Early Stages Of Metastasis Research Articles

Abstract P3-01-01: A dual role of natural killer cells in breast cancer metastasis

Abstract BACKGROUND: Metastatic disease drives breast cancer mortality. We recently discovered that leading cells at the invasive edge of mammary tumor organoids retain a conserved basal epithelial program defined by their expression of keratin-14 (K14), establishing K14 as a good marker of invasive breast cancer cells. K14-positive invasive cells also exhibit characteristics that make them targets of immunosurveillance by natural killer (NK) cells. While NK cells are key immune mediators in the control of metastasis, our understanding of the specific mechanisms behind this regulation and its eventual evasion by metastatic cells remains incomplete. METHODS: We have developed a novel preclinical 3D co-culture assay to discover mechanisms behind interactions between K14+ invasive breast cancer cells and NK cells. Combined with in vivo assays of metastasis, we are able to determine how NK cells limit the early stages of metastasis and also how tumor cells can influence key NK cell properties. RESULTS: In ex vivo co-culture assays of NK cells isolated from healthy mouse donors and mammary tumor organoids from MMTV-PyMT and C31T mouse models of breast cancer, we demonstrate that NK cells limit the early stages of metastasis, namely invasion and colony formation. Antibodies to invasive K14+ cells were able to enhance the ability of NK cells to limit colony formation, suggesting antibody-dependent cell mediated cytotoxicity. Surprisingly, when isolated from tumor bearing mice, NK cells did not limit invasion and instead promoted colony formation. The in vivo adoptive transfer of NK cells from healthy donors prevents the progression of early lung metastatic seeds to macrometastases, while the adoptive transfer of cells isolated from tumor bearing donors promotes macrometastatic development. This growth promoting phenotype can be neutralized with antibodies targeting inhibitory cell surface receptors. CONCLUSIONS: Our ex vivo and in vivo data demonstrate that healthy donor NK cells can limit metastasis through the directed cytotoxicity against pioneering K14+ invasive cells. However, prolonged exposure to tumors reprogram NK cells from tumor killing to tumor promoting, specifically in promoting the outgrowth of macrometastases. Further, we can neutralize this effect using NK cell specific inhibitory antibodies. This is the first time inhibitory signaling on NK cells have been linked with a growth promoting phenotype. These data can provide insight into when the use of NK cell directed therapies can be used to treat or prevent clinically relevant metastatic disease. Citation Format: Isaac S. Chan, Hildur Knútsdoóttir, Gayathri Ramakrishnan, Veena Padmanaban, Joel S. Bader, Elizabeth M. Jaffee, Andrew J. Ewald. A dual role of natural killer cells in breast cancer metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-01.

Effect of Varying Mechanical Environment in 2D Culture on Subsequent Metastasis Process of Ovarian Cancer

The epithelial ovarian cancer metastasizes through a unique transcoelomic routes, wherein cancer cells crumble out to body cavity, survive in an anchorage-independent manner as spheroids, and then finally stick to the outermost layer of another organ to develop a secondary tumor. The later stage mechanisms have already been elucidated in vitro and in vivo recently. Comparatively, only few clues exist to explain what happens during the early stage of metastasis. Recent studies reported marked heterogeneities in junctional proteins and cytoskeleton composition even within a single spheroid. Also, some of these intra-tumor heterogeneities are known to be sustained throughout the whole metastasis steps. Since the intra-tumor heterogeneity arises at that stage, herein, we focused on varying the mechanical environment of early-stage to induce cancer cells to have different cytoskeletal composition and so on. We cultured a non-serous high-grade ovarian cancer cell line, SK-OV-3, on polyacrylamide gels of different stiffness to establish two groups of distinct phenotypes. The difference between these two population of cells was characterized by immunostaining and migratory phenotype. To mimic the floating situation in the body cavity, we adopted the hanging drop method to generate the cellular spheroids. Afterward, the cells cultured with different stiffness were made into cellular spheroids, together or separately. The resultant spheroids were also tested with the adhesiveness and spreading ability on a physiologically relevant environment. Through these experimental setting, we examined whether different mechanical microenvironmental conditions in 2D culture, mimicking the heterogeneous conditions of the primary tumor, affect the subsequent spheroidal compaction and dissemination.

Boron nitride nanotube-CREKA peptide as an effective target system to metastatic breast cancer

The development of nanomaterials that are capable of recognizing disease-specific biomarkers with high sensitivity and specificity is related to several advances in the field of nanomedicine. Furthermore, the targeted delivery of anticancer agents to tumor tissues enhances their efficiency and reduces their toxic side effects. Boron nitride nanotubes (BNNTs) are nanostructured materials, analog to carbon nanotubes, which present good biocompatibility and morphology suitable for tumor cell internalization. CREKA is a pentapeptide that has a high affinity to fibrin, a protein found in the new tumor vessels in the early stages of metastasis and in thrombosis regions. In this study BNNTs were chemically functionalized with the peptide CREKA, and this system was characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), zeta potential, scanning electron microscopy, and transmission electron microscopy. After the mentioned chemical steps, the FTIR analysis shows an organic phase related to the CREKA, TGA indicates that about 10% of the peptide is firmly attached to BNNT surface. In addition, the radiolabeling process was successful, achieving the purity required for the biodistribution study. In vivo experiments showed that a considerable amount of BNNT-CREKA was accumulated at the tumor and metastasis sites. The present results indicate an effective targeting of the system to tumor and metastasis sites. Further studies can reveal potential applications of functionalized BNNTs in cancer treatment.

