apoptosis. BTZ also increased mitochondrial superoxide levels in KG-1a cells, and BTZ-induced apoptosis was partially prevented by pretreatment with the antioxidant N-acetylcysteine, indicating that BTZ induces oxidative stress-mediated apoptosis in KG-1a cells. At a dosage of 0.1 mg/kg every other day for 2 weeks, BTZ significantly reduced the percentage of hCD45-positive cells in the bone marrow and peripheral blood of NSG mice engrafted with KG-1a cells with tolerable toxicity. Taken together, these data indicate that the anti-LSC potential of BTZ appears to be an important strategy for AML treatment.

Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma.

BACKGROUND: EGFR and/or HER2 expression in pancreatic cancers is correlated with poor prognoses. We generated homodimeric (EGFRxEGFR or HER2xHER2) and heterodimeric (EGFRxHER2) T cell-engaging bispecific antibodies (T-BsAbs) to direct polyclonal T cells to these antigens on pancreatic tumors. METHODS: EGFR and HER2 T-BsAbs were constructed using the 2 + 2 IgG-[L]-scFv T-BsAbs format bearing two anti-CD3 scFvs attached to the light chains of an IgG to engage T cells while retaining bivalent binding to tumor antigens with both Fab arms. A Fab arm exchange strategy was used to generate EGFRxHER2 heterodimeric T-BsAb carrying one Fab specific for EGFR and one for HER2. EGFR and HER2 T-BsAbs were also heterodimerized with a CD33 control T-BsAb to generate 'tumor-monovalent' EGFRxCD33 and HER2xCD33 T-BsAbs. T-BsAb avidity for tumor cells was studied by flow cytometry, cytotoxicity by T-cell mediated 51Chromium release, and in vivo efficacy against cell line-derived xenografts (CDX) or patient-derived xenografts (PDX). Tumor infiltration by T cells transduced with luciferase reporter was quantified by bioluminescence. RESULTS: The EGFRxEGFR, HER2xHER2, and EGFRxHER2 T-BsAbs demonstrated high avidity and T cell-mediated cytotoxicity against human pancreatic ductal adenocarcinoma (PDAC) cell lines in vitro with EC50s in the picomolar range (0.17pM to 18pM). They were highly efficient in driving human polyclonal T cells into subcutaneous PDAC xenografts and mediated potent T cell-mediated anti-tumor effects. Both EGFRxCD33 and HER2xCD33 tumor-monovalent T-BsAbs displayed substantially reduced avidity by SPR when compared to homodimeric EGFRxEGFR or HER2xHER2 T-BsAbs (∼150-fold and ∼6000-fold respectively), tumor binding by FACS (8.0-fold and 63.6-fold), and T-cell mediated cytotoxicity (7.7-fold and 47.2-fold), while showing no efficacy against CDX or PDX. However, if either EGFR or HER2 was removed from SW1990 by CRISPR-mediated knockout, the in vivo efficacy of heterodimeric EGFRxHER2 T-BsAb was lost. CONCLUSION: EGFR and HER2 were useful targets for driving T cell infiltration and tumor ablation. Two arm Fab binding to either one or both targets was critical for robust anti-tumor effect in vivo. By engaging both targets, EGFRxHER2 heterodimeric T-BsAb exhibited potent anti-tumor effects if CDX or PDX were EGFR+HER2+ double-positive with the potential to spare single-positive normal tissue.

PUMC-MB1 is a novel group 3 medulloblastoma preclinical model, sensitive to PI3K/mTOR dual inhibitor.

