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S100A7/psoriasin, a member of the epidermal differentiation complex, is widely overexpressed in invasive estrogen receptor (ER)α-negative breast cancers. However, it has not been established whether S100A7 contributes to breast cancer growth or metastasis. Here, we report the consequences of its expression on inflammatory pathways that impact breast cancer growth. Overexpression of human S100A7 or its murine homologue mS100a7a15 enhanced cell proliferation and upregulated various proinflammatory molecules in ERα-negative breast cancer cells. To examine in vivo effects, we generated mice with an inducible form of mS100a7a15 (MMTV-mS100a7a15 mice). Orthotopic implantation of MVT-1 breast tumor cells into the mammary glands of these mice enhanced tumor growth and metastasis. Compared with uninduced transgenic control mice, the mammary glands of mice where mS100a7a15 was induced exhibited increased ductal hyperplasia and expression of molecules involved in proliferation, signaling, tissue remodeling, and macrophage recruitment. Furthermore, tumors and lung tissues obtained from these mice showed further increases in prometastatic gene expression and recruitment of tumor-associated macrophages (TAM). Notably, in vivo depletion of TAM inhibited the effects of mS100a7a15 induction on tumor growth and angiogenesis. Furthermore, introduction of soluble hS100A7 or mS100a7a15 enhanced chemotaxis of macrophages via activation of RAGE receptors. In summary, our work used a powerful new model system to show that S100A7 enhances breast tumor growth and metastasis by activating proinflammatory and metastatic pathways. Cancer Res; 72(3); 604–15. ©2011 AACR.

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Increasing evidence suggesting breast cancer stem cells (BCSCs) drive metastasis and evade traditional therapies underscores a critical need to exploit the untapped potential of nanotechnology to develop innovative therapies that will significantly improve patient survival. Photothermal therapy (PTT) to induce localized hyperthermia is one of few nanoparticle-based treatments to enter clinical trials in human cancer patients, and has recently gained attention for its ability to induce a systemic immune response targeting distal cancer cells in mouse models. Here, we first conduct classic cancer stem cell (CSC) assays, both in vitro and in immune-compromised mice, to demonstrate that PTT mediated by highly crystallized iron oxide nanoparticles effectively eliminates BCSCs in translational models of triple negative breast cancer. PTT in vitro preferentially targets epithelial-like ALDH + BCSCs, followed by mesenchymal-like CD44+/CD24− BCSCs, compared to bulk cancer cells. PTT inhibits BCSC self-renewal through reduction of mammosphere formation in primary and secondary generations. Secondary implantation in NOD/SCID mice reveals the ability of PTT to impede BCSC-driven tumor formation. Next, we explore the translational potential of PTT using metastatic and immune-competent mouse models. PTT to inhibit BCSCs significantly reduces metastasis to the lung and lymph nodes. In immune-competent BALB/c mice, PTT effectively eliminates ALDH + BCSCs. These results suggest the feasibility of incorporating PTT into standard clinical treatments such as surgery to enhance BCSC destruction and inhibit metastasis, and the potential of such combination therapy to improve long-term survival in patients with metastatic breast cancer.

Accumulating evidence has demonstrated that breast cancers are initiated and develop from a small population of stem-like cells termed cancer stem cells (CSCs). These cells are hypothesized to mediate tumor metastasis and contribute to therapeutic resistance. However, the molecular regulatory networks responsible for maintaining CSCs in an undifferentiated state have yet to be elucidated. In this study, we used CSC markers to isolate pure breast CSCs fractions (ALDH+ and CD44+CD24− cell populations) and the mature luminal cells (CD49f− EpCAM+) from the MCF7 cell line. Proteomic analysis was performed on these samples and a total of 3304 proteins were identified. A label-free quantitative method was applied to analyze differentially expressed proteins. Using the criteria of greater than twofold changes and p value

Psoriasin (S100A7) is a calcium-binding protein that has shown to be highly expressed in high-grade ductal carcinoma in situ (DCIS) and a subset of invasive breast cancers. However, its role in invasion and metastasis is not very well known. In this study, we have shown that S100A7 differentially regulates epidermal growth factor (EGF)-induced cell migration and invasion in ERα(-) MDA-MB-231 cells and ERα(+) MCF-7 and T47D breast cancer cells. Further signaling studies revealed that S100A7 enhances EGF-induced EGFR phosphorylation and actin remodeling that seems to favor lamellipodia formation in ERα(-) cells. In addition, S100A7 overexpression enhanced NF-κB-mediated matrix metalloproteinase-9 (MMP-9) secretion in MDA-MB-231 cells indicating its role in enhanced invasiveness. However, S100A7 overexpression inhibited migration and invasion of MCF-7 cells by inactivating Rac-1 pathway and MMP-9 secretion. Moreover, S100A7 overexpressing MDA-MB-231 cells showed enhanced metastasis compared to vector control in in vivo nude mice as detected by bioluminescence imaging. Our tissue microarray data also revealed predominant expression of S100A7 in ERα(-) metastatic carcinoma, especially in lymph node regions. Overall these studies suggest that S100A7 may enhance metastasis in ERα(-) breast cancer cells by a novel mechanism through regulation of actin cytoskeleton and MMP-9 secretion.

