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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 pre-metastatic 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 pre-metastatic 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 pre-metastatic niche in liver for TNBC.

Background: We had identified somatic mutations of the NAD(P) Dependent Steroid Dehydrogenase-Like (NSDHL) gene from breast tumors of patients with distant metastasis using whole-exome sequencing. This study aimed to investigate the function of NSDHL mutants in breast cancer. Methods: ER-positive and tamoxifen-resistant human breast cancer cell line (ZR75-1) and mouse macrophage cell line (RAW264,7) were used. GFP-tagged NSDHL mutants were generated by subcloning the mutant NDHL into a lentiviral vector yielding an in-frame fusion of 3′ to GFP. ZR75-1 cells expressing the GFP-tagged NSDHL mutants were selected by puromycin with subsequent FACS analysis. The following experiments were performed: western blot, immunofluorescence staining, qRT-PCR, CellTiter-Glo assay, cell cycle assay, transwell migration assay, wound healing assay, tumor spheroid formation assay, and total cellular cholesterol measurement. An orthotopic breast tumor mouse model by injection of ZR75.1 cells into mammary gland fat pad was used in vivo. Results: Four novel NSDHL somatic mutations were discovered in the breast tumor tissues of patients. While total cholesterol levels in NSDHL mutant-transduced cells did not significantly increase, they were notably higher in wild-type NSDHL-transduced cells than in parent ZR75-1 cells. When compared to the parent and wild-type NSDHL-transduced cells, there was a minor increase in growth rate and a large increase in migratory capacities in NSDHL mutant-transduced cells (P< 0.05). The size of tumor spheroids is significantly larger in NSDHL mutant-transduced cells than in wild-type NSDHL-transduced cells. Both EGFR expression and epithelial to mesenchymal transition (EMT) markers were higher in NSDHL mutant-transduced cells than in parent and wild-type NSDHL-transduced cells. The NSDHL mutant-transduced cells’ conditioned media promoted the migratory ability and M2 polarization of RAW264.7 cells. In an orthotopic xenograft mouse model, NSDHL mutant-transduced cells were shown to promote tumor growth better than parent and wild-type NSDHL-transduced cells, suggesting that somatic mutation of the NSDHL contributes to the malignant progression of breast cancer cells. Conclusion: The present data indicate that breast cancer cells harboring somatic mutants of the NSDHL gene seem to display more aggressive behavior by gaining biological worse phenotype both in vitro and in vivo. More investigation is required into the mechanisms behind the function and accumulation of mutant NSDHL proteins to speed the development of therapies for patients with breast cancer harboring mutant NSDHL. Citation Format: Moonjou Baek, Hoe Suk Kim, So-Hyun Yoon, Seungyeon Ryu, Sangeun Lee, Kyoungsook Park, Han-Byoel Lee, Wonshik Han. Overexpression of somatic mutation in NSDHL promotes breast cancer cell migration and tumor growth [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-13-06.

Background: Statins were suggested for repurposed drugs, having multifaceted effects which include anti-tumor activities by modulating the immune response. Here, we aimed to demonstrate the effect of statins on programmed death-ligand 1 (PD-L1) expression in triple-negative breast cancer (TNBC) cells. Methods: Thirteen human TNBC cell lines, mouse macrophage cell line (RAW 264.7), cholesterol and 27-hydroxycholesterol, and clinically approved lovastatin and simvastatin were used. Flow cytometry, Annexin V/propidium iodide assay, western blot, qRT-PCR, transwell migration assay, and immunohistochemistry were employed. A co-culture of macrophages with various breast cancer cells was performed. An orthotopic breast tumor mouse model and metastasis model by injection of GFP-tagged MDA-MB-231 cells into mammary gland fat pad and the tail vein injection were produced. In tumor model mice, lovastatin(10mg/kg) was daily injected intraperitoneally. In vivo fluorescent imaging was used to identify primary tumor development and lung metastasis. Results: Among thirteen TNBC cell lines, MDA-MB-231, HCC38, and HCC70 highly expressed endogenous/constitutive PD-L1. Statins reduced PD-L1 expression and exerted anti-proliferative and apoptotic effects in MDA-MB-231, HCC38, HCC70, and Raw264.7 in a dose- and time-dependent manner (p< 0.05). Meanwhile, statins increased the expression of PD-L1 in Hs578T and MDA-MB-468. STAT3 phosphorylation was inhibited in MDA-MB-231 and HCC70, but not in Hs578T, while AKT phosphorylation was reduced in MDA-MB-231, HCC70, Hs578T, and MDA-MB-468 when statins were treated. The migration of MDA-MB-231 and Raw264.7 in statin-treated conditioned media was decreased (p< 0.05). Cholesterol and 27-hydroxy cholesterol did not restore PD-L1 expression in statin-treated MDA-MB-231. Statins suppressed the expression of M2 markers (PD-L1, CD206, YM-1, Fizz1, arginase-1) in RAW26437 stimulated by a conditioned medium of MDA-MB-231. Lovastatin suppressed the primary tumor growth and metastasis in xenograft tumor mice. Conclusions: Our findings show that statins have an anti-tumor effect, which kills breast cancer cells and triggers macrophage reprogramming by reducing PD-L1 expression, impairing the AKT, ERK, and STAT3 signal pathways, and decreasing M2 markers. Further study is needed to investigate an in-depth molecular mechanism study by which statins regulate PD-L1 expression in TNBC and to confirm the safe and effective use of statins as adjuvant therapy in TNBC. Citation Format: Sangeun Lee, Hoe Suk Kim, So-Hyun Yoon, Seungyeon Ryu, Moonjou Baek, A Young Park, Han-Byoel Lee, Wonshik Han. Statins exhibit an anti-tumor effect by attenuating PD-L1 in breast cancer cells and macrophages and reducing breast tumor progression in xenograft mouse model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-10.

