Cancer Metastasis In Mice Research Articles

Moderate Static Magnet Fields Suppress Ovarian Cancer Metastasis via ROS-Mediated Oxidative Stress.

Metastasis is the leading cause of cancer patient death, which is closely correlated with reactive oxygen species (ROS) levels. It is well known that the effects of ROS on tumors are diverse, depending on ROS concentration and cell type. We found that ovarian cancer cells have significantly lower levels of ROS than normal ovarian cells. Moreover, increased ROS levels in ovarian cancer cells can substantially inhibit their migration and invasion ability. Furthermore, the results show that moderate static magnetic field (SMF) can inhibit ovarian cancer cell migration, invasion, and stemness in a ROS-dependent manner. RNA sequencing results confirm that SMFs increased the oxidative stress level and reduced the stemness of ovarian cancer cells. Consistently, the expressions of stemness-related genes were significantly decreased, including hyaluronan receptor (CD44), SRY-box transcription factor 2 (Sox2), and cell myc proto-oncogene protein (C-myc). Furthermore, moderate SMFs provided by a superconducting magnet and permanent magnet have good biosafety and can both inhibit ovarian cancer metastasis in mice. Therefore, our study demonstrates the effects of SMFs on oxidative stress and metastasis in the ovarian cancer cells, which reveals the potential of applying SMF as a physical method in cancer therapy in the future.

Emerging roles of Cxcl12/Cxcr4 signaling axis in breast cancer metastasis

Abstract The Cxcl12/Cxcr4 axis promotes breast cancer metastasis in mice and humans. Prior studies have linked Cxcl12/Cxcr4 to cancer cell homing, survival and proliferation at distant sites secreting Cxcl12, such as lungs and liver. However, the mechanism via which Cxcr4+ cancer cells escape primary tumors in the first place (which also highly express Cxcl12), remains elusive. Here, by measuring chemotactic gradients using immunofluorescence, we show that Cxcl12 gradients are concentrically expressed around cancer cell intravasation sites, and spatially associate with Cxcr4+ tumor cells. Without affecting the gradient itself, pharmacological inhibition of Cxcl12 suppresses the translocation of Cxcr4+ cancer cells to intravasation sites. Paradoxically, tumor cell-specific deletion of Cxcr4 results in a suboptimal prometastatic response. We theorized that Cxcr4+ tumor-associated macrophages (TAMs), previously shown to accompany migratory tumor cells, could instead read the Cxcl12 chemotactic gradient, thus partially rescuing the prometastatic phenotype. Indeed, clodronate-mediated TAM depletion abrogates Cxcr4+ cancer cell translocation to intravasation sites and subsequent dissemination to secondary sites. We also found that breast cancer endothelium is lined by Cxcl12+Pdgfrb+ stromal cells; however, Cxcl12 gradients originating from intravasation sites are specifically tied to a subset of Cxcl12High perivascular TAMs. As such, pharmacological inhibition of Pdgfrb depletes Pdgfrb+Cxcl12+ stromal cells, but does not affect Cxcl12+ TAM-dependent translocation of Cxcr4+ tumor cells to intravasation sites. Overall, our data support a new model for the implication of Cxcl12/Cxcr4 in the early stages of metastasis.

CXCR1 and CXCR2 Chemokine Receptor Agonists Produced by Tumors Induce Neutrophil Extracellular Traps that Interfere with Immune Cytotoxicity

Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ Tcells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.

Proteomic characterization of early lung response to breast cancer metastasis in mice

