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.

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.

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.

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.

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.

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.

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.

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.

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.

Bisphenol A Promotes the Invasive and Metastatic Potential of Ductal Carcinoma In Situ and Protumorigenic Polarization of Macrophages.

Increased cancer risk and immune disorders linked with exposure to environmental endocrine disruptors like bisphenol A (BPA) have been steadily reported. Nevertheless, the impacts of BPA on the breast ductal carcinoma in situ (DCIS) progression and macrophage polarization remain to be elucidated. Here, we analyzed the differentially expressed genes in BPA-exposed DCIS cells and explored BPA effects on DCIS progression and macrophage polarization in vitro and in vivo. Two hundred and ninety-one genes were differentially expressed in 10-8 M BPA-exposed DCIS cells, in which the gene ontology terms of biological processes associated with negative regulation of cell death, cell adhesion, and immune response was enriched. 10-8 M BPA promoted the proliferation and migration of DCIS cells and the migration of macrophages, and upregulated the expression of M1 (NOS2) or M2 markers (Arg-1 and CD206) in macrophages. In coculture system, the migratory capacity of both cells and the expression levels of NOS2, Arg-1, and CD206 in macrophages were significantly enhanced upon 10-8 M BPA. In a DCIS xenograft model, oral exposure to an environmentally human-relevant low dose of 2.5 µg/l BPA for 70 days via drinking water led to an approximately 2-fold promotion in the primary tumor growth rate and a significant enhancement of lymph node metastasis along with increased protumorigenic CD206+ M2 polarization of macrophages. These results demonstrate that BPA acts as an accelerator to promote DCIS progression to invasive breast cancer by affecting DCIS cell proliferation and migration as well macrophage polarization toward a protumorigenic phenotype.

Transplantation of human mobilized mononuclear cells improved diabetic neuropathy.

Rodent stem cells demonstrated regenerative effects in diabetic neuropathy via improvement in nerve perfusion. As a pre-clinical step, we explored if human mobilized mononuclear cells (hMNC) would have the same effects in rats. hMNC were injected into Rt. hind-limb muscles of streptozotocin-induced diabetic nude rats, and the grafts were monitored using with MRI. After 4 weeks, the effects were compared with those in the vehicle-injected Lt. hind limbs. Nerve conduction, muscle perfusion and gene expression of sciatic nerves were assessed. Induction of diabetes decreased nerve function and expression of Mpz and Met in the sciatic nerves, which are related with myelination. hMNC injection significantly improved the amplitude of compound muscle action potentials along with muscle perfusion and sciatic nerve Mpz expression. On MRI, hypointense signals were observed for 4 weeks at the graft site, but their correlation with the presence of hMNC was detectable for only 1 week. To evaluate paracrine effects of hMNC, IMS32 cells were tested with hepatocyte growth factor (HGF), which had been reported as a myelination-related factor from stem cells. We could observe that HGF enhanced Mpz expression in the IMS32 cells. Because hMNC secreted HGF, IMS32 cells were co-cultured with hMNC, and the expression of Mpz increased along with morphologic maturation. The hMNC-induced Mpz expression was abrogated by treatment of anti-HGF. These results suggest that hMNC could improve diabetic neuropathy, possibly through enhancement of myelination as well as perfusion. According to in vitro studies, HGF was involved in the hMNC-induced myelination activity, at least in part.

Magnetic Resonance Imaging-Guided Drug Delivery to Breast Cancer Stem-Like Cells.

