Mouse Model Of Pulmonary Metastasis Research Articles

Identification and Characterization of Metastasis-Initiating Cells in ESCC in a Multi-Timepoint Pulmonary Metastasis Mouse Model.

Metastasis is the biggest obstacle to esophageal squamous cell carcinoma (ESCC) treatment. Single-cell RNA sequencing analyses are applied to investigate lung metastatic ESCC cells isolated from pulmonary metastasis mouse model at multiple timepoints to characterize early metastatic microenvironment. A small population of parental KYSE30 cell line (Cluster S) resembling metastasis-initiating cells (MICs) is identified because they survive and colonize at lung metastatic sites. Differential expression profile comparisons between Cluster S and other subpopulations identified a panel of 7 metastasis-initiating signature genes (MIS), including CD44 and TACSTD2, to represent MICs in ESCC. Functional studies demonstrated MICs (CD44high) exhibited significantly enhanced cell survival (resistances to oxidative stress and apoptosis), migration, invasion, stemness, and in vivo lung metastasis capabilities, while bioinformatics analyses revealed enhanced organ development, stress responses, and neuron development, potentially remodel early metastasis microenvironment. Meanwhile, early metastasizing cells demonstrate quasi-epithelial-mesenchymal phenotype to support both invasion and anchorage. Multiplex immunohistochemistry (mIHC) staining of 4 MISs (CD44, S100A14, RHOD, and TACSTD2) in ESCC clinical samples demonstrated differential MIS expression scores (dMISs) predict lymph node metastasis, overall survival, and risk of carcinothrombosis.

Abstract 729: IDO1 inhibition enables IFNγ-elicited responsiveness of pulmonary breast cancer metastases to hypoxia-directed tumoricidal agents

Abstract In mouse models of lung carcinoma and pulmonary breast cancer metastasis, genetic ablation of the tryptophan catabolizing enzyme IDO1 (indoleamine 2,3-dioxygenase 1) resulted in restricted tumor outgrowth associated with attenuated induction of the inflammatory cytokine IL6 (interleukin 6). Utilizing the 4T1 breast cancer metastasis model to further elucidate the mechanistic basis underlying IDO1’s pro-tumor activity in the lungs established that IDO1 acts as an integral determinant of a tumor-promoting inflammatory state that supports neovascular retention. Ido1−/− (nullizygous) mice exhibited impaired pulmonary metastases neovascularization and outgrowth that was driven by IFNγ (interferon gamma) in the absence of IDO1-dependent IL6 production. Hypoxia was likewise elevated, and these stress conditions increased the susceptibility of the metastatic tumor cells to cytotoxicity associated with administration of GSK2656157, an inhibitor of PERK (protein kinase RNA-like ER kinase), which acts as a primary sensor for activating the UPR (unfolded protein response) survival pathway. In WT (wild type) mice with established 4T1 lung metastases, administration of the IDO1 inhibitors epacadostat, navoximod or indoximod each similarly resulted in rapid collapse of the metastatic tumor neovasculature and concomitantly increased intratumoral hypoxia and sensitivity to PERK inhibition. Therefore, while IDO1 inhibition does not directly promote effective tumor cell killing in this context, the disruption of blood supply due to IFNγ-mediated neovascular regression subjects the tumor cells to hypoxic environmental stress, raising the possibility for therapeutic intervention with selectively targeted agents. Combining chemotherapy with immune checkpoint blockade (ICB) is recognized as an effective strategy for treating lung cancer, with first-line therapy for some patients including the alkylating agent carboplatin in conjunction with pembrolizumab. The challenge remains, however, that chemotherapy causes systemic side effects that can negatively impact quality of life and limit efficacy. Evofosfamide is an alkylating prodrug that was designed to be selectively activated in regions of hypoxia, thereby limiting systemic exposure. We report that enhanced metastatic tumor cell killing with evofosfamide can be achieved by co-administration of an IDO1 inhibitor. Elevated intratumoral hypoxia has also previously been linked to increased expression levels of PDL1, a favorable indicator of ICB responsiveness, and we have confirmed the upregulation of PDL1 in 4T1 lung metastases following IDO1 inhibitor administration. Our data in the lung metastasis model support future studies to evaluate whether IDO1 inhibition can be effectively combined with evofosfamide and anti-PD1 treatment to improve therapeutic outcomes. Citation Format: Shih-Chun Shen, Souvik Dey, James B. DuHadaway, Erika Sutanto-Ward, George C. Prendergast, Alexander J. Muller. IDO1 inhibition enables IFNγ-elicited responsiveness of pulmonary breast cancer metastases to hypoxia-directed tumoricidal agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 729.

