We thank the animal facilities of the Cordelier Research Center (CEF, Paris, France), Genethon (Evry, France) and Genopole (CERFE, Evry, France).&#160;We thank Jo Ann Cahn for her careful reading of the manuscript. This research was supported by the Institut National de la Sant&#233; Et de la Rechercher M&#233;dicale, Paris Descartes University, Pierre &#38; Marie Curie University, the Association pour la Recherche contre le Cancer, the Tunisian Direction G&#233;n&#233;rale de la Recherche Scientifique, the Franco-Tunisian CMCU project, and the Actions Th&#233;matiques Incitatives de Genopole (ATIGE) funds. J.C was supported by the Frontiers in Life Science doctoral school and by a fellowship from the INCA (Institut National du Cancer). R.B.A. was a recipient of grants from the DGRS-INSERM and the CMCU. S.D. received a grant from the Institut National du Cancer.

All procedures involving mice complied with European Union guidelines, French regulations for animal experimentation (Ministry of Agriculture Act No. 2001-464, May 2001), and the guidelines of the Institut National de la Sant&#233; et de la Recherche M&#233;dicale (INSERM) Committee on Animal Research, and were approved by the relevant local committee (the Charles Darwin Ethics Committee for Animal Experiments, Paris, France; Permit Number: p3/2009/004).
1. Cell Preparation
<ol>
	<li>Grow mouse B lymphoma cell line A20.IIA-luc2 in RPMI-1640 Glutamax medium supplemented with 10% fetal calf serum, 100 &#181;g/ml penicillin, 100 &#181;g/ml streptomycin, 10 mM sodium pyruvate, 50 &#181;M 2-mercaptoethanol, and 0.50 mg/ml hygromycin B.</li>
	<li>Maintain cell culture at 37 &#176;C, 5% CO2 and change medium every two to three days. Harvest 5 ml of the cell suspension with a pipette one day after changing the medium.</li>
	<li>Spin cells at 300 &#215; g for 10 min and suspend the cells in 3 ml sterile phosphate-buffered saline (PBS). Repeat this step twice to wash the cells.</li>
	<li>Mix 15 &#181;l of cell suspension with 30 &#181;l Trypan Blue labeling before loading a Malassez counting chamber. Calculate the cell concentration with the formula: Concentration (cells/ml) = Number of cells in the counting grid * 3 * 1000.</li>
	<li>Spin the cells one more time at 300 x g for 10 min. Remove supernatant with a pipette. With C the concentration calculated at step 1.4), the number of cells is N = C*3.</li>
	<li>Calculate the volume of sterile PBS 1x required to obtain cell suspension A at a concentration of 5 x 107 cells/ml with the following formula: PBS Volume (ml) = N / (5 x 107). Suspend the cell pellet (from step 1.5)) in the volume of sterile PBS 1x calculated in the previous sentence. Cell suspension A is to be used in the SCL model (in vivo injected volume is 100 &#181;l: 5 x 106 cells).</li>
	<li>Pipette 10 &#181;l of cell suspension A and add 90 &#181;l of sterile PBS in a 1.5 ml tube to obtain 100 &#181;l of cell suspension B at 5 x 106 cells per ml. Cell suspension B is to be used in the PIOL model (in vivo injected volume is 2 &#181;l: 1 x 104 cells).</li>
</ol>
2. Luciferin
<ol>
	<li>Dilute 1 g of D-luciferin potassium salt powder in 30 ml of sterile PBS 1x in a 50-ml tube and shake for a few seconds to dissolve the aggregates. 
	NOTE: Because luciferin is light-sensitive, prepare 500 &#181;l aliquots in dark 1.5-ml microcentrifuge tubes.</li>
	<li>Store the aliquots at -20 &#176;C. 
	NOTE: The aliquots can be stored for several months. 
	After thawing, the aliquots must not be stored for more than 1 day at + 4 &#176;C. Freezing-thawing cycles are preferable to storage at 4 &#176;C.</li>
	<li>Inject 100 &#181;l of D-luciferin potassium salt solution intraperitoneally for each imaging assay. 
	NOTE: This solution corresponds to 3.3 mg per mouse for a dose of 150 mg/kg.</li>
</ol>
3. Anesthetic Mixture and Anesthetization
<ol>
	<li>Prepare an anesthetic solution by mixing ketamine 120 mg/kg and xylazine 6 mg/kg in sterile PBS 1x.</li>
	<li>Inject 60 &#181;l of anesthetic mixture intraperitoneally for each imaging assay with a 25 G needle. For surgery (with the PIOL or SCL model), inject 80 &#181;l of the mixture to obtain a deeper anesthesia. Put the mouse back in its cage.</li>