Supplementary Table 1. Information about patients derived xenograft models and derived patient. Supplementary Table 2. Ontology of differentially expressed genes in metastasis-harboring liver of PDX models. Supplementary Table 3. Differentially expressed genes in liver and lung of metastasis-harboring PDX models. Supplementary Table 4. Human ratio in lung and liver of additional PDX models identified by RNA sequencing. Supplementary Table 5. Gene ontology of co-expressed genes with Cx3cr1 in liver of PDX models identified by RNA sequencing. Supplementary Table 6. Top ranked genes having significant correlation with Cx3cr1 in liver of PDX models identified by RNA sequencing. Supplementary Table 7. Demographic and clinical characteristics of all patients. Supplementary Table 8. Information about used primer sequences for quantitative real-time PCR Supplementary Figure 1. Liver and lung H&E staining pictures of eight nonmetastatic PDX models a. Microscopic images of non-metastasis harboring PDX mouse model's liver (left) and lung (right) taken at 400x magnification. Supplementary Figure 2. Immunohistochemistry about CX3CR1 in liver and lung of breast cancer metastasis patients. Supplementary Figure 3. Hepatic metastasis after four weeks after orthotopic injection of 4T1 breast cancer cells identified by H&E staining. Supplementary Figure 4. Identification of pre-metastatic niche in liver tissues of 4T1 bearing mice a. mRNA expression of inflammation associated genes identified by real-time PCR in liver tissues of 4T1 bearing mice. Supplementary Figure 5. Identification of CX3CR1 positive cell population. Supplementary Figure 6. FACS analysis with anti-CX3CR1 of Raw264.7 macrophage cell lines. Supplementary Figure 7. Trans-well migration assay of BMDM with or without CX3CL1. Supplementary Figure 8. Mmp9 mRNA expression having significant correlation with Cx3cr1 mRNA expression in liver of PDX models. Supplementary Figure 9. MMP9 expression of macrophages with CX3CL1 treatment. Supplementary Figure 10. Signaling pathway of CX3CL1 treated macrophages. Supplementary Figure 11. Splenic injection of 4T1 cells inducing hepatic metastasis within two weeks in most cases. Supplementary Figure 12. mRNA expression about Cx3cr1 and Mmp9 in liver of vehicle or AZD8797 treated BALB/C mice identified by real-time PCR. Supplementary Figure 13. Correlation plot of Tnfa and Cx3cr1 mRNA expression in liver and lung of PDX models. Supplementary Figure 14. EVs isolation of CD63-mRuby-4T1.