Analysis of tumor vascularization in a mouse model of metastatic lung cancer

Therapies targeting tumor vasculature would improve the treatment of lung metastasis, although the early changes in vascular structure are incompletely understood. Here, we show that obstructive metastatic foci in lung arterioles decrease the pulmonary vascular network. To generate a mouse model of lung metastasis activation, luciferase-expressing tumor cells were inoculated into the subiliac lymph node (SiLN) of an MXH10/Mo-lpr/lpr mouse, and metastatic tumor cells in the lungs were activated by SiLN resection. Activation of metastases was monitored by in vivo bioluminescence imaging. Pulmonary blood vessel characteristics were analyzed using ex vivo micro-computed tomography. The enhanced permeability and retention (EPR) effect in neovasculature after tumor cell activation was evaluated from the accumulation of intravenously injected indocyanine green (ICG) liposomes. Metastatic foci in lung arterioles were investigated histologically. Micro-computed tomography revealed decreases in pulmonary blood vessel length, volume and number of branching nodes during the early stage of metastasis caused by metastatic foci. ICG liposome accumulation by the EPR effect was not detected. Histology identified metastatic foci in lung arterioles. The lack of an EPR effect after the formation of metastatic foci in lung arterioles makes conventional systemic chemotherapy ineffective for lung metastasis. Thus, alternative therapeutic methods of drug delivery are needed.

Predictors of non-sentinel lymph node metastasis in clinical early stage (cT1-2N0) breast cancer patients with 1-2 metastatic sentinel lymph nodes

The purpose of this study was to determine the risk factors that caused non-sentinel lymph nodes (nonSLNs) metastasis by considering the clinicopathological characteristics of patients who have 1-2 sentinel lymph node (SLN) metastasis in the clinical early stage (T1-2, N0) breast cancer. The demographic and clinicopathological characteristics of the patients were recorded retrospectively. Among these, age, size of the primary breast tumor, tumor localization and multifocality/multicentricity status, preoperative serum Neutrophil/Lymphocyte rate (NLR), c-erbB2/HER2-neu status, Estrogen Receptor (ER) and Progesterone Receptor (PR) status, primary tumor proliferation index (Ki-67), histopathological grade, molecular subtypes, histopathological subtypes, nipple/areola infiltration, Lymphatic Invasion (LI), Vascular Invasion (VI), Perineural Invasion (PNI), number of metastatic SLN m(SLN), mSLN diameter, SLN Extranodal Extension (ENE) status, and number of metastatic nonSLNs were recorded. According to the univariate analysis, the HER2 positivity, Ki-67≥%20, mSLN diameter, LI, VI, PNI, ENE and molecular subtypes were found to be significant. However, the age, tumor localization, multifocality/multicentricity, T stage, ER and PR status, tumor size, histopathological grade and subtypes, nipple/areola infiltration and NLR were not found to be significant. In the multivariate analysis, significant independent predictors in nonSLN metastasis development were found to be HER2 positivity, PNI, mSLN diameter ≥10,5mm and ENE. The HER2 positivity, ENE, PNI and mSLN diameter ≥10,5 mm were found to be very strong predictors in nonSLN metastasis development. The findings of this study have the potential to be a guideline for surgeons and oncologists when determining their patients' treatment plan. These components are candidates for inclusion among the clinicopathological factors that may be used in the new nomograms due to their higher sensitivity and specificity.

Abstract 1949: Mouse model of metastatic colorectal cancer by orthotopic transplantation of patient derived organoids

Abstract Cancer metastasis proceeds through multiple steps and metastatic colonization is formed by disseminated tumor cells with proliferative capacity in distant tissues. In the analysis of metastasis, macroscopically completed lesions are often used for subjects, which is attributed to the difficulty to capture the early process occurring in living body and the analysis using human specimen is particularly limited. To capture the metastatic process of colorectal cancer (CRC) and to identify cell population involved in it, we produced the mouse model for CRC by orthotopic transplantation of patient derived organoids (PDOs). Unlike ectopic transplantation models, this model precisely recapitulates the metastasis process in human patients. To evaluate the metastasis potentials, the patient matched organoids from the primary, the synchronous liver metastasis and the post-chemotherapy liver recurrence lesion were established. No significant difference in macroscopic morphology or pathogenic mutations were noted among the organoids. The organoids were marked by fluorescent protein and luciferase fusion gene to be visualized the process over time by in vivo imaging system and injected under rectal mucosa on prolapse. Each graft reflected histopathological feature of the surgical specimen. At the results of observation, the lung metastases were frequently occurred in recurrence organoids mouse models, but little occurred in primary or metastasis ones. The lung metastases were considered to be in the early stage of metastasis, because they spread diffusely in each lung lobes, and could not be confirmed macroscopically. They were forming two state; one appeared to be the embolism in blood vessels around the bronchus and others were the micro-metastases in lung tissue. The orthotropic transplantation of patient-matched PDOs provides unique opportunities to explore the metastasis process of CRC. Citation Format: Takuya Okamoto, Katsuyuki Yaginuma, Satoshi Nagayama, Ryoji Yao. Mouse model of metastatic colorectal cancer by orthotopic transplantation of patient derived organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1949.

