SerpinB2 deficiency is associated with delayed mammary tumor development and decreased pro-tumorigenic macrophage polarization.

The in vivo functions of SerpinB2 in tumor cells and tumor-associated macrophages (TAMs) during breast cancer development and metastasis remain elusive. SerpinB2-deficient MMTV-PyMT mice (PyMTSB2-/-) were previously produced to explore the biological roles of SerpinB2 in breast cancer. Compared with MMTV-PyMT wild-type (PyMTWT) mice, PyMTSB2-/- mice showed delayed tumor progression and reduced CK8 + tumor cell dissemination to lymph nodes. RNA-Seq data revealed significantly enriched genes associated with inflammatory responses, especially upregulated M1 and downregulated M2 macrophage marker genes in PyMTSB2-/- tumors. Decreased CD206+M2 and increased NOS2+M1 markers were detected in the primary tumors and metastatic lymph nodes of PyMTSB2-/- mice. In an in vitro study, SerpinB2 knockdown decreased the sphere formation and migration of MDA-MB-231 cells and suppressed protumorigenic M2 polarization of RAW264.7 cells. The combination of low SerpinB2, high NOS2, and low CD206 expression was favorable for survival in patients with breast cancer, as assessed in the BreastMark dataset. Our study demonstrates that SerpinB2 deficiency delays mammary tumor development and metastasis in PyMTWT mice, along with reduced sphere formation and migration abilities of tumor cells and decreased macrophage protumorigenic polarization.

Impact of media compositions and culture systems on the immunophenotypes of patient-derived breast cancer cells.

BACKGROUND: Heterogeneous tumor cells are thought to be a significant factor in the failure of endocrine therapy in estrogen receptor-positive (ER+) cancers. Culturing patient-derived breast cancer cells (PDBCCs) provides an invaluable tool in pre-clinical and translational research for the heterogeneity of cancer cells. This study aimed to investigate the effects of different media components and culture methods on the BCSC-associated immunophenotypes and gene expression in ER + PDBCCs. METHODS: Ten patients with ER + breast cancer were employed in this study, six of whom had neoadjuvant chemotherapy and four of whom did not. PDBCCs were isolated by enzymatic methods using collagen I and hyaluronidase. PDBCCs were grown as monolayers in mediums with different compositions and as multicellular spheroid in a suspended condition. Collagen I-coated plate and ultralow attachment plate coated with polymer-X were used for monolayer and spheroid culture. Flow cytometry, immunofluorescent staining, RT-PCR, and RNA-sequencing were employed to examine the immunophenotype and genetic profile of PDBCCs. RESULTS: More than 95% of PDBCCs sustain EpCAM high/+/fibroblast marker- phenotypes in monolayer conditions by subculturing 3-4 times. A83-01 removal induced senescent cells with high ß-galactosidase activity. PDBCCs grown as monolayers were characterized by the majority of cells having an EpCAM+/CD49f + phenotype. Compared to full media in monolayer culture, EGF removal increased EpCAM+/CD49f - phenotype (13.8-fold, p = 0.028), whereas R-spondin removal reduced it (0.8-fold, p = 0.02). A83-01 removal increased EpCAM+/CD24 + phenotype (1.82-fold, p = 0.023) and decreased EpCAM low/-/CD44+/CD24- phenotype (0.45-fold, p = 0.026). Compared to monolayer, spheroid resulted in a significant increase in the population with EpCAM-/CD49+ (14.6-fold, p = 0.006) and EpCAM low/-/CD44+/CD24- phenotypes (4.16-fold, p = 0.022) and ALDH high activity (9.66-fold, p = 0.037). ALDH1A and EMT-related genes were upregulated. In RNA-sequencing analysis between spheroids and monolayers, a total of 561 differentially expressed genes (2-fold change, p < 0.05) were enriched in 27 KEGG pathways including signaling pathways regulating pluripotency of stem cells. In a recurrence-free survival analysis based on the Kaplan-Meier Plotter database of the up-and down-regulated genes identified in spheroids, 15 up-, and 14 down-regulated genes were associated with poor prognosis of breast cancer patients. CONCLUSION: The media composition and spheroid culture method change in the BCSCs and EMT markers of PDBCCs, implying the importance of defining the media composition and culture method for studying PDBCCs in vitro.

