W.P.S. was supported in part by grants from the National Institutes of Health (CA129359), the Susan G. Komen for the Cure Foundation (BCTR0706967), the Department of Defense (BC084561), and the Seidman Cancer Center, while M.K.W. was supported by a fellowship from the American Cancer Society (PF-09-120-01). Additional support for this work was provided by the Case Center for Imaging Research, the Case Comprehensive Cancer Center (P30 CA43703), and the Cystic Fibrosis Center (P30 DK027651).

1. Stable Expression of CMV-Driven Renilla Luciferase
<ol>
	<li>Transfection and selection of a stable clonal population is the preferred method for expression of this renilla luciferase reporter. This approach yields a more consistent and uniform renilla luciferase expression following the subsequent introduction of additional secondary reporter constructs (e.g., firefly luciferase or fluorescent proteins).</li>
	<li>Transfect malignant cells of interest with the expression vector encoding renilla luciferase, such as pcDNA3.1-Hygro or another plasmid harboring a selectable marker.</li>
	<li>Following transfection, place the transfectants under an optimized antibiotic concentration for several days-to-weeks to facilitate the isolation of individual colonies, which are subsequently isolated individually and subcultured.</li>
	<li>Select &ge;10 individual renilla luciferase-expressing colonies to monitor the extent of renilla expression.</li>
	<li>Colonies expressing high quantities of renilla luciferase are subsequently subjected to functional analyzes to ensure that the (i) pathophysiological properties of their parental counterparts are retained, and (ii) renilla expression values do not deviate or change over time. These steps are absolutely essential to avoid isolating and studying clonal variants/deviants, and to validate proper integration of the renilla construct into the genome.</li>
	<li>Once a stable transfectant is isolated and verified, one can remove the selective pressure and be assured that the expression of renilla luciferase will remain constant over extended lengths of time both in vitro and in vivo .</li>
</ol>
2. Expression, Selection, and Functional Verification of Inducible Promoter-Driven Firefly Luciferase
<ol>
	<li>Subclone the promoter of interest into a pGL4-luciferase reporter plasmid harboring a selectable marker (e.g., puromycin) that is distinct from that used to select for stable renilla expression (e.g., hygromycin).</li>
	<li>Transfect malignant cells as in Step I, and subsequently select for a stable polyclonal population of firefly luciferase-expressing cells. Because the location where a report gene integrates into the genome can elicit erroneous effects on its expression and regulation, it is strongly recommended to select for polyclonal populations of firefly luciferase-expressing cells as opposed to clonal populations to ensure that (i) reporter gene expression more accurately reflects that of the endogenous gene in parental cells; and (ii) integration effects on gene expression are averaged and diminished across the heterogeneous cell population.</li>
	<li>Malignant cells engineered to stably express both renilla and firefly luciferases are collectively referred to as dual bioluminescent reporter cells, or DBR cells.</li>
	<li>Using traditional in vitro cell-based luciferase reporter gene assays, verify that the DBR cells regulate firefly luciferase expression, either positively or negatively, in a manner concordant with that observed in parental cells transiently transfected with these vectors. Likewise, verify that CMV-driven expression of renilla is not regulated by various cell stimulations or treatment conditions.</li>
	<li>To do so, culture DBR and parental cells in 24-well plates, and subsequently transiently co-transfect parental cells with the original CMV-renilla and promoter-firefly constructs used to generate DBR cells.</li>
	<li>Afterward, treat DBR and transfected parental cells with factors or pharmacological agents known to regulate the promoter of interest, and subsequently quantitate firefly and renilla luminescence using the Promega Dual Luciferase Assay Kit.</li>