Abstract 2649: A novel approach to fate-map hypoxic cells during tumor progression uncovers metastatic potency of post-hypoxic cells

Abstract Hypoxia occurs in 90% of solid tumors due to inefficient vasculature and has been demonstrated to promote a more aggressive phenotype, correlating with worse patient prognosis. Current methods to measure hypoxia in human tumors only identify cells that are hypoxic at the time the tissue is collected and do not delineate cells that were transiently exposed to hypoxia but survived and adapted. To overcome this challenge, we generated a Lox-Cre dual-vector oxygen-dependent reporter system. Under hypoxia, Cre expression leads to the cleavage of a DsRed reporter and permanent expression of a GFP reporter. We stably transduced breast cancer cell lines and demonstrated an irreversible reporter-switch that occurs when cells are exposed to 0.5% O2, the typical O2 level in human breast tumors, but does not occur under physiological conditions (8% O2). To explore the utility of our system, we embedded spheroids derived from MDA-MB-231 reporter cells in collagen and cultured them under 20% O2 conditions. A defined hypoxic core (GFP+) was visible after 15 days in culture and it correlated with hypoxic O2 measurements. We then generated orthotopic breast tumors by transplanting MDA-MB-231 reporter cells into the mammary fat pad of mice. Hypoxic cells (GFP+) were localized in the peri-necrotic region of the tumor and co-localized with hypoxyprobe and HIF1α labeling. RNA sequencing of GFP+ compared to DsRed+ tumor cells demonstrated that GFP+ cells were enriched for the hallmark signature of hypoxia. Additionally, we developed a transgenic mouse expressing the hypoxia-driven construct that was bred to a tdTomato-floxed GFP mouse. A female double fluorescent/hypoxia-reporter mouse was then bred to a male FVB/N-Tg(MMTV-PyMT) mouse in order to generate triple-transgenic mice that form breast tumors that spontaneously metastasize. We systematically studied hypoxia in spontaneous breast tumors from the development of ductal hyperplasia to late stage invasive carcinoma. Tumor hypoxia was first detected in early stage ductal carcinoma in situ (DCIS) lesions. Similarly to the spheroid model, mammary organoids derived from triple-transgenic mice tumors cultured under 20% O2 conditions also exhibited a defined hypoxic core after 15 days in culture. Both orthotopic and transgenic mouse models revealed an increased potency of post-hypoxic cells at arriving to distant sites in early stage metastasis. We utilized RNA sequencing to identify potential mechanisms of enhanced metastatic capacity. Ultimately, our system allowed the fate-map of hypoxic cells during tumor progression to metastasis. We show for the first time that 3D in vitro organoids derived from PyMT tumors develop regions of hypoxia under normal tissue culture conditions. In vivo, we show that hypoxic cells are more potent at arriving at distant sites and that they retain a different gene expression that potentially confers a metastatic advantage. Citation Format: Inês Godet, Yu Jung Shin, Julia A. Ju, Soumitra Bhoyar, I Chae Ye, Guannan Wang, Saraswati Sukumar, Daniele M. Gilkes. A novel approach to fate-map hypoxic cells during tumor progression uncovers metastatic potency of post-hypoxic cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2649.

Abstract 1112: CCDC102B promotes metastatic cascade in breast cancer by activating NF-κB via down-regulating RACK1