Transcriptome analysis of SerpinB2-deficient breast tumors provides insight into deciphering SerpinB2-mediated roles in breast cancer progression.

BACKGROUND: SerpinB2 is highly expressed in immune and tumor cells and is involved in multiple biological functions, including cell survival and remodeling for disease progression. This study prepared SerpinB2-deficient mice and analyzed the differentially expressed genes (DEGs) to determine if loss of this protein delays mammary tumor progression. RESULTS: A total of 305 DEGs (75 upregulated and 230 downregulated; > 1.5-fold difference, P < 0.05) were identified in SB2-/-;PyMT tumors compared with PyMT tumors. The DEGs were mainly involved in immune and inflammatory responses related to T cell differentiation, IFN-γ production, and lymphocyte chemotaxis based on 61 enriched GO terms, hierarchical clustering, KEGG pathways, and a functionally grouped annotation network. The significantly changed DEGs (Anxa3, Ccl17, Cxcl13, Cxcr3, IFN-γ, Nr4a1, and Sema3a) annotated with at least two GO categories in SB2-/-;PyMT tumors was validated by qRT-PCR. CONCLUSIONS: SerpinB2 deficiency alters the expression of multiple genes in mammary tumors, which might cause a delay in PyMT-induced mammary tumor progression.

Cancer-associated fibroblasts induce an aggressive phenotypic shift in non-malignant breast epithelial cells via interleukin-8 and S100A8.

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment have been associated with tumor progression in breast cancer. Although crosstalk between breast cancer cells and CAFs has been studied, the effect of CAFs on non-neoplastic breast epithelial cells is not fully understood to date. Here, we investigated the effect of CAFs on aggressive phenotypes in non-neoplastic MCF10A breast epithelial cells. CAFs induced epithelial-to-mesenchymal transition (EMT) and invasive phenotype in MCF10A cells. S100A8, a potential prognostic marker in several cancers, was markedly increased in MCF10A cells by CAFs. S100A8 was crucial for CAFs-induced invasive phenotype of MCF10A cells. Among cytokines increased by CAFs, interleukin (IL)-8 induced S100A8 through transcription factors p65 NF-κB and C/EBPß. In a xenograft mouse model with MCF10A cells and CAFs, tumor was not developed, suggesting that coinjection with CAFs may not be sufficient for in vivo tumorigenicity of MCF10A cells. Xenograft mouse tumor models with MDA-MB-231 breast carcinoma cells provided an in vivo evidence for the effect of CAFs on breast cancer progression as well as a crucial role of IL-8 in tumor growth and S100A8 expression in vivo. Using a tissue microarray of human breast cancer, we showed that S100A8 expression was correlated with poor outcomes. S100A8 expression was more frequently detected in cancer-adjacent normal human breast tissues than in normal breast tissues. Together, this study elucidated a novel mechanism for the acquisition of invasive phenotype of non-neoplastic breast cells induced by CAFs, suggesting that targeting IL-8 and S100A8 may be an effective strategy against breast cancer.

Theranostics for Breast Cancer Stem Cells.

Effectively targeting and treating breast cancer stem cells (BCSCs), which have been linked to tumor development and metastasis, and recurrence still remains a challenging issue in preclinic and clinic. Screening and identifying characteristic BCSC biomarkers is important for distinguishing BCSCs from differentiated tumor cells within the tumor mass. Molecular imaging and nanotechnology are evolving as new fields that have a potentially high research and clinical impact. Developing the biocompatible contrast agents conjugated with high-affinity biomarker to selectively target BCSCs and is one of the key prerequisites for image-guided diagnosis and monitoring therapy of BCSCs. Very recently, we documented the extra domain-B fibronectin (EDB-FN), which is considered as a new putative biomarker for BCSCs (NDY-1 cell) derived from human breast carcinosarcoma. We here review BCSC-targeted theranostics in vitro and in vivo by delivering siRNA or drug using the nanoparticles conjugated with a small peptide specific to EDB-FN.

