Experimental Metastasis Assay Research Articles

Microtubule interacting and trafficking domain containing 1 deficiency leads to poor survival via tissue factor-mediated coagulation in bladder cancer

BackgroundPatients with cancer are at an increased risk of developing a hypercoagulative phenotype and venous thromboembolism. However, no clinical trial has yet confirmed that anticoagulant therapy improves cancer prognosis, and the mechanism underlying hypercoagulation in patients with bladder cancer is not well understood. ObjectivesWe hypothesized that the prognostic genes affect tumor progression via tumor-mediated coagulation. MethodsWe detected the most significant prognostic genes of bladder cancer with The Cancer Genome Atlas dataset and validated them in 2 Gene Expression Omnibus datasets and 1 ArrayExpress dataset. Immunohistochemical tests were performed on a cohort of 80 individuals to further examine the prognostic genes. For the most reliable prognostic gene, its influence on coagulation was evaluated with gene knockdown followed by next-generation sequencing and cellular and animal experiments. ResultsDepletion of microtubule interacting and trafficking domain containing 1 (MITD1), a major prognostic gene of bladder cancer, significantly increased the tissue factor (TF) expression. MITD1 deficiency led to cytokinesis arrest, which, in turn, promoted the TF expression via unfolded protein response and c-Jun. The knockdown of IRE1, an essential kinase of unfolded protein response or the inactivation of c-Jun using c-Jun N-terminal kinase inhibitors weakened MITD1 deficiency– or dithiothreitol-induced TF upregulation. Cells lacking MITD1 promoted coagulation and metastasis in the experimental metastasis assay. ConclusionOur findings suggest the novel role of tumor prognostic genes upon the development of hypercoagulative phenotype and venous thromboembolism, thereby underlining the importance of anticoagulant therapy and shedding light on the therapeutic value of targeting MITD1 in bladder cancer.

Heat-shock protein 90α protects NME1 against degradation and suppresses metastasis of breast cancer.

NME1 has been exploited as a potential translational target for decades. Substantial efforts have been made to upregulate the expression of NME1 and restore its anti-metastasis function in metastatic cancer. Cycloheximide (CHX) chase assay was used to measure the steady-state protein stability of NME1 and HSP90α. The NME1-associating proteins were identified by immunoprecipitation combined with mass spectrometric analysis. Gene knockdown and overexpression were employed to examine the impact of HSP90AA1 on intracellular NME1 degradation. The motility and invasiveness of breast cancer cells were examined in vitro using wound healing and transwell invasion assays. The orthotopic spontaneous metastasis and intra-venous experimental metastasis assays were used to test the formation of metastasis in vivo, respectively. HSP90α interacts with NME1 and increases NME1 lifetime by impeding its ubiquitin-proteasome-mediated degradation. HSP90α overexpression significantly inhibits the metastatic potential of breast cancer cells in vitro and in vivo. A novel cell-permeable peptide, OPT22 successfully mimics the HSP90α function and prolongs the life span of endogenous NME1, resulting in reduced metastasis of breast cancer. These results not only reveal a new mechanism of NME1 degradation but also pave the way for the development of new and effective approaches to metastatic cancer therapy.

Experimental and spontaneous metastasis assays can result in divergence in clonal architecture

Intratumoural heterogeneity is associated with poor outcomes in breast cancer. To understand how malignant clones survive and grow in metastatic niches, in vivo models using cell lines and patient-derived xenografts (PDX) have become the gold standard. Injections of cancer cells in orthotopic sites (spontaneous metastasis assays) or into the vasculature (experimental metastasis assays) have been used interchangeably to study the metastatic cascade from early events or post-intravasation, respectively. However, less is known about how these different routes of injection impact heterogeneity. Herein we directly compared the clonality of spontaneous and experimental metastatic assays using the human cell line MDA-MB-231 and a PDX model. Genetic barcoding was used to study the fitness of the subclones in primary and metastatic sites. Using spontaneous assays, we found that intraductal injections resulted in less diverse tumours compared to other routes of injections. Using experimental metastasis assays via tail vein injection of barcoded MDA-MB-231 cells, we also observed an asymmetry in metastatic heterogeneity between lung and liver that was not observed using spontaneous metastasis assays. These results demonstrate that these assays can result in divergent clonal outputs in terms of metastatic heterogeneity and provide a better understanding of the biases inherent to each technique.

