Inflammatory Breast Cancer Tumor Emboli Research Articles

Florid Lymphovascular Invasion of Inflammatory Breast Cancer is Due to Geometric Embolic Budding

The sina qua non of inflammatory breast cancer (IBC) is numerous tumor emboli especially within overlying dermal lymphatics. The explanation remains a mystery. In human studies comparing IBC with non‐IBC, although common tumor parameters such as Ki‐67 index, mitotic count and nuclear size show a close overlap, there is a dramatic exponential difference in embolic density in IBC v non‐IBC. Using contrasting properties of two IBC xenografts, Mary‐X and Karen‐X, this study offers a novel explanation and proposed mechanism to explain the very high embolic numbers in IBC. The explanation is geometric budding of emboli. Mary‐X exhibits florid lymphovascular invasion in vivo which give rise to high numbers of CTCs and pulmonary metastases whereas Karen‐X lacks these features. Mary‐X also gives rise to compact spheroids in vitrowhich exhibit dramatic budding whereas Karen‐X exhibits only loose non‐budding aggregates. Furthermore Mary‐X emboli also bud into daughter emboli in vivo. The mechanism that regulates the compactness of the spheroids as well as the emboli involves the generation of E‐cad/NTF1, a calpain‐mediated cleavage product of membrane E‐cadherin. Inhibiting the generation of E‐cad/NTF1 by blocking either the calpain site of cleavage (SC) or the site of binding (SB) with specific decapeptides both reduces spheroid compactness and decreases budding. Since E‐cad/NFT1 retains the p120ctn binding site but loses both the β‐catenin and α‐binding sites, promoting its 360° distribution around the cell’s membrane, the varying levels of expression of this truncated molecule may trigger budding of both the spheroids as well as the emboli. Recurrent and geometric budding of parental emboli into daughter emboli then would account for the plethora of IBC tumor emboli seen in patients.

Abstract 4854: Targeting ribonucleotide reductase M2 using didox causes inhibition of estrogen receptor-negative, inflammatory breast cancer cell proliferation and tumor emboli formation in culture

Abstract Ribonucleotide reductase (RR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides during the rate-limiting step of DNA synthesis. Given its significant role in cell cycle regulation, RR and its catalytic subunits are significant molecular targets for inhibiting tumor cell growth and proliferation. Didox (3,4-dihydroxybenzohydroxamic acid) is a known inhibitor of the catalytic RR subunit of RRM2 and overall DNA synthesis. Previous meta-analyses have revealed that RRM2 is highly expressed in estrogen receptor-negative (ER-) breast cancer tumors. As ER negativity is frequent in inflammatory breast cancer (IBC), a distinct and the most aggressive breast cancer subtype, we proposed to evaluate efficacy of didox in IBC cell models. ER- inflammatory breast cancer (IBC) cell lines SUM149 (ER-, PR-, EGFR-activated), lapatinib-resistant SUM149 (rSUM149), and non-IBC BT474M1 metastatic subline of BT474 (ER+, PR+, HER2+) were compared for the cytotoxic and cytostatic effects of didox using cell viability (trypan blue exclusion) and proliferation (MTT) assays. Protein profiling was performed to evaluate changes in molecular expression of key proteins involved in cellular signaling pathways for apoptosis and proliferation. Only ER- cell lines exhibited decreased dose-dependent decrease in viability following didox treatment. In addition, didox inhibited tumor emboli formation in a novel ex vivo tumor emboli in culture assay that mimics the clinicopathologic hallmark of IBC disease in patients. Moreover, didox decreased proliferation in lapatinib-resistant rSUM149, suggesting that the drug may help circumvent lapatinib resistance. Studies are ongoing to evaluate the mechanism of didox in overcoming lapatinib resistance and evaluation in an in vivo IBC invasion model. IBC is rare but accounts for 10% of breast cancer deaths, and despite multimodal therapy the 5-yr survival is only 25-55%. Further, there is a need to identify new therapies that can inhibit IBC tumor emboli formation and migration. Didox has demonstrated minimal toxicity in human phase I/II cancer clinical trials and therefore is an attractive strategy for inflammatory breast cancer therapy. Support in part from the Development funds (GRD) of the Duke Cancer Institute as part of the P30 Cancer Center Support Grant NIH CA014236 and Department of Defense Breakthrough Level 2 W81XWH-17-1-0297 (GRD). Citation Format: Pranalee Patel, Ben Mirman, Renzhi Zhan, Gayathri R. Devi. Targeting ribonucleotide reductase M2 using didox causes inhibition of estrogen receptor-negative, inflammatory breast cancer cell proliferation and tumor emboli formation in culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4854.