The feasibility of detecting breast cancer stem-like cells (BCSCs) with magnetic resonance imaging using extradomain-B of fibronectin (EDB-FN)-specific peptide (APTEDB )-conjugated thermally cross-linked superparamagnetic iron oxide nanoparticles (APTEDB -TCL-SPIONs) is previously demonstrated. Here, doxorubicin (Dox)-loaded APTEDB -TCL-SPIONs (Dox@APTEDB -TCL-SPIONs) are generated and their theranostic ability in a BCSC xenograft mouse model is assessed. The Dox@APTEDB -TCL-SPIONs enable more efficient delivery of Dox to tumors than nontargeted Dox@TCL-SPIONs. Much greater inhibition of BCSC tumor growth is observed after treatment with the Dox@APTEDB -TCL-SPIONs than with either Dox@TCL-SPIONs or free Dox. Hypointense signals are observed in the majority of the mice in postcontrast but not precontrast T2*-weighted MR images of tumors 7 days after treatment with Dox@APTEDB -TCL-SPIONs. An inverse correlation is observed between signal intensity and both EDB-FN expression and response to chemotherapy. The data indicate Dox@APTEDB -TCL-SPIONs can detect BCSCs within tumors by targeting EDB-FN-expressing cells. These nanoparticles thus have theranostic potential in breast cancer.

IL-6-mediated cross-talk between human preadipocytes and ductal carcinoma in situ in breast cancer progression.

BACKGROUND: The function of preadipocytes in the progression of early stage breast cancer has not been fully elucidated at the molecular level. To delineate the role of preadipocytes in breast cancer progression, we investigated the cross-talk between human breast ductal carcinoma in situ (DCIS) cells and preadipocytes with both an in vitro culture and xenograft tumor model. METHODS: GFP or RFP was transduced into human DCIS cell line MCF10DCIS.com cells or preadipocytes using lentivirus. Cell sorter was used to separate pure, viable populations of GFP- or RFP-transduced cells. Cell viability and proliferation was assessed by crystal violet assays and cell migration and invasion capability was assayed by the transwell strategy. Gene and protein levels were measured by western blot, RT-PCR and immunostaining. Adipokines and cytokines were quantified using ELISA. Human tumor xenografts in a nude mice model were used. Ultrasound imaging of tumors was performed to evaluate the therapeutic potential of a IL-6 neutralizing antibody. RESULTS: In the co-culture system with the MCF10DCIS.com and preadipocytes, MCF10DCIS.com proliferation, migration and invasion were enhanced by preadipocytes. Preadipocytes exhibited in an increased IL-6 secretion and cancer-associated fibroblast markers expression, FSP1 and α-SMC in co-culture with MCF10DCIS.com or in MCF10DCIS.com conditioned media, whereas the adipocyte differentiation capacity was suppressed by co-culture with MCF10DCIS.com. A neutralizing antibody of IL-6 or IL-6R suppressed the promotion of MCF10DCIS.com proliferation and migration by co-culture with preadipocytes. In the xenograft tumor model, the tumor growth of MCF10DCIS.com was enhanced by the co-injection of preadipocytes, and the administration of IL-6 neutralizing antibodies resulted in potent effects on tumor inhibition. CONCLUSIONS: Our findings suggest that IL-6-mediated cross-talk between preadipocytes and breast DCIS cells can promote the progression of early stage breast cancer. Therefore, blocking IL-6 signaling might be a potential therapeutic strategy for breast DCIS characterized by pathological IL-6 overproduction.

Breast cancer cell-derived exosomes and macrophage polarization are associated with lymph node metastasis.

Crosstalk between breast cancer and macrophages has potential implications for tumor metastasis. This study investigates macrophage polarization induced by triple-negative breast cancer (TNBC) cell-derived exosomes that promote lymph node (LN) metastasis in orthotopic TNBC models. The MDA-MB-231 cancer cell line expressing the exosomal CD63-red fluorescence (RFP) fusion protein was generated to noninvasively visualize exosome transfer into cancer cells and macrophages. Administration of RFP-tagged exosomes enhanced migration of macrophages and induced macrophage polarization in vitro. In orthotopic TNBC models, noninvasive bioluminescent imaging, ultrasound-guided photoacoustic imaging, and histological analysis revealed that intravenous injection of RFP-tagged exosomes promoted primary tumor growth and axillary LN metastasis in which expression of CD206, a marker or alternatively activated type 2 (M2) macrophages, was significantly higher than expression of NOS2, a marker of classically activated type 1 (M1) macrophages. These results suggest breast cancer cell-derived exosomes stimulate macrophage polarization that creates favorable conditions for LN metastatic processes in TNBC.

