Hematogenous Dissemination Of Cancer Cells Research Articles

Choroidal Metastasis as a First Sign of Recurrence in a Patient with Breast Cancer: A Case Report

Choroidal metastasis is the most frequent ocular tumor. Due to its high vascularity, the choroid is particularly susceptible to hematogenous dissemination of cancer cells. The most common primary tumors in choroidal metastasis are located in the lung for men and the breast for women. We report a patient followed for breast cancer treated in June 2005. She was asymptomatic until February 2016, when she presented a progressive decrease in left eye’s visual acuity and headache. Funduscopic examination showed a left exudative retinal detachment. The brain Magnetic resonance imaging revealed a left choroid metastasis. The total body computer tomography scan showed a many lung, liver, peritoneal and bone metastases. The symptoms were improved after whole-brain radiotherapy and first line systemic chemotherapy based on Bevacizumab and weekly Paclitaxel.

The Moth-Eaten Lung: Lung Adenocarcinoma with Cavitating Miliary intrapulmonary Carcinomatosis

1375 Journal of Thoracic Oncology ® • Volume 10, Number 9, September 2015 CASE PRESENTATION A 64-year-old man of Asian descent presented to our clinic because of dry cough and shortness of breath for 2 months. He was a never-smoker and denied tuberculosis exposure. His chest radiography showed diffuse miliary nodules distributed at bilateral lungs. The computed tomography of chest showed a 2.5-cm nodule at the superior segment of right lower lung and widespread of cavitating and noncavitating miliary nodules at bilateral lungs (Fig. 1). A diagnosis of stage IV lung adenocarcinoma with EGFR exon-19 deletion mutation was made. The patient was treated with afatinib 40 mg per day. On afatinib treatment at 3 months, the chest computed tomography showed shrinkage of the main tumor and regression of the cavitating and noncavitating miliary nodules (Fig. 2). The patient is currently undergoing afatinib treatment with good partial response. Lung is frequently a metastatic site for non–small-cell lung cancer. It can present as multiple lung nodules, lymphadenopathy, and effusion on chest imaging. Nevertheless, lung cancer with cavitating miliary carcinomatosis is extremely uncommon. It indicates a hematogenous dissemination of cancer cells and usually carries a rapidly fatal clinical course. Interestingly, this patient population has been shown to harbor a higher rate of adenocarcinoma and EGFR mutation, especially EGFR exon-19 deletion mutation.

Abstract 1593: Label-free high throughput microfluidic device for the isolation of circulating tumor cells from breast cancer patients

Abstract Introduction and Objective: A necessary step in distant metastasis is the hematogenous dissemination of cancer cells from the primary tumor site to remote sites. The presence of circulating tumor cells (CTCs) in the peripheral blood represents a strong and independent prognostic factor for decreased disease-free and overall survival in many solid malignancies. Immune-affinity based capture is the most commonly used method for the isolation of CTCs which utilizes antibodies to capture tumor cells expressing specific proteins. However, immune-affinity based approaches offer low throughput (∼1mL/hr) and considerable cell loss (∼20-40%) resulting from heterogeneous expression of biomarkers on CTCs. Various label-free approaches utilizing physical properties of CTCs have been developed to overcome the limitations of immune-affinity based isolation techniques, including micro-filters, microscale laminar vortices, inertial migration of particles, and integrated systems. Here we present an inertial microfluidic-based separation technique for high throughput and label-free isolation of CTCs that yields the highest throughput with high CTC recovery and high blood cell removal among all the label-free technologies. Methods: The PDMS-made microfluidic device has 637 mm in length with 56 corners, 500 μm in width, and 100 μm in height. The separation is driven by two main forces: (i) inertial force that focuses the cells into streamlines, and (ii) drag force from Dean flow that migrates the focused cells to various positions based on size. The device was optimized with MCF-7 and Panc-1 cell lines spiked into PBS buffer and also diluted blood. It was then tested on 10 mL blood samples from patients with metastatic breast cancer. The separated cells were cytospun and stained to identify CTCs as cytokeratin positive, DAPI positive, CD45 negative cells. Results: Samples were processed through the inertial microfluidic device that enriches CTCs in the second outlet based on size difference between CTCs and blood cells. The device was optimized to operate at an extremely high throughput of 2500 μL/min with high recovery (92% for both spiked samples of MCF-7 and PANC-1 cell lines) and high white blood cells (WBCs) removal (91%). To determine the efficiency of capture of rare cell populations, healthy donor blood samples were spiked with MCF-7/GFP at 100 cells and 95% recovery was obtained. In patient samples, we identified CTCs in 24 of 27 (89%) breast patients with metastatic disease (4.3±4.8 CTCs/mL) with low WBCs contamination (465±473 WBCs/mL). Conclusion: The study of CTCs could have a direct impact upon patient care by presenting a novel CTC isolation method. This technology may be applicable to early detection, and also for monitoring response to treatment. Our approach is superior to current strategies because it is independent of cell surface markers, which may be varied in tumor cells, and may exclude cancer stem cells. Citation Format: Eric Lin, Lianette Rivera, Hyeun Joong Yoon, Shamileh Fouladdel, Jacob Wieger, Stephanie Guthrie, Yadwinder S. Deol, Shawn G. Clouthier, Tahra K. Luther, Diane M. Simeone, Monika L. Burness, Ebrahim Azizi, Max S. Wicha, Sunitha Nagrath. Label-free high throughput microfluidic device for the isolation of circulating tumor cells from breast cancer patients. [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 1593. doi:10.1158/1538-7445.AM2015-1593