NAD(P)-dependent steroid dehydrogenase-like is involved in breast cancer cell growth and metastasis.

BACKGROUND: The cholesterol biosynthesis pathway is typically upregulated in breast cancer. The role of NAD(P)-dependent steroid dehydrogenase-like (NSDHL) gene, which is involved in cholesterol biosynthesis, in breast cancer remains unknown. This study aimed to uncover the role of NSDHL in the growth and metastasis of breast cancer. METHODS: After NSDHL knockdown by transfection of short interfering RNA into human breast cancer cell lines (MCF-7, MDA-MB-231 and BT-20) and human breast epithelial cell line (MCF10A), cell proliferation assay, cell cycle analysis, three-dimensional cell culture, clonogenic assay, transwell migration and invasion assays, and wound healing assay were performed. Erlotinib was used as the target drug for epidermal growth factor receptor. Immunodeficient mice (NOD.Cg-Prkdcscid Il2rgtm1wjl /SzJ) were used as orthotropic breast tumor models by injecting them with NSDHL-knockdown MDA-MB-231 cells using lentivirus-carrying NSDHL short hairpin RNA. Clinical data from 3951 breast cancer patients in Gene Expression Omnibus databases were used to investigate the potential prognostic role of NSDHL by survival analysis. RESULTS: NSDHL knockdown in BT-20, and MDA-MB-231 resulted in a significant decrease in their viability, colony formation, migration, and invasion abilities (p < 0.05). Total cholesterol levels were observed to be significantly decreased in NSDHL-knockdown BT-20 and MDA-MB-231 (p < 0.0001). NSDHL knockdown significantly increased the rate of erlotinib-induced cell death, especially in MDA-MB-231 (p = 0.01). NSDHL knockdown led to significantly decreased tumor growth and lung metastasis in the MDA-MB-231 xenograft model (p < 0.01). Clinically, high NSDHL expression in tumors of patients with breast cancer was associated with significantly reduced recurrence-free survival (p < 0.0001). CONCLUSIONS: NSDHL might have a role in promoting breast cancer progression. The usage of NSDHL as a therapeutic target in breast cancer needs to be clarified in further studies.

Comparison of transcriptome expression alterations by chronic exposure to low-dose bisphenol A in different subtypes of breast cancer cells.

The impacts of chronic bisphenol A (BPA) exposure suspected to be a potential risk factor for breast cancer progression are not thoroughly understood in different subtypes of breast cancer cells (BCCs). This study aimed to compare the differentially expressed genes (DEGs) and biological functions in MCF-7 (luminal A), SK-BR3 (HER2-enriched) and MDA-MB-231 (triple-negative) cells exposed to BPA at an environmentally human-relevant low dose (10-8 M) for 30 days, by using the approach of RNA sequencing and online informatics tools. BPA-exposure resulted in 172, 137, and 139 DEGs in MCF-7/BPA, SK-BR3/BPA, and MDA-MB-231/BPA, respectively. The significantly enriched gene ontology terms of DEGs in each cell were different: cellular response to gonadotropin-releasing hormone, negative regulation of fibrinolysis, choline metabolism, glutamate signaling pathways and coagulation pathway in MCF-7/BPA; positive regulation of inflammatory response and VEGF/VEGFR signaling pathways in SK-BR3/BPA; negative regulation of keratinocyte proliferation and HIF signaling pathways in MDA-MB-231/BPA cells. The immune network analysis of DEGs across the breast cancer cells indicated NKT, NK and T cell activation and dendritic cell migration by regulating the expression of immunomodulatory genes. High expression of IL19, CA9 and SPARC identified in MCF-7/BPA, SK-BR3/BPA, and MDA-MB-231/BPA are detrimental gene signatures to predict poor overall survival in luminal A, HER2-enriched and triple-negative breast cancer patients, respectively. These findings indicate chronic BPA exposure has dissimilar impacts on the regulation of gene expression and diverse biological functions, including immune modulation, in different subtypes of BCCs.