	<li>When the mouse appears immobile, remove it from its cage and gently squeeze the mouse&#39;s leg between fingers. If the mouse reacts with an escape reflex, wait several min. Repeat the action until the mouse does not react, which confirms satisfactory anesthetization.</li>
	<li>Place the mouse on a warming plate or under a warming light.</li>
	<li>Apply eye ointment to avoid eye dryness during anesthetization for the imaging assay or for SCL surgery. Apply the eye ointment after surgery for the PIOL model.</li>
</ol>
4. Surgery and Cell Inoculation
NOTE: Perform all surgical procedures on a warming plate or under a warming light, in a type 1 microbiological safety cabinet in an animal biosafety level 2 facility. All surgical tools used in this section were autoclaved before use.&#160;
<ol>
	<li>Subcutaneous Lymphoma Model:
	<ol>
		<li>Prepare 100 &#181;l of the cell suspension obtained in step 1.6 in a 1-ml syringe with a 25 G needle. Gently squeeze the mouse skin on the flank between fingers, at the injection site. Insert the needle exactly into the skin fold. To ensure subcutaneous injection, do not place the needle deep into the tissue.
		<ol>
			<li>Inject the cells into the skinfold. Observe whether a little liquid ball appears under the skin to confirm that the injection was performed correctly.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>PIOL model: 
	NOTE: This procedure requires 2 operators, referred to here as operator 1 and operator 2.
	<ol>
		<li>Removal of conjunctiva:
		<ol>
			<li>Have Operator 1 place the mouse under a dissecting microscope. Press gently with the fingers at each side of the eye to clear it. Maintain this position.</li>
			<li>Have Operator 2 look through the dissecting microscope. Grip the conjunctiva with a small pair of pliers in one hand; with the other hand, cut the conjunctiva just below the pliers with a small pair of surgical scissors.</li>
			<li>Have Operator 1 release the eye of the mouse pressed at step 4.2.1.1)</li>
		</ol>
		</li>
		<li>Cell injection:
		<ol>
			<li>Prepare a 10-&#181;l blunt precision syringe. Wash it by pumping sterile deionized water through it. Repeat twice or three times to ensure there are no bubbles in the syringe. Then prepare 2 &#181;l of cell suspension for injection.</li>
			<li>Have Operator 2 press gently with the fingers of one hand at each side of the eye to clear it. Maintain this position.</li>
			<li>Have Operator 1 grip the edge of the eye with a small pair of pliers in one hand and pull it gently backwards to stretch the tissue.</li>
			<li>Have Operator 2 look through the dissecting microscope. With the other hand, make a small hole in the inferior eyeball of the mouse with a 32 G needle.</li>
			<li>Have Operator 2 put down the needle and pick up (with the same hand) the precision syringe and insert the needle into the hole made at step 4.2.2.4.</li>
			<li>Have Operator 1 push on the syringe plunger with the other hand.</li>
			<li>Have Operator 2 verify with the dissecting microscope that the cell suspension in the syringe has been correctly injected inside the eyeball (the liquid flow is easily observable).</li>
			<li>Have Operator 2 remove the precision syringe.</li>
			<li>Have Operator 1 release the edge of the animal eye gripped at step 4.2.2.3)</li>
			<li>Have Operator 2 release the sides of the eye pressed at step 4.2.2.2)</li>
			<li>Apply the eye ointment immediately. 
			NOTE: If all steps were performed correctly, the mouse should not bleed at all during this procedure.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Bioluminescence Imaging &#8211; Day 0&#160;
NOTE: All products injected into the mouse must be at RT before injection. After the tumor cells have been inoculated, and while the animal is still anesthetized, proceed to these steps for the imaging.
<ol>
	<li>Turn on the imager and open the acquisition software. Initialize the camera, the stages, and the lenses by clicking on the &#34;Initialize&#34; button. The initialization will take 10 to 15 min to be complete.</li>