Vitamin C Inhibited Pulmonary Metastasis through Activating Nrf2/HO-1 Pathway.

As an important nutritional component, vitamin C (Vc) shows good antitumor activity in a variety of cancer, but there are few studies in pulmonary metastasis. In order to verify its anticancer and antimetastatic effect, the study sets up H22 pulmonary metastasis mouse model. The results show that intraperitoneal injection of Vc inhibits pulmonary metastasis through up-regulating the expression of Nrf2, HO-1, cleaved caspases 3 and 9, and causing DNA damage and apoptosis which is similar to the pro-oxidant effect of Vc in p53 null cells (H1299 cells). Meanwhile, oral administration of Vc up-regulates the expression of p53, directly activates Nrf2/HO-1 pathway, increases expression of cleaved caspases 3 and 9, and ultimately inhibits pulmonary metastasis, which is the same as the antioxidant result of Vc in p53 wild-type cells. In addition, Vc inhibits the proliferation and migration of lung cancer cells in a concentration-dependent manner and has little cytotoxic effects on normal cells. Notably, the experiment further illustrates that besides intravenous Vc, oral Vc significantly inhibits the pulmonary metastasis in mice. All in all, these findings provide new clues for Vc-treated pulmonary metastasis in clinical research.

The Combination of SHOX2 and RASSF1A DNA Methylation Had a Diagnostic Value in Pulmonary Nodules and Early Lung Cancer

Introduction: The study explored the effects of SHOX2 and RASSF1A DNA methylation in lung cancer (LC). Method: Bronchoalveolar lavage fluid (BALF) samples as well as LC and normal adjacent tissues were collected from 72 LC patients and 35 patients with benign pulmonary nodules. Quantitative analysis of SHOX2 and RASSF1A DNA methylation was performed in benign pulmonary nodules and different stages of LC. The diagnostic value of SHOX2 and RASSF1A DNA methylation in LC and benign pulmonary nodules was determined by receiver operating characteristics analysis. Gain/loss-of-function experiments were constructed in LC cells and mouse models of xenograft and pulmonary nodule metastasis. The levels of SHOX2 and transfer-associated genes were tested through quantitative reverse transcription polymerase chain reaction and Western blot. Malignant phenotype of LC cells was assessed by functional experiment. The tumor volume and weight of mice in xenograft models were measured. Pulmonary nodule metastasis was determined through HE staining assay. 5-azacytidine appeared as a positive control drug. Result: SHOX2 DNA methylation or RASSF1A DNA methylation had diagnostic efficiency in pulmonary nodules and early LC, with the two combined having better diagnostic value. SHOX2 expression was upregulated in LC. Similar to 5-azacytidine, SHOX2 knockdown inhibited LC cell viability, migration, and invasion in vitro as well as restrained LC tumorigenesis and pulmonary nodule metastasis in vivo, whereas overexpressed SHOX2 had the opposite effects. Conclusion: The combination of SHOX2 and RASSF1A DNA methylation had a diagnostic value in pulmonary nodules and early LC. SHOX2 positively modulated the tumorigenesis and metastasis of LC by regulating DNA methylation processes.

Abstract B009: Tumoral activation of endothelium drives macrophages-mediated metastatic niche formation and promotes lung metastasis

Abstract Primary tumor induces macrophages-enriched environment to establish metastatic niche which enhances homing and colonization of circulating tumor cells. However, the molecular mechanism underlying endothelial remodeling that favors macrophages infiltration and subsequent niche formation is not fully understood. Here, we found that tumor-derived factors activated endothelial cells to increase adhesive efficacy of monocytes to the endothelium, leading to macrophage infiltration. In mouse models of pulmonary metastasis, this endothelial activation triggered formation of premetastatic niche via accumulation of macrophages in the lungs. Of note, macrophages primed the metastatic microenvironment through enhancing expression of niche-related genes including S100A8, S100A9, MMP9 and fibronectin within the premetastatic lungs and directly transmitted survival signal to tumor cells in a contact-dependent manner. Furthermore, we demonstrated that depletion of macrophages specifically during premetastatic stages reduced niche formation and also resulted in reduced metastatic burden. These findings suggest that endothelial remodeling at metastatic site is a key step for initiation of premetastatic niche formation supported by macrophages. Targeting this step could present an opportunity for therapeutic intervention of metastatic spread in patients with malignant cancers. Citation Format: Hyewon Chung, Sang Wha Kim, Seung Hyeok Seok. Tumoral activation of endothelium drives macrophages-mediated metastatic niche formation and promotes lung metastasis [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B009.

