Name: Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer (Video) | JoVE Journal
Uploaded: 2013-03-24T14:00:00.000+00:00
Duration: 8 min 26 s
Description: Explore live imaging of drug responses in breast cancer using spinning disc confocal microscopy in mouse models. This method enables real-time tracking of therapeutic penetration and cellular outcomes at single-cell resolution.

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5 minutes

We thank J. Cappellani and J. Qiu for technical support. This work was supported by funds from the National Cancer Institute (U01 CA141451), the Starr Cancer Consortium, Susan G. Komen for the Cure, Long Island 2 Day Walk to Fight Breast Cancer, Manhasset Women's Coalition Against Breast Cancer to M.E., and a pre-doctoral fellowship from the Congressionally Directed Breast Cancer Research Program, U.S. to E.S.N. E.S.N. is also the recipient of the Leslie C. Quick and William Randolph Hearst Foundation Fellowships from the Watson School of Biological Sciences. H.A.A. was supported by funds from the Research Council of Norway (160698/V40 and 151882), and Southeastern Regional Health Authorities (2007060).

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The authors have no conflicts of interest to disclose. All animal experiments were conducted in accordance with IACUC approved protocols.

The responses of tumors to systemic therapies in vivo can be dramatically different to those of cancer cells in vitro, as the microenvironment influences both the acute response and relapse5. One of the biggest obstacles to explicating in vivo chemoresistance pathways is the complexity of the interactions between cancer cells and their microenvironment. In particular, because solid tumors have developed a balance between cancer and stromal cells, perturbations of one component, such as the cell death that occurs during anti-cancer treatment, can result in dramatic effects on tissue organization.
The intravital imaging technique provided here allows for the direct visualization of cancer cell-stroma interactions within the tumors of live, anesthetized mice during treatment with anti-cancer drugs. We use spinning disk confocal microscopy, which has a relative limited penetration depth (see 17), but our surgical and labeling techniques can be used with other types of microscopy. Movies acquired from these experiments can be used to track processes that include changes in fluorescence intensity (e.g. due to infiltration of a labeled population or induction of cell death), cell motility, distribution of injectables (e.g. to track vascular leakage), and co-localization of fluorescent signals5,12,17,20.
We routinely image mice for over 6 hours and also performed imaging for over 18 hours. This requires maintaining the mice continuously under anesthesia for these long time periods. With proper anesthesia, over 90% of our animals survive 6 hours, and about 80% survive over 18 hours. Details on how to perform long-term anesthesia have been published previously19. In our experience, five factors are critical: avoiding hypothermia, avoiding dehydration, maintaining physiological blood oxygen saturation levels, using humidified carrier gas for isoflurane, and keeping the animals at the lowest level of anesthesia at which they do not show signs of pain. To maintain body temperature, we keep mice under a heated blanket (at 38 &deg;C). To avoid dehydration, we inject small volumes of saline i.p. throughout the entire imaging procedure. To maintain physiological blood oxygen saturation levels, we start with 21% oxygen in the carrier gas (rather than the commonly used 100%). This enables us to increase inhaled oxygen levels if a mouse - hours into an experiment - shows a decrease in blood oxygen saturation. In our experience, increasing the inhaled oxygen levels at such time (e.g. to 30-40%) almost always will stabilize the animal allowing for hours of additional imaging. The optimal level of anesthesia is for most mice achieved with 1-1.5% isoflurane and results in breath rates of 60-65/min and pulse rates of 400-450/min. In our imaging experiments, we primarily use transgenic mouse models (MMTV-PyMT and C3[1]-Tag) in which tumors are multifocal, allowing for imaging of multiple lesions at different stages of tumor progression in parallel at multiple x,y positions during a single imaging session. This decreases concerns regarding mouse-to-mouse variation with respect to experiments aimed at identifying stage-dependent therapeutic responses, and reduces the number of animals required for imaging.