	<li>Inject 100 &#181;l of D-luciferin potassium salt solution intraperitoneally with a 25 G needle. Do not administer it intravenously. If intravenous administration is required for any reason, the D-luciferin sodium salt must be used rather than D-luciferin potassium salt. 
	NOTE: Luciferin is the excess reactant at this concentration; therefore the bioluminescence signal reaches a plateau after 3 to 7 min and persists for more than 30 min.</li>
	<li>10 min after D-luciferin injection13, place the subject mouse in the imager. Place the mouse in its natural position, its back towards the camera, in as flat a position as possible. This position is natural and easily reproducible.</li>
	<li>Tick the auto-exposure feature, and click on Acquire to acquire the back (posterior) image of the animal, with the auto-exposure feature. 
	NOTE: The auto-exposure feature optimizes exposure time by calculating it from a 1-sec exposure image. If the mouse&#39;s bioluminescence signal is negative or very low, the optimal exposure time might be automatically set to more than 20 min. In this case, an exposure time of 8 to 10 min may be a good compromise. Exposure time may be set manually, but the images must not contain saturated pixels.</li>
	<li>Turn the mouse over to expose the front of the mouse to the camera. Try to flatten the mouse and spread its anterior limbs so that they do not block the chest.</li>
	<li>Acquire a front image of the animal. Verify that the auto-exposure checkbox is still ticked and click again on the &#34;Acquire&#34; button. 
	NOTE: The front image can be acquired before the back image and vice versa. The exposure time of front and back images can be different depending on the relative intensity of each side of the mouse. It is calculated automatically when using the auto-exposure feature. Quantification uses only the photon flux (photons per sec) and does not depend on exposure time.</li>
	<li>Place the mouse on a warming plate or under a warming light until it recovers from the anesthetic and then place it back in its cage.</li>
</ol>
6. Bioluminescence Imaging &#8211; After Day 0
<ol>
	<li>Turn on the imager, initialize the camera, the stages, and the lenses as in step 5.1).</li>
	<li>Anesthetize the mouse with an intraperitoneal injection of 60 &#181;l of the ketamine (120 mg/kg) / xylazine (6 mg/kg) mixture (see steps 3.1 to 3.5). 
	NOTE: This method of anesthetization allows imaging every 5 days. To perform daily imaging, a method that uses isoflurane as the anesthetic and a bioluminescence imager suitable for use with this anesthetic is required.</li>
	<li>Acquire front and back images of the animal, using the auto-exposure feature, as in steps 5.5) and 5.6). Handle the mouse as in step 5.7).</li>
</ol>
7. Bioluminescence Quantification and Image Analysis
NOTE: The luminoscore method is based on image analysis. Once the images have been acquired according to the steps above, the quantification can be performed at any time (including immediately after acquisition), to associate a luminoscore to each mouse at each time point.&#160;
<ol>
	<li>Display the &#34;Tool Palette&#34; by clicking on the &#34;View&#34; menu and then click on &#34;Tool Palette&#34; if not already displayed. Click on the &#34;ROI Tool&#34; tab of the Tool Palette. Choose the &#34;Contour&#34; button and then select &#34;Free draw&#34;.</li>
	<li>Manually mark the Region of Interest (ROI) around the mouse by following the edges of the mouse on the front view. Close the contour with a right click on the computer mouse.</li>
	<li>Click on the &#34;View&#34; menu and then &#34;ROI Measurements&#34;. Make sure &#34;Radiance (Photons)&#34; is selected in the &#34;Measurement Types&#34; scrolling menu on the bottom left of the &#34;ROI Measurements&#34; window. If not, select it. Then measure the photon flux (ph/s) by recording the value in the &#34;Total Flux [p/s]&#34; box. 
	NOTE: Do not use Radiance or any other unit that is relative to the ROI surface area.</li>
	<li>Repeat steps 7.2) and 7.3) for the back view.</li>
	<li>Sum the two photon flux values obtained from front and back view. The result is the luminoscore.</li>
	<li>Compare the values for each group using an appropriate statistic test (non-parametric two-tailed Mann-Whitney test, for instance)14.</li>
</ol>