Background: Breast cancer is one of the leading causes of cancer death among women and the pathological status of axillary lymph nodes is an important predictor of prognosis. However, the mechanism involved in this early stage of metastasis remains largely unknown. Methods: We used microarray analysis to carry out differential genomics analyses between matched pairs of sentinel lymph node metastatic tissues and breast primary tumors. We found 182 up-regulated genes in metastatic tissues (fold change≥1.5). We used CRISPR/Cas9 gene editing system to knockout these genes, each with 6 unique sgRNAs, and screened in vivo by transplanting loss-of-function cell pool into immunocompromised mice. MAGeCK was used to analyze the screening. Immunohistochemistry was performed to examine protein expressions in 212 cases of breast cancer tissues using tissue microarray (TMA) technology. Gene and protein expressions were monitored by qPCR and western blot. The relationship between coiled-coil domain containing 102B (CCDC102B) and receptor for activated C kinase 1 (RACK1) was examined using Co-IP, mass spectrometry, nuclear protein extraction assay and dual-luciferase reporter assay. Functional studies were detected in vitro by cell proliferation, wound-healing and migration, and in vivo by lung metastasis in immunocompromised mice injected CCDC102B-overexpressed cells into tail vein. The primary biological functions and pathways related to CCDC102B were enriched by RNA sequencing. The regulation of NF-κB signaling by CCDC102B and RACK1 was detected using dual-luciferase reporter assay and western blot. Results: We identified CCDC102B through screening and found that it was significantly up-regulated in breast cancer patients with poor prognosis. The expression of CCDC102B were independent predictors in both DFS and OS ( P =0.009; P =0.014). CCDC102B expression was found to promote the proliferation and migration of breast cancer cells MDA-MB-231 and BT549, and lung metastasis in mice. Moreover, we found that overexpression of CCDC102B decreased RACK1 expression whereas knockdown of CCDC102B increased RACK1 expression at both protein and mRNA levels, and the aggressive behavior of CCDC102B in breast cancer cells could be reversed by the expression of RACK1. According to gene set enrichment analysis (GSEA), CCDC102B was correlated with significant enrichment focused on NF-κB pathway. The mechanistic study revealed that gain of CCDC102B resulted in decreased expression of RACK1, which increased levels of epithelial mesenchymal transition (EMT) and phosphorylated Rel-A (p65), leading to the activation of NF-κB pathway and progression of breast cancer metastasis. Conclusion: Taken together, our findings suggest that CCDC102B is strongly associated with negative clinical outcomes and may serve as a potential metastasis promotor by regulating the activation of NF-κB pathway via RACK1. Citation Format: Jing Si, Rong Guo. CCDC102B promotes metastatic cascade in breast cancer by activating NF-κB via down-regulating RACK1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1112.

Abstract 1112: CCDC102B promotes metastatic cascade in breast cancer by activating NF-κB via down-regulating RACK1

Abstract Background: Breast cancer is one of the leading causes of cancer death among women and the pathological status of axillary lymph nodes is an important predictor of prognosis. However, the mechanism involved in this early stage of metastasis remains largely unknown. Methods: We used microarray analysis to carry out differential genomics analyses between matched pairs of sentinel lymph node metastatic tissues and breast primary tumors. We found 182 up-regulated genes in metastatic tissues (fold change≥1.5). We used CRISPR/Cas9 gene editing system to knockout these genes, each with 6 unique sgRNAs, and screened in vivo by transplanting loss-of-function cell pool into immunocompromised mice. MAGeCK was used to analyze the screening. Immunohistochemistry was performed to examine protein expressions in 212 cases of breast cancer tissues using tissue microarray (TMA) technology. Gene and protein expressions were monitored by qPCR and western blot. The relationship between coiled-coil domain containing 102B (CCDC102B) and receptor for activated C kinase 1 (RACK1) was examined using Co-IP, mass spectrometry, nuclear protein extraction assay and dual-luciferase reporter assay. Functional studies were detected in vitro by cell proliferation, wound-healing and migration, and in vivo by lung metastasis in immunocompromised mice injected CCDC102B-overexpressed cells into tail vein. The primary biological functions and pathways related to CCDC102B were enriched by RNA sequencing. The regulation of NF-κB signaling by CCDC102B and RACK1 was detected using dual-luciferase reporter assay and western blot. Results: We identified CCDC102B through screening and found that it was significantly up-regulated in breast cancer patients with poor prognosis. The expression of CCDC102B were independent predictors in both DFS and OS (P=0.009; P=0.014). CCDC102B expression was found to promote the proliferation and migration of breast cancer cells MDA-MB-231 and BT549, and lung metastasis in mice. Moreover, we found that overexpression of CCDC102B decreased RACK1 expression whereas knockdown of CCDC102B increased RACK1 expression at both protein and mRNA levels, and the aggressive behavior of CCDC102B in breast cancer cells could be reversed by the expression of RACK1. According to gene set enrichment analysis (GSEA), CCDC102B was correlated with significant enrichment focused on NF-κB pathway. The mechanistic study revealed that gain of CCDC102B resulted in decreased expression of RACK1, which increased levels of epithelial mesenchymal transition (EMT) and phosphorylated Rel-A (p65), leading to the activation of NF-κB pathway and progression of breast cancer metastasis. Conclusion: Taken together, our findings suggest that CCDC102B is strongly associated with negative clinical outcomes and may serve as a potential metastasis promotor by regulating the activation of NF-κB pathway via RACK1. Citation Format: Jing Si, Rong Guo. CCDC102B promotes metastatic cascade in breast cancer by activating NF-κB via down-regulating RACK1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1112.

ILF2 promotes anchorage independence through direct regulation of PTEN.

