NK4, an HGF antagonist, prevents hematogenous pulmonary metastasis by inhibiting adhesion of CT26 cells to endothelial cells

Hepatocyte growth factor (HGF) plays a definitive role in invasive, angiogenic, and metastatic activities of tumor cells by binding to the c-Met receptor. NK4, a competitive antagonist for HGF and the c-Met receptor, prevents tumor cell growth and metastasis via its bifunctional properties to act as an HGF antagonist and angiogenesis inhibitor. In the present study, we investigated the inhibitory effectiveness of NK4 on hematogenous pulmonary metastasis of the CT26 murine colon cancer cell line, focusing on tumor cell adhesion to endothelial cells. In an in vitro adhesion assay, HGF facilitated adhesion of CT26 cells to a murine endothelial cell line (F-2) in a dose-dependent manner. Furthermore, the enhancing effect of HGF on CT26-F-2 cell interaction was blocked by NK4 as well as by anti-HGF antibody. Similarly, HGF-induced phosphorylation of focal adhesion kinase (FAK), downstream of integrin signaling, was reduced by NK4 and by anti-HGF antibody. However, distinct integrin expression on the surface of CT26 cells was not altered by HGF. In an in vivo experimental pulmonary metastasis assay, stable NK4 expression potently decreased the number of pulmonary metastatic foci. The NK4-induced suppression of pulmonary metastasis was partially reversed when HGF was intraperitoneally administered in an adhesive phase. These results suggest that NK4 could act on tumor cells to inhibit CT26 adhesion to endothelial cells by reducing FAK phosphorylation, which is regulated by inside-out HGF/c-Met signaling, and thereby suppress hematogenous pulmonary metastasis.     

Toll-like receptor (TLR) expression of immune system cells from metastatic breast cancer patients with circulating tumor cells

The risk posed by breast cancer represents a complex interaction among factors affecting tumor immunity of the host. Toll-like receptors (TLRs) are members of the innate immune system and generally function to attract host immune cells upon activation. However, the good intentions of TLRs are sometimes not transferred to positive long-term effects, due to their involvement in exacerbating inflammatory effects and even contributing to continued inflammation. Chronic inflammatory states are considered to favor an increased predisposition to cancer, with continuous activation of inflammatory cytokines and other hallmarks of inflammation exerting a deleterious effect. Circulating tumor cells (CTCs) are neoplastic cells present in the peripheral blood circulation that have been found to be an indicator of disease progression and long-term survival. In the present study, we examined the expression of TLRs on dendritic cells, which play a major role in eliciting anti-tumor immunity, in metastatic breast cancer patients with CTCs. Flow cytometric data showed significant differences between circulating tumor cell (CTC) positive patients and CTC negative patients in their expression of TLR2 by CD8 positive cytotoxic T cells and TLR2, TLR4, TLR3, and TLR8 by CD11c positive dendritic cells (p < 0.05). Expression of TLR2, TLR4, and TLR8 was increased in CTC positive patients, whereas TLR3 expression was decreased in the dendritic cell population.     

应用微流控芯片富集循环肿瘤细胞的发展现状及展望

循环肿瘤细胞(circulating tumor cell,CTCs)存在于肿瘤患者的外周血中,与恶性肿瘤的转移、预后评价以及临床的个体化治疗均密切相关。相比于组织活检,CTCs的检测仅需抽取静脉血即可进行,因而具有取样简单、可重复性、无创等优点。CTCs的富集方法有很多,近年来兴起的微流控芯片分选技术具有高通量、低消耗、可集成等特点。基于CTCs的物理性质和生物性质所设计的不同芯片在分选速度、效率、纯度以及细胞活性保持方面各有优势,本文将对此加以重点阐述。     

Tungsten treatment prevents tumor necrosis factor-induced injury of brain endothelial cells

Exposure to recombinant human tumor necrosis factor-alpha (TNF-) or calcium ionophore (A23187) for 4 h increased (P< 0.05) lactate dehydrogenase (LDH) release from cultured bovine brain endothelial cells (EC). In contrast, treatment with endotoxin or interleukin-1 did not increase (P> 0.05) LDH release from brain EC. Pretreatment with tungsten decreased (P< 0.05) xanthine oxidase activity in brain EC and decreased (P< 0.05) LDH release from brain EC following exposure to TNF. Our results suggest that TNF- injures brain microvascular EC and that this effect may be mediated by xanthine oxidase.     