Abstract P4-08-10: Azeliragon (TTP488), an orally- available small molecule RAGE inhibitor, reduces metastasis in preclinical mouse models of breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype with a high metastatic rate. Despite significant advances in breast cancer therapeutics, due to the lack of specific therapeutic targets in TNBC, cytotoxic chemotherapy is still the mainstay of treatment for this BC subtype. Preclinical studies have shown that the Receptor for Advanced Glycation End-products (RAGE) drives the progression and metastasis of aggressive cancer subtypes, including TNBC. RAGE plays a multifaceted role in driving tumorigenesis and metastasis through tumor cell-intrinsic mechanisms, such as cancer cell invasion, migration and epithelial-mesenchymal transition, and tumor cell-extrinsic mechanisms. This multifaceted role in cancer progression and metastasis makes RAGE a promising therapeutic target in the prevention and treatment of breast cancer. Here we tested the preclinical anti-metastasic efficacy of two small molecule RAGE inhibitors; TTP48 (Azeliragon) and FPS-ZM1. Importantly, TTP488 displays a high safety profile in human trials and has previously undergone Phase 3 clinical trials for Alzheimer’s disease. While FPS-ZM1 is a well-known RAGE inhibitor in preclinical cancer models, TTP488 has not been tested for its anti-cancer activity in breast cancer. Methods: We tested the in vitro anti-metastatic effect of TTP488 and FPS-ZM1 on cancer cell migration and invasion in Boyden chamber assays with TNBC cell lines (MDA-MB-231 and 4T-1). We used the 4175 highly metastatic MDA-MB-231 variant in xenograft studies in NSG mice to test the efficacy of the RAGE inhibitors in vivo on tumor progression and metastasis. Experimental metastasis assays were performed with tail-vein injection of 4T-1 cells into BALBc mice. We performed bulk RNA sequencing on the MDA-MB-231/4175/NSG tumors to unveil and compare the mechanism of action of the two small molecule RAGE inhibitors. Results: Our results showed that TTP488 and FPS-ZM1 impaired mechanisms of metastasis in vitro with both MDA-MB231/4175 and 4T-1 cells. TTP488 and FPS-ZM1 significantly inhibited MDA-MB231/4175 cell metastasis from the orthotopic site in NSG mice without displaying any deleterious effects on mouse health. In the syngeneic 4T-1/BALBc model, both TTP488 and FPS-ZM1 impaired metastasis in tail-vein injected experimental metastasis assays. Transcriptomic analysis of primary xenograft tumors from NSG mice revealed that TTP488 and FPS-ZM1 displayed high concordance in gene expression changes. Pathway enrichment analysis showed that both RAGE inhibitors affected metastatic pathways, including focal adhesion, ECM-receptor interaction, cell cycle, and DNA replication. Conclusions: These results show that TTP488 impairs metastasis of multiple highly aggressive TNBC models for the first time. Importantly, as TTP488 displays a high safety profile in human trials, this study provides the rationale for evaluating TTP488 in clinical trials to treat or prevent metastatic breast cancer. Citation Format: Melinda Magna, Gyong Ha Hwang, Alec McIntosh, Katherine Drews-Elger, Masaru Takabatake, Barbara Mera, Taekyoung Kwak, Philip Miller, Marc Lippman, Barry I. Hudson. Azeliragon (TTP488), an orally- available small molecule RAGE inhibitor, reduces metastasis in preclinical mouse models of breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-10.

Targeting ARF1-IQGAP1 interaction to suppress colorectal cancer metastasis and vemurafenib resistance

IntroductionAcquired resistance to BRAF inhibitor vemurafenib is frequently observed in metastatic colorectal cancer (CRC), and it is a thorny issue that results in treatment failure. As adaptive responses for vemurafenib treatment, a series of cellular bypasses are response for the adaptive feedback reactivation of ERK signaling, which warrant further investigation. ObjectivesWe identified ARF1 (ADP-ribosylation factor 1) as a novel regulator of both vemurafenib resistance and cancer metastasis, its molecular mechanism and potential inhibitor were investigated in this study. MethodsDIA-based quantitative proteomics and RNA-seq were performed to systematic analyze the profiling of vemurafenib-resistant RKO cells (RKO-VR) and highly invasive RKO cells (RKO-I8), respectively. Co‑immunoprecipitation assay was performed to detect the interaction of ARF1 and IQGAP1 (IQ-domain GTPase activating protein 1). An ELISA-based drug screen system on FDA-approved drug library was established to screen the compounds against the interaction of ARF1-IQGAP1.The biological functions of ARF1 and LY2835219 were determined by transwell, western blotting, Annexin V-FITC/PI staining and in vivo experimental metastasis assays. ResultsWe found that ARF1 strongly interacted with IQGAP1 to activate ERK signaling in VR and I8 CRC cells. Deletion of IQGAP1 or inactivation of ARF1 (ARF-T48S) restored the invasive ability induced by ARF1. As ARF1-IQGAP1 interaction is essential for ERK activation, we screened LY2835219 as novel inhibitor of ARF1-IQGAP1 interaction, which inactivated ERK signaling and suppressed CRC metastasis and vemurafenib-resistance in vitro and in vivo with no observed side effect. Furthermore, LY2835219 in combined treatment with vemurafenib exerted significantly inhibitory effect on ARF1-mediated cancer metastasis than used independently. ConclusionThis study uncovers that ARF1-IQGAP1 interaction-mediated ERK signaling reactivation is critical for vemurafenib resistance and cancer metastasis, and that LY2835219 is a promising therapeutic agent for CRC both as a single agent and in combination with vemurafenib.

Metastasis suppressor NME1 in exosomes or liposomes conveys motility and migration inhibition in breast cancer model systems.

