共查询到20条相似文献,搜索用时 15 毫秒
1.
Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment. 相似文献
2.
Recent advances in the field of intravital imaging have for the first time allowed us to conduct pharmacokinetic and pharmacodynamic studies at the single cell level in live animal models. Due to these advances, there is now a critical need for automated analysis of pharmacokinetic data. To address this, we began by surveying common thresholding methods to determine which would be most appropriate for identifying fluorescently labeled drugs in intravital imaging. We then developed a segmentation algorithm that allows semi-automated analysis of pharmacokinetic data at the single cell level. Ultimately, we were able to show that drug concentrations can indeed be extracted from serial intravital imaging in an automated fashion. We believe that the application of this algorithm will be of value to the analysis of intravital microscopy imaging particularly when imaging drug action at the single cell level. 相似文献
3.
《European journal of cell biology》2020,99(6):151098
Metastasis, a process that requires tumor cell dissemination followed by tumor growth, is the primary cause of death in cancer patients. An essential step of tumor cell dissemination is intravasation, a process by which tumor cells cross the blood vessel endothelium and disseminate to distant sites. Studying this process is of utmost importance given that intravasation in the primary tumor, as well as the secondary and tertiary metastases, is the key step in the systemic spread of tumor cells, and that this process continues even after removal of the primary tumor. High-resolution intravital imaging of the tumor microenvironment of breast carcinoma has revealed that tumor cell intravasation exclusively occurs at doorways, termed “Tumor MicroEnvironment of Metastasis” (TMEM), composed of three different cell types: a Tie2high/VEGFhigh perivascular macrophage, a Mena overexpressing tumor cell, and an endothelial cell, all in direct contact. In this review article, we discuss the interactions between these cell types, the subsequent signaling events which lead to tumor cell intravasation, and the role of invadopodia in supporting tumor cell invasion and dissemination. We end our review by discussing how the knowledge acquired from the use of intravital imaging is now leading to new clinical trials targeting tumor cell dissemination and preventing metastatic progression. 相似文献
4.
Bojana Gligorijevic Dmitriy Kedrin Jeffrey E Segall John Condeelis Jacco van Rheenen 《Journal of visualized experiments : JoVE》2009,(28)
In the last decade, intravital microscopy of breast tumors in mice and rats at single-cell resolution1-4 has resulted in important insights into mechanisms of metastatic behavior such as migration, invasion and intravasation of tumor cells5, 6, angiogenesis3 and immune cells response7-9. We have recently reported a technique to image orthotopic mammary carcinomas over multiple intravital imaging sessions in living mice10. For this, we have developed a Mammary Imaging Window (MIW) and optimized imaging parameters for Dendra211 photoswitching and imaging in vivo. Here, we describe the protocol for the manufacturing of MIW, insertion of the MIW on top of a tumor and imaging of the Dendra2- labeled tumor cells using a custom built imaging box. This protocol can be used to image the metastatic behavior of tumor cells in distinct microenvironments in tumors and allows for long term imaging of blood vessels, tumor cells and host cells.Open in a separate windowClick here to view.(56M, flv) 相似文献
5.
Lathia JD Gallagher J Myers JT Li M Vasanji A McLendon RE Hjelmeland AB Huang AY Rich JN 《PloS one》2011,6(9):e24807
High-grade gliomas (World Health Organization grade III anaplastic astrocytoma and grade IV glioblastoma multiforme), the most prevalent primary malignant brain tumors, display a cellular hierarchy with self-renewing, tumorigenic cancer stem cells (CSCs) at the apex. While the CSC hypothesis has been an attractive model to describe many aspects of tumor behavior, it remains controversial due to unresolved issues including the use of ex vivo analyses with differential growth conditions. A CSC population has been confirmed in malignant gliomas by preferential tumor formation from cells directly isolated from patient biopsy specimens. However, direct comparison of multiple tumor cell populations with analysis of the resulting phenotypes of each population within a representative tumor environment has not been clearly described. To directly test the relative tumorigenic potential of CSCs and non-stem tumor cells in the same microenvironment, we interrogated matched tumor populations purified from a primary human tumor transplanted into a xenograft mouse model and monitored competitive in vivo tumor growth studies using serial in vivo intravital microscopy. While CSCs were a small minority of the initial transplanted cancer cell population, the CSCs, not the non-stem tumor cells, drove tumor formation and yielded tumors displaying a cellular hierarchy. In the resulting tumors, a fraction of the initial transplanted CSCs maintained expression of stem cell and proliferation markers, which were significantly higher compared to the non-stem tumor cell population and demonstrated that CSCs generated cellular heterogeneity within the tumor. These head-to-head comparisons between matched CSCs and non-stem tumor cells provide the first functional evidence using live imaging that in the same microenvironment, CSCs more than non-stem tumor cells are responsible for tumor propagation, confirming the functional definition of a CSC. 相似文献
6.
