Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.     

综述: 整合素相关微环境调控肿瘤转移

整合素是肿瘤微环境的重要组成部分,是广泛存在于细胞膜表面的黏附分子,可以识别并结合细胞外基质中相应的配体,参与许多重要的生理过程,包括肿瘤转移．整合素可以促进肿瘤转移的各个阶段,肿瘤微环境也会反过来影响整合素的表达,从而促进癌症的发生发展．     

前列腺癌骨转移：肿瘤细胞与骨微环境之间的相互作用

  肿瘤转移包括一系列复杂的过程,主要涉及肿瘤细胞由原发部位脱离开始,到肿瘤细胞在其它转移部位——比如骨骼,生长增殖的多个关键性步骤.“种子和土壤学说”预示骨微环境中表达许多因子,吸引多种肿瘤细胞的迁移以及促进肿瘤的增殖.通过肿瘤细胞与其所处生长环境中的双向和动态的作用,促进肿瘤在骨骼中的发展.因此,骨微环境中产生的因子对肿瘤骨转移具有重要意义.本综述以前列腺癌为例,总结了肿瘤转移的机理研究概况,特别强调了目前有关肿瘤细胞与骨微环境之间相互作用研究的重要性,并提出了未来的研究方向     

CPNE8 Promotes Gastric Cancer Metastasis by Modulating Focal Adhesion Pathway and Tumor Microenvironment

Little is known about the oncogenic role or biological function of copine Ⅷ (CPNE8) in gastric cancer (GC). Based on TCGA database, we screened for CPNE8 and analyzed the expression of CPNE8 in GC. The correlations between CPNE8 and clinical features were analyzed using TCGA and GEO databases. The prognostic value of CPNE8 was assessed using Cox analysis and Kaplan-Meier curves. The results showed that increased expression of CPNE8 was positively correlated with metastasis and can be considered an independent prognostic risk factor for poor survival. We found that CPNE8 can promote cell proliferation, migration, and invasiveness in GC using in vitro and in vivo experiments. Our study demonstrated that CPNE8 promotes tumor progression via regulation of focal adhesion, and these effects can be rescued by focal adhesion kinase (FAK) inhibitor GSK2256098 or knockdown of FAK. In addition, CPNE8 was correlated significantly with the infiltration of cancer-associated fibroblasts and immune cells, as demonstrated by various algorithms, and high CPNE8 expression predicted poor efficacy of immune checkpoint therapy. Our findings suggest that CPNE8 modulates focal adhesion and tumor microenvironment to promote GC progression and invasiveness and could serve as a novel prognostic biomarker in GC.     

78例肺癌脑转移患者临床分析

目的：探讨与肺癌脑转移预后相关的影响因素,为更好的治疗肺癌脑转移提供参考依据。方法：回顾性分析2002年~2009年唐都医院收治的78例肺癌脑转移的临床资料。结果：患者年龄、性别、病理类型、脑转移灶数目、颅外有无转移性病灶、患者的行为状态（PS评分）对患者生存期无显著影响（P〉0.05）。但放疗＋化疗治疗组疗效显著优于单纯放疗组及单纯化疗组（P〈0.05）,症状消失者（CR）或明显缓解者（PR）生存较无明显改善者（SD）或加重者（PD）生存期显著延长。结论：对肺癌脑转移患者应采取放疗联合化疗综合方案治疗,同时,应及时关注其症状缓解情况并指导治疗。     

78 例肺癌脑转移患者临床分析

目的:探讨与肺癌脑转移预后相关的影响因素,为更好的治疗肺癌脑转移提供参考依据。方法:回顾性分析2002年~2009年唐都医院收治的78例肺癌脑转移的临床资料。结果:患者年龄、性别、病理类型、脑转移灶数目、颅外有无转移性病灶、患者的行为状态(PS评分)对患者生存期无显著影响(P>0.05)。但放疗+化疗治疗组疗效显著优于单纯放疗组及单纯化疗组(P<0.05),症状消失者(CR)或明显缓解者(PR)生存较无明显改善者(SD)或加重者(PD)生存期显著延长。结论:对肺癌脑转移患者应采取放疗联合化疗综合方案治疗,同时,应及时关注其症状缓解情况并指导治疗。     

Suppressing MDSC Infiltration in Tumor Microenvironment Serves as an Option for Treating Ovarian Cancer Metastasis