PURPOSE: Medulloblastoma (MB), a common and heterogeneous posterior fossa tumor in pediatric patients, presents diverse prognostic outcomes. To advance our understanding of MB's intricate biology, the development of novel patient tumor-derived culture MB models with necessary data is still an essential requirement. METHODS: We continuously passaged PUMC-MB1 in vitro in order to establish a continuous cell line. We examined the in vitro growth using Cell Counting Kit-8 (CCK-8) and in vivo growth with subcutaneous and intracranial xenograft models. The xenografts were investigated histopathologically with Hematoxylin and Eosin (HE) staining and immunohistochemistry (IHC). Concurrently, we explored its molecular features using Whole Genome Sequencing (WGS), targeted sequencing, and RNA sequecing. Guided by bioinformatics analysis, we validated PUMC-MB1's drug sensitivity in vitro and in vivo. RESULTS: PUMC-MB1, derived from a high-risk MB patient, displayed a population doubling time (PDT) of 48.18 h and achieved 100% tumor growth in SCID mice within 20 days. HE and Immunohistochemical examination of the original tumor and xenografts confirmed the classification of PUMC-MB1 as a classic MB. Genomic analysis via WGS revealed concurrent MYC and OTX2 amplifications. The RNA-seq data classified it within the Group 3 MB subgroup, while according to the WHO classification, it fell under the Non-WNT/Non-SHH MB. Comparative analysis with D283 and D341med identified 4065 differentially expressed genes, with notable enrichment in the PI3K-AKT pathway. Cisplatin, 4-hydroperoxy cyclophosphamide/cyclophosphamide, vincristine, and dactolisib (a selective PI3K/mTOR dual inhibitor) significantly inhibited PUMC-MB1 proliferation in vitro and in vivo. CONCLUSIONS: PUMC-MB1, a novel Group 3 (Non-WNT/Non-SHH) MB cell line, is comprehensively characterized for its growth, pathology, and molecular characteristics. Notably, dactolisib demonstrated potent anti-proliferative effects with minimal toxicity, promising a potential therapeutic avenue. PUMC-MB1 could serve as a valuable tool for unraveling MB mechanisms and innovative treatment strategies.

Tumor promoting effect of spheroids in an orthotopic prostate cancer mouse model.

In this study, we aimed to establish a technique for intraprostatic implantation of prostate cancer (PCa) spheroids and to identify the impact of three-dimensional organization of PCa cells on tumor progression and metastasis in a representative in vivo model. 40,000 LNCaP cells were implanted into the prostate of immunodeficient SCID mice either as single cells (n = 8) or as preformed 3D spheroids (n = 8). For a follow up of 20 weeks, tumor growth was monitored by serum PSA and high-resolution 3D ultrasonography. Eventually, animals were sacrificed and autopsied. The organ dissects were analyzed for the presence of metastases by histology (H&E) and immunohistochemistry (AMACR, AR, Ki-67, CK5, CK8, E-Cadherin, Vimentin). Solid intraprostatic tumors developed in 50% of mice after spheroid implantation and in 50% of mice after implantation of a single cells. Primary tumors of LNCaP spheroids evolved earlier, exhibiting a shorter tumor doubling time whilst developing larger tumor volumes, which was reflected by a higher immunohistochemical expression of Ki-67 and AR, too. Spheroid tumors established lung and lymph node metastases in 75% of mice, in contrast to 50% of mice after single cell implantation. Our technique enables a variety of studies regarding the influence of the tumor microenvironment on PCa progression.

A Humanized Patient-Derived Xenograft Model for Pancreatic Cancer.

Pancreatic cancer is associated with a high mortality rate, and there are still very few effective treatment options. Patient-derived xenografts have proven to be invaluable preclinical disease models to study cancer biology and facilitate testing of novel therapeutics. However, the severely immune-deficient mice used to generate standard models lack any functional immune system, thereby limiting their utility as a tool to investigate the tumor-immune cell interface. This chapter will outline a method for establishment of "humanized" patient-derived xenografts, which are reconstituted with human immune cells to imitate the immune-rich microenvironment of pancreatic cancer.

Transient receptor potential vanilloid-1 (TRPV1) channels act as suppressors of the growth of glioma.

The aim of this study was to investigate the expression and function of the transient receptor potential vanilloid 1 (TRPV1) in glioma. We found that the expression of TRPV1 mRNA and protein were upregulated in glioma compared with normal brain by qPCR and western blot analysis. In order to investigate the function of TRPV1 in glioma, short hairpin RNA (shRNA) and the inhibitor of TRPV1 were used. In vitro, the activation of TRPV1 induced cell apoptosis with decreased migration capability and inhibited proliferation, which was abolished upon TRPV1 pharmacological inhibition and silencing. Mechanistically, TRPV1 modulated glioma proliferation through the protein kinase B (Akt) signaling pathway. More importantly, in immunodeficient (NOD-SCID) mouse xenograft models, tumor size was significantly increased when TRPV1 expression was disrupted by a shRNA knockdown approach in vivo. Altogether, our findings indicate that TRPV1 negatively controls glioma cell proliferation in an Akt-dependent manner, which suggests that targeting TRPV1 may be a potential therapeutic strategy for glioma.

