The microscopy facilities used are part of the Erasmus Optical Imaging Center, and we would like to thank the staff of the OIC for their services.

1. Preparation
<ol>
	<li>Assemble the imaging chamber, which consists of two parts.
	<ol>
		<li>Place a 25-mm cover glass (Figure 1A-1) at the bottom part of the chamber (Figure 1A-2) and screw the top part into the bottom part (Figure 1A-3). Do not tighten too hard, as the glass can break. Autoclave the entire chamber (Figure 1A-4).</li>
	</ol>
	</li>
	<li>Prepare cell culture medium by supplementing Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) without phenol red with 10% heat-inactivated fetal calf serum (FCS) and 1 mM L-glutamine under sterile conditions. Store at 4 &#176;C and warm to 37 &#176;C before use.</li>
	<li>Maintain the cells with cell culture medium in culturing flasks using standard cell culture techniques. 
	NOTE: Here, two mouse cell lines, melanoma B16BL611 and Lewis lung carcinoma (LLC), and two human melanomas, BLM and 1F612, were used.</li>
	<li>Make 0.1% gelatin by weighing and dissolving gelatin in phosphate-buffered saline (PBS) at 37 &#176;C overnight. Sterilize the solution by filtering it through a 0.22-&#181;m filter and store it at 4 &#176;C.</li>
</ol>
2. Plate Cells
<ol>
	<li>Remove the imaging chamber from the sterilization bag in a laminar flow cabinet, carefully tighten the chamber, and place it in a petri dish. 
	NOTE: To avoid contamination, keep the imaging chamber in a petri dish until the chamber can be placed under the microscope.</li>
	<li>Coat the glass of the imaging chamber with 1 mL of 0.1% sterile gelatin for 20 min at 37 &#176;C and 5% CO2. 
	NOTE: This will allow better cell attachment.</li>
	<li>Remove the medium from the cell culture flasks (see steps 1.2 and 1.3), wash once with 1x PBS, and detach the cells using 0.25% trypsin. Inactivate the trypsin by adding cell culture medium, collect the cells, spin them down at 1,100 x g for 5 min, and resuspend the pellet in 5 mL of cell culture medium.</li>
	<li>Dilute 20 &#181;L of cell suspension with 20 &#181;L of trypan blue (which will be taken up by dead cells). Count the number of living and dead cells using a hemocytometer; the number of dead cells should not exceed 10%.</li>
	<li>Aspirate the gelatin and plate the cells in a dilution that allows for a maximum of 70% coverage within 24 h. For example, use a dilution of 5 x 104 or 10 x 104 cells in 1 mL. Allow the cells to adhere at 5% CO2 and 37 &#176;C for 24 h.</li>
</ol>
3. Time-lapse Microscopy (Figure 2)
NOTE: Here, a confocal microscope with a custom-made stage holder was used, although most manufactures offer a range of incubators for live-cell imaging that are also suitable to investigate nanoparticulated drug delivery.
<ol>
	<li>Evaluate the cells under a microscope for confluency, cellular morphology, and contamination. Place the chamber back in the CO2 incubator. 
	NOTE: The confluency should not exceed 70%. If the cellular morphology is inconsistent (i.e., the cells do not adhere) or contamination is detected, discard the imaging chamber.</li>
	<li>Switch on the confocal microscope (Figure 1E-1), fluorescent light (Figure 1E-2), and computer (Figure 1E-3).</li>
	<li>Connect the lens heater to the objective and set it to 35 &#176;C (Figure 1E-4 + insert).</li>
	<li>Prepare the incubation unit, as shown in Figure 1B. 
	NOTE: This unit consists of a heated stage (Figure 1B-1), a flow stage with a connector for the CO2 tubes (Figure 1B-2) and CO2 probe (Figure 1B-3), and a red closing patch (Figure 1B-4). This patch is used to temporarily close the unit until the imaging chamber is placed.</li>
	<li>Place the incubation unit on the microscope stage and connect the in- and out-flow CO2 tubes (Figure 1E-5). Open the main CO2 valve (Figure 1E-6) and switch on the CO2 controller (Figure 1E-7). Check that the controller is set to 5% CO2. 
	NOTE: For humidity, CO2 passes through a humidifier (Figure 1E-8). For hypoxia experiments, a separate O2 regulator (Figure 1E-9) is included in the setup.</li>
	<li>Set the stage temperature to 37 &#176;C (Figure 1E-10). For hyperthermia experiments, set the temperature to 42 &#176;C. Allow the incubation unit to reach the selected temperature and CO2 percentage.</li>
	<li>Take the imaging chamber from the main CO2 incubator and transport the chamber in a petri dish to the microscopy room.</li>
	<li>Place the imaging chamber (Figure 1A-4) in a custom-made, 35 mm-diameter stage holder (Figure 1A-5) and close the chamber with a custom-made sealing lid (Figure 1A-6). 
	NOTE: The lid allows CO2 to pass and prevents evaporation and contamination.
	<ol>
		<li>Open the incubation unit, replace the red patch with the imaging chamber, and close the unit (Figure 1C). Do this as fast as possible to prevent the cooling down of the cells. Make sure that no cables are stuck between stage and the microscope.</li>