The MMTV-PyMT model (and other spontaneous cancer models) go through progressive histopathological stages that can be recognized during intravital imaging, although the pathological staging of tumor lesions that can be done during intravital imaging is different from, and less detailed, than that of histological sections5,17. In contrast, transplantation models tend to reflect late stage tumors best, as the cancer cells usually are isolated from advanced tumors. Such models are thus more amenable to studying tumor-stroma interactions of late stage tumors than differences in these interactions between different stages.
We show examples of how to image nuclear structural changes that occur after cell death induced by therapy. In the absence of anti-cancer treatment, this labeling strategy may instead be used to image cell division in situ, elucidating, for example, how cell proliferation and dormancy is influenced by the microenvironment. In the future, fluorescent labeling of mitochondrial components may make it possible to image mitochondrial changes that occur during apoptotic cell death.
In this protocol, we have also shown examples of how to image cancer cell-immune cell interactions after therapy, but other microenvironmental components can also be visualized and imaged. Existing transgenic reporter lines for stromal components include those for fibroblasts (e.g., FSP1-EGFP17, &alpha;SMA-RFP21, COL1A1-EGFP21) or cells of the vasculature (e.g. Tie2-GFP22; VEGF-GFP23).
Intravital imaging allows for the real-time visualization of the interactions and processes that occur in vivo following chemotherapy administration. With the technology currently available, we have made major progress in understanding how myeloid cells influence therapeutic response; however, because the tumor microenvironment is so complex, major advancements are still needed to begin to delve mechanistically into the processes underlying environment-mediated drug resistance. One of the most important advancements still required is the identification of better markers for specific cell populations. The importance of better markers is well illustrated with two of the most prominent stromal cell components of the breast tumor microenvironment, fibroblasts and immune cells. &alpha;-Smooth muscle actin (&alpha;SMA) is frequently used to identify fibroblasts, but the protein is also expressed by vascular smooth muscle cells and myoepithelial cells24. Thus, although &alpha;SMA-GFP reporter mice exist, determining the specific cell type being following during an intravital imaging experiment is challenging. Similarly, a single fluorescent marker such as EGFP expressed under the c-fms promoter will often identify multiple subpopulations of immune cells12,17. Thus, although one would ideally like to use a single marker to identify a specific cell type the complexity of the underlying biology poses a major challenge in using this approach.
One partial solution to this problem is to use injectable labels to further differentiate subpopulations of cells expressing the same fluorescent reporter. For example, fluorescent dextrans are taken up by macrophages and can be used to label the macrophage subpopulations that are labeled by the c-fms-EGFP and Fsp1-EGFP transgenic reporter mice17. Antibodies conjugated to fluorescent probes have also been used to identify specific subpopulations (e.g. the Gr1-positive population of myeloid cells labeled by the c-fms-EGFP reporter17).
Unlike tumor response curves generated by caliper measurement, which provide little mechanistic information about how or why tumors respond to the administered therapeutic, or histological series derived from tissues harvested at different time points that require large cohorts of animals, our technique provides direct, quantifiable information on the dynamics of cell death and on the interactions of stromal cells with cancer cells in small cohorts. Thus, the advantage of using the procedure reported here is that it provides dynamic information on drug responses in the context of an intact tumor microenvironment in real-time. Such information can lead to important insights into the underlying processes that drive therapeutic responses5.