Tumor-derived exosomes have documented roles in accelerating the initiation and outgrowth of metastases, as well as in therapy resistance. Little information supports the converse, that exosomes or similar vesicles can suppress metastasis. We investigated the NME1 (Nm23-H1) metastasis suppressor as a candidate for metastasis suppression by extracellular vesicles. Exosomes derived from two cancer cell lines (MDA-MB-231T and MDA-MB-435), when transfected with the NME1 (Nm23-H1) metastasis suppressor, secreted exosomes with NME1 as the predominant constituent. These exosomes entered recipient tumor cells, altered their endocytic patterns in agreement with NME1 function, and suppressed in vitro tumor cell motility and migration compared to exosomes from control transfectants. Proteomic analysis of exosomes revealed multiple differentially expressed proteins that could exert biological functions. Therefore, we also prepared and investigated liposomes, empty or containing partially purified rNME1. rNME1 containing liposomes recapitulated the effects of exosomes from NME1 transfectants in vitro. In an experimental lung metastasis assay the median lung metastases per histologic section was 158 using control liposomes and 15 in the rNME1 liposome group, 90.5% lower than the control liposome group (P = 0.016). The data expand the exosome/liposome field to include metastasis suppressive functions and describe a new translational approach to prevent metastasis.

Abstract 962: A hybrid epithelial-mesenchymal phenotype identifies lung cancer cells with high metastatic potential due to increased stemness properties and escape from NK immunosurveillance

Abstract Introduction: Epithelial to mesenchymal transition (EMT) is essential for cancer dissemination but its role in overt metastasis establishment remains an open issue. Multiple EMT states and great phenotypic plasticity have also been described, indicating the need for in-depth characterization of biological properties associated with different EMT phenotypes. The so-called hybrid phenotype, characterized by presence of both mesenchymal and epithelial markers, might in fact identify cancer cells with enhanced metastatic propensity. Materials and Methods: To assess the metastatic potential of different EMT phenotypes, we exploited two lung cancer cell lines with different properties (A549/epithelial and LT73/hybrid) and generated their respective sublines through four sequential rounds of in vitro migration assay (named M4). TGFβ treatment was also used to induce EMT. With a previously validated score based on the ratio between expression of epithelial (CDH1) or mesenchymal genes (SNAI2), cells were classified as: epithelial (A549)>hybrid (A549-M4, LT73)>mesenchymal (LT73-M4, A549-TGFβ). The disseminating/metastatic properties of the different lines were tested in vivo by subcutaneous and tail-vein injections in SCID mice. Interaction with NK cells (NKs), known to specifically target EMT cancer cells, was also assessed. Results and Discussion: Compared to parental cells, M4 cells showed up-regulation of EMT-associated genes, expansion of the subset of CD133+ lung cancer stem cells (CSC) and displayed in vivo higher ability to disseminate from primary tumors to murine lungs (2-fold change, p=0.004). However, hybrid cells (LT73 and A549-M4) showed an increased ability to generate overt lung metastases in experimental metastasis assays compared to either epithelial (A549) or mesenchymal cells (LT73-M4 and A549-TGFβ). The higher metastatic ability of hybrid cells was correlated with CSCs expansion and reduced NKs infiltration to the metastatic lungs. Expression of IL-8, a cytokine involved in NKs recruitment, was decreased in hybrid cells and they were less prone to chemoattract NKs in vitro compared to both epithelial and mesenchymal cells. Finally, in vivo prevention of NKs recruitment to the lungs using a neutralizing antibody, massively increased metastasis formation and expansion of CSCs in mice injected i.v. with mesenchymal A549-TGFβ (p=0.0006), but had limited effects in groups injected with both epithelial A549 and hybrid A549-M4. Conclusions: Our data indicate that the hybrid phenotype correlates with the highest metastatic potential related to efficient cancer cell dissemination, increased induction of stemness features and ability to avoid NKs recruitment. Conversely, fully mesenchymal cells show limited metastatic potential, possibly due the control exerted by NKs on their CSC subsets. Citation Format: Giulia Bertolini, Monica Parodi, Massimo Milione, Fabio Murianni, Giovanni Centonze, Giulia Taiè, Gabriella Sozzi, Massimo Vitale, Luca Roz. A hybrid epithelial-mesenchymal phenotype identifies lung cancer cells with high metastatic potential due to increased stemness properties and escape from NK immunosurveillance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 962.

Abstract 6336: Regulation of tissue factor dependent procoagulant properties by CD44: Implication for metastasis of breast tumor cells