肿瘤微环境中的基质细胞在转移形成中的角色

传统观念认为癌症的进展仅仅是由癌细胞基因和表型变化的多个过程所致.但最近20年的研究显示肿瘤微环境(tumor microenvironment,TME)对于肿瘤行为的影响是同等重要的.TME的组成包括局部的基质细胞,如定植的成纤维细胞(cancer-associated fibroblasts,CAF)和巨噬细胞,远处招募的细胞如内皮细胞、免疫细胞包括髓系和淋巴系细胞、骨髓来源的前体细胞和循环中的血小板.TME能够分泌影响并调控肿瘤表型的分子,如能揭示成瘤细胞与微环境之间的关系,必定能够为肿瘤的发生发展及治疗等一系列难题提供全新的视角.     

Combined use of positive and negative immunomagnetic isolation followed by real-time RT-PCR for detection of the circulating tumor cells in patients with colorectal cancers

To establish a novel molecular diagnostic method of detecting circulating tumor cells (CTCs) LS174T colon cancer cells were serially diluted with normal blood. Additional peripheral blood samples were collected from 25 patients with colorectal carcinoma. Mononuclear cells (MNCs) were collected, equally divided into four parts, and then cancer cells were enriched by four methods: method A, nonimmunobead method; method B, negative immunobead method: CD45 immunomagnetic beads were used to deplete the leukocytes; method C, positive immunobead method: Ber-EP4 immunomagnetic beads were used to enrich cancer cells; method D, negative-and-positive immunobead method: CD45 immunomagnetic beads were first used to deplete the leukocytes from MNC and then Ber-EP4 immunomagnetic beads were used to enrich cancer cells. Finally, real-time quantitative RT-PCR was used to monitor mRNA expression of ₂-mircoglobulin (2M) and carcinoembryonic antigen (CEA). The relative CEA mRNA values were corrected with reference to 2M mRNA, to CEA mRNA/2M mRNA ratios according to a CEA mRNA external standards prepared with tenfold serial dilutions (1–10⁴ IS174T cells) of cDNA and 2M mRNA external standards prepared with tenfold serial dilutions (10²–10⁷ leukocytes) of cDNA. In recovery experiments a significant correlation between the number of cancer cells and CEA mRNA expression was found when CD45 or Ber-EP4 immunomagnetic beads were used alone. A highly significant correlation was found when CD45 and Ber-EP4 immunomagnetic beads were used successively. The sensitivity of method D was one cancer cell per milliliter of blood. Circulating cancer cells were detected in 19 of 25 patients with colorectal cancers. The relative CEA mRNA value obtained by method D was the smallest. The positive detection rate of circulating cancer cells in patients at Dukes B, C, and D stages were 25.0% (1/4), 83.3% (10/12), and 88.9% (8/9). Combinative use of immunomagnetic isolation followed by real-time RT-PCR is a useful technique to detect circulating tumor cells in patients with colorectal carcinomas. Applying negative and positive immunomagnetic beads successively yields the highest correlation with amount of tumor cells.     