	</ol>
	</li>
	<li>Leave the unit to acclimatize for 30 min.</li>
	<li>Open the software and switch on the lasers. 
	NOTE: Here, 488 nm argon, 543 nm helium-neon, and 633-nm helium-neon lasers were used.</li>
	<li>Make a new database by clicking &#34;New&#34; under the File tab. Name and save the database.</li>
	<li>Set the configuration by clicking &#34;Config&#34; under the Acquire tab. Select &#34;Channel mode&#34; and &#34;Single track&#34; to evaluate a single fluorophore or &#34;Multi track&#34; for several fluorophores. Select the appropriate band-pass filters matching the fluorophore (e.g., 560 to 615 nm band-pass filter for dox and Dox-NP; see the Representative Results for more examples). Select the bright-field channel in one of the tracks.</li>
	<li>Set the scan control by clicking &#34;Scan&#34; under the Acquire tab. Set the frame size (1,024 x 1,024), scan speed (optimal), scan direction (8-bit, single), and scan average (4) by clicking &#34;Mode.&#34; Set the pinhole (200 &#181;m), gain and offset (e.g., 580, 700, and 835; see Figure 3), and laser power (488 = 10%, 543 = 100%, 633 = 100%) by clicking &#34;Channels.&#34;</li>
	<li>Save these settings by clicking &#34;Config&#34; in the Configuration tab and save them in the configuration database.</li>
	<li>Set the time-lapse by clicking &#34;Multi Time X&#34; (Figure 1D) in the Macro tab. Click &#34;Single location&#34;.</li>
	<li>To keep the cells in focus, select the automated focus in the macro by clicking &#34;Auto Focus.&#34; 
	NOTE: Autofocus is achieved with the 633-nm laser using the reflection of the glass as a reference point.</li>
	<li>Apply the saved configuration by clicking &#34;Single track&#34; or &#34;Multi track,&#34; scrolling to the required configuration in the configuration database, setting the time interval (e.g., 15 min), number of scans (e.g., 97) and clicking &#34;Load Config.&#34; 
	Note: Using these settings, a time-series of 97 x 15 min = 24 h will be made.</li>
	<li>Name the time-lapse in the &#34;Base File Name&#34; and select the database (made in step 3.11) by clicking &#34;Image Data Base&#34; to save the time-lapse. Set a temporary folder by clicking &#34;Tmp Image Folder.&#34; 
	NOTE: If the system stops or crashes for any reason, the images are located in this folder.</li>
	<li>Open the incubating unit, lift the imaging ring, add a drop of immersion oil to the objective, and close the unit as fast as possible. Focus and select a position in the imaging chamber. 
	NOTE: This position contains cells in a monolayer, with a minimum of 70% confluency, allowing for the imaging of individual cells.</li>
	<li>Check the configuration settings for the background and correct if necessary. 
	NOTE: The background can be corrected by reducing the gain or laser power. Any change in the configuration needs to be saved (see step 3.17) and loaded (see step 3.18) again in the macro.</li>
	<li>In the laminar flow cabinet, dilute free drug or nanoparticles at a concentration of 5 &#181;g/mL drug or 0.05 &#181;mol of lipids in cell culture medium. Filter through a 0.22-&#181;m syringe filter if the drug/nanoparticles are not sterile.</li>
	<li>Open the incubating chamber, lift the sealing lid, remove the medium from the cells, add 1 mL of diluted drug/nanoparticles, and close the unit as fast as possible.</li>
	<li>Focus on the cells and start the time-lapse by clicking &#34;Start Time&#34; in the Multi Time X macro.</li>
	<li>Check regularly to see if the cells are still in focus or use the autofocus (see step 3.16). The program automatically saves the time-lapse in the database; do not close the database while the program is running.</li>
</ol>
4. Data Analysis
<ol>
	<li>Depending on the original research question, use software programs such as ImageJ for data analysis (see the Results section for examples).</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pegylated+liposomal+doxorubicin:+a+review+of+its+use+in+metastatic+breast+cancer,+ovarian+cancer,+multiple+myeloma+and+AIDS-related+Kaposi's+sarcoma.">Pegylated liposomal doxorubicin: a review of its use in metastatic breast cancer, ovarian cancer, multiple myeloma and AIDS-related Kaposi's sarcoma.</a> Drugs. 71 (18), 2531-2558 (2011).</li><li>Gabizon, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+circulation+time+and+enhanced+accumulation+in+malignant+exudates+of+doxorubicin+encapsulated+in+polyethylene-glycol+coated+liposomes.">Prolonged circulation time and enhanced accumulation in malignant exudates of doxorubicin encapsulated in polyethylene-glycol coated liposomes.</a> Cancer Res. 54 (4), 987-992 (1994).