Solid tumors are comprised of two major compartments: cancer cells and the stromal constituents (both cellular and non-cellular) that assist in maintaining an appropriate environment for tumor growth1. These stromal constituents play critical roles in the development, progression, and metastasis of many types of cancers, including those of the breast2-5. The stromal milieu also influences therapeutic responses2,4,5. Thus, determining how cancer cells respond to conventional and novel therapies within the context of the intact microenvironment is important for furthering our understanding of cancer biology and improving current therapeutic strategies. Furthermore, defining how cancer cell-stroma interactions change following the administration of therapy is critical for understanding the biology of tumor relapse.
Organotypic co-culturing systems have been useful for studying tumor-stroma interactions6, but it is evident that the methods currently available cannot successfully recapitulate the intact tumor microenvironment in vitro, in particular with respect to vascular function and recruitment of immune cells. Indeed, cancer cell responses to therapies that are observed in vitro are often significantly different from those observed in vivo, where the microenvironment is intact5. Thus, in vivo models for studying tumor-stroma interactions and their impact on therapeutic response may better reflect clinically relevant mechanisms7.
The primary means of studying responses to anti-cancer therapy in vivo have been through measurements of tumor size and histological evaluation of tissues derived from treated animals or patients. However, advancements in microscopy and fluorescent reporters over the past two decades have enabled the visualization of inter- and intracellular processes at high resolution in live, anesthetized animals (intravital imaging)8. These intravital microscopy technologies have proven to be especially valuable for spatially and temporally dissecting the in vivo dynamics of tumor-stroma interactions at cellular and sub-cellular resolution8,9.
Processes that have been unraveled by intravital imaging include anti-tumor T cell cytotoxicity10,11, interactions between T cells and myeloid cells12, the dynamics of changes in collagen composition and organization following therapeutic treatment13, macrophage-dependent cancer cell intravasation and metastasis14, and vascular permeability15.
There are three major requirements for intravital imaging. These include appropriate preparation and exposure of tissues for imaging, fluorescent labeling of the tissue components of interest, and a paired microscopy-camera system capable of acquiring images16. The most common strategies used to address these requirements include: 1) heterotopic preparations (e.g., inoculation of the ear or eye orbital), permanent imaging windows, or exteriorized preparations (e.g., dorsal skin flap); 2) transgenic fluorescent reporters and fluorescent injectables to label tissue components; and 3) multiphoton or confocal microscopy systems paired with photomultiplier tubes or charge-coupled device (CCD) cameras, respectively9. We use a surgically prepared skin flap model to expose the inguinal mammary gland for imaging in anesthetized animals that express transgenes encoding fluorescent reporters. Images are obtained over a period of 12 to 40 hours using an intensified CCD (ICCD) camera paired with a four-laser spinning disk confocal microscope system17. Imaging in this manner has allowed us to study such processes as in vivo drug distribution, stage-dependent chemotherapeutic responses, type of chemotherapy-induced cell death, and myeloid cell behavior5.
We provide a protocol for the intravital imaging of cancer cell responses to anti-cancer therapy and tumor-stroma interactions in mouse models of breast cancer. This protocol can be used to track the behaviors and death of both cancer cells and stromal components with a wide variety of transgenic and injectable fluorescent labels in both transgenic and transplantation models for periods of up to 40 hours in a single imaging session.