Summary We present "Labyrinth," a label-free microfluidic device to isolate circulating tumor cells (CTCs) using the combination of long loops and sharp corners to focus both CTCs and white blood cells (WBCs) at a high throughput of 2.5 mL/min. The high yield (>90%) and purity (600 WBCs/mL) of Labyrinth enabled us to profile gene expression in CTCs. As proof of principle, we used previously established cancer stem cell gene signatures to profile single cells isolated from the blood of breast cancer patients. We observed heterogeneous subpopulations of CTCs expressing genes for stem cells, epithelial cells, mesenchymal cells, and cells transitioning between epithelial and mesenchymal. Labyrinth offers a cell-surface marker-independent single-cell isolation platform to study heterogeneous CTC subpopulations.

To assess the potential for marketing pre-amended plant growing media, we compared survival of the entomopathogenic nematodes, Steinernema carpocapsae, delivered via Galleria mellonella and Tenebrio molitor cadavers in the Scotts commercial growing medium, Miracle-Gro®. Six pouches, each filled with 10 g of the growing medium containing 6 nematode-infected cadavers of either insect species were placed in each 1 kg Miracle-Gro® bag. The nematode survival was assessed in the cadavers, pouches and the bags. Although, overall survival of S. carpocapsae declined within 90 days in both cadaver types, survival was significantly higher in G. mellonella than T. molitor cadavers at 15, 45, 60 and 90 days most likely due to the higher reproduction in G. mellonella cadavers. Similarly, survival (assessed as the number of baited G. mellonella killed) remained consistently higher both in the pouches and bags in the G. mellonella cadaver treatment compared to the T. molitor treatment. In another experiment in which bags were not opened until 180 days, all G. mellonella cadavers disintegrated within 180 days of storage; however, the number of baited G. mellonella larvae killed in the pouch was similar in both the cadaver treatments indicating equal number of surviving nematodes. Further, we evaluated the effect of initial moisture content of the growing medium on the survival and virulence of nematodes when delivered through cadavers and aqueous suspension. The results showed that survival and virulence of S. carpocapsae was higher in G. mellonella cadaver treatment than in the aqueous suspension treatment at all sampling times, and reducing the initial moisture content of the medium from 50% to 20–30% enhanced nematode survival in the medium. This is the first report demonstrating the possibility of directly delivering entomopathogenic nematodes through commercial growing medium via infected insect cadavers, a method in which nematode survival could be further enhanced by reducing the initial moisture content of the growing medium.

There is substantial evidence that many cancers, including breast cancer, are driven by a population of cells that display stem cell properties. These cells, termed cancer stem cells (CSCs) or tumor initiating cells, not only drive tumor initiation and growth but also mediate tumor metastasis and therapeutic resistance. In this chapter, we summarize current advances in CSC research with a major focus on breast CSCs (BCSCs). We review the prevailing methods to isolate and characterize BCSCs and recent evidence documenting their cellular origins and phenotypic plasticity that enables them to transition between mesenchymal and epithelial-like states. We describe in vitro and clinical evidence that these cells mediate metastasis and treatment resistance in breast cancer, the development of novel strategies to isolate circulating tumor cells (CTCs) that contain CSCs and the use of patient-derived xenograft (PDX) models in preclinical breast cancer research. Lastly, we highlight several signaling pathways that regulate BCSC self-renewal and describe clinical implications of targeting these cells for breast cancer treatment. The development of strategies to effectively target BCSCs has the potential to significantly improve the outcomes for patients with breast cancer.