</li><li>Ten Hagen, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-dose+tumor+necrosis+factor-alpha+augments+antitumor+activity+of+stealth+liposomal+doxorubicin+(DOXIL)+in+soft+tissue+sarcoma-bearing+rats.">Low-dose tumor necrosis factor-alpha augments antitumor activity of stealth liposomal doxorubicin (DOXIL) in soft tissue sarcoma-bearing rats.</a> Int J Cancer. 87, 829-837 (2000).</li><li>Hoving, S., Seynhaeve, A. L., van Tiel, S. T., Eggermont, A. M., Ten Hagen, T. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+encapsulated+anthracyclines:+new+therapeutic+horizons.">Liposomal encapsulated anthracyclines: new therapeutic horizons.</a> Curr Oncol Rep. 3 (2), 156-162 (2001).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triggered+content+release+from+optimized+stealth+thermosensitive+liposomes+using+mild+hyperthermia.">Triggered content release from optimized stealth thermosensitive liposomes using mild hyperthermia.</a> J Control Release. 143 (2), 274-279 (2010).</li><li>Lu, T., Lokerse, W. J., Seynhaeve, A. L., Koning, G. A., ten Hagen, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formulation+and+optimization+of+idarubicin+thermosensitive+liposomes+provides+ultrafast+triggered+release+at+mild+hyperthermia+and+improves+tumor+response.">Formulation and optimization of idarubicin thermosensitive liposomes provides ultrafast triggered release at mild hyperthermia and improves tumor response.</a> J Control Release. 220, 425-437 (2015).</li><li>Seynhaeve, A. L., Dicheva, B. M., Hoving, S., Koning, G. A., Ten Hagen, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intact+Doxil+is+taken+up+intracellularly+and+released+doxorubicin+sequesters+in+the+lysosome:+Evaluated+by+in+vitro/in+vivo+live+cell+imaging.">Intact Doxil is taken up intracellularly and released doxorubicin sequesters in the lysosome: Evaluated by in vitro/in vivo live cell imaging.</a> J Control Release. 172 (1), 330-340 (2013).</li><li>Dicheva, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+Thermosensitive+Liposomes:+A+Novel+Dual+Targeted+Heat-Triggered+Drug+Delivery+Approach+for+Endothelial+and+Tumor+Cells.">Cationic Thermosensitive Liposomes: A Novel Dual Targeted Heat-Triggered Drug Delivery Approach for Endothelial and Tumor Cells.</a> Nano Lett. 13 (6), 2324-2331 (2012).</li><li>Brouckaert, P. G., Leroux-Roels, G. G., Guisez, Y., Tavernier, J., Fiers, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+anti-tumour+activity+of+recombinant+human+and+murine+TNF,+alone+and+in+combination+with+murine+IFN-gamma,+on+a+syngeneic+murine+melanoma.">In vivo anti-tumour activity of recombinant human and murine TNF, alone and in combination with murine IFN-gamma, on a syngeneic murine melanoma.</a> Int J Cancer. 38 (5), 763-769 (1986).</li><li>Van Muijen, G. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+expression+of+metastasizing+and+non-metastasizing+human+melanoma+cells+xenografted+into+nude+mice.">Antigen expression of metastasizing and non-metastasizing human melanoma cells xenografted into nude mice.</a> Clin Exp Metastasis. 9 (3), 259-272 (1991).</li><li>Das, A. 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Y., Posada, M. M., Zhao, L., Rosania, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+doxorubicin+efflux+from+the+nucleus+of+drug-resistant+cancer+cells+following+extracellular+drug+clearance.">Rapid doxorubicin efflux from the nucleus of drug-resistant cancer cells following extracellular drug clearance.</a> Pharm Res. 24 (11), 2156-2167 (2007).</li><li>Parslow, A., Cardona, A., Bryson-Richardson, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+drift+correction+following+4D+confocal+time-lapse+imaging.">Sample drift correction following 4D confocal time-lapse imaging.</a> J Vis Exp. (86), (2014).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mild+hyperthermia+triggered+doxorubicin+release+from+optimized+stealth+thermosensitive+liposomes+improves+intratumoral+drug+delivery+and+efficacy.">Mild hyperthermia triggered doxorubicin release from optimized stealth thermosensitive liposomes improves intratumoral drug delivery and efficacy.</a> J Control Release. 168 (2), 142-150 (2013).</li><li>Pedrosa, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+intracellular+Doxorubicin+delivery+through+nanoliposomes+equipped+with+selective+tumor+cell+membrane+permeabilizing+short-chain+sphingolipids.">Improving intracellular Doxorubicin delivery through nanoliposomes equipped with selective tumor cell membrane permeabilizing short-chain sphingolipids.</a> Pharm Res. 30 (7), 1883-1895 (2013).</li><li>Pedrosa, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-chain+glycoceramides+promote+intracellular+mitoxantrone+delivery+from+novel+nanoliposomes+into+breast+cancer+cells.">Short-chain glycoceramides promote intracellular mitoxantrone delivery from novel nanoliposomes into breast cancer cells.</a> Pharm Res. 32 (4), 1354-1367 (2015).</li></ol>

