Dear Simona,

There are a variety for Spheroids cryosectioning. I have copied a protocol which I think will be a good to start with. The other protocols are described in the attached files.

Materials and Methods

Cell Lines and Culture

GFP-4T1 [10] is a green fluorescent protein (GFP)-expressing mouse metastatic mammary epithelial cell line that is resistant to Taxol [11]. The 2H11 cell line was validated as a tumor-like endothelial cell line by Walter-Yohrling et al. [12]. Most endothelial cell lines being used to study angiogenesis have been immortalized using SV40 and express the SV40 T antigen, with the assumption that SV40 is nonpermissive in murine cells. Although transformed, these cell lines tend to retain most of the normal cellular physiology and functional characteristics of endothelial cells. 2H11 is one such endothelial cell line originally generated by O'Connell and Rudmann [13], O'Connell and Edidin [14], and O'Connell et al. [15] from endothelial cells isolated from lymph nodes of adult C3H/HeJ mice transformed using SV40 displaying characteristics of Kaposi sarcoma. This cell line is most well characterized by the presence of several attributes typical of normal endothelial cells and those of endothelial cells directly isolated from tumors. Walter-Yohrling et al. [12] thus termed this cell type as tumor-endothelial cells. The standard endothelial cell markers found in this cell type, some of which we have also verified in this article, include sialomucin/CD34, GPIIIB/CD36, endogolin/CD105, P1H12/CD146, VCAM1/106, Tie-1, and Tie-2. The tumor-endothelial markers that are expressed in relatively high levels include mTEM1, mTEM5, mTEM7, and mTEM8. This murine endothelial cell line also responds to antiangiogenic agents by inhibition of proliferation and tube formation. It has been identified as a murine endothelial cell line to model tumor endothelium for studying the antiangiogenic activity of therapeutic compounds in vitro and is therefore our choice for representing the endothelial component in our model. The cell lines were maintained as monolayer culture at 37°C and 5% CO2 in Dulbecco modified Eagle medium (Mediatech, Manassas, VA), supplemented with 10% fetal bovine serum (Atlas, Fort Collins, CO), 100 U/ml penicillin, and 100 µg/ml streptomycin (HyClone, South Logan, UT).

Chemicals and Reagents

Paclitaxel (Taxol), dimethyl sulfoxide (DMSO), and MitoTracker Red CMXRos were purchased from Sigma-Aldrich, Inc (St Louis, MO), Research Organics (Cleveland, OH), and Invitrogen (Carlsbad, CA) respectively. Antibodies to murine NOV/CCN3, murine interleukin 1α, and rat matrix metalloproteinase 8 (MMP-8) for reverse-phase immunoblot analysis were obtained from R&D Systems (Minneapolis, MN). The glyceraldehyde-3-phosphate dehydrogenase antibody was from Cell Signaling Technology (Danvers, MA). The MMP-9 antibody was obtained from Abcam (Cambridge, MA). The antibodies galectin-1 (Santa Cruz Biotechnology, Santa Cruz, CA), CD34 (BD Pharmingen, Sparks, MD), and human von Willebrand factor (vWF; Dako Denmark A/S, Glostrup, Denmark) were used for immunoblot analysis and immunostaining to identify the tumor-endothelial cells in spheroids.

Spheroid Culture in “Hanging Drop”

GFP-4T1 tumor cells and 2H11 endothelial cells were used to generate multicellular spheroids by growing them as “hanging drops” of medium (in Dulbecco modified Eagle medium with 10% fetal bovine serum and antibiotic/antimycotic mix) [16]. Briefly, single-cell suspension of GFP-4T1 cells (1000 cells/20 µl) was dispensed on the inside of the lid of each well of a 48-well cell culture plate (Greiner Cellstar, BioExpress, Kaysville, UT). Gravity-enforced self-aggregation of cells was facilitated by inverting the lid and setting it on the plate and incubating under standard conditions. At day 3, the lids were set upright, and 2000 cells in a volume of 5 µl, 2H11 endothelial, or additional GFP-4T1 tumor cells were introduced to the existing culture. These cultures were reinverted and left as hanging drops and incubated further for 10 to 14 days. The day tumor or endothelial cells were reintroduced to the tumor cell aggregates in the hanging drop of medium was taken as day 1 in all experiments. Introduction of 1/10th the number of tumor or endothelial cells to the existing hanging drop culture of 4T1 tumor cells did not change the pattern of growth and morphology of the spheroids (Figure W1). Because the proliferation of cells in spheroids is not as fast as that observed in monolayer cell cultures and to generate adequate numbers of each cell type from the spheroids to assess the treatment response when tumor and endothelial cells were in contact, we opted to do our experiments using spheroids prepared by the introduction of 2000 cells to the existing culture (1:1 ratio). The three-dimensional spheroids composed of GFP-4T1 cells alone are referred to as tumor cell-only spheroids and those formed by the coculture of 4T1 and 2H11 cells types are referred to as tumor-endothelial cell spheroids in the rest of this report. Both tumor cell-only and tumorendothelial cell spheroids remain intact and viable at day 10, and therefore, all in vitro and in vivo experiments have been done using spheroids at day 10 of culture in hanging drops.