Development of a compact bidirectional promoter-driven dual chimeric antigen receptor (CAR) construct targeting CD19 and CD20 in the Sleeping Beauty (SB) transposon system.

BACKGROUND: A bidirectional promoter-driven chimeric antigen receptor (CAR) cassette provides the simultaneous expression of two CARs, which significantly enhances dual antigen-targeted CAR T-cell therapy. METHODS: We developed a second-generation CAR directing CD19 and CD20 antigens, incorporating them in a head-to-head orientation from a bidirectional promoter using a single Sleeping Beauty transposon system. The efficacy of bidirectional promoter-driven dual CD19 and CD20 CAR T cells was determined in vitro against cell lines expressing either, or both, CD19 and CD20 antigens. In vivo antitumor activity was tested in Raji lymphoma-bearing immunodeficient NOD-scid IL2Rgammanull (NSG) mice. RESULTS: Of all tested promoters, the bidirectional EF-1α promoter optimally expressed transcripts from both sense (CD19-CAR) and antisense (GFP.CD20-CAR) directions. Superior cytotoxicity, cytokine production and antigen-specific activation were observed in vitro in the bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells. In contrast, a unidirectional construct driven by the EF-1α promoter, but using self-cleaving peptide-linked CD19 and CD20 CARs, showed inferior expression and in vitro function. Treatment of mice bearing advanced Raji lymphomas with bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells effectively controlled tumor growth and extended the survival of mice compared with group treated with single antigen targeted CAR T cells. CONCLUSION: The use of bidirectional promoters in a single vector offers advantages of size and robust CAR expression with the potential to expand use in other forms of gene therapies like CAR T cells.

The histone lysine acetyltransferase KAT2B inhibits cholangiocarcinoma growth: evidence for interaction with SP1 to regulate NF2-YAP signaling.

BACKGROUND: Cholangiocarcinoma (CCA) is a highly malignant cancer of the biliary tract with poor prognosis. Further mechanistic insights into the molecular mechanisms of CCA are needed to develop more effective target therapy. METHODS: The expression of the histone lysine acetyltransferase KAT2B in human CCA was analyzed in human CCA tissues. CCA xenograft was developed by inoculation of human CCA cells with or without KAT2B overexpression into SCID mice. Western blotting, ChIP-qPCR, qRT-PCR, protein immunoprecipitation, GST pull-down and RNA-seq were performed to delineate KAT2B mechanisms of action in CCA. RESULTS: We identified KAT2B as a frequently downregulated histone acetyltransferase in human CCA. Downregulation of KAT2B was significantly associated with CCA disease progression and poor prognosis of CCA patients. The reduction of KAT2B expression in human CCA was attributed to gene copy number loss. In experimental systems, we demonstrated that overexpression of KAT2B suppressed CCA cell proliferation and colony formation in vitro and inhibits CCA growth in mice. Mechanistically, forced overexpression of KAT2B enhanced the expression of the tumor suppressor gene NF2, which is independent of its histone acetyltransferase activity. We showed that KAT2B was recruited to the promoter region of the NF2 gene via interaction with the transcription factor SP1, which led to enhanced transcription of the NF2 gene. KAT2B-induced NF2 resulted in subsequent inhibition of YAP activity, as reflected by reduced nuclear accumulation of oncogenic YAP and inhibition of YAP downstream genes. Depletion of NF2 was able to reverse KAT2B-induced reduction of nuclear YAP and subvert KAT2B-induced inhibition of CCA cell growth. CONCLUSIONS: This study provides the first evidence for an important tumor inhibitory effect of KAT2B in CCA through regulation of NF2-YAP signaling and suggests that this signaling cascade may be therapeutically targeted for CCA treatment.

Everolimus decreases [U-13C]glucose utilization by pyruvate carboxylase in breast cancer cells in vitro and in vivo.