Abstract The aim here is to study the implication of the stem cell marker and fibrin receptor, CD44, in the regulation of tissue factor and its associated pro-coagulant and pro-metastatic properties in breast tumor cells. We examined this CD44/TF/coagulation axis in breast cancer EMT+ (MDA-MB-231, Hs578T), EMT- (MCF7, T47D) and EMT-inducible (EGF-treated MDA-MB-468, MDA-MB-468 harboring a doxycycline-inducible snail expression vector) cellular models. We used siRNA to inhibit CD44 expression and realized RT-qPCR, western blot, flow cytometry analyzes and promoter reporter assays to monitor regulations of our gene of interest. Furthermore, we realized in vitro clotting assays and TF activity assays to examine coagulant properties, and in vivo experimental metastasis assays to assess early metastatic potential. We first confirmed by WB, RT-qPCR and FACS analyzes that CD44 is over-expressed in our EMT+ and EMT-induced (EMTi) cellular models, concomitantly with TF expression. Using siRNAs, we showed that CD44 silencing in these same EMT+ and EMTi cellular models decreased both mRNA and protein levels of TF. Clotting assays and TF activity assays further showed that CD44 silencing decreased pro-coagulant properties of our EMT+ and EMTi cellular models in vitro and of their pro-metastatic properties in vivo. Examining further the molecular mechanisms of TF regulation by CD44, we employed truncated TF promoter reporter vectors to identify key transcription factors linking CD44 to TF transcription. These experiments revealed that a TF promoter region containing Sp1 binding sites is sufficient to mediate the diminution of TF promoter activity seen after CD44 silencing. Mutations in the Sp1 binding sites abrogated the activity of the TF reporter vector. Further pointing to Sp1 as a key transcription factor implicated in TF regulation by CD44, we observed that silencing CD44 inhibited Sp1 expression and that silencing Sp1 decreased TF mRNA levels. Our results thus demonstrate a key role of CD44 in regulating TF expression in EMT+ cancer cells, and point to Sp1 as a transcriptional mediator of this regulation. This modulation of TF expression by CD44 in EMT+ cells led to a concomitant regulation of pro-coagulant properties, and impacts metastatic potential. Citation Format: Amélie V. Villard, Anthony Genna, Christine Gilles. Regulation of tissue factor dependent procoagulant properties by CD44: Implication for metastasis of breast tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6336.

Abstract PD3-05: The paradoxical role of RalA and RalB in triple negative breast cancer

Abstract Background: Breast Cancer (BC) is the most common cancer and leading cause of cancer associated mortality in women worldwide. TNBC patients have the highest mortality mainly due to lack of receptors for targeted therapies. RalA and RalB are small GTPases that are known to regulate growth and metastasis in several cancers. However, roles for these GTPases in BC is poorly understood. The goal of this study was to investigate the contributions of RalA and RalB in TNBC. Methods: Control, RalA or RalB CRISPR knockout (KO) MDA-MB-231 cells were injected into the mammary gland of nod-skid-gamma (NSG) mice. Tumor growth was monitored and groups were taken as they met early removal criteria. Tumors and lungs were formalin-fixed and paraffin embedded. Tumors underwent immunohistochemical staining for Ki-67 and Cleaved caspase-3 and lungs were stained for hematoxylin and eosin and imaged on a Leica Aperio ScanScope XT to calculate lung metastasis. RalA or RalB were also depleted in MDA-MB-231 and MVT1 cells by shRNA. In addition, RalA depleted MDA-MB-231 cells were labeled with luciferase and injected into the tail vein of NSG mice and imaged on an IVIS spectrum to test seeding and lung colonization. Immunohistochemistry of patient TMAs was preformed on a Bond RX autostainer using RalA (Abcam, ab126627, 1:2000). Immunohistochemical stains were imaged on a PerkinElmer’s Vectra® Automatic Imaging System and quantified using inForm® Advanced Image Analysis software. Statistical significance of Kaplan-Meier survival curves were determined by log rank. Results: RalA knockout and depletion slowed primary orthotopic tumor growth in MDA-MB-231 and MVT1 cells. RalB KO had the opposite effect and increased growth rate compared to controls and RalA KO cells. Ki67 and cleaved caspase 3 IHC staining of tumors indicate KO of RalA decreased proliferation, whereas KO RalB increased proliferation with no change in apoptosis. RalA KO decreased the number and area of lung metastasis in both spontaneous and experimental metastasis assays. RalB KO or depletion caused an increase in the area and number of metastasis. Utilizing data from the METABRIC and TCGA BC datasets, elevated RALA, but not RALB, was prognostic of worse outcome in the overall BC populations and the TNBC populations specifically. RALA was shown to be more highly expressed in BC, particularly TNBC, relative to normal mammary tissue whereas RalB was decreased in BC and TNBC. IHC staining of a TMA comprised of all BC subtypes and a TMA of only TNBC samples confirmed RalA as a prognostic marker of patient outcome. Conclusions: RalA and RalB have important but paradoxical roles in TNBC. Citation Format: Jonathan M. Spehar, Katie A. Thies, Matthew W. Cole, Rachel E. Schafer, Dillon S. Richardson, Sarah A. Steck, Manjusri Das, Arthur W. Lian, Alo Ray, Sue E. Knoblaugh, Cynthia D. Trimmers, Gina M. Sizemore, Steven T. Sizemore. The paradoxical role of RalA and RalB in triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-05.

Patient-derived xenograft culture-transplant system for investigation of human breast cancer metastasis

Metastasis is a fatal disease where research progress has been hindered by a lack of authentic experimental models. Here, we develop a 3D tumor sphere culture-transplant system that facilitates the growth and engineering of patient-derived xenograft (PDX) tumor cells for functional metastasis assays in vivo. Orthotopic transplantation and RNA sequencing (RNA-seq) analyses show that PDX tumor spheres maintain tumorigenic potential, and the molecular marker and global transcriptome signatures of native tumor cells. Tumor spheres display robust capacity for lentiviral engineering and dissemination in spontaneous and experimental metastasis assays in vivo. Inhibition of pathways previously reported to attenuate metastasis also inhibit metastasis after sphere culture, validating our approach for authentic investigations of metastasis. Finally, we demonstrate a new role for the metabolic enzyme NME1 in promoting breast cancer metastasis, providing proof-of-principle that our culture-transplant system can be used for authentic propagation and engineering of patient tumor cells for functional studies of metastasis.