A novel role for flotillin-1 in H-Ras-regulated breast cancer aggressiveness.

Elevated expression and aberrant activation of Ras have been implicated in breast cancer aggressiveness. H-Ras, but not N-Ras, induces breast cell invasion. A crucial link between lipid rafts and H-Ras function has been suggested. This study sought to identify the lipid raft protein(s) responsible for H-Ras-induced tumorigenicity and invasiveness of breast cancer. We conducted a comparative proteomic analysis of lipid raft proteins from invasive MCF10A human breast epithelial cells engineered to express active H-Ras and non-invasive cells expressing active N-Ras. Here, we identified a lipid raft protein flotillin-1 as an important regulator of H-Ras activation and breast cell invasion. Flotillin-1 was required for epidermal growth factor-induced activation of H-Ras, but not that of N-Ras, in MDA-MB-231 triple-negative breast cancer (TNBC) cells. Flotillin-1 knockdown inhibited the invasiveness of MDA-MB-231 and Hs578T TNBC cells in vitro and in vivo. In xenograft mouse tumor models of these TNBC cell lines, we showed that flotillin-1 played a critical role in tumor growth. Using human breast cancer samples, we provided clinical evidence for the metastatic potential of flotillin-1. Membrane staining of flotillin-1 was positively correlated with metastatic spread (p = 0.013) and inversely correlated with patient disease-free survival rates (p = 0.005). Expression of flotillin-1 was associated with H-Ras in breast cancer, especially in TNBC (p < 0.001). Our findings provide insight into the molecular basis of Ras isoform-specific interplay with flotillin-1, leading to tumorigenicity and aggressiveness of breast cancer.

Overexpression of the miR-141/200c cluster promotes the migratory and invasive ability of triple-negative breast cancer cells through the activation of the FAK and PI3K/AKT signaling pathways by secreting VEGF-A.

BACKGROUND: The role of microRNA-200 (miR-200) family members in the migration and invasion of breast cancer is controversial. This study investigated the mechanisms by which the miR-200 family members modulated the migratory and invasive abilities of an aggressive triple-negative breast cancer (TNBC) cell line, MDA-MB-231. METHODS: The miR-200 family (miR-200b/200a/429 and miR-141/200c clusters) and green fluorescence protein (GFP) were transduced into MDA-MB-231 cells using a lentiviral system. Stable cells highly expressing the miR-200 family and GFP were isolated by puromycin selection and fluorescence-activated cell sorting. Gene expression was evaluated using real-time polymerase chain reaction (PCR) and reverse transcriptase-PCR (RT-PCR). The migratory and invasive abilities were assessed using trans-well and wound-healing assays. The secreted cytokines and growth factors in cultured media were quantified using a Bio-Plex200 multiplex array system. Western blot assays and immunofluorescence staining were conducted to investigate miR-200 family-regulated signaling pathways. The entire dataset obtained in this study was statistically evaluated using a one-way ANOVA followed by a t-test. RESULTS: The stable overexpression of the miR-200b/200a/429 or miR-141/200c cluster suppressed cell growth and significantly increased migration and invasion of MDA-MB-231 cells. miR-141/200c overexpression was more effective in decreasing cell growth and promoting migration and invasion of MDA-MB-231 cells than was miR-200b/200a/429 overexpression. In addition, the overexpression of the miR-200b/200a/429 or miR-141/200c cluster led to an increase in the phosphorylation of focal adhesion kinase (FAK) and protein kinase B (AKT). Chemical inhibitors of FAK and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT suppressed the migration and invasion of MDA-MB-231 cells that was enhanced by the overexpression of the miR-200b/200a/429 or miR-141/200c cluster. Compared to the miR-200b/200a/429 cluster-transduced MDA-MB-231 cells, the miR-141/200c cluster-transduced MDA-MB-231 cells exhibited a significant increase in vascular endothelial growth factor (VEGF)-A secretion and integrin-alphaV (integrin-αV) expression. Treatment with an anti-VEGF-A-neutralizing antibody inhibited the increase in migration and invasion in both the miR-200b/200a/429- and miR-141/200c-transduced MDA-MB-231 cells but significantly reduced the phosphorylation of FAK and AKT in only the miR-141/200c cluster-transduced MDA-MB-231 cells. CONCLUSIONS: Taken together, our data demonstrate a mechanism in which the miR-141/200c cluster, through FAK- and PI3K/AKT-mediated signaling by means of increased VEGF-A secretion, promotes the migratory and invasive abilities of MDA-MB-231 cells.