<ol>
	<li>Rehemtulla, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+Quantitative+Assessment+of+Cancer+Treatment+Response+Using+In+Vivo+Bioluminescence+Imaging.">Rapid and Quantitative Assessment of Cancer Treatment Response Using In Vivo Bioluminescence Imaging.</a> Neoplasia. 2 (6), 491-495 (2000).</li><li>Edinger, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancing+animal+models+of+neoplasia+through+in+vivo+bioluminescence+imaging.">Advancing animal models of neoplasia through in vivo bioluminescence imaging.</a> European Journal of Cancer. 38 (16), 2128-2136 (2002).</li><li>Hastings, J. W., Gibson, Q. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Oxygen+in+the+Photoexcited+Luminescence+of+Bacterial+Luciferase.">The Role of Oxygen in the Photoexcited Luminescence of Bacterial Luciferase.</a> Journal of Biological Chemistry. 242 (4), 720-726 (1967).</li><li>Inouye, S., Shimomura, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Use+of+Renilla+Luciferase,+Oplophorus+Luciferase,+and+Apoaequorin+as+Bioluminescent+Reporter+Protein+in+the+Presence+of+Coelenterazine+Analogues+as+Substrate.">The Use of Renilla Luciferase, Oplophorus Luciferase, and Apoaequorin as Bioluminescent Reporter Protein in the Presence of Coelenterazine Analogues as Substrate.</a> Biochemical and Biophysical Research Communications. 233 (2), 349-353 (1997).</li><li>Pesnel, 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=Quantitation+in+Bioluminescence+Imaging+by+Correction+of+Tissue+Absorption+for+Experimental+Oncology.">Quantitation in Bioluminescence Imaging by Correction of Tissue Absorption for Experimental Oncology.</a> Molecular Imaging and Biology. 13 (4), 646-652 (2010).</li><li>Corre, G., 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=Stochastic+Fluctuations+and+Distributed+Control+of+Gene+Expression+Impact+Cellular+Memory.">Stochastic Fluctuations and Distributed Control of Gene Expression Impact Cellular Memory.</a> PLoS ONE. 9 (12), (2014).</li><li>Ben Abdelwahed, 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=Lymphoma+B-cell+responsiveness+to+CpG-DNA+depends+on+the+tumor+microenvironment.">Lymphoma B-cell responsiveness to CpG-DNA depends on the tumor microenvironment.</a> Journal of Experimental &amp; Clinical Cancer Research CR. 32 (1), 18 (2013).</li><li>Abdelwahed, R. B., 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=Preclinical+Study+of+Ublituximab,+a+Glycoengineered+Anti-Human+CD20+Antibody,+in+Murine+Models+of+Primary+Cerebral+and+Intraocular+B-Cell+Lymphomas.">Preclinical Study of Ublituximab, a Glycoengineered Anti-Human CD20 Antibody, in Murine Models of Primary Cerebral and Intraocular B-Cell Lymphomas.</a> Investigative Ophthalmology &amp; Visual Science. 54 (5), 3657-3665 (2013).</li><li>Donnou, S., Galand, C., Touitou, V., Saut&#232;s-Fridman, C., Fabry, Z., Fisson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+Models+of+B-Cell+Lymphomas:+Promising+Tools+for+Designing+Cancer+Therapies.">Murine Models of B-Cell Lymphomas: Promising Tools for Designing Cancer Therapies.</a> Advances in Hematology. 2012, (2012).</li><li>Qi, X. -. F., 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=CpG+oligodeoxynucleotide+induces+apoptosis+and+cell+cycle+arrest+in+A20+lymphoma+cells+via+TLR9-mediated+pathways.">CpG oligodeoxynucleotide induces apoptosis and cell cycle arrest in A20 lymphoma cells via TLR9-mediated pathways.</a> Molecular Immunology. 54 (3-4), 327-337 (2013).</li><li>Houot, R., Levy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+modulation+combined+with+intratumoral+CpG+cures+lymphoma+in+a+mouse+model+without+the+need+for+chemotherapy.">T-cell modulation combined with intratumoral CpG cures lymphoma in a mouse model without the need for chemotherapy.</a> Blood. 113 (15), 3546-3552 (2009).</li><li>Krieg, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+9+(TLR9)+agonists+in+the+treatment+of+cancer.">Toll-like receptor 9 (TLR9) agonists in the treatment of cancer.</a> Oncogene. 27 (2), 161-167 (2008).</li><li>Inoue, Y., Kiryu, S., Watanabe, M., Tojo, A., Ohtomo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+of+Imaging+after+D-Luciferin+Injection+Affects+the+Longitudinal+Assessment+of+Tumor+Growth+Using+In+Vivo+Bioluminescence+Imaging.">Timing of Imaging after D-Luciferin Injection Affects the Longitudinal Assessment of Tumor Growth Using In Vivo Bioluminescence Imaging.</a> International Journal of Biomedical Imaging. 2010, (2010).</li><li>Mann, H. B., Whitney, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+a+Test+of+Whether+one+of+Two+Random+Variables+is+Stochastically+Larger+than+the+Other.">On a Test of Whether one of Two Random Variables is Stochastically Larger than the Other.</a> Annals of Mathematical Statistics. 18 (1), 50-60 (1947).</li><li>Darne, C., Lu, Y., Sevick-Muraca, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+fluorescence+and+bioluminescence+tomography:+a+review+of+approaches,+algorithms+and+technology+update.">Small animal fluorescence and bioluminescence tomography: a review of approaches, algorithms and technology update.</a> Physics in Medicine and Biology. 59 (1), 1 (2014).</li></ol>