The Initial Hours of Metastasis: The Importance of Cooperative Host–Tumor Cell Interactions during Hematogenous Dissemination

Tumor cells transit from the primary tumor via the blood circulation to form metastases in distant organs. During this process, tumor cells encounter a number of environmental challenges and stimuli that profoundly impact their metastatic potential. Here, we review the cooperative and dynamic host-tumor cell interactions that support and promote the hematogenous dissemination of cancer cells to sites of distant metastasis. In particular, we discuss what is known about the cross-talk occurring among tumor cells, platelets, leukocytes, and endothelial cells and how these cell-cell interactions are organized both temporally and spatially at sites of extravasation and in the early metastatic niche. Metastasis is a function not only of tumor cells but also involves cooperative interactions of those cells with normal cells of the body, in particular platelets and leukocytes. These other cell types alter the behavior of the tumor cells themselves and of endothelial cells lining the vasculature and assist in tumor cell arrest and extravasation at sites of metastasis and subsequently in the establishment of tumor cells in the early metastatic niche. A better understanding of the important role that these contact and paracrine interactions play during metastasis will offer new opportunities for therapeutic intervention.

The ARF Tumor Suppressor Inhibits Tumor Cell Colonization Independent of p53 in a Novel Mouse Model of Pancreatic Ductal Adenocarcinoma Metastasis

Pancreatic ductal adenocarcinoma (PDAC) is an incurable, highly metastatic disease that is largely resistant to existing treatments. A better understanding of the genetic basis of PDAC metastasis should facilitate development of improved therapies. To that end, we developed a novel mouse xenograft model of PDAC metastasis to expedite testing of candidate genes associated with the disease. Human PDAC cell lines BxPC-3, MiaPaCa-2, and Panc-1 stably expressing luciferase were generated and introduced by intracardiac injections into immunodeficient mice to model hematogenous dissemination of cancer cells. Tumor development was monitored by bioluminescence imaging. Bioluminescent MiaPaCa-2 cells most effectively recapitulated PDAC tumor development and metastatic distribution in vivo. Tumors formed in nearly 90% of mice and in multiple tissues, including normal sites of PDAC metastasis. Effects of p14ARF, a known suppressor of PDAC, were tested to validate the model. In vitro, p14ARF acted through a CtBP2-dependent, p53-independent pathway to inhibit MiaPaCa-2-invasive phenotypes, which correlated with reduced tumor cell colonization in vivo. These findings establish a new bioluminescent mouse tumor model for rapidly assessing the biological significance of suspected PDAC metastasis genes. This system may also provide a valuable platform for testing innovative therapies.

