Specific Metastatic Sites Research Articles

CXCR4 Regulates the Early Extravasation of Metastatic Tumor Cells In Vivo

Recent studies have demonstrated that the chemokine receptor CXCR4 plays a crucial role in organ-specific metastasis formation. Although a variety of studies showed the expression of chemokine receptors, in particular, CXCR4, by gastrointestinal tumors, the precise mechanisms of chemokine receptor-mediated homing of cancer cells to specific sites of metastasis remained elusive. Here, we used liver metastatic human HEP-G2 hepatoma and HT-29LMM colon cancer cells expressing functional CXCR4 to dissect the metastatic cascade by intravital fluorescence microscopy. Immunohistochemistry revealed that the CXCR4 ligand CXCL12 is expressed by endothelial cells and likely Kupffer cells lining the liver sinusoids. Tumor cell adhesion and extravasation in vivo was quantitatively analyzed using intravital fluorescence microscopy. Treatment of cells with an anti-CXCR4 antibody did not affect cell adhesion but significantly impaired tumor cell extravasation (HEP-G2; isotype control: 22.3% ± 4.3% vs anti-CXCR4: 6.0% ± 5.0%, P < .001). In addition, pretreatment of tumor cells with the ligand CXCL12 enhanced the activation of the small GTPases Rho, Rac, and cdc42 as well as tumor cell extravasation without affecting tumor cell adhesion within liver sinusoids. Taken together, the findings of the present study provide first in vivo insights into the early events of chemokine ligand/receptor-mediated liver metastasis formation of tumor cells and define tumor cell extravasation rather than tumor cell arrest as the rate-limiting event.

Reassessment of CXCR4 Chemokine Receptor Expression in Human Normal and Neoplastic Tissues Using the Novel Rabbit Monoclonal Antibody UMB-2

BackgroundThe CXCR4 chemokine receptor regulates migration and homing of cancer cells to specific metastatic sites. Determination of the CXCR4 receptor status will provide predictive information for disease prognosis and possible therapeutic intervention. However, previous attempts to localize CXCR4 using poorly characterized mouse monoclonal or rabbit polyclonal antibodies have produced predominant nuclear and occasional cytoplasmic staining but did not result in the identification of bona fide cell surface receptors.Methodology/Principal FindingsIn the present study, we extensively characterized the novel rabbit monoclonal anti-CXCR4 antibody (clone UMB-2) using transfected cells and tissues from CXCR4-deficient mice. Specificity of UMB-2 was demonstrated by cell surface staining of CXCR4-transfected cells; translocation of CXCR4 immunostaining after agonist exposure; detection of a broad band migrating at M r 38,000–43,000 in Western blots of homogenates from CXCR4-expressing cells; selective detection of the receptor in tissues from CXCR4+/+ but not from CXCR4−/− mice; and abolition of tissue immunostaining by preadsorption of UMB-2 with its immunizing peptide. In formalin-fixed, paraffin-embedded human tumor tissues, UMB-2 yielded highly effective plasma membrane staining of a subpopulation of tumor cells, which were often heterogeneously distributed throughout the tumor. A comparative analysis of the mouse monoclonal antibody 12G5 and other frequently used commercially available antibodies revealed that none of these was able to detect CXCR4 under otherwise identical conditions.Conclusions/SignificanceThus, the rabbit monoclonal antibody UMB-2 may prove of great value in the assessment of the CXCR4 receptor status in a variety of human tumors during routine histopathological examination.

A rationale for the targeted treatment of oligometastases with radiotherapy

An oligometastatic state has been proposed wherein patients with metastases limited in number and location may benefit from local therapy directed at all known sites of metastases. We describe here the clinical and biological basis for the oligometastatic state. We present evidence for a potentially curative approach to patients with oligometastases using stereotactic body radiotherapy (SBRT) and we review the literature for SBRT directed at specific metastatic sites in the lungs, liver and multiple organs.