BRMS1 suppresses breast cancer experimental metastasis to multiple organs by inhibiting several steps of the metastatic process

Breast cancer metastasis suppressor 1 (BRMS1) inhibits formation of macroscopic lung metastases in breast, ovary, and melanoma xenograft models. Because it is unclear which step(s) of the metastatic cascade are affected by BRMS1, the major aim of this study was to determine when and how BRMS1 acts to suppress metastasis. We also examined whether BRMS1 expression globally blocks metastasis or selectively inhibits metastatic outgrowths in specific tissues. Metastatic human breast carcinoma cell lines MDA-MB-231 and -435 expressing enhanced green fluorescent protein (GFP; 231 GFP and 435 GFP) and cell lines transduced with the BRMS1 gene (231 GFP-BRMS1 and 435 GFP-BRMS1) were injected into the left cardiac ventricle to achieve the widest possible cellular distribution, by minimizing first-pass clearance in the lungs. Compared with parental cells, BRMS1-expressing clones formed significantly fewer metastases in all organs tested. When cells were injected directly into the vasculature, fewer of the BRMS1-expressing cells reached lungs or bone compared with parental cells, suggesting that restoration of BRMS1 expression increased cell death during transit. Susceptibility to anoikis was verified in vitro by demonstrating decreased survival on poly-hydroxyethyl methacrylate-coated dishes. Most of the BRMS1-expressing cells reaching secondary sites failed to proliferate, suggesting that BRMS1 also inhibits colonization. Coupled with previous reports showing modest effects of BRMS1 on adhesion and invasion, our results indicate that BRMS1 inhibits metastases in multiple organs by blocking several steps in the metastatic cascade.     

Mast cells impair melanoma cell homing and metastasis by inhibiting HMGA1 secretion

Metastatic disease is the major cause of death from cancer. From the primary tumour, cells remotely prepare the environment of the future metastatic sites by secreted factors and extracellular vesicles. During this process, known as pre-metastatic niche formation, immune cells play a crucial role. Mast cells are haematopoietic bone marrow-derived innate immune cells whose function in lung immune response to invading tumours remains to be defined. We found reduced melanoma lung metastasis in mast cell-deficient mouse models (Wsh and MCTP5-Cre-RDTR), supporting a pro-metastatic role for mast cells in vivo. However, due to evidence pointing to their antitumorigenic role, we studied the impact of mast cells in melanoma cell function in vitro. Surprisingly, in vitro co-culture of bone-marrow-derived mast cells with melanoma cells showed that they have an intrinsic anti-metastatic activity. Mass spectrometry analysis of melanoma-mast cell co-cultures secretome showed that HMGA1 secretion by melanoma cells was significantly impaired. Consistently, HMGA1 knockdown in B16-F10 cells reduced their metastatic capacity in vivo. Importantly, analysis of HMGA1 expression in human melanoma tumours showed that metastatic tumours with high HMGA1 expression are associated with reduced overall and disease-free survival. Moreover, we show that HMGA1 is reduced in the nuclei and enriched in the cytoplasm of melanoma metastatic lesions when compared to primary tumours. These data suggest that high HMGA1 expression and secretion from melanoma cells promote metastatic behaviour. Targeting HMGA1 expression intrinsically or extrinsically by mast cells actions reduce melanoma metastasis. Our results pave the way to the use of HMGA1 as anti-metastatic target in melanoma as previously suggested in other cancer types.     

Generation of a complement-derived chemotactic factor for tumor cells in experimentally induced peritoneal exudates and its effect on the local metastasis of circulating tumor cells.

A chemotactic factor for tumor cells was found in inflammatory exudate fluids induced by giving intraperitoneal injections of glycogen to Sprague-Dawley rats. The quantity of chemotactic activity and the period of time during which it could be detected correlated with the inflammatory reaction, measured by the cellular composition of the exudates and their content of protein and lysosomal enzymes. In gel filtration the chemotactic factor behaved mainly as a molecule having a molecular weight of approximately 6000 daltons. Its biologic activity was blocked by antiserums directed against C5 but not by antiserums against C3 or C4. In these two respects, the factor generated in vivo has the same properties as a previously described chemotactic factor that can be generated in vitro by proteolysis of purified C5 or C5a. Chemotactic activity was not detected in the glycogen-induced peritoneal exudates of rats depleted of serum complement by cobra venom factor. Intravenously injected Walker tumor cells arrested and formed metastases in the mesenteries of rats with peritonitis in greater numbers than in normal controls, animals depleted of complement during the experimental period, or animals given intraperitoneal injections of the vasopermeability agent, histamine. The growth of tumor cells in vitro was not promoted by peritoneal exudate fluids, nor was the number of metastases on vivo greater than in negative controls, in animals in which peritonitis was induced 24 hours after the intravenous injection of tumor cells. It is argued that chemotactic mechanisms can contribute to the formation of metastases at sites of tissue injury.     