Noninvasive MRI and multilineage differentiation capability of ferritin-transduced human mesenchymal stem cells.

Molecular imaging can be a breakthrough tool for the investigation of the behavior and ultimate feasibility of transplanted human mesenchymal stem cells (hMSCs) inside the body, and for the development of guidelines and recommendations based on the treatment and evaluation of stem cell therapy for patients. The goals of this study were to evaluate the multilineage differentiation ability of hMSCs expressing an MRI reporter, human ferritin heavy chain (FTH) and to investigate the feasibility of using FTH-based MRI to provide noninvasive imaging of transplanted hMSCs. The transduction of FTH and green fluorescence protein (GFP) did not influence the expression of the mesenchymal stem cell surface markers (CD29+/CD105+/CD34-/CD45-) or the self-renewal marker genes [octamer-binding transcription factor 4 (OCT-4) and SRY (sex determining region Y)-box 2 (Sox-2)], cell viability, migration ability and the release of cytokines [interleukin-5 (IL-5), IL-10, IL-12p70, tumor necrosis factor-α (TNF-α)]. FTH-hMSCs retained the capacity to differentiate into adipogenic, chondrogenic, osteogenic and neurogenic lineages. The transduction of FTH led to a significant enhancement in cellular iron storage capacity and caused hypointensity and a significant increase in R2 * values of FTH-hMSC-collected phantoms and FTH-hMSC-transplanted sites of the brain, as shown by in vitro and in vivo MRI performed at 9.4 T, compared with control hMSCs. This study revealed no differences in biological characteristics between hMSCs and FTH-hMSCs and, therefore, these cells could be used for noninvasive monitoring with MRI during stem cell therapy for brain injury. Our study suggests the use of FTH for in vivo long-term tracking and ultimate fate of hMSCs without alteration of their characteristics and multidifferentiation potential.

Changes in metabolic markers in insulin-producing ß-cells during hypoxia-induced cell death as studied by NMR metabolomics.

This study was designed to investigate changes in the metabolites in the intracellular fluid of the pancreatic ß-cell line INS-1 to identify potential early and late biomarkers for predicting hypoxia-induced cell death. INS-1 cells were incubated under normoxic conditions (95% air, 5% CO2) or hypoxic conditions (1% O2, 5% CO2, 95% N2) for 2, 4, 6, 12, or 24 h. The biological changes indicating the process of cell death were analyzed using the MTT assay, flow cytometry, Western blotting, and immunostaining. Changes in the metabolic profiles from cell lysates were identified using ¹H nuclear magnetic resonance (¹H NMR) spectroscopy, and the spectra were analyzed by the multivariate model Orthogonal Projections to Latent Structure-Discriminant Analysis. Cell viability decreased approximately 40% after 12-24 h of hypoxia, coincident with a high level of cleaved caspase-3. A high level of HIF-1α was detected in the 12-24 h hypoxic conditions. The metabolite profiles were altered according to the degree of exposure to hypoxia. A spectral analysis showed significant differences in creatine-containing compounds at the early stage (2-6 h) and taurine-containing compounds at the late stage (12-24 h), with the detection of HIF-1α and cleaved caspase-3 in cells exposed to hypoxia compared to normoxia. Glycerophosphocholine decreased during the early stage hypoxia. The change in taurine- and creatine-containing compounds and choline species could be involved in the ß-cell death process as inhibitors or activators of cell death. Our results imply that assessment by ¹H NMR spectroscopy would be a useful tool to predict the cell death process and to identify molecules regulating hypoxia-induced cell death mechanisms.

