Name: live imaging of drug responses in the tumor microenvironment in mouse models of breast cancer
Uploaded: 2013-03-24T14:00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer at JoVE.com

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).

<ol>
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J., Boldajipour, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marginating+dendritic+cells+of+the+tumor+microenvironment+cross-present+tumor+antigens+and+stably+engage+tumor-specific+T+cells.">Marginating dendritic cells of the tumor microenvironment cross-present tumor antigens and stably engage tumor-specific T cells.</a> Cancer Cell. 21, 402-417 (2012).</li><li>Brown, E., McKee, T., 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=Dynamic+imaging+of+collagen+and+its+modulation+in+tumors+in+vivo+using+second-harmonic+generation.">Dynamic imaging of collagen and its modulation in tumors in vivo using second-harmonic generation.</a> Nat. Med. 9, 796-800 (2003).</li><li>Wyckoff, J. 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L., Fukumura, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+tumour+pathophysiology+using+intravital+microscopy.">Dissecting tumour pathophysiology using intravital microscopy.</a> Nat. Rev. Cancer. 2, 266-276 (2002).</li><li>Egeblad, M., Ewald, A. J., 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=Visualizing+stromal+cell+dynamics+in+different+tumor+microenvironments+by+spinning+disk+confocal+microscopy.">Visualizing stromal cell dynamics in different tumor microenvironments by spinning disk confocal microscopy.</a> Dis Model Mech. 1, 155-167 (2008).</li><li>Cheng, N., Lambert, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+Transplantation+of+Stromal+Cells+and+Carcinoma+Cells+in+C57BL/6J.">Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J.</a> Mice. J. Vis. Exp. (54), ee2716 (2011).</li><li>Ewald, A. J., Werb, Z., Egeblad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+vital+signs+for+long-term+survival+of+mice+under+anesthesia.">Monitoring of vital signs for long-term survival of mice under anesthesia.</a> Cold Spring Harb. Protoc. 2011, pdb.prot5563 (2011).</li><li>Sounni, N. E., Dehne, K., 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=Stromal+regulation+of+vessel+stability+by+MMP14+and+TGFbeta.">Stromal regulation of vessel stability by MMP14 and TGFbeta.</a> Dis. Model Mech. 3, 317-332 (2010).</li><li>Magness, S. T., Bataller, R., Yang, L., Brenner, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dual+reporter+gene+transgenic+mouse+demonstrates+heterogeneity+in+hepatic+fibrogenic+cell+populations.">A dual reporter gene transgenic mouse demonstrates heterogeneity in hepatic fibrogenic cell populations.</a> Hepatology. 40, 1151-1159 (2004).</li><li>Motoike, T., Loughna, S., 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=Universal+GFP+reporter+for+the+study+of+vascular+development.">Universal GFP reporter for the study of vascular development.</a> Genesis. 28, 75-81 (2000).</li><li>Fukumura, D., Xavier, 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=Tumor+induction+of+VEGF+promoter+activity+in+stromal+cells.">Tumor induction of VEGF promoter activity in stromal cells.</a> Cell. 94, 715-725 (1998).</li><li>Sugimoto, H., Mundel, T. 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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 border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>MMTV-PyMT mice</td>
			<td>Jackson Laboratory</td>
			<td>2374</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>C3(1)-Tag mice</td>
			<td>Jackson Laboratory</td>
			<td>13591</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ACTB-ECFP mice</td>
			<td>Jackson Laboratory</td>
			<td>3773</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ACTB-H2B-EGFP mice</td>
			<td>Jackson Laboratory</td>
			<td>5418</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1 x PBS</td>
			<td>Made in-house</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2 MD Dextran, Fluorescein-conjugated</td>
			<td>Invitrogen</td>
			<td>D-7137</td>
			<td>Reconstitute in dH2O (4 mg/ml, store at -20&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 dH2O (4 mg/ml, store at -20&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 dH2O and store at 4 &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>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Oxygen</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>18G x 1&frac12;" regular bevel needle</td>
			<td>BD</td>
			<td>305196</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&mu;Manager</td>
			<td>Vale Lab, UCSF</td>
			<td><a href="www.micro-manager.org" target="_blank">www.micro-manager.org</a></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>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>Corning</td>
			<td>2940-245</td>
			<td>No. 1&frac12;, 24x50 mm; disinfect with 70% isopropanol wipes</td>
		</tr>
		<tr>
			<td>Curity gauze sponges (sterile)</td>
			<td>Kendall</td>
			<td>6939</td>
			<td>&nbsp;</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>&nbsp;</td>
			<td>&nbsp;</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 &gt;200 &deg;C for 30 sec</td>
		</tr>
		<tr>
			<td>Imaris</td>
			<td>Bitplane</td>
			<td><a href="www.bitplane.com" target="_blank">www.bitplane.com</a></td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Krazy Glue</td>
			<td>Elmer's Products</td>
			<td>KG484</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laboratory tape ( &frac12;")</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laboratory tape (1")</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lid to a Styrofoam shipping cooler</td>
			<td>&nbsp;</td>
			<td>&nbsp;</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" 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" length</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Spinning-disk confocal, XYZ Piezo stage, epifluourescence capablility17,25</td>
		</tr>
		<tr>
			<td>Microscope stage insert</td>
			<td>Applied Scientific Instrumentation</td>
			<td>&nbsp;</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><a href="www.starrlifesciences.com" target="_blank">www.starrlifesciences.com</a></td>
			<td>&nbsp;</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>&nbsp;</td>
		</tr>
		<tr>
			<td>Surflo winged infusion set</td>
			<td>Terumo</td>
			<td>SV-23BLK</td>
			<td>23G x &frac34;" ultra thin needle, 12" tubing</td>
		</tr>
		<tr>
			<td>Betadine Spray</td>
			<td>Purdue Pharma</td>
			<td>BASP3H</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer at JoVE.com

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

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 ...

live-imaging-drug-responses-tumor-microenvironment-mouse-models

Research

JoVE Journal

Medicine

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

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 ...

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 ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...