Myeloid-derived suppressor cells (MDSC) facilitate distant metastasis of malignancies by shielding circulating tumor cells (CTC) from immune surveillance

The mechanisms of distant metastasis of malignancies largely remain unknown. Circulating tumor cells (CTC) derived from the primary cancer initiate distant metastasis by entering and traversing the bloodstream. Current methods to detect CTC are based on the notion that CTC do not express the common leukocyte antigen CD45. However, these methods neglect the fact that CTC can directly adhere to platelets and immune cells and therefore appear to be CD45-positive. The potential effects of interactions between CTC and adhesive immune cells have been largely overlooked, despite the fact that most CTC are killed by immune effector cells and only those that evade immune surveillance result in clonal expansion and metastatic lesions. It is crucial to define the characteristics that allow a select CTC population to escape immune surveillance; particularly, it must be determined whether interactions between CTC and adhesive immune cells provide a protective effect on CTC survival. If interactions between CTC and adhesive immune cells offer a selective advantage to those CTC cells, the next consideration is which characteristics of a CTC–immune cell population allow sufficient protection to facilitate immune evasion. Myeloid-derived suppressor cells (MDSC) are a large heterogeneous population of immature myeloid cells that accumulate during cancer progression to induce extensively systemic and local immunosuppression, a phenomenon that has been demonstrated to facilitate cancer distant metastasis. We hypothesize, therefore, that CTC populations interacting with adhesive immune cells will have different biological behavior than CTC populations alone. Further, we hypothesize that CTC can create a defensive shield consisting of adhesive MDSC, which allows evasion of immune surveillance and therefore facilitates distant metastatic lesions. This possibility highlights the importance of direct interactions between CTC and adhesive immune cells and suggests the potential target that the CTC–MDSC cluster represents for prevention and treatment of distant metastasis of malignancies.     

Comparative analysis of innate immune system function in metastatic breast,colorectal, and prostate cancer patients with circulating tumor cells

In recent years, circulating tumor cells (CTCs) in metastatic cancer patients have been found to be a promising biomarker to predict overall survival and tumor progression in these patients. A relatively high number of CTCs has been correlated with disease progression and poorer prognosis. This study was designed to assess innate immune system function, known to be responsible for the immune defense against developing neoplasms, in metastatic cancer patients with CTCs. Our aim is to provide a link between indication of poorer prognosis, represented by the number of CTCs to the cytotoxic activity of natural killer cells, an important component of the innate immune system, and to represent a promising expanded approach to management of metastatic cancer patients with CTCs. Seventy-four patients, with metastatic breast, colorectal, or prostate cancer, were recruited for this study. Using a flow cytometric assay, we measured natural killer (NK) cell cytotoxicity against K562 target cells; and CTCs were enumerated using the CellSearch System. Toll-like receptors 2 and 4 expression was also determined by flow cytometry.     

Assessment of the role of circulating breast cancer cells in tumor formation and metastatic potential using in vivo flow cytometry

The identification of breast cancer patients who will ultimately progress to metastatic disease is of significant clinical importance. The quantification and assessment of circulating tumor cells (CTCs) has been proposed as one strategy to monitor treatment effectiveness and disease prognosis. However, CTCs have been an elusive population of cells to study because of their small number and difficulties associated with isolation protocols. In vivo flow cytometry (IVFC) can overcome these limitations and provide insights in the role these cells play during primary and metastatic tumor growth. In this study, we used two-color IVFC to examine, for up to ten weeks following orthotopic implantation, changes in the number of circulating human breast cells expressing GFP and a population of circulating hematopoietic cells with strong autofluorescence. We found that the number of detected CTCs in combination with the number of red autofluorescent cells (650 to 690 nm) during the first seven days following implantation was predictive in development of tumor formation and metastasis eight weeks later. These results suggest that the combined detection of these two cell populations could offer a novel approach in the monitoring and prognosis of breast cancer progression, which in turn could aid significantly in their effective treatment.     