Photothermal ablation of inflammatory breast cancer tumor emboli using plasmonic gold nanostars.

Inflammatory breast cancer (IBC) is rare, but it is the most aggressive subtype of breast cancer. IBC has a unique presentation of diffuse tumor cell clusters called tumor emboli in the dermis of the chest wall that block lymph vessels causing a painful, erythematous, and edematous breast. Lack of effective therapeutic treatments has caused mortality rates of this cancer to reach 20%–30% in case of women with stage III–IV disease. Plasmonic nanoparticles, via photothermal ablation, are emerging as lead candidates in next-generation cancer treatment for site-specific cell death. Plasmonic gold nanostars (GNS) have an extremely large two-photon luminescence cross-section that allows real-time imaging through multiphoton microscopy, as well as superior photothermal conversion efficiency with highly concentrated heating due to its tip-enhanced plasmonic effect. To effectively study the use of GNS as a clinically plausible treatment of IBC, accurate three-dimensional (3D) preclinical models are needed. Here, we demonstrate a unique in vitro preclinical model that mimics the tumor emboli structures assumed by IBC in vivo using IBC cell lines SUM149 and SUM190. Furthermore, we demonstrate that GNS are endocytosed into multiple cancer cell lines irrespective of receptor status or drug resistance and that these nanoparticles penetrate the tumor embolic core in 3D culture, allowing effective photothermal ablation of the IBC tumor emboli. These results not only provide an avenue for optimizing the diagnostic and therapeutic application of GNS in the treatment of IBC but also support the continuous development of 3D in vitro models for investigating the efficacy of photothermal therapy as well as to further evaluate photothermal therapy in an IBC in vivo model.

Inflammatory breast cancer tumor emboli express high levels of anti-apoptotic proteins: use of a quantitative high content and high-throughput 3D IBC spheroid assay to identify targeting strategies.

Inflammatory breast cancer (IBC) is one of the most lethal breast cancer variants; with existing therapy, 5-yr survival rate is only 35%. Current barriers to successful treatment of IBC include frequent infiltration and the presence of tumor cell clusters, termed tumor emboli, within the breast parenchyma and lymphatics. Prior studies have identified the role of anti-apoptotic signaling, in particular hyperactivation of NFκB and its target genes, in IBC pathobiology and therapeutic resistance. The objectives of this study were to: (1) determine if IBC tumor emboli express anti-apoptotic proteins and (2) develop a high content, multiparametric assay to assess the morphology of the IBC 3D spheroids and to optimize a high throughput format to screen for compounds that can inhibit the formation of the IBC tumor clusters/embolic structures. Immunohistochemical analysis of IBC patient tumor samples with documented tumor emboli revealed high NFκB (p65) staining along with expression of XIAP, a potent anti-apoptotic protein known to interact with NFκB signaling in enhancing survival of malignant cells. Subsequently, the high content assay developed allowed for simultaneous imaging and morphometric analysis, including count and viability of spheroids derived from SUM149, rSUM149 and SUM190 cells and its application to evaluate XIAP and NFκB inhibitory agents. We demonstrate the efficacy of the off-patent drug disulfiram when chelated with copper, which we had previously reported to inhibit NFκB signaling, was highly effective in disrupting both IBC spheroids and emboli grown in vitro. Taken together, these results identify a high-throughput approach to target tumor spheroid formation for drug discovery. Finally, disulfiram is a safe and approved drug for management of alcohol abuse, warranting its evaluation for repurposing in IBC therapy.