Anoikis is a specific form of programmed cell death induced by loss of contact between cells and extracellular matrices or other cells. Only tumor cells that are resistant to anoikis can survive in the state of detachment from the primary tissue during the early stages of metastasis. The ability to resist anoikis is crucial for cancer cell metastasis. ILF2 is a proto-oncogene previously studied in glioma, NSCLC, esophageal cancer and pancreatic ductal carcinoma. The results from the present study revealed that the transcription factor interleukin enhancer-binding factor 2 (ILF2) was highly expressed in non-small cell lung cancer (NSCLC) cell lines compared with in normal cell lines. ChIP and luciferase reporter gene assays demonstrated that ILF2 inhibited the expression level of the tumor suppressor gene phosphatase and tensin homolog (PTEN) by directly binding to its upstream regulatory region. Furthermore, the results from the detection of cell adhesion and apoptosis in cell suspension culture demonstrated that this mechanism enabled NSCLC cells to reduce adherence to the matrix and to survive in this abnormal state. These results suggested that ILF2 may promote the anchorage-independence of NSCLC cells through the suppression of PTEN.

Detection of circulating cancer stem cells (cCSCs) as a predictive biomarker for breast cancer relapse or metastasis.

e14559 Background: Breast cancer is one of the most common types of cancer in women worldwide. It has been demonstrated that even localized tumors without clinically apparent metastases give rise to circulating epithelial tumor cells (CETCs). Recent studies have provided strong support for the cCSC hypothesis, which suggests that a more aggressive subpopulation of circulating tumor cells is the source of metastatic spread from the primary tumor. Measuring CETCs in blood of patients has emerged as a non-invasive diagnostic procedure for screening patients who may be at high risk for developing metastatic cancers. However, accurate detection of CETCs may provide erroneous results, since CETCs undergoing EMT during metastasis are down-regulated for the expression of epithelial cell markers. Methods: 26 breast cancer patients were included into the study. The determination of CETCs and cCSC was performed using maintrac method and tumorsphere forming assay, respectively. Cell viability, surface marker expression and ALDH 1 activity of the cells in the spheres were evaluated by fluorescence scanning microscope. Results: Sphere formation was observed in 80 % of patients. We found that the number of tumorspheres depended on stage of disease. The number of tumorspheres increased significantly with tumor progression, especially with the presence of metastases. Tumorsphere formation was observed in all metastatic patients (median of 30 tumorspheres/100µl of blood), although only 27% of them had detectable CETCs. Analysis of surface marker expression profile showed that the cells in the spheres had typical phenotype of cancer stem cells. Sphere formation was not observed in healthy subjects (n = 20). Conclusions: There is a high correlation between the numbers of tumorspheres cultured from peripheral blood with clinical stage of disease. Identifying cCSC in blood sample can help clinicians to monitor breast cancer patients and to detect metastasis in early stages.

Cancer cells in the tumor core exhibit spatially coordinated migration patterns.

In the early stages of metastasis, cancer cells exit the primary tumor and enter the vasculature. Although most studies have focused on the tumor invasive front, cancer cells from the tumor core can also potentially metastasize. To address cell motility in the tumor core, we imaged tumor explants from spontaneously forming tumors in mice in real time using long-term two-photon microscopy. Cancer cells in the tumor core are remarkably dynamic and exhibit correlated migration patterns, giving rise to local 'currents' and large-scale tissue dynamics. Although cells exhibit stop-and-start migration with intermittent pauses, pausing does not appear to be required during division. Use of pharmacological inhibitors indicates that migration patterns in tumors are actively driven by the actin cytoskeleton. Under these conditions, we also observed a relationship between migration speed and correlation length, suggesting that cells in tumors are near a jamming transition. Our study provides new insight into the dynamics of cancer cells in the tumor core, opening new avenues of research in understanding the migratory properties of cancer cells and later metastasis.This article has an associated First Person interview with the first author of the paper.

Regulation of miRNAs by Snail during epithelial-to-mesenchymal transition in HT29 colon cancer cells

Epithelial-to-mesenchymal transition (EMT) in cancer cells, represents early stages of metastasis and is a promising target in colorectal cancer (CRC) therapy. There have been many attempts to identify markers and key pathways induced throughout EMT but the process is complex and depends on the cancer type and tumour microenvironment. Here we used the colon cancer cell line HT29, which stably overexpressed Snail, the key transcription factor in early EMT, as a model for colorectal adenocarcinoma cells with a pro-metastatic phenotype. We investigated miRNA expression regulation during that phenotypic switching. We found that overexpression of Snail in HT29 cells triggered significant changes in individual miRNA levels but did not change the global efficiency of miRNA processing. Snail abundance repressed the expression of miR-192 and miR-194 and increased miR-205, let-7i and SNORD13 levels. These identified changes correlated with the reported transcriptomic alterations in Snail-overexpressing HT29 cells. We also investigated how Snail affected the miRNA content of extracellular vesicles (EVs) released from HT29 cells. Our data suggest that the presence of Snail significantly alters the complex mRNA/miRNA interactions in the early steps of metastasis and also has an impact on the content of EVs released from HT29 cells.

Can Neutrophil to Lymphocyte Ratio Be a Predictor Tool for The Non-Sentinel Lymph Node Metastasis in Breast Cancer?