Organ-specific metastatic tumor cell adhesion and extravasation of colon carcinoma cells with different metastatic potential

Adhesive and invasive characteristics appear to be crucial for organ-specific metastasis formation. Using intravital microscopy we investigated the relation between the metastatic potential of colon carcinoma cells and their adhesive and invasive behavior during early steps of metastasis within microvasculatures of rat liver, lung, intestine, skin, muscle, spleen, and kidney in vivo. Colon carcinoma cells with low (HT-29P), intermediate (KM-12C), and high (HT-29LMM, KM-12L4) metastatic potential were injected into nude or Sprague-Dawley rats. Initial interactions with host organ microvasculatures were semiquantitatively analyzed throughout 20 to 30 minutes. Circulating cells passed microvessels in all observed organs without size restriction. All cell lines showed high adhesion rates, independent from their metastatic potential, within liver and lung but very rarely in other organs. Diameters of involved microvessels were larger than diameters of adherent tumor cells. Cell extravasation of highly metastatic HT-29LMM and KM-12L4 cells into liver parenchyma was significantly higher compared to low metastatic cells (P<0.05). Our results indicate that colon carcinoma cells can arrest in target organs without size restriction. Cell adhesion of circulating tumor cells occurred in metastatic target organs only, likely attributable to specific interactions. Migration into target organs correlated with their metastatic potential.     

In vitro metastatic colonization of human ovarian cancer cells to the omentum

Despite the potentially crucial contributions of the omentum in the regulation of ovarian cancer metastatic growth, it remains a poorly understood organ. Due to its anatomic location and structural fragility, the omentum presents inherent challenges to mechanism-based in vivo studies. Thus, the availability of an ex vivo omental model would, in part, address some of these difficulties posed. Here we describe a technique for identifying, isolating and maintaining ex vivo cultures of omenta from immune-compromised and -competent mice. Ex vivo culture conditions were developed that maintain tissue viability, architecture, and function for up to 10 days. Further experiments demonstrate that the ex vivo culture conditions allow for the proliferation of ovarian cancer cells in vitro and support a similar pattern of microscopic lesions after either intraperitoneal injection of ovarian cancer cells or co-culture of ovarian cancer cells with the omentum. In agreement with previous studies from our laboratory, histologic evaluation of these specimens found that ovarian cancer cells, as well as other peritoneal cancer cells, preferentially accumulate in, and colonize, omental areas rich in immune cells. We now recognize that these are specific, functional structures referred to as milky spots. In sum, these are foundational studies of a readily accessible model, which is easily manipulated and can be immediately used to study the dynamic process of omental colonization. It is hoped that investigators will use the data herein as a starting point for refinements and modifications which will enable them to tailor the model to the specific needs of the experimental question(s) they wish to pursue.     

Photodynamic ablation of lymphatic vessels and intralymphatic cancer cells prevents metastasis

The dissemination of tumor cells to sites far from the primary tumor (metastasis) is the principal cause of death in cancer patients. Tumor-associated lymphatic vessels are a key conduit for metastatic tumor cells, which typically first colonize the lymph nodes. Although the primary tumor and affected lymph nodes can be removed during surgery, tumor cells inside lymphatic vessels are left behind. Here, we show that in-transit tumor cells inside lymphatic vessels in mice bearing mouse melanomas or human lung tumors give rise to metastases. Using photodynamic therapy with the benzoporphyrin derivative verteporfin, we selectively destroyed lymphatic vessels in mice and pigs. Destruction of tumor-associated lymphatic vessels also eradicated intralymphatic tumor cells and prevented metastasis of mouse melanoma cells and subsequent relapse. Photodynamic therapy, when combined with anti-lymphangiogenic therapy, prevented further tumor invasion of lymphatic vessels. These findings highlight the potential of targeting in-transit tumor cells in patients.     