Irreversible cardiac remodeling promotes cancer progression via secreted factors in ATF3 transgenic mouse model

Abstract Cardiac hypertrophy and cancer are the leading cause of deaths worldwide. While heart failure and cancer have been considered separate diseases, it is becoming evident that they are highly connected and affect each other's outcome. Recent studies using mouse models mimicking myocardial infraction and aortic stenosis were shown to promote cancer progression. These two mouse models involve major irreversible surgeries. Here, we employed a transgenic mouse model for heart hypertrophy and examined the crosstalk between the hypertrophied heart and cancer progression. Heart hypertrophy was induced by adult ATF3 expression in cardiomyocytes followed by either PyMT, breast cancer cells or Lewis lung carcinoma (LLC) cells implantation. PyMT and LLC tumors grow larger in size and display increased cell proliferation in ATF3-transgenic mice as compared with control mice. In addition, pulmonary experimental metastasis assay revealed augmented number and size of metastasis lesions in the lungs. Subsequently, we identified five putative cardiac secreted factors that are highly expressed in the hearts derived from ATF3-transgenic that may be involved in tumor progression. These include Fibronectin, CP, serpin3, CTgF and PSTN. Next, we addressed whether the cause or the effect are necessary to promote tumor growth. Towards this end, ATF3 expression was turned-off after cardiac hypertrophy development followed by cancer cell implantation. The tumor promotion phenotype as well as enhancement of metastatic seeding in these mice was fully preserved, suggesting the generation of an irreversible viscous cycle that lead to cancer progression. Significantly, the expression of Fibronectin and CTgF was dampened in the presence of doxycycline, thus exclude their putative role in mediating the tumor promotion and metastatic seeding phenotypes. Collectively, heart hypertrophy and cancer diseases communicate via secreted factors that result in cancer promotion and disease deterioration. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Israel Science Foundation

Mechanisms Linking Fibrin(ogen) Structure/Function to Tumor Metastasis

Previous studies established that the platelet/fibrin(ogen) axis promotes metastatic potential by impeding the clearance of newly formed micrometastases by natural killer (NK) cells. However, multiple important questions remain, including the potential of fibrin(ogen) to promote metastasis through interactions with cells other than platelets (e.g., inflammatory cells), and the fundamental question of whether fibrin polymerization is required for metastasis. It also remains to be determined if the link between fibrin(ogen) and innate tumor immunity is relevant to tumor growth in some contexts, or is strictly limited to early events important in metastasis.To test the hypothesis that fibrin(ogen) promotes metastasis by distinct mechanisms linked to specific structure/function interactions with several cell types, we performed experimental metastasis assays in immunocompetent mice carrying specific fibrinogen structure/function alterations. Expression of a mutant fibrinogen lacking the binding motif for the leukocyte integrin αMβ2 (Fibγ390-396A) significantly decreased metastatic potential relative to wildtype fibrinogen, suggesting a role for fibrin(ogen)-inflammatory cell interactions mediated by αMβ2 in metastasis. To directly determine the importance of thrombin-mediated fibrin polymerization in metastasis, we analyzed metastatic potential in FibAEK mice, which carry a form of fibrinogen essentially “locked” in the soluble state due to a germ-line mutation in the Aα chain thrombin cleavage site. Metastatic potential in FibAEK mice was diminished relative to control mice, speaking to the importance of thrombin-mediated fibrin polymer formation in this process. However, the FibAEK mice retained significant metastatic potential relative to complete fibrinogen deficiency, indicating that fibrinogen monomer retains some significant prometastatic properties. In order to better define the role of fibrin(ogen)-platelet interactions in metastasis, we compared metastatic potential in control and FibγΔ5 mice, carrying a form of fibrinogen lacking the γ chain binding motif for the platelet integrin αIIbβ3. Surprisingly, this mutation had no impact on metastatic potential. Given the established importance of platelets in metastasis, these findings suggest that fibrin is capable of platelet stabilization through mechanism(s) independent of this αIIbβ3 binding motif.Fibrin(ogen) appears to be important in metastasis across multiple tumor types, but its role in tumor growth is context dependent. Previous studies established that fibrin(ogen) promotes the growth of colonic adenocarcinoma in mice. To determine if fibrin(ogen) is mechanistically coupled to NK cell functions in this context, we depleted NK cells in cohorts of Fib-/- and control mice with an anti-NK1.1 antibody prior to subcutaneous inoculation of MC38 cells (a C57Bl/6-derived colonic adenocarcinoma). Depletion of NK cells had no impact on tumor growth in Fib+ mice. In mice with intact NK cells (i.e., inoculated with a nonspecific IgG), fibrinogen deficiency significantly limited tumor growth. However, depletion of NK cells resulted in a restoration of tumor growth in Fib-/- mice that was comparable to that observed in Fib+ mice.Together, these studies suggest that both fibrin and fibrinogen play a significant role in metastasis. In addition to interactions with platelets, fibrin(ogen) appears to support metastasis through leukocyte interactions mediated by αMβ2. These studies also reveal an important context where fibrin(ogen) promotes tumor growth via mechanisms coupled to NK cell functions, opening the door to the possibility that targeting these interactions could provide a means to limit the progression of established tumors. DisclosuresNo relevant conflicts of interest to declare.