Wang W Goswami S Sahai E Wyckoff JB Segall JE Condeelis JS 《Trends in cell biology》2005,15(3):138-145
Invasion of neighboring extracellular matrix tissue, the lymphatic system and blood vessels is a key element of tumor cell metastasis in many epithelial tumors. Understanding the cell motility pathways that contribute to invasion can provide new approaches and targets for anticancer therapy. The recent convergence of technologies for expression profiling and intravital imaging has revealed the identities of some of the genes that contribute to motility and chemotaxis of cancer cells in tumors. In particular, the genes encoding a minimum motility machine are coordinately upregulated in tumor cells collected by an in vivo invasion assay. These results support a "tumor microenvironment invasion model" and provide new target opportunities for cancer therapy. 相似文献
7.
Leong HS Lizardo MM Ablack A McPherson VA Wandless TJ Chambers AF Lewis JD 《PloS one》2012,7(1):e30177
The analysis of dynamic events in the tumor microenvironment during cancer progression is limited by the complexity of current in vivo imaging models. This is coupled with an inability to rapidly modulate and visualize protein activity in real time and to understand the consequence of these perturbations in vivo. We developed an intravital imaging approach that allows the rapid induction and subsequent depletion of target protein levels within human cancer xenografts while assessing the impact on cell behavior and morphology in real time. A conditionally stabilized fluorescent E-cadherin chimera was expressed in metastatic breast cancer cells, and the impact of E-cadherin induction and depletion was visualized using real-time confocal microscopy in a xenograft avian embryo model. We demonstrate the assessment of protein localization, cell morphology and migration in cells undergoing epithelial-mesenchymal and mesenchymal-epithelial transitions in breast tumors. This technique allows for precise control over protein activity in vivo while permitting the temporal analysis of dynamic biophysical parameters. 相似文献
8.
9.
目的:制备对硝基苯硫酚(4-Nitrobenzenethiol,4-NBT)分子内嵌的星形表面增强拉曼散射(Surface enhanced Raman Scattering,SERS)金"套娃"纳米颗粒,测定其拉曼增强效果和应用于细胞以及活体肿瘤拉曼影像的可行性。方法:以种子介导法先后制备金纳米星及星形SERS金"套娃"纳米颗粒,采用透射电镜观察其形貌,激光粒度分析仪测定其粒径及Zeta电位,拉曼光谱仪测定其拉曼光谱,考察其对A549细胞的拉曼成像效果,建立A549皮下瘤模型,考察其对活体皮下瘤的成像效果。结果:制备并优化的金纳米星粒径较小,为60.5 nm,其针尖密度较高,以此为核心制备的星形SERS金"套娃"纳米颗粒形态规整,粒径约为66.7nm,Zeta电位约为-16.6 m V,拉曼增强效果提升至其前驱体金纳米星的5.3倍,能够实现对A549细胞及A549皮下瘤的拉曼成像。结论:所制备的星形SERS金"套娃"纳米颗粒形态规整均一,拉曼增强效果较好,能实现对细胞及活体肿瘤的拉曼影像。 相似文献
10.
