Murine Lung Metastasis Model Research Articles

Buddlejasaponin IV induces apoptotic cell death by activating the mitochondrial‑dependent apoptotic pathway and reducing α2β1 integrin‑mediated adhesion in HT‑29 human colorectal cancer cells.

Colon cancer is one of the most frequent malignant neoplasms worldwide. Epidemiological studies suggested that the development of colon cancer can be prevented by plant‑derived ingredients. In the present study, the chemopreventive activity of buddlejasaponin IV (BS‑IV), isolated from the aerial part of Pleurospermumkamtschaticum, was investigated using cell viability, DNA fragmentation, caspase‑3 activity, anoikis, cell adhesion, and flow cytometry assays and a murine lung metastasis model. Protein expression levels were detected by western blotting. Treatment with BS‑IV significantly reduced cell viability and caused DNA fragmentation in HT‑29 human colorectal cancer cells. BS‑IV increased the ratio of Bax to Bcl‑2 by significantly inhibiting Bcl‑2 expression levels. BS‑IV reduced expression levels of procaspase‑9, procaspase‑3, and full‑length poly (ADP‑ribose) polymerase (PARP) and increased cleaved PARP and nonsteroidal anti‑inflammatory drug activated gene‑1 expression levels and caspase‑3 activity. In addition, BS‑IV decreased the attachment of HT‑29 cells to the extracellular matrix proteins collagen typeI and IV and downregulated cell surface expression of α2β1 integrin by inhibiting its glycosylation. BS‑IV also reduced the expression and phosphorylation levels of focal adhesion kinase (FAK) and Akt, and the reduced FAK and Akt levels were rescued by treatment with a caspase‑3 inhibitor Z‑VAD‑FMK. Furthermore, orally administered BS‑IV inhibited the formation of tumor nodules in Balb/C mice intravenously injected with CT‑26 murine colorectal cancer cells. Collectively, these findings indicated that BS‑IV induces apoptosis via the mitochondrial‑dependent pathway by increasing the ratio of Bax to Bcl‑2 and activating caspases. BS‑IV also induces anoikis by inhibiting α2β1 integrin‑mediated cell adhesion and signaling and inhibits the lung metastasis of colon cancer cells. Therefore, BS‑IV may serve as a promising cancer chemopreventive agent.

SMARCC1 Suppresses Tumor Progression by Inhibiting the PI3K/AKT Signaling Pathway in Prostate Cancer.

BackgroundSWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin subfamily C member 1 (SMARCC1) protein is a potential tumor suppressor in various cancers. However, its role in prostate cancer (PCa) remains controversial. The aim of this study was to determine the biological function of SMARCC1 in PCa and explore the underlying regulatory mechanisms.MethodsThe expression of SMARCC1 was validated in PCa tissues by immunohistochemistry. Meanwhile, function experiments were used to evaluate the regulatory role on cell proliferation and metastasis in PCa cells with SMARCC1 depletion both in vitro and in vivo. The expression levels of relevant proteins were detected by Western blotting.ResultsOur finding showed that SMARCC1 was significantly downregulated in prostate adenocarcinoma, with a higher Gleason score (GS) than that in low GS. The decreased expression of SMARCC1 was significantly correlated with a higher GS and poor prognosis. Additionally, we found that silencing of SMARCC1 dramatically accelerated cell proliferation by promoting cell cycle progression and enhancing cell migration by inducing epithelial mesenchymal transition (EMT). Furthermore, depletion of SMARCC1 facilitated PCa xenograft growth and lung metastasis in murine models. Mechanistically, the loss of SMARCC1 activated the PI3K/AKT pathway in PCa cells.ConclusionSMARCC1 suppresses PCa cell proliferation and metastasis via the PI3K/AKT signaling pathway and is a novel therapeutic target.