The authors have nothing to disclose.

Light absorption by organs and tissues remains a limitation of bioluminescence imaging, although this limitation is intrinsic to any optical imaging modality. In the context of our approach, the effects of anatomic structures on the luminoscore are expected to have low variability provided that the studies are performed in a given model (location and mouse strand), allowing then comparisons. Bioluminescence does not need excitation light and thus is more adapted to whole body in vivo imaging than fluorescence.
Spatial resolution is also a limitation of bioluminescence imaging, and precise location of the organ from which bioluminescence photons are emitted remains difficult. However, good knowledge of the model can help in the qualitative location of the tumor sites. The only output in this bioluminescence-based method is the luminoscore. Location does not influence the luminoscore because the manual mark-up Region of Interest (ROI) covers the whole mouse. Finally, firefly luciferase requires oxygen. Accordingly, bioluminescence imaging usually underestimates necrotic tumors. Once again, a good knowledge of this aspect of the model is required.
In this paper, we are not aiming at assessing the efficacy of CpG as an anti-tumor drug (which has already been demonstrated7,9,11) but to describe a method allowing comparison of bioluminescence datasets. We indeed describe a method to quantify tumor burden intended to help standardize the acquisition protocol for comparing different assays in different places at different times and that does not require computer calculations. To ensure reproducibility and correct photon flux quantification, the imaging device must be calibrated with a light reference for home-made devices or as recommended by the provider for commercial devices.
Our protocol requires careful attention to several critical points: (i) First, the quality of the mouse anesthesia is crucial for obtaining clear images, especially in cases with long exposure times. (ii) The quantification unit must be always be the photon flux because radiance depends on the surface area of each mouse and might be irrelevant for comparing different mice. (iii) The bioluminescence images must not contain saturated pixels, because these would bias the luminoscore. (iv) Back and front acquisition are required to collect all photons emitted from the tumor site (i.e., front acquisition may not necessarily detect photons from the back of the tumor). Various different ROI drawings were tested during the development of the luminoscore method. Only the manual mark-up yielded satisfactory results that are more likely to be statistically significant (data not shown).
Inoue et al. recommend a luciferin dose of 75 mg/kg13. By using a dose of 150 mg/kg instead, the timing of imaging after the luciferin administration remains unchanged and we ensure that the plateau lasts throughout a long exposure acquisition. Luciferin must be the excess reactant throughout the acquisition process. Depending on the model, the region of interest can be adapted, as we showed in the SCL model where we drew two ROI per animal. In the SCL model, the treatment is injected when the tumor reaches 0.5 cm in its largest diameter to limit variability of engraftment. Depending on the mice, the tumor might have grown differently. To standardize and compare mice, we decided to use a ratio between treated and non-treated side that reveals the relative growth of both flanks tumors.
If no signal is observed from an injected mouse expected to be positive, either (i) the number of cells is very low and the signal is below the detection threshold; or (ii) the mouse lacks oxygen and requires immediate care.
Several of the quantitative bioluminescence analyses described by various authors require complex calculations and instruments (e.g., 3D bioluminescence tomography) to approach the absolute quantification of emitted bioluminescence photons5,15. There is no consensus on a method for quantifying bioluminescence reproducibly, especially in tumor models, with a 2D bioluminescence imager. Our aim was to standardize the image acquisition protocol to limit user-dependence.
The injection of CpG in situ after tumor inoculation reduced tumor burden in both models. The luminoscore method can serve as a tool to monitor tumor burden in other models of tumor immunotherapies. Monitoring tumor burden provides a noninvasive method that can improve our understanding of tumor growth and metastasizing mechanisms without interfering with the tumor microenvironment. The identification of animals that do and do not respond to treatment with this method is reinforced by the verification of successful tumor cell injection at the beginning of the experiment.
We here showed the adaptability of the luminoscore method in a SCL model using A20.IIA-luc2 cells. However, this method can be adapted to any other tumor model using other cell lines provided that they express luciferase, or in the context of T-cell studies (tumor specific cytotoxic T-cells, regulatory T-cells, etc.). The monitoring of in vivo gene transfer in the context of gene therapy of rare disease could also be done using the luminoscore method.
Finally the data shows that the bioluminescence-based luminoscore method enables comparisons between experiments, offers flexibility and adaptability to specific experimental needs, and is a useful tool for noninvasive longitudinal preclinical studies.

