Establishment Of Metastatic Foci Research Articles

S-Nitrosocaptopril prevents cancer metastasis in vivo by creating the hostile bloodstream microenvironment against circulating tumor cells.

A perfect microenvironment facilitates the activated circulating tumor cells (CTCs) to spark the adhesion-invasion-extravasation metastatic cascade in their premetastatic niche. Platelet-CTC interaction contributes to the progression of tumor malignancy by protecting CTCs from shear stress and immunological assault, aiding CTCs entrapment in the capillary bed, enabling CTCs to successfully exit the bloodstream and enter the tissue, inducing epithelial-mesenchymal-like transition (EMT), and assisting in the establishment of metastatic foci. To prevent the cascade from sparking, we show that, the multifunctional S-nitrosocaptopril (CapNO) acts on both CTCs and platelets to interrupt platelet/CTCs interplay and adhesion to endothelium, thus inhibiting CTC-based pulmonary metastasis in vivo. The activated platelets cloak cancer HT29 cells, resulting in HT29-exhibiting platelet biomarkers CD61 and P-selectin positive. CapNO inhibits both sialyl Lewisx (Slex) expression on HT29 and ADP-induced activation of platelets through P-selectin- and GPIIb/IIIa-dependent mechanisms, confirmed by the corresponding antibody assay. CapNO inhibits platelet- or interleukin (IL)-1β-mediated adhesion between HT29 and endothelial cells, and micrometastatic formation in the lungs of immunocompetent syngeneic mouse models. CapNO have also shown the effects of vasodilation, anticoagulation, inhibition of matrix metalloproteinase-2 (MMP2) expression on cancer cells, and inhibition of cell adhesion molecules (CAMs) expression on vascular endothelium. Due to a series of the beneficial effects of CapNO, CTCs remain exposed to the hostile bloodstream environment and are vulnerable to death induced by shear stress and immune elimination. This new discovery provides a basis for CapNO used for cancer metastatic chemoprevention, and might suggest regulation of the CTCs bloodstream microenvironment as a new avenue for cancer metastatic prevention.

Abstract 4140: Secreted protein acidic and rich in cysteine (SPARC) promotes mammary tumor growth and dissemination through immune and non-immune mediated mechanisms

Abstract Secreted protein and rich in cysteine (SPARC) is a matricellular glycoprotein which has been extensively associated with breast cancer progression, both in human tumors and in experimental models. However, the molecular mechanisms responsible for this effect are still unclear in particular because most studies were performed in immunocompromised nude mice. We undertook a comprehensive study of SPARC role in this disease by using the highly metastatic and poorly immunogenic 4T1 and LM3 mammary tumor cells syngeneic in Balb/c mice. Knock down of SPARC expression using a targeted shRNA in both cell lines greatly reduced primary tumor growth and completely obliterated the establishment of metastatic foci in lung. We performed global gene expression analysis comparing primary tumors and metastatic foci of control 4T1 cells with those arisen from shRNA-treated, SPARC-deficient 4T1 cells. From these studies, a potential role of the inflammatory/immune response in controlling tumor evasion and metastasis, more specifically through genes associated with the prostaglandin pathway, was suggested. As we explored this hypothesis, we observed that knock down of SPARC expression in 4T1 3D spheroids, but not in 2D monolayers, downregulated COX-2 mRNA and protein levels. Moreover, exogenous addition of SPARC in SPARC-deficient spheroids of 4T1 and MDA-MB-231 human breast cancer cells increased COX-2 expression. Interestingly, the re-expression of COX-2 in SPARC-deficient 4T1 cells partially restored the in vivo primary tumor growth with no effect on the establishment of metastatic foci. COX-2 plays an important role in inducing myeloid-derived suppressor cells (MDSCs, CD11b+Gr-1+ cells), which promotes breast cancer outgrowth through immunosuppression. Flow cytometry analyses demonstrated that SPARC-deficient 4T1 tumor-bearing mice exhibited reduced levels of CD11b+Gr-1+ cells compared to control 4T1 tumor-bearing mice in the spleen (6,9±3,2 vs. 25,3±2,0 p<0,001), lungs (0,7±0,2 vs. 6,2±0,9 p<0,001) and bone marrow (48,0±6,4% vs. 62,1±5,3 p>0,05). COX-2 re-expression restored systemic MDSCs expansion in SPARC-deficient 4T1 tumor-bearing mice. The present data suggest that COX-2 expression is under SPARC control in breast cancer cells; moreover, COX-2 could mediate SPARC effects on tumor evasion from immune surveillance. Citation Format: Leandro N. Güttlein, Lorena G. Benedetti, Raúl G. Spallanzani, Cecilia Rotondaro, Ximena L. Raffo Iraolagoitia, Leonardo Sganga, Edgardo Salvatierra, Andrea S. Llera, Norberto W. Zwirner, Osvaldo L. Podhajcer. Secreted protein acidic and rich in cysteine (SPARC) promotes mammary tumor growth and dissemination through immune and non-immune mediated mechanisms. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4140.