The Cholesterol Metabolite 27HC Increases Secretion of Extracellular Vesicles Which Promote Breast Cancer Progression

Cholesterol has been implicated in the clinical progression of breast cancer, a disease that continues to be the most commonly diagnosed cancer in women. Previous work has identified the cholesterol metabolite 27-hydroxycholesterol (27HC) as a major mediator of the effects of cholesterol on breast tumor growth and progression. 27HC can act as an estrogen receptor (ER) modulator to promote the growth of ERα+ tumors, and as a liver X receptor (LXR) ligand in myeloid immune cells to establish an immune-suppressive program. In fact, the metastatic properties of 27HC require the presence of myeloid cells with neutrophils (polymorphonuclear neutrophils; PMNs) being essential for the increase in lung metastasis in murine models. In an effort to further elucidate the mechanisms by which 27HC alters breast cancer progression, we made the striking finding that 27HC promoted the secretion of extracellular vesicles (EVs), a diverse assortment of membrane bound particles that includes exosomes. The resulting EVs had a size distribution that was skewed slightly larger than EVs generated by treating cells with vehicle. The increase in EV secretion and size was consistent across 3 different subtypes: primary murine PMNs, RAW264.7 monocytic cells, and 4T1 murine mammary cancer cells. Label-free analysis of 27HC-EVs indicated that they had a different metabolite composition to those from vehicle-treated cells. Importantly, 27HC-EVs from primary PMNs promoted tumor growth and metastasis in 2 different syngeneic models, demonstrating the potential role of 27HC-induced EVs in the progression of breast cancer. EVs from PMNs were taken up by cancer cells, macrophages, and PMNs, but not T cells. Since EVs did not alter proliferation of cancer cells, it is likely that their protumor effects are mediated through interactions with myeloid cells. Interestingly, RNA-seq analysis of tumors from 27HC-EV-treated mice do not display significantly altered transcriptomes, suggesting that the effects of 27HC-EVs occur early on in tumor establishment and growth. Future work will be required to elucidate the mechanisms by which 27HC increases EV secretion, and how these EVs promote breast cancer progression. Collectively, however, our data indicate that EV secretion and content can be regulated by a cholesterol metabolite, which may have detrimental effects in terms of disease progression, important findings given the prevalence of both breast cancer and hypercholesterolemia.

Controlling cancer-induced inflammation with a nucleic acid scavenger prevents lung metastasis in murine models of breast cancer

Tumor cells release nucleic acid-containing proinflammatory complexes, termed nucleic acid-containing damage-associated molecular patterns (NA DAMPs), passively upon death and actively during stress. NA DAMPs activate pattern recognition receptors on cells in the tumor microenvironment leading to prolonged and intensified inflammation that potentiates metastasis. No strategy exists to control endogenous or therapy-induced inflammation in cancer patients. We discovered that the generation 3.0 polyamidoamine dendrimer (PAMAM-G3) scavenges NA DAMPs and mitigates their proinflammatory effects. In this study, we tested if the nucleic acid scavenger (NAS) PAMAM-G3 reduces lung metastasis in murine models of breast cancer. Our data indicate that PAMAM-G3 treatment decreases cell-free DNA levels and reduces lung metastasis in the experimental intravenous tumor-injection model and the postsurgical tumor-resection model of 4T1 breast cancer. Reduction in lung metastasis is associated with reduction in inflammatory immune cell subsets and proinflammatory cytokine levels in the tumor and the periphery. This study is the first example of NAS-mediated inhibition of metastasis to the lung. The study results provide a strong rationale for inclusion of NAS therapy in women with breast cancer undergoing standard-of-care surgery.

The therapeutic potential of losartan in lung metastasis of colorectal cancer.

Colorectal cancer (CRC) is a common cancer with a high incidence rate. Components of the renin-angiotensin system (RAS) have been reported to be dysregulated in several malignancies including CRC. Here, we have explored the potential anti-metastatic effects of a RAS inhibitor, losartan, in an experimental model of lung metastasis in CRC. A murine model of lung metastasis of CRC was used, which involved the intravenous injection of CT26 cells via a tail vein. Four experimental groups comprised: an untreated group; a group that received 5-FU which was administered intraperitoneally; a losartan group and a combination group that received 5-FU plus losartan. We evaluated the anti-inflammatory effects of losartan by histopathological method, and the measurement of oxidative or antioxidant markers including malondialdehyde (MDA) and total thiol (T-SH) tissue levels, superoxide dismutase (SOD) and catalase activity. We found that losartan inhibited lung metastasis of CRC and there was a reduction of the IL-6 expression level in the tissue sample. It was also associated with reduced levels of the anti-angiogenic factor vascular endothelial growth factor (VEGF). Furthermore, we found that losartan induced oxidative stress as assessed by an elevation of MDA level, reduction of T-SH, SOD and catalase activities in lung tissue. Our findings demonstrated that losartan ameliorates angiogenesis, inflammation and the induction of oxidative stress via angiotensin II type I receptor (AT1R). This may shine some lights on targeting the RAS pathway as a potential therapeutic approach in the treatment of metastatic CRC patients.