The authors have nothing to disclose.

As observed in the past, fixation can destroy liposomal nanocarriers almost immediately&#160;and DXR released form these destroyed liposomes still had the time to enter the nucleus, making the interpretation of data very difficult and even misleading. With some microscopic adjustments, the chemotherapeutic fate in living cells and tissues can be routinely investigated to confirm or overthrow histological observations. A recent paper showed the uptake, release, and localization of DXR in cells treated with free and encapsulated dox using live-cell time-lapse imaging9. This work describes the experimental setup in more detail. Using this model, it is possible to visualize the immediate transportation of dox to the nucleus, where it continuously accumulates until the cell eventually dies. In contrast, when Dox-NP is used, only small amounts of released dox are found in the nucleus. Although continuous exposure results in continuous DXR buildup, the fluorescent signal is much lower in Dox-NP- versus dox-treated cells, indicating a difference in cellular concentration and subsequent cytotoxicity. Furthermore, by using live-cell markers, it was shown that, when exposing cells to Dox-NP, DXR and the nanocarrier are located in the lysosome. This indicates that the complete liposome is taken up by the cell and demonstrates the involvement of an endocytic pathway during the uptake of these nanoparticles. The DXR in the nucleus is clearly from released dox. However, using this method, as both the DXR and carrier co-localize and seem to be entrapped in lysosomes, it is still inconclusive whether this is encapsulated or released dox. It is possible that the intrinsic stability of the nanoparticle prevents dox release when localized in the lysosome.
In this protocol, live-cell imaging is demonstrated using a specific microscopic setup, with a custom-made stage holder (the specifications can be given on request). However, most confocal microscope manufactures offer a range of incubators that perform live-cell imaging. Preserving cell culture conditions (i.e., temperature, CO2, pH, humidity, and sterility) is the main criterion of these incubators and requires some preliminary testing to determine the proper conditions, which is further explained. Automated data acquisition programs can also be delivered by the same manufactures. A &#34;Multi location&#34; option makes it possible to image several positions in the same chamber, refining statistical analysis. However, this option in the macro mentioned in this manuscript requires fixed XYZ positions, by which the possibility to correct for Z-drift is lost. Scanning the Z-dimension can be included in this macro and uses a focus plane for the analysis. However, this requires extra scanning, increasing the possibility of phototoxicity and bleaching.
As with all fluorescent measurements, overexposure is a problem, especially when comparing two compounds with a different cellular uptake. The fluorescent intensity is not only dependent upon the intrinsic signal of the compound, but also upon the rate of uptake, the concentration, and the drug exposure time. As shown in Figure 3, the nuclear DXR content of dox-treated cells is already visible, whereas no signal is seen in Dox-NP-treated cells. Only by increasing the gain can the DXR in Dox-NP-treated cells be visualized, leading to overexposure in dox-treated cells. Moreover, even intracellularly, Dox-NP is heterogeneously distributed, which can result in unavoidable under- or overexposure. Especially when investigating the cellular DXR entrapment, the gain was reduced significantly to distinguish individual organelles (Figures 5B and 5C). Thus, the measurements represented by pixel density must be accompanied by the representative images for the proper interpretation of results.
Phototoxicity, bleaching, and a low signal-to-noise ratio are also important issues in fluorescent microscopy, and a compromise between the quality of the image and the image acquisition must be established. However, even when the microscope setup is optimal, the image quality is also largely determined by the quality of the cells and the added compound. DXR gives a strong signal and, with the proper precautions, bleaching was not observed, even after longer periods (up to 72 h). However, the reduction of the fluorescent signal can also be a result of efflux. Efflux is an important phenomenon in drug resistance15 and occurs when cells pump out the drug from the intracellular site of action. A decrease of the fluorescent signal over time can therefore also be a result of dox efflux9. Comparing the fluorescent signal of a non-scanned location at the end of the experiment with the last image from the time-lapse series will demonstrate bleaching (i.e., the signal will be higher in the non-scanned location) or efflux (i.e., both signals will be identical). Several correctional options (e.g., background, drift, bleaching, etc.) are available in programs like ImageJ16. Also, as the fluorescent intensity increases over time, the configuration settings can be pre-evaluated in a pilot experiment to minimize overexposure at the end of the time-lapse (step 3.21). Alternatively, an imaging chamber with cells already exposed to the drug for the desired time-period can be used to initialize the settings. Finally, for this type of analysis, toxic drug concentrations (step 3.22) should be avoided and pre-established using conventional bio-assays.
Cells are continuously cultured and maintained in medium without phenol red. Phenol red fluoresces in the red part of the spectrum and can be observed in cellular organelles, most likely lysosomes, presenting false-positive information (step 1.2). To allow for the monitoring of individual cells, cell confluence should not exceed 70% (step 3.1.). The CO2 flow-speed (step 3.5) needs to be regulated in a validation experiment when the equipment is purchased or after maintenance. When the speed is too high, medium will evaporate. When the speed is too low, the pH of the medium will increase, which can affect cell growth. As the medium is without the phenol red pH indicator changes in pH cannot be observed and are therefore easily overlooked. Once the CO2 flow is validated, these settings can be used in all future experiments.
Using the method described here, the fate of nanoparticles can easily be investigated using live-cell imaging and time-lapse microscopy. In this procedure, commercially available nanoparticles were used. However, the fate of thermosensitive17, cationic liposomes10; liposomes enriched with short-chain shingolipids18; and nanoparticles encapsulating other drugs8,19 were also investigated in this manner in tumor and normal cells.

The ability to follow a biological process, such as cell-cell interactions, inter- and intracellular transport, cytotoxic compound uptake, and protein-protein interaction of single molecules in living cells and tissues, has gained great interest over the last decade, as it provides the extra dimension of &#34;real time.&#34; In this manuscript, a method to follow the intracellular fate of free dox and dox incorporated in nanoparticles (Dox-NP) is described.
Nanoparticles have been used for decades to treat certain cancers. Dox-NP has been approved for the treatment of AIDS-related Kaposi sarcoma, advanced ovarian and breast cancer, and multiple myeloma1. It was one of the first liposomal nanoparticles developed and introduced in the clinical setting. The free form, dox, is vastly cleared from the blood circulation, limiting its capacity to reach the tumor site in sufficient amounts. Furthermore, treatment is accompanied by severe and dose-limiting side-effects, such as stomatitis and heart failure. When encapsulated into pegylated liposomal nanoparticles the circulation time increases from minutes to days2. This type of cholesterol-containing liposome is quite stable and this stability determines the pharmacokinetics of encapsulated drug.
However, this rigidity has a downside. The cytotoxic ability of a compound towards tumor cells is first evaluated in vitro, and it has been shown that dox is more potent than Dox-NP in cytotoxic assays3,4. It was hypothesized that the stability of the carrier prevents the release and cellular uptake of the drug, and it is worthwhile to investigate this phenomenon further. The intrinsic liposomal properties, built to last the aggressive elements in the blood stream and to reach the tumor site intact, resulted in the impaired bioavailability of the cytotoxic component (i.e., dox) at the target site (i.e., the tumor cell nucleus). Although Dox-NP hardily improved the response compared to the free form, it was still of clinical value, as encapsulation significantly reduced the cardiotoxic effects5. In the following years, other compounds were encapsulated in nano-carriers6. Also, modifying the carriers resulted in triggered drug release (i.e., at the tumor site) but maintained stability in the blood circulation7,8. Despite years of investigation and clinical use, the intratumoral fates of nanocarriers like Dox-NP are still not entirely clear. In a recent publication, it was shown that the nanoparticle is taken up as a whole while the dox remains trapped in the lysosomes9.
Cellular uptake of a nanoparticle and drug release are dynamic processes that can best be monitored using live-cell imaging. Moreover, classical histology, in which cells are incubated and thereafter fixed, can give false results if the fixation destroys the liposomal carrier and introduces artifacts. The main requirement for live-cell imaging is visualization, preferably by fluorescence, of the desired process. Certain compounds, like dox, have an intrinsic red fluorescence. Also, fluorescent markers can be introduced into the carrier, and cell organelles can be visualized using live-cell markers. In this way, an array of parameters, like uptake of the carrier, drug release, and cellular localization of the carrier and drug, can be imaged and analyzed. Here, a method is described in which the intercellular fate of dox and Dox-NP in several tumor cells is followed in real-time. Also, this method can be easily adapted to create the ideal conditions for targeted (e.g., charged nanoparticles10) and triggered (e.g., hyperthermia7) drug release.