Modification of p53 with O-linked N-acetylglucosamine regulates p53 activity and stability.

Post-translational addition of O-linked N-acetylglucosamine (O-GlcNAc) to p53 is known to occur, but the site of O-GlcNAcylation and its effects on p53 are not understood. Here, we show that Ser 149 of p53 is O-GlcNAcylated and that this modification is associated with decreased phosphorylation of p53 at Thr 155, which is a site that is targeted by the COP9 signalosome, resulting in decreased p53 ubiquitination. Accordingly, O-GlcNAcylation at Ser 149 stabilizes p53 by blocking ubiquitin-dependent proteolysis. Our results indicate that the dynamic interplay between O-GlcNAc and O-phosphate modifications coordinately regulate p53 stability and activity.

Triple-Negative Breast Cancer-Derived Extracellular Vesicles Promote a Hepatic Premetastatic Niche via a Cascade of Microenvironment Remodeling.

Patients with triple-negative breast cancer (TNBC) often develop metastases in visceral organs including the liver, but the detailed molecular mechanisms of TNBC liver metastasis is not clearly understood. In this study, we tried to dissect the process of premetastatic niche formation in the liver by using patient-derived xenograft (PDX) models of TNBC with different metastatic propensity. RNA sequencing of TNBC PDX models that successfully metastasized to liver showed upregulation of the Cx3cr1 gene in the liver microenvironment. In syngeneic breast cancer models, the Cx3cr1 upregulation in liver preceded the development of cancer cell metastasis and was the result of recruitment of CX3CR1-expressing macrophages. The recruitment was induced by the CX3CL1 production from the liver endothelial cells and this CX3CL1-CX3CR1 signaling in the premetastatic niche resulted in upregulation of MMP9 that promoted macrophage migration and cancer cell invasion. In addition, our data suggest that the extracellular vesicles derived from the breast cancer cells induced the TNFα expression in liver, which leads to the CX3CL1 upregulation. Lastly, the plasma CX3CL1 levels in 155 patients with breast cancer were significantly associated with development of liver metastasis. IMPLICATIONS: Our data provides previously unknown cascades regarding the molecular education of premetastatic niche in liver for TNBC.

O-GlcNAc modification of PPARγ reduces its transcriptional activity.

The peroxisome proliferator-activated receptor γ (PPARγ), a member of the nuclear receptor superfamily, is a key regulator of adipogenesis and is important for the homeostasis of the adipose tissue. The ß-O-linked N-acetylglucosamine (O-GlcNAc) modification, a posttranslational modification on various nuclear and cytoplasmic proteins, is involved in the regulation of protein function. Here, we report that PPARγ is modified by O-GlcNAc in 3T3-L1 adipocytes. Mass spectrometric analysis and mutant studies revealed that the threonine 54 of the N-terminal AF-1 domain of PPARγ is the major O-GlcNAc site. Transcriptional activity of wild type PPARγ was decreased 30% by treatment with the specific O-GlcNAcase (OGA) inhibitor, but the T54A mutant of PPARγ did not respond to inhibitor treatment. In 3T3-L1 cells, an increase in O-GlcNAc modification by OGA inhibitor reduced PPARγ transcriptional activity and terminal adipocyte differentiation. Our results suggest that the O-GlcNAc state of PPARγ influences its transcriptional activity and is involved in adipocyte differentiation.

Imaging and quantification of metastatic melanoma cells in lymph nodes with a ferritin MR reporter in living mice.