Early tumor cell detection remains a challenge and is crucial for enhancing cancer treatment efficacy. In vivo bioluminescence imaging (BLI) is a very sensitive, noninvasive optical technique, widely used for monitoring tumors in small animals. Firefly luciferase-expressing cells are commonly used for such experiments1,2. This oxygenase oxidizes D-luciferin with molecular oxygen but requires two cofactors - Mg2+ and adenosine triphosphate3. Firefly luciferase is more suitable for in vivo imaging than renilla luciferase4 because its quantum yield is higher.
The oxidized substrate - oxyluciferin - spontaneously emits a photon to return to its fundamental state and then becomes inactive. The emitted photons have a maximum wavelength around 530 nm. A high-sensitivity camera can detect the luminescent photons from the inside of a small animal and provide images that make it possible to locate tumor cells.
The ability to quantify tumor burden accurately by photon counts could serve as a powerful and sensitive tool for quantifying treatment efficacy. Because treatment effects can be detected sooner, this sensitivity might make it possible to determine the exact moment at which a treatment becomes effective.
Absolute quantification of total emitted photons is very complex. The number of photons gathered depends on the depth of the tumor and on the organs the photons are emitted through. Correction coefficients based on tissue absorption coefficients can be calculated5, but the absolute quantification of tumor cell numbers requires knowing the number of photons emitted by each tumor cell. Moreover, luciferase expression, like that of many reporter genes (e.g., fluorescent proteins) is not homogeneous, even in a cell population derived from a single clone6. The number of luciferase proteins in cells cannot be calculated exactly. The establishment of standardized experimental conditions thus appears crucial for a reliable semi-quantitative analysis.
We applied the luminoscore method to two different mouse lymphoma models7,8,9. In these models, syngeneic tumor cells are injected into the eye or under the skin to obtain, respectively, a primary intraocular lymphoma (PIOL) model and a subcutaneous lymphoma (SCL) model. In each of these orthotopic models, the treatment is administered in situ, seven days after the tumor inoculation for PIOL and when the tumor has reached 0.5 to 0.7 cm in its largest diameter for SCL.
We used the luminoscore method to monitor the effects of in&#160;situ CpG therapy, previously shown to be effective7,10,11. CpG is an oligonucleotide sequence and a ligand of TLR9, which in turn is an intracellular receptor expressed by numerous cells of the immune system, including dendritic cells, B lymphocytes, monocytes, and natural killer cells. CpG-DNA is a 20-mer DNA sequence that contains the CpG (CG) immunostimulatory motif; the control (ODN-control) is the same 20-mer DNA sequence, except that the immunostimulatory CG sequence is inverted (GC). TLR9 engagement in the murine lymphoma we are studying induces apoptosis10, activates the immune system12, and thereby significantly reduces tumor burden7,11.
Here we describe a standardized method for quantifying tumor burden and treatment response through bioluminescent images. This method relies on different aspects of the imaging procedure, from acquisition to analysis, to optimize reliability, reproducibility, non-user dependence, and statistical significance. A bioluminescence quantification index is attributed to each mouse; this value, which we call a luminoscore, can be compared not only between animals but also between experiments.
In this work, we focus on the bioluminescence imaging procedure as well as the image quantification by the luminoscore method. We show the effectiveness of this method for verifying the injection, monitoring tumor burden, and assessing the efficacy of in situ cancer treatment. Each of these points is illustrated in representative results from experiments using different mouse models to highlight the adaptability of the luminoscore method.

<table><tbody><tr><td>CpG 1826</td><td>Invivogen</td><td>Sequence: 5_-TCCATGACGTTCCTGACGTT&lt;br/&gt; Catalog number: tlrl-1826</td><td></td></tr><tr><td>ODN 1826 control</td><td>Invivogen</td><td>Sequence: 5_-TCCATGAGCTTCCTGAGCTT&lt;br/&gt; Catalog number: tlrl-1826c</td><td></td></tr><tr><td>D-luciferin potassium salt</td><td>Interchim</td><td>Catalog number: FP-M1224D</td><td></td></tr><tr><td>Ketamin</td><td>Virbac, France</td><td></td><td></td></tr><tr><td>Xylazin</td><td>Bayer Healthcare</td><td>Rompun 2%</td><td></td></tr><tr><td>A20.IIA-luc2 cell line</td><td></td><td>A20.IIA transfected with pGL4.50[luc2/CMV/hygro]&lt;sup&gt;7&lt;/sup&gt;&lt;br/&gt; (Promega E1310)</td><td></td></tr><tr><td>Mice</td><td>Balb/cByj</td><td>Six-weeks old females from Charles River</td><td></td></tr><tr><td>IVIS lumina Biolumienscence imager</td><td>Perkin Elmer</td><td></td><td></td></tr><tr><td>Living Image software</td><td>Perkin Elmer</td><td>Used for measuring photon flux on images and drawing ROIs</td><td></td></tr><tr><td>R software (opensource)</td><td>R-project</td><td>Used for statistic tests</td><td></td></tr><tr><td>Hamilton Precision Serynge 10 &amp;micro;l&amp;nbsp;</td><td>Hamilton</td><td>Product number 7642-01100</td><td></td></tr><tr><td>Eye ointment</td><td>Lacrinorm</td><td></td><td></td></tr><tr><td>Dissecting microscope</td><td>ZEISS</td><td>Stemi 305</td><td></td></tr></tbody></table>

bioluminescence-based tumor quantification method for monitoring tumor progression and treatment effects in mouse lymphoma models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