The effects of microwave ablation and surgical resection on hematogenous dissemination of cancer cells in treating patients with small primary hepatocellular carcinoma

OBJECTIVE To conduct a comparative study of the effects of treatment using microwave ablation versus surgical resection on hematogenous dissemination of cancer cells, and on the level of immune cells of the peripheral blood in patients with small primary hepatocellular carcinoma (PHC, ≤5 cm).

Assessing Tumor Growth and Distribution in a Model of Prostate Cancer Metastasis using Bioluminescence Imaging

Bioluminescence imaging (BLI) has greatly facilitated the development of animal models of cancer, allowing sensitive detection of luciferase-expressing cancer cells in living mice. Previous efforts characterizing such models have involved small numbers of animals, limiting understanding of their performance features. We employed BLI to serially image the growth and distribution of a prostate cancer cell line, 22Rv1, after intracardiac injection into scid mice (n = 85). This approach models hematogenous dissemination of cancer cells and allows inquiry of the process of metastatic colonization at various organ sites, although accurately injecting cancer cells into the left ventricle remains challenging. Therefore, to predict injection success we measured the ratio of the thoracic bioluminescence signal to the whole body bioluminescence signal (T/WB ratio) immediately following intracardiac injection. A T/WB ratio less than 0.50 predicted the development of tumors outside of the thoracic cavity while a T/WB greater than 0.50 predicted the development of tumors entirely within the thoracic cavity, suggestive of a failed injection. Progressive tumor growth was quantified using BLI. Tumors colonized multiple organ sites including bone, liver, and adrenal glands resembling the spectrum of metastases in autopsy studies of patients with prostate cancer. Tumors growing in bone exhibited mixed osteolytic and osteoblastic features, eliciting a spiculated periosteal response. With the ability to more accurately predict injection success, we can now monitor efficacy of intracardiac injections facilitating the performance of this model.

PD-1 blockade inhibits hematogenous spread of poorly immunogenic tumor cells by enhanced recruitment of effector T cells

Since metastasis is the major cause of death for cancer patients, there is an urgent need to develop new therapies to control hematogenous dissemination of cancer cells. Previously we and others demonstrated a novel mechanism that allows tumors to escape from the host immune response by expressing PD-L1 which can negatively regulate immune response through the interaction with PD-1, an immunoinhibitory receptor belonging to the CD28 family. In this study, we report that hematogenous spread of poorly immunogenic B16 melanoma cells to the liver was inhibited in PD-1-deficient mice. After inoculation to spleen, PD-L1 was induced on tumor cells, which did not express PD-L1 in vitro. As compared with wild-type mice, intrasplenic injection of B16 cells into PD-1-deficient mice showed enhanced induction of effector T cells in spleen, prolonged T cell proliferation and cytokine production, and augmented homing of effector T cells to tumor sites in the liver, resulting in accumulation of effector T cells in the tumor sites. PD-1 blockade by genetic manipulation or antibody treatment inhibited not only hematogenous dissemination of B16 melanoma cells to the liver on the C57BL/6 background, but also dissemination of CT26 colon cancer cells to the lung on the BALB/c background. These results suggest that PD-1 blockade may be a powerful tool for treatment of hematogenous spread of various tumor cells.

Understanding Hematogenous Breast Cancer Metastasis: Tumor Cell Integrin Activation Matters.