<table border="0" cellpadding="0" cellspacing="0" width="1013">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">DMEM (no phenol-red)</td>
			<td style="width:263px;">Sigma</td>
			<td style="width:115px;">D1145</td>
			<td style="width:384px;">Supplemented with 1 mM L-glutamine and 10 % FCS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Trypsin-versene (EDTA)</td>
			<td style="width:263px;">Lonza</td>
			<td style="width:115px;">BE17-161E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Fetal calf serum</td>
			<td style="width:263px;">Sigma</td>
			<td>F7524</td>
			<td>Heat inactivate for 30 min at 55 &#186;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">L-Glutamin</td>
			<td style="width:263px;">Lonza</td>
			<td>BE17-605E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Gelatin from bovine skin</td>
			<td style="width:263px;">Sigma</td>
			<td style="width:115px;">G9391</td>
			<td>Make a 0,1% solution in PBS, sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Phosphate buffered saline (PBS)</td>
			<td style="width:263px;">Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Stericup 0,22 &#181;m filter - 500 ml&#160;</td>
			<td style="width:263px;">Millipore</td>
			<td style="width:115px;">SCGPU05RE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Trypan-blue</td>
			<td style="width:263px;">Sigma</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:252px;">Cells: Lewis lung carcimoma</td>
			<td style="width:263px;">ATCC</td>
			<td>CRL-1642</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:252px;">Cells: B16BL6</td>
			<td style="width:263px;">Dr. P. Brouckaert, Ghent University</td>
			<td>donated</td>
			<td>Ref.10</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:21px;width:252px;">Cells: BLM and 1F6</td>
			<td style="width:263px;">Dr. van Muijen, University of Nijmegen</td>
			<td>donated</td>
			<td>Ref.11</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Petri dish</td>
			<td style="width:263px;">Greiner</td>
			<td style="width:115px;">664160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Doxorubicin</td>
			<td style="width:263px;">Actavis&#160;</td>
			<td style="width:115px;"></td>
			<td>mentioned as dox in the manuscript</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Doxil/Caelyx</td>
			<td style="width:263px;">Janssen-Cilag</td>
			<td style="width:115px;"></td>
			<td>mentioned as Dox-NP in the manuscript</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Syringe filter 0.2 &#181;m</td>
			<td style="width:263px;">VWR international</td>
			<td style="width:115px;">10462200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Lysotracker-green DND-26</td>
			<td style="width:263px;">Invitrogen</td>
			<td style="width:115px;">L7526</td>
			<td>mentioned as lyosomal marker (LM)-green in manuscript</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Lysotracker-red DND-99</td>
			<td style="width:263px;">Invitrogen</td>
			<td style="width:115px;">L7528</td>
			<td>mentioned as lyosomal marker (LM)-red in manuscript</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:252px;">Attofluor cell ring</td>
			<td style="width:263px;">Invitrogen</td>
			<td style="width:115px;">A7816</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:252px;">Cover glass 25 mm #1</td>
			<td style="width:263px;">Thermo scientific</td>
			<td style="width:115px;">CB00250RA1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Stage holder + sealing lid</td>
			<td style="width:263px;">Custom made</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ZEISS LSM 510 microscope</td>
			<td style="width:263px;">Zeiss</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Software LSM 510 version 3.2 SP2</td>
			<td style="width:263px;">Zeiss</td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:252px;">Image J</td>
			<td style="width:263px;">NIH</td>
			<td style="width:115px;"></td>
			<td>https://imagej.nih.gov/ij/index.html</td>
		</tr>
	</tbody>
</table>