Abstract P3-16-02: Nanotheranostics using plasmonic gold nanostars to target inflammatory breast cancer cells and tumor emboli

Abstract Introduction: Due to the aggressive nature of inflammatory breast cancer (IBC) and insensitive imaging techniques, patients diagnosed with IBC have a poor prognosis. IBC's unique ability to form emboli leads to rapid growth and distant invasion. New therapies aimed at disrupting the formation of tumor emboli are needed. Gold nanostars (GNS) are unique nanoparticles that can be imaged in real time with high sensitivity, and are rapidly endocytosed into cancer cells. Due to their sharp spikes, GNS exhibit plasmonic properties, which strongly enhance the electromagnetic field upon laser excitation. In addition to their use as highly intense and stable fluorescent labels, these nanoparticles can be photothermally activated to trigger cellular ablation. The use of GNS as an imaging and therapeutic modality for IBC should be investigated. Methods: IBC (SUM-149/SUM190), non-IBC (BT474M1/MD-MBA-231), and drug resistant isotype variant (rSUM-149) cell lines were incubated with GNS, stained with Hoechst33342, and imaged with multiphoton microscopy (MPM). The dose and time dependent effects of GNS on SUM-149 proliferation was assessed with MTT assays. Photothermal treatment was performed on GNS-labeled IBC, non-IBC, and drug resistant cancer cell lines. The effects of photothermal therapy on cell viability were assessed using fluorescein diacetate and propidium iodide. Using a tumor emboli model, SUM-149, rSUM-149, and SUM-190 tumor emboli were labeled with GNS on the 3rd day of embolic maturation. Emboli were sectioned, stained with H/E, and imaged with MPM to demonstrate the depth of GNS penetrance. The potential for photothermal ablation of the GNS-labeled tumor emboli was assessed using various laser intensities. Propidium iodide was used to examine emboli viability following treatment. Results: In all cell lines, GNS displayed rapid cellular uptake without nuclear involvement. MTT assay showed that GNS concentrations of 0.15 and 0.20 nM caused decreases in cell proliferation at 6 and 12 hours. Proliferative capacity was unaltered at 24 hours for all concentrations, however this may be due to spectrophotometric interference with high intracellular GNS concentrations. Live/dead staining confirmed effective photothermal treatment in all cultures with a clear zone of cellular death. For tumor emboli studies, GNS allowed bright fluorescent monitoring of tumor emboli using MPM, and cross sectional imaging demonstrated GNS penetrance into the embolic core. Photothermal ablation of GNS-labeled tumor emboli was successfully demonstrated following laser irradiation. Cell death was confirmed with propidium iodide. Conclusion: Gold nanostars provide a highly fluorescent intracellular label for cancer cell lines, and tumor emboli without significantly altering cell proliferation. Furthermore, the inherent optical properties of the GNS allows for a combined therapeutic application following phototoactivation. This is the first study to demonstrate the nanothernostic application of GNS in IBC tumor emboli. Prior studies have shown the effectiveness of photothermal ablation in in vivo sarcoma models; we are currently extending our studies to an IBC mouse model. Supported by the P30 Cancer Center Support Grant (GRD) and Duke Exploratory Funds (TVD). Citation Format: Shammas RL, Fales AM, Crawford BM, Hollenbeck ST, Vo-Dinh T, Devi GR. Nanotheranostics using plasmonic gold nanostars to target inflammatory breast cancer cells and tumor emboli [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-16-02.

Abstract B44: Immune pathway dysregulations in inflammatory breast cancer health disparity