Background: Cholesterol biosynthesis pathway is commonly elevated or dysregulated in cancer cells and high cholesterol levels are associated with cancer progression. NAD(P)-dependent steroid dehydrogenase-like (NSDHL), an enzyme involved in cholesterol biosynthesis, has been implicated in maintaining cancer stem cell, but its role in regulating breast cancer stem cells (BCSC) remains unclear. This study aimed to uncover the molecular mechanisms by which NSDHL regulates BCSC properties.Methods: The knockdown of NSDHL was induced by an introduction of siRNA/shRNA into MCF-7 cells. A non-adherent 3D culture method based on polymer-X coating was applied for growing tumorspheres under serum-free condition. 3’ quant mRNA-sequencing, qRT-PCR, western blot, immunofluorescent staining, ELISA, flow cytometry and ALDEFLUOR assay were carried out. Immunodeficient NSG mice were used for orthotropic xenograft tumor models by injecting tumorspheres.Result: NSDHL knockdown reduced a tightly cohesive sphere formation. RNA-seq data revealed a significant upregulation of 253 DEGs and downregulation of 364 DEGs with >2-fold change in NSDHL knockdown tumorsphere. The KEGG pathway analysis showed that DEGs were mainly enriched in TGF-β signaling pathway and cell cycle. NSDHL knockdown reduced the population of BCSCs with CD44+/CD24- phenotype and high ALDH activity as well as luminal progenitors with CD49f+/EpCAM+ phenotype along with decrease in TGF-β 1 and 3 and GLI1 in tumorsphere. In orthotropic xenograft tumor models, NSDHL knockdown strongly suppressed the tumor initiation and growth.Conclusions: Our findings reveal that NSDHL has an important role in maintaining BCSC population and tumor initiating capacity, suggesting NSDHL as an attractive therapeutic target to eliminate BCSCs, and thus preventing breast cancer initiation and progression. Citation Format: So-Hyun Yoon, Hoe Suk Kim, Junhyuk Song, Sunkyu Kwon, Seungyeon Ryu, Moonjou Baek, Sangeun Lee, Han-Byoel Lee, Hyeong-Gon Moon, Dong-Young Noh, Sangyong Jon, Wonshik Han. NSDHL knockdown decreases tightly cohesive tumorsphere formation and breast cancer stem cell population [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-12-11.

Purpose: Immunophenotyping is a useful tool for diagnostic and therapeutic assessment of heterogenous breast cancer cells that depend on external factors and growth conditions. The objective of this study was to characterize the phenotype of patient-derived breast cancer cells (PDBCCs) grown in different culture medium and under different conditions including 2D monolayer and non-adherent 3D spheroid culture by using flow cytometry. Methods: PDBCCs and normal breast cells were harvested from fresh surgical specimens (N=11) of breast tumor and normal tissues. PDBCCs were grown in various culture media under monolayer, spheroid and organoid conditions. Flow cytometry, qRT-PCR, ALDEFLUOR assay, β-galactosidase staining and RNA-seq were performed. Results: PDBCC growth rates were different for each patient case. As compared with full media, A83-01 removal increased EpCAM+CD24+ cells (1.8-fold, p Clinically poor prognosis-associated DEGs based on recurrence-free survival analysisGenes symbolLog2(FC) P Citation Format: Seungyeon RYU, Hoe Suk Kim, So-Hyun Yoon, Sangeun Lee, Junhyuk Song, Moonjou Baek, Han-Byoel Lee, Sangyong Jon, Wonshik Han. Analysis of immunophenotyping of patient-derived primary breast cancer cells according to the medium composition and culture method [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-05-09.

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 p p = 0.01). NSDHL knockdown led to significantly decreased tumor growth and lung metastasis in the MDA-MB-231 xenograft model (p p 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.

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 were able to analyse the cancer stem cell-like microniches in relation to the tumour microenvironment of triple-negative breast cancer (TNBC) patients. We identified alternative splice variants, performed complementarity-determining region analysis of infiltrating T cells and B cells, and assessed adenosine-to-inosine (A-to-I) base editing in tumour tissue sections. Especially, in TNBC microniches, A-to-I editome specific to different microniche groups was identified.

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). 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. 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. 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.

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.