Reprogrammed metabolism is a hallmark of cancer, but notoriously difficult to target due to metabolic plasticity, especially in response to single metabolic interventions. Combining mTOR inhibitor everolimus and mitochondrial complex 1 inhibitor metformin results in metabolic synergy in in vitro models of triple-negative breast cancer. Here, we investigated whether the effect of this drug combination on tumor size is reflected in changes in tumor metabolism using [U-13C]glucose labeling in an MDA-MB-231 triple negative breast cancer xenograft model. The in vitro effects of everolimus and metformin treatment on oxidative phosphorylation and glycolysis reflected changes in 13C-labeling of metabolites in MDA-MB-231 cells. Treatment of MDA-MB-231 xenografts in SCID/Beige mice with everolimus resulted in slower tumor growth and reduced tumor size and tumor viability by 35%. Metformin treatment moderately inhibited tumor growth but did not enhance everolimus-induced effects. High serum levels of everolimus were reached, whereas levels of metformin were relatively low. Everolimus decreased TCA cycle metabolite labeling and inhibited pyruvate carboxylase activity. Metformin only caused a mild reduction in glycolytic metabolite labeling and did not affect pyruvate carboxylase activity or TCA cycle metabolite labeling. In conclusion, treatment with everolimus, but not metformin, decreased tumor size and viability. Furthermore, the efficacy of everolimus was reflected in reduced 13C-labeling of TCA cycle intermediates and reduced pyruvate carboxylase activity. By using in-depth analysis of drug-induced changes in glucose metabolism in combination with measurement of drug levels in tumor and plasma, effects of metabolically targeted drugs can be explained, and novel targets can be identified.

Dual-specificity tyrosine-regulated kinase 2 exerts anti-tumor effects by induction of G1 arrest in lung adenocarcinoma.

OBJECTIVES: Lung cancer is a leading cause of cancer-related mortality and remains one of the most poorly prognosed disease worldwide. Therefore, it is necessary to identify novel molecular markers with potential therapeutic effects. Recent findings have suggested that dual-specificity tyrosine-regulated kinase 2 (DYRK2) plays a tumor suppressive role in colorectal, breast, and hepatic cancers; however, its effect and mechanism in lung cancer remain poorly understood. Therefore, this study aimed to investigate the tumor-suppressive role and molecular mechanism of DYRK2 in lung adenocarcinoma (LUAD) by in vitro experiments and xenograft models. MATERIALS AND METHODS: The evaluation of DYRK2 expression was carried out using lung cancer cell lines and normal bronchial epithelial cells. Overexpression of DYRK2 was induced by an adenovirus vector, and cell proliferation was assessed through MTS assay and Colony Formation Assay. Cell cycle analysis was performed using flow cytometry. Additionally, proliferative capacity was evaluated in a xenograft model by subcutaneously implanting A549 cells into SCID mice (C·B17/Icr-scidjcl-scid/scid). RESULTS: Immunoblotting assays showed that DYRK2 was downregulated in most LUAD cell lines. DYRK2 overexpression using adenovirus vectors significantly suppressed cell proliferation compared with that in the control group. Additionally, DYRK2 overexpression suppressed tumor growth in a murine subcutaneous xenograft model. Mechanistically, DYRK2 overexpression inhibited the proliferation of LUAD cells via p21-mediated G1 arrest, which was contingent on p53. CONCLUSION: Taken together, these findings suggest that DYRK2 may serve as potential prognostic biomarker and therapeutic target for LUAD.

Long-term di-(2-ethylhexyl) phthalate exposure reduces sorafenib treatment efficacy by enhancing mesenchymal transition in hepatocellular carcinoma.

Di(2-ethylhexyl) phthalate (DEHP) is a worldwide common plasticizer. Nevertheless, DEHP is easily leached out to the environment due to the lack of covalent bonds with plastic. High dose of DEHP exposure is often observed in hemodialysis patients because of the continual usage of plastic medical devices. Although the liver is the major organ that catabolizes DEHP, the impact of long-term DEHP exposure on the sensitivity of liver cancer to chemotherapy remains unclear. In this study, we established long-term DEHP-exposed hepatocellular carcinoma (HCC) cells and two NOD/SCID mice models to investigate the effects and the underlying mechanisms of long-term DEHP exposure on chemosensitivity of HCC. The results showed long-term DEHP exposure potentially increased epithelial-mesenchymal transition (EMT) in HCC cells. Next generation sequencing showed that long-term DEHP exposure increased cell adhesion/migratory related genes expression and blunted sorafenib treatment induced genes alterations. Long-term exposure to DEHP reduced the sensitivity of HCC cells to sorafenib-induced anti-migratory effect by enhancing the EMT transcription factors (slug, twist, and ZEB1) and mesenchymal protein (vimentin) expression. In NOD/SCID mice model, we showed that long-term DEHP-exposed HCC cells exhibited higher growth rate. Regarding the anti-HCC effects of sorafenib, subcutaneous HuH7 tumor grew slowly in sorafenib-treated mice. Nonetheless, the anti-tumor growth effect of sorafenib was not observed in long-term DEHP-exposed mice. Higher mesenchymal markers and proliferating cell nuclear antigen (PCNA) expression were found in sorafenib-treated long-term DEHP-exposed mice. In conclusion, long-term DEHP exposure promoted migratory activity in HCC cells and decreased sorafenib sensitivity in tumor-bearing mice.