Abstract Background: Inflammatory breast cancer (IBC) is a distinct, aggressive and the most lethal form of breast cancer. Furthermore, data from population-based registries, when stratified by race and compared to other locally advanced breast cancers, suggest that IBC has a disproportionately high incidence, prevalence & shorter median survival time in African Americans (AA) compared to Caucasian American (CA) patients. IBC health disparities that are independent of external factors like differences in income, screening rates, and access to health care suggest a biological component to poor outcomes. IBC pathobiology is also unique wherein instead of a solid tumor, tightly packed tumor cell clusters/emboli are formed with a propensity for lymphatic dissemination. The underlying mechanisms for this extremely aggressive breast cancer phenotype are largely unknown and, therefore, present a major impediment to identifying the molecular underpinnings for health disparities in IBC patients. Methods and Results: In order to understand how IBC cells evade cell death signals in the host microenvironment, we conducted comparative analysis of the largest collection of untreated primary tumor samples of IBC (N=137), along with stage- and subtype-matched non-IBC (N=252) and normal breast tissue. Results reveal that pretreatment tumor tissue samples from IBC patients have a heightened expression of adaptive stress response genes comprised of high levels of the nuclear transcription factor, NFkappaB, and its target genes in the anti-apoptotic and immune/inflammatory pathways that correlate with cell survival. In addition, guided by analysis based on presence of tumor infiltrating leukocytes in IBC (N=69) and non-IBC (N=62) patient samples, we have identified a set of pathways and genes that have the ability to distinguish the immune responses in IBC and non-IBC patients in a subtype independent manner. In particular, interferon alpha/beta and NFκB signaling pathways were differentially regulated in IBC vs. non-IBC. Gene expression driven analysis of enrichment of leukocyte subsets reveal specific differences in plasma cells and effector memory CD8 cells in IBC bulk tumor samples. Preliminary data analysis of gene expression data sets from laser microdissected epithelium and stromal compartments of AA and CA breast cancer patients also show enrichment of plasma cells, effector memory CD4+, and M1 macrophages. Studies are ongoing to characterize these immune profiles that are unique and distinguishable in IBC patients and further identify race-related differences. Most importantly, we discovered that the constitutive NFκB hyperactivation observed in IBC tumor cells is driven by stress-mediated translational upregulation of the most potent anti-apoptotic protein, XIAP. Interestingly, XIAP:NFκB overexpressing cells are highly resistant to immunotherapy mediated cell death. These data strongly suggest that in IBC, host immune phenotype promotes outgrowth of cell death resistant population and specific tumor cell pro-survival signaling mechanisms that enhances invasion and lymphatic dissemination. We have developed novel in vitro IBC tumor emboli culture model and a transgenic mouse bearing red fluorescent lymphatic vasculature that allow recapitulating host factors to define tumor cell signaling mechanisms on immune effector function and the resultant invasive phenotype. Conclusions: As the incidence is rising and targets for therapy are scant, IBC has the potential to become a major public health disparity concern. This study reveals that the immune factors in the host microenvironment may interact with underlying IBC genetics to promote the aggressive nature of the tumor, and more specifically, these factors may be one of the sources of biologic variation between patients of different ethnicities leading to IBC disparity outcomes. Supported in part by Duke IBC Consortium, DCI NIH CAO14236 development funds (GRD), DoD W81XWH-13-1-0047 (GRD). Citation Format: Gayathri R. Devi, Eun-Sil Shelley Hwang, John Stewart, Michael A. Morse, Steven Van Laere. Immune pathway dysregulations in inflammatory breast cancer health disparity. [abstract]. In: Proceedings of the Ninth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2016 Sep 25-28; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(2 Suppl):Abstract nr B44.

Modeling and characterization of inflammatory breast cancer emboli grown in vitro

Inflammatory breast cancer (IBC) is the deadliest form of breast cancer, presenting as intralymphatic emboli. Emboli within the dermal lymphatic vessels are thought to contribute to rapid metastasis. The lack of appropriate in vitro models has made it difficult to accurately study how IBC emboli metastasize. To date, attempts at creating IBC tumor emboli in vitro have used 3D culture on a solid layer of Matrigel(TM) , which does not resemble the physical properties of the lymphatic system. Dermal lymphatic fluid produces oscillatory fluid shear forces and is 1.5-1.7-fold more viscous than water with a pH range of 7.5-7.7. We have established a method for forming tumor emboli by culturing the IBC cell lines in suspension with either polyethylene glycol- or hyaluronic acid-containing medium and oscillatory fluid shear forces. Non-IBC cells do not form emboli under identical conditions. In vitro IBC emboli were analyzed for expression of markers associated with patient emboli and their ability to undergo invasion. In a direct comparison, the in vitro IBC emboli closely resemble IBC patient emboli with respect to size, composition and E-cadherin expression. Further, cells from the emboli are able to invade in clusters via RhoC GTPase-dependent amoeboid movement. Invasion by clusters of IBC cells is disrupted by exposure to TGFβ. This study provides a biologically relevant in vitro model to accurately grow and study inflammatory breast cancer biology and metastasis.