Background: Cancer stem-like cells (CSCs) represent a critical subset of the tumor population, which contributes to tumor recurrence, metastasis and therapy resistance. Spheroid culture is unique method for CSC enrichment. Patient-derived breast cancer cells (PDBCCs) has provided an invaluable tool in preclinical and translational research. The objective of this study is to characterize the immunophenotype and transcriptomes of PDBCCs grown in the adherent monolayer and spheroid condition. Methods: PDBCCs were harvested from residual tumor of fresh surgical specimens of patients with ER-positive subtype (8 cases). The adherent monolayer(M) and spheroid(S) culture of PDBCCs were applied on collagen I-coated plate and an ultra-low attachment polymer-X coated plate. Flow cytometry and immunocytochemistry was performed by using fluorescent-dye conjugated antibodies for EpCAM, CD49f, CD24, CD44, and cytokeratin. ALDEFLUOR® assay to detect ALDH activity was used. Total RNA-sequencing was applied to investigate differentially expressed genes (DEGs), followed by GO and KEGG pathway enrichment analysis. Real-time RT-PCR was performed to evaluate BCSC-related RNA levels. The Kaplan-Meier Plotter online database was used to evaluate the prognostic value of DEGs (2-fold change, p Results: Under adherent monolayer condition, more than 95% of PDBCCs passaged for up to 5 passages on collagen I-coated plate sustained EpCAM+/cytokeratin+/fibroblast marker_ phenotype. On polymer-X coated plates, PDBCCs formed compact multicellular spheroids with a diameter of less than 300 µm. As compared with monolayer, CSCs with EpCAM-/CD49f+ phenotype (M vs S; 0.16±0.06 vs 2.34±0.65, p=0.015) and high ALDH activity (M vs S; 0.73±0.43 vs 7.05±1.48, p=0.038) were significantly increased by spheroid culture. In whole-transcriptome analysis (3 cases) between spheroid and monolayer, a total of 561 DEGs were identified, including 290 upregulated and 271 downregulated DEGs (2- fold change, p Conclusion: Spheroid culture of PDBCCs with ER+ subtype enriches CSCs and poor prognostic gene signatures. Future studies to explore the specific molecular mechanisms underlying these observations may provide new molecular targets for the development of therapies for targeting CSCs in breast cancer patient. Citation Format: Hoe Suk Kim, Seungyeon Ryu, So-Hyun Yoon, Sangeun Lee, Junhyuk Song, Yoonjung Choi, Moonjou Baek, Han-Byoel Lee, Sangyong Jon, Daeyoup Lee, Wonshik Han. Spheroid culture of ER+ breast cancer patient-derived tumor cells enriches cancer stem-likecells with EpCAM-/CD49f+and high ALDH activity and poor prognostic gene signatures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6373.

Background: NAD(P) Dependent Steroid Dehydrogenase-Like (NSDHL) is an enzyme involved in post-squalene cholesterol biosynthesis. We recently suggest new insights into the critical role of the NSDHL involved in breast tumor growth and metastasis and its potential for targeted cancer therapy. Compound 9, which is a novel inhibitor of NSDHL, was kindly provided by Dr. Lee B-J in Seoul National University College of Pharmacy. This study aimed to investigate the anti-cancer potential of Compound 9 in breast cancer. Methods: Therapeutic potential of Compound 9 (10~100 μM) was investigated in several human breast cancer cell lines. Total intracellular cholesterol level was measured using Total Cholesterol Assay Kit. Cell cycle analysis using DNA staining with PI, apoptosis analysis using Annexin V/PI-labeling, and transwell migration assay were performed. The Synergistic anticancer effect of compound-9 and paclitaxel was evaluated by crystal violet assay. Western blot and immunofluorescent staining were assessed for investigating the mechanism by which Compound 9 exerts the anti-cancer effect. Results: Time-dependent intracellular uptake of Compound 9 by breast cancer cells was observed. Compound 9 induced cytotoxic, anti-proliferative, and apoptotic effects and cell cycle arrest (G0/G1), and suppressed migration activity on diverse breast cancer cell lines in a time- and dose-dependent manner. Total intracellular cholesterol level was reduced by Compound 9. Combination with Compound-9 and Paclitaxel led to synergistic cytotoxicity. Compound-9 increased the autophagy-related proteins (P62, LC3II) but decreased the anti-apoptotic proteins (XIAP, BCL2). Conclusion: Our data show that Compound 9 exerts strong anti-proliferative and cytotoxic activity in breast cancer cells, suggesting potential additional utility as an anti-cancer drug. Further studies are necessary to validate the potential anti-cancer effect of Compound-9 in breast tumor mice models. Citation Format: So-Hyun Yoon, Hoe Suk Kim, Moonjou Baek, Seungyeon Ryu, Sangeun Lee, Han-Byoel Lee, Bong-Jin Lee, Wonshik Han. Novel NSDHL inhibitor (Compound 9) exerts anti-proliferative and cytotoxic activity in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1800.