Introduction and Objective: The metastasis of cancer is preceded by the dissemination of cancer cells from the primary tumor site to remote sites via the blood circulation. The presence of circulating tumor cells (CTCs) in the peripheral blood represents a strong and independent prognostic factor for decreased disease-free and overall survival. Immune-affinity based capture, although being the most commonly used method for the isolation of CTCs, offers low throughput (∼1mL/hr) and have considerably cell loss caused by the heterogeneous expression of biomarkers on CTCs. Various label-free approaches utilizing the physical properties of CTCs have been developed to overcome the limitations, such as micro-filters, microscale laminar vortices, inertial migration of particles, and integrated systems. Here we present an inertial microfluidic-based separation technique for high throughput and label-free isolation of CTCs yielding the highest throughput with high CTC recovery and high blood cell removal among all the label-free technologies. The isolated CTC populations were further analyzed for single cell multiplex gene expression analysis. Methods: The PDMS-made inertial microfluidic device has 637 mm in length with 56 corners, 500 μm in width, and 100 μm in height. The separation of CTCs is driven by two main forces: (i) inertial force that focuses the cells into streamlines, and (ii) drag force from Dean flow that migrates the focused cells to various positions based on size. Device is optimized with MCF-7 and Panc-1 cell line within PBS buffer solution and diluted blood, and is tested in patients with breast cancer on an average of 5 mL of whole blood processed through double devices in series. CTCs isolated were analyzed for tumor specific protein markers and genomic characterization is done using singe cell analysis techniques. Results: Samples are processed through the inertial microfluidic device and CTCs are enriched in second outlet based on size difference between CTCs and blood cells. Device is optimized to operate at an extremely high throughput of 2500 μL/min with high recovery (greater than 90%) and high white blood cells (WBCs) removal (5 log orders). In patient samples, we identified CTCs in 38 of 40 (95%) breast patients with metastatic disease (5.4±4.6 CTC/mL) with low WBC contamination (663±647 WBC/mL). Based on the gene expression, both inter and intra patient heterogeneity of CTCs at the single cell level were discovered among the tested patient samples. Conclusion: The study of CTCs could have a direct impact upon society by presenting novel ways to address one of the major hurdles in cancer research - early detection - and will foster the advancement of science and engineering via the exploration of new druggable targets approaches and the further understanding of the pharmacodynamics. Citation Format: Eric Lin, Lianette Rivera, Shamileh Fouladdel, Hyeun Joong Yoon, Stephanie Guthrie, Jacob Weiner, Yadwinder S. Deol, Evan Keller, Vaibhav Sahai, Diane M. Simeone, Monika L. Burness, Ebrahim Azizi, Max S. Wicha, Sunitha Nagrath. Label-free high throughput microfluidic device for the isolation and single cell multiplex gene expression analysis of circulating tumor cells from breast cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3307.

S100A7/psoriasin, a member of the epidermal differentiation complex, is widely overexpressed in invasive estrogen receptor (ER)α-negative breast cancers. However, it has not been established whether S100A7 contributes to breast cancer growth or metastasis. Here, we report the consequences of its expression on inflammatory pathways that impact breast cancer growth. Overexpression of human S100A7 or its murine homologue mS100a7a15 enhanced cell proliferation and upregulated various proinflammatory molecules in ERα-negative breast cancer cells. To examine in vivo effects, we generated mice with an inducible form of mS100a7a15 (MMTV-mS100a7a15 mice). Orthotopic implantation of MVT-1 breast tumor cells into the mammary glands of these mice enhanced tumor growth and metastasis. Compared with uninduced transgenic control mice, the mammary glands of mice where mS100a7a15 was induced exhibited increased ductal hyperplasia and expression of molecules involved in proliferation, signaling, tissue remodeling, and macrophage recruitment. Furthermore, tumors and lung tissues obtained from these mice showed further increases in prometastatic gene expression and recruitment of tumor-associated macrophages (TAM). Notably, in vivo depletion of TAM inhibited the effects of mS100a7a15 induction on tumor growth and angiogenesis. Furthermore, introduction of soluble hS100A7 or mS100a7a15 enhanced chemotaxis of macrophages via activation of RAGE receptors. In summary, our work used a powerful new model system to show that S100A7 enhances breast tumor growth and metastasis by activating proinflammatory and metastatic pathways. Cancer Res; 72(3); 604–15. ©2011 AACR.

Psoriasin (S100A7) is expressed in several epithelial malignancies including breast cancer. Although S100A7 is associated with the worst prognosis in estrogen receptor α-negative (ERα(-)) invasive breast cancers, its role in ERα-positive (ERα(+)) breast cancers is relatively unknown. We investigated the significance of S100A7 in ERα(+) breast cancer cells and observed that S100A7 overexpression in ERα(+) breast cancer cells, MCF7 and T47D, exhibited decreased migration, proliferation, and wound healing. These results were confirmed in vivo in nude mouse model system. Mice injected with S100A7-overexpressing MCF7 cells showed significant reduction in tumor size compared with mice injected with vector control cells. Further mechanistic studies revealed that S100A7 mediates the tumor-suppressive effects via a coordinated regulation of the β-catenin/TCF4 pathway and an enhanced interaction of β-catenin and E-cadherin in S100A7-overexpressing ERα(+) breast cancer cells. We observed down-regulation of β-catenin, p-GSK3β, TCF4, cyclin D1, and c-myc in S100A7-overexpressing ERα(+) breast cancer cells. In addition, we observed increased expression of GSK3β. Treatment with GSK3β inhibitor CHIR 99021 increased the expression of β-catenin and its downstream target c-myc in S100A7-overexpressing cells. Tumors derived from mice injected with S100A7-overexpressing MCF7 cells also showed reduced activation of the β-catenin/TCF4 pathway. Therefore, our studies reveal for the first time that S100A7-overexpressing ERα(+) breast cancer cells exhibit tumor suppressor capabilities through down-modulation of the β-catenin/TCF4 pathway both in vitro and in vivo. Because S100A7 has been shown to enhance tumorigenicity in ERα(-) cells, our studies suggest that S100A7 may possess differential activities in ERα(+) compared with ERα(-) cells.