Modulatory effects of heparin and short-length oligosaccharides of heparin on the metastasis and growth of LMD MDA-MB 231 breast cancer cells in vivo

Expression of the chemokine receptor CXCR4 allows breast cancer cells to migrate towards specific metastatic target sites which constitutively express CXCL12. In this study, we determined whether this interaction could be disrupted using short-chain length heparin oligosaccharides. Radioligand competition binding assays were performed using a range of heparin oligosaccharides to compete with polymeric heparin or heparan sulphate binding to I125 CXCL12. Heparin dodecasaccharides were found to be the minimal chain length required to efficiently bind CXCL12 (71% inhibition; P<0.001). These oligosaccharides also significantly inhibited CXCL12-induced migration of CXCR4-expressing LMD MDA-MB 231 breast cancer cells. In addition, heparin dodecasaccharides were found to have less anticoagulant activity than either a smaller quantity of polymeric heparin or a similar amount of the low molecular weight heparin pharmaceutical product, Tinzaparin. When given subcutaneously in a SCID mouse model of human breast cancer, heparin dodecasaccharides had no effect on the number of lung metastases, but did however inhibit (P<0.05) tumour growth (lesion area) compared to control groups. In contrast, polymeric heparin significantly inhibited both the number (P<0.001) and area of metastases, suggesting a differing mechanism for the action of polymeric and heparin-derived oligosaccharides in the inhibition of tumour growth and metastases.

Prognostic factors in metastatic nasopharyngeal carcinoma

Background The current retrospective study aimed to identify some determinants of survival in metastatic NPC. Methods The study concerned 95 patients with metastatic nasopharyngeal carcinoma treated between 1993 and 2001. Statistical comparison between patients subgroups survival was carried out employing the log-Rank test (statistical significance was defined as p ≤ 0.05). Multivariable analysis was performed using the Cox model (p ≤ 0.05 was used as the cut-off value of statistical significance). Factors that were considered included: age group(≤ 45 years or > 45 years and ≤ 25 years or > 25 years), gender, performance status at diagnosis of metastatic disease (PS 0-1 or 2-3), time of metastasis diagnosis(at presentation or later), number of metastatic sites (single or multiple), specific metastatic sites(bone, liver, lung, distant nodes), number of bone metastasis(single or multiple),disease free survival(DFI) ( ≤ or > 6 months), prior chemotherapy, radiotherapy of metastatic sites. Results Negative prognostic factors in univariate analysis were: poor PS ( ≥ 1), multiple metastatic sites, multiple bone metastasis, previous chemotherapy, visceral or node metastasis and non irradiated metastasis. Poor PS, multiple metastatic sites, and prior chemotherapy were independently significant negative prognostic factors in multivariable analysis. Conclusions In this study we identified new prognostic factors in univariate and multivariate analysis. . A regular and careful follow-up of patients treated for NPC is then recommended in order to detect early metastatic dissemination (with minimal localizations) while patients have still a good PS.

The multifaceted roles of chemokines in malignancy

Tumor development and progression are multifactorial processes, regulated by a large variety of intrinsic and microenvironmental factors. A key role in cancer is played by members of the chemokine superfamily. Chemokines and their receptors are expressed by tumor cells and by host cells, in primary tumors and in specific metastatic loci. The effects of chemokines on tumorigenesis are diverse: While some members of the superfamily significantly support this process, others inhibit fundamental events required for tumor establishment and metastasis. The current review describes the multifaceted roles of chemokines in malignancy, addressing four major aspects of their activities: (1) inducing leukocyte infiltration to tumors and regulating immune functions, with emphasis on tumor-associated macrophages (and the chemokines CCL2, CCL5), T cells (and the chemokines CXCL9, CXCL10) and dendritic cells (and the chemokines CCL19, CCL20, CCL21); (2) directing the homing of tumor cells to specific metastatic sites (the CXCL12-CXCR4 axis); (3) regulating angiogenic processes (mainly the ELR(+)-CXC and non-ELR-CXC chemokines); (4) acting directly on the tumor cells to control their malignancy-related functions. Together, these different chemokine functions establish a net of interactions between the tumor cells and their microenvironment, and partly dictate the fate of the malignancy cascade.

This Month in Gastroenterology

This Month in Gastroenterology

High expression of the chemokine receptor CCR7 is associated with worse outcome in breast cancer