ZIM3 activation of CCL25 expression in pulmonary metastatic nodules of osteosarcoma recruits M2 macrophages to promote metastatic growth.

Tumor-associated macrophages (TAMs) play an important role in tumor growth and metastasis. However, the involvement of TAMs infiltration in pulmonary osteosarcoma (OS) metastasis remains poorly understood. Therefore, the effect of OS cells on macrophages migration was investigated by in vivo and in vitro experiments to evaluate the infiltration and mechanism of TAMs in pulmonary OS metastases. The results showed that the zinc finger protein ZIM3 was upregulated in OS cells than in osteoblasts and activated the expression of CCL25, which subsequently promoted the migration of M2 macrophages. CCL25 or ZIM3 silencing in OS cells inhibited the infiltration of M2 macrophages and the formation of pulmonary metastatic nodules in a mouse model of pulmonary OS metastasis and prolonged the survival of the mice. Furthermore, bioinformatics analyses revealed that CCL25 and ZIM3 expressions are negatively correlated with the prognosis of OS patients. In conclusion, this study found that a large number of M2 TAMs were recruited into pulmonary metastatic nodules of OS through the activation of the ZIM3-CCL25 axis in OS cells, thereby facilitating OS metastasis. Therefore, the suppression of ZIM3-CCL25-induced recruitment of M2 TAMs to the metastatic sites might be considered as a therapeutic approach to inhibit the growth of pulmonary OS metastases.

Silencing of UBE2D1 inhibited cell migration in gastric cancer, decreasing ubiquitination of SMAD4

BackgroundGastric cancer (GC) is the second leading cause of cancer-related deaths. Because it is hard to diagnose at early stage, the overall 5 years survival rate is lower than 25%. High migration is the main hallmark of malignant cells at advanced stage of GC. Thus, it is urgent to find biomarkers for early diagnosis and more effective therapy of GC.MethodsIn this study, lentivirus-mediated silencing and overexpression lentiviruses targeting the ubiquitin-conjugating enzyme E2 D1 (UBE2D1), transwell, wound healing, and pulmonary metastasis mouse model were applied to analyze the function of UBE2D1 in vitro and in vivo. Real-time PCR and immunohistochemistry were used to elucidate the level of UBE2D1 in GC samples.ResultsSilencing of UBE2D1 inhibited cell migration and the levels of epithelial-mesenchymal transition makers (MMP2 and MMP9) in AGS and MKN45 cells. Silencing of UBE2D1 inhibited cell metastasis in mouse model. On the contrary, UBE2D1 overexpression increased cell migration and the levels of MMP2 and MMP9 in MGC-803 cells. Further, silencing of UBE2D1 decreased the ubiquitination level of mothers against decapentaplegic homolog 4 (SMAD4), and the increase of cell migration induced by UBE2D1 overexpression could be reversed by SMAD4.ConclusionSilencing of UBE2D1 inhibited cell migration through transforming growth factor β (TGF-β)/SMAD4 signaling pathway in GC.

Baicalin, a Potent Inhibitor of NF-κB Signaling Pathway, Enhances Chemosensitivity of Breast Cancer Cells to Docetaxel and Inhibits Tumor Growth and Metastasis Both In Vitro and In Vivo.

ObjectiveThe aim of this study is to investigate the anti-cancer activity and sensibilization of baicalin (BA) against breast cancer (BC) cells.MethodsThe anti-proliferation of BA in BC cell lines was evaluated by MTT and colony formation assays. Apoptotic induction of BA was measured by flow cytometry. Wound-healing and transwell assays were exploited to assess migrated and invasive inhibition of BA. Western-blot and immunofluorescence were used to study mechanisms of anti-migration and sensibilization of BA. Anti-tumor and anti-metastasis effects of BA were evaluated in subcutaneous and pulmonary metastasis mouse model of BC cells.ResultsBA significantly suppressed proliferation and induced apoptosis of BC cells in a concentration- and time-dependent manner. Additionally, BA induced cell apoptosis via the mitochondria-mediated pathway, as evidenced by cellular induction of reactive oxygen species and upregulated expression of the Bax/Bcl-2 ratio. The overall expression and nuclear translocation of NF-κB signaling pathway in BC cells were dramatically inhibited by treatment with BA. BA significantly suppressed abilities of migration and invasion in BC cells. Notably, BA sensitized BC cells to docetaxel (DXL) by suppressing the expression of survivin/Bcl-2. BA also retarded tumor growth and triggered apoptosis of tumor cells in a tumor mouse model of 4T1 cells. Furthermore, pulmonary metastasis of BC cells was distinctly suppressed by BA in a tumor mouse model of 4T1 cells.ConclusionBA effectively triggered apoptosis, inhibited metastasis, and enhanced chemosensitivity of BC, implying that BA might serve as a promising agent for the treatment of BC.