Background Cannabinoids bind to cannabinoid receptors CB1 and CB2 and have been reported to possess anti-tumorigenic activity in various cancers. However, the mechanisms through which cannabinoids modulate tumor growth are not well known. In this study, we report that a synthetic non-psychoactive cannabinoid that specifically binds to cannabinoid receptor CB2 may modulate breast tumor growth and metastasis by inhibiting signaling of the chemokine receptor CXCR4 and its ligand CXCL12. This signaling pathway has been shown to play an important role in regulating breast cancer progression and metastasis. Methodology/Principal Findings We observed high expression of both CB2 and CXCR4 receptors in breast cancer patient tissues by immunohistochemical analysis. We further found that CB2-specific agonist JWH-015 inhibits the CXCL12-induced chemotaxis and wound healing of MCF7 overexpressing CXCR4 (MCF7/CXCR4), highly metastatic clone of MDA-MB-231 (SCP2) and NT 2.5 cells (derived from MMTV-neu) by using chemotactic and wound healing assays. Elucidation of the molecular mechanisms using various biochemical techniques and confocal microscopy revealed that JWH-015 treatment inhibited CXCL12-induced P44/P42 ERK activation, cytoskeletal focal adhesion and stress fiber formation, which play a critical role in breast cancer invasion and metastasis. In addition, we have shown that JWH-015 significantly inhibits orthotopic tumor growth in syngenic mice in vivo using NT 2.5 cells. Furthermore, our studies have revealed that JWH-015 significantly inhibits phosphorylation of CXCR4 and its downstream signaling in vivo in orthotopic and spontaneous breast cancer MMTV-PyMT mouse model systems. Conclusions/Significance This study provides novel insights into the crosstalk between CB2 and CXCR4/CXCL12-signaling pathways in the modulation of breast tumor growth and metastasis. Furthermore, these studies indicate that CB2 receptors could be used for developing innovative therapeutic strategies against breast cancer.

Chemokine receptor CXCR4 and its cognate ligand CXCL12, also known as stromal derived factor-1 (SDF-1), have been shown to play an important role in growth and metastasis of various tumors. CXCR4 is the most common chemokine receptor that has been demonstrated to be overexpressed in several cancers. Its overexpression is also correlated with poor clinical outcomes and survival in various cancers, including breast, prostate, and lung. CXCR4/CXCL12 signaling axis plays an important role in organ selective metastasis since CXCR4 overexpressing cancer cells have been shown to metastasize to organs such as bone, lymph nodes, liver, lung, and brain, which produce high amounts of CXCL12 and thus provide a favorable microenvironment. CXCR4/CXCL12 axis has been shown to enhance tumor growth and metastasis by maintaining cancer stem cells and modulating tumor stroma through activation of cancer-associated fibroblasts and recruitment of CXCR4+ endothelial precursor cells, thereby enhancing angiogenesis. CXCR4/CXCL12 axis has been shown to mediate both pro-tumorigenesis and metastasis by activating multiple signaling pathways, including protein tyrosine pathways through activation of Pyk2. Several CXCR4/CXCL12 antagonists/agonists have been shown to have potential therapeutic effects as they significantly inhibit tumor growth and metastasis of various cancers in both in vitro and in vivo mouse models. These studies suggest that the CXCR4/CXCL12 signaling axis is an important target for developing innovative therapies against various cancers.

Targeting dendritic cell (DC) functions such as migration is a pivotal mechanism used by HIV-1 to disseminate within the host. The HIV-1 envelope protein is the most important of the virally encoded proteins that exploits the migratory capacity of DCs. In the present study, we elucidated the signaling machinery involved in migration of immature DCs (iDCs) in response to HIV-1 envelope protein. We observed that M-tropic HIV-1 glycoprotein 120 (gp120) induces phosphorylation of the nonreceptor tyrosine kinase, Pyk2. Inhibition of Pyk2 activity using a pharmacologic inhibitor, kinase-inactive Pyk2 mutant, and Pyk2-specific small interfering RNA blocked gp120-induced chemotaxis, confirming the role of Pyk2 in iDC migration. In addition, we also illustrated the importance of Pyk2 in iDC migration induced by virion-associated envelope protein, using aldithriol-2–inactivated M-tropic HIV-1 virus. Further analysis of the downstream signaling mechanisms involved in gp120-induced migration revealed that Pyk2 activates p38 mitogen-activated protein kinase, which in turn activates the F-actin–binding protein, leukocyte-specific protein 1, and enhances its association with actin. Taken together, our studies provide an insight into a novel gp120-mediated pathway that regulates DC chemotaxis and contributes to the dissemination of HIV-1 within an infected person.