A Chinese Herbal Formula Suppresses Colorectal Cancer Migration and Vasculogenic Mimicry Through ROS/HIF-1α/MMP2 Pathway in Hypoxic Microenvironment.

Various malignant tumors, including colorectal cancer, have the ability to form functional blood vessels for tumor growth and metastasis. Vasculogenic mimicry (VM) refers to the ability of highly invasive tumor cells to link each other to form vessels, which is associated with poor cancer prognosis. However, the antitumor VM agents are still lacking in the clinic. Astragalus Atractylodes mixture (AAM), a traditional Chinese medicine, has shown to inhibit VM formation; however the exact mechanism is not completely clarified. In this study, we found that HCT-116 and LoVo could form a VM network. Additionally, hypoxia increases the intracellular reactive oxygen species (ROS) level and accelerates migration, VM formation in colorectal cancer cells, while N-Acetylcysteine (NAC) could reverse these phenomena. Notably, further mechanical exploration confirmed that the matrix metalloprotease 2 (MMP2) induction is ROS dependent under hypoxic condition. On the basis, we found that AAM could effectively inhibit hypoxia-induced ROS generation, migration, VM formation as well as HIF-1α and MMP2 expression. In vivo, AAM significantly inhibits metastasis of colorectal cancer in murine lung-metastasis model. Taken together, these results verified that AAM effectively inhibits migration and VM formation by suppressing ROS/HIF-1α/MMP2 pathway in colorectal cancer under hypoxic condition, suggesting AAM could serve as a therapeutic agent to inhibit VM formation in human colorectal cancer.

Identification of novel ligands that alter the function of type II NKT cells

Abstract Natural killer T (NKT) cells are distinct innate lymphocytes which recognize lipid antigens in the context of the major histocompatibility complex-like molecule CD1d. When activated, these cells can rapidly produce a variety of cytokines and modulate innate and adaptive immune responses. There are two main subsets of NKT cells, termed type I and type II NKT cells. Type I NKT cells are characterized primarily on the basis of their invariant TCRα expression and reactivity to the glycolipid α-galactosyl ceramide (αGalCer), whereas type II NKT cells express a different and more diverse TCR repertoire than type I NKT cells. Currently, the most widely studied antigen for type II NKT cells is sulfatide. Sulfatide-reactive type II NKT cells are reported to have the immunosuppressive function in autoimmune diseases and cancer. We previously showed a role for sulfatide-reactive type II NKT cells in suppressing tumor immunosurveillance in a murine model of lung metastasis. The activation of type II NKT cells in this model by the injection of sulfatide increased the development of lung metastasis. We synthesized diverse sulfatide-analogues varying in the number of double bonds and hydroxyl groups in the ceramide. In vitro, analogues having a phytosphingosine sphingoid base activated both type II and type I NKT cell hybridomas. On the other hand, the analogue C24:2 with a sphingosine base activated only the type II NKT cell hybridoma. In vivo, the C24:2 analogue significantly reduced the development of lung metastasis, showing distinct and opposite effects from sulfatide. The identification of new ligands inducing altered functional activity of type II NKT cells could be of great interest for developing new anti-tumor immunotherapies.

Anti-Metastatic Effect of Semi-Purified Nuphar Lutea Leaf Extracts.

Nuphar lutea L. SM., leaf and rhizome extracts (NUP), contain nupharidines as active components. Nupharidines belong to the sesquiterpene lactones class of a naturally occurring plant terpenoids. This family of compounds has gained considerable interest for treating infection, inflammation and cancer.NF-κB is a central, downstream regulator of inflammation, cell proliferation and apoptosis. In our previous work we demonstrated strong inhibition of NF-κB activity and induction of apoptosis by NUP. In addition, NUP exhibited anti-inflammatory properties and partial protection from LPS-induced septic shock by modulating ERK pathway and cytokine secretion in macrophages.In the present study, we examined the effect of NUP in a B16 melanoma experimental murine lung metastasis model and its ability to affect the ERK and NF-κB pathways in variety of cell lines.We showed that NUP and cisplatin combined treatment was synergistic and reduced the lung metastatic load. In addition NUP treatment inhibited TNFα-induced IκBα degradation and NF- κB nuclear translocation. We also observed that NUP induced ERK activation. Furthermore, ERK inhibition prevented NF-κB inactivation by NUP.Overall, our work implies that co-administration of NF-κB inhibitors such as NUP, with standard anti-cancer drugs, may act as “sensitizers” for more effective chemotherapy.