Expression of new antigens on tumor cells by inhibiting nonsense-mediated mRNA decay

Immunologic Research - The main reason why tumors are not controlled by the immune system of the cancer patient is that tumors do not express potent tumor antigens that can be recognized by the...     

Discordant and heterogeneous clinically relevant genomic alterations in circulating tumor cells vs plasma DNA from men with metastatic castration resistant prostate cancer

Circulating tumor cell (CTC) and cell‐free (cf) DNA‐based genomic alterations are increasingly being used for clinical decision‐making in oncology. However, the concordance and discordance between paired CTC and cfDNA genomic profiles remain largely unknown. We performed comparative genomic hybridization (CGH) on CTCs and cfDNA, and low‐pass whole genome sequencing (lpWGS) on cfDNA to characterize genomic alterations (CNA) and tumor content in two independent prospective studies of 93 men with mCRPC treated with enzalutamide/abiraterone, or radium‐223. Comprehensive analysis of 69 patient CTCs and 72 cfDNA samples from 93 men with mCRPC, including 64 paired samples, identified common concordant gains in FOXA1, AR, and MYC, and losses in BRCA1, PTEN, and RB1 between CTCs and cfDNA. Concordant PTEN loss and discordant BRCA2 gain were associated with significantly worse outcomes in Epic AR‐V7 negative men with mCRPC treated with abiraterone/enzalutamide. We identified and externally validated CTC‐specific genomic alternations that were discordant in paired cfDNA, even in samples with high tumor content. These CTC/cfDNA‐discordant regions included key genomic regulators of lineage plasticity, osteomimicry, and cellular differentiation, including MYCN gain in CTCs (31%) that was rarely detected in cfDNA. CTC MYCN gain was associated with poor clinical outcomes in AR‐V7 negative men and small cell transformation. In conclusion, we demonstrated concordance of multiple genomic alterations across CTC and cfDNA platforms; however, some genomic alterations displayed substantial discordance between CTC DNA and cfDNA despite the use of identical copy number analysis methods, suggesting tumor heterogeneity and divergent evolution associated with poor clinical outcomes.     

Chronic psychological stress promotes lung metastatic colonization of circulating breast cancer cells by decorating a pre‐metastatic niche through activating β‐adrenergic signaling

Numerous studies have indicated that primary tumors induce the formation of a pre‐metastatic niche in distant organs by secreting tumor‐derived factors. The present study shows that pre‐exposure to chronic stress enhanced lung colonization efficiency by circulating tumor cells, suggesting that chronic stress critically influences pre‐metastatic lungs before the arrival of disseminated tumor cells. Ablation of the sympathetic nerve function by 6‐OHDA or blockage of the β‐adrenergic signaling by propranolol remarkably suppressed stress‐induced lung metastasis. Depletion of circulating monocytes or lung macrophages strongly abolished stress‐induced lung seeding by tumor cells, whereas treatment of mice with the β‐adrenergic agonist isoproterenol (ISO) during the pre‐metastatic phase promoted the infiltration of macrophages to the lung. Meanwhile, the numbers of monocytes in peripheral blood, spleen, and bone marrow were remarkably increased in response to ISO stimulation. These data indicate that the β‐adrenergic signaling promotes lung metastatic colonization by tumor cells through increased output of monocytes in the pre‐metastatic phase and infiltration of macrophages into the pre‐metastatic lung. Mechanistic studies revealed that ISO stimulation upregulated the expression of CCL2 in pulmonary stromal cells and CCR2 in monocytes/macrophages, leading to the recruitment and infiltration of macrophages into the pre‐metastatic lung. By inducing a response of monocytes/macrophages driven by the CCL2/CCR2 axis, stress‐related catecholamine may act as a crucial factor in regulating the pre‐metastatic niche for and lung colonization by tumor cells. Our data demonstrate that disturbance of host macro‐environmental homeostasis has an influence on future metastatic organs. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.