Near-infrared photothermal therapy using anti-EGFR-gold nanorod conjugates for triple negative breast cancer.

Current EGFR-targeted therapy for triple negative breast cancer (TNBC) has produced disappointing results. A rational therapeutic strategy to improve EGFR-targeted treatment for TNBC is therefore needed. In this study we evaluated the feasibility of treating TNBC using photoacoustic imaging (PAI)-guided near-infrared photothermal therapy (NIR-PTT) with anti-EGFR-conjugated gold nanorods (anti-EGFR-GN). NIR-PTT combined with anti-EGFR-GN exerted synergistic anti-proliferative and apoptotic actions through upregulation of HSP70 and cleaved caspase-3, downregulation of Ki-67 and EGFR, and inhibition of several intracellular signaling molecules (mTOR, AKT, ERK1/2 and FAK). These combined effects give this approach significant efficacy. Our findings suggest PAI-guided NIR-PTT using anti-EGFR-GN represent a novel and effective strategy for EGFR-targeted therapy in TNBC.

Gene expression profiling of calcifications in breast cancer.

We investigated the gene expression profiles of calcifications in breast cancer. Gene expression analysis of surgical specimen was performed using Affymetrix GeneChip® Human Gene 2.0 ST arrays in 168 breast cancer patients. The mammographic calcifications were reviewed by three radiologists and classified into three groups according to malignancy probability: breast cancers without suspicious calcifications; breast cancers with low-to-intermediate suspicious calcifications; and breast cancers with highly suspicious calcifications. To identify differentially expressed genes (DEGs) between these three groups, a one-way analysis of variance was performed with post hoc comparisons with Tukey's honest significant difference test. To explore the biological significance of DEGs, we used DAVID for gene ontology analysis and BioLattice for clustering analysis. A total of 2551 genes showed differential expression among the three groups. ERBB2 genes are up-regulated in breast cancers with highly suspicious calcifications (fold change 2.474, p < 0.001). Gene ontology analysis revealed that the immune, defense and inflammatory responses were decreased in breast cancers with highly suspicious calcifications compared to breast cancers without suspicious calcifications (p from 10-23 to 10-8). The clustering analysis also demonstrated that the immune system is associated with mammographic calcifications (p < 0.001). Our study showed calcifications in breast cancers are associated with high levels of mRNA expression of ERBB2 and decreased immune system activity.

Investigation of discriminant metabolites in tamoxifen-resistant and choline kinase-alpha-downregulated breast cancer cells using 1H-nuclear magnetic resonance spectroscopy.

Metabolites linked to changes in choline kinase-α (CK-α) expression and drug resistance, which contribute to survival and autophagy mechanisms, are attractive targets for breast cancer therapies. We previously reported that autophagy played a causative role in driving tamoxifen (TAM) resistance of breast cancer cells (BCCs) and was also promoted by CK-α knockdown, resulting in the survival of TAM-resistant BCCs. There is no comparative study yet about the metabolites resulting from BCCs with TAM-resistance and CK-α knockdown. Therefore, the aim of this study was to explore the discriminant metabolic biomarkers responsible for TAM resistance as well as CK-α expression, which might be linked with autophagy through a protective role. A total of 33 intracellular metabolites, including a range of amino acids, energy metabolism-related molecules and others from cell extracts of the parental cells (MCF-7), TAM-resistant cells (MCF-7/TAM) and CK-α knockdown cells (MCF-7/shCK-α, MCF-7/TAM/shCK-α) were analyzed by proton nuclear magnetic resonance spectroscopy (1H-NMRS). Principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) revealed the existence of differences in the intracellular metabolites to separate the 4 groups: MCF-7 cells, MCF-7/TAM cells, MCF-7-shCK-α cells, and MCF-7/TAM/shCK-α cells. The metabolites with VIP>1 contributed most to the differentiation of the cell groups, and they included fumarate, UA (unknown A), lactate, myo-inositol, glycine, phosphocholine, UE (unknown E), glutamine, formate, and AXP (AMP/ADP/ATP). Our results suggest that these altered metabolites would be promising metabolic biomarkers for a targeted therapeutic strategy in BCCs that exhibit TAM-resistance and aberrant CK-α expression, which triggers a survival and drug resistance mechanism.