IntroductionThe tumor-promoting rearrangement of the lungs facilitates the process of cancer cell survival in a foreign microenvironment and enables their protection against immune defense. The study aimed to define the fingerprint of the early rearrangement of the lungs via the proteomic profiling of the lung tissue in the experimental model of tumor metastasis in a murine 4T1 mammary adenocarcinoma. Materials and methodsThe studies were performed on 7-8-week-old BALB/c female mice. Viable 4T1 cancer cells were orthotopically inoculated into the right mammary fat pad. The experiment was performed in the early phase of the tumor metastasis one and two weeks after cancer cell inoculation. The comparative analysis of protein profiles was carried out with the aid of the two-dimensional difference in gel electrophoresis (2D-DIGE). Proteins, of which expression differed significantly, were identified using nano-liquid chromatography coupled to a high-resolution mass spectrometry (nanoLC/hybrid ion trap- Orbitrap XL Discovery). ResultsPalpable primary tumors were noted in the 2nd week after cancer cell inoculation. The investigated period preceded the formation of numerous macrometastases in the lungs, however the metastasis-promoting changes were visible very early. Primary tumor-induced inflammation developed in the lungs as early as after the 1st week and progressed during the 2nd week, accompanied by increased concentration of 2-OH-E+, an oxidative stress marker, and imbalance in nitric oxide metabolites, pointing to endothelium dysfunction. The early proteomic changes in the lungs in the 1st week after 4T1 cell inoculation resulted in the reorganization of lung tissue structure [actin, cytoplasmic 1 (Actb), tubulin beta chain (Tubb5), lamin-B1 (Lmnb1), serine protease inhibitor A3K (Serpina3k)] and activation of defense mechanisms [selenium-binding protein 1 (Selenbp1), endoplasmin (Hsp90b1), stress 70 protein, mitochondrial (Hspa9), heat shock protein HSP 90-beta (Hsp90ab1)], but also modifications in metabolic pathways [glucose-6-phosphate 1-dehydrogenase X (G6pdx), ATP synthase subunit beta, mitochondrial (Atp5b), L-lactate dehydrogenase B chain (Ldhb)]. Further development of the solid tumor after the 2nd week following cancer cell inoculation, secretion of prolific tumor-derived factors as well as the presence of the increasing number of circulating cancer cells and extravasation processes further impose reorganization of the lung tissue [Actb, vimentin (Vim), clathrin light chain A (Clta)], altering additional metabolic pathways [annexin A5 (Anxa5), Rho GDP-dissociation inhibitor 2 (Arhgdib), complement 1 Q subcomponent-binding protein, mitochondrial (C1qbp), 14-3-3 protein zeta/delta (Ywhaz), peroxiredoxin-6 (Prdx6), chitinase-like protein 4 (Chi3l4), reticulocalbin-1 (Rcn1), EF-hand domain-containing protein D2 (Efhd2), calumenin (Calu)]. Interestingly, many of differentially expressed proteins were involved in calcium homeostasis (Rcn1, Efhd2, Calu, Actb, Vim, Lmnb1, Clta, Tubb5, Serpina3k, Hsp90b1, Hsp90ab1, Hspa9. G6pdx, Atp5b, Anxa5, Arhgdib, Ywhaz). ConclusionThe analysis enabled revealing the importance of calcium signaling during the early phase of metastasis development, early cytoskeleton and extracellular matrix reorganization, activation of defense mechanisms and metabolic adaptations. It seems that the tissue response is an interplay between pro- and anti-metastatic mechanisms accompanied by inflammation, oxidative stress and dysfunction of the barrier endothelial cells.

Neutrophil extracellular traps promote peritoneal metastasis of colon cancer cells.

Cytoreductive surgery is the only curative option for patients with peritoneal carcinomatosis, however, intraperitoneal recurrence rate is high making new ways to prevent cancer recurrence an urgent need. Recent evidence suggests that neutrophils are involved in cancer progression. The purpose of our study was to examine the role of neutrophils in the spread of colon cancer cells in the peritoneal cavity.The number of metastatic noduli in the peritoneal cavity was quantified in mice injected with murine colon cancer cells (CT-26) intraperitoneally after surgical laparotomy and treated with a neutrophil depleting antibody or DNase I. In addition, peritoneal metastases were harvested from patients with peritoneal carcinomatosis. Scanning and transmission electron microscopy showed extensive neutrophil extracellular trap (NET) formation in peritoneal colon cancer metastases in mice and patients. Neutrophil depletion markedly reduced the number of metastases in laparotomised animals. Administration of DNase I decreased the number of metastatic nodules by 88% in laparotomised animals as well as NET-induced chemokine-dependent colon cancer cell migration and adhesion in vitro. Finally, CT-26 cancer cells were found to express the αvβ3 integrin and inhibition of αv integrin abolished NET-induced adhesion of colon cancer cells to vitronectin. Taken together, our data show that NETs play an important role in colon cancer cell metastasis in the peritoneal cavity and regulate colon cancer cell migration and adhesion to extracellular matrix proteins. These novel findings suggest that targeting NETs might be an effective strategy to antagonize intrabdominal recurrences of colon cancer after cytoreductive surgery in patients with peritoneal carcinomatosis.