IRX1 hypomethylation promotes osteosarcoma metastasis via induction of CXCL14/NF-κB signaling.

Osteosarcoma is a common malignant bone tumor with a propensity to metastasize to the lungs. Epigenetic abnormalities have been demonstrated to underlie osteosarcoma development; however, the epigenetic mechanisms that are involved in metastasis are not yet clear. Here, we analyzed 2 syngeneic primary human osteosarcoma cell lines that exhibit disparate metastatic potential for differences in epigenetic modifications and expression. Using methylated DNA immunoprecipitation (MeDIP) and microarray expression analysis to screen for metastasis-associated genes, we identified Iroquois homeobox 1 (IRX1). In both human osteosarcoma cell lines and clinical osteosarcoma tissues, IRX1 overexpression was strongly associated with hypomethylation of its own promoter. Furthermore, experimental modulation of IRX1 in osteosarcoma cell lines profoundly altered metastatic activity, including migration, invasion, and resistance to anoikis in vitro, and influenced lung metastasis in murine models. These prometastatic effects of IRX1 were mediated by upregulation of CXCL14/NF-κB signaling. In serum from osteosarcoma patients, the presence of IRX1 hypomethylation in circulating tumor DNA reduced lung metastasis-free survival. Together, these results identify IRX1 as a prometastatic gene, implicate IRX1 hypomethylation as a potential molecular marker for lung metastasis, and suggest that epigenetic reversion of IRX1 activation may be beneficial for controlling osteosarcoma metastasis.

Type I IFN signaling on dendritic cells is required for NK cell-mediated anti-tumor immunity.

NK cells play a vital role in innate anti-tumor immunity. Crosstalk between NK cells and dendritic cells (DCs) has come to the forefront in protection against tumors in the context of DC vaccines. We previously discovered that NK cell activation mediates the anti-tumor activity elicited by DC vaccines in response to melanoma tumor challenge in a murine lung metastasis model. In this study, we sought to explore the mechanism behind this NK-DC communication, specifically looking at the involvement of IL-15 and type I IFN signaling. Using DCs from IL-15(-/-) and IL-15Rα(-/-) mice, we found that the anti-tumor effect of the vaccine remained comparable with DCs from wild type mice. Moreover, DCs derived from IFN-α/βR(-/-) mice also maintained their anti-tumor effect. Interestingly, endogenous DCs were found to accumulate in the draining lymph nodes post-immunization and their depletion abolished the anti-tumor effect of the vaccine. Our findings suggest the important role that type I IFN signaling and endogenous DCs play in DC vaccine-mediated anti-tumor protection. Our data suggest that type I IFNs from vaccine DCs activate host DCs to provide NK cell-mediated anti-tumor immunity.

Targeted delivery of a sialic acid-blocking glycomimetic to cancer cells inhibits metastatic spread.

Sialic acid sugars are overexpressed by cancer cells and contribute to the metastatic cascade at multiple levels. Therapeutic interference of sialic acids, however, has been difficult to pursue because of the absence of dedicated tools. Here we show that a rationally designed sialic acid-blocking glycomimetic (P-3F(ax)-Neu5Ac) successfully prevents cancer metastasis. Formulation of P-3F(ax)--Neu5Ac into poly(lactic-co-glycolic acid nanoparticles coated with antityrosinase-related protein-1 antibodies allowed targeted delivery of P-3F(ax)--Neu5Ac into melanoma cells, slow release, and long-term sialic acid blockade. Most importantly, intravenous injections of melanoma-targeting P-3F(ax)--Neu5Ac nanoparticles prevented metastasis formation in a murine lung metastasis model. These findings stress the importance of sialoglycans in cancer metastasis and advocate that sialic acid blockade using rationally designed glycomimetics targeted to cancer cells can effectively prevent cancer metastases. This targeting strategy to interfere with sialic acid-dependent processes is broadly applicable not only for different types of cancer but also in infection and inflammation.

Expression and function of the ACE2/angiotensin(1-7)/Mas axis in osteosarcoma cell lines U-2 OS and MNNG-HOS.