Near-infrared photothermal therapy using EGFR-targeted gold nanoparticles increases autophagic cell death in breast cancer.

Although triple negative breast cancer (TNBC) is a small percentage of all breast cancers, to date, TNBC is one of the most challenging types of breast cancer for basic and clinic research because TNBC patients display a high risk of relapse, shorter overall survival and limited therapeutic options after completion of conventional chemotherapy compared with patients with other breast cancer subtypes. The epidermal growth factor receptor (EGFR) is a promising target for TNBC treatment. Although near infrared-photothermal therapy (NIR-PTT) using anti-EGFR antibody-conjugated gold nanorods (anti-EGFR-GNs), has attracted considerable interest for non-invasive and targeted TNBC treatment through an activation of apoptotic pathway, it is unclear whether anti-EGFR-GNs-combined NIR-PTT modulates the induction of autophagy contributing to cell death. Therefore, we investigated the autophagic cell death in cultured TNBC cells and mouse xenograft tumors during anti-EGFR-GNs-combined NIR-PTT. We here found that the cytotoxicity induced by anti-EGFR-GNs-combined NIR-PTT was rescued by treatment with autophagy inhibitor, 3-methyladenine (3-MA). Anti-EGFR-GNs-combined NIR-PTT induced remarkable levels of autophagy activity as evidenced by a large number of autophagic vesicles and a significant increase in autophagy-specific proteins; microtubule-associated protein light chain 3 (LC3), p62, beclin-1, and autophagy-related gene5 (Atg5), accompanying the inhibition of AKT-mTOR signaling pathway responsible for inducing autophagy. Moreover, in mouse xenograft tumors, anti-EGFR-GNs-combined NIR-PTT also increased LC3 and beclin-1 levels. Our findings, for the first time, demonstrate that anti-EGFR-GNs-combined NIR-PTT remarkably induces autophagy leading to EGFR-targeted cancer cell death.

LOXL4 knockdown enhances tumor growth and lung metastasis through collagen-dependent extracellular matrix changes in triple-negative breast cancer.

Lysyl oxidase (LOX) family genes catalyze collagen cross-link formation. To determine the effects of lysyl oxidase-like 4 (LOXL4) expression on breast tumor formation and metastasis, we evaluated primary tumor growth and lung metastasis in mice injected with LOXL4-knockdown MDA-MB-231 triple-negative human breast cancer cells. In addition, we analyzed overall survival in breast cancer patients based on LOXL4 expression using a public online database. In the mouse xenograft model, LOXL4 knockdown increased primary tumor growth and lung colonization as well as collagen I and IV, lysine hydroxylase 1 and 2, and prolyl 4-hydroxylase subunit alpha 1 and 2 levels. Second harmonic generation imaging revealed that LOXL4 knockdown resulted in the thickening of collagen bundles within tumors. In addition, weak LOXL4 expression was associated with poor overall survival in breast cancer patients from the BreastMark dataset, and this association was strongest in triple-negative breast cancer patients. These results demonstrate that weak LOXL4 expression leads to remodeling of the extracellular matrix through induction of collagen synthesis, deposition, and structural changes. These alterations in turn promote tumor growth and metastasis and are associated with poor clinical outcomes in triple-negative breast cancer.