using in vitro live-cell imaging to explore chemotherapeutics delivered by lipid-based nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Labeling liposomal carriers with different markers has been previously described9. Carriers labeled with far-red dioctadecyl tetramethylindotricarbocyanine perchlorate (DiD; Ex = 644 nm; Em = 665 nm) or green 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein) (CF-PE; Ex = 490 nm; Em = 515 nm) are presented in this manuscript. To show intercellular similarities and differences in liposomal uptake, release, and intracellular localization, several tumor types were studied. These include two mouse lines, the B16BL6 melanoma11 and the Lewis lung carcinoma (LLC), and two human melanomas, a highly metastatic (BLM) and a non-metastatic (1F6) melanoma12,13. All experiments were performed using living cells. In all experiments, a concentration of 5 &#181;g/mL dox, 5 &#181;g/mL Dox-NP, or 0.05 &#181;mol of nanoparticles were administered for 3 or 24 h. Measured and analyzed dox (in free, released, encapsulated, sequestered, or intercalated form) were referred to as DXR. A 40X (numerical aperture: 1.3) oil objective lens was used, and imaging was performed with a 488 nm argon laser (10%/0.2 mW power) and a 505 to 550 nm band-pass filter for CF-PE and lysosomal marker (LM)-green. A 543 nm helium-neon laser (100%/ 0.2 mW power) and a 560 to 615 nm band-pass filter was used for dox, Dox-NP, and lysosomal marker-red. A 633 nm helium-neon laser (100%/0.5 mW power) and a 640 nm long-pass filter was used for DiD.
Because of its encapsulation, the in vitro cytotoxicity, bioavailability, and pharmacokinetics of dox were significantly altered3,9,14. The difference between the bioavailability of the drug when administered in free and encapsulated form is demonstrated in Figure 3. Dox is an intercalating agent and must enter the cell nucleus to become cytotoxic. Figure 3 and related Movies 1 and 2 show a 3 h time-lapse of BLM cells treated with dox or Dox-NP under identical conditions. Within minutes of dox exposure, nuclear DXR can be observed (Figure 3A, Movie 1) and, using a lower gain (insert), can be seen intercalating in the nucleus. In contrast, when exposed to Dox-NP, intracellular DXR is hardly visible within this time-frame (Figure 3B, Movie 2). Only by increasing the photomultiplier gain, DXR in the cytoplasm can be observed (insert). Using ImageJ, the pixel densities of cytoplasmic and nuclear DXR were analyzed. As cells are in motion during time-lapse evaluation, it is important to combine fluorescent with bright-field images. This presents the possibility of tracking individual cells and stabilizing the XY-drift using specific plugins in ImageJ. In the analysis presented in Figure 3, the bright-field image was used to draw a region of interest (ROI) around the cytoplasm (solid line) and nucleus (dotted line). The ROI manager in ImageJ can be found in the menu item Analyze &#62; Tools &#62; ROI manager. These ROIs are used in the fluorescent image to analyze pixel density by using the menu item Analyze &#62; Measure. The difference between the pixel density in the nucleus (Figure 3C) and cytoplasm (Figure 3D) of dox- or Dox-NP-treated cells confirms what is seen in the images. Also, cells were incubated for 24 h with dox or Dox-NP, after which the compound was replaced by medium and immediately imaged with increased sensitivity of the photomultiplier gain (Figure 4). Other settings, such as laser power, offset, pinhole, and image display, were identical. The low DXR signal in Dox-NP-treated compared to dox-treated cells is striking. Using a gain of 700, DXR in the Dox-NP-treated cells becomes visible, whereas the image of dox is already overexposed. This simple in vitro experiment visualizes a significant difference in the bioavailability of free versus encapsulated drug.
When cells are continuously exposed to Dox-NP for 24 h, DXR builds up over time, as shown in Figure 5A and Movies 3 and 4. The signal increases in the cytoplasm, and later, DXR is also found intercalated in the nucleus. These observations are confirmed by the pixel density graphs (Figure 5B). The measured cytoplasmic pixel density is lower in BLM compared to 1F6 cells because of the difference in intracellular distribution. Whereas DXR in 1F6 cells is distributed throughout the cell, DXR in BLM cells is more concentrated in an organelle close to the nucleus (Figure 5C).
Therefore, the localization of drug and carrier in different cell lines (Figure 6) was investigated. Cells were incubated for 24 h with Dox-NP/DiD and imaged. Using the bright-field image, an overlay of the cell membrane (solid line) and nucleus (dotted line) was drawn in the fluorescent image of three different cell lines. It is demonstrated here that the carrier, labeled with DiD, follows the same cytoplasmic pattern as DXR: concentrated in an organelle close to the nucleus in BLM, around the entire nucleus in LLC, and randomly scattered throughout the cytoplasm in B16BL6 cells. In the nucleus, only DXR and no DiD can be seen, indicating released dox. By reducing the photomultiplier gain, individual cellular vesicles containing DXR and carrier, labelled with DiD, as well as with CF-PE (Figures 6B and 6C), can be seen.
Cytoplasmic DXR sequesters in small vesicles, and cells were labeled with a live-cell lysosomal marker in green or red. The movement of these lysosomes can be followed in the cells (Movie 5). Cytoplasmic DXR and the nanocarrier colocalize within these structures, and the intercellular difference in the DXR pattern, as seen in Figure 6, is explained here. Lysosomes are randomly distributed throughout the cytoplasm in B16BL6 and are located next to the nucleus in BLM cells (Figure 7A). Using ImageJ, the colocalization between DXR and carrier in the lysosome was calculated (Figure 7B). The color images were made binary (Process &#62; Binary &#62; Make Binary) and, using the colocalization plug-in (Analyze &#62; Colocalization), a colocalization image was made of DXR with DiD. This creates a green-red-white colored image, with white pixels representing the colocalized pixels of both images. Via the menu item Process &#62; Binary &#62; Make Binary, the white pixels will be separated and converted to black pixels, from which the pixel density is measured (Analyze &#62; Measure). Thereafter, a colocalization image was made between this DXR-DiD image and the binary image of the lysosomal marker (LM) and pixel density measured. The colocalization data is the percentage of pixels positive for DXR-DiD and DXR-DiD/lysosomal marker (Figure 7C). That the carrier, labeled with CF-PE as well as DiD, is located in the lysosome is also shown in Figure 7D.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55405/55405fig1.jpg" /> 