Background: To investigate the relationship between neutrophil to lymphocyte ratio (NLR) and non-sentinel lymph node (SLN) metastasis in early stages of breast cancer. Methods: Records of patients with clinically early stage (cT1-T3, N0) invasive breast cancer who underwent SLN biopsy in combination with blue dye and lymphoscintigraphy between April 2015 and January 2017 were noted retrospectively. Pre-treatment NLR of the patients with and without non-SLN metastasis were compared. Results: A total of 178 patients with a mean age of 51,65+-10,93 years were included. NLR was statistically significant in the non-SLN positive patients (p=0.011). Lymphovascular invasion and extracapsular extension of SLN were more common in the non-SLN positive group (all p 0.05). The ODDS value was found to be 3,733 (95% CI: 1,05-13,24) and the risk of metastasis in those with lymphovascular invasion was 3.73 fold. The ODDS value was found to be 10,267 (95% CI: 2,63- 40,12) and the risk of metastasis was 10,267 times greater in those with capsule spread. Conclusion: Lymphovascular invasion, extracapsular extension and NLR are the predictive parameters of non-SLN positivity. Pre-surgical NLR is a useful diagnostic tool for predicting non-SLN metastasis.

Alterations in arginine and energy metabolism, structural and signalling lipids in metastatic breast cancer in mice detected in plasma by targeted metabolomics and lipidomics

BackgroundThe early detection of metastasis based on biomarkers in plasma may improve cancer prognosis and guide treatment. The aim of this work was to characterize alterations in metabolites of the arginine pathway, energy metabolism, and structural and signalling lipids in plasma in the early and late stages of murine breast cancer metastasis.MethodsMice were orthotopically inoculated with 4T1 metastatic breast cancer cells, and plasma was analysed along the pulmonary metastasis progression using LC-MS/MS-based targeted metabolomics and lipidomics.ResultsBased on primary tumour growth and pulmonary metastases, 1–2 weeks after 4T1 cancer cell inoculation was defined as an early metastatic stage, and 3–4 weeks after 4T1 cancer cell inoculation was defined as a late metastatic stage. Early metastasis was featured in plasma by a shift of L-arginine metabolism towards arginase (increased ornithine/arginine ratio) and polyamine synthesis (increased putrescine). Late metastasis was reflected in plasma by further progression of changes in the arginine pathway with an additional increase in asymmetric dimethylarginine plasma concentration, as well as by a profound energy metabolism reprogramming towards glycolysis, an accelerated pentose phosphate pathway and a concomitant decrease in tricarboxylic cycle rate (“Warburg effect”). The late but not the early phase of metastasis was also characterized by a different lipid profile pattern in plasma, including a decrease in total phosphatidylcholines, a decrease in diester-bound phospholipid fraction and an increase in lysophospholipids associated with an increase in total sphingomyelins.ConclusionsThe early phase of metastasis in murine 4T1 metastatic breast cancer was associated with plasma metabolome changes characteristic of arginase activation and polyamine synthesis. The late metastasis was reflected in plasma not only by the alterations in arginine pathways but also by a shift towards glycolysis and the pentose pathway, remodelling of structural lipids and activation of lipid signalling, all of which coincided with metastasis progression.

Exploring protocol for breast cancer xenograft model using endothelial colony-forming cells

Background: Triple-negative breast cancer (TNBC) has poor prognosis because it tends to be metastatic and invasive. To improve TNBC therapeutic strategies, a xenograft model mimicking the early stage of metastasis is needed. Angiogenesis plays an important role in tumor progression, including tumor growth and metastasis. In Angiogenesis is a key requirement in TNBC xenograft models, thus, the cells forming blood vessels are needed. Circulating endothelial progenitors and endothelial colony-forming cells (ECFCs) have a high proliferative capacity and form new blood vessels by differentiating into the endothelium. In this study, a xenograft model was established for the evaluation of tumor progression based on the orthotopic implantation of MDA-MB-231 cells with the ECFCs. Methods: The human breast cancer MDA-MB-231 cells with or without ECFCs were transplanted into fat pads of female Balb/c-nude mice using two protocols that differed only in terms of the number of MDA-MB-231 cells implanted. Then, two protocols were evaluated using tumorigenicity, tumor growth, and tumor weight as parameters of tumor progression. Furthermore, the existence of ECFCs and their role were studied in the tumor tissues. Results: The results indicated that ECFCs increased the incidence and growth of MDA-MB-231 cell-derived tumors. The effects of ECFCs on tumor progression were particularly striking when 300,000 MDA-MB-231 cells were implanted using protocol two. Conclusions: This protocol could be utilized for fundamental studies on TNBC tumor progression and provides a means for evaluating chemotherapeutic candidates for the treatment of TNBC.