It is still a big puzzle how ovarian cancer cells and the tumor microenvironment (TME) attract lymphocytes infiltration for facilitating metastasis, a leading cause of death from gynecological malignancies. Using genome-wide LncRNA microarray assay, here we report that a LncRNA associated with ovarian cancer metastasis (LncOVM) is highly correlated with poor prognosis and survival. LncOVM interacts with and stabilizes PPIP5K2 by suppressing ubiquitinated degradation to promote complement C5 secretion from ovarian cancer cells. The TME-enriched complement C5 attracts myeloid-derived suppressor cells (MDSCs) infiltration in TME to facilitate metastasis. Knockdown of LncOVM or PPIP5K2 inhibits tumor progression in xenograft models. Application of C5aR antibody or inhibitor (CCX168) inhibits MDSC recruitment and restores the suppression of tumorigenesis and metastasis in vivo. Our study reveals that suppression of ovarian cancer metastasis can be achieved by targeting MDSC infiltration in TME through disrupting LncOVM-PPIP5K2-complement axis, providing an option for treating ovarian cancer patients.     

Integrated Genomic and Epigenomic Analysis of Breast Cancer Brain Metastasis

The brain is a common site of metastatic disease in patients with breast cancer, which has few therapeutic options and dismal outcomes. The purpose of our study was to identify common and rare events that underlie breast cancer brain metastasis. We performed deep genomic profiling, which integrated gene copy number, gene expression and DNA methylation datasets on a collection of breast brain metastases. We identified frequent large chromosomal gains in 1q, 5p, 8q, 11q, and 20q and frequent broad-level deletions involving 8p, 17p, 21p and Xq. Frequently amplified and overexpressed genes included ATAD2, BRAF, DERL1, DNMTRB and NEK2A. The ATM, CRYAB and HSPB2 genes were commonly deleted and underexpressed. Knowledge mining revealed enrichment in cell cycle and G2/M transition pathways, which contained AURKA, AURKB and FOXM1. Using the PAM50 breast cancer intrinsic classifier, Luminal B, Her2+/ER negative, and basal-like tumors were identified as the most commonly represented breast cancer subtypes in our brain metastasis cohort. While overall methylation levels were increased in breast cancer brain metastasis, basal-like brain metastases were associated with significantly lower levels of methylation. Integrating DNA methylation data with gene expression revealed defects in cell migration and adhesion due to hypermethylation and downregulation of PENK, EDN3, and ITGAM. Hypomethylation and upregulation of KRT8 likely affects adhesion and permeability. Genomic and epigenomic profiling of breast brain metastasis has provided insight into the somatic events underlying this disease, which have potential in forming the basis of future therapeutic strategies.     

Determinants of Maximal Force Transmission in a Motor-Clutch Model of Cell Traction in a Compliant Microenvironment

The mechanical stiffness of a cell’s environment exerts a strong, but variable, influence on cell behavior and fate. For example, different cell types cultured on compliant substrates have opposite trends of cell migration and traction as a function of substrate stiffness. Here, we describe how a motor-clutch model of cell traction, which exhibits a maximum in traction force with respect to substrate stiffness, may provide a mechanistic basis for understanding how cells are tuned to sense the stiffness of specific microenvironments. We find that the optimal stiffness is generally more sensitive to clutch parameters than to motor parameters, but that single parameter changes are generally only effective over a small range of values. By contrast, dual parameter changes, such as coordinately increasing the numbers of both motors and clutches offer a larger dynamic range for tuning the optimum. The model exhibits distinct regimes: at high substrate stiffness, clutches quickly build force and fail (so-called frictional slippage), whereas at low substrate stiffness, clutches fail spontaneously before the motors can load the substrate appreciably (a second regime of frictional slippage). Between the two extremes, we find the maximum traction force, which occurs when the substrate load-and-fail cycle time equals the expected time for all clutches to bind. At this stiffness, clutches are used to their fullest extent, and motors are therefore resisted to their fullest extent. The analysis suggests that coordinate parameter shifts, such as increasing the numbers of motors and clutches, could underlie tumor progression and collective cell migration.     

