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Method Article
Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.
This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.
The premetastatic niche is an important process in cancer metastasis defined as a set of alterations that occur in the organs that are targets for metastases prior to arrival of tumor cells1,2. Therefore, therapeutic targeting of this step of cancer progression may prevent metastases to the vital organs that cause approximately 90% of cancer-associated deaths. Although the concept of the premetastatic niche, also known as the "seed and soil" theory, was introduced more than a century ago, no experimental proof has been provided until recently, when the bone marrow-derived cells were demonstrated to contribute to the premetastatic soil1,3-7. Despite these developments, the premetastatic niche remains a largely understudied aspect of cancer pathophysiology and further research to identify cellular players and mechanisms involved is needed.
Here we report the in vivo approaches to study the role of alveolar macrophages in breast cancer metastases and the lung premetastatic niche. The alveolar macrophages arrive to the lungs early during the embryonic development and self-renew there during adulthood8. They also have important immunomodulatory and homeostatic functions including the protection of this organ from undesired inflammatory responses to the environmental innocuous antigens9. Therefore, we hypothesize that tumors exploit this physiological immunosuppression, imposed by alveolar macrophages, and, consequently, alveolar macrophages contribute to the lung premetastatic niche by suppressing antitumor immunity. This hypothesis is supported by our recent report demonstrating that the specific depletion of these cells reduces lung metastases and enhances antitumor T cell responses10.
For these studies we apply a well-established syngeneic model of breast cancer (4T1), which mimics stage IV metastatic breast cancer11; and has been previously reported in studies of the premetastatic niche6. To track metastasizing tumor cells in vivo we use 4T1 cells expressing GFP (4T1-GFP) in conjunction with animal imaging and confocal microscopy. We focus on the lung premetastatic niche, since this organ is one of the most common targets of hematogenous metastases of human malignancies12. To investigate functions of alveolar macrophages in the premetastatic niche, we use clodronate liposomes to deplete these cells13; and evaluate impact of this depletion on lung metastases. Of note, this method specifically depletes alveolar macrophages but no other phagocytic cells in the lungs or in circulation10.
所有动物研究已经批准了机构动物护理和德州理工大学健康科学中心使用委员会,并遵循"指南实验动物的护理和使用"由美国国立卫生研究院发表在列出的各项规定。用八到十二周龄的雌性BALB / c小鼠是市售的。注射1×10 5个 4T1或1×10 5个 4T1细胞表达GFP,可从不同的供应商处购买,到乳腺脂肪垫。
1. 4T1文化和4T1-GFP的细胞和肿瘤细胞悬浮用于注射14准备
2.脂质体的10鼻腔给药
注:除非另有规定执行在层流(LAF)生物安全柜使用无菌溶液的所有步骤。
3.鼠标牺牲和组织收集
注:使用高压灭菌和鼠标清扫消毒器械。安乐死而下,通过重要器官的放血和去除小鼠麻醉。
4.肺转移瘤的评价
5.流式细胞仪(FACS)肺泡巨噬细胞在肺内分析
6. FACS数据分析:门控策略及细胞亚群的鉴定
4T1-GFP肿瘤细胞注射到乳腺脂肪垫导致了概括人乳腺癌的转移扩散小鼠肿瘤( 图1A)的形成,如转移灶在肺中( 图2),肝脏,骨骼迅速地形成和老鼠11大脑。 4T1细胞用GFP稳定转染促进肿瘤生长的监测( 图1B),跟踪转移性肿瘤细胞和定量转移负担( 图2B,3B)。另外,肿瘤的成像提供关于几种病理特征如肿瘤细胞...
The recent insights into cancer biology and causative factors involved in carcinogenesis and tumor progression lead to development of genetically engineered mouse (GEM) models of cancer, in which tumors grow spontaneously, usually over a period of several months15. Although these tumor models appear to reflect better the natural history of human malignancies than xenografts or syngeneic models, much time required for tumor development and various degrees of malignant phenotype penetrance limit the use of these...