A novel CXCR4 antagonist counteracts paradoxical generation of cisplatin-induced pro-metastatic niches in lung cancer

Platinum-based chemotherapy remains widely used in advanced non-small cell lung cancer (NSCLC) despite experimental evidence of its potential to induce long-term detrimental effects, including the promotion of pro-metastatic microenvironments. In this study, we investigated the interconnected pathways underlying the promotion of cisplatin-induced metastases.In tumor-free mice, cisplatin treatment resulted in an expansion in the bone marrow of CCR2+CXCR4+Ly6Chigh inflammatory monocytes (IMs) and an increase in lung levels of stromal SDF-1, the CXCR4 ligand. In experimental lung metastasis assays, cisplatin-induced IMs promoted the extravasation of tumor cells and the expansion of CD133+CXCR4+ metastasis-initiating cells (MICs). Peptide R, a novel CXCR4 inhibitor designed as an SDF-1 mimetic peptide, prevented cisplatin-induced IM expansion, the recruitment of IMs into the lungs, and the promotion of metastasis. At the primary tumor site, cisplatin treatment reduced tumor size while simultaneously inducing tumor release of SDF-1, MIC expansion, and recruitment of pro-invasive CXCR4+ macrophages. Co-recruitment of MICs and CCR2+CXCR4+ IMs to distant SDF-1-enriched sites also promoted spontaneous metastases that were prevented by CXCR4 blockade. In clinical specimens from NSCLC patients SDF-1 levels were found to be higher in platinum-treated samples and related to a worse clinical outcome. Our findings reveal that activation of the CXCR4/SDF-1 axis specifically mediates the pro-metastatic effects of cisplatin and suggest CXCR4 blockade as a possible novel combination strategy to control metastatic disease.

Unbiased Phenotype-Based Screen Identifies Therapeutic Agents Selective for Metastatic Prostate Cancer.

In American men, prostate cancer is the second leading cause of cancer-related death. Dissemination of prostate cancer cells to distant organs significantly worsens patients’ prognosis, and currently there are no effective treatment options that can cure advanced-stage prostate cancer. In an effort to identify compounds selective for metastatic prostate cancer cells over benign prostate cancer cells or normal prostate epithelial cells, we applied a phenotype-based in vitro drug screening method utilizing multiple prostate cancer cell lines to test 1,120 different compounds from a commercial drug library. Top drug candidates were then examined in multiple mouse xenograft models including subcutaneous tumor growth, experimental lung metastasis, and experimental bone metastasis assays. A subset of compounds including fenbendazole, fluspirilene, clofazimine, niclosamide, and suloctidil showed preferential cytotoxicity and apoptosis towards metastatic prostate cancer cells in vitro and in vivo. The bioavailability of the most discerning agents, especially fenbendazole and albendazole, was improved by formulating as micelles or nanoparticles. The enhanced forms of fenbendazole and albendazole significantly prolonged survival in mice bearing metastases, and albendazole-treated mice displayed significantly longer median survival times than paclitaxel-treated mice. Importantly, these drugs effectively targeted taxane-resistant tumors and bone metastases – two common clinical conditions in patients with aggressive prostate cancer. In summary, we find that metastatic prostate tumor cells differ from benign prostate tumor cells in their sensitivity to certain drug classes. Taken together, our results strongly suggest that albendazole, an anthelmintic medication, may represent a potential adjuvant or neoadjuvant to standard therapy in the treatment of disseminated prostate cancer.

A Genome-Wide Screen in Mice To Identify Cell-Extrinsic Regulators of Pulmonary Metastatic Colonisation.

Metastatic colonization, whereby a disseminated tumor cell is able to survive and proliferate at a secondary site, involves both tumor cell-intrinsic and -extrinsic factors. To identify tumor cell-extrinsic (microenvironmental) factors that regulate the ability of metastatic tumor cells to effectively colonize a tissue, we performed a genome-wide screen utilizing the experimental metastasis assay on mutant mice. Mutant and wildtype (control) mice were tail vein-dosed with murine metastatic melanoma B16-F10 cells and 10 days later the number of pulmonary metastatic colonies were counted. Of the 1,300 genes/genetic locations (1,344 alleles) assessed in the screen 34 genes were determined to significantly regulate pulmonary metastatic colonization (15 increased and 19 decreased; P < 0.005 and genotype effect <-55 or >+55). While several of these genes have known roles in immune system regulation (Bach2, Cyba, Cybb, Cybc1, Id2, Igh-6, Irf1, Irf7, Ncf1, Ncf2, Ncf4 and Pik3cg) most are involved in a disparate range of biological processes, ranging from ubiquitination (Herc1) to diphthamide synthesis (Dph6) to Rho GTPase-activation (Arhgap30 and Fgd4), with no previous reports of a role in the regulation of metastasis. Thus, we have identified numerous novel regulators of pulmonary metastatic colonization, which may represent potential therapeutic targets.