The renin-angiotensin-system (RAS), via its classical angiotensin-converting enzyme (ACE)/angiotensinII/angiotensinII type1 receptor (AT1R)-axis, is associated with proliferation and metastasis of numerous types of solid tumor. AT1R blockers reduce tumor volume and decrease liver and lung metastasis in murine models of osteosarcoma. Expression and function of the alternative ACE2/Ang(1-7)/Mas axis in osteosarcoma is yet to be studied. In the present study, the basic and interleukin (IL)-1β-stimulated expression of components of this alternative RAS axis were analyzed and the impact of Mas on proliferation and/or migration of U-2 OS and MNNG-HOS osteosarcoma cells was studied. Quantitative polymerase chain reaction revealed that the two cell lines expressed the Ang(1‑7)-generating peptidases ACE2, neutral endopeptidase 24.11 and prolyl-endopeptidase together with the putative receptor for Ang(1-7), Mas. IL-1β provoked an induction of Mas mRNA and protein expression which was associated with a reduction of proliferation and migration. By contrast, small interfering RNA-mediated knockdown of Mas expression led to increased cell proliferation. In conclusion, osteosarcoma cells express a functional active alternative ACE2/Ang(1-7)/Mas axis. The induction and reinforcement of this axis may be beneficial for the treatment of osteosarcoma by reducing growth and preventing cancer metastasis. These effects may be achieved directly by the administration of Mas agonists or, indirectly, via blocking the classical AngII RAS axis via ACE inhibitors or AT1R antagonists.

Nicotinic Receptor β2 Determines NK Cell-Dependent Metastasis in a Murine Model of Metastatic Lung Cancer

Cigarette smoke exposure markedly compromises the ability of the immune system to protect against invading pathogens and tumorigenesis. Nicotine is a psychoactive component of tobacco products that acts as does the natural neurotransmitter, acetylcholine, on nicotinic receptors (nAChRs). Here we demonstrate that natural killer (NK) cells strongly express nAChR β2. Nicotine exposure impairs the ability of NK cells to kill target cells and release cytokines, a process that is largely abrogated by nAChR β2 deficiency. Further, nicotinic suppression of NF-κB-induced transcriptional activity in NK cells is dependent on nAChR β2. This nAChR subtype also plays a large role in the NK cell-mediated control of melanoma lung metastasis, in a murine lung metastasis model exposed to nicotine. Our findings suggest nAChR β2 as a prominent pathway for nicotine induced impairment of NK cell functions which contributes to the occurrence of smoking-related pathologies.

Abstract 1365: The PAMM (pulmonary assessment of metastatic morbidity) diagnostic: A novel non-invasive method for evaluating lung metastasis in murine models

Abstract Metastasis continues to be the most significant problem in the field of cancer. The biological complexity that characterizes metastasis requires relevant experimental systems for its study, necessitating the use of animal models. When using murine models for pulmonary metastasis, it is valuable to conduct longitudinal studies during which progression of metastatic burden in the lung occurs. Current methods for evaluating pulmonary metastatic burden in experimental mice are limited to advanced imaging approaches or to the development of morbidity linked to pulmonary metastasis. Unfortunately, clinical signs of morbidity are often vague and unpredictable. A novel, inexpensive, clinical method to detect advanced metastatic burden in mice before the development of significant morbidity or mortality would be beneficial to the study of metastasis in mice. With this goal in mind we have developed a novel, non-invasive technique for assessing pulmonary metastatic burden in laboratory mice. The PAMM (Pulmonary Assessment of Metastatic Morbidity) test is performed by restraining an awake mouse and gently applying pressure with the index finger under the xiphoid process. This pressure reduces the diaphragmatic component to respiration. Mice with advanced lung metastases will show signs of respiratory distress within 3 seconds of applying this pressure. Application of this method in 4 models of pulmonary metastasis (n=27 mice including sarcoma and mammary carcinoma histologies), amongst 3 independent “users,” yielded 100% positive and 100% negative predictive values that were validated by post mortem examination. In summary, the PAMM method is a simple, inexpensive, reproducible and efficient method to assist in the detection of advanced pulmonary metastasis. This method contributes to the humane care of mice included in longitudinal studies of pulmonary metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1365. doi:1538-7445.AM2012-1365