P1-01-09: A Focused Immune Response Targeting the Homotypic Binding Domain of the Carcinoembryonic Antigen Blocks the Establishment of Tumor Foci In Vivo.

Abstract Background: The carcinoembryonic antigen (CEA) is over-expressed in ∼56% of breast cancer biopsies but is absent on normal breast tissues. Clinically, high preoperative serum concentrations of CEA in breast cancer patients correlate with metastasis, treatment failure and poor overall prognosis. More importantly, the homotypic cell adhesion functions of CEA have been associated with cancer progression and tumor metastasis. One approach to prevent the establishment of CEA-dependent metastatic foci in breast cancer patients would thus be to interfere with the cell adhesion functions of this cell surface antigen. We hypothesize that vaccinating CEA-expressing transgenic (CEA. Tg) mice with a recombinant, altered-self form of the Ig V-like N domain of CEA, involved in homotypic interactions, should result in a focused immune response able to block the CEA-mediated adhesion of murine carcinoma cells expressing CEA (MC38.CEA) and the establishment of metastatic foci in this transgenic mouse model. Material and Methods: A recombinant form of the CEA N domain was expressed in E.coli, purified and shown in cell-based assays to bind to the A3 domain of human CEA and to block cell adhesion in CEA-expressing tumor cell lines. CEA-expressing transgenic mice were vaccinated i.p. with the recombinant CEA N domain protein and poly I:C. Resulting serum Ig [IgG1 and IgG2a] as well as cytokine expression levels [IFN-g, IL-4 and IL10 responses] were measured by ELISA and ELISPOT assays. The CEA-expressing MC38 murine tumor cells were implanted s.c. into CEA.Tg mice [hind leg; primary site model; tumor growth within 7 days] or administered i.v. to generate lung tumor metastases within 60 days or injected i.p. to generate peritoneal tumor nodules within 35 days in unvaccinated animals. Results: The recombinant folded, deglycosylated N domain of human CEA is perceived as an altered self-antigen by CEA.Tg mice when administered i.p. with poly I:C acting as an adjuvant. The vaccinated mice develop strong IgG1 and IgG2a responses able to kill CEA-expressing human breast cancer cell lines [MCF-7 and MDA-MB231] in vitro by both Antibody-Dependent Cellular Cytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC) mechanisms. The sera of such animals also inhibited cell-cell aggregation mediated by CEA expression. Vaccination of CEA.tg mice following the establishment of CEA-expressing MC38 murine hind leg tumor mass resulted in a significant delay in tumor growth. More importantly, animals pre-vaccinated with the recombinant CEA N domain did not display lung or peritoneal tumor foci following the i.v. or i.p. injection of CEA-expressing murine tumor cells. Discussion: A simple vaccination protocol using a recombinant form of the N domain of CEA as an immunogen is sufficient to engender an immune response that can block the development of CEA-expressing tumor foci in the lungs and peritoneal cavity of CEA.tg mice and destroys human breast cancer cells expressing CEA. These results suggest that mounting a focused antibody response to the N domain of CEA may represent a simple therapeutic strategy to control the establishment of metastatic foci in breast cancer patients expression high levels of the antigen CEA. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-09.