Microenvironmental cues in tumors induce in a wide variety of cellular states that subsequently lead to cancer cells with distinct cellular identity, behavior, and fate. Recent literature suggests that the ability to change cellular states, a process defined as cell state plasticity, enable cells to rapidly adapt to their changing environment during tumor progression and metastasis. In this review, we will discuss how recent high-resolution intravital microscopy studies have been instrumental to reveal the real-time dynamics of tumor cell state plasticity during the different steps of the metastatic cascade. In addition, we will highlight the role of tumor plasticity during anticancer treatment response, and how plasticity can be used as a potential druggable target. 相似文献
11.
Cell migration in tumors 总被引:14,自引:0,他引:14
Invasion of cancer cells into surrounding tissue and the vasculature is an initial step in tumor metastasis. This requires chemotactic migration of cancer cells, steered by protrusive activity of the cell membrane and its attachment to the extracellular matrix. Recent advances in intravital imaging and the development of an in vivo invasion assay have provided new insights into how cancer cell migration is regulated by elements of the local microenvironment, including the extracellular matrix architecture and other cell types found in primary tumors. These results, combined with new findings from in vitro studies, have led to new insights into the molecular mechanisms of cell protrusive activity and chemotactic migration during invasion and metastasis. 相似文献
12.
Esra Gü? Manuel Fankhauser Amanda W. Lund Melody A. Swartz Witold W. Kilarski 《Journal of visualized experiments : JoVE》2014,(86)
Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy. 相似文献
13.
H. L. Rachel Liou Jay T. Myers Deborah S. Barkauskas Alex Y. Huang 《Journal of visualized experiments : JoVE》2012,(60)
Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal''s environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging. 相似文献
14.
Since the time it was pioneered in 1992, intravital imaging of tumors at cellular resolution has offered us the extremely important opportunity of “seeing biology.” However, until now, most studies were monitoring tumor cell behavior in the same animal over short times, requiring the combining of acquired data into a hypothesis via statistical analysis. In the last year, different groups have independently developed techniques to extend the time scale of intravital imaging to several days. This improvement allows one to address the connection between tumor cell behavior and the microenvironment which surrounds them. We can now assess dynamics of the cell-cell interactions in tumors, analyze tumor cell fate and changes in the tumor extracellular matrix which accompany tumor progression.Key words: intravital, multiphoton, spinning disc, microenvironment, second harmonic generation, mammary imaging window, dorsal skinfold chamber, photoswitchingIntravital imaging of tumors at cellular resolution offers insight into the physiology of cells in vivo in real time. The first published study which included injectable dyes to monitor tumor metastasis inside the embryo was done by the group of Groom.1 Some years later, Farina,2 and then Naumov,3 and co-workers, used GFP-labeled tumor cells to study tumors by confocal scanning microscopy. Soon after, Brown,4 and Wang,5 and co-workers, introduced two-photon microscopes into their studies.Until recently, single cell-resolved intravital imaging in tumors commonly involved recording movies 4D (3D through time) with one or two channels, collecting data via multiphoton microscopy from one region at a time.6–8 The inner side of the orthotopic tumor is exposed by making a small incision in the skin and skin folding. This technique, termed ‘skin-flap’, allows for several hours of imaging in one animal. Data from several animals are combined into the final result averaging measurements as well as differences in tumor preparation, animal condition and genotype. Some low-resolution studies have proposed a reversible flap9 on the tumor tissue implanted several days earlier. However, visualized areas were not the same at each of the timepoints. Also, as skin flaps were opened repeatedly, they were potentially influencing the microenvironment by surgery-related immune/inflamatory-responses. In addition, several groups have been using a dorsal skinfold chamber10 in which the tumor is grown ectopically, in the space between the skin and glass coverslip on the back of the mouse. This preparation could be used for either low resolution measurements over several days, or short-term measurements at cellular resolution.In the last few months, several studies have included techniques which extend the time-scale of intravital imaging in tumors from hours to days (Technique MIW + photoswitching12 Dorsal skinfold + SHG recognition13 Extended skinflap15 Orthotopic tumors Yes No Yes Long-term anesthesia needed No No Yes Multiple imaging sessions available Yes Yes No Microscopy Confocal and multiphoton Multiphoton Spinning disc confocal Depth of imaging ∼120 µm12 ∼100 µm13 <70 µm15 Detectors PMT (1 for each channel) PMT (1 for each channel) Camera Number of channels 4 2 4