Abstract P4-06-03: Zinc Finger Nuclease Genome Engineering Reveal Multiple Functions of Secretory Leukocyte Peptidase Inhibitor in Regulating Pleuripotency of Cancer Stem Cells in Inflammatory Breast Cancer

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive and lethal variant of this disease and is known to be enriched for cells with a cancer stem cell phenotype. IBC is characterized by the presence of cell aggregates, defined as tumor emboli, that metastasize into skin and chest wall. The only documented biomarker of tumor emboli is the surface glycoprotein, E-cadherin. We previously demonstrated that IBC tumor emboli express the alarm anti-protease, secretory leukocyte peptidase inhibitor (SLPI), a metastasis related gene highly expressed in IBC patient tumors. Since the function of SLPI in IBC is unknown, the present studies used zinc finger technology to knockout (KO) copies of SLPI in the SUM149 IBC cell line to define the role of SLPI in IBC. Materials and Methods: Using CompoZr zinc finger nuclease (ZFN) technology (Sigma-Aldrich), SLPI KO cell lines were generated by disrupting all alleles (3) in exon 1 of SUM149 cells derived from an IBC primary tumor with a high CD44+cell population. The target-specific ZFNs bound DNA at a sequence-specific location and created double strand breaks repaired by non-homologous end joining, resulting in deletions at the SLPI locus. Single cell SLPI KO clones were isolated and serially passaged to establish stable cell lines. Functional assays were used to assess proliferation, invasion, tube formation and clonogenicity. Global transcriptional profiling was performed to identify genes and signaling pathways directly regulated by SLPI. Results: SUM149 SLPI KO clones did not produce SLPI protein and had a significantly slower turn-over time of 75 hrs compared with 24 hrs in SUM149 wild type clones. Loss of SLPI blocked invasion by 50%, and completely inhibited formation of tube-like structures, an activity defined as vasculogenic mimicry, characteristic of IBC. The loss of only 1 SLPI allele resulted in the inability of SUM149 cells to grow as anchorage independent clones in soft agar, commonly used as a predictor of in vivo tumorigenicity. SLPI directly regulated expression of multiple genes within the embryonic stem cell pleuripotency canonical pathway, including WNT, Frizzled, PDK-1, platelet derived growth factor receptor and sphingosine-1-phosphate receptor. Studies are underway to determine the specific role of SLPI in IBC tumor growth and metastasis. Conclusions: Our previous studies demonstrated that SLPI is expressed by IBC tumor emboli and can be used as a biomarker of tumor emboli in IBC core and skin punch biopsies. SLPI was found to not only regulate critical functional activities of IBC tumor cells but also to directly regulate genes within pathways critically important to maintenance of pleuripotency of tumors with a cancer stem cell phenotype. Collectively, these studies demonstrate the power and utility of the zinc finger technology, which enables the interrogation of tumor cells to discern the direct function and role of specific genes of interest. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-03.

Abstract P6-10-04: The Presence of Anaplastic Lymphoma Kinase Recapitulates Formation of Breast Tumor Emboli with Encircling Lymphovasculogenesis