Early and late endothelial response in breast cancer metastasis in mice: simultaneous quantification of endothelial biomarkers using a mass spectrometry-based method.

ABSTRACTThe endothelium plays an important role in cancer metastasis, but the mechanisms involved are still not clear. In the present work, we characterised the changes in endothelial function at early and late stages of breast cancer progression in an orthotopic model of murine mammary carcinoma (4T1 cells). Endothelial function was analysed based on simultaneous microflow liquid chromatography–tandem mass spectrometry using multiple reaction monitoring (microLC/MS-MRM) quantification of 12 endothelium-related biomarkers, including those reflecting glycocalyx disruption – syndecan-1 (SDC-1), endocan (ESM-1); endothelial inflammation – vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), E-selectin (E-sel); endothelial permeability – fms-like tyrosine kinase 1 (FLT-1), angiopoietin 2 (Angpt-2); and haemostasis – von Willebrand factor (vWF), tissue plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1), as well as those that are pathophysiologically linked to endothelial function – adrenomedullin (ADM) and adiponectin (ADN). The early phase of metastasis in mouse plasma was associated with glycocalyx disruption (increased SDC-1 and ESM-1), endothelial inflammation [increased soluble VCAM-1 (sVCAM-1)] and increased vascular permeability (Angpt-2). During the late phase of metastasis, additional alterations in haemostasis (increased PAI-1 and vWF), as well as a rise in ADM and substantial fall in ADN concentration, were observed. In conclusion, in a murine model of breast cancer metastasis, we identified glycocalyx disruption, endothelial inflammation and increased endothelial permeability as important events in early metastasis, while the late phase of metastasis was additionally characterised by alterations in haemostasis.

Specific Inhibition of Heparanase by a Glycopolymer with Well-Defined Sulfation Pattern Prevents Breast Cancer Metastasis in Mice.

Heparanase, the heparan sulfate polysaccharide degrading endoglycosidase enzyme, has been correlated with tumor angiogenesis and metastasis and therefore has become a potential target for anticancer drug development. In this systematic study, the sulfation pattern of the pendant disaccharide moiety on synthetic glycopolymers was synthetically manipulated to achieve optimal heparanase inhibition. Upon evaluation, a glycopolymer with 12 repeating units was determined to be the most potent inhibitor of heparanase (IC50 = 0.10 ± 0.36 nM). This glycopolymer was further examined for cross-bioactivity using a solution-based competitive biolayer interferometry assay with other HS-binding proteins (growth factors, P-selectin, and platelet factor 4), which are responsible for mediating angiogenic activity, cell metastasis, and antibody-induced thrombocytopenia. The synthetic glycopolymer has low affinity for these HS-binding proteins in comparison to natural heparin. In addition, the glycopolymer possessed no proliferative properties toward human umbilical endothelial cells (HUVECs) and a potent antimetastatic effect against 4T1 mammary carcinoma cells. Thus, our study not only establishes a specific inhibitor of heparanase with high affinity but also illustrates the high effectiveness of this multivalent heparanase inhibitor in inhibiting experimental metastasis in vivo.

USP1 inhibition destabilizes KPNA2 and suppresses breast cancer metastasis.

Metastatic progression is the main cause of mortality in breast cancer, necessitating the determination of the molecular events driving this process for the development of new therapeutic approaches. Here, we demonstrate that hyperactivation of the deubiquitinase USP1 contributes to breast cancer metastasis. Upregulated USP1 expression in primary breast cancer specimens correlates with metastatic progression and poor prognosis in breast cancer patients. USP1 enhances the expression of a number of pro-metastatic genes in breast cancer cells, promotes cell migration and invasion in vitro, and facilitates lung metastasis of breast cancer cells. Moreover, USP1-mediated deubiquitination and stabilization of KPNA2 are revealed as the downstream events crucial for USP1-pro-metastatic function. Most importantly, pharmacological intervention of USP1 function by pimozide or ML323 significantly represses breast cancer metastasis in mice, suggesting a rationale for using USP1 inhibitors for treatment of patients with breast cancer. Taken together, our results establish USP1 as a promoter of breast cancer metastasis and provide evidence for the potential practice of USP1 targeting in the treatment of breast cancer.