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>MMTV-PyMT mice</td><td>Jackson Laboratory</td><td>2374</td><td></td></tr><tr><td>C3(1)-Tag mice</td><td>Jackson Laboratory</td><td>13591</td><td></td></tr><tr><td>ACTB-ECFP mice</td><td>Jackson Laboratory</td><td>3773</td><td></td></tr><tr><td>ACTB-H2B-EGFP mice</td><td>Jackson Laboratory</td><td>5418</td><td></td></tr><tr><td>1 x PBS</td><td>Made in-house</td><td></td><td></td></tr><tr><td>2 MD Dextran, Fluorescein-conjugated</td><td>Invitrogen</td><td>D-7137</td><td>Reconstitute in dH&lt;sub&gt;2&lt;/sub&gt;O (4 mg/mL, store at -20 &amp;deg;C); dilute to 1 mg/ml in 1 x PBS</td></tr><tr><td>10 kD Dextran, Alexa Fluor 647-conjugated</td><td>Invitrogen</td><td>D-22914</td><td>Reconstitute in dH&lt;sub&gt;2&lt;/sub&gt;O (4 mg/mL, store at -20 &amp;deg;C); dilute to 1 mg/ml in 1 x PBS</td></tr><tr><td>Doxorubicin hydrochloride</td><td>Sigma-Aldrich</td><td>44583</td><td>Reconstitute in dH&lt;sub&gt;2&lt;/sub&gt;O and store at 4 &amp;deg;C; dilute in 1 x PBS</td></tr><tr><td>Isothesia (Isoflurane)</td><td>Butler Animal Health Supply</td><td>029450</td><td>250 ml</td></tr><tr><td>Propidium iodide</td><td>Invitrogen</td><td>P3566</td><td>1 mg/ml; dilute 1:15 in 1 x PBS</td></tr><tr><td>Nitrogen</td><td></td><td></td><td></td></tr><tr><td>Oxygen</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>18G x 1&amp;frac12;&quot; regular bevel needle</td><td>BD</td><td>305196</td><td></td></tr><tr><td>&amp;mu;Manager</td><td>Vale Lab, UCSF</td><td>&lt;a href=&quot;http://www.micro-manager.org&quot; target=&quot;_blank&quot;&gt;www.micro-manager.org&lt;/a&gt;</td><td>Open-source software</td></tr><tr><td>Alcohol swab</td><td>BD</td><td>326895</td><td>70% isopropyl alcohol swabs</td></tr><tr><td>Anesthesia system</td><td>Molecular Imaging Products, Co.</td><td></td><td></td></tr><tr><td>Cover glass</td><td>Corning</td><td>2940-245</td><td>No. 1&amp;#189;, 24x50 mm; disinfect with 70% isopropanol wipes</td></tr><tr><td>Curity gauze sponges (sterile)</td><td>Kendall</td><td>6939</td><td></td></tr><tr><td>Glass microscope slides</td><td>Corning</td><td>2948-75x25</td><td>Pre-cleaned; if not pre-cleaned, disinfect with 70% isopropanol wipes</td></tr><tr><td>Hardened fine scissors</td><td>Fine Science Tools</td><td>14090-11</td><td>2 pairs; stainless steel, sharp-sharp tips, straight tip, 26 mm cutting edge, 11 cm length</td></tr><tr><td>Heated blanket</td><td>Gaymar Industries</td><td></td><td></td></tr><tr><td>Hot bead sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td>Turn on approximately 30 min before use; sterilize tools at &amp;gt;200 &amp;deg;C for 30 sec</td></tr><tr><td>Imaris</td><td>Bitplane</td><td>&lt;a href=&quot;http://www.bitplane.com&quot; target=&quot;_blank&quot;&gt;www.bitplane.com&lt;/a&gt;</td><td></td></tr><tr><td>Krazy Glue</td><td>Elmer&amp;#8217;s Products</td><td>KG484</td><td></td></tr><tr><td>Laboratory tape ( &amp;frac12;&quot;)</td><td></td><td></td><td></td></tr><tr><td>Laboratory tape (1&quot;)</td><td></td><td></td><td></td></tr><tr><td>Lid to a Styrofoam shipping cooler</td><td></td><td></td><td>This will be used as the surgical platform</td></tr><tr><td>Micro dissecting forceps</td><td>Roboz</td><td>RS-5153</td><td>1x2 teeth, slight curve, 0.8 mm tip width, 4&quot; length</td></tr><tr><td>Micro dissecting forceps</td><td>Roboz</td><td>RS-5135</td><td>Serrated, slight curve, 0.8 mm tip width; 4&quot; length</td></tr><tr><td>Microscope</td><td></td><td></td><td>Spinning-disk confocal, XYZ Piezo stage, epifluourescence capablility&lt;sup&gt;17,25&lt;/sup&gt;</td></tr><tr><td>Microscope stage insert</td><td>Applied Scientific Instrumentation</td><td></td><td>Custom fabricated, with two circular imaging ports</td></tr><tr><td>MouseOx oximeter, software, and sensors</td><td>STARR Life Sciences</td><td>&lt;a href=&quot;http://www.starrlifesciences.com&quot; target=&quot;_blank&quot;&gt;www.starrlifesciences.com&lt;/a&gt;</td><td></td></tr><tr><td>Nalgene Super Versi-Dry lab soakers</td><td>Thermo Scientific</td><td>74218-00</td><td>Cut into fourths for lining surgical platform</td></tr><tr><td>Nebulizer</td><td>Salter Labs</td><td>8901</td><td>Used to humidify gases; prevents irritation of lung tissues</td></tr><tr><td>Slip-tip disposable tuberculin syringe (1 ml)</td><td>BD</td><td>309659</td><td></td></tr><tr><td>Surflo winged infusion set</td><td>Terumo</td><td>SV-23BLK</td><td>23G x &amp;#190;&quot; ultra thin needle, 12&quot; tubing</td></tr><tr><td>Betadine Spray</td><td>Purdue Pharma</td><td>BASP3H</td><td></td></tr></tbody></table>