Cellular MRI with a reporter gene offers the opportunity to track small numbers of tumor cells and to study metastatic processes in their earliest developmental stages in the target organs of interest. This study demonstrates the feasibility of using the MR reporter ferritin for the noninvasive imaging and quantification of metastatic melanoma cells in the lymph nodes (LNs) of living mice. A B16F10 murine melanoma cell line expressing human ferritin heavy chain (hFTH) and green fluorescent protein (GFP) was constructed to allow the detection of cells by MRI and fluorescence imaging. Stable overexpression of hFTH and GFP in B16F10 murine melanoma cells was feasible and showed no cellular toxicity. In addition, hFTH cells were detectable by 9.4-T MRI in vitro and in vivo, yielding significant changes in T(2)* relative to control cells. In BALB/c nude mice, the presence of hFTH- and GFP-expressing metastatic melanoma cells in deep-seated axillary LNs was demonstrated as areas of low T(2)* on MRI, but the same LNs were not visible by fluorescence imaging because the light was unable to penetrate the tissue. Furthermore, the metastatic volume of each LN, which was assessed by cumulative histogram analysis of the T(2)* MRI data, correlated well with tumor burden, which was determined by histology (r = -0.8773, p = 0.0001). This study is the first to use MRI and an MR reporter gene for both the visualization and quantification of metastatic cancer cells in LNs.

Detection of prostaglandin E2-induced dendritic cell migration into the lymph nodes of mice using a 1.5 T clinical MR scanner.

The control of dendritic cell (DC) migration into lymph nodes (LNs) is important for the development of more effective DC-based immunotherapies. This study was undertaken to evaluate, dynamically and noninvasively, prostaglandin E2 (PGE2)-enhanced migration of DCs using a 1.5 T clinical MR scanner. DC2.4 cells were labeled with superparamagnetic iron oxide (SPIO), a clinically approved MRI contrast agent. DCs were stimulated with tumor necrosis factor-α and interferon-γ in the presence or absence of PGE2. Before and after subcutaneous injection of labeled DCs into the hind leg footpads of mice, MRI detailing the extent of DC migration into popliteal LNs was performed using a 1.5 T clinical MR scanner. SPIO labeling did not influence the viability, endocytic activity, migratory ability and/or co-stimulatory molecule expression of DCs. PGE2 enhanced significantly chemokine receptor-7 expression and the migration of DCs (p < 0.05). After subcutaneous injection of DCs, there were decreases in MR signal intensity in popliteal LNs at 24 h post-injection; in PGE2-treated cells, the MR signal intensity was significantly lower (decrease of 86.6 ± 2.5%) than in PGE2-untreated cells (decrease of 70.0 ± 4.2%) (p < 0.05). Histological analyses with the conventionally used Prussian blue stain demonstrated that the PGE2-treated DCs migrated more deeply into the center of LNs. PGE2-enhanced migration of SPIO-labeled DCs into LNs can be detected using a 1.5 T clinical MR scanner. Our results suggest that in vivo MRI of DC migration is a useful imaging method to predict DC therapy with a high rate of efficacy and to improve DC-based immunotherapy, thereby reducing costs compared with current treatments in clinical trials.

Spatial epitranscriptomics reveals A-to-I editome specific to cancer stem cell microniches.

Epitranscriptomic features, such as single-base RNA editing, are sources of transcript diversity in cancer, but little is understood in terms of their spatial context in the tumour microenvironment. Here, we introduce spatial-histopathological examination-linked epitranscriptomics converged to transcriptomics with sequencing (Select-seq), which isolates regions of interest from immunofluorescence-stained tissue and obtains transcriptomic and epitranscriptomic data. With Select-seq, we analyse the cancer stem cell-like microniches in relation to the tumour microenvironment of triple-negative breast cancer patients. We identify alternative splice variants, perform complementarity-determining region analysis of infiltrating T cells and B cells, and assess adenosine-to-inosine base editing in tumour tissue sections. Especially, in triple-negative breast cancer microniches, adenosine-to-inosine editome specific to different microniche groups is identified.

Application of immunotherapy based on dendritic cells stimulated by tumor cell-derived exosomes in a syngeneic breast tumor mouse model.