Abstract 3149: Collagen XIII induces β1-integrin activation and triple-negative breast cancer metastasis

Abstract Increased collagen expression and deposition is a risk factor for breast cancer development. Collagen XIII is a type II transmembrane protein within the collagen superfamily. Experiments in tissue culture and knockout mouse model show that collagen XIII is involved in cell adhesion and differentiation of certain cell types. However, function of collagen XIII in cancer development and progression largely remains to be determined. We found that collagen XIII expression is significantly induced during breast cancer development, especially in triple negative breast cancer. Increased collagen XIII mRNA level is associated with poor prognosis. Importantly, silencing collagen XIII expression in MDA MB 231 cells slow down xenograft growth and significantly reduced cancer metastasis in mice. Using gain- and loss-of function approaches, we showed that collagen XIII enhanced cell migration, invasion, and mammosphere/tumorsphere formation. We also found that collagen XIII induced integrin-β1 activation, and subsequently enhanced TGF-β signaling. Blocking integrin-β1 activation with AIIB2 antibody significantly reduced collagen XIII-induced cell migration and mammosphere formation. These findings demonstrate a novel function of collagen XIII in promoting cancer metastasis through enhancing β1 integrin-dependent cell migration and invasion Citation Format: Hui Zhang, Tricia Fredericks, Ren Xu. Collagen XIII induces β1-integrin activation and triple-negative breast cancer metastasis [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 3149.

Complement 5a stimulates macrophage polarization and contributes to tumor metastases of colon cancer

Inflammatory cells such as macrophages can play a pro-tumorigenic role in the tumor stroma. Tumor-associated macrophages (TAMs) generally display an M2 phenotype with tumor-promoting activity; however, the mechanisms regulating the TAM phenotype remain unclear. Complement 5a (C5a) is a cytokine-like polypeptide that is generated during complement system activation and is known to promote tumor growth. Herein, we investigated the role of C5a on macrophage polarization in colon cancer metastasis in mice. We found that deficiency of the C5a receptor (C5aR) severely impairs the metastatic ability of implanted colon cancer cells. C5aR was expressed on TAMs, which exhibited an M2-like functional profile in colon cancer liver metastatic lesions. Furthermore, C5a mediated macrophage polarization and this process relied substantially on activation of the nuclear factor-kappa B (NF-κB) pathway. Finally, analysis of human colon carcinoma indicated that C5aR expression is negatively associated with tumor differentiation grade. Our results demonstrate that C5aR has a central role in regulating the M2 phenotype of TAMs, which in turn, contributes to hepatic metastasis of colon cancer through NF-κB signaling. C5a is a potential novel marker for cancer prognosis and drugs targeting complement system activation, specifically the C5aR pathway, may offer new therapeutic opportunities for colon cancer management.

Abstract 3908: The mechanism of premetastatic niche formation through FBXW7/NOTCH/CCL2 pathway and clinical trial using CCL2 inhibitor “SK-818” for breast cancer patients