Breast cancer stem cells are responsible for tumor recurrence, metastasis and drug resistance. One of the important steps in metastasis is entry of tumor cells into the blood circulation. These circulating tumor cells (CTCs) may serve as markers of cancer progression and as a “liquid biopsy” to provide information on tumor biology at single cell resolution. Current methods of CTC capture are not fully inclusive to isolate all phenotypic variations of cancer stem cells (CSCs) among the CTCs. Using newly developed microfluidic methods to isolate CTCs from blood samples of metastatic breast cancer patients, we assessed gene expression profiles in these cells. The gene expression signatures of isolated CTCs were determined using a highly sensitive microfluidic-based 96-plex RT-qPCR method. Analyzed multiplex RT-qPCR data revealed distinct gene expression patterns among CTCs isolated from different patients. Cancer stem cell markers such as ALDH1a1, ALDH1a2 and ALDH1a3 as well as CD44 and CD24 genes were differentially expressed in isolated CTCs. In addition, markers of the epithelial to mesenchymal (EMT) and mesenchymal to epithelial (MET) phenotypes such as Vimentin, EpCAM, HER2, CDH1, CDH2, TGFb1 and cytokeratins were also expressed variably in the CTC samples. Furthermore, MMP9, TIMP1, CD90, CD14, SPARC, BRAF, KRAS, PI3K and ERCC1 genes which play a role in cancer initiation, promotion, metastasis and drug resistance were also differentially expressed in the isolated CTCs. Overall, these studies further our understanding into the heterogeneity of CTCs and CSCs as well as providing a potential tool for real-time monitoring of cancer patients on clinical trials. Citation Format: Shamileh Fouladdel, Hyeun Joong Yoon, Eric Lin, Tae Hyun Kim, Yadwinder S. Deol, Tahra K. Luther, Shawn G. Clouthier, Hui Jiang, Monika L. Burness, Sunitha Nagrath, Ebrahim Azizi, Max S. Wicha. Gene expression signatures of isolated circulating tumor cells from metastatic breast cancer patients reveal presence of breast cancer stem cells with EMT or MET phenotypes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4726. doi:10.1158/1538-7445.AM2015-4726

Background: Many cancers, including breast cancer, are hierarchically organized and contain a subpopulation of self-renewing cells that display stem cell properties. Breast cancer stem cells (BCSCs) drive tumor growth and metastasis and contribute to therapeutic resistance. Recent studies suggest that BCSCs exist in alternative mesenchymal-like (EMT) and epithelial-like (MET) states which are characterized by expression of the BCSC markers CD44+/CD24- and aldehyde dehydrogenase (ALDH), respectively. BCSCs maintain the plasticity to transition between EMT and MET-like states in a process regulated by the tumor microenvironment. However, the pathways involved in regulation of these stem cell states remain undefined. Approach and Results: In order to elucidate the mechanisms which regulate EMT and MET BCSCs, we performed gene expression and proteomic analysis of BCSC populations enriched for EMT (CD44+/CD24-) or MET, ALDH (Aldefluor-positive). Among the genes enriched in the MET BCSC populations were Annexin A3 (ANXA3). Validation by qPCR and western blotting confirmed increased ANXA3 mRNA and protein expression in the ALDH+ fractions of MCF7, SUM 159, T47D and SUM 149 breast cancer cells, as well as normal mammary epithelial cells. Immunohistochemical analysis revealed that ANXA3 expression was significantly higher in breast cancers compared to normal breast tissue with over 75% of primary human tumors demonstrating significant ANXA3 expression. To determine the functional role of ANXA3 in regulating BCSCs we established breast cancer cell lines with inducible knockdown of ANXA3 utilizing a doxycycline (DOX) inducible shRNA. ANXA3 knockdown reduced cell proliferation and decreased colony formation in soft agar. In addition, ANXA3 knockdown significantly decreased the proportion of ALDH+ BCSCs. In contrast, ANXA3 knockdown increased the relative proportion of EMT-like (i.e. CD44+/CD24-) cells as well as expression of EMT regulatory genes including Twist1 and Snail1. In addition, ANXA3 knocked down significantly decreased tumorsphere formation in vitro and tumor initiating capacity of breast cancer cells in NOD/SCID mice. Conclusions: These results suggest that ANXA3 may play a significant role in regulating the self-renewal of the MET-like ALDH+ BCSC. Studies are underway to determine the mechanisms of this regulation and to assess the potential of ANXA3 as a cancer stem cell therapeutic target. Citation Format: Yadwinder S. Deol, Sean P. McDermott, David M. Lubman, Jenny C. Chang, Song Nie, Tahra K. Luther, Yang Cong, Ebrahim Azizi, Justin Colacino, Shawn G. Clouthier, Max Wicha. Annexin A3 regulates MET-like aldehyde dehydrogenase positive breast cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2220. doi:10.1158/1538-7445.AM2015-2220