Figure 1: Instruments and equipment setup. A) Imaging chamber + necessities. 25-mm #1 cover glass (1), bottom part of the chamber (2), top part of the chamber (placed upside down) and O-ring (3), stage holder (4), and sealing lid (5). Scale bar: 2 cm. B) Incubation unit. Heated microscope stage (1), flow stage with in- and out-flow for CO2 (2), a CO2 probe (3), and a closing patch (4). Scale bar: 2 cm. C) Imaging chamber in the incubator unit, as seen under the microscope. D) Multi-time series macro. E) Equipment for imaging. Inverted microscope (1), fluorescent light (2), computer (3), ring heater (4, insert) and controller (4, main figure), CO2 flow tubing (5), CO2 valve (6), CO2 controller (7), CO2 humidifier (8), O2 valve and controller (9), and stage temperature controller (10). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55405/55405fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55405/55405fig2.jpg" /> 
Figure 2: Schematic of the microscope parameters detailed in Protocol section 3. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55405/55405fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55405/55405fig3.jpg" /> 
Figure 3: Investigating the short-term uptake of dox and Dox-NP.&#160; BLM cells were continuously exposed to dox or Dox-NP for 3 h, and a time-lapse was taken. (A) Still pictures of the time-lapse of cells incubated with dox. (B) Still pictures of the time-lapse of cells incubated with Dox-NP. Scale bar = 50 &#181;m. (C) Pixel density increase of nuclear DXR in dox- and Dox-NP-treated cells. (D) Pixel density increase of cytoplasmic DXR in dox- and Dox-NP-treated cells. The data represent the mean &#177; SD of 3 cells per field. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55405/55405fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55405/55405fig4.jpg" /> 
Figure 4: Investigating the long-term uptake of dox and Dox-NP in different cell lines. Cells were exposed to dox or Dox-NP and imaged 24 h later. Images were taken immediately after the removal of the drugs, with 3 different gains. Scale bar = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55405/55405fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55405/55405fig5.jpg" /> 
Figure 5: Investigating the cellular uptake and release of liposomal agents. Cells were continuously exposed to Dox-NP for 24 h, and a time-lapse was made. (A) Still pictures of the time-lapse. Scale bar = 50 &#181;m. (B) Pixel density increase of DXR in the nucleus and cytoplasm. The data represent the mean &#177; SD of 3 cells per field. (C) Images demonstrating the localization of DXR within the cell. Solid line: cell membrane, dotted line: nucleus. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55405/55405fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55405/55405fig6.jpg" /> 
Figure 6: Investigating the localization of drug and carrier in different cell lines. (A) Cells were exposed to Dox-NP/DiD for 24 h and imaged. Scale bar = 50 &#181;m. (B) Cells were exposed to Dox-NP/DiD/CF-PE for 24 h and imaged with a lower intensity gain to distinguish individual vesicles. (C) Bright-field and DXR overlay showing DXR in cytoplasmic vesicles (arrow). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55405/55405fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/55405/55405fig7.jpg" /> 
Figure 7: Investigation of the intracellular localization of the drug and carrier. Cells were exposed to Dox-NP/DiD or CF-PE/DiD for 24 h, and lysosomes are visualized using the live-cell lysosomal marker (LM) in green or red. (A) Intracellular localization of DXR and the carrier (DiD). The arrows represent 3 examples of DXR and DiD localized in the lysosome. Scale bar = 50 &#181;m. (B) Co-localization analysis of DXR and the carrier (DiD) in lysosomes (LM) using ImageJ. (C) Percentage of co-localization of DXR and the carrier in lysosomes. The data represent the mean &#177; SEM of 3 individual experiments. (D) Lysosomal sequestering of the carrier visualized using two different markers. Scale bar = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55405/55405fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Movie 1" src="/files/ftp_upload/55405/55405mov1.jpg" /> 
Movie 1: Related to Figure 3: BLM cells were incubated with dox for 3 h. Time-lapse: 19 images, with an interval of 10 min. Frame rate: 3 fps. Scale bar = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55405/Movie_1_BLM_Doxorubicin_for_3h.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie 2" src="/files/ftp_upload/55405/55405mov2.jpg" /> 
Movie 2: Related to Figure 3: BLM cells were incubated with Dox-NP for 3 h. Time-lapse: 19 images, with an interval of 10 min. Frame rate: 3 fps. Scale bar = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55405/Movie_2_BLM_Doxil_for_3h.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie 3" src="/files/ftp_upload/55405/55405mov3v3.jpg" /> 
Movie 3: Related to Figure 5: BLM cells were incubated with Dox-NP for 24 h. Time-lapse: 96 images, with an interval of 15 min. Frame rate: 8 fps. Scale bar = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55405/Movie_3,_BLM_Doxil_for_24h_correct2.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie 4" src="/files/ftp_upload/55405/55405mov4.jpg" /> 
Movie 4: Related to Figure 5: 1F6 cells were incubated with Dox-NP for 24 h. Time-lapse: 96 images, with an interval of 15 min. Frame rate: 8 fps. Scale bar = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55405/Movie_4_1F6_Doxil_for_24h.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>
<img alt="Movie 5" src="/files/ftp_upload/55405/55405mov5.jpg" /> 
Movie 5: Related to Figure 7: Lysosomes of B16BL6 cells were stained with a live-cell marker. Time-lapse: 10 images, with an interval of 10 s. Frame rate: 3fps. Scale bar = 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55405/Movie_5_B16BL6_lysotracker.avi" target="_blank">Please click here to view this video. (Right-click to download.)</a>