Abstract Background Metastasis is a major cause of death in cancer patients, & elucidation of the mechanisms is expected to provide a basis for the development of new cancer treatment. Premetastatic niche whereby tumors prepare defined organs for metastasized cells is considered to be crucial for the development of metastasis, which consists of host cells such as monocytes, macrophages, & other inflammatory cells. However, the mechanism of how tumor cells communicate with host cells to develop a premetastatic niche remains unclear. FBXW7 is the F-box protein component of an SCF-type ubiquitin ligase, in which it functions as a receptor responsible for substrate recognition followed by the degradation of the substrate. Some part of the substrates of FBXW7 regulates the recruitment of monocytes, macrophages, & other inflammatory cells. Therefore, we hypothesized that expression of FBXW7 in the host cells is a key determinant of the formation of premetastatic niches. Materials & Methods To investigate the role of FBXW7 in the formation of premetastatic niche, we developed bone marrow-specific FBXW7-deficient mice. 1. We transferred B16F10 melanoma cells or E0771 mouse breast cancer cells into the tail vein or mammary fat pad of the mice & the wild type mice to evaluate the metastatic potential to lung. 2. We examined the serum concentrations of various cytokines or chemokines in the mice before & after E0771 cells transplantation using cytokine array. 3. To examine whether the enhanced metastasis apparent in FBXW7-deficient mice is dependent on the CCL2/CCR2 pathway, we treated FBXW7-deficient mice with SK-818, a CCR2 antagonist. 4. We measured the expression of FBXW7 mRNA in peripheral blood of breast cancer patients by qRT-PCR & examined the relationship between FBXW7 mRNA expression & prognosis. Results 1. The extent of lung metastasis of B16F10 or E0771 cell was markedly enhanced in FBXW7-deficient mice. 2. FBXW7-deficient mice exhibited increased serum levels of CCL2. Moreover, accumulation of NOTCH & consequent transcriptional activation of CCL2 was observed in bone marrow-derived stromal cells of FBXW7-deficient mice. 3. Administration of SK-818 blocked the enhancement of lung metastasis in FBXW7-deficient mice. 4. In human breast cancer patients, low FBXW7 mRNA expression in peripheral blood showed high concentration of CCL2 in serum & poor prognosis. Conclusions We found that the FBXW7/NOTCH/CCL2 pathway play a key role in the regulation of cancer metastasis through the formation of premetastatic niches, & that SK-818 inhibit cancer metastasis in mice. SK-818 is currently administered clinically for the treatment of patients with hepatitis B virus infection in Japan. Based on these findings, we have been conducting Phase I clinical trial to assess the safety of SK-818 for breast cancer patients. Citation Format: Miwa Noda, Takaaki Masuda, Naoki Hayashi, Takahiro Kogawa, Shinji Ohno, Keiichi Nakayama, Yohsuke Kuroda, Hidetoshi Eguchi, Shuhei Ito, Masafumi Inomata, Koshi Mimori. The mechanism of premetastatic niche formation through FBXW7/NOTCH/CCL2 pathway and clinical trial using CCL2 inhibitor “SK-818” for breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3908. doi:10.1158/1538-7445.AM2017-3908

Aspirin prevents colorectal cancer metastasis in mice by splitting the crosstalk between platelets and tumor cells.

We investigated whether platelets prime colon cancer cells for metastasis and whether pharmacological inhibition of platelet function may prevent it. Coculturing HT29 human colon carcinoma cells with human platelets led to the induction of mesenchymal-like cancer cells characterized by downregulation of E-cadherin and upregulation of Twist1, enhanced cell mobility and a proaggregatory action on platelets. These changes were prevented by different antiplatelet agents, aspirin[an inhibitor of cyclooxygenase(COX)-1], DG-041[an antagonist of prostaglandin(PG)E2 EP3 receptor] and ticagrelor (a P2Y12 receptor antagonist). The injection of HT29 cells, exposed to platelets in vitro, into the tail vein of humanized immunodeficient mice led to higher incidence of lung metastasis compared to the injection of untreated HT29 cells. This effect was associated with enhanced systemic biosynthesis of thromboxane(TX)A2 and PGE2 in vivo. Platelet COX-1 inhibition by aspirin administration to mice prevented the increased rate of metastasis as well as the enhanced production of TXA2 and PGE2 induced by the in vitro priming of HT29 cells by platelets. In conclusion, targeting platelet COX-1 with low-dose aspirin exerts an antimetastatic action by averting the stem cell mimicry of cancer cells associated with enhanced proaggregatory effects induced by platelet-tumor cell interactions. These effects may be shared by other antiplatelet drugs.

Abstract B53: LKB1 kinase-dependent and -independent defects disrupt polarity and adhesion signaling to create a uniquely invasive amoeboid cancer cell