9593 Background: Breast cancers can express chemokine receptors to varying degrees, notably CCR7 and CXCR4, whereas their ligands can be expressed on organs that represent important sites of breast cancer metastasis. These findings have implicated chemokines and their receptors in directing breast cancer cells to specific sites of metastasis. Using a breast cancer tissue microarray (TMA), we analyzed the protein expression and sub-cellular localization of CCR7 and CXCR4. Methods: Our TMA consisted of 659 cases of primary breast cancers, nearly equally divided between node negative and node positive cancers. We used our automated quantitative image analysis software (AQUA), which uses a modified immuno-histochemistry technique with fluorochromes as a detection method. Primary antibodies were monoclonal CCR7 (BD Pharmagin, Minneapolis, MN) and CXCR4 (R&D Systems, Minneapolis, MN). All statistical analyses were performed using Statview software (version 5.0.1; SAS Institute Inc., Cary, NC). The total tumor and sub-cellular distribution pattern of CCR7 and CXCR4 were then correlated with clinico-pathologic factors and outcome. Results: CCR7 stained predominantly in a membrane/cytoplasmic distribution, whereas CXCR4 stained more diffusely throughout the entire tumor. We chose to divide the AQUA continuous data into traditional quartiles. This showed that the highest 25% of CCR7 expressers had a worse disease-specific survival when compared to the lowest 25% expressers (p=0.03). No significant correlation was identified between CCR7 expression and ER/PR, HER2/neu, nodal status, tumor size or nuclear grade. CXRC4 expression within the entire tumor or within the subcellular compartments was not associated with any of the clinico-pathologic variables or clinical outcomes. Conclusions: CCR7 and CXCR4 are variably expressed in breast cancers. Highest expression of the chemokine receptor CCR7 correlates with poor survival, without significant correlation with other clinico-pathologic factors. No significant financial relationships to disclose.

CXCR4 is expressed in ductal carcinoma in situ of the breast and in atypical ductal hyperplasia.

Recent evidence attributed important influence of chemokines and their receptors on motility, homing, and proliferation of cancer cells at specific metastatic sites. Here we report that the CXCL12 (SDF-1alpha) chemokine receptor CXCR4 is expressed in human ductal carcinoma in situ (DCIS) as well as in atypical ductal hyperplasia. CXCR4 was expressed in pure DCIS and DCIS with concurrent invasive disease. In 66% of the samples, atypical ductal hyperplasia was present, and > 92% exhibited positive CXCR4-staining. Expression of CXCR4 at this very early step of tumor development indicates a role of this receptor in providing a selective advantage to such cells on their way to metastasizing carcinomas. These results strengthen the ideas to target chemokine networks involved in tumor progression and metastatis as a therapeutic approach in malignant disease or as a chemoprevention strategy, blocking the transition from premalignancy to malignancy.

Design of a prognostic index score for metastatic nasopharyngeal carcinoma

The survival outcome of patients with systemic cancer differs significantly between individuals even within the same tumour type. We set out to illustrate this by analysing the factors determining survival in patients with metastatic disease from nasopharyngeal carcinoma (NPC) and to design a scoring system based on these prognostic factors. Patients referred between January 1994 and December 1999 were retrospectively analysed. Factors analysed included patient (age group, gender, performance status (BS) at diagnosis of metastases), disease (number of metastatic sites, specific metastatic sites, disease-free interval (DFI), metastases at presentation, presence of locoregional recurrence), and laboratory factors (leucocyte count, haemoglobin level, albumin level). Univariate and multivariable analyses were performed using the Cox proportion hazards model. A numerical score was derived from the regression coefficients of each independent prognostic variable. The prognostic index score (PIS) of each patient was calculated by totalling up the scores of each independent variable. Independently significant, negative prognostic factors were liver metastasis, lung metastasis, anaemia, poor PS, distant metastasis at initial diagnosis, and a DFI of <6 months. Three prognostic groups based on the PIS were obtained: (i) good risk (PIS=0–6); (ii) intermediate risk (7–10); (iii) poor risk (⩾11). The median survivals for these groups were 19.5, 10, and 5.8, months, respectively, (log rank test: P<0.0001). The variable prognosis of patients with disseminated NPC can be assessed by using easily available clinical information (patient, disease and laboratory factors). The PIS system will need to be validated on prospectively collected data of another cohort of patients.

NF-κ B Promotes Breast Cancer Cell Migration and Metastasis by Inducing the Expression of the Chemokine Receptor CXCR4

Metastasis of cancer cells is a complex process involving multiple steps including invasion, angiogenesis, and trafficking of cancer cells through blood vessels, extravasations, organ-specific homing, and growth. While matrix metalloproteinases, urokinase-type plasminogen activator, and cytokines play a major role in invasion and angiogenesis, chemokines such as stromal derived factor-1alpha (SDF-1alpha) and their receptors such as CXCR4 are thought to play a critical role in motility, homing, and proliferation of cancer cells at specific metastatic sites. We and others have previously reported that the extracellular signal-activated transcription factor NF-kappaB up-regulates the expression of matrix metalloproteinases, urokinase-type plasminogen activator, and cytokines in highly metastatic breast cancer cell lines. In this report, we demonstrate that NF-kappaB regulates the motility of breast cancer cells by directly up-regulating the expression of CXCR4. Overexpression of the inhibitor of kappaB (IkappaB) in breast cancer cells with constitutive NF-kappaB activity resulted in reduced expression of CXCR4 and a corresponding loss of SDF-1alpha-mediated migration in vitro. Introduction of CXCR4 cDNA into IkappaB-expressing cells restored SDF-1alpha-mediated migration. Electrophoretic mobility shift assays and transient transfection assays revealed that the NF-kappaB subunits p65 and p50 bind directly to sequences within the -66 to +7 region of the CXCR4 promoter and activate transcription. We also show that the cell surface expression of CXCR4 and the SDF-1alpha-mediated migration are enhanced in breast cancer cells isolated from mammary fat pad xenografts compared with parental cells grown in culture. A further increase in CXCR4 cell surface expression and SDF-1alpha-mediated migration was observed with cancer cells that metastasized to the lungs. Taken together, these results implicate NF-kappaB in the migration and the organ-specific homing of metastatic breast cancer cells.