Scutellarin, a flavonoid compound from Scutellaria barbata, suppresses growth of breast cancer stem cells in vitro and in tumor-bearing mice.

BACKGROUND: Scutellaria barbata D. Don (SB), commonly known as Ban Zhi Lian and firstly documented by Shigong Chen, is a dried whole plant that has been studied for its therapeutic effects on breast cancer, colon cancer, and prostate cancer. Among its various compounds, scutellarin (SCU) has been demonstrated with anti-tumor effects. PURPOSE: This study aimed to evaluate the effects of SB water extract (SBW) and scutellarin on breast cancer stem cells (BCSCs), and to investigate their potential therapeutic effects on breast tumors in mice. METHODS: BCSCs were enriched from human breast cancer cells (MDA-MB-231 and MDA-MB-361) and their characteristics were analyzed. The effects of varying concentrations of SBW and scutellarin on cell viability, proliferation, self-renewal, and migration abilities were studied, along with the underlying mechanisms. The in vivo anti-tumor effects of scutellarin were further evaluated in SCID/NOD mice. Firstly, mice were inoculated with naïve BCSCs and subjected to treatment with scutellarin or vehicle. Secondly, BCSCs were pre-treated with scutellarin or vehicle prior to inoculation into mice. RESULTS: The derived BCSCs expressed CD44, CD133 and ALDH1, but not CD24, indicating that BCSCs have been successfully induced from both MDA-MB-231 and MDA-MB-361 cells. Both SBW and scutellarin reduced the viability, proliferation, sphere and colony formation, and migration of BCSCs. In mice with tumors derived from naïve BCSCs, scutellarin significantly reduced tumor growth, expression of proliferative (Ki67) and stem cell markers (CD44), and lung metastasis. In addition, pre-treatment with scutellarin also slowed tumor growth. Western blot results suggested the involvement of Wnt/ß-catenin, NF-κB, and PTEN/Akt/mTOR signaling pathways underlying the inhibitory effects of scutellarin. CONCLUSION: Our study demonstrated for the first time that both SB water extract and scutellarin could reduce the proliferation and migration of BCSCs in vitro. Scutellarin was shown to possess novel inhibitory activities in BCSCs progression. These findings suggest that Scutellaria barbata water extract, in particular, scutellarin, may have potential to be further developed as an adjuvant therapy for reducing breast cancer recurrence.

Fatty Acids Play a Critical Role in Mitochondrial Oxidative Phosphorylation in Effector T Cells in Graft-versus-Host Disease.

Although the role of aerobic glycolysis in activated T cells has been well characterized, whether and how fatty acids (FAs) contribute to donor T cell function in allogeneic hematopoietic stem cell transplantation is unclear. Using xenogeneic graft-versus-host disease (GVHD) models, this study demonstrated that exogenous FAs serve as a crucial source of mitochondrial respiration in donor T cells in humans. By comparing human T cells isolated from wild-type NOD/Shi-scid-IL2rγnull (NOG) mice with those from MHC class I/II-deficient NOG mice, we found that donor T cells increased extracellular FA uptake, the extent of which correlates with their proliferation, and continued to increase FA uptake during effector differentiation. Gene expression analysis showed the upregulation of a wide range of lipid metabolism-related genes, including lipid hydrolysis, mitochondrial FA transport, and FA oxidation. Extracellular flux analysis demonstrated that mitochondrial FA transport was required to fully achieve the mitochondrial maximal respiration rate and spare respiratory capacity, whereas the substantial disruption of glucose supply by either glucose deprivation or mitochondrial pyruvate transport blockade did not impair oxidative phosphorylation. Taken together, FA-driven mitochondrial respiration is a hallmark that differentiates TCR-dependent T cell activation from TCR-independent immune response after hematopoietic stem cell transplant.

T-cell help in the tumor microenvironment enhances rituximab-mediated NK-cell ADCC.