Serum proangiogenic factors in patients with testicular germ cell tumors

16050 Background: Angiogenesis has an established role in the initiation and maintenance of carcinogenesis, as well as a certain role in the establishment of metastatic foci. The role of angiogenesis in testicular germ cell tumors, although obvious, is not fully elucidated. The present study aims to clarify the role of serum proangiogenic factors in patients with testicular germ cell tumors. Methods: We measured serum basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet derived growth factor BB (PDGF-BB) and angiopoietin-2 (Ang-2), using solid phase Elisa in 50 patients (mean age 32,3±9,1 years) with testicular cancer who underwent chemotherapy and in 16 healthy male, age-matched (mean age 31,8±7,12 years) controls. Three serum samples were obtained from patients with testicular cancer: prior to chemotherapy (baseline), before cycle 3, and 9–14 months after the completion of chemotherapy. One serum sample was obtained from controls. Informed consent was taken before the obtainment of the samples. Results: Baseline mean serum bFGF, VEGF, PDGF-BB, and Ang-2 were 666,5 ±550pg/ml, 228,3±181pg/ml, 10623,8pg/ml, 4089,82±3312pg/ml in patients and 1153,6±967pg/ml, 158,9±137pg/ml, 8726,1±4318pg/ml, 2647,2±1186pg/ml in controls (p=0,1, p=0,8, p=0,3 and p=0,013). Mean serum values 1 year after the completion of chemotherapy were 310,8±298pg/ml, 139±127pg/ml, 7500±3726pg/ml and 6275±5600pg/ml (p=0,044, p=0,198, p<0,001, p=0,008). As we can see, there was a trend toward normalization of proangiogenic factors values, especially for PDGF-BB and Ang-2, and there was a difference between patients and controls for Ang-2. Ang-2 was also statistically significant different between good (n=8) and intermediate-bad prognostic groups (n=42) (mean values 3413±2100 and 5291±3200, p=0,03). Conclusions: These results support the notion about the role of proangiogenic factors in germ cell carcinogenesis. Especially angiopoietin-2 seems to play a significant role, as it was the only factor with statistically significant difference between patients and controls and the only factor with difference between the various prognostic groups. Further study of angiopoietin-2 and its role in germ cell tumors is warranted. No significant financial relationships to disclose.

Matrix metalloproteinases and tumor metastasis

Functions of individual matrix metalloproteinases (MMPs) differentially expressed by tumor cells and stromal cells, are finely regulated by their spatial as well as temporal interactions with distinct cellular and extracellular components of the tumor microenvironment and also distant pre-metastatic sites. Certain aspects of MMP involvement in tumor metastasis such as tumor-induced angiogenesis, tumor invasion, and establishment of metastatic foci at the secondary site, have received extensive attention that resulted in an overwhelming amount of experimental and observational data in favor of critical roles of MMPs in these processes. In particular, dependency of tumor angiogenesis on the activity of MMPs, especially that of MMP-9, renders this step possibly the most effective target of synthetic MMP inhibitors. MMP functioning in other stages of metastasis, including the escape of individual tumor cells from the primary tumor, their intravasation, survival in circulation, and extravasation at the secondary site, have not yet received enough consideration, resulting in insufficient or controversial data. The major pieces of evidence that are most compelling and clearly determine the role and involvement of MMPs in the metastatic cascade are provided by molecular genetic studies employing knock-out or transgenic animals and tumor cell lines, modified to overexpress or downregulate a specific MMP. Findings from all of these studies implicate different functional mechanisms for both tumor and stromal MMPs during distinct steps of the metastatic cascade and indicate that MMPs can exhibit pro-metastatic as well as anti-metastatic roles depending on their nature and the experimental setting. This dual function of individual MMPs in metastasis has become a major focus of this review.

This Month in Gastroenterology

This Month in Gastroenterology

Matrix Metalloproteases and Tumor Dissemination

In order for primary tumors to disseminate to secondary organs, they must undergo a number of prerequisite processes and molecular events. These events and processes include growth and establishment of the primary tumor, angiogenesis and vascular infiltration of the primary tumor, invasion and entry of tumor cells into the vascular circulation (a process known as intravasation), survival of tumor cells in the circulation, arrest in the microcirculation of the secondary organ, exit from the vasculature in the secondary site (a process known as extravasation), and finally growth and establishment of metastatic foci. The relevant molecular events that occur within this metastatic cascade can accurately be studied only with in vivo animal model systems. Quantification of the in vivo model systems is often difficult. Our laboratory has recently established quantitative in vivo assays for angiogenesis [1] and human tumor cell metastasis [2] employing the developing chick embryo as the host animal. These assays are more facile (allowing for molecular intervention) and more rapid (occurring over a 3–7 day time period) than the standard in vivo mouse assays which often require 2 weeks to 2 months duration. One of the protein families that we have examined for their possible functional role in our angiogenesis and/or metastasis models are the matrix metalloproteinases (MMPs). These enzymes, secreted or membrane anchored, are produced by distinct cell types and have been shown to cleave a variety of extracellular matrix components and also to activate by select cleavage other MMPs, growth factors and cytokines. In the angiogenesis model system we have shown that a sub-set of the MMPs, namely the interstitial collagenases, contribute a rate-limiting reaction to the vascular remodeling events that precede new blood vessel formation. We have been able to demonstrate the specific involvement of these collagenases by taking advantage of a genetically modified type I collagen (r/r collagen). This collagen is mutated at a single locus such that it is resistant specifically to cleavage by interstitial collagenases but is equal to wild type in its susceptibility to the gelatinases. When this mutant collagen is used in vivo as the underlying substratum, the sprouting endothelial cells exhibit a dramatically reduced ability to form new blood vessels when compared to their ability to do so on wild type collagen. The distinct MMPs that contribute to the specific cleavage of collagen in the angiogenesis model system are being identified and characterized. In the metastasis model system, specific MMP inhibitors have been shown to block some of the aforementioned steps in the metastatic cascade. One step that appears to be Matrix Metalloproteases and Tumor Dissemination