Background: Breast cancer Patient Derived Organoids (PDOs) and patient-derived xenografts (PDXs) which recapitulate the tumor heterogeneity and molecular signatures of original tumor tissue, are useful tools to enable greater precision of both approved and experimental medicines through personalized therapeutic approaches. We focus on use of ex vivo PDOs, PDX-derived organoids (PDXOs), and vivo PDXs models for drug testing and personalized therapy. In this study, we investigated the oncogenic role and therapeutic potency of targeting WEE1 in breast cancer. Methods: Fresh surgical specimens of primary TNBC breast tumor (N=3) were used for PDOs. PDOs and PDX-derived organoids (PDXOs) were cultured in previously reported media compositions based on ECM hydrogel. PODs and PDXOs were subjected to drug screening for 22 anticancer drugs including chemoreagents and targeted drugs in the 3D HTS system. Drug sensitivity was tested in triplicate in different concentration ranges for 7 days. The IC50 for each drug was calculated by a sigmoidal dose-response curve, using the GraphPad Prism 9 program. For drug screening in PDXs models, PDOs were injected into the fourth mammary fat pads of NOD.Cg-Prkdcscid Il2rgtm1wjl/SzJ mice. WEE1 inhibitor (AZD1775) was administered by oral gavage (AZD1775, 30 mg/kg) for 3 weeks. Immunostaining of ER, PR, Her2, CK5 and Ki67 was performed in PODs, PODXs and PDX tumor tissues. Results: All PDOs, PDXs, and PDXOs were ER-, PR- and HER2- negative. PDOs and PDXOs (172T, 185T, 207T) showed differential response to 22 anticancer drugs. In PDOs and PDXOs models, 172T and 207T PDOs and PDXOs are highly sensitive to AZD1775, but 185T PDOs and PDXOs less sensitive or never respond to treatment with AZD1775 (10 µM). In PDX models, treatment with AZ1775 decreased tumor growth (P Conclusion: Consequently, PDOs, PDXOs and PDXs recapitulated immunohistological signatures of original tumor tissues. The results of WEE1 inhibitor response between PDOs, PDXOs and PDXs were consistent. This study may provide a preclinical tool to screen drug responses to standard-of-care and newly identified drugs. Citation Format: Seungyeon Ryu, Hoe Suk Kim, Jung Eun KIM, So-Hyun Yoon, Sangeun Lee, Moonjou Baek, Han-Byoel Lee, Dong Woo Lee, Bosung Ku, Wonshik Han. Application of triple negative breast cancer patient-derived organoid and xenograft model for drug screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6039.

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.

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 (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/EBPβ. 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.

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.

Background Patient-derived cancer organoids, which reliably conserve original features of tumors, are emerging as an excellent model for predicting therapy response and drug screening. Developing optimized 3D high-throughput drug screening platform to establish patient-derived cancer organoids and simultaneously perform drug screening is essential for personalized medicine. Methods We established normal breast organoids (n=4) and breast cancer organoids (n=10) from 20 fresh surgical specimen (normal 7, tumor 13 cases). A number (500-2000) of normal and cancer cells were automatically dispensed with the ASFA™ Spotter ST and organoids were generated by hydrogel hanging-drop culture on Cellvitro™ Pillar platform (Medical & Bio Decision, South Korea). Organoids were subjected to drug screening for 17 anticancer drugs including chemoreagents and targeted drugs in the 3D HTS system. Drug sensitivity was tested in triplicate in different concentration ranges for 5 days. Drug cytotoxic effect was assessed by calcein AM staining. Acquisition and analysis of high-content 3D organoid images were peformed using ASFA™ SCANNER (Medical & Bio Decision, South Korea). The IC50 for each drug was calculated by a sigmoidal dose-response curve, using the GraphPad Prism 9 program. We analyzed a drug response index (DRI) using a prediction alogorithm to evalute drug sensitivity (DRI0.5). Results We summarized the DRI value of patient-derived breast organoids of 7 drugs (Table 1). 6 tumor organoids (2T, 6T, 8T, 10T, 13T, 15T) showed high sensitivity to Docetaxel, Doxorubicin, Paclitaxel, and Gemcitabin while 4 tumor organoids (1T, 9T, 14T, 16T) were less sensitive and resistant. 2 tumor organoids (10T, 16T) were sensitive to Tamoxifen and 2 tumor organoids (6T, 8T) show high sensitivity to palbociclib and erlotinib. Normal organoids show less sensitivity and resistance to chemotherapeutic drugs. Drug response index >0.5 : resistancy top 30%, Drug response index Table 1.DRI values of patient-derived breast organoids of 7 drugs.Patient No.Tumor/NormalDocetaxelPaclitaxelDoxorubicinTamoxifenGemcitabinePalbociclibErlotinib1TTumor1.051.261.080.060.590.26-2TTumor-0.64-0.72-0.60-0.08-0.79--6TTumor-0.98-0.99-2.330.00-1.44-2.31-1.608TTumor-0.32-0.50-0.250.64-1.07-1.47-0.669TTumor1.050.980.610.870.430.610.4410TTumor-0.84-0.77-0.78-1.680.790.610.0913TTumor-0.87-0.430.560.70-0.510.611.1714TTumor0.861.261.180.481.81--15TTumor-1.59-1.45-1.41--0.71-0.13-16TTumor1.051.261.18-1.70-1.22-2NNormal1.051.260.650.741.810.610.7810NNormal1.05-0.910.810.870.880.610.084NNormal-0.31-0.25-0.530.25-0.190.61-1.575NNormal1.051.130.380.75-0.630.610.10 Citation Format: Jungeun Kim, Hoe Suk Kim, Ga Yeon Kim, Kyung hyeun Park, Seung yeon Ryu, Sangeun Lee, Dong Woo Lee, Bosung Ku, Han-Byoel Lee, Wonshik Han. Development of automated 3D high-throughput drug screening platform for patient-derived breast cancer organoids [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-02-02.