</ol>
3. Establishing 4T1 Primary Mammary Tumors
<ol>
	<li>Metastatic 4T1 mammary carcinoma cells that were found to lack adventitial rodent pathogens were engineered to stably express a CMV-driven renilla luciferase (pcDNA3.1-CMV-renilla luciferase-hygro) and a SBE-driven firefly luciferase (pGL4.2-SBE-firefly luciferase-puro) as described above. Orthotopic engraftment of these breast cancer cells results in the formation of spontaneous metastases primarily in the lungs.</li>
	<li>4T1 cells should not be allowed to reach confluence during their propagation in traditional 2-dimensional tissue culture systems, and they should be passaged the day before their in vivo inoculation.</li>
	<li>4T1 cells should be dissociated by trypsinization, washed thoroughly in growth media, and diluted in PBS to a concentration of 2x105 cells/ml and immediately stored on ice.</li>
	<li>Female Balb/C mice (4-6 weeks old) should be anesthetized with an induction dose of 3% isoflurane and maintained under anesthesia at a dose of 1% isoflurane. Prepare the injection site by swabbing with 70% isopropyl alcohol. Using a forceps, gently grasp and lift the 4th inguinal mammary pad. Carefully, place a 27.5 gauge needle bevel side up and inject into the mammary fat pad directly under the nipple, taking special care not push the needle into the abdominal cavity. Release the gland and slowly inject 50 &mu;l of the cell suspension (1x104 cells) into the mammary fat pad.</li>
</ol>
4. Initial Dual Luminescent Imaging
<ol>
	<li>Immediately after engrafting 4T1 cells onto the mammary fat pad and while the mice are still anesthetized, inject 100 &mu;l of RediJect Coelenterazine into the lateral tail vein. This is the optimal substrate concentration recommended by the vendor and the best tolerated by the animal.</li>
	<li>Immediately place the mouse into the isoflurane nose cone within the IVIS-200 imaging system and acquire a 0.5-1 minute luminescent image. Efficiency between injection and image acquisition is very important, as the signal for renilla luciferase drops precipitously ~30 seconds post-injection. Place the mouse back into its cage and allow it to recover for &ge;1 hour, which ensures that any residual renilla luciferase signal has dissipated and that the mouse has fully recovered from the anesthesia.</li>
	<li>Administer 150 mg/kg of D-luciferin potassium salt via I.P. injection and wait 5 minutes. This is the optimal concentration of D-luciferin, which provides a stable luminescent signal for 5-15 minutes following its injection into test animals. Anesthetize the mouse using isoflourane and replace in the IVIS-200, taking care to position the animal in a very similar position relative to the original renilla acquisition. The overall intensity of this firefly signal will depend on the activity of the promoter of interest, and as such, visualizing firefly luciferase activity may require extended acquisition times.</li>
	<li>Using the IVIS Living Image software set to "photons" mode, establish values for both the renilla and firefly acquisitions as a means to establish baseline relative luminescence ratios (RLR).</li>
</ol>
5. Longitudinal Luminescent Imaging
<ol>
	<li>4T1 cells are highly aggressive, such that inoculation of 1x104 cells typically leads to palpable tumor formation within 1 week, and to the lethality of the animal within 4-5 weeks. 4T1 tumors have an inherent propensity ulcerate in week 4. The growth and maintenance of ulcerated tumors may require separate IACUC approval. The 4T1 tumor studies shown herein have been approved by the IACUC at Case Western Reserve University.</li>
	<li>As described above, mice should be injected weekly with RediJect Coelenterazine to monitor general tumor growth and metastasis, as well as with D-luciferin to monitor pathway-specific signaling during various stages of tumor development. Take care each time to ensure the renilla luciferase signal has completely dissipated prior to acquisition of the firefly luciferase signal.</li>