Ophiopogon Saponin C1 Inhibits Lung Tumors by Stabilizing Endothelium Permeability via Inhibition of PKCδ.

As the most frequent cause of cancer-related death worldwide, lung cancer is closely related to inflammation. The interaction between tumor cells and inflammatory cells promotes tumor development and metastasis. During tumor development, vascular endothelial cells form the most important barrier to prevent tumor cell migration to the blood and tissue. Increased vascular permeability provides favorable conditions for the migration of tumor cells, and endothelial tight junctions are an important component of the vascular barrier. Protein kinase C δ is involved in the occurrence of non-small cell lung cancer and regulates vascular permeability and tight junction protein expression. Src kinase was reported to play an important role in TNF-α-induced endothelial inflammation. Ophiopogon Saponin C1 is a new chemical compound isolated from Liriope muscari, but its pharmacological activities have not been fully elucidated. Therefore, we tested the protective effects of C1 on endothelial permeability in a model of TNF-α-induced endothelial inflammation by transendothelial electrical resistance and sodium fluorescein assays and verified these results in a nude mouse model of experimental pulmonary adenocarcinoma metastasis. We further elucidated the mechanism of C1, which was based on the PKCδ and Src proteins, by Western blotting. C1 can inhibit lung cancer in vivo, regulate the level of plasma inflammation in tumor-bearing mice, and protect the pulmonary vascular barrier against injury induced by cancer. It was investigated the expression and distribution of the TJ index protein ZO-1 in mouse vascular endothelium and HUVECs and found that C1 could inhibit the degradation and breakage of the ZO-1 protein. Related signaling experiments confirmed that C1 can inhibit TNF-α and activation of PKCδ and Src kinase. This study laid the foundation for further analysis of new drugs with clear mechanisms and independent intellectual property rights of traditional Chinese medicines.

Garcinol inhibits esophageal cancer metastasis by suppressing the p300 and TGF-β1 signaling pathways

Metastasis causes the main lethality in esophageal cancer patient. Garcinol, a natural compound extracted from Gambogic genera, is a histone acetyltransferase (HAT) inhibitor that has shown anticancer activities such as cell cycle arrest and apoptosis induction. In this study, we investigated the effects of garcinol on the metastasis of esophageal cancer in vitro and in vivo. We found that garcinol (5–15 μM) dose-dependently inhibited the migration and invasion of human esophageal cancer cell lines KYSE150 and KYSE450 in wound healing, transwell migration, and Matrigel invasion assays. Furthermore, garcinol treatment dose-dependently decreased the protein levels of p300/CBP (transcriptional cofactors and HATs) and p-Smad2/3 expression in the nucleus, thus impeding tumor cell proliferation and metastasis. Knockdown of p300 could inhibit cell metastasis, but CBP knockdown did not affect the cell mobility. It has been reported that TGF-β1 stimulated the phosphorylation of Smad2/3, which directly interact with p300/CBP in the nucleus, and upregulating HAT activity of p300. We showed that garcinol treatment dose-dependently suppressed TGF-β1-activated Smad and non-Smad pathway, inhibiting esophageal cancer cell metastasis. In a tail vein injection pulmonary metastasis mouse model, intraperitoneal administration of garcinol (20 mg/kg) or 5-FU (20 mg/kg) significantly decreased the number of lung tumor nodules and the expression levels of Ki-67, p300, and p-Smad2/3 in lung tissues. In conclusion, our study demonstrates that garcinol inhibits esophageal cancer metastasis in vitro and in vivo, which might be related to the suppression of p300 and TGF-β1 signaling pathways, suggesting the therapeutic potential of Garcinol for metastatic tumors.

MicroRNA-98-5p inhibits proliferation and metastasis in non-small cell lung cancer by targeting TGFBR1.