Determinants of Maximal Force Transmission in a Motor-Clutch Model of Cell Traction in a Compliant Microenvironment

The mechanical stiffness of a cell’s environment exerts a strong, but variable, influence on cell behavior and fate. For example, different cell types cultured on compliant substrates have opposite trends of cell migration and traction as a function of substrate stiffness. Here, we describe how a motor-clutch model of cell traction, which exhibits a maximum in traction force with respect to substrate stiffness, may provide a mechanistic basis for understanding how cells are tuned to sense the stiffness of specific microenvironments. We find that the optimal stiffness is generally more sensitive to clutch parameters than to motor parameters, but that single parameter changes are generally only effective over a small range of values. By contrast, dual parameter changes, such as coordinately increasing the numbers of both motors and clutches offer a larger dynamic range for tuning the optimum. The model exhibits distinct regimes: at high substrate stiffness, clutches quickly build force and fail (so-called frictional slippage), whereas at low substrate stiffness, clutches fail spontaneously before the motors can load the substrate appreciably (a second regime of frictional slippage). Between the two extremes, we find the maximum traction force, which occurs when the substrate load-and-fail cycle time equals the expected time for all clutches to bind. At this stiffness, clutches are used to their fullest extent, and motors are therefore resisted to their fullest extent. The analysis suggests that coordinate parameter shifts, such as increasing the numbers of motors and clutches, could underlie tumor progression and collective cell migration.     

The Microenvironment of Cervical Carcinoma Xenografts: Associations with Lymph Node Metastasis and Its Assessment by DCE-MRI

Poor disease-free and overall survival rates in locally advanced cervical cancer are associated with a tumor micro-environment characterized by extensive hypoxia, interstitial hypertension, and high lactate concentrations. The potential of gadolinium diethylenetriamine pentaacetic acid-based dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) in assessing the microenvironment and microenvironment-associated aggressiveness of cervical carcinomas was investigated in this preclinical study. CK-160 and TS-415 cervical carcinoma xenografts were used as tumor models. DCE-MRI was carried out at 1.5 T, and parametric images of K^trans and v_e were produced by pharmacokinetic analysis of the DCE-MRI series. Pimonidazole was used as a marker of hypoxia. A Millar catheter was used to measure tumor interstitial fluid pressure (IFP). The concentrations of glucose, adenosine triphosphate (ATP), and lactate were measured by induced metabolic bioluminescence imaging. High incidence of lymph node metastases was associated with high hypoxic fraction and high lactate concentration in CK-160 tumors and with high IFP and high lactate concentration in TS-415 tumors. Low K^trans was associated with high hypoxic fraction, low glucose concentration, and high lactate concentration in tumors of both lines and with high incidence of metastases in CK-160 tumors. Associations between v_e and microenvironmental parameters or metastatic propensity were not detected in any of the tumor lines. Taken together, this preclinical study suggests that K^trans is a potentially useful biomarker for poor outcome of treatment in advanced cervical carcinoma. The possibility that K^trans may be used to identify patients with cervical cancer who are likely to benefit from particularly aggressive treatment merits thorough clinical investigations.     

Exercise Modulates Redox-Sensitive Small GTPase Activity in the Brain Microvasculature in a Model of Brain Metastasis Formation

Tumor cell extravasation into the brain requires passage through the blood-brain barrier (BBB). There is evidence that exercise can alter the oxidation status of the brain microvasculature and protect against tumor cell invasion into the brain, although the mechanisms are not well understood. In the current study, we focused on the role of microenvironment generated by exercise and metastasizing tumor cells at the levels of brain microvessels, influencing oxidative stress-mediated responses and activation of redox-sensitive small GTPases. Mature male mice were exercised for four weeks using a running wheel with the average voluntary running distance 9.0±0.3 km/day. Mice were then infused with 1.0×10⁶ D122 (murine Lewis lung carcinoma) cells into the brain microvasculature, and euthanized either 48 hours (in short-term studies) or 2–3 weeks (in long-term studies) post tumor cell administration. A significant increase in the level of reactive oxygen species was observed following 48 hours or 3 weeks of tumor cells growth, which was accompanied by a reduction in MnSOD expression in the exercised mice. Activation of the small GTPase Rho was negatively correlated with running distance in the tumor cell infused mice. Together, these data suggest that exercise may play a significant role during aggressive metastatic invasion, especially at higher intensities in pre-trained individuals.     