Abstract The tumor suppressor LKB1 is a serine/threonine kinase and the 3rd most commonly mutated gene in lung adenocarcinoma. The majority of LKB1 mutations are truncations that predictably disrupt its kinase activity and remove its C-terminal domain (CTD). Since LKB1 inactivation drives cancer metastasis in mice and leads to aberrant cell invasion in vitro, we sought to determine how domain-specific LKB1 inactivation impacts lung cancer cell adhesion and polarity during 3D invasion. We generated lung cancer cell lines stably expressing empty GFP or GFP-tagged LKB1: wildtype, a farnesylation motif mutant, a kinase dead mutant, CTD (lacking kinase activity) and the CTD containing the farnesylation motif mutant, and then embedded spheroids of these cells in a 3D collagen matrix. Using confocal immunofluorescence and live cell imaging, we show cells re-expressing either wildtype LKB1 or the LKB1 CTD alone exhibited a mesenchymal polarity with strong directional persistence, which is completely abrogated upon loss of farnesylation. Given that the CTD lacks kinase activity, these data highlight a farnesylation-dependent but kinase-independent regulation of mesenchymal polarity and directional persistence during invasion. We next examined Rho-GTPase activity in response to LKB1 farnesylation and show that the LKB1 CTD alone is sufficient to activate RhoA. This activation is again completely abrogated upon loss of LKB1 farnesylation, highlighting another farnesylation-dependent but kinase-independent regulation. Given that farnesylation regulates both mesenchymal polarity and RhoA activity, we next probed the relationship between mesenchymal polarity and RhoA by creating cell lines re-expressing either constitutively active RhoA or cdc42. We show that restoring RhoA activity, but not cdc42, in farnesylation-compromised cells restores mesenchymal polarization and directional persistence during invasion. Importantly, restoring RhoA activity in the absence of LKB1 fails to restore mesenchymal polarity, indicating that a region of LKB1 must be necessary for the regulation of polarity through RhoA. We next examined the role of LKB1 in regulating focal adhesion kinase (FAK) activity during invasion. While farnesylation does not affect FAK activity, LKB1 signals to MARK1 in a kinase-dependent manner to repress FAK, which then represses collagen remodeling during 3D invasion. Since LKB1 frequently undergoes truncating mutations that predictably affect both farnesylation and kinase activity, cancer cells with LKB1 loss would exhibit a unique amoeboid morphology with both hyperactive FAK and the ability to remodel collagen. Together, these data suggest that it is a combination of kinase-dependent and -independent defects that create a uniquely invasive cell upon LKB1 inactivation. Citation Format: Scott Wilkinson, Jessica Konen, Adam I. Marcus. LKB1 kinase-dependent and -independent defects disrupt polarity and adhesion signaling to create a uniquely invasive amoeboid cancer cell. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B53.

LKB1 kinase-dependent and -independent defects disrupt polarity and adhesion signaling to drive collagen remodeling during invasion.

LKB1 is a serine/threonine kinase and a commonly mutated gene in lung adenocarcinoma. The majority of LKB1 mutations are truncations that disrupt its kinase activity and remove its C-terminal domain (CTD). Because LKB1 inactivation drives cancer metastasis in mice and leads to aberrant cell invasion in vitro, we sought to determine how compromised LKB1 function affects lung cancer cell polarity and invasion. Using three-dimensional models, we show that LKB1 kinase activity is essential for focal adhesion kinase-mediated cell adhesion and subsequent collagen remodeling but not cell polarity. Instead, cell polarity is overseen by the kinase-independent function of its CTD and more specifically its farnesylation. This occurs through a mesenchymal-amoeboid morphological switch that signals through the Rho-GTPase RhoA. These data suggest that a combination of kinase-dependent and -independent defects by LKB1 inactivation creates a uniquely invasive cell with aberrant polarity and adhesion signaling that drives invasion into the microenvironment.

A monoclonal antibody against KCNK9 K(+) channel extracellular domain inhibits tumour growth and metastasis.

Two-pore domain potassium (K2P) channels act to maintain cell resting membrane potential—a prerequisite for many biological processes. KCNK9, a member of K2P family, is implicated in cancer, owing to its overexpression in human tumours and its ability to promote neoplastic cell survival and growth. However, KCNK9's underlying contributions to malignancy remain elusive due to the absence of specific modulators. Here we describe the development of monoclonal antibodies against the KCNK9 extracellular domain and their functional effects. We show that one antibody (Y4) with the highest affinity binding induces channel internalization. The addition of Y4 to KCNK9-expressing carcinoma cells reduces cell viability and increases cell death. Systemic administration of Y4 effectively inhibits growth of human lung cancer xenografts and murine breast cancer metastasis in mice. Evidence for Y4-mediated carcinoma cell autonomous and immune-dependent cytotoxicity is presented. Our study reveals that antibody-based KCNK9 targeting is a promising therapeutic strategy in KCNK9-expressing malignancies.