PRIMARY ADENOSQUAMOUS LUNG CARCINOMA TRANSFORMING TO PURE SQUAMOUS CELL BRAIN METASTASIS

SESSION TITLE: Lung Cancer SESSION TYPE: Fellow Case Report Posters PRESENTED ON: 10/09/2018 01:15 PM - 02:15 PM INTRODUCTION: Adenosquamous lung cancer (ASLC) comprises only 0.4% to 4.0% of all primary lung cancers and carries the worst prognosis when comparing to individual histology, owing its high mortality to brain metastases as the major cause of death. Our case illustrates the severity and rapidity of such a disease; however it also demonstrates a unique metastatic histological transformation. CASE PRESENTATION: 38 year old Jamaican male with no past medical history presented complaining of worsening right side weakness for 1 month. The ascending hemiparesis started in the right lower extremity and eventually progressed to the right upper extremity sparing the intrinsic muscles of the hand. Reflexes were normal throughout and there were no signs of sensory deficits. Review of systems was positive for night sweats and unintentional weight loss of 5 lbs in the past 2 months. Social history was significant for smoking 20 pack years as well as daily use of marijuana. CT thorax revealed a spiculated nodule in the right upper lobe measuring 1.7 x 1.9 x 2.1 cm with mild superior pleural retraction and right paratracheal lymphadenopathy. CT brain showed large areas of white matter edema in the frontal lobes bilaterally in conjunction with bilateral hypodense cystic masses with the largest measuring 4.2 x 2.5cm with a midline shift of 2.5mm. Left sided craniotomy was performed to resect the cystic frontal lobe tumor. Histology was notable for metastatic squamous cell carcinoma with the following supportive immunohistochemistry: positive CK5, CK6, CK7, P63, and Ki-67. Furthermore, a right upper lobe wedge resection revealed histology positive for adenosquamous carcinoma with lymphovascular invasion and positive mucin stains. EGFR, KRAS, and ALK immunohistochemistry came back negative. Upon discharge, the patient was followed by oncology and only pursued radiation treatment. DISCUSSION: In order to make a diagnosis of ASC, each histological component must make up at least 10% of the tumor. The histology along with the immunochemistry and mucin stain findings is what led us to diagnose the patient with ASC lung carcinoma. The histogenesis of ASC neoplasm has been investigated for many years; however, it is still unclear. The ASC tumor contains two phenotypes with two different lineages, one being columnar cells and the other being squamous cells. The two theories that exist are monoclonality and polyclonality. Polyclonality is described as the theory of the collision of two histologically independent tumors, while monoclonality signifies a single lineage. CONCLUSIONS: ASC lung cancer is known to be invasive with early stage metastasis and a prognosis of 6 month mortality. The histogenesis of this malignancy has long been debated. Our case demonstrates the fact that the metastatic site was only found to be composed of purely squamous cell; therefore, our case supports the polyclonal theory. Reference #1: Min Kong, Jiang Jin, Xiuyu Cai. Characteristics of lymph node mestastasis in resected adenosquamous lung cancer. Medicine (Baltimore). 2017 Dec; 96(48); e8870. Reference #2: Pan Yang, Wei-Lang Li, Jeff-X Zhou. Peritoneum as the sole distant metastatic site on lung adenosquamous carcinoma: a case report. J Med Case Rep. 2017; 11: 274. Reference #3: Jarred Burkart, Konstantin Shilo, Metastatic Squamous Cell Carcinoma Component from an Adenosquamous Carcinoma of the Lung with Identical Epidermal Growth Factor Receptor Mutations. Case Rep Pulonology. 2015; 2015: 283875. DISCLOSURES: No relevant relationships by Artur Alaverdian, source=Web Response No relevant relationships by Theresa Henson, source=Web Response No relevant relationships by Elyana Matayeva, source=Web Response

PO-172 Zerumbone inhibits epithelial-mesenchymal transition and cancer stem cells properties by inhibiting β-catenin pathway through miR-200c

IntroductionTNS4 (tensin 4 or Cten) is a putative novel oncogene in colorectal, breast and pancreatic cancer, but a tumour suppressor in prostate cancer. A tensin switch has been described in breast cancer where EGF (epidermal growth factor) activates TNS4 and down-regulates TNS3 which results in capping the tail of ITGB1 (integrin B1). However in colorectal cancer, the same tensin switch has not been verified. We have explored effects of TNS4 upregulation by EGF stimulation in colorectal cancer cell lines and how the interaction between ITGB1 and TNS4 might drive the epithelial to mesenchymal transition (EMT).Material and methodsUpon EGF stimulation (20 ng/mL) of several colorectal cancer cell lines of known KRAS mutant status, TNS4 and ITGB1 levels were detected by western blot, together with other EMT markers. Co-Immunoprecipitation was performed to detect possible interactions between TNS4 and ITGB1 upon stimulation and confocal imaging of immunofluorescence stainings were carried out to confirm co-localization.Results and discussionsAfter 20 ng/ml EGF stimulation for 24 hour, TNS4 and ITGB1 upregulation occurs in cell lines with wild-type KRAS allele, but not in mutant KRAS cell lines. This is not accompanied by an upregulation in TNS3, as described before. IGTB1 co-immunoprecipitates with TNS4 after EGF stimulation, but not in untreated controls. This is accompanied by alteration of EMT markers expression, and is mediated by TNS4, in accordance with recent reports.ConclusionWe may have identified a mechanism of interaction between ITGB1 and TNS4 that is implicated in the epithelial to mesenchymal transition by driving phenotypical changes at early metastatic stages. These results still warrant confirmation and further investigation to understand how tensins and integrins regulate cell motility during metastasis.