Abstract P3-01-11: Targeting RAGE inhibits breast cancer invasion and metastasis

Abstract Background: Breast cancer (BC) is treatable with early detection, but once metastasis occurs, there is no cure. Therefore, it is of upmost importance to find targetable biomarkers that are involved in modulating the multi-step metastatic process. The Receptor for Advanced Glycation Endproducts (RAGE) and its ligands are an inflammatory pathway that are involved in modulating breast cancer progression and metastasis. We recently demonstrated that genetic or pharmacological interruption of RAGE signaling affected tumor progression and metastasis. However, no studies have dissected the role of RAGE in tumor growth from the metastatic cascade. Here, we show for the first time in multiple metastatic breast cancer models that targeting RAGE impairs BC metastasis. Methods: We tested the anti-metastatic effect of RAGE in vitro using the RAGE inhibitor FPS-ZM1 or RAGE shRNA knockdown in cell invasion, proliferation, migration and sphere formation assays (with 4T1, Py8119 and E0771 mouse BC cells). For in vivo assays we used orthotopic BC models (4T1/BALB/c and E0771 &amp; Py8119/C57BL6) and experimental metastasis assays (tail vein injection of 4T1/BALB/c and Py8119/C57BL6). To target RAGE, we used genetic (shRNA in 4T1 cells and RAGE knockout in C57BL6 mice) and pharmacological approaches (I.P. injection of FPS-ZM1). To investigate the synergistic effects of RAGE inhibition on progression and metastasis, we tested combination therapy of low-dose doxorubicin and FPS-ZM1. Results: Inhibition of RAGE with FPS-ZM1 (1-5uM) impaired tumor cell invasion of 4T1, E0771 and Py8119 cells. Similarly, in spheroid assays, treatment of cells with FPS-ZM1 resulted in fewer and smaller colonies. However, FPS-ZM1 treatment did not affect cell proliferation. In vivo studies revealed that FPS-ZM1 (1mg/kg) has a modest effect on tumor growth in 4T1/BALB/c injected mice but displayed a dramatic inhibitory effect on metastasis to the lungs. In experimental metastasis assays, tail-vein injection of 4T1 cells in BALB/c mice demonstrated that FPS-ZM1 treatment strongly impaired metastatic disease in mice compared to controls. To dissect the genetic role of RAGE in the tumor versus host, we test the effect of RAGE knockdown in 4T1 cells in experimental metastasis assays. RAGE shRNA impaired metastasis to the lungs, albeit to a lower degree seen with the RAGE inhibitor. To test the role of the host, we injected Py8119 cells into wild-type and RAGE knockout (RKO) mice. RKO mice displayed fewer metastatic burden compared to wild-type mice. Finally, in our combination treatment experiments, treatment of 4T1-injected BALB/c mice with Doxorubicin and FPS-ZM1 (alone and in combination), demonstrated that drug combination was more effective in inhibiting lung metastasis than either reagent alone. We are currently assessing how RAGE mechanistically drives these metastatic changes. Conclusion: Our data strongly suggests RAGE plays an important role in breast cancer metastasis, with less of an effect on tumor growth. Ongoing studies in our lab are testing which stage of the metastatic cascade RAGE is involved in, and the underlying mechanisms driving these processes. In conclusion, the use of RAGE inhibitors could represent a novel therapeutic approach for metastatic breast cancer. Citation Format: Gyong Ha Hwang, Melinda Magna, Barbara Mera, Toni Yeasky, Taekyoung Kwak, Lucas Outcault, Masaru Takabatake, Thuy-Mai Le, Marc E. Lippman, Barry I. Hudson. Targeting RAGE inhibits breast cancer invasion and 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 P3-01-11.

P66ShcA functions as a contextual promoter of breast cancer metastasis

BackgroundThe p66ShcA redox protein is the longest isoform of the Shc1 gene and is variably expressed in breast cancers. In response to a variety of stress stimuli, p66ShcA becomes phosphorylated on serine 36, which allows it to translocate from the cytoplasm to the mitochondria where it stimulates the formation of reactive oxygen species (ROS). Conflicting studies suggest both pro- and anti-tumorigenic functions for p66ShcA, which prompted us to examine the contribution of tumor cell-intrinsic functions of p66ShcA during breast cancer metastasis.MethodsWe tested whether p66ShcA impacts the lung-metastatic ability of breast cancer cells. Breast cancer cells characteristic of the ErbB2+/luminal (NIC) or basal (4T1) subtypes were engineered to overexpress p66ShcA. In addition, lung-metastatic 4T1 variants (4T1-537) were engineered to lack endogenous p66ShcA via Crispr/Cas9 genomic editing. p66ShcA null cells were then reconstituted with wild-type p66ShcA or a mutant (S36A) that cannot translocate to the mitochondria, thereby lacking the ability to stimulate mitochondrial-dependent ROS production. These cells were tested for their ability to form spontaneous metastases from the primary site or seed and colonize the lung in experimental (tail vein) metastasis assays. These cells were further characterized with respect to their migration rates, focal adhesion dynamics, and resistance to anoikis in vitro. Finally, their ability to survive in circulation and seed the lungs of mice was assessed in vivo.ResultsWe show that p66ShcA increases the lung-metastatic potential of breast cancer cells by augmenting their ability to navigate each stage of the metastatic cascade. A non-phosphorylatable p66ShcA-S36A mutant, which cannot translocate to the mitochondria, still potentiated breast cancer cell migration, lung colonization, and growth of secondary lung metastases. However, breast cancer cell survival in the circulation uniquely required an intact p66ShcA S36 phosphorylation site.ConclusionThis study provides the first evidence that both mitochondrial and non-mitochondrial p66ShcA pools collaborate in breast cancer cells to promote their maximal metastatic fitness.