Abstract Background: Genetic abnormalities in the anaplastic lymphoma kinase (ALK) gene result in activation of signaling pathways including Akt, mTor, and JAK/Stat3. ALK has been shown to be a primary oncogenic driver in a variety of human tumors, including both hematologic malignancies such as anaplastic large cell lymphoma, as well as solid tumors including neuroblastoma, non-small cell lung cancer, myofibroblastic tumors and most recently, high grade serous ovarian carcinoma. While only ∼3% of all breast cancers have been reported to have ALK genetic abnormalities, our studies revealed that inflammatory breast cancer (IBC), the most lethal variant of breast cancer, is characterized by prevalent ALK gene amplification with activated ALK signaling. The present studies investigated the role of ALK in breast cancer by expressing full-length wild type ALK in MCF-7 cells. Materials and Methods: Clones of MCF-7 breast cancer cells expressing wild type ALK or non-target vector were produced by lentivirus infection and selection of single cell clones. MCF-7 ALK clones were evaluated using live cell and phase contrast imaging, immunofluorescent staining with confocal imaging, gene profiling, phospho-protein array analysis, western blot and ELISA validation. In vivo studies were performed by injection of MCF-7 ALK clones into using NOD. Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice using IACUC approved animal protocols. Results: When cultured on plastic substrates, MCF-7 ALK clones formed tumor cell aggregates instead of monolayer cultures, and when cultured as tumor spheroids under non-adherent 3D conditions had a distinct cellular phenotype with significantly greater clonogenicity than either non-target vector MCF-7 clones or the parental cell line. Whole transcriptome analysis, with validation using protein arrays, western blots and ELISA analysis revealed that the presence of ALK up-regulated phospho-src. In vivo studies revealed that ALK expressing MCF-7 clones formed tumor emboli that were enwrapped by dermal lymphatic vessels, essentially recapitulating the phenotype of IBC tumor emboli that exhibit encircling lymphovasculogenesis. Enforced expression of wild type ALK in another breast cancer cell line resulted in similar formation of tumor emboli. Discussion: These studies provide first time evidence for the association between full length ALK and formation of highly invasive tumor emboli enwrapped by lymphatic vessels, which is a primary characteristic of IBC. These studies, taken together with discovery of the prevalence of ALK gene amplification in IBC patients, indicate that ALK represents an important therapeutic target for IBC, with the availability of new ALK targeted therapies to evaluate as single agents and in combinations with other agents that may effectively target IBC tumor emboli that we have now linked to ALK and which represent the metastatic lesion of this lethal variant of breast cancer. Funding by Susan G. Komen Organization Promise Grant KG081287 (FMR and MC). Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-10-04.

Inflammatory Breast Cancer Stem Cells: Contributors to Aggressiveness, Metastatic Spread and Dormancy

Cancer stem cell populations have been identified for several types of cancers and suggest a way for tumor cells to be resistant to therapies. Further, because of the longevity, endurance and replicative potential of cancer cells with stem-like properties, other malignant attributes such as recurrence after long periods of dormancy can also be explained. Inflammatory breast cancer (IBC) is a unique and aggressive form of breast cancer that has a clinical course unlike other forms of breast cancer. The main hallmark of IBC is prolific invasion of the dermal lymphatic vessels by tumor emboli leading to rapid metastasis of the disease. Despite an extremely aggressive treatment approach, the majority of women with IBC present with disease recurrence suggesting the presence of chemo resistant and/or dormant breast cancer cells. Current evidence suggests that IBC tumor emboli contain distinct populations of cells with stem cell-like properties. Thus, specific targeting of these stem cell-like cancer cells may be the key to effectively treating IBC.

P4-09-08: Secretory Leukocyte Protease Inhibitor as a Differential Diagnostic Biomarker of Tumor Emboli in Inflammatory Breast Cancer.