Immunohistochemistry

Spheroids were harvested and frozen in OCT (Tissue-Tek, Sakura Finetek USA, Inc, Torrance, CA). A total of 6 to 10 spheroids were embedded in each OCT block. Five-micrometer cryostat sections were immunohistochemically analyzed using standard protocols. The primary antibodies used recognized CD34 at a dilution of 1:50 or vWF at a dilution of 1:200. Fluorescent visualization was performed with 1:200 Northern Lights antirat IG-NL557 (R&D Systems) and 1:200 Cy 3 (Jackson ImmunoResearch Laboratories, Inc, West Grove, PA) antibody, respectively, and counterstained with Vectashield Mounting Medium with 4′,6-diamidino-2-phenylindole (Vector, Burlingame, CA). Slides were observed at 10x, 20x, or 40x using Olympus IX71 microscope and images taken by an Olympus DP72 camera (Olympus, Tokyo, Japan).

Hematoxylin and Eosin Staining and Imaging

Hematoxylin and eosin (H&E) staining was performed by staining the cryostat sections with Harris hematoxylin (aluminum potassium sulfate, hematoxylin, absolute alcohol, mercuric oxide, and glacial acetic acid) followed by 1% acid alcohol and, subsequently, 1% eosin. Images of spheroids were taken at 10x using Olympus IX71 microscope and Olympus DP72 camera. Tissue imaging was performed using an Aperio ScanScope (Aperio, Vista, CA) at 20x magnifications and analyzed using ImageScope software (Aperio).

Stabilization of Spheroids and Confocal Microscopy

Tumor-endothelial cell spheroids consisting of the GFP-4T1 murine mammary epithelial cancer cells stably expressing GFP and the 2H11 murine tumor-endothelial cells and the tumor cell-only (GFP-4T1) spheroids were incubated with 1 µM of MitoSOX Red (Invitrogen) at 37°C for 10 minutes followed by washing twice with phosphate-buffered saline (PBS). The spheroids were then fixed for 3 hours at 4°C in 1% parafomaldehyde (EUROMEDEX, Mundolsheim, France) and washed twice with PBS. SeaPlaque low-melt agarose (0.8%; FMC BioProducts, Rockland, ME) was prepared for use as described by the manufacturer, cooled on ice, and made isotonic by the addition of 40x PBS. SeaPlaque low-melt agarose was added to the spheroids, and then they were loaded onto cooled slides. Double-sided tape was used between the slide and coverslipped to maintain the shape of the tumor spheroids. Imaging was performed using a confocal laser scanning system (LSM 510; Carl Zeiss, Peabody, MA).

Cell Death Assay

At day 10, the spheroids were transferred to a low-binding 96-well plate (Nalgene, Nunc, Japan) and subjected to treatment with drug for 48 hours. After drug treatment, spheroids were dissociated using trypsin. The three-dimensional spheroids developed an extracellular matrix, making it extremely difficult to dissociate the cells; we therefore passed the cells after dissociation through a 40-µm filter to avoid the presence of cell clusters. The suspension is diluted to a concentration of 5 x 104/ml for measurement of apoptosis using a cell death detection ELISA kit (Roche, Indianapolis, IN). This is a quantitative photometric immunoassay for the determination of cytoplasmic histone-associated oligosomes generated during apoptosis. After dilution, cells were centrifuged at 200g for 5 minutes, and the cell pellet was resuspended in 0.5 ml of incubation buffer and incubated at room temperature for 30 minutes. After centrifugation at 16,000g for 10 minutes, 0.4 ml of supernatant was removed and diluted 1:10 in incubation buffer for analysis. The ELISA plate was prepared according to the manufacturer's instructions, and 0.1 ml of sample was added to the appropriate wells and incubated at room temperature for 90 minutes. After conjugation and incubation with substrate solution, the plate was shaken on an orbital shaker at 250 rpm for 15 minutes, and the absorbance at 405 nm was determined using an ELx800 microplate reader (BioTek, Winooski, VT).

Fluorescence Assay

The tumor cell-only and the tumor-endothelial cell spheroids were transferred to low binding 96-well plates at day 10, and emission of fluorescence was read at 510 nm using a Synergy 2 Multi-Mode Microplate Reader (BioTek). The spheroids were subjected to treatment with Taxol for 48 hours after which the fluorescence intensity was read again. The relative decrease in fluorescence was calculated to assess the response of 4T1 tumor cells to Taxol in the presence of 2H11 endothelial cells.

Survival Assay

The tumor cell-only and tumor-endothelial cell spheroids were transferred to low-binding 96-well plates and irradiated at 2 Gy. Spheroids of each type were pooled and trypsinized, cell clusters were removed by passing through a 40-µm filter, and 200 to 8000 cells of each treatment were plated in triplicate onto a six-well culture dish. Phase-contrast and fluorescence images were taken 2 weeks after plating. A final cell count after 2 weeks for each set was done to estimate the overall survival.