live imaging of drug responses in the tumor microenvironment in mouse models of breast cancer

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

The overall goal of this procedure is to track in real time drug penetration and therapeutic response to the administration of a therapeutic drug. This is accomplished by first treating the animal with the drug. Next, the inguinal mammary gland containing the tumor is exposed by creating a skin flap.Then the mouse is transferred to the microscopic stage and blood oxygen saturation and pulse rates are measured and an indwelling line for fluid and drug or dye administration is inserted. Finally, time-lapse images are acquired for later analyses. Ultimately, results can be obtained that show changes in cell death or tumor stroma interactions in response to a drug through live spinning disc confocal microscopy of mouse mammary tumors.This method can help answer key questions in the tumor microenvironment field, such as how various stroma components of a tumor influence the response to therapy. Before beginning, see the written protocol for typical schedules for drug administration and imaging. Turn on a hot bead sterilizer and let it reach at least 200 degrees Celsius.After washing the surgical tools in soap and water, including a pair of forceps with teeth, a pair of serrated forceps, and two pairs of scissors, use the hot bead sterilizer to sterilize them for at least 30 seconds. Then let them.Cool. Place a lab soaker on top of the styrofoam lid that will be used as the surgical platform.Use laboratory tape to affix a nose cone with a line to the anesthesia system to the lab soaker and certain 18 gauge by one and a half inch needle through the tape into the styrofoam lid on either side of the nose cone to keep the nose cone secured in place. Set aside Betadine sterile gauze. Two 70%isopropanol wipes a microscope slide, super glue, and four pieces of half inch wide lab tape.After anesthetizing the animal in an induction chamber, when it is breathing deeply and slowly transfer the mouse to the surgical platform, ventral side facing up. To avoid high pressure, open the anesthesia line to the surgical platform and then close the anesthesia line to the induction chamber. Reduce the concentration of isoflurane from 4%to 2.5%Check that the depth of anesthesia is sufficient for surgery by performing a footpad pinch.If the animal does not react, use four pieces of lab tape to secure its limbs to the surgical platform and use an electric razor to remove the hair from the ventral surface. Use 70%isopropanol wipes, followed by Betadine applied to sterile gauze to disinfect the ventral surface of the animal using scissors and forceps with teeth. Make a subcutaneous ventral midline incision from about three millimeters above the urethra to the xiphoid process.Take care to avoid puncturing or cutting through the peritoneum with the scissors and serrated forceps. Gently detached the skin from the peritoneal cavity with the inguinal mammary gland containing the tumor to be imaged Still attached. Position a glass microscope slide against the skin flap so that the bulk of the tumor sits flat once the mouse is placed on the stage, and so that it is not obstructed by the hind limbs.Once the microscope slide has been properly positioned, use super glue to attach the slide to the external surface of the skin. Remove the laboratory tape securing the limbs of the animal. Open the anesthesia line to the microscope stage, and then close the anesthesia line to the surgical platform.Quickly transfer the animal to the microscope stage Once the animal has been transferred, position and secure the anesthesia line and nose cone to ensure that the mouse remains anesthetized and is in a comfortable position. Reduce the concentration of isoflurane to 1.5%Expose the tumor so that it is positioned on top and in the center of one of the glass covered imaging ports on the microscope stage. Insert the indwelling intraperitoneal line with winged infusion set attached to the one milliliter syringe containing sterile PBS saline or saline with propidium iodide.When the intraperitoneal line is inserted, inject the animal with 100 microliters saline with propidium iodide and continue to inject 50 microliters of saline with propidium iodide every hour until the imaging session is complete. Using the eye pieces, verify that the tumor is correctly positioned and gently taped down the microscope slide and the intraperitoneal line. The slide should be secured loosely enough that blood flow to the tissue is not obstructed.Taping down the microscope slide helps to minimize imaging artifacts introduced by the animal's breathing to acquire time-lapse images. Use the open source software micromanager, compile and analyze the raw data using IRIS or other image analysis software. Fluorescently conjugated DExT strands can be used as a surrogate marker for drug delivery into tissues.Shown here is the arrival of fitzy conjugated two megadalton dextran IV injected into a mouse expressing blue fluorescent protein in breast cancer cells. Following the arrival of the drug into the tumor, extravasation and diffusion can be imaged to determine intravascular half-life and drug distribution. Here, the extravasation and distribution of LOR conjugated 10 kilodalton dextran IV injected into a mouse expressing blue fluorescent protein in breast cancer cells and green fluorescent protein in myeloid cells is shown.Imaging can provide information regarding the type of cell death that cancer cells undergo. Here, structural nuclear changes indicative of necrosis or apoptosis are observed in a mouse expressing green fluorescent protein conjugated to hisone two b. After doxorubicin treatment, chemotherapy induced cancer cell death frequently results in a reactive recruitment of immune cells into tumors.Here is an example of this stromal response to acute treatment with doxorubicin in a mouse expressing blue and green fluorescent proteins in breast cancer and myeloid cells respectively. In addition to visualizing the overall stromal response to chemotherapy, specialized stromal responses can also be visualized here. The phagocytosis of necrotic cell material by a neighboring cell is shown Once mastered.This technique can be done in 30 to 60 minutes and animals can be imaged for 12 to 40 hours if it is performed properly.

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Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

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