MicroRNAs (miRNAs or miRs) have recently emerged as key regulators of various types of cancer, including non-small cell lung cancer (NSCLC). The disrupted expression of miRNAs is associated with tumorigenesis and metastasis; however, the underlying mechanisms remain unclear. In this study, we demonstrate that miR-98-5p is downregulated in NSCLC and that miR-98-5p deficiency is associated with an advanced clinical stage and metastasis. A dual-luciferase reporter assay was performed to confirm that transforming growth factor beta receptor 1 (TGFBR1), a key stimulator of tumor proliferation and metastasis, was a direct target of miR-98-5p. miR-98-5p overexpression resulted in the downregulation of TGFBR1 and the suppression of the viability, proliferation, migration and invasion of A549 and H1299 cells. Furthermore, miR-98-5p was demonstrated to be an efficient suppressor of tumor growth in an A549 subcutaneous xenograft tumor mouse model. Finally, miR-98-5p overexpression exerted a significant anti-metastatic effect in a mouse model of pulmonary metastasis. On the whole, the results of the present study suggest that miR-98-5p/TGFBR1 may serve as promising targets for NSCLC therapy.

The Role of PPARβ/δ in Melanoma Metastasis.

Background: Peroxisome proliferator–activated receptor (PPAR) β/δ, a ligand-activated transcription factor, is involved in diverse biological processes including cell proliferation, cell differentiation, inflammation and energy homeostasis. Besides its well-established roles in metabolic disorders, PPARβ/δ has been linked to carcinogenesis and was reported to inhibit melanoma cell proliferation, anchorage-dependent clonogenicity and ectopic xenograft tumorigenicity. However, PPARβ/δ’s role in tumour progression and metastasis remains controversial. Methods: In the present studies, the consequence of PPARβ/δ inhibition either by global genetic deletion or by a specific PPARβ/δ antagonist, 10h, on malignant transformation of melanoma cells and melanoma metastasis was examined using both in vitro and in vivo models. Results: Our study showed that 10h promotes epithelial-mesenchymal transition (EMT), migration, adhesion, invasion and trans-endothelial migration of mouse melanoma B16/F10 cells. We further demonstrated an increased tumour cell extravasation in the lungs of wild-type mice subjected to 10h treatment and in Pparβ/δ−/− mice in an experimental mouse model of blood-borne pulmonary metastasis by tail vein injection. This observation was further supported by an increased tumour burden in the lungs of Pparβ/δ−/− mice as demonstrated in the same animal model. Conclusion: These results indicated a protective role of PPARβ/δ in melanoma progression and metastasis.

Redox- and pH-Responsive Nanoparticles Release Piperlongumine in a Stimuli-Sensitive Manner to Inhibit Pulmonary Metastasis of Colorectal Carcinoma Cells

Redox-responsive nanoparticles having a diselenide linkage were synthesized to target pulmonary metastasis of cancer cells. Methoxy poly(ethylene glycol)-grafted chitosan (ChitoPEG) was crosslinked using selenocystine-acetyl histidine (Ac-histidine) conjugates (ChitoPEGse) for stimuli-responsive delivery of piperlongumine (PL). ChitoPEGse nanoparticles swelled in an acidic environment and became partially disintegrated in the presence of H2O2, resulting in an increase of particle size and in a size distribution having multimodal pattern. PL release increased under acidic conditions and in the presence of H2O2. Uptake of ChitoPEGse nanoparticles by CT26 cells significantly increased in acidic and redox state. PL-incorporated ChitoPEGse nanoparticles (PL NPs) showed similar anticancer activity in vitro against A549 and CT26 cells compared to PL itself. PL NP showed superior anticancer and antimetastatic activity in an in vivo CT26 cell pulmonary metastasis mouse model. Furthermore, an immunofluorescence imaging study demonstrated that PL NP conjugates were specifically delivered to the tumor mass in the lung. Conclusively, ChitoPEGse nanoparticles were able to be delivered to cancer cells with an acidic- or redox state-sensitive manner and then efficiently targeted pulmonary metastasis of cancer cells since ChitoPEGse nanoparticles have dual pH- and redox-responsiveness.