Resistance to chemotherapy via Stat3-dependent overexpression of Bcl-2 in metastatic breast cancer cells.

Disruption of apoptosis may allow metastatic cell survival and confer resistance to chemotherapeutic drugs. We have analysed the molecular pathways that activate these survival genes in specific sites of metastasis. Estrogen receptor-negative breast cancer cell line MDA-MB435 and two metastatic sublines derived from lung (435L) and brain (435B) were analysed for the expression of members of the Bcl-2 family of apoptosis regulators. The levels of Bcl-2 were higher in the metastatic sublines than in parental cells, which correlated with the activation of Stat3, but not with the expression and/or activation of known bcl-2 transcription factors (CREB and WT1). In the brain subline, both expression of Bcl-2 and Stat3 activation were induced by epidermal growth factor and abrogated after treatment with kinase inhibitors specific for epidermal growth factor receptor or Jak2. Furthermore, transfection of 435B with a dominant-negative Stat3 markedly reduced the expression of Bcl-2 protein, whereas transient expression of a constitutively active Stat3 increased Bcl-2 in parental 435 cells. In addition, blockade of Stat3 activation by treatment with epidermal growth factor receptor and Jak2 kinase inhibitors or transfection with a dominant negative Stat3, sensitizes 435B cells to chemotherapy-induced apoptosis. Our data suggest that an increased activation of the Stat3-Bcl-2 pathway in estrogen receptor-negative metastatic breast cancer cell lines confer a survival advantage to these cells and contribute to their chemoresistance.

The relative value of conventional staging procedures for developing prognostic models in extensive-stage small-cell lung cancer.

Published prognostic models for small-cell lung cancer (SCLC) have either combined limited- and extensive-stage patients or have not included standard anatomic staging information to assess the relative value of the knowledge of specific sites and number of sites of metastases in predicting survival in extensive-stage disease. We studied 136 extensive-stage patients in whom traditional staging procedures were performed and in whom other previously demonstrated significant pretreatment variables were determined. Using the Cox proportional hazards model, when all data were included, three variables were significant: performance status (PS) (P = .0001), number of sites of metastases (P = .0010), and age (P = .0029). A prognostic algorithm was developed using these variables, which divided the patients into three distinct groups. When the anatomic staging data were omitted, the serum albumin (P = .0313) was the only variable in addition to PS (P = .0001) and age (P = .0064) that was significant. An alternative algorithm using these three variables was nearly as predictive as the original. Therefore, in extensive-stage patients, reasonable pretreatment prognostic information can be obtained without using the number or specific sites of metastases as variables once the presence of distant metastases has been demonstrated.

Correlation of growth capacity of human tumor cells in hard agarose with their in vivo proliferative capacity at specific metastatic sites.

The purpose of this study was to determine whether the degree of anchorage-independent growth of human tumor cells in increasing concentrations of agarose correlated with the capacity of the cells to produce experimental metastases in nude mice. Human melanoma, breast carcinoma, and colon carcinoma cells from parental lines and variants selected in vivo for metastasis and in vitro cloned lines were plated into medium containing 0.3%, 0.6%, 0.9%, or 1.2% of agarose. These cells were also injected into nude mice: intravenously for melanoma, into the mammary fat pad for breast carcinoma, and into the spleen for colon carcinoma. Production of tumor cell colonies in dense agarose (greater than 0.6%) correlated with production of experimental metastases in the lung (melanoma, breast carcinoma) or liver (colon carcinoma). We conclude that the degree of anchorage-independent growth of tumor cells can predict their biological behavior and metastatic potential in vivo. Thus, this technique may be useful for the isolation of metastatic cells from heterogeneous human neoplasms.