ABSTRACT: Rituximab (RTX) and other monoclonal antibodies (mAbs) that bind directly to malignant cells are of great clinical value but are not effective for all patients. A major mechanism of action of RTX is antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells. Prior in vitro studies in our laboratory demonstrated that T cells contribute to maintaining the viability and cytotoxic potential of NK cells activated by anti-CD20-coated target B cells. Here, we conducted studies using a novel mouse model and clinical correlative analysis to assess whether T-cell help contribute to RTX-mediated NK-cell ADCC in the tumor microenvironment (TME) in vivo. A humanized mouse model was developed using Raji lymphoma cells and normal donor peripheral blood mononuclear cells that allows for control of T-cell numbers in the lymphoma TME. In this model, NK-cell viability and CD16 and CD25 expression dropped after RTX in the absence of T cells but increased in the presence of T cells. RTX therapy was more effective when T cells were present and was ineffective when NK cells were depleted. In patients with indolent lymphoma, fine needle aspirates were obtained before and ∼1 week after treatment with a RTX-containing regimen. There was a strong correlation between CD4+ T cells as well as total T cells in the pretherapy TME and an increase in NK-cell CD16 and CD25 expression after RTX. We conclude that T-cell help in the TME enhances RTX-mediated NK-cell viability and ADCC.

Toxicology, pharmacokinetics, and immunogenicity studies of CCR4-IL2 bispecific immunotoxin in rats and minipigs.

We have developed a diphtheria toxin-based recombinant human CCR4-IL2 bispecific immunotoxin (CCR4-IL2-IT) for targeted therapy of cutaneous T-cell lymphoma (CTCL). CCR4-IL2-IT demonstrated superior efficacy in an immunodeficient mouse CTCL model. Recently, we have compared the in vivo efficacy of CCR4-IL2-IT versus Brentuximab (FDA approved leading drug in CTCL market) in the same immunodeficient mouse CTCL model. The comparison demonstrated that CCR4-IL2-IT was significantly more effective than Brentuximab. In this study, we have performed non-GLP (Good Laboratory Practice) toxicology, pharmacokinetics, immunogenicity studies of CCR4-IL2-IT in both rats and minipigs. CCR4-IL2-IT demonstrated excellent safety profiles in both rats and minipigs. The maximum tolerated dose of CCR4-IL2-IT was determined as 0.4 mg/kg in both rats and minipigs. Complete blood count and chemistry analysis did not show significant difference for all measured parameters between the blood samples of pre-injection versus post-injection from the five-day toxicology studies of CCT4-IL2-IT in both rats and minipigs. Histology analysis did not show difference between the PBS treatment group versus CCR4-IL2-IT treatment group at 50 µg/kg in both rats and minipigs. The half-life of CCR4-IL2-IT was determined as about 45 min in rats and 30 min in minipigs. The antibodies against CCR4-IL2-IT were detected in about two weeks after CCR4-IL2-IT treatment. CCR4-IL2-IT did not induce cytokine release syndrome in a peripheral blood mononuclear cell derived humanized mouse model. The depletion of CCR4+ cell and CD25+ cell (two target cell populations of CCR4-IL2-IT) was observed in minipigs. The excellent safety profile promoted us to further develop CCR4-IL2-IT towards clinical trials.

Targeting inhibition of TCTP could inhibit proliferation and induce apoptosis in AML cells.

Translationally controlled tumor protein (TCTP) is a highly conserved multifunctional protein, which participates in many important physiological processes. Recently, the roles of TCTP in cell proliferation and apoptosis, especially its close relationship with various tumors, have attracted widespread attention. In this study, we found that the protein level of TCTP was significantly reduced in acute promyelocytic leukemia cell line NB4 transfected with retinoic acid-induced gene G (RIG-G). The RIG-G was found in our previous work as a key mediator of anti-proliferative activity in retinoid/interferon-related pathways. Here, we tried to further explore the function of TCTP in the development of acute myeloid leukemia (AML) from different levels. Our results showed that inhibiting TCTP expression could attenuate AML cells proliferation and induce apoptosis both in AML cell lines and in xenograft of NOD-SCID mice. In addition, either compared with patients in complete remission or non-leukemia patients, we detected that the expression of TCTP was generally high in the fresh bone marrow of AML patients, suggesting that there was a certain correlation between TCTP and AML disease progression. Taken together, our study revealed the role of TCTP in AML development, and provided a potential target for AML treatment.