Extracellular matrix (ECM)-based hydrogels can serve as scaffolds in reconstruction of physiologically relevant three-dimensional (3D) in vitro models. Biocompatible or integrated hydrogels can be used to modulate ECM properties such as stiffness and composition for studies of cell−ECM interactions and morphogenesis. To this end, we developed a new type of spatially modified collagen type 1 hydrogel by convective addition of collagen type 1 solution. The matrix displayed properties that were distinct from those of a collagen type 1 hydrogel and recapitulated the morphology and function of mammary epithelium in a 3D microfluidic platform. In this ECM, mammary epithelial cells secreted laminin and exhibited self-assembly into a basement membrane. Thus, this spatially modified ECM offers biophysical features that can facilitate the construction of mammary epithelium and, by extension, that of various other epithelial types. Additionally, our reconstructed mammary duct can be used as an in vitro model for the study of early-stage breast cancer. In this study, we present a new type of spatially modified ECM composed of type 1 collagen whose properties differ from those of a collagen matrix. Using this ECM, we developed a stable human mammary epithelium with a lumen structure that exhibited a barrier function in a microfluidic platform, produced laminin, and formed a basement membrane. This reconstructed mammary duct can serve as a model for investigating breast cancer and may be adapted to other types of epithelium for in vitro studies. A biomaterial that mimics human mammary ducts has been developed by scientists in South Korea and could be used to investigate early-stage breast cancer. Hydrogels are networks of polymer molecules that have a physical texture similar to that of the extracellular matrix surrounding and supporting cells in the human body, making them ideal scaffolds for building artificial tissue. Seok Chung from Korea University in Seoul, Su Hyun Lee from the Seoul National University Hospital and their co-workers made a hydrogel with the structure of mammary ducts using collagen, and then deposited mammary epithelial cells throughout it. The hydrogel was incorporated into a microfluidic device, providing a way of introducing the various biological components found in a natural extracellular matrix into it, and thereby a method to test how these components interact with the cells.

// Yin Ji Piao 1, 2, * , Hoe Suk Kim 1, * , Eun Hye Hwang 1 , Jisu Woo 1 , Meihua Zhang 1, 3 and Woo Kyung Moon 1, 2 1 Department of Radiology, Seoul National University Hospital and Seoul National University College of Medicine, Jongno-Gu, Seoul 03080, Korea 2 Department of Biomedical Sciences, Seoul National University College of Medicine, Jongno-Gu, Seoul 03080, Korea 3 Department of Radiology, Yanbian University Hospital, Yanji City, Jilin Province 133000, China * These authors contributed equally to this work Correspondence to: Woo Kyung Moon, email: moonwk@snu.ac.kr Keywords: exosome; triple-negative breast cancer; lymph node; metastasis; macrophage Received: September 15, 2017 Accepted: December 01, 2017 Published: December 13, 2017 ABSTRACT 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.