An Intrasplenic Injection Model for the Study of Cancer Stem Cell Seeding Capacity.

In many tumor types, only a minor pool of cancer cells-the so-called cancer stem cells-is able to colonize distant organs and give rise to secondary tumors. In humans, the liver is one of the main target organs for many metastatic tumor types, including colorectal cancer. However, mouse tumour models only rarely spontaneously metastasize to the liver. Therefore, reliable in vivo experimental metastasis assays are crucial to study cell seeding capacity and the mechanisms controlling these metastatic stem cell properties. Here, we describe an intrasplenic injection model that mimics the process of liver metastasis occurring in cancer patients.

Identification of GOLM1 as a positively regulator of tumor metastasis by regulating MMP13 in gastric carcinoma.

Metastatic gastric carcinoma (GC) is a typically incurable disease. The progression of anti-metastatic treatment is hampered because the underlying mechanisms regulating the metastasis of GC cell are not well illuminated. Therefore, further elucidation of the molecular mechanism behind the metastatic traits of GC cells is needed for optimizing GC treatment. The levels of GOLM1 and MMP13 in GC cells and tissues were measured by using qPCR assay. The growth of GC cells in vitro was detected using MTS and colony formation assays. The migration and invasion of GC cells was analyzed using wound healing test and Transwell invasion assay. The level of MMP13 in GC cell was measured using immunoblotting and the level of GOLM1 was measured using immunofluorescence staining. The role of GOLM1 on the distant metastasis of GC SGC7910 cell was analyzed using experimental metastasis assay. Transplanted tumor model was constructed to analyze the influence of GOLM1 on GC cell growth in vivo. Here, we report that GOLM1 is over-expressed in GC and knockdown GOLM1 impairs the aggressive phenotypes of GC cell in vitro. Furthermore, downregulation of GOLM1 restrains the tumor growth of GC cell in nude mice. Nevertheless, upregulation of GOLM1 distinctly elevated the growth, migration ability and invasiveness of GC SGC7910 cell. Finally, GOLM1 increases the metastatic phenotypes of GC cell in a MMP13-dependent manner. Altogether, this investigation demonstrates the crucial function of GOLM1 in the progression of GC, which indicating GOLM1 as a potential target for GC treatment.

Fibg390-396A Limits Metastatic Potential Via a Mechanism Independent of Fibrin-Myeloid Cell Interactions, but Prospectively through the Disruption of FXIII-Mediated Fibrin Crosslinking

Previous studies have shown that fibrin(ogen) supports metastasis. In order to better define the mechanisms coupling fibrin(ogen) to metastasis, we compared metastatic potential in immunocompetent mice carrying specific structure/function alterations in fibrinogen and controls. FibrinogenAEK mice, which have a germ-line mutation in the thrombin cleavage site that essentially "locks" fibrinogen in its soluble state, exhibited diminished metastatic potential relative to FibWT mice. Notably, previous studies have established that factor XIII is also a significant determinant of metastasis. Taken together, these studies suggest that stable fibrin polymer formation is important in the metastatic process. However, FibAEK retained significant metastatic potential relative to mice with complete fibrinogen deficiency, indicating that fibrinogen possesses prometastatic properties in the absence of polymer formation. Fibrin(ogen) has also been shown to mediate inflammatory cell functions through direct engagement of leukocyte integrins independently of its role in platelet aggregation. Given previous studies demonstrating that myeloid cells are promote metastatic potential, we hypothesized that fibrin(ogen)-mediated leukocyte engagement represents one mechanism by which fibrinogen drives metastasis. Consistent with this view, mice expressing a mutant fibrinogen lacking the y chain binding motif for the leukocyte integrin αMβ2 (Fibγ390-396A) had diminished metastatic potential in both spontaneous and experimental metastasis assays. Furthermore, fate analyses revealed that Fibγ390-396A results in diminished survival of newly-formed metastatic foci. The early reduction in metastatic potential observed in Fibγ390-396A mice suggests that fibrin(ogen) promotes metastasis by recruiting myeloid cells to the early metastatic niche. In order to explore this hypothesis, we performed experimental metastasis assays in immunocompetent mice in which macrophages or neutrophils were specifically depleted using either clodronate or an anti-Ly6G antibody. In contrast to mice carrying the Fibγ390-396A mutation, depletion of macrophages or neutrophils had no significant impact on the early survival of metastatic foci. These studies suggest that expression Fibγ390-396A limits metastatic potential via a mechanism independent of interactions with myeloid cells. In addition to disrupting fibrin-αMβ2 interactions, elimination of the γ chain 390-396a binding motif has also been shown to limit factor XIII binding to fibrinogen. Given the established importance of FXIII in metastasis, it is conceivable that even subtle alterations in the kinetics of fibrin cross-linking resulting from expression of Fibγ390-396A limits metastatic potential. Disclosures Palumbo: Ionis Pharmaceuticals: Research Funding.