作者宣称,他们没有竞争的经济利益。
这项研究是由美国国防部支持的授予TSA 140010到MK和BC 111038到MMM由作者本人观点和意见,推荐并不会反映这些美国陆军和国防部的。
Name | Company | Catalog Number | Comments |
4T1 cell line | American Type Culture Collection, Manassas, VA, USA | CRL 2539 | Tumor cells |
4T1-GFP cell line | Caliper life sciences/ Perkin Elmer, Waltham, MA, USA | BW128090 | Tumor cells |
RPMI | Corning, Corning, NY, USA | 10-040-CM | Media |
Heat inactivated FBS | Gibco (Thermo Scientific), USA | 10082147 | Media |
Penicillin Streptomycin | Fisher Scientific, Waltham, MA, USA | MT-300-02-CI | Media |
PBS | Fisher Scientific, Waltham, MA, USA | BP399-20 | Dilute with distilled water |
Trypsin 0.25% with EDTA | Hyclone, Logan, Utah, USA | SH30042.02 | Tissue culture supplies |
T75 cm2 flask | Fisher Scientific, Waltham, MA, USA | 12-565-32 | Tissue culture supplies |
15 ml conical tube | BD falcon, Franklin Lakes, NJ, USA | 352096 | Tissue culture supplies |
50 ml conical tube | BD falcon, Franklin Lakes, NJ, USA | 352098 | Tissue culture supplies |
60 mm2 Petri dish | Fisher Scientific, Waltham, MA, USA | AS4052 | For lung imaging |
Isoflurane (Isothesia) | Butler Schein Animal health, Dublin, OH, USA | NDC 11695-6776-2 | Mouse anesthesia |
Clodronate liposomes | Formumax Scientific Inc, Palo Alto, CA, USA | F70101C-N | Macrophages depletion |
Control liposomes | Formumax Scientific Inc, Palo Alto, CA, USA | F70101-N | Control PBS-liposomes |
29 gauge insulin syringes (12.7 mm and 0.5 ml capacity)- Reli-On | Walmart, Bentonville, AR, USA | For tumor cell injection | |
Hair removal cream (Nair) | Walmart, Bentonville, AR, USA | ||
Paraformaldehyde solution (4%) | Affymetrix, Santa Clara, CA, USA | 19943-I Lt | Dilute to 4% or 1% using 1x PBS |
OCT compound | Fisher Scientific, Waltham, MA, USA | 230-730-571 | For freezing tissue in cryomolds |
Fluoro-Gel-II with DAPI | Electron Microscopy Sciences, Hatfield, PA, USA | 17985-51 | Mounting medium |
Sucrose | Sigma, St. Louis, MO, USA | S-9378 | Cryopreservation |
Collagenase P | Roche, Basel, Switzerland | 11249002001 | Components of tissue digestion buffer |
Dnase I | Roche, Basel, Switzerland | 10104159001 | Components of tissue digestion buffer |
Trypsin inhibitor | Sigma, St. Louis, MO, USA | T9253 | Components of tissue digestion buffer |
40 micron cell strainers | Fisher Scientific, Waltham, MA, USA | 22-363-547 | Used in tissue digestion to remove clumps |
Trustain FcX-Fc Block (CD16/CD32) | Biolegend, San Diego, CA, USA | 101320 | Antibodies for flow cytometry |
BV605 CD45 | Biolegend, San Diego, CA, USA | 103139 | Antibodies for flow cytometry |
PE CD11b | Biolegend, San Diego, CA, USA | 101207 | Antibodies for flow cytometry |
PE Cy7 F4/80 | Biolegend, San Diego, CA, USA | 123113 | Antibodies for flow cytometry |
APC/Cy7 CD11c | Biolegend, San Diego, CA, USA | 117323 | Antibodies for flow cytometry |
PerCPcy5.5 IA/IE (MHCII) | Biolegend, San Diego, CA, USA | 107625 | Antibodies for flow cytometry |
PE CD80 | Biolegend, San Diego, CA, USA | 104707 | Antibodies for flow cytometry |
AF647 CD86 | Biolegend, San Diego, CA, USA | 105019 | Antibodies for flow cytometry |
Fixable viability Dye eflour 506 | eBioscience, San Diego, CA,USA | 65-0866 | Antibodies for flow cytometry |
Cryostat | Leica Biosystems, Buffalo Grove, IL, USA | CM1850 | Cryosectioning |
UVP iBox Explorer | UVP Inc, Upland, CA, USA | Mouse and lung fluorescent imaging | |
Aperio Scanscope CS | Leica Biosystems, Buffalo Grove, IL, USA | Digital pathology | |
BD LSRFortessa | BD Biosciences, Franklin Lakes, NJ, USA | Flow cytometry/data acquisition | |
Nikon A1 confocal TE2000 microscope | Nikon Instruments Inc., Melville, NY 11747-3064, U.S.A. | Imaging and quantifying GFP fluorescence in lung cryosections | |
UVP visionworks software (Version 7.1RC3.38) | UVP Inc, Upland, CA, USA | Imaging software for iBOX | |
Aperio Imagescope software (v12.1.0.5029) | Leica Biosystems, Buffalo Grove, IL, USA | Imaging software for analysis of digital slides | |
Flow JO software (version 9.8.1) | Flow JO LLC, Ashland, OR, USA | Analysis of flow cytometric data | |
NIS Elements AR (version 4.20.01) 64 Bit | Nikon Instruments Inc., Melville, NY 11747-3064, U.S.A. | Acquisition and analysis of lung cryosections for GFP |
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