Metastatic disease is the leading cause of morbidity and mortality in patients with breast cancer. Elucidating the mechanisms that govern the hematogenous spread of cancer cells would provide novel targets for the development of new therapeutic strategies.Our group has previously demonstrated that the activated form of integrin specifically promotes breast cancer metastasis. Here, we have investigated the mechanisms by which activated av33 directly impacts defined steps in hematogenous dissemination of cancer cells in vivo.The lung colonization assay was used as a model to analyze tumor cell arrest in the vasculature, extravasation and secondary tumor cell growth at the target site. To address a role of the activation state of adhesion receptor integrin av33 in these steps of the metastatic cascade, we genetically tagged variants of the MDA-MB 435 human breast cancer cell model with dsRed2 or luciferase, and followed cells that express either non-activated 33wild type (WT) or constitutively activated mutant 33D723R, upon injection into the circulation of SCID mice. The presence of the cells and metastatic burden was analyzed by fluorescence imaging of lung whole mounts, supported by real time PCR quantification of human Alu sequences, and by non-invasive whole body imaging based on bioluminescence detection.Within 24 hours after injecting 1 million tumor cells into the tail vein, 90% of 33D723R expressing cells and 99.5% of 33WT expressing cells were cleared from the lungs. In both cell variants, the cleared cells undergo apoptosis as shown by immunohistochemical analysis. However, significantly more 33D723R than 33WT expressing cells consistently arrested and survived in the pulmonary vascular bed. A hallmark of constitutively activated integrin av33 is the ability of the receptor to bind soluble plasma protein ligands and thereby promote tumor cell interaction with platelets, a mechanism that supports tumor cell arrest during blood flow in-vitro. The contribution of platelets to the initial steps of tumor cell target organ colonization was evaluated in mice with experimental thrombocytopenia compared to mice with normal platelet counts. Following initial tumor cell arrest, 33D723R expressing cells showed a significantly shorter lag phase and enhanced proliferation than 33WT expressing cells, and this further determined the development of metastatic foci in the lungs. Slower growth of 33D723R expressing cells in-vitro indicates a specific advantage that these cells have in the microenvironment of the lung tissue. For both cell variants, target organ colonization was restricted to the lungs, as demonstrated using imaging of bioluminescence signal.Thus, we demonstrated that the activated form of integrin av33 confers an advantage on breast cancer cells at two distinct steps within the metastatic cascade. The first step is specific arrest within the pulmonary vascular bed, followed by a critical second step where expression of activated av33 is associated with increased tumor cell survival and a faster transition from a lag phase to rapid proliferation leading to the successful generation of multicellular lesions in the lungs and the establishment of clinically relevant metastatic disease.

Anti-tumor immunoglobulin M increases lung metastasis in an experimental model of malignant melanoma.

Cancer metastasis involves distinct steps that depend on complicated tumor-host interactions. The hematogenous dissemination of tumor cells may be facilitated by factors that promote the arrest and adherence of cancer cells in capillaries. We examined whether anti-tumor monoclonal immunoglobulin M (IgM) antibodies promoted the hematogenous dissemination of B16 melanoma cells in syngeneic mice. IgM monoclonal antibodies were generated that selectively bind to B16 melanoma cells as compared to syngeneic fibroblasts, lymphocytes or Lewis lung carcinoma cells. Incubation of B16-BL6 or B16-F0 melanoma cells with these IgM anti-tumor antibodies significantly increased the number of lung colonies as compared with control antibodies. Moreover, intraperitoneal injection of specific antibody also significantly increased lung colonization. All anti-tumor antibodies promoted the aggregation of B16 melanoma cells. A chemically generated immunoglobulin G (IgG)-like fragment of an anti-tumor IgM antibody displayed greatly reduced tumor aggregation and, in contrast to intact IgM, did not significantly increase lung colonization of B16 melanoma cells. Neither intact IgM nor the IgG-like fragment enhanced the in vitro invasiveness of B16 melanoma cells across Matrigel-coated membranes. Our results, therefore, suggest that besides their beneficial anti-tumor effects, anti-tumor IgM antibodies may also promote the hematogenous dissemination of cancer cells.

Hematogenous dissemination of lung cancer cells during surgery: quantitative detection by flow cytometry and prognostic significance