SPON2, a newly identified target gene of MACC1, drives colorectal cancer metastasis in mice and is prognostic for colorectal cancer patient survival.

MACC1 (metastasis associated in colon cancer 1) is a prognostic biomarker for tumor progression, metastasis and survival of a variety of solid cancers including colorectal cancer (CRC). Here we aimed to identify the MACC1-induced transcriptome and key players mediating the MACC1-induced effects in CRC. We performed microarray analyses using CRC cells ectopically overexpressing MACC1. We identified more than 1300 genes at least twofold differentially expressed, including the gene SPON2 (Spondin 2) as 90-fold upregulated transcriptional target of MACC1. MACC1-dependent SPON2 expression regulation was validated on mRNA and protein levels in MACC1 high (endogenously or ectopically) and low (endogenously or by knockdown) expressing cells. Chromatin immunoprecipitation analysis demonstrated the binding of MACC1 to the gene promoter of SPON2. In cell culture, ectopic SPON2 overexpression induced cell viability, migration, invasion and colony formation in endogenously MACC1 and SPON2 low expressing cells, whereas SPON2 knockdown reduced proliferative, migratory and invasive abilities in CRC cells with high endogenous MACC1 and SPON2 expression. In intrasplenically transplanted NOD/SCID mice, metastasis induction was analyzed with control or SPON2-overexpressing CRC cells. Tumors with SPON2 overexpression induced liver metastasis (vs control animals without any metastases, P=0.0036). In CRC patients, SPON2 expression was determined in primary tumors (stages I-III), and survival time was analyzed by Kaplan-Meier method. CRC patients with high SPON2 expressing primary tumors demonstrated 8 months shorter metastasis-free survival (MFS) compared with patients with low SPON2 levels (P=0.053). Combining high levels of SPON2 and MACC1 improved the identification of high-risk patients with a 20-month shorter MFS vs patients with low biomarker expression. In summary, SPON2 is a transcriptional target of the metastasis gene MACC1. SPON2 induces cell motility in vitro and CRC metastasis in mice. In patients, SPON2 serves as prognostic indicator for CRC metastasis and survival, and might represent a promising target for therapeutic approaches.

Safety evaluation of intravenously administered mono-thioated aptamer against E-selectin in mice

The medical applications of aptamers have recently emerged. We developed an antagonistic thioaptamer (ESTA) against E-selectin. Previously, we showed that a single injection of ESTA at a dose of 100μg inhibits breast cancer metastasis in mice through the functional blockade of E-selectin. In the present study, we evaluated the safety of different doses of intravenously administered ESTA in single-dose acute and repeat-dose subacute studies in ICR mice. Our data indicated that intravenous administration of up to 500μg ESTA did not result in hematologic abnormality in either study. Additionally, intravenous injection of ESTA did not affect the levels of plasma cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, GM-CSF, IFN-γ, and TNF-α) or complement split products (C3a and C5a) in either study. However, repeated injections of ESTA slightly increased plasma ALT and AST activities, in accordance with the appearance of small necrotic areas in the liver. In conclusion, our data demonstrated that intravenous administration of ESTA does not cause overt hematologic, organs, and immunologic responses under the experimental conditions.

Natural killer T cell activation overcomes immunosuppression to enhance clearance of postsurgical breast cancer metastasis in mice

Metastatic lesions are responsible for over 90% of breast cancer associated deaths. Therefore, strategies that target metastasis are of particular interest. This study examined the efficacy of natural killer T (NKT) cell activation as a post-surgical immunotherapy in a mouse model of metastatic breast cancer. Following surgical resection of orthotopic 4T1 mammary carcinoma tumors, BALB/c mice were treated with NKT cell activating glycolipid antigens (α-GalCer, α-C-GalCer or OCH) or α-GalCer-loaded dendritic cells (DCs). Low doses of glycolipids transiently reduced metastasis but did not increase survival. A high dose of α-GalCer enhanced overall survival, but was associated with increased toxicity and mortality at early time points. Treatment with α-GalCer-loaded DCs limited tumor metastasis, prolonged survival, and provided curative outcomes in ∼45% of mice. However, survival was not increased further by additional DC treatments or co-transfer of expanded NKT cells. NKT cell activation via glycolipid-loaded DCs decreased the frequency and immunosuppressive activity of myeloid derived suppressor cells (MDSCs) in tumor-resected mice. In vitro, NKT cells were resistant to the immunosuppressive effects of MDSCs and were able to reverse the inhibitory effects of MDSCs on T cell proliferation. NKT cell activation enhanced antitumor immunity in tumor-resected mice, increasing 4T1-specific cytotoxic responses and IFNγ production from natural killer (NK) cells and CD8+ T cells. Consistent with increased tumor immunity, mice surviving to day 150 were resistant to a second tumor challenge. This work provides a clear rationale for manipulating NKT cells to target metastatic disease.