Loss of Caveolin-1 in Metastasis-Associated Macrophages Drives Lung Metastatic Growth through Increased Angiogenesis

SummaryAlthough it is well established that tumor-associated macrophages take part in each step of cancer progression, less is known about the distinct role of the so-called metastasis-associated macrophages (MAMs) at the metastatic site. Previous studies reported that Caveolin-1 (Cav1) has both tumor-promoting and tumor-suppressive functions. However, the role of Cav1 in bone-marrow-derived cells is unknown. Here, we describe Cav1 as an anti-metastatic regulator in mouse models of lung and breast cancer pulmonary metastasis. Among all the recruited inflammatory cell populations, we show that MAMs uniquely express abundant levels of Cav1. Using clodronate depletion of macrophages, we demonstrate that macrophage Cav1 signaling is critical for metastasis and not for primary tumor growth. In particular, Cav1 inhibition does not affect MAM recruitment to the metastatic site but, in turn, favors angiogenesis. We describe a mechanism by which Cav1 in MAMs specifically restrains vascular endothelial growth factor A/vascular endothelial growth factor receptor 1 (VEGF-A/VEGFR1) signaling and its downstream effectors, matrix metallopeptidase 9 (MMP9) and colony-stimulating factor 1 (CSF1).

Nicotinic Acetylcholine Receptor Subtype Alpha-9 Mediates Triple-Negative Breast Cancers Based on a Spontaneous Pulmonary Metastasis Mouse Model.

Triple-negative breast cancer (TNBC) subtype is associated with poor prognosis and a high risk of recurrence-related death in women. Despite the aggressiveness of TNBCs, targeted TNBC therapy is not yet available in the clinic. To overcome this challenge, we generated highly metastatic TNBC cells (LM) derived from metastasized lung cells via a serial spontaneous pulmonary metastasis animal model to identify targetable molecules for attenuating the progression of TNBC metastasis. Gene analysis of primary tumor (P), first-round (1LM) and second-round (2LM) metastasized lung cells revealed that mesenchymal-related genes were significantly expressed in LM cells, especially in 2LM cells. Interestingly, α9-nAChR gene expression was also dramatically induced in LM cells, confirming our previous finding that α9-nAChR plays important roles in receptor-mediated carcinogenic signals in human breast cancer development. Using α9-nAChR as a biomarker, we transfected 2LM cells with CRISPR/Cas9 lentivirus targeting the α9-nAChR genomic region (2LM-α9-nAChR-null), showing that mesenchymal markers and the migration and invasion abilities of 2LM cells were significantly attenuated in 2LM-α9-nAChR-null cells both in vitro and in vivo. In addition, the high efficiency of editing the α9-nAChR gene using a CRISPR/Cas9 lentivirus was demonstrated by gene sequencing, genomic indel frequency and protein expression analyses. Collectively, these results confirmed those of our previous study that advanced-stage breast tumors are associated with substantially higher levels of α9-nAChR gene expression, indicating that α9-nAChR expression is essential for mediating TNBC metastasis during cancer development and may potentially act as a biomarker for targeted therapy in clinical investigations.

Vimentin is a crucial target for anti-metastasis therapy of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a unique subtype of head and neck cancer, with tendency to spread to regional lymph nodes and distant organs at early stage. Vimentin, a major cytoskeletal protein constituent of the intermediate filament, plays a critical role in the epithelial-mesenchymal transition. Overexpression of vimentin is considered to be a critical prerequisite for metastasis in numerous human cancers. Therefore, targeting vimentin for cancer therapy has gained a lot of interest. In the present study, we detected vimentin expression in NPC tissues and found that overexpression of vimentin is associated with poor prognosis of NPC patients. Silencing of vimentin in NPC CNE2 cells by RNAi suppresses cells migration and invasion in vitro. However, blocking vimentin did not affect cell proliferation of CNE2 cells. In addition, the in vivo metastatic potential of CNE2 cells transfected with Vimentin shRNA was suppressed in a nude mouse model of pulmonary metastasis. Silencing of Vimentin in CNE2 cells leads to a decrease of microvessel density and VEGF, CD31, MMP2, and MMP9 expressions in pulmonary metastatic tumors. Importantly, we found that it is easier for the tumor cells from the high vimentin-expressing pulmonary metastatic tumors to reinvade the microvessel and to form stable tumor plaques attached to the endothelial cells, which resemble the resource of circulating tumor cells and are very hard to eliminate. However, depletion of vimentin inhibits the formation of vascular tumor plaques. Our findings suggest that RNAi-based vimentin silencing may be a potential and promising anti-metastatic therapeutic strategy for NPC.