The Luminoscore Method can Serve to Verify the Tumor Cell Injection
In models involving the injection of a small number of tumor cells or when the injection site does not permit visual verification of the injection, it can be very difficult to ensure the quality of the injection. The luminoscore method serves as a fast and convenient tool to verify the quality of the procedure and ascertain easily and instantaneously that everything went right. In both models, the tumor is detectable as early as 10 min after the injection (Figure 1A). The images alone suffice to verify that tumor cells are present in the correct location. Nonetheless, quantifying the images provides an idea of the heterogeneity of the injection. The dot plot (Figure 1B) clearly shows a difference between animals that received (black dots) and did not receive (red line) injections. Interestingly, the signal obtained in the SCL model is 100 times higher than in the PIOL model; this finding is consistent with the number of cells injected (5 x 106 and 1 x 104 cells, respectively).
<img alt="Figure 1" src="/files/ftp_upload/53609/53609fig1.jpg" /> 
Figure 1.&#160;Verification of Injection in Different Mouse Lymphoma Models. The luminoscores measured 10 min after tumor cell inoculation in different lymphoma models. (A)&#160;Representative images of both models. (B) Luminoscore of each animal in the different models. The red line corresponds to the mean background noise measured on 10 animals before tumor cell inoculation; the dotted lines are the mean +/- standard deviation. For each model, all the animals can be distinguished from background noise. In the PIOL model, 1 x 104 cells were inoculated in the vitreous of the right eye, and in the SCL model, 5 x 106 cells subcutaneously into each flank of the mouse. <a href="https://www.jove.com/files/ftp_upload/53609/53609fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Monitoring Tumor Burden and Treatment Response
The luminoscore is also a powerful tool for studying tumor growth and treatment efficacy. Figure 2A shows representative images of the monitoring of tumor growth in the control and the CpG treated PIOL groups. The mice were inoculated with 1 x 104 tumor cells each, and treatment was administered in situ on Day 7. The images show that after a sufficient time (28 days), metastases began to appear in the control group. Although the primary tumor did not appear sensitive to treatment, fewer metastases were observed in the CpG-treated group (Table 1). The quantitative analysis of the tumor burden in each group (Figure 2B) shows that CpG slowed tumor development, producing a statistically significant difference between the treated and the control group after 28 days (non-parametric two-tailed Mann-Whitney test p = 0.0079). Nonetheless, the tumors still grew in the treated mice, and none of them survived (data not shown). This finding is consistent with previous reports: CpG has no significant effects on primary ocular tumors10. The significant effect that we have here between the ODN and the CpG group comes from the metastasis growth inhibition.
<img alt="Figure 2" src="/files/ftp_upload/53609/53609fig2.jpg" /> 
Figure 2.&#160;Monitoring Tumor Burden and Treatment Response. (A) Representative images of the two groups (ODN-control and CpG-treated) of PIOL-bearing mice. (B) Monitoring tumor burden with the luminoscore. As seen on the dot plot, the effect of CpG on the luminoscore is significant on day 28. In those two groups, this method made it possible to monitor tumor burden and to measure the slight but significant (p = 0.0079) effects of CpG-DNA, which could have not been detected by images alone. <a href="https://www.jove.com/files/ftp_upload/53609/53609fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Groups</td>
			<td>Mouse</td>
			<td>Presence of metastases</td>
			<td>Location</td>
			<td>Photon flux (ph/s)</td>
		</tr>
		<tr>
			<td rowspan="5">CpG</td>
			<td>1</td>
			<td>No</td>
			<td>X</td>
			<td>X</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Yes</td>
			<td>Eye-draining lymp node</td>
			<td>9.32E+05</td>
		</tr>
		<tr>
			<td>3</td>
			<td>No</td>
			<td>X</td>
			<td>X</td>
		</tr>
		<tr>
			<td>4</td>
			<td>No</td>
			<td>X</td>
			<td>X</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Yes</td>
			<td>Eye-draining lymp node</td>
			<td>2.21E+07</td>
		</tr>
		<tr>
			<td rowspan="5">ODN</td>
			<td>1</td>
			<td>Yes</td>
			<td>Eye-draining lymp node</td>
			<td>1.06E+07</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Yes</td>
			<td>Eye-draining lymp node</td>
			<td>7.25E+07</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Yes</td>
			<td>Eye-draining lymp node + contralateral</td>
			<td>7.64E+08</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Yes</td>
			<td>Eye-draining lymp node + contralateral</td>
			<td>1.74E+09</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Yes</td>
			<td>Eye-draining lymp node + contralateral</td>
			<td>9.76E+07</td>
		</tr>
	</tbody>
</table>
Table 1.&#160;The Luminoscore Method can be Adapted to Different Approaches
The luminoscore method is not limited to a single type of mouse model. Figure 3 shows that it can be adapted to different mouse models. In the SCL model, two tumors are grafted on each side of the mouse. The treatment is administered in situ to a single side on Day 0, and the contralateral tumor serves as its control (Figure 3A). Therefore, two ROI were drawn per mouse: one for each side (Figure 3C). The ratio between the luminoscore on the treated and control sides describes the relative course of each tumor. This ratio was set to 1 on Day 0, when the treatment was administered. We observed a significant decrease in this ratio 13 days after treatment administration (Figure 3D; non-parametric two-tailed Mann-Whitney test p = 0.004). This decrease reveals that the treated-side tumor has been resorbed, as shown on the representative bioluminescence images (Figure 3B).
<img alt="Figure 3" src="/files/ftp_upload/53609/53609fig3.jpg" /> 
Figure 3. Adaptation of the Luminoscore Method to a Model with Two Primary Tumor Sites. (A) Experimental design of an SCL assay. The mice were injected with two primary tumors in each flank. One side is injected with either CpG-DNA or ODN-control, and the other side with PBS, to serve as its control. (B) Representative images of CpG-treated and control mice at day 0 and day 13. The treatment was administered on day 0. Tumor growth was inhibited on the CpG-treated side of the mouse. (C) The manually marked up regions of interest per animal for two primary tumor sites. (D) The ratio between the luminoscores of the CpG-treated or ODN-control side and the PBS control side is an index that reflects the relative growth of each tumor within the same animal. This ratio was set to 1 on day 0. On day 13, the ratio of the CpG-treated group had decreased significantly (p = 0.004), revealing inhibition of tumor growth by in situ administration of CpG-DNA. <a href="https://www.jove.com/files/ftp_upload/53609/53609fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models at JoVE.com