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive and lethal variant of locally advanced breast cancer. Pathologically, IBC is characterized by the presence of nests of cells, defined as tumor emboli, that undergo lymphovascular invasion into the skin and chest wall and represent the metastatic lesion of IBC. Due to the distinct presentation of IBC, it is often misdiagnosed due to a lack of defined biomarkers that are specific for tumor emboli in biopsy samples of IBC patients. To date, the only validated marker that has shown to consistently identify IBC tumor emboli is the surface glycoprotein E-cadherin. Materials and Methods: With the goal of identifying genes specifically expressed by IBC tumor emboli, the present studies performed whole transcriptome analysis of tumor emboli from skin punch biopsy samples from IBC patients and tumor emboli from the Mary-X pre-clinical model of IBC which were isolated using laser capture microdissection. Results: Secretory leukocyte peptidase inhibitor (SLPI) was a gene identified as being highly expressed by tumor emboli. This gene was previously identified as a metastasis related gene, has been reported to be associated with poor prognosis in serous ovarian carcinoma and is implicated in mediating resistance to paclitaxel. Immunofluorescence staining and confocal microscopy revealed that SLPI is a robust marker of tumor emboli in skin punch biopsies from IBC patient and in Mary-X tumor emboli. SLPI differentially delineates between IBC tumor emboli and hair follicles present in the skin, which also stain with E-cadherin. An additional marker, Cytokeratin 15 (CK15), can be used in combination with SLPI to differentially identify the components of the skin that are CK15 positive, and IBC tumor emboli that reside within the skin, which are CK15 negative. Use of podoplanin antibodies, which stain lymphovascular endothelium, demonstrate that IBC tumor emboli that express SLPI protein, are completely encircled within the dermal lymphovasculature. Conclusions: These studies are among the first to identify SLPI as a potential biomarker of tumor emboli in skin punch biopsy samples from IBC patients and in pre-clinical models of IBC. Studies are ongoing to establish the molecular function and role of SLPI in mediating the survival and invasion of IBC tumor emboli. Collectively, these studies are the first to identify SLPI as a potential biomarker of IBC tumor emboli in skin punch biopsy samples. In addition to serving as a potential biomarker of tumor emboli, SLPI may also serve as a therapeutic target for development of approaches to eradicate IBC tumor emboli. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-09-08.

P2-05-04: Mapping the Specific Gene Families Activated in the Lymphangiogenesis and Vasculogenic Mimicry Exhibited by Inflammatory Breast Cancer.

Abstract Background: Inflammatory breast cancer (IBC) is the most metastatic variant of locally advanced breast cancer. Although IBC is diagnosed less commonly than other types of breast cancer, it is extremely aggressive, and accounts for a disproportionate number of breast cancer related deaths annually. IBC exhibits very specific patterns of lymphangiogenesis and vasculogenic mimicry, however detailed studies of the genes and proteins involved in these angiogenic processes are lacking. This study performed whole unbiased gene transcription studies with validation by protein arrays using all available pre-clinical cell lines and in vivo xenograft models of IBC, including a new model of IBC, FC-IBC01, which exhibits lymphovascular invasion, to identify the specific pathways involved in the distinctive angiogenesis observed in IBC. Materials and Methods: Real-time quantitative RT-PCR, cDNA microarray gene profiling, immunofluorescence with confocal imaging and protein arrays were used to examine differential expression of specific angiogenic gene families including VEGFA,B,C,D, VEGF Receptor genes, and ANG/TIE genes linked to angiogenesis and lymphangiogenesis. Results: Activity of the matrix metalloproteinase, MMP-2, is required for IBC tumor cells to undergo vasculogenic mimicry (VM), which is associated with a loss of TIMP-2, a well known inhibitor of angiogenesis. Therapeutics that target MMP activity can successfully inhibit this VM. Furthermore, pre-clinical models of IBC that form IBC tumor emboli exhibit lymphovascular invasion that is associated with distinct patterns of expression of genes that encode for distinct receptor tyrosine kinases that may represent important therapeutic targets for IBC. Discussion: Identification of the distinct angiogenic pathways that are activated in IBC provides insight into the therapeutic targets that may abrogate the distinct lymphovascular invasion and vasculogenic mimicry that are linked to the aggressive metastasis of IBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-05-04.

P1-02-03: The Reciprocal Roles of E-Cadherin and ZEB1 Demonstrate the Mesenchymal-Epithelial Transition as a Primary Characteristic of Inflammatory Breast Cancer.