// Meihua Zhang 1, 2, 3, * , Hoe Suk Kim 1, * , Tiefeng Jin 1, 4 , Jisu Woo 1 , Yin Ji Piao 1, 2 and Woo Kyung Moon 1, 2 1 Department of Radiology, Seoul National University Hospital, Jongno-gu, Seoul 03080, Korea 2 Department of Biomedical Sciences, Seoul National University College of Medicine, Jongno-gu, Seoul 110-799, Korea 3 Department of Radiology, Yanbian University Hospital, JiLin Province 133000, China 4 Department of Pathology and Cancer Research Center, Yanbian University Medical College, Yanji 133002, China * These authors have contributed equally to this work Correspondence to: Woo Kyung Moon, email: moonwk@snu.ac.kr Keywords: near-infrared photothermal therapy, photoacoustic imaging, breast cancer, gold nanorod, epidermal growth factor receptor Received: May 14, 2017 Accepted: August 17, 2017 Published: September 23, 2017 ABSTRACT 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.

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.

// Sul Ki Choi 1, 2, * , Hoe Suk Kim 1, * , Tiefeng Jin 1, 3 , Woo Kyung Moon 1, 2 1 Department of Radiology, Seoul National University Hospital, Jongno-gu, Seoul 03080, Korea 2 Department of Biomedical Sciences, Seoul National University College of Medicine, Jongno-gu, Seoul 03080, Korea 3 Department of Pathology and Cancer Research Center, Yanbian University Medical College, Yanji 133002, China * These authors have contributed equally to this work Correspondence to: Woo Kyung Moon, email: moonwk@snu.ac.kr Keywords: triple-negative breast cancer (TNBC), lysyl oxidase-like 4 (LOXL4), collagen, tumor progression, overall survival Received: September 24, 2016 Accepted: December 20, 2016 Published: January 02, 2017 ABSTRACT 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.

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.

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. Electronic supplementary material The online version of this article (10.1186/s13046-018-0867-3) contains supplementary material, which is available to authorized users.

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.

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

This study evaluated the ability of a non-invasive cardiac output monitoring device (NICOM) to predict fluid responsiveness in paediatric patients undergoing cardiac surgery.Children aged5 yr undergoing congenital heart surgery were included. Once the sternum had been closed after repair of the congenital heart defect, 10 ml kg(-1) colloid solution was administered for volume expansion. Transoesophageal echocardiography (TOE) was performed to measure stroke volume (SV) and respiratory variation in aortic blood flow peak velocity (ΔV(peak)) before and after volume expansion. Haemodynamic and NICOM variables, including SV(NICOM), stroke volume variance (SVV(NICOM)), cardiac index (CI(NICOM)), and percentage change in thoracic fluid content compared with baseline (TFCd0%), were also recorded. Patients in whom the stroke volume index (SVI), measured using TOE, increased by15% were defined as fluid responders.Twenty-nine patients were included (13 responders and 16 non-responders). Before volume expansion, only ΔV(peak) differed between groups (P=0.036). The SVV(NICOM), HR, and central venous pressure did not predict fluid responsiveness, but ΔV(peak) did. The CI(NICOM) was not correlated with CI(TOE) (r=0.107, P=0.43). Using Bland-Altman analysis, the mean bias between CI(TOE) and CI(NICOM) was 0.89 litre min(-1) m(-2), with a precision of 1.14 litre min(-1) m(-2). Trending ability of NICOM for SVI and CI was poor when TOE was a reference method.The SVV(NICOM) did not predict fluid responsiveness in paediatric patients during cardiac surgery. In addition, there was no correlation between CI(TOE) and CI(NICOM). Fluid management guided by NICOM should be performed carefully.ClinicalTrials.gov NCT01996956.

Targeted therapy for breast cancer stem cell (BCSC): A novel liposomal system (APTEDB -LS-siRNA(EDB) ) that enables simultaneous targeting and knockdown of extra domain B of fibronectin (EDB-FN) shows potent therapeutic efficacy in the BCSC-derived tumors in vivo.

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. Copyright © 2014 John Wiley & Sons, Ltd.

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 p from 10−23 to 10−8). The clustering analysis also demonstrated that the immune system is associated with mammographic calcifications (p

This study was designed to investigate changes in the metabolites in the intracellular fluid of the pancreatic β-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 β-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.