Animals

Female, athymic, nude mice (8–9 weeks old, 20–22 g) were purchased from Charles River Laboratories (Wilmington, MA) and housed in the animal care facility of the University of Arkansas for Medical Sciences. All experiments were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences. The mice were given clean water ad libitum and 2016 Teklad Global 16% Protein Rodent Diet (Harlan Feeds, Woodland, CA).

Animal Model and Surgical Techniques

All surgical procedures were performed in a sterile field. Dorsal skin window chambers and surgical instruments were autoclaved before use. Saline used to keep tissue moist during surgical preparation was mixed with gentamicin (50 µl/ml).

Dorsal skinfold window chamber model. The dorsal skin window chamber in the mouse was prepared as described [17,18]. Briefly, female mice (20–22 g body weight) were anesthetized (inhalant isoflurane at 3% maintenance) and placed on a heating pad. Two symmetrical titanium frames were implanted into a dorsal skinfold, to sandwich the extended double layer of skin. An approximately 15 x 15-mm2 layer was excised. Two three-dimensional spheroids of each type at day 10 after coculture in hanging drops of medium were placed on the underlying cutaneous maximus muscle and subcutaneous tissues of each mouse, which were subsequently covered with a glass coverslip incorporated in one of the frames. After a recovery period of 1 to 2 days, the windows were imaged for neovascularization using an Olympus IX71 microscope and an Olympus DP72 camera.

Blood Vessel Extraction Using MATLAB

Using a computer program created in house, two-dimensionally projected vascular networks were produced from the region of interest selected from images recorded with the 4x objective. The algorithms used for identification of the vascular networks were implemented by use of the MATLAB software (MathWorks, Natick, MA). The extraction technique involved a series of filtering, threshold, and other morphologic image analysis procedures to get vascular tree structure. Finally, binary images of blood vessel structure were assigned the intensity of the input image [19].

Rear limb model. A small incision was made in the right hind limb above the ankle and two three-dimensional spheroids at day 10 after coculture in hanging drops of medium were implanted in the exposed subcutaneous tissue of each mouse. The incision was then sutured with tissue adhesive (3M VetBond, St Paul, MN). Tumor growth was recorded by caliper measurement. The mean of two perpendicular diameters was obtained. Tumor diameters were measured each day. The animals were killed at the end of 33 days. Lung and spleen were excised to observe green fluorescence (GFP-4T1) indicative of possible metastasis.

Intravital Fluorescence Microscopy

The dorsal skinfold window chamber model allows for intravital imaging of the tumor growth and neovascularization. Because the 4T1 tumor cells were GFP positive, the normal and tumor tissue could be easily differentiated. Fluorescence microscopy was performed using an Olympus IX71 microscope, and digital images were taken every 4 days after the window chambers were implanted using an Olympus DP72 camera.

Mouse Angiogenesis Antibody Array

The mice were killed on day 16 after implantation. Tumors originating from tumor cell-only and tumor-endothelial cell three-dimensional spheroids were excised and homogenized in 400 µl of lysis buffer (25 mM HEPES, pH 7.5, 0.5% sodium deoxycholate, 5 mM EDTA, 5 mM dithiothreitol, 20 mM glycerophosphate, 1 mM Na3 VO4, 50 mM NaF, 1% Triton X-100, 20 µg/ml aprotinin, 50 µg/ml leupeptin, 10 µM pepstatin, 1 µM okadaic acid, and 1 mM phenylmethylsulfonyl fluoride). The lysates were incubated at 4°C and passed through a 21-gauge syringe 10 times, and the insoluble material was removed by centrifugation (15 minutes at 12,000g). The protein concentration was determined using the Bio-Rad protein assay. Two hundred micrograms of lysate was hybridized to a mouse angiogenesis antibody array (R&D Systems) as instructed.

Pathway Analysis

The Ingenuity Pathway Analysis Tool was used to generate significant gene networks and examine the functional associations between differentially expressed proteins in the angiogenesis arrays obtained from incubation with lysates of tumors originating from GFP-4T1 tumor and GFP-4T1-2H11 tumor-endothelial spheroids (www.ingenuity.com).

Immunoblot Analysis

Lysates from the three-dimensional spheroid of each type were prepared in the lysis buffer as described. About 10 to 20 spheroids were incubated in the lysis buffer described above at 4°C for 1 hour on NUTATOR (Benchmark Scientific, Inc, Edison, NJ). Cell debris was removed by centrifugation (15 minutes at 12,000g). Immunoblot analysis was performed using 50 µg of protein/lane and standard polyvinylidene fluoride membrane transfer followed by probing with the respective antibody and chemiluminescent detection of the bands.

Statistical Analysis

Data are expressed as mean ± 1 SD of at least three different experiments unless otherwise mentioned. Statistical significance of difference in means was performed using the parametric two-sample t test with unequal variance (MathWorks). The corresponding P values are shown for each of the comparisons under the respective sections.

Hoping these publications will be helpful,

Rafik

thanks so much!

thanks so much!