Introduction and Objective: A necessary step in distant metastasis is the hematogenous dissemination of cancer cells from the primary tumor site to remote sites. The presence of circulating tumor cells (CTCs) in the peripheral blood represents a strong and independent prognostic factor for decreased disease-free and overall survival in many solid malignancies. Immune-affinity based capture is the most commonly used method for the isolation of CTCs which utilizes antibodies to capture tumor cells expressing specific proteins. However, immune-affinity based approaches offer low throughput (∼1mL/hr) and considerable cell loss (∼20-40%) resulting from heterogeneous expression of biomarkers on CTCs. Various label-free approaches utilizing physical properties of CTCs have been developed to overcome the limitations of immune-affinity based isolation techniques, including micro-filters, microscale laminar vortices, inertial migration of particles, and integrated systems. Here we present an inertial microfluidic-based separation technique for high throughput and label-free isolation of CTCs that yields the highest throughput with high CTC recovery and high blood cell removal among all the label-free technologies. Methods: The PDMS-made microfluidic device has 637 mm in length with 56 corners, 500 μm in width, and 100 μm in height. The separation is driven by two main forces: (i) inertial force that focuses the cells into streamlines, and (ii) drag force from Dean flow that migrates the focused cells to various positions based on size. The device was optimized with MCF-7 and Panc-1 cell lines spiked into PBS buffer and also diluted blood. It was then tested on 10 mL blood samples from patients with metastatic breast cancer. The separated cells were cytospun and stained to identify CTCs as cytokeratin positive, DAPI positive, CD45 negative cells. Results: Samples were processed through the inertial microfluidic device that enriches CTCs in the second outlet based on size difference between CTCs and blood cells. The device was optimized to operate at an extremely high throughput of 2500 μL/min with high recovery (92% for both spiked samples of MCF-7 and PANC-1 cell lines) and high white blood cells (WBCs) removal (91%). To determine the efficiency of capture of rare cell populations, healthy donor blood samples were spiked with MCF-7/GFP at 100 cells and 95% recovery was obtained. In patient samples, we identified CTCs in 24 of 27 (89%) breast patients with metastatic disease (4.3±4.8 CTCs/mL) with low WBCs contamination (465±473 WBCs/mL). Conclusion: The study of CTCs could have a direct impact upon patient care by presenting a novel CTC isolation method. This technology may be applicable to early detection, and also for monitoring response to treatment. Our approach is superior to current strategies because it is independent of cell surface markers, which may be varied in tumor cells, and may exclude cancer stem cells. Citation Format: Eric Lin, Lianette Rivera, Hyeun Joong Yoon, Shamileh Fouladdel, Jacob Wieger, Stephanie Guthrie, Yadwinder S. Deol, Shawn G. Clouthier, Tahra K. Luther, Diane M. Simeone, Monika L. Burness, Ebrahim Azizi, Max S. Wicha, Sunitha Nagrath. Label-free high throughput microfluidic device for the isolation of circulating tumor cells from breast cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1593. doi:10.1158/1538-7445.AM2015-1593

A considerable body of evidence indicates that many cancers are hierarchically organized and driven by cancer stem cells (CSCs) that have the ability to self-renew and to generate heterogeneous populations of tumor bulk. Preclinical studies have also demonstrated that CSCs mediate tumor metastasis and resistance to chemotherapy and radiation therapy. As a result of substantial studies to identify CSCs markers and related pathways, agents developed to target CSCs are in early stage clinical trials. Given the clinical importance of CSCs, there is an urgency to develop the methodology to assay these cells. In this study, we explored CSCs heterogeneity at the single cell level using C1 and BioMark HD technologies. Cancer stem cells from MCF7 and SUM159 breast cancer cell lines were sorted by MoFlo XDP based CSCs marker including CD44, CD24 and ALDH1 immunophenotyping. Sorted cells were then separately loaded onto the C1 chip and studied by C1 instrument that isolates up to 96 single cells and performs lysis, RNA release, reverse transcription, and finally cDNA preamplification of up to 96 gene transcripts for each single cell. We selected the 96 genes based on important genetic pathways, EMT and CSCs markers, and also genes identified in the expression array data to be differentially expressed in CD44+/CD24- and or ALDH+ primary breast tumor samples. Following C1 preamplification, we analyzed the mRNA expression levels of 96 genes on each of the isolated single cells by BioMark HD instrument which generates nearly 10,000 qPCR data points in a single run using a 96x96 chip. Results showed that more than one population exist with significant differences in mRNA expression levels of certain genes such as CAPRIN2, TM4SF1, NECAB3, PPL, TSPAN6, STAP2, FAM46C, TCEA3, TPSB2 and FCN1, as well as IL6, GATA3, KI67, PCNA, EZH2, and MET in seemingly homogenous populations of ALDH+ and ALDH-(CD44+/CD24- in MCF7 cells) CSCs. In addition, a distinct pattern of gene expression for sorted luminal MCF7 in comparison to basal SUM159 cells was observed for epithelial versus mesenchymal markers such as CDH1, CDH2, KRT7, KRT19, EpCAM, and Vimentin genes. We have recently reported that breast cancer stem cells (BCSCs) exist in inter-convertible EMT/MET states characterized by distinct genes and biomarker expression. Using this methodology of single cell analysis, we found that these CSC populations still display significant heterogeneity in mRNA expression. This technique of single cell multiplex gene expression analysis, along with a microfluidic method for a label-free cell sorting is currently under optimization to be used for isolation and molecular characterization of circulating tumor cells (CTCs) from blood samples of breast cancer patients as a means of liquid biopsy. Altogether, results of this and similar studies on single CSCs have considerable clinical implications in cancer diagnosis and prognosis, as well as in the successful targeted therapy of cancer stem cells. Citation Format: Ebrahim Azizi, Shamileh Fouladdel, Yadwinder S. Deol, Jonathan Bender, Sean McDermott, Hui Jiang, Mary Sehl, Shawn G. Clouthier, Sunitha Nagrath, Max S. Wicha. Exploring cancer stem cells heterogeneity via single cell multiplex gene expression analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1943. doi:10.1158/1538-7445.AM2014-1943