High-Contrast PET of Melanoma Using 18F-MEL050, a Selective Probe for Melanin with Predominantly Renal Clearance

The aim of this study was to evaluate the novel probe (18)F-6-fluoro-N-[2-(diethylamino)ethyl] pyridine-3-carboxamide ((18)F-MEL050) for the imaging of primary and metastatic melanoma. PET using (18)F-MEL050 was performed in murine models of melanoma. The specificity of (18)F-MEL050 was studied by comparing its accumulation in pigmented B16-F0 allograft tumors with that of human amelanotic A375 xenografts using PET and high-resolution autoradiography. (18)F-MEL050 PET results were compared with (18)F-FDG PET, the current standard in melanoma molecular imaging. To test the ability of (18)F-MEL050 to assess the metastatic spread of melanoma, a murine model of lung metastasis was imaged by PET/CT, and results correlated with physical assessment of tumor burden in the lungs. In pigmented B16-F0 grafts, (18)F-MEL050 PET yielded a tumor-to-background ratio of approximately 20:1 at 1 h and greater than 50:1 at 2 and 3 h. In the B16-F0 melanoma allograft model, tumor-to-background ratio was more than 9-fold higher for (18)F-MEL050 than for (18)F-FDG (50.9 +/- 6.9 vs. 5.8 +/- 0.5). No uptake was observed in the amelanotic melanoma xenografts. Intense uptake of (18)F-MEL050 was evident in metastatic lesions in the lungs of B16-BL6 tumor-bearing mice on PET at 2 h after tracer injection, with high concordance between (18)F-MEL050 accumulation on PET/CT and tumor burden determined at necroscopy. (18)F-MEL050 has a rapid tumor uptake and high retention with specificity for melanin, suggesting great potential for noninvasive clinical evaluation of suspected metastatic melanoma.

Sustained and NK/CD4+ T Cell-Dependent Efficient Prevention of Lung Metastasis Induced by Dendritic Cells Harboring Recombinant Sendai Virus

We recently demonstrated efficient antitumor immunity against murine tumors using dendritic cells (DCs) activated by recombinant Sendai viruses (rSeVs), and proposed a new concept, "immunostimulatory virotherapy," for cancer immunotherapy. However, there has been little information on the efficacy of this method in preventing metastatic diseases. In this study, we investigated the efficacy of vaccinating DCs activated by fusion gene-deleted nontransmissible rSeV (rSeV/dF) using a murine model of lung metastasis. Bolus and i.v. administration of DCs harboring rSeV/dF-expressing GFP without pulsation of tumor Ag (DC-rSeV/dF-GFP) 2 days before tumor inoculation showed efficient prevention against lung metastasis of c1300 neuroblastoma, but not of RM-9 prostatic cancer. We found that the timing of DC therapy was critical for the inhibition of pulmonary metastasis of RM-9, and that the optimal effect of DCs was seen 28 days before tumor inoculation. Interestingly, the antimetastatic effect was sustained for over 3 mo, even when administered DCs were already cleared from the lung and organs related to the immune system. Although NK cell activity had already declined to baseline at the time of tumor inoculation, Ab-mediated depletion studies revealed that CD4+ cells as well as the presence of, but not the activation of, NK cells were crucial to the prevention of lung metastasis. These results are the first demonstration of efficient inhibition of lung metastasis via bolus administration of virally activated DCs that was sustained and NK/CD4+ cell-dependent, and may suggest a potentially new mechanism of DC-based immunotherapy for advanced malignancies.

Inhibitory Effect of MD-Fraction on Tumor Metastasis: Involvement of NK Cell Activation and Suppression of Intercellular Adhesion Molecule (ICAM)-1 Expression in Lung Vascular Endothelial Cells

The anti-metastatic activity of MD-Fraction extracted from the maitake mushroom (Grifola frondosa) was examined in an experimental murine model of lung metastasis. Intraperitoneal administration of MD-Fraction 2 d before tumor implantation significantly inhibited lung metastasis of colon-26 carcinoma and B16/BL6 melanoma cells. In this model, MD-Fraction enhanced IL-12 production from antigen presenting cells (APCs). MD-Fraction treatment activated NK cells and increased cytotoxicity against YAC-1 and colon-26 carcinoma cells. Furthermore, the depletion of NK cells with anti-asialo GM1 abolished the inhibitory effect of MD-Fraction on lung metastasis of colon-26 cells. Ex vivo, B16/BL6 cell adhesion to LPS-activated murine lung vascular endothelial cells was inhibited by MD-Fraction and anti-intercellular adhesion molecule (ICAM)-1 antibody. These results suggest that MD-Fraction inhibits tumor metastasis by activating NK cells and APCs, and by suppressing of ICAM-1 leading to the inhibition of tumor cell adhesion to vascular endothelial cells.