CD90(+) Mesothelial-Like Cells in Peritoneal Fluid Promote Peritoneal Metastasis by Forming a Tumor Permissive Microenvironment

The peritoneal cavity is a common target of metastatic gastrointestinal and ovarian cancer cells, but the mechanisms leading to peritoneal metastasis have not been fully elucidated. In this study, we examined the roles of cells in peritoneal fluids on the development of peritoneal metastasis. We found that a minor subset of human intraperitoneal cells with CD90(+)/CD45(−) phenotype vigorously grew in culture with mesothelial-like appearance. The mesothelial-like cells (MLC) displayed the characteristics of mesenchymal stem cell, such as differentiating into adipocytes, osteocytes, and chondrocytes, and suppressing T cell proliferation. These cells highly expressed type I collagen, vimentin, α-smooth muscle actin and fibroblast activated protein-α by the stimulation with TGF-β, which is characteristic of activated myofibroblasts. Intraperitoneal co-injection of MLCs with the human gastric cancer cell line, MKN45, significantly enhanced the rate of metastatic formation in the peritoneum of nude mice. Histological examination revealed that many MLCs were engrafted in metastatic nodules and were mainly located at the fibrous area. Dasatinib, a potent tyrosine kinase inhibitor, strongly inhibited the proliferation of MLCs but not MKN45 in vitro. Nevertheless, oral administration of Dasatinib significantly inhibited the development of peritoneal metastasis of MKN45, and resulted in reduced fibrillar formation of metastatic nodules. These results suggest floating MLCs in the peritoneal fluids support the development of peritoneal metastasis possibly through the production of the permissive microenvironment, and thus the functional blockade of MLCs is a reasonable strategy to treat recurrent abdominal malignancies.     

The Vascular Basement Membrane as “Soil” in Brain Metastasis

Brain-specific homing and direct interactions with the neural substance are prominent hypotheses for brain metastasis formation and a modern manifestation of Paget''s “seed and soil” concept. However, there is little direct evidence for this “neurotropic” growth in vivo. In contrast, many experimental studies have anecdotally noted the propensity of metastatic cells to grow along the exterior of pre-existing vessels of the CNS, a process termed vascular cooption. These observations suggest the “soil” for malignant cells in the CNS may well be vascular, rather than neuronal. We used in vivo experimental models of brain metastasis and analysis of human clinical specimens to test this hypothesis. Indeed, over 95% of early micrometastases examined demonstrated vascular cooption with little evidence for isolated neurotropic growth. This vessel interaction was adhesive in nature implicating the vascular basement membrane (VBM) as the active substrate for tumor cell growth in the brain. Accordingly, VBM promoted adhesion and invasion of malignant cells and was sufficient for tumor growth prior to any evidence of angiogenesis. Blockade or loss of the β1 integrin subunit in tumor cells prevented adhesion to VBM and attenuated metastasis establishment and growth in vivo. Our data establishes a new understanding of CNS metastasis formation and identifies the neurovasculature as the critical partner for such growth. Further, we have elucidated the mechanism of vascular cooption for the first time. These findings may help inform the design of effective molecular therapies for patients with fatal CNS malignancies.     

Metastasis of Breast Tumor Cells to Brain Is Suppressed by Phenethyl Isothiocyanate in a Novel In Vivo Metastasis Model

Breast tumor metastasis is a leading cause of cancer-related deaths worldwide. Breast tumor cells frequently metastasize to brain and initiate severe therapeutic complications. The chances of brain metastasis are further elevated in patients with HER2 overexpression. In the current study, we evaluated the anti-metastatic effects of phenethyl isothiocyanate (PEITC) in a novel murine model of breast tumor metastasis. The MDA-MB-231-BR (BR-brain seeking) breast tumor cells stably transfected with luciferase were injected into the left ventricle of mouse heart and the migration of cells to brain was monitored using a non-invasive IVIS bio-luminescent imaging system. In order to study the efficacy of PEITC in preventing the number of tumor cells migrating to brain, mice were given 10 µmol PEITC by oral gavage for ten days prior to intra-cardiac injection of tumor cells labeled with quantum dots. To evaluate the tumor growth suppressive effects, 10 µmol PEITC was given to mice every day starting 14^th day after intra-cardiac cell injection. Based on the presence of quantum dots in the brain section of control and treated mice, our results reveal that PEITC significantly prevented the metastasis of breast cancer cells to brain. Our results demonstrate that the growth of metastatic brain tumors in PEITC treated mice was about 50% less than that of control. According to Kaplan Meir’s curve, median survival of tumor bearing mice treated with PEITC was prolonged by 20.5%. Furthermore as compared to controls, we observed reduced HER2, EGFR and VEGF expression in the brain sections of PEITC treated mice. To the best of our knowledge, our study for the first time demonstrates the anti-metastatic effects of PEITC in vivo in a novel breast tumor metastasis model and provides the rationale for further clinical investigation.