PO-169 Rethinking the EGF-mediated tensin switch in colorectal cancer

IntroductionTNS4 (tensin 4 or Cten) is a putative novel oncogene in colorectal, breast and pancreatic cancer, but a tumour suppressor in prostate cancer. A tensin switch has been described in breast cancer where EGF (epidermal growth factor) activates TNS4 and down-regulates TNS3 which results in capping the tail of ITGB1 (integrin B1). However in colorectal cancer, the same tensin switch has not been verified. We have explored effects of TNS4 upregulation by EGF stimulation in colorectal cancer cell lines and how the interaction between ITGB1 and TNS4 might drive the epithelial to mesenchymal transition (EMT).Material and methodsUpon EGF stimulation (20 ng/mL) of several colorectal cancer cell lines of known KRAS mutant status, TNS4 and ITGB1 levels were detected by western blot, together with other EMT markers. Co-Immunoprecipitation was performed to detect possible interactions between TNS4 and ITGB1 upon stimulation and confocal imaging of immunofluorescence stainings were carried out to confirm co-localization.Results and discussionsAfter 20 ng/ml EGF stimulation for 24 hour, TNS4 and ITGB1 upregulation occurs in cell lines with wild-type KRAS allele, but not in mutant KRAS cell lines. This is not accompanied by an upregulation in TNS3, as described before. IGTB1 co-immunoprecipitates with TNS4 after EGF stimulation, but not in untreated controls. This is accompanied by alteration of EMT markers expression, and is mediated by TNS4, in accordance with recent reports.ConclusionWe may have identified a mechanism of interaction between ITGB1 and TNS4 that is implicated in the epithelial to mesenchymal transition by driving phenotypical changes at early metastatic stages. These results still warrant confirmation and further investigation to understand how tensins and integrins regulate cell motility during metastasis.

Abstract 5001: Identification of a Six2/Sox2/Nanog stem cell axis that promotes breast cancer metastatic colonization

Abstract Although significant progress has been made in understanding the molecular mechanisms that lead to metastatic breast cancer, it remains the overwhelming cause of death for patients. Current studies primarily focus on the prevention of early stages of metastasis, such as migration and invasion. But at the time of diagnosis tumor cells have likely left the primary tumor, suggesting that inhibition of the early stages of metastasis may not be the most effective means of inhibiting metastatic burden. Instead, the identification and targeting of molecules required for establishment and survival of cells at secondary sites is imperative for advancing therapies. We are examining the role of the developmental transcription factor Six2 in promoting metastatic burden. Six2 is a member of the Six family of transcription factors and is responsible for the maintenance of stem/progenitor cells during nephrogenesis. Using knockdown (KD) and overexpression (OE) models in murine mammary cells, we demonstrated that Six2 promotes specifically late-stage metastasis (growth at the secondary site). RNA-Seq analysis on control and Six2 OE cells showed a dramatic enrichment in stem cell transcriptional programs downstream of Six2 in mammary/breast cancer cells. Flow cytometry analyses and tumorsphere assays demonstrate that Six2 OE leads to an increase, whereas Six2 KD leads to a decrease, in the mammary stem cell population and tumorsphere formation, respectively. Using an immune competent mouse model, in vivo orthotopic limiting-dilution experiments reveal that Six2 regulates tumor initiation. In addition, we show that Six2 regulates late-stage metastasis of human breast cancer cells in vivo, as measured by metastatic burden after tail vein injection. Thus, our data demonstrate that Six2 similarly promotes stem phenotypes in breast cancer cells as it does during kidney development, and suggest that this attribute may be critical for its ability to promote metastatic outgrowth. To determine the molecular mechanism by which Six2 mediates stem phenotypes and metastasis in breast cancer, we interrogated our RNA-seq data, which suggested that Six2 may control master regulators of stemness. We demonstrate that both Sox2 and Nanog are regulated by Six2. In human breast cancer gene expression datasets, Six2 significantly positively correlates with both Sox2 and Nanog, and combined OE of Six2 with either Sox2 or Nanog results in poor prognosis. Preliminary data suggest that loss of Sox2 downstream of Six2 inhibits metastatic outgrowth, and that Sox2 is upstream of Nanog in the pathway. Together, our data demonstrate that Six2 regulates cancer stem phenotypes to promote metastatic outgrowth, uncovering a novel Six2/Sox2/Nanog axis that is critical for metastatic colonization. Citation Format: Michael U. Oliphant, Ahwan Pandey, Katherine Johnson, Rani Powers, Matthew Galbraith, James Costello, Heide L. Ford. Identification of a Six2/Sox2/Nanog stem cell axis that promotes breast cancer metastatic colonization [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 5001.