Background: Psoriasin (S100A7) is a low molecular weight protein associated with several epithelial malignancies including psoriasis, breast cancer, oral squamous cell carcinoma, etc. It is highly expressed in ductal carcinoma in situ and is mainly associated with the worst prognosis in invasive estrogen receptor α negative (ERα−) breast cancers; however, its exact role in ERα positive (ERα+) breast cancers is not known. Therefore, we investigated the tumorigenic potential of S100A7 over expression in ERα+ breast cancer cells both in vitro and in vivo. Methods: The tumorigenicity of stably over-expressing S100A7 and vector control ERα+ cell lines was assessed using proliferation, migration and wound healing assays. Microarray analysis was carried out to determine the tumorigenic mechanisms associated with S100A7 over-expression and the validation of the targets was performed using real time PCR and immunoblotting. In vivo effect of S100A7 over-expression on tumorigenicity was analyzed using xenograft mouse models and the tumors derived from these mice were analyzed using immunohistochemistry. Results: S100A7 over-expressing ERα+ cells showed significantly reduced EGF-induced proliferation, migration and wound healing compared to vector control cells. Characterization of the EGF-mediated signaling pathways revealed reduced phosphorylation of EGFR, AKT and ERK in S100A7 over-expressing cells. We also observed decreased mRNA and protein levels of β-catenin pathway associated genes -TCF4 and cyclinD1 in S100A7 over-expressing cells. Further analysis of β-catenin /TCF4 pathway showed reduced expression of β-catenin and increased expression of GSK3β and phospho β-catenin in S100A7 over-expressing cells. In addition, we found decreased expression of c-myc and cyclin D1 which are downstream targets of β-catenin/TCF4 pathway. Next, we observed enhanced interaction of E-cadherin and β-catenin in the membranes of S100A7 over-expressing cells. We also observed decreased Rac activation and actin polymerization in S100A7 over-expressing cells which may account for their reduced migration. Finally, we demonstrated that S100A7 over-expressing cells decreased tumor formation in nude mouse model. Furthermore, the immunohistochemical analysis showed down regulation of Cyclin D1 and β-catenin and increased expression of E-cadherin in S100A7 over-expressing tumors, thus confirming the reduced activation of β-catenin/TCF4 pathway in vivo. Conclusions: We conclude that S100A7 decreases the tumorigenicity of ERα+ cells by down modulating β-catenin/TCF4 pathway both in vitro and in vivo. S100A7 is normally associated with increased tumorigenicity in ERα− breast cancer but our results suggest that S100A7 may have a differential role in ERα+ and ERα− cells. Our studies indicate that S100A7 could act as a novel therapeutic target for drug resistant ERα+ breast tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1024. doi:10.1158/1538-7445.AM2011-1024