Abstract Background: Inflammatory breast cancer (IBC) is a rare but very aggressive form of breast cancer. IBC is characterized by nests of tightly aggregated cells, defined as tumor emboli, that exhibit characteristics of cancer stem cells (CSCs). IBC tumor emboli express E-cadherin which is required to maintain their integrity and our recent evidence demonstrates that expression of E-cadherin by tumor emboli is associated with lack of ZEB1 expression, a transcriptional repressor of E-cadherin. This is at odds with the current hypothesis that metastatic progression is associated with the process of epithelial mesenchymal transition (EMT), with loss of E-cadherin and gain of transcription factors including ZEB1, acquisition of CSC characteristics and enhanced invasive capabilities. Materials and Methods: shRNA knockdown and over-expression methods, real time PCR arrays, western blotting, and in vitro assays to evaluate proliferation, invasion, growth in soft agar and clonogenicity and in vivo animal studies were used. Results: Expression of E-cadherin was reduced by shRNA and ZEB1 was expressed in SUM149 IBC tumor cells. Numerous EMT-related genes were upregulated with loss of E-cadherin and gain of ZEB1, including N-cadherin and vimentin. However, there were marginal differences in the in vitro parameters of proliferation, Matrigel invasion and anchorage independent growth in soft agar between SUM149-shECad or SUM149-ZEB1 clones and their respective vector control cells. The loss of E-cadherin and gain of ZEB1 altered the morphology of SUM149 cells when cultured under low adherence conditions permissive for the enrichment of CSC, exhibiting a reversion in grape-like morphology to more well defined spheres, which was accompanied by increased clonogenicity in both SUM149-shECad and SUM149-ZEB1 cells. The loss of E-cadherin and the gain of ZEB1 significantly inhibited tumor growth of cells injected in the mammary fat pad of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Tumor volume at 56 days for E-cadherin vector control cells was 771.9 mm3 +/− 185.6 compared to shECadherin tumors, which was 13.6 mm3 +/− 7.2. Tumor volume of ZEB1 vector control tumors was 346.1 mm3 +/− 96 compared to volume of ZEB1 expressing tumors, which was 21.5 mm3 +/− 7.2.Conclusions: E-cadherin with lack of ZEB1 expression in IBC is consistent with a mesenchymal-epithelial transition (MET), consistent with the retention of the epithelial phenotype while maintaining a program of rapid metastasis and colonization of lymph nodes and distant organ sites. Furthermore, we demonstrate that the E-cadherin-ZEB1 axis is critical for the in vivo growth of IBC tumor cells. Although SUM149 cells are fully capable of undergoing an EMT process, which is under negative regulation by E-cadherin, the process of EMT does not drive in vivo tumor growth in IBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-03.

Suberoylanilide hydroxamic acid blocks self‐renewal and homotypic aggregation of inflammatory breast cancer spheroids

Inflammatory breast cancer (IBC) is the most aggressive form of locally advanced breast cancer (LABC). Patients with IBC commonly present with skin metastasis, which are observed microscopically as tumor emboli within dermal lymphatics. These metastatic tumor cells aberrantly overexpress E-cadherin and exhibit the ability to undergo self-renewal and are highly invasive. There are no therapeutics yet identified that target the structure and functions of IBC tumor emboli. The present studies evaluated the effects of the pan-histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) using IBC tumor spheroids derived from established IBC cell lines and tumor spheroids derived from pleural effusion (PE) aspirates of patients with IBC and LABC, designated as PE-IBC and PE-LABC. Methods used are as follows: culture of IBC cells from clonal density single cells in low adherence culture conditions that promote formation of IBC tumor spheroids; clonogenic assays; cell fractionation and Western blotting; confocal microscropy; and modified Boyden chamber invasion assays. SAHA inhibited self-renewal of IBC tumor spheroids from established IBC cell lines and PE-IBC and PE-LABC, as assessed by decreased clonogenic growth. SAHA blocked homotypic aggregation of the cells that comprised the IBC tumor spheroids leading to loss of their 3-dimensional (3D) structure, which was associated with a change in location of E-cadherin protein from the plasma membrane in untreated IBC tumor spheroids to the cytoplasm of cells within IBC tumor spheroids with SAHA treatment. In addition, SAHA blocked the robust invasion exhibited by IBC tumor spheroids of established cell lines as well as by tumor spheroids derived from PE-IBC and PE-LABC. SAHA targets the integrity and biological activities of IBC tumor spheroids and may be a promising agent to evaluate for its effectiveness in treatment of IBC.