Redistribution of fibroblasts and macrophages as micrometastases develop into established liver metastases.

Fibroblastic tissue is an important component of malignant tumors, involved in the establishment of metastatic foci from micrometastases, and thought to prevent invasion of metastatic tumor cells into surrounding tissue. However, experimental models of fibrosis during the growth of micrometastasis into established metastases were not previously available. In the present paper, we performed immunohistochemical studies on experimental hepatic metastasis with colon 38 mouse colon carcinoma cells injected into syngeneic C57BL/6 mice. Early and late stages of metastatic nodules were examined for the distribution of endothelial cells, fibroblasts, and macrophages by the use of markers of these cells. One week after intrasplenic injection of colon 38 cells, micrometastases mainly appeared in the region of sinusoids accompanied with invasion of F4/80-positive Kupffer cells. Transitional metastases can be defined based on the histological appearance and intensive infiltration of both macrophages and fibroblasts. These transitional metastases were connected by protrusions of fibroblast-rich tissues co-localized with collagen-rich matrix and CD31-positive cells. This protrusion preceded fibrosis formation characteristics to established metastases associated with angiogenesis and segregation of tumor cells from host cells. Three stages can thus be classified during the development of hepatic metastasis in this syngeneic experimental system: micrometastasis, transitional metastasis, and established metastasis.

Motility of leukemic lymphocytes

In Acute Lymphocytic Leukemia motility of lymphocytes is associated with dissemination of malignancy and establishment of metastatic foci. Normal and leukemic lymphocytes in circulation reach solid tissues where due to in adequate perfusion some cells get trapped among tissue spaces. Although normal lymphocytes reenter into circulation leukemic lymphocytes are thought to remain entrapped owing to reduced mobility and form secondary metastasis. Cell surface, transmembrane interactions, cytoskeleton and level of cell differentiation are implicated in lymphocyte mobility. An attempt has been made to correlate ultrastructural information with quantitative data obtained by Laser Doppler Velocimetry (LDV). TEM of normal &amp; leukemic lymphocytes revealed heterogeneity in cell populations ranging from well differentiated (Fig. 1) to poorly differentiated cells (Fig. 2). Unlike other cells, surface extensions in differentiated lymphocytes appear to originate by extrusion of large vesicles in to extra cellular space (Fig. 3). This results in persistent unevenness on lymphocyte surface which occurs due to a phenomenon different from that producing surface extensions in other cells.

Effects of incision and irradiation on regional lymph node metastasis in carcinoma of the hamster tongue

The effects of incision and irradiation on regional lymph node metastasis in DMBA-induced squamous cell carcinomas of the hamster tongue are reported. Metastasis to the submandibular lymph nodes was confirmed histologically in 48.0% of the animals. The incidence of lymph node metastasis was significantly increased (65.9%) after repeated incisions of tongue carcinomas. Three gray whole-body irradiation also increased the rate of metastasis from 31.0% to 46.3%. Higher incidences of lymphatic vessel invasion after incision and concomitant lymph node metastasis in the lymphatic invasion-positive group indicated a stepwise relationship leading to an increase in lymph node metastasis after incision. Because of the high incidence of metastases and close resemblance to human carcinomas in the tumor cell deposition and establishment of metastatic foci, DMBA-induced tongue carcinoma with invasion may serve as an experimental model of human oral carcinomas.