Shedding of neoplastic cells into the circulation is an essential event for the hematogenous metastasis of solid tumors. Recently, several studies reported that a high frequency of cancer cells could be detected in the bloodstream during surgery. The intraoperative detection of hematogenous dissemination of cancer cells was able to identify a subset of patients with malignant diseases at high risk for postoperative metastasis and to predict a poor prognosis. In order to evaluate the association between intraoperative dissemination of cancer cells and postsurgical survival of patients with non-small cell lung cancer (NSCLC), we developed a flow cytometric assay for specific detection of lung cancer cells in the blood. The monocyte-enriched population in the blood was separated by a modified Ficoll–Hypaque density centrifugation and then labeled with a combination of monoclonal antibodies specific for CD45, cytokeratin (CK) and two antigens expressed on lung cancer cells (2F7 and S5A). The assay could detect quantitatively lung cancer cells (defined as CD45 −1 CK + 2F7/S5A + cells), with the sensitivity limit of one cancer cell in 10 5 normal leukocytes. The specificity for lung cancer was 97%, which was calculated from the results of healthy subjects (20 cases) and patients affected with benign pulmonary diseases (26 cases) or esophageal cancer (14 cases). Blood samples of 31 NSCLC patients were collected from pulmonary vein during open thoracic surgery. Fifteen of them (48.4%) were found to have positive test results. The average cancer cell counts in these cases were 0.306×10 6/l. Patients under 55 years of age had a significantly higher percentage of positive findings than those over 55 years of age ( P<0.05). The positive rate increased over the stages and lymph node status, but the differences were not statistically significant. Moreover, patients with squamous cell carcinoma at later stages (stages III and IV) had an increased frequency of positive test results than those at earlier stages (stages I and II, P<0.05). In contrast, no such a difference was found in cases with adenocarcinoma. On the basis of 30-months follow-up date, the median survival time and 2-year survival rate for patients with positive and negative findings were 11 vs. 27 months, and 26.7 vs. 62.5%, respectively. There was a statistically significant difference between overall survival curves that favored the patients with negative test results ( P=0.023). Multivariate analysis indicated the stage of disease and the positive test results as two independent factors that affected survival time ( P=0.017 and 0.027). When a comparison was made within the patients at stages III and IV, the presence of cancer cells in blood was associated with a significantly shorter survival. These data indicate that the hematogenous dissemination of lung cancer cells during surgery would be one of the mechanisms of postoperative tumor metastasis. The detection of these cells may help to identify patients with poor prognosis.

Molecular detection of circulating cancer cells during surgery in patients with biliary-pancreatic cancer

Background: It remains unclear whether surgical treatment for biliary-pancreatic cancers provokes the hematogenous dissemination of cancer cells. The aim of this study was to detect circulating cancer cells in the blood stream before and during tumor resection for biliary-pancreatic cancer.Methods: We analyzed blood samples obtained perioperatively from the portal vein, peripheral artery, and superior vena cava, using a carcinoembryonic antigen (CEA)-specific nested reverse transcriptase-polymerase chain reaction.Results: CEA-mRNA expression was detected in the blood of 21 (52.5%) of 40 patients with biliary-pancreatic cancer. The patients with detectable CEA-mRNA expression included 8 (42.1%) of 19 with bile duct cancers and 13 (61.9%) of 21 with pancreatic cancers. CEA-mRNA expression was not detected in blood obtained from 15 healthy volunteers and 15 patients with benign disease. The positive rate of CEA-mRNA of advanced clinical stage (TNM pStage III and IV) showed higher than that of early stage (pStage I and II; P <0.05). Tumor resection increased significantly the positive rates of CEA-mRNA in the blood stream of three kinds of vessel.Conclusions: Surgical procedures provoke the hematogenous dissemination of cancer cells perioperatively. Therefore, new strategies during operations to prevent liver metastases are needed to improve the survival of patients with biliary-pancreatic cancer.

Immunocytologic detection of isolated tumor cells in bone marrow of patients with squamous cell carcinomas of the head and neck region

The well-documented specificity of anticytokeratin monoclonal antibodies for detection of epithelial micrometastatic cancer cells in bone marrow as a prognostic indicator inspired us to apply this approach to patients with squamous cell carcinomas (SSC) of the head and neck region. The sensitivity of the broad-spectrum anticytokeratin monoclonal antibody (mAb) A45-B/B3 used for tumor cell detection was demonstrated by immunostaining of cryostat sections from the respective primary tumors. Analysis of 31 patients with SSC revealed A45-B/B3-positive cells in 10 cases (32.3%) at frequencies of 1–207 per 1×10 6 mononuclear cells. Most specimens displayed isolated tumor cells, while cell clusters were found in only two cases (6.5%). The present data suggest that hematogenous dissemination of cancer cells is more frequent than expected from clinicopathologic staging of patients with SSC of the head and neck region.