Interruption of CCL2 Inhibition Enhances Metastatic Disease

Abstract Discontinuation of anti-CCL2 therapy augments lethal breast cancer metastasis in mice.

Abstract PR14: Quantitative and multiparameter analysis of tumor cell intravasation

Abstract Multiple steps in cancer metastasis, including tumorigenesis, tumor angiogenesis, invasion and colonization, can be successfully examined with well-established models. However, due to deficiency in experimental model systems that would accurately recapitulate tumor cell entry into the host vasculature, intravasation remains the least studied step in the metastatic cascade. Our laboratory has developed several independent avian and murine model systems to investigate the complex process of intravasation in a quantifiable and mechanistic manner. Chick embryo models of intravasation are based on the ability of a highly vascularized tissue, the chorioallantoic membrane (CAM), to sustain primary tumor development and spontaneous metastasis. Furthermore, the CAM serves as a repository of newly intravasated cells, which are quantified by human-specific Alu-qPCR. By employing different CAM models, the early events in spontaneous metastasis that ultimately lead to intravasation can be dissected and their individual input to overall intravasation can be quantified. These early processes include cell escape from the primary tumor, establishment of intratumoral and extratumoral angiogenic networks, invasion of adjacent stroma by escaped tumor cells, and migration of escaped tumor cells along tumor-associated blood vessels. In conjunction with confirmatory models of cancer metastasis in mice, we have identified several molecular systems that functionally contribute to the intravasation process. To investigate the specific mechanisms whereby tumor cells enter the angiogenic blood vessels, we have developed a new high-resolution confocal microscopy model based on acquisition and multiparameter analysis of primary microtumors imaged as entire units. This model was adapted to primary tumors developing within the mesoderm of the CAM as well as primary tumors developing in the dermis of mouse ear. In both avian and murine models, the volume of microtumors, the number of tumor cells per tumor, and the total volume of tumor-associated vasculature can be determined from 3D-reconstructed Z-stacks. Most importantly, the volume of intravascular tumor cells can be quantified by colocalizing tumor-vascular signals with a stringent threshold of a minimum volume of an individual tumor cell so that the cells that are localized within the vessels could be discriminated from the cells adherent to the abluminal vessel surface. In addition, high-resolution imaging of whole microtumors allows to visualize and quantify the tumor cells that appear as entering into the vasculature or in close association with the abluminal surface of the vessels. This approach allowed us for the fist time to visualize the intravasation events in numbers rendering statistical significance. By employing unique cell and protein markers in this novel system, the vascular cells interacting with the intravasating tumor cells can be identified and the candidate molecules involved in the close tumor-vascular interactions can be probed by treating the developing microtumors with specific reagents and small molecule inhibitors. By using these novel high-resolution models for intravasation, we have documented a majority of intravasation events within the primary tumor or in the nearest proximity to the primary tumor border. These data challenge the widely accepted concept that tumor cell escape, stromal invasion, and entry of escaped tumor cells into blood vessels adjacent to the primary tumor, are prerequisites of tumor cell intravasation. In contrast, our findings indicate that the bulk of cell intravasation events can take place within the primary tumor and prior to significant stromal invasion and migration of escaped tumor cells towards and along tumor-associated blood vessels. Our data suggest that tumor cell intravasation and metastatic spread can occur much earlier than commonly acknowledged by oncologists based on histological examination of resected tumors. This abstract is also presented as Poster A24. Citation Format: Elena I. Deryugina, William B. Kiosses, James P. Quigley. Quantitative and multiparameter analysis of tumor cell intravasation. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr PR14.