// Tiefeng Jin 1, 2, * , Hoe Suk Kim 1, * , Sul Ki Choi 1, 3 , Eun Hye Hwang 1 , Jisu Woo 1 , Han Suk Ryu 4 , Kwangsoo Kim 5 , Aree Moon 6 and Woo Kyung Moon 1, 3 1 Department of Radiology, Seoul National University Hospital, Jongno-gu, Seoul 03080, Korea 2 Department of Pathology & Cancer Research Center, Yanbian University Medical College, Yanji 133002, China 3 Department of Biomedical Science, Seoul National University College of Medicine, Seoul National University, Jongno-gu, Seoul 03080, Korea 4 Department of Pathology, Seoul National University Hospital, Jongno-gu, Seoul 03080, Korea 5 Division of Clinical Bioinformatics, Biomedical Research Institute, Seoul National University Hospital, Jongno-gu, Seoul 03080, Korea 6 Duksung Innovative Drug Center College of Pharmacy, Duksung Women's University, Dobong-gu, Seoul 01369, Korea * These authors have contributed equally to this work Correspondence to: Woo Kyung Moon, email: moonwk@snu.ac.kr Keywords: SerpinB2, SerpinE1, metastasis, microRNA-200c/141, triple negative breast cancer Received: June 06, 2016 Accepted: February 12, 2017 Published: February 24, 2017 ABSTRACT The microRNA-200 (miR-200) family is associated with tumor metastasis and poor patient prognosis. We found that miR-200c/141 cluster overexpression upregulated SerpinB2 in the MDA-MB-231 triple-negative (TN) breast cancer cell line. We observed transcription factor (c-Jun, c-Fos, and FosB) upregulation, nuclear localization of c-Jun, and increased SerpinB2 promoter-directed chloramphenicol acetyltransferase activity in miR-200c/141 cluster-overexpressing cells relative to controls. Additionally, miR-124a and miR-26b, which directly target SepinB2, were downregulated compared to controls. In mouse xenograft models, miR-200c/141 cluster overexpression promoted lymph node and lung metastasis, and siRNA-mediated SerpinB2 knockdown decreased lung metastasis, suggesting that SerpinB2 mediates miR-200c/141-induced lung metastasis. We also explored the clinical significance of SerpinB2 protein status through analysis of primary breast tumor samples and The Cancer Genome Atlas (TCGA) data. High SerpinB2 levels were associated with reduced survival and increased lymph node metastasis in breast cancer patients. SerpinB2 was overexpressed in the TN breast cancer subtype as compared to the luminal subtype. The present study demonstrates that SerpinB2 promotes miR-200c/141 cluster overexpression-induced breast cancer cell metastasis, and SerpinB2 overexpression correlates with increased metastatic potential and unfavorable outcomes in breast cancer patients. SerpinB2 may be a useful biomarker for assessing metastasis risk in breast cancer patients.

We assessed the use of ultrasound (US)-guided photoacoustic imaging (PAI) and anti-EGFR antibody-conjugated gold nanorods (anti-EGFR-GNs) to non-invasively detect EGFR-expressing primary tumor masses and regional lymph node (LN) metastases in breast tumor mice generated by injecting MCF-7 (EGFR-negative) or MDA-MB-231 (EGFR-positive) human breast cells using a preclinical Vevo 2100 LAZR Imaging system. Anti-EGFR-GNs provided a significant enhancement in the PA signal in MDA-MB-231 tumor and the axillary LN metastases relative to MCF-7 tumor and non-LN metastases. We demonstrated that US-guided PAI using anti-EGFR-GNs is highly sensitive for the selective visualization of EGFR-expressing breast primary tumors as well as LN micrometastases. (C) 2016 Optical Society of America

This study was undertaken to investigate the effect of a magnetic resonance imaging (MRI) contrast agent, superparamagnetic iron oxide nanoparticle (SPIO), on signal transduction by glucose and insulin in pancreatic β-cells. INS-1 cells were labeled in culture medium containing clinically approved SPIO for 24 h. Labeled and unlabeled cells were stimulated with glucose (25 mM) or insulin (0.1-1 µM) for 12 h. The phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2) and protein kinase B (AKT) and intracellular insulin protein levels were assessed by Western blotting. After labeling with increasing amounts of SPIO, cytotoxicity was not observed, yet the intracellular iron concentration increased in a dose-dependent manner. SPIO labeling (200 µg Fe ml(-1)) induced a significant increase in ERK1/2 and AKT phosphorylation (labeled vs unlabeled, p < 0.05), but significantly reduced the glucose-stimulated phosphorylation of ERK1/2 and AKT and insulin-stimulated phosphorylation of AKT (labeled vs unlabeled, p < 0.05). The level of intracellular insulin protein was found to be lower in labeled cells than unlabeled cells (labeled vs unlabeled, p < 0.05). This study demonstrates that SPIO labeling alters some fundamental functional variables, at least in INS-1 cells, through modulation of the glucose- or insulin-induced activation of ERK1/2 and AKT, which leads to insulin biosynthesis.