Antimetastatic activities of pegylated liposomal doxorubicin in a murine metastatic lung cancer model

To determine whether pegylated liposomal doxorubicin (PLD) could control the metastasis beyond primary tumor, the efficacy of PLD was evaluated in terms of metastatic growth inhibition and increasing life span in a murine lung metastasis model. As early as 20 days after C57BL/6 mice were inoculated with 2 × 106 murine Lewis lung carcinoma cells into the right legs, metastases could be observed in the lungs. Comparing the metastatic status of the PLD treatment conducted one day with one week after the tumor implantation, pathological study of the metastasis in the lungs indicated that without the removal of primary tumor, PLD could prevent metastasis by suppressing the growth of primary tumor. To evaluate the direct antimetastasis ability of PLD with clinical relevance, a surgery was performed to resect the tumor-bearing limb. The treatment was started 3 days after the amputation with free doxorubicin or PLD. In this therapeutic model, PLD targeted directly to the metastasis in the lungs, which resulted in substantially longer survival time than free doxorubicin. Despite the superiority of PLD over free doxorubicin in treating pulmonary metastasis, our observation suggested that without the removal of primary tumor, the effect of PLD was only modest, and surgery plus multiple injections of PLD will be the best choice for patients with metastatic disease.

Cigarette Smoke Impairs NK Cell-Dependent Tumor Immune Surveillance

In this study, we investigated the impact of cigarette smoke on tumor immune surveillance and its consequences to lung tumor burden in a murine lung metastasis model. Cigarette smoke exposure significantly increased the numbers of lung metastases following B16-MO5 melanoma challenge. This effect was reversible; we observed significantly fewer tumor nodules following smoking cessation. Using RAG2(-/-) and RAG2(-/-)gamma(c)(-/-) mice, we provide strong evidence that increased tumor incidence was NK cell dependent. Furthermore, we show that cigarette smoke suppressed NK activation and attenuated NK CTL activity, without apparent effect on activating or inhibitory receptor expression. Finally, activation of NK cells through bone marrow-derived dendritic cells conferred protection against lung metastases in smoke-exposed mice; however, protection was not as efficacious as in sham-exposed mice. To our knowledge, this is the first experimental evidence showing that cigarette smoke impairs NK cell-dependent tumor immune surveillance and that altered immunity is associated with increased tumor burden. Our findings suggest that altered innate immunity may contribute to the increased risk of cancer in smokers.

Guanylyl cyclase C prevents colon carcinoma cell metastasis

Background/Aims Of patients with colorectal cancer, >50% die from metastatic progression of their disease. A novel paradigm is emerging in which guanylyl cyclase C (GC-C), the intestinal receptor for the diarrheagenic heat-stable enterotoxin (ST), serves as a tumor suppressor. While the importance of GC-C in regulating tumor cell proliferation has been defined, its role in mechanisms underlying metastasis remains unexplored. Methods The role of ST and cGMP, the intracellular mediator of GC-C, in regulating key in vitro indices of metastasis, including cell spreading, actin polymerization, detachment from collagen substrates, and matrix metalloproteinase-9 (MMP-9) activity was examined. The suitability of MMP-9 as a target for GC-C signaling in human colon tumors was confirmed, ex vivo, by RT-PCR employing laser capture microdissection and electron microscopy. Further, the impact of GC-C activation on the metastatic potential of human colon cancer cells was explored in vivo employing a murine model of lung metastasis. Results ST and cGMP negatively regulated all indices of metastasis examined, including tumor cell MMP-9 activity. Importantly, GC-C activation by ST remarkably reduced “seeding” of colon cancer cell metastases in mouse lung, in part, through an α3β1 integrin-dependent mechanism. Conclusions GC-C and its downstream effectors regulate the malignant progression of human colorectal tumors by inhibiting key processes underlying metastasis. Clinical Pharmacology & Therapeutics (2005) 77, P3–P3; doi: 10.1016/j.clpt.2004.11.016