Objective: Psoriasin (S100A7), small 11 kDa calcium binding protein, has been shown to be highly expressed in ER-alpha-negative (ERα−) invasive breast carcinomas with poor patient outcome. However, the role of S100A7 in breast cancer progression and metastasis is not well understood. Here we elucidate S100A7-mediated effects in breast tumorogenesis. Methods: We studied EGF-mediated cell migration and cell motility in vitro in S100A7 overexpressing MDA-MB-231 (231-S100A7) cells by standard assays. We generated bi-transgenic MMTV-rtTA; mS100A7/A15 mice which expressed mS100A7/A15 in mammary glands under doxycycline (Dox) (1g/kg) treatment. Mammary glands were isolated and stained for carmine, H&E and immunostaining according to the standard protocols. We also injected MVT1 cells (derived from MMTV-c-Myc; MMTV-VEGF) orthotopically into the mammary glands of bi-transgenic mice to study the role of mS100A7/A15 in mammary tumor growth and metastasis. Results: In the present investigation, we have shown that S100A7 overexpression in ERα− breast cancer cell line enhanced EGF-induced migration and wound healing compared to vector control cells. Further elucidation of signaling mechanisms reveal that S100A7 enhanced EGF-induced EGFR and Stat3 activation. Microarray and real time PCR analysis revealed upregulation of pro-inflammatory molecules such as CXCL8, CXCL1, IL-1α, serum amyloid A2 (SAA2) and MMP9 in 231-S100A7 cells compared to 231-Vec. These results were further confirmed with cytokine array analysis in the conditioned media (CM) of 231-S100A7 cells compared to 231-Vec. To further characterize the role of S100A7, we targeted the expression of murine homologue of S100A7, also known as mS100A7/A15, to mammary epithelial cells by generating bi-transgenic MMTV-rtTA; mS100A7/A15 mice. Morphological examination of the mammary glands after three months of Dox treatment revealed ductal hyperplasia, enhanced mS100A7/A15, Ki67 and cyclin D1 expression compared to uninduced mice. We also showed enhanced activation of Stat3, AKT, ERK1/2, MMP2 and SAA2 in the mammary glands of these mice. We further observed enhanced tumor growth and metastasis of orthotopically injected MVT1 cells in Dox treated compared to untreated mice. Tumors and lung tissues obtained from Dox treated bi-transgenic mice showed increased expression of prometastatic genes and enhanced recruitment of tumor-associated macrophages (TAM). Furthermore, CM from 231-S100A7 cells significantly enhanced migration of THP1-differentiated macrophages. Conclusion: These findings suggest that S100A7 may regulate breast cancer progression and metastasis by enhancing inflammatory signals that result in enhanced recruitment of TAM. Furthermore, these studies suggest that S100A7 is a novel therapeutic target for the treatment of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 403. doi:10.1158/1538-7445.AM2011-403

Introduction S100A7 (Psoriasin) is an inflammatory protein known to be upregulated in breast cancer. However, the role of S100A7 in breast cancer has been elusive, since both pro- and anti-proliferative roles have been reported in different types of breast cancer cells and animal models. To date, the mechanism by which S100A7 differentially regulates breast cancer cell proliferation is still not clear. Methods We used Gene Functional Enrichment Analysis to search for the determining factor of S100A7 differential regulation. We confirmed the factor and elaborated its regulating mechanism using in vitro cell culture. We further verified the findings using xenografts of human breast cancer cells in nude mice. Results In the present study, we show that S100A7 significantly downregulates the expression of miR-29b in Estrogen Receptor (ER)-positive breast cancer cells (represented by MCF7), and significantly upregulates miR-29b in ER-negative cells (represented by MDA-MB-231). The differential regulation of miR-29b by S100A7 in ER-positive and ER-negative breast cancer is supported by the gene expression analysis of TCGA invasive breast cancer dataset. miR-29b transcription is inhibited by NF-κB, and NF-κB activation is differentially regulated by S100A7 in ER-positive and ER-negative breast cancer cells. This further leads to differential regulation of PI3K p85α and CDC42 expression, p53 activation and p53-associated anti-proliferative pathways. Reversing the S100A7-caused changes of miR-29b expression by transfecting exogenous miR-29b or miR-29b-Decoy can inhibit the effects of S100A7 on in vitro cell proliferation and tumor growth in nude mice. Conclusions The distinct modulations of the NF-κB – miR-29b – p53 pathway make S100A7 an oncogene in ER-negative and a cancer-suppressing gene in ER-positive breast cancer cells, with miR-29b being the determining regulatory factor. Electronic supplementary material The online version of this article (doi:10.1186/s12943-014-0275-z) contains supplementary material, which is available to authorized users.

S100A7 (Psoriasin) is an inflammatory protein known to be upregulated in breast cancer. However, the role of S100A7 in breast cancer has been elusive, since both pro- and anti-proliferative roles have been reported in different types of breast cancer cells and animal models. To date, the mechanism by which S100A7 differentially regulates breast cancer cell proliferation is still not clear.We used Gene Functional Enrichment Analysis to search for the determining factor of S100A7 differential regulation. We confirmed the factor and elaborated its regulating mechanism using in vitro cell culture. We further verified the findings using xenografts of human breast cancer cells in nude mice.In the present study, we show that S100A7 significantly upregulates the expression of miR-29b in Estrogen Receptor (ER)-positive breast cancer cells (represented by MCF7), and significantly downregulates miR-29b in ER-negative cells (represented by MDA-MB-231) [Corrected]. The differential regulation of miR-29b by S100A7 in ER-positive and ER-negative breast cancer is supported by the gene expression analysis of TCGA invasive breast cancer dataset. miR-29b transcription is inhibited by NF-κB, and NF-κB activation is differentially regulated by S100A7 in ER-positive and ER-negative breast cancer cells. This further leads to differential regulation of PI3K p85α and CDC42 expression, p53 activation and p53-associated anti-proliferative pathways. Reversing the S100A7-caused changes of miR-29b expression by transfecting exogenous miR-29b or miR-29b-Decoy can inhibit the effects of S100A7 on in vitro cell proliferation and tumor growth in nude mice.The distinct modulations of the NF-κB - miR-29b - p53 pathway make S100A7 an oncogene in ER-negative and a cancer-suppressing gene in ER-positive breast cancer cells, with miR-29b being the determining regulatory factor.