We here evaluated the therapeutic effect of tumor cell-derived exosomes (TEXs)-stimulated dendritic cells (DCs) in a syngeneic orthotopic breast tumor model. The DC line DC2.4 and breast cancer cell line E0771 originally isolated from C57BL/6 mice were used. E0771 cells stably expressing the exosomal CD63-RFP or luciferase (Luc) and DC2.4 cells stably expressing GFP were produced using lentivirus. TEXs were purified from conditioned medium of E0771/CD63-RFP cells. Breast tumor model was established by injecting E0771/Luc cells into mammary gland fat pad of mice. TEXs contained immune modulatory molecules such as HSP70, HSP90, MHC I, MHC II, TGF-ß, and PD-L1. TEXs were easily taken by DC2.4 cells, resulting in a significant increase in the in vitro proliferation and migration abilities of DC2.4 cells, accompanied by the upregulation of CD40. TEX-DC-treated group exhibited a decreased tumor growth compared with control group. CD8+ cells were more abundant in the tumors and lymph nodes of TEX-DC-treated group than in those of control group, whereas many CD4+ or FOXP3+ cells were localized in those of control group. Our results suggest a potential application of TEX-DC-based cancer immunotherapy.

The effects of clinically used MRI contrast agents on the biological properties of human mesenchymal stem cells.

This study was undertaken to compare the labeling efficiencies of three iron-oxide based MRI contrast agents [Feridex, Resovist and monocrystalline iron oxide (MION)] and to evaluate their effects on the biological properties of human mesenchymal stem cells (hMSCs). The hMSCs were cultivated for 1 and 7 days after 24-h labeling with iron oxide nanoparticles (12.5 microg Fe/mL) in the presence of poly-L-lysine (0.75 microg/mL). The hMSCs were labeled more efficiently with use of Feridex, Resovist as compared to MION. No significant differences were observed in terms of viability and proliferation of labeled hMSCs. The level of Oct-4 mRNA increased in labeled hMSCs at day 1 and the cellular phenotype changed from CD45-/CD44+/CD29+ to CD45low/CD44+/CD29+ at day 7, which closely resembles the phenotype of fresh bone marrow-derived hMSCs. Our study has demonstrated that the Feridex or Resovist is the preferred labeling agent for hMSCs. There was a change in Oct-4 and CD45 expression after labeling.

Noninvasive Photoacoustic Imaging of Dendritic Cell Stimulated with Tumor Cell-Derived Exosome.

PURPOSE: The tools to trigger dendritic cell (DC) activation and to verify DC migration in vivo are important for directing DC immunotherapy toward successful treatment. We evaluated whether tumor cell-derived exosome (TEX)-stimulated DC migration into lymph node (LN) in mouse could be tracked using gold nanoparticle (GN)-labeling and ultrasound (US)-guided photoacoustic imaging (PAI). PROCEDURES: GFP-transduced DC2.4 cells were used. RFP-tagged TEXs were purified from a stable 4T1 cell line overexpressing the exosomal CD63-RFP fusion protein. The TEX uptake by DCs was visualized using confocal laser scanning microscopy. GNs with surface plasmon resonance at 808 nm were used for DC-labeling. DCs that migrated into axillary LN were longitudinally monitored by US-guided PAI and analyzed by silver staining and immunohistochemistry. RESULTS: TEXs were easily internalized in DCs, increased proliferation and migration capacities, and upregulated co-stimulatory molecules, CCR7 and TNF-α without cytotoxicity. The GN-labeling exerted no adverse effects on the biological functions of DCs. US-guided PAI and DC-labeling allowed for sensitive and longitudinal monitoring of TEX-stimulated DC migration toaxillary LN. CONCLUSIONS: TEXs efficiently activated DCs and GN-labeled DC migration into LN was successfully monitored using US-guided PAI, suggesting that TEXs are a good source for DC activation and US-guided PAI is a cost-effective and easy-to-use imaging modality for noninvasive tracking of DCs.