	<li>As 4T1 tumors develop and progress, renilla luciferase acquisitions should be normalized to the initial renilla values measured at the time of tumor cell inoculation as a means to normalize and track primary tumor growth. More importantly, calculating RLR values over time will establish the temporal regulation of individual signaling systems relative to tumor growth and progression.</li>
	<li>Overt pulmonary metastasis becomes apparent within 3-4 weeks following 4T1 cell engraftment onto the mammary fat pad. Comparing pulmonary RLR values versus those calculated for the primary tumor establishes the spatial regulation of individual signaling systems relative to tumor growth and metastasis.</li>
</ol>
6. Dual Bioluminescent Imaging of Two Unique Cell Types in a Single Animal
<ol>
	<li>Engineer one breast cancer cell line to stably expression CMV-driven renilla luciferase as described above. Repeat this engineering process using CMV-driven firefly luciferase on a second distinct breast cancer cell line.</li>
	<li>Herein we mixed renilla luciferase-expressing 4T1 cells with their nonmetastatic and isogenic firefly luciferase-expressing 4T07 counterparts 10, 11. Varying ratios of these mixed breast cancer cell populations are subsequently injected into the mammary fat pad as described above.</li>
	<li>Immediately acquire both firefly and renilla luciferase images to establish initial RLRs representative of a given inoculated cell mixture.</li>
	<li>Longitudinal dual bioluminescent imaging will visualize and track changes in the cellular composition within the primary tumor, as well as the spatiotemporal metastasis of each breast cancer derivative relative to tumor growth and progression.</li>
	<li>Alternatively, individual breast cancer cell populations can be inoculated into different locations in the mouse ( i.e. the right and left abdominal mammary fat pads) to assess the systemic influences these two populations exhibit over one another during various stages of tumor growth and metastasis.</li>
</ol>
7. Representative Results:
A major strength of dual bioluminescent imaging lies in the fact that each image is internally consistent and controlled, such that the continued signals derived from the primary tumor function as a important metric to gauge the overall success or failure of each renilla and firefly acquisition. This aspect of the imaging procedure is particularly important during acquisitions of the activity of renilla luciferase, whose Coelenterazine substrate is highly sensitive to oxidation that results in its ability to auto-luminescence. Figure 1A shows an example of this side effect, which immediately manifests as nonspecific luminescent signals derived from the intestinal tract, not the established primary tumor. Typically, these nonspecific Coelenterazine signals transpire following failed intravenous injections of this renilla luciferase substrate. However, once robust primary tumor-derived renilla signals have been obtained (Figure 1B, left image), it is safe to proceed in capturing pathway-specific signaling measurements derived from imaging firefly luciferase (Figure 1B, right panel), whose D-luciferin substrate is highly stable upon I.P. injection and produces negligible levels of background auto-luminescence.
<img alt="Figure 1" src="/files/ftp_upload/3245/3245fig1.jpg" /> 
Figure 1. Acquiring renilla and firefly-derived bioluminescent images to calculate in vivo RLRs. (A) An example of a failed I.V. injection of Coelenterazine resulting in nonspecific signal from the intestinal tract. Circle and PT indicate the approximate location of the primary tumor. (B) A successful renilla acquisition of a mouse bearing a 4T1 primary tumor and pulmonary metastases (left panel; 4wk after fat pad engraftment). The corresponding firefly acquisition allows for calculation of RLRs from both the primary tumor and its pulmonary metastases. (C) Graphical representation of the primary tumor and pulmonary metastasis RLRs calculated from mice shown in panel B (n=4).