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Bioluminescence imaging is a well-known tool for localizing tumors and metastases, but quantification of these images often requires complex calculations and particular instruments. We describe the easy-to-use luminoscore method, based on precise acquisition conditions, requiring no calculations, and enabling tumor burden and treatment response to be monitored in mouse models.

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. ...

bioluminescence-based-tumor-quantification-method-for-monitoring

Research

JoVE Journal

Medicine

20.2K Views.  Genethon.  Bioluminescentie beeldvorming is een bekend hulpmiddel voor het lokaliseren van tumoren en metastasen, maar kwantificering van deze beelden zijn vaak complexe berekeningen en bijzondere instrumenten. We beschrijven de easy-to-use luminoscore methode, gebaseerd op een nauwkeurige overname voorwaarden, vereist geen berekeningen, en het mogelijk maken tumorlast en de respons op de behandeling te kunnen houden in muismodellen. 

Video: Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

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 ...

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most commonly used procedure for human tumor xenograft establishment consisted of the collection of cells from culture flasks, followed by the subcutaneous injection of the collected cells in immunocompromised mice. Whereas this approach still sees frequent use in many laboratories, there has been a significant shift in emphasis over the past decade towards orthotopic xenograft establishment, which, in the instance of brain tumors, requires tumor cell injection into appropriate neuroanatomical structures. Because intracranial xenograft establishment eliminates the ability to monitor tumor growth through direct measurement, such as by use of calipers, the shift in emphasis towards orthotopic brain tumor xenograft models has necessitated the utilization of non-invasive imaging for assessing tumor burden in host animals. Of the currently available imaging methods, bioluminescence monitoring is generally considered to offer the best combination of sensitivity, expediency, and cost. Here, we will demonstrate procedures for orthotopic brain tumor establishment, and for monitoring tumor growth and response to treatment when testing experimental therapies.

Luciferase-modified human brain tumor xenografts can be established intracranially in athymic mice, with subsequent monitoring of tumor growth and response to therapy using bioluminescence imaging. In combination with survival analysis, bioluminescence monitoring is an essential research tool for pre-clinical testing of therapies being considered for treating brain tumors.

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most ...

Neuroscience

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

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 ...

Multimodal Bioluminescent and Positronic-emission Tomography/Computational Tomography Imaging of Multiple Myeloma Bone Marrow Xenografts in NOG Mice

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular interactions that exist within this microenvironment. Yet the BM microenvironment cannot be easily replicated in vitro, which potentially limits the physiologic relevance of many in vitro and ex vivo experimental models. These issues can be overcome by utilizing a xenograft model in which luciferase (LUC)-transfected 8226 MM cells will specifically engraft in the mouse skeleton. When these mice are given the appropriate substrate, D-luciferin, the effects of therapