This work was financially funded by Geran Putra Berimpak (GBP), Universiti Putra Malaysia: 9542800. We would like to thank the Agro-Biotechnology Institute, Malaysia (ABI) laboratories, and microscopy imaging facility. Special thanks to Mohd Daniel Hazim for help with editing and recording the experimental video for this work.

NOTE: Sections 1&#8722;6 describe the coculture model of 4T1 mouse mammary carcinoma cells and RAW 264.7 mouse macrophages. Section 7 describes the time-lapse assessment of M1 macrophages phagocyted 4T1 cells.
1. Culturing 4T1 mouse mammary carcinoma cells and RAW 264.7 mouse macrophages
<ol>
	<li>Thaw a vial of cryogenically preserved 4T1 and RAW 264.7 passage 6 cells by gentle agitation in a water bath at 37 &#176;C or the normal growth temperature for the cell lines. 
	NOTE: Thawing should be done rapidly, approximately within 2 min. To avoid contamination, remove the vial from the water bath and decontaminate it by spraying with 70% ethanol. To avoid genetic drift, especially for the macrophages, use a low passage number (i.e., less than 20).</li>
	<li>Dilute the thawed cells in 10 mL of complete DMEM medium in a 15 mL conical tube and centrifuge the cells at 600 x g for 5 min to obtain a cell pellet. 
	NOTE: Complete DMEM medium supplemented with 10% (v/v) fetal bovine serum (FBS), 200 U/mL penicillin/streptomycin, and 2 mM L-glutamine is used throughout the protocol.</li>
	<li>Carefully aspirate the media, resuspend the cells in 8 mL of complete medium and transfer into a 25 cm2 tissue culture treated flask. Culture both 4T1 and RAW 264.7 cells in complete DMEM medium at 37 &#176;C with 5% CO2. 
	NOTE: Resuspend the cell pellets gently and add the cell suspension into the culture flask drop by drop to avoid killing the cells.</li>
	<li>After 1 week of culturing to allow cell adherence, monitor the flask daily and add 8 mL of complete DMEM medium as needed.</li>
</ol>
2. Immunostaining of M1 polarized RAW 264.7 macrophages
<ol>
	<li>As the confluency of RAW 264.7 cells reaches 70&#8722;80%, discard the culture media and gently rinse 2x with at least 2 mL of PBS. 
	NOTE: Before seeding RAW 264.7 cells, make sure no cell differentiation has occurred (i.e., dendrite-like phenotype and/or increased cell size). If cell morphology changes are visible, discard the culture and thaw a new cell line from the earlier passage vial.</li>
	<li>Prepare the macrophages for collection by gently dislodging the adherent cells using a cell scraper and transfer them into a 15 mL conical tube.</li>
	<li>Count the cells using a hemocytometer and prepare a cell suspension of 1 x 105 cells/dish using complete DMEM medium.</li>
	<li>Seed 2 mL of the macrophages suspension into two imaging dishes. Incubate the cells 2&#8722;3 h at 37 &#176;C with 5% CO2 to allow cell adherence.</li>
	<li>Prepare M1 polarization media by adding 100 ng/mL LPS and 20 ng/mL IFN-&#947; into complete DMEM medium10,11.</li>
	<li>Discard the culture supernatant from the macrophages and carefully wash the monolayers in the dish 2x with at least 1 mL of PBS.</li>
	<li>Add 2 mL of M1 polarization media into one of the imaging dishes, allow the RAW 264.7 cells to induce the M1 macrophage phenotype, and incubate 2&#8211;3 h at 37 &#176;C with 5% CO2. 
	NOTE: Seed RAW 264.7 macrophages in an imaging dish with DMEM supplemented with 10% FBS as a control for the anti-iNOS antibody staining. Label the imaging dishes RAW (control) and M1 macrophages (LPS and IFN-&#947; polarized), respectively.</li>
	<li>Before immunostaining, fix the RAW 264.7 and M1 macrophages cells using 2 mL of 4% paraformaldehyde and incubate for 15 min at 37 &#176;C (room temperature, RT).</li>
	<li>Remove the supernatant and wash the cell monolayer with 1 mL of PBS at least 3x.</li>
	<li>Quench with 2 mL of 50 mM ammonium chloride in PBS and incubate for 15 min at RT.</li>
	<li>Permeabilize using 2 mL of 0.3% Triton X-100 in PBS for 15 min at RT.</li>
	<li>Remove the supernatant and wash the cell monolayer with 1 mL of PBS at least 3x.</li>
	<li>Block using 2 mL of 10% FBS in PBS for 30 min at RT. 
	NOTE: The blocking step in immunostaining is to minimize the non-specific binding of antibody within the cell.</li>
	<li>Remove the supernatant and wash the cell monolayer with 1 mL of PBS at least 3x.</li>
	<li>Incubate with 2 mL of anti-iNOS-FITC in PBS and 1% FBS overnight at 4 &#176;C. 
	NOTE: Label the cells using the appropriate antibody dilution according to the manufacturer's recommendations. Protect the dishes from light by covering them with aluminum foil from this point on.</li>
	<li>Remove the supernatant and wash the cell monolayer with 1 mL of PBS at least 3x.</li>
	<li>Incubate 1 mL of 300 nM 6-diamidino-2-phenylindole (DAPI) into imaging dishes for 10 min in 37 &#176;C, in the dark by covering with aluminum foil.</li>
	<li>Wash cells with 1 mL of PBS at least 3x and add 1 mL of complete DMEM medium into the imaging dishes. 
	NOTE: The cells are now ready for fluorescence imaging. The microscope setup will need an inverted stage and excitation filters for the chosen stains (in this case, FITC and DAPI).</li>
	<li>Turn on the microscope and load the imaging software. Mount the imaging dish on the microscope and adjust the focus to observe RAW 264.7 and M1 macrophages. Optimize the appearance of phase-contrast FITC and DAPI images by adjusting the transmitted light and exposure times. 
	NOTE: Begin imaging when all points are in focus and the channels are optimized.</li>
	<li>Capture phase-contrast FITC and DAPI images (Figure 1).</li>
</ol>
3. Seeding 4T1 mouse mammary carcinoma cells
<ol>
	<li>As the confluency of 4T1 cells reaches 70&#8722;80%, discard the culture media and gently rinse 2x with at least 2 mL of PBS. Add 1 mL of prewarmed trypsin to dissociate the cells and incubate for up to 20 min at 37 &#176;C. 
	NOTE: Observe the cells under a microscope. Detached cells will be rounded.</li>
	<li>Once the cells detach, transfer them to a 15 mL conical tube and add 2 mL of prewarmed complete DMEM medium to inactivate the trypsin. Gently disperse the medium by pipetting the cell layer surface to recover the cells. Then, centrifuge at 600 x g for 5 min to obtain a cell pellet.</li>
	<li>Remove the supernatant and resuspend the pellet in 10 mL of prewarmed complete DMEM medium.</li>
	<li>Count the cells using a hemocytometer and seed 1 x 105 4T1 cells in 2 mL of complete DMEM medium in each of two 35 mm glass-bottom imaging dishes treated with tissue culture. Incubate the cells overnight at 37 &#176;C with 5% CO2. 
	NOTE: Culture 4T1 cells in one of the imaging dishes with M1 polarization media and label 4T1-Inducer (control). This is to ensure normal growth of 4T1 cells when exposed to the inducers.</li>
</ol>
4. Labeling living 4T1 mouse mammary carcinoma cells using CFSE staining
<ol>
	<li>First, remove the existing media from the 4T1 cells imaging dishes. Wash the cell monolayer at least 2x with 1 mL of PBS.</li>
	<li>Prepare 5 &#181;M of CFSE staining solution by diluting CFSE in 1 mL of PBS. Add 1 mL of 5 &#181;M CFSE staining solution into each imaging dish and incubate the cells for 20 min at RT or 37 &#176;C, in the dark. 
	NOTE: Label the cells using appropriate CFSE working concentration according to the manufacturer's recommendations and preliminary experiments to obtain the optimum CFSE staining concentration. Labeling efficiency will be higher if CFSE is diluted in PBS.</li>
	<li>Quench the staining by adding an equal volume of complete DMEM medium containing 10% FBS and staining solution to the cells and incubate for 5 min at RT in the dark.</li>
	<li>Discard the CFSE-containing solution and wash the cells 1x with an equal volume of culture media. 
	NOTE: 4T1 cells are now fluorescently labeled.</li>
	<li>Return the 4T1-CFSE cells to standard culture conditions at RT with 5% CO2.</li>
</ol>
5. Seeding RAW 264.7 mouse macrophages and coculture with 4T1
<ol>
	<li>As confluency of RAW 264.7 reach 70&#8722;80%, discard the culture media and gently rinse 2x with at least 2 mL of PBS.</li>
	<li>Prepare the macrophages for collection by gently dislodging the adherent cells using a cell scraper and transfer them into a 15 mL conical tube.</li>
	<li>Count the cells using a hemocytometer and prepare a cell suspension of 1 x 105 cells/mL by diluting the remaining solution with a complete DMEM medium according to the seeding density. 
	NOTE: Overly confluent cells in cocultures may severely reduce phagocytosis. In this study, the seeding ratio of macrophages: cancer cells was adjusted to a 1:1 ratio. The ratio can be adjusted depending on the aggressiveness of the tumor cells and the origin of the macrophages.</li>
	<li>Before coculturing, discard the culture supernatant from the 4T1 cells seeded a day before (step 4.5) and carefully wash the monolayers 2x with at least 1 mL of PBS.</li>
	<li>Seed 1 x 105 RAW 264.7 cells per 2 mL of complete DMEM medium into one of the 4T1 imaging dishes. Incubate the cells 2&#8722;3 h at 37 &#176;C with 5% CO2 to allow cell adherence. Label the imaging dish 4T1-M1 coculture. 
	NOTE: The 4T1 (control) cells were not cocultured with macrophages.</li>
</ol>
6. M1 polarization of RAW 264.7 macrophages
<ol>
	<li>Prepare M1 polarization media by adding 100 ng/mL LPS and 20 ng/mL IFN-&#947; into complete DMEM medium supplemented with 10% (v/v) FBS10,11.</li>
	<li>Discard the culture supernatant from the macrophages incubated before (step 5.5) and carefully wash the monolayers in the dish 2x with at least 1 mL of PBS.</li>
	<li>Then, add 2 mL of M1 polarization media into the imaging dish and allow the RAW 264.7 cells to induce into the M1 macrophage phenotype by incubating at 37 &#176;C with 5% CO2. 
	NOTE: M1 macrophage cells may take up 2&#8722;3 h of incubation to achieve complete polarization. Preliminary experiments need to be done to ensure the suitable incubation period to allow complete polarization of macrophages.</li>
</ol>
7. Live-cell video microscopy of phagocytosis
NOTE: Many factors need to be considered when performing live-cell imaging to obtain a producible video, including optimization of image exposure, time measurement, and automatic focus correction.
<ol>
	<li>Before the experiment, set up the cell culture incubator by turning on thermostat at 37 &#176;C and supplying 5% CO2. 
	NOTE: The microscope setup requires an inverted stage, a stage top incubator, humidity control (95%), and gas incubation system. This setup is crucial to maintain the cells for long-term live-cell video microscopy assessment.</li>
	<li>Turn on the microscope including the piezo stage and load the microscope imaging software.</li>
	<li>Position the seeded coculture cells in a 35 mm glass-bottom imaging dish in the center of the stage and gently screw on the top chamber. Use the joystick to motorize the stage by selecting the position to be imaged.</li>
	<li>To view the sample, select the phase-contrast filter on the turret. After initially viewing the sample, locate the appropriate fields and bring the sample into focus. 
	NOTE: Imaging software allows use of fully automated objective selection, Perfect Focus System, time-lapse series, multichannel image acquisition, and multipoint image acquisition.</li>
	<li>To set up multichannel fluorescence for CFSE labeling and differential interference contrast acquisition, open the imaging software acquisition dialog box and select the [Lambda] tab checkbox (Figure 2). 
	NOTE: For a 13 h time-lapse imaging, a phase-contrast channel should be used.</li>
	<li>Select [Lambda] tab | [Optical Configuration] and select [10X DIC] for differential interference contrast and [GFP-R] for the green fluorescence filter checkbox. Under the [Lambda] | [Focus] column, select the [10X DIC] checkbox to set as the focus reference.</li>
	<li>Open the imaging software acquisition dialog box and click the [XYZ Time &#8230;] button and select the [XY] tab.</li>
	<li>Move to the image acquisition point using the joystick on the motorized stage while checking the live image or select a different position through the eyepieces. Then click on the checkbox under the [Point Name] column to set each point in each location for image capture (see Figure 3). 
	NOTE: The automated stage allows multipoint image acquisition of different XY coordinates to capture multiple fields.</li>
	<li>Fine-tune the focus for each sample viewed on the screen. Use the controls on Auto-focus correction.</li>
	<li>Use the software to set up time-lapse image capture. In the imaging software acquisition dialog box, check the [Time] tab (see Figure 4).</li>
	<li>Determine the [Interval] (the delay between the beginning of one time point to another time point) and [Duration] (the total length of time of the experiment). Units of time vary and can be selected in milliseconds (ms), seconds (s), minutes (m), or hours (h) from the dropdown menu. 
	NOTE: The imaging period length for time-lapse imaging of fluorescence and DIC time-lapse multichannel acquisition may vary depending on the fluorescent dye used in this assay. Photobleaching may occur if the labeled cells are exposed for too long.</li>
	<li>Check the [Save to File] checkbox to save acquired images. Under the [Time schedule] tab click the [Run now] button to acquire multipoint time-series images (see Figure 5). 
	NOTE: In the imaging software, images are saved in nd2 file format. Each time-lapse can be individually saved in an MP4 file format later.</li>
</ol>

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The authors have nothing to disclose.

The protocol described requires two steps: 1) coculture of 4T1 mouse mammary carcinoma cells and M1-polarized macrophages, and 2) assessment of the macrophage phagocytic activity using time-lapse microscopy. Live cell coculture is widely used in phagocytosis and migration assays. The live cell coculture model here is a simple, adaptable in vitro procedure (Figure 2) that utilizes a fluorescent dye, CFSE, which is used to label the 4T1 targeted cells. This is used for proper tracking of the c...

Macrophages are the first line of immune defense and play a role in orchestrating immune responses against pathogens and foreign materials, including cancer cells. They are a specialized phagocyte that destroys and gets rid of unwanted particles in the body. Macrophages contribute defensive functions such as the clearance of apoptotic cells and microorganisms and the recruitment of other immune cells1. Macrophages can differentiate into two different types, M1 and M2 macrophages, in response to environmental signals2. M1-polarized macrophages (i.e., classically activated macrophages) are activated by the cytokine interferon-&#947; (IFN-&#947;) and lipopolysaccharides (LPS) and are involved in the inflammatory response, pathogen clearance, efficient phagocytosis, and tumoricidal immunity3,4. The M2 macrophages are closely related to tumor-associated macrophages (TAMs) and have anti-inflammatory and tumor promotion properties4.
Phagocytosis, derived from Ancient Greek (phagein), meaning "to devour," (kytos), meaning "cell," and -osis, meaning &#34;process&#34;5. Phagocytosis is a receptor-mediated process where phagocytes (including macrophages, monocytes, and neutrophils) kill and engulf invading pathogens, clean up foreign particles, and clear apoptotic cell debris. Tumor-associated macrophages (TAMs) are found in the stroma in different tumors, including breast cancer, and have pro-tumor functions6,7, resulting in resistance to phagocytosis. The detailed mechanism of tumor cell phagocytosis by macrophages is not yet understood.
This study presents a two-step method: 1) 4T1 mouse mammary carcinoma cells and M1-polarized macrophages are cocultured, and 2) the phagocytic activity of the macrophages is assessed using live-cell video microscopy. CFSE fluorescent dye was used to stain the 4T1 mouse mammary carcinoma cells. The stain labels 4T1 cells to distinguish them from the cocultured M1 macrophages within a single imaging dish. RAW 264.7 macrophages are polarized with LPS and IFN-&#947; into an M1 phenotype. To ensure a complete polarization, immunostaining with anti-iNOS antibody conjugated to FITC was performed. Subsequently, a multipoint series of time-lapse images was acquired to observe multiple events, including phagocytosis, within the coculture.
A better understanding of the interactions between tumor cells and macrophages may lead to potential cancer immunotherapy. Live-cell imaging offers a detailed view of cellular dynamics in a real-time setting and has been used to study cell migration, phenotypic screening, apoptosis, and cytotoxicity8,9 in neuroscience, developmental biology, and drug discovery. Although the proposed tumor in this study is breast cancer, the method can also be applied to multiple target cells and distinct effector cells.

<table>
	<tbody>
		<tr>
			<td>Anti-INOS antibody conjugated FITC</td>
			<td>Miltenyi Biotec</td>
			<td>REA982</td>
			<td>150 &#181;g in 1 mL</td>
		</tr>
		<tr>
			<td>Carboxyfluorescein succinimidyl ester (CFSE)</td>
			<td>Invitrogen</td>
			<td>C1157</td>
			<td>25 mg</td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>10 mg</td>
		</tr>
		<tr>
			<td>DMEM/high glucose</td>
			<td>HyClone</td>
			<td>SH30003.04</td>
			<td>670.0 g</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Tico Europe</td>
			<td>FBSEU500</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Lipopolysaccharides</td>
			<td>Sigma</td>
			<td>L4516-1MG</td>
			<td>1 mg</td>
		</tr>
		<tr>
			<td>Mouse recombinant interferon-gamma</td>
			<td>Stemcell Technologies</td>
			<td>78021</td>
			<td>100 &#181;g</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin solution</td>
			<td>Cellgro</td>
			<td>30-003-CI</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P5368-10PAK</td>
			<td>10 pack</td>
		</tr>
		<tr>
			<td>Trypsin EDTA</td>
			<td>Cellgro</td>
			<td>25-052-CI</td>
			<td>1X, 100 mL</td>
		</tr>
		<tr>
			<td>1000 &#181;L pipette tips</td>
			<td>WhiteBox</td>
			<td>WB-301-01-052</td>
			<td>5000 tips/case</td>
		</tr>
		<tr>
			<td>2 mL serological pipette</td>
			<td>JET BIOFIL</td>
			<td>GSP012002</td>
			<td>Non-pryogenic</td>
		</tr>
		<tr>
			<td>200 &#181;L pipette tips</td>
			<td>WhiteBox</td>
			<td>WB-301-02-302</td>
			<td>20,000 tps/case</td>
		</tr>
		<tr>
			<td>25 cm2 cell culture flask</td>
			<td>Corning</td>
			<td>CLS430639</td>
			<td>Tissue culture treated</td>
		</tr>
		<tr>
			<td>Bench top centrifuge</td>
			<td>Dynamica</td>
			<td>FA15C</td>
			<td>Model: Velocity 14R</td>
		</tr>
		<tr>
			<td>Biological safety fume hood</td>
			<td>Nuaire</td>
			<td>NU-565-400</td>
			<td>Model: Home/ LabGard&#174; ES TE NU-565 Class II, Type B2 Biosafety Fume Hood</td>
		</tr>
		<tr>
			<td>CO2/air mixer</td>
			<td>Chamlide (Live Cell Instrument)</td>
			<td>FC-R-20</td>
			<td>FC-5 (CO2/Air Mixer) with the flow meter</td>
		</tr>
		<tr>
			<td>Cell scrapper</td>
			<td>NEST</td>
			<td>710001</td>
			<td>220 mm</td>
		</tr>
		<tr>
			<td>CO2 cell incubator</td>
			<td>Panasonic</td>
			<td>N/A</td>
			<td>Model: MCO-19M(UV)</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Nikon Instruments Inc.</td>
			<td>N/A</td>
			<td>Nikon Ti-Eclipse</td>
		</tr>
		<tr>
			<td>Glass bottom dish</td>
			<td>Ibidi</td>
			<td>81218-200</td>
			<td>35 mm</td>
		</tr>
		<tr>
			<td>NIS elements software</td>
			<td>Nikon Instruments Inc.</td>
			<td>Available online download</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette controller</td>
			<td>CappAid</td>
			<td>PA-100</td>
			<td>CappController pipette controller, 0.1-100ml</td>
		</tr>
		<tr>
			<td>Thermostat</td>
			<td>Shinko</td>
			<td>Discontinued</td>
			<td>JCS-33A 48 x 48 x 96.5mm</td>
		</tr>
	</tbody>
</table>

time-lapse 2d imaging of phagocytic activity in m1 macrophage-4t1 mouse mammary carcinoma cells in co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

The time-lapse two-dimensional (2D) images of the coculture model of 4T1 mouse mammary carcinoma cell lines show the 4T1 cells being engulfed by M1 macrophages during a 13 h period. It is important to ensure a complete polarization of the M1 macrophages by performing immunostaining. The results (Figure 1) show that the concentration of 100 ng/mL lipopolysaccharides (LPS) and 20 ng/mL IFN-&#947; polarized RAW 264.7 macrophages into the M1 state. Labeling the targeted cells with a fluorescent ...

Watch this Scientific Journal Video about Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures at JoVE.com

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

time-lapse-2d-imaging-phagocytic-activity-m1-macrophage-4t1-mouse

Research

JoVE Journal

Cancer Research

9.0K Views.  Universiti Putra Malaysia. Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

Video: Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&#38;E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&#38;E section.&#160; The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&#38;E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&#38;E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&#38;E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated cancer associated fibroblasts are a key component of the tumor stroma that interact with cancer cells and support their growth and survival. Models that recapitulate the interaction of cancer cells and activated fibroblasts are important tools for studying the stromal biology and for development of antitumor agents. Here, a method is described for the rapid generation of robust 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and activated pancreatic fibroblasts that can be used for subsequent biological studies. Additionally, described is the use of 3D spheroids in carrying out functional metabolic assays to probe cellular bioenergetics pathways using an extracellular flux analyzer paired with a spheroid microplate. Pancreatic cancer cells (Patu8902) and activated pancreatic fibroblast cells (PS1) were co-cultured and magnetized using a biocompatible nanoparticle assembly. Magnetized cells were rapidly bioprinted using magnetic drives in a 96 well format, in growth media to generate spheroids with a diameter ranging between 400-600 &#181;m within 5-7 days of culture. Functional metabolic assays using Patu8902-PS1 spheroids were then carried out using the extracellular flux technology to probe cellular energetic pathways. The method herein is simple, allows consistent generation of cancer cell-fibroblast spheroid co-cultures and can be potentially adapted to other cancer cell types upon optimization of the current described methodology.

Here, a method is described for the preparation of 3-dimensional (3D) spheroid co-culture of pancreatic cancer cells and fibroblasts, followed by measurement of metabolic functions using an extracellular flux analyzer.

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells

The acquisition of the angiogenic phenotype is an essential component of the escape from tumor dormancy. Although several classic in vitro assays (e.g., proliferation, migration, and others) and in vivo models have been developed to investigate and characterize angiogenic and non-angiogenic cell phenotypes, these methods are time and labor intensive, and often require expensive reagents and instruments, as well as significant expertise. In a recent study, we used a novel quantitative phase imaging (QPI) technique to conduct time-lapse and labeling-free characterizations of angiogenic and non-angiogenic human osteosarcoma KHOS cells. A panel of cellular parameters, including cell morphology, proliferation, and motility, were quantitatively measured and analyzed using QPI. This novel and quantitative approach provides the opportunity to continuously and non-invasively study relevant cellular processes, behaviors, and characteristics of cancer cells and other cell types in a simple and integrated manner. This report describes our experimental protocol, including cell preparation, QPI acquisition, and data analysis.

Dormant and active cancer cell phenotypes were characterized using quantitative phase imaging. Cell proliferation, migration, and morphology assays were integrated and analyzed in one simple method.

The acquisition of the angiogenic phenotype is an essential component of the escape from tumor dormancy. Although several classic in vitro assays (e.g., ...

Transradial Access Chemoembolization for Hepatocellular Carcinoma Patients

Transarterial chemoembolization (TACE) is the most common modality for treatment of hepatocellular carcinoma (HCC) at the intermediate stage. TACE is typically performed via transfemoral access (TFA). However, transradial access (TRA) is preferred in coronary artery interventions due to decreased complications and mortality. Whether the advantages of TRA can be applied to TACE required investigation.
Patients receiving TRA TACE at a single center were retrospectively enrolled for study. Procedural details, technical success, radial artery occlusion (RAO) rate, and access site-related bleeding complications were evaluated. From October 2017 to October 2018, 112 patients underwent 160 TRA TACE procedures. The overall technical success rate was 95.0% (152/160). The rate of crossover from TRA to TFA was 1.9%. No access site-related bleeding complications were found in any cases. Asymptomatic RA occlusion occurred in three patients (2.7%). Compared with TFA, TRA can increase safety and patient satisfaction while decreasing access site-related bleeding complications. Moreover, TRA interventions can benefit patients with advanced age, obesity, or a high risk of bleeding complications.

Transarterial chemoembolization (TACE) is the standard therapy for patients in the intermediate stage of hepatocellular carcinoma and is typically performed through femoral artery access. Compared with transfemoral access, transradial access (TRA) can decrease the rate of bleeding complications and improve patient tolerance. A method is presented here to perform transarterial chemoembolization via radial artery access.

Transarterial chemoembolization (TACE) is the most common modality for treatment of hepatocellular carcinoma (HCC) at the intermediate stage. TACE is ...

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In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Longitudinal Intravital Imaging Through Clear Silicone Windows

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging windows is particularly powerful because it allows longitudinal observation of the same tissue over days to weeks. Typical imaging windows comprise a glass coverslip in a biocompatible metal frame sutured to the mouse&#8217;s skin. These windows can interfere with the free movement of the mice, elicit a strong inflammatory response, and fail due to broken glass or torn sutures, any of which may necessitate euthanasia. To address these issues, windows for long-term abdominal organ and mammary gland imaging&#160;were developed from a thin film of polydimethylsiloxane (PDMS), an optically clear silicone polymer previously used for cranial imaging windows. These windows can be glued directly to the tissues, reducing the time needed for insertion. PDMS is flexible, contributing to its durability in mice over time&#8212;up to 35 days have been tested. Longitudinal imaging is imaging of the same tissue region during separate sessions. A stainless-steel grid was embedded within the windows to localize the same region, allowing the visualization of dynamic processes (like mammary gland involution) at the same locations, days apart. This silicone window also allowed monitoring of single disseminated cancer cells developing into micro-metastases over time. The silicone windows used in this study are simpler to insert than metal-framed glass windows and cause limited inflammation of the imaged tissues. Moreover, embedded grids allow for straightforward tracking of the same tissue region in repeated imaging sessions.

An approach is here presented for long-term intravital imaging using optically clear, silicone windows that can be glued directly to the tissue/organ of interest and the skin.&#160;These windows are cheaper and more versatile than others currently used in the field, and the surgical insertion causes limited inflammation and distress to the animals.

Intravital microscopy (IVM) enables visualization of cell movement, division, and death at single-cell resolution. IVM through surgically inserted imaging ...

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

The intravital imaging method described here utilizes collagen second harmonic generation and endogenous fluorescence from the metabolic co-factor NAD(P)H to non-invasively segment an unlabeled tumor microenvironment into tumor, stromal, and vascular compartments for in-depth analysis of 4D intravital images.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor ...

Real-Time Detection and Capture of Invasive Cell Subpopulations from Co-Cultures

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of cancer cell populations typically generate a single endpoint measurement that does not distinguish between cancer cell subpopulations with different invasive potential. Also, the tumor microenvironment consists of different resident stromal and immune cells that alter and participate in the invasive behavior of cancer cells. Invasion into tissues also plays a role in immune cell subpopulations fending off microorganisms or eliminating diseased cells from the parenchyma and endothelial cells during tissue remodeling and angiogenesis. Real-Time Cellular Analysis (RTCA) that utilizes impedance biosensors to monitor cell invasion was a major step forward beyond endpoint measurement of invasion: this provides continuous measurements over time and thus can reveal differences in invasion rates that are lost in the endpoint assay. Using current RTCA technology, we expanded dual-chamber arrays by adding a further chamber that can contain stromal and/or immune cells and allows measuring the rate of invasion under the influence of secreted factors from co-cultured stromal or immune cells over time. Beyond this, the unique design allows for detaching chambers at any time and isolating of the most invasive cancer cell, or other cell subpopulations that are present in heterogeneous mixes of tumor isolates tested. These most invasive cancer cells and other cell subpopulations drive malignant progression to metastatic disease, and their molecular characteristics are important for in-depth mechanistic studies, the development of diagnostic probes for their detection, and the assessment of vulnerabilities. Thus, the inclusion of small- or large-molecule drugs can be used to test the potential of therapies that target cancer and/or stromal cell subpopulations with the goal of inhibiting (e.g., cancer cells) or enhancing (e.g., immune cells) invasive behavior.

We describe an approach to detect and capture invasive cell subpopulations in real-time. The experimental design uses Real-Time Cellular Analysis by monitoring changes in the electric impedance of cells. Invasive cancer, immune, endothelial or stromal cells in complex tissues can be captured, and the impact of co-cultures can be assessed.

Invasion and metastatic spread of cancer cells are the major cause of death from cancer. Assays developed early on to measure the invasive potential of ...

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1&#945; expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...