Natural Killer Cell (NK-92MI)-Based Therapy for Pulmonary Metastasis of Anaplastic Thyroid Cancer in a Nude Mouse Model.

Natural killer (NK) cells represent the third largest population of lymphocytes, and they play an important role in immune surveillance against tumors. The lungs are a common metastatic site for anaplastic thyroid cancer (ATC), and metastasis is one of the most frequent causes of mortality in this type of cancer. In the current study, we evaluated the effects of NK cell-based immunotherapy for pulmonary metastasis of ATC and determined how it affects the effector molecules of NK cells. Human NK cells (NK-92MI) were retrovirally transduced to express the effluc gene. Human ATC cells (CAL-62) were transduced with the effluc and Rluc genes. The cytotoxicity of NK cells against CAL-62 cells was assessed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay system. Pulmonary metastases of ATC were developed by i.v. injection of CAL-62, and metastasis growth was monitored using bioluminescence imaging (BLI). To treat the metastases, five million NK-92MI cells were injected twice into the caudal vein of nude mice. To assess the targetability of NK cells to ATC tumors, NK-92MI cells expressing the effluc gene (NK/F) were administered through the tail vein of nude mice with a pulmonary metastasis or tumor xenograft. BLI was subsequently performed at 1, 3, 24, and 48 h. NK/F and CAL-62 cells expressing the effluc or Rluc gene (CAL-62/F, CAL-62/R) were successfully established. Expression of the effluc and Rluc genes in NK/F, CAL-62/F, and CAL-62/R cells was verified by RT-polymerase chain reaction, western blotting, and luciferase assay. After coculture of NK-92MI and CAL-62/F cells for 24 h, the BLI signal intensity of CAL-62/F cells proportionally decreased with the number of cocultured NK cells. An ATC pulmonary metastasis mouse model was successfully generated, and NK cells significantly inhibited the growth of the metastasis (p < 0.01). The NK/F cells exhibited targetability to the pulmonary metastasis and tumor xenograft in the mouse model. The results of present study suggest that NK cells are able to target ATC tumors and that NK cell-based immunotherapy may serve as an effective therapeutic approach for pulmonary metastases of ATC.

Abstract 2680: A novel pro-tumorigenic role for IDO1 in inflammatory neovascularization

Abstract Small molecule inhibitors of the tryptophan catabolizing enzyme IDO1 (indoleamine 2,3-dioxygenase 1) have shown early promise in clinical trials as immuno-oncology agents. While the tolerogenic activity of IDO1 has been well established, we have recently identified an additional role for IDO1 in supporting neovascularization at the regulatory interface between the inflammatory cytokines IFNγ (interferon γ) and IL6 (interleukin 6). IFNγ is a primary inducer of IDO1, but is also a key mediator of immune-based tumor suppression, which studies have associated with its anti-angiogenic activity. Conversely, genetic studies in mice have clearly established the tumor-promoting role of IDO1, suggesting that it may act in a negative feedback capacity. Targeted disruption of the Ido1 gene in mice resulted in enhanced resistance to lung tumor and metastasis development. This corresponded with attenuated induction of the pro-angiogenic cytokine IL6, which, when provided through ectopic expression, was able to restore pulmonary metastasis susceptibility to Ido1-/- mice. These initial findings led us to hypothesize that IDO1 might contribute to cancer promotion by countering the anti-neovascular effect of IFNγ, possibly through IDO1-potentiated elevation of IL6. Testing this hypothesis in mouse models of oxygen-induced retinopathy and pulmonary metastasis, we determined that loss of IDO1 did indeed result in reduced neovascularization in conjunction with impaired metastasis outgrowth, effects that were completely reversed by the concurrent elimination of IFNγ. Loss of IL6 was likewise associated with IFNγ-dependent reductions in neovascularization and impaired metastasis outgrowth, as predicted. Having established a novel role for IDO1 in inflammatory neovascularization, current investigations are focused on the underlying molecular and cellular mechanisms involved. At the molecular level, one of the consequences of tryptophan catabolism by IDO1 can be to trigger the ISR (integrated stress response) through activation of the GCN2/CHOP pathway that has previously been linked to the downstream induction of IL6. Thus far, data collected in both the oxygen-induced retinopathy and pulmonary metastasis models are consistent with the ISR being the relevant downstream signaling pathway from IDO1 in this biological context. At the cellular level, we have detected the incorporation of non-endothelial, IDO1-positive cells into the vessels that comprise the neovascular tufts, implicating IDO1 in the process of vasculogenesis. These insights into this unrecognized aspect of IDO1 biology are likely to have important ramifications for IDO1 inhibitor development, not only in cancer where clinical trials are currently ongoing, but in other disease indications involving neovascularization as well. Citation Format: Arpita Mondal, Erika Sutanto-Ward, James B. DuHadaway, Arturo Bravo-Nuevo, Sunil Thomas, George C. Prendergast, Alexander J. Muller. A novel pro-tumorigenic role for IDO1 in inflammatory neovascularization [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 2680. doi:10.1158/1538-7445.AM2017-2680