<ol>
	<li>Wetterwald, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+cancer+metastasis+to+bone+marrow:+a+mouse+model+of+minimal+residual+disease.">Optical imaging of cancer metastasis to bone marrow: a mouse model of minimal residual disease.</a> Am. J. Pathol. 160, 1143-1153 (2002).</li><li>Wendt, M. K., Drury, L. J., Vongsa, R. A., Dwinell, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+CXCL12+Expression+Induces+Anoikis+in+Colorectal+Carcinoma+Cells.">Constitutive CXCL12 Expression Induces Anoikis in Colorectal Carcinoma Cells.</a> Gastroenterology. 135, 508-508 (2008).</li><li>Wendt, M., Schiemann, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+targeting+of+the+focal+adhesion+complex+prevents+oncogenic+TGF-beta+signaling+and+metastasis.">Therapeutic targeting of the focal adhesion complex prevents oncogenic TGF-beta signaling and metastasis.</a> Breast Cancer Research. 11, R68-R68 (2009).</li><li>Korpal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+transforming+growth+factor-beta+signaling+dynamics+and+therapeutic+response+in+breast+cancer+bone+metastasis.">Imaging transforming growth factor-beta signaling dynamics and therapeutic response in breast cancer bone metastasis.</a> Nat Med. , (2009).</li><li>Giampieri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+and+reversible+TGFbeta+signalling+switches+breast+cancer+cells+from+cohesive+to+single+cell+motility.">Localized and reversible TGFbeta signalling switches breast cancer cells from cohesive to single cell motility.</a> Nat Cell Biol. 11, 1287-1296 (2009).</li><li>Giampieri, S., Pinner, S., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+illuminates+transforming+growth+factor+beta+signaling+switches+during+metastasis.">Intravital imaging illuminates transforming growth factor beta signaling switches during metastasis.</a> Cancer Res. 70, 3435-3439 (2010).</li><li>DeNardo, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD4(+)+T+cells+regulate+pulmonary+metastasis+of+mammary+carcinomas+by+enhancing+protumor+properties+of+macrophages.">CD4(+) T cells regulate pulmonary metastasis of mammary carcinomas by enhancing protumor properties of macrophages.</a> Cancer Cell. 16, 91-102 (2009).</li><li>Wyckoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Paracrine+Loop+between+Tumor+Cells+and+Macrophages+Is+Required+for+Tumor+Cell+Migration+in+Mammary+Tumors.">A Paracrine Loop between Tumor Cells and Macrophages Is Required for Tumor Cell Migration in Mammary Tumors.</a> Cancer Res. 64, 7022-7029 (2004).</li><li>Kim, M. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+self-seeding+by+circulating+cancer+cells.">Tumor self-seeding by circulating cancer cells.</a> Cell. 139, 1315-1326 (2009).</li><li>Aslakson, C. J., Miller, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+events+in+the+metastatic+process+defined+by+analysis+of+the+sequential+dissemination+of+subpopulations+of+a+mouse+mammary+tumor.">Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor.</a> Cancer Res. 52, 1399-1405 (1992).</li><li>Wendt, M. K., Smith, J. A., Schiemann, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor-beta-induced+epithelial-mesenchymal+transition+facilitates+epidermal+growth+factor-dependent+breast+cancer+progression.">Transforming growth factor-beta-induced epithelial-mesenchymal transition facilitates epidermal growth factor-dependent breast cancer progression.</a> Oncogene. 29, 6485-6498 (2010).</li><li>Hawkins, E., Unch, J., Murhpy, N., Vidugiriene, J., Scurria, M., Klaubert, D., Wood, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright+Light,+No+Lysis.">Bright Light, No Lysis.</a> Measuring Renilla Luciferase Luminescence in Living Cells. , (2005).</li><li>Roncali, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+device+for+real-time+bioluminescence+imaging+in+moving+rodents.">New device for real-time bioluminescence imaging in moving rodents.</a> J. Biomed. Opt. 13, 054035-054035 (2008).</li></ol>

No conflicts of interest declared.

The absolute power of bioluminescent imaging techniques lies in their ability to quantify tumor growth and metastasis in complex longitudinal studies, which herein involved the use of aggressive 4T1 breast cancer cells. Because these procedures rely on the stable integration of both luciferase reporter constructs, these techniques can be readily adapted and translated to other cancer cell lines of varying tumor latencies and metastatic capabilities. Similar to the results shown herein, calculating specific RLRs within slowly developing primary tumors and their eventual metastases allows for the real-time spatiotemporal identification of specific signaling events that transpire during the metastatic progression of tumors irrespective of their duration of latency. Upon identification of specific promoter regulatory events, it is important to excise both the primary tumor and its metastatic lesions for the performance of standard immunohistochemical and differential gene expression analyses to verify similar regulation of the endogenous gene and/or protein.
Technically speaking, the primary challenge of dual bioluminescent analyses lies in the relatively short duration of the renilla luciferase signals. As such, this imaging technique requires significant optimization of the tail vein injection procedure, as well as the immediate imaging of individually injected animals, a process that is time consuming and somewhat inefficient relative to imaging firefly luciferase. Recently, Promega introduced "Viviren," which represents a second generation luciferase substrate whose sites of oxidation are blocked by esterification until this molecule gains entry into cells, at which point the molecule is rapidly de-esterified. Collectively, this novel substrate effectively lowers auto-luminescence and nonspecific modification and/or degradation coupled to renilla-induced auto-luminescence 12. In doing so, this new renilla luciferase substrate produces brighter luminescent signals; however, the excessive costs associated with the acquisition and use of "Viviren" have provided relatively few studies necessary to evaluate the overall utility of this substrate in dual bioluminescent analyses. Finally, the sensitivity of any bioluminescent assay is critically dependent upon the CCD camera operant in acquiring individual light units. Indeed, as these technologies improve, we foresee a point in the future where our ability to visualize complex light-mediated bioluminescent reactions may transpire efficiently in free moving animals 13.

<table border="1">
	<tbody>
		<tr>
			<th>Name of reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
		</tr>
		<tr>
			<td>pGL4.2-Puro [luc2/Puro] Vector		</td>
			<td>Promega</td>
			<td>E6751</td>
		</tr>
		<tr>
			<td>Glomax Mult-idection system</td>
			<td>Promega</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>IVIS 200 series</td>
			<td>Caliper Life Sciences</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dual luciferase reporter Assay system</td>
			<td>Promega</td>
			<td>E1960</td>
		</tr>
		<tr>
			<td>Rediject coelenterazine-h</td>
			<td>Caliper Life Sciences</td>
			<td>760506</td>
		</tr>
		<tr>
			<td>D-Luciferin K-Salt</td>
			<td>Caliper Life Sciences</td>
			<td>122796</td>
		</tr>
	</tbody>
</table>

in vivo dual substrate bioluminescent imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

Watch this Scientific Journal Video about In vivo Dual Substrate Bioluminescent Imaging at JoVE.com

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

in-vivo-dual-substrate-bioluminescent-imaging

Research

JoVE Journal

Medicine

15.3K ビュー.  Case Western Reserve University. ここに我々は彼らの成長と転移時に転移性乳癌細胞で発現ホタルおよびウミシイタケルシフェラーゼ酵素の両方の生物発光反応を構築可視化し、定量化するための方法を説明。

ビデオ: In vivo Dual Substrate Bioluminescent Imaging

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

Dual-mode Imaging of Cutaneous Tissue Oxygenation and Vascular Function

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health disorders such as chronic wounds. We report the development of a dual-mode imaging system for non-invasive and non-contact imaging of cutaneous tissue oxygenation and vascular function. The imaging system integrated an infrared camera, a CCD camera, a liquid crystal tunable filter and a high intensity fiber light source. A Labview interface was programmed for equipment control, synchronization, image acquisition, processing, and visualization. Multispectral images captured by the CCD camera were used to reconstruct the tissue oxygenation map. Dynamic thermographic images captured by the infrared camera were used to reconstruct the vascular function map. Cutaneous tissue oxygenation and vascular function images were co-registered through fiduciary markers. The performance characteristics of the dual-mode image system were tested in humans.

A dual-mode imaging system was developed for non-contact assessment of cutaneous tissue oxygenation and vascular function.

Accurate assessment of cutaneous tissue oxygenation and vascular function is important for appropriate detection, staging, and treatment of many health ...

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...