Watch this Scientific Journal Video about Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles at JoVE.com

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an ...

using-vitro-live-cell-imaging-to-explore-chemotherapeutics-delivered

Nanyang Technological University

Research

JoVE Journal

Medicine

8.3K Views.  Erasmus MC. Live-cell imaging is a powerful tool to visualize dynamic processes. The examination of fixed cells provides only static pictures, which can lead to misinterpretation and confusion about the process. This work presents a method to study uptake, drug release, and intracellular localization of liposomal nanoparticles in living cells. 

Video: Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel underlying neurobiological mechanisms behind drug action and neurotransmitter-related disorders, such as depression and ADHD. Like most of the imaging methods (PET, SPECT, CT) it represents a progress in the investigation of brain disorders and the related function of neurotransmitter pathways in a non-invasive way with respect of the overall neuronal connectivity. Moreover it also provides the ideal tool for translation to clinical investigations. MRI, while still behind in molecular imaging strategies compared to PET and SPECT, has the great advantage to have a high spatial resolution and no need for the injection of a contrast-agent or radio-labeled molecules, thereby avoiding the repetitive exposure to ionizing radiations. Functional MRI (fMRI) is extensively used in research and clinical setting, where it is generally combined with a psycho-motor task. phMRI is an adaptation of fMRI enabling the investigation of a specific neurotransmitter system, such as serotonin (5-HT), under physiological or pathological conditions following activation via administration of a specific challenging drug.
The aim of the method described here is to assess brain 5-HT function in free-breathing animals. By challenging the 5-HT system while simultaneously acquiring functional MR images over time, the response of the brain to this challenge can be visualized. Several studies in animals have already demonstrated that drug-induced increases in extracellular levels of e.g. 5-HT (releasing agents, selective re-uptake blockers, etc) evoke region-specific changes in blood oxygenation level dependent (BOLD) MRI signals (signal due to a change of the oxygenated/deoxygenated hemoglobin levels occurring during brain activation through an increase of the blood supply to supply the oxygen and glucose to the demanding neurons) providing an index of neurotransmitter function. It has also been shown that these effects can be reversed by treatments that decrease 5-HT availability16,13,18,7. In adult rats, BOLD signal changes following acute SSRI administration have been described in several 5-HT related brain regions, i.e. cortical areas, hippocampus, hypothalamus and thalamus9,16,15. Stimulation of the 5-HT system and its response to this challenge can be thus used as a measure of its function in both animals and humans2,11.

The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and bronchodilator drugs. Despite the availability of multiple treatments, both diseases are growing public health concerns. The majority of asthma patients are well controlled on current inhaled therapies but a substantial number of patients with severe asthma are not. Asthma affects an estimated 300 million people worldwide and approximately 20 percent have a severe form of the disease. In contrast to asthma, there are few effective therapies for COPD. An estimated 10% of the population has COPD and the trend in death rates is increasing for COPD while decreasing for other major diseases. Although developing drugs for inhaled delivery is challenging, the nose-only inhalation unit enables direct delivery of novel drugs to the lung of rodents for pre-clinical efficacy and safety/toxicology studies. Inhaled drug delivery has multiple advantages for respiratory diseases, where high concentration in the lung improves efficacy and minimizes systemic side effects. Inhaled corticosteroids and bronchodilators benefit from these advantages and inhaled delivery may also hold potential for future biologic therapies. The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs of rodents, key data required before initiating clinical development.

The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs; key data enabling clinical inhaled drug development.

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and ...

Using Retinal Imaging to Study Dementia

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central nervous system (CNS) and shares prominent similarities with the brain in terms of embryological origin, anatomical features and physiological properties.&#160; The vascular and neuronal structure in the retina can now be visualized easily and non-invasively using retinal imaging techniques, including fundus photography and optical coherence tomography (OCT), and quantified semi-automatically using computer-assisted analysis programs. Studying the associations between vascular and neuronal changes in the retina and dementia could improve our understanding of dementia and, potentially, aid in diagnosis and risk assessment.&#160; This protocol aims to describe a method of quantifying and analyzing retinal vasculature and neuronal structure, which are potentially associated with dementia. This protocol also provides examples of retinal changes in subjects with dementia, and discusses technical issues and current limitations of retinal imaging.

The retina shares prominent similarities with the brain and thus represents a unique window to study vasculature and neuronal structure in the brain non-invasively. This protocol describes a method to study dementia using retinal imaging techniques. This method can potentially aid in diagnosis and risk assessment of dementia.

The retina offers a unique &#8220;window&#8221; to study pathophysiological processes of dementia in the brain, as it is an extension of the central ...

Quantification of Levator Ani Hiatus Enlargement by Magnetic Resonance Imaging in Males and Females with Pelvic Organ Prolapse

Here we present a protocol to examine the levator ani hiatus in males and females with pelvic organ prolapse, during the Valsalva maneuver and while evacuating acoustic gel, using a horizontally oriented 1.5 T magnetic resonance (MR) scanner. On midsagittal images, the vertical distance of pelvic organs is measured in millimeters relative to the hymen plane (female) and to the lower border of the symphysis pubis (male), preceded by - (above) or + ( below) signs. On axial images, the levator ani area is calculated in square centimeters with a free-hand tracing method from three key images, passing through the midsymphysis (level I), tangential to the lower border of the symphysis (level II), and at the maximal anterior rectal wall bulging (level III). Areas at rest and strained are compared to find evidence of a percentage of increase. The purpose is to provide objective evidence of the maximal extent of pelvic organs descent and hiatus enlargement without the interference of foreign objects or the examiner&#39;s proximity, so as to overcome the limitations of pelvic examination and transperineal sonography (i.e., subjectivity and sex-related limitations [female only]).

Here we present a protocol to standardize the measurement of the levator ani hiatus size&#160;by magnetic resonance imaging. The purpose is to extract biomechanical inferences from image analysis by comparing resting and straining values in patients of both sexes with pelvic prolapse, using consistent anatomical bony landmarks.

Here we present a protocol to examine the levator ani hiatus in males and females with pelvic organ prolapse, during the Valsalva maneuver and while ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...