FLT1 signaling in metastasis-associated macrophages activates an inflammatory signature that promotes breast cancer metastasis.

Although the link between inflammation and cancer initiation is well established, its role in metastatic diseases, the primary cause of cancer deaths, has been poorly explored. Our previous studies identified a population of metastasis-associated macrophages (MAMs) recruited to the lung that promote tumor cell seeding and growth. Here we show that FMS-like tyrosine kinase 1 (Flt1, also known as VEGFR1) labels a subset of macrophages in human breast cancers that are significantly enriched in metastatic sites. In mouse models of breast cancer pulmonary metastasis, MAMs uniquely express FLT1. Using several genetic models, we show that macrophage FLT1 signaling is critical for metastasis. FLT1 inhibition does not affect MAM recruitment to metastatic lesions but regulates a set of inflammatory response genes, including colony-stimulating factor 1 (CSF1), a central regulator of macrophage biology. Using a gain-of-function approach, we show that CSF1-mediated autocrine signaling in MAMs is downstream of FLT1 and can restore the tumor-promoting activity of FLT1-inhibited MAMs. Thus, CSF1 is epistatic to FLT1, establishing a link between FLT1 and inflammatory responses within breast tumor metastases. Importantly, FLT1 inhibition reduces tumor metastatic efficiency even after initial seeding, suggesting that these pathways represent therapeutic targets in metastatic disease.

Abstract 1340: Antitumor effector B cells directly kill tumor cells involving the CXCL12/CXCR4 pathway and their therapeutic efficacy is enhanced by IL-2

Abstract In cancer immunotherapy, effector B cells can inhibit spontaneous tumor metastases as well as local tumor growth in animal models. We have previously reported that antitumor effector B cells generated from tumor-draining lymph node (TDLN) could directly kill tumor cells via the Fas/FasL pathway and are regulated by IL-10. Trafficking of TDLN B cells in vivo suggested that they were recruited to the tumor and lung as well as secondary lymphoid organs. The number of TDLN B cells increased following transfer. As a result, adoptive transfer of TDLN B cells conferred tumor regression in a spontaneous pulmonary metastasis mouse model of breast cancer, 4T1. Removal of IL-10, either by using IL-10-/- TDLN B cells or by systemic neutralization of IL-10 using anti-IL-10 antibody, significantly augmented the therapeutic efficacy of adoptively transferred TDLN B cells. In recent studies, we found that tumor cell death mediated by effector B cells involves the CXCR4/CXCL12 as well as Fas/FasL interactions. Blockade of CXCR4 and FasL using a CXCR4-specific inhibitor, AMD3100, and anti-FasL antibody, respectively, reduced the killing of 4T1 tumor cells by 4T1 TDLN B cells. Of note, blockade of CXCR4 and FasL concurrently inhibited B cell-mediated direct killing of tumor cells in an additive fashion, suggesting that both CXCL12/CXCR4 and Fas/FasL pathways are involved in the direct killing of 4T1 tumor cells by 4T1 TDLN B cells. In other experiments, we found that TDLN B cells express IL-2 receptors (IL-2R) and the ability of the TDLN B cells to inhibit metastasis was significantly enhanced with exogenous IL-2 administration. Besides our previously reported Fas/FasL pathway and IL-10-mediated suppression, our recently identified CXCL12/CXCR4 pathway and IL-2-mediated augmentation of the biological function of effector B cells suggests these B cells may represent an important component of the host immune response against tumor. Citation Format: Yang Xia, Huimin Tao, Yangyang Hu, Li Zhou, Yangyi Bao, Steven K Lundy, Shiang Huang, Qiao Li, Alfred E Chang. Antitumor effector B cells directly kill tumor cells involving the CXCL12/CXCR4 pathway and their therapeutic efficacy is enhanced by IL-2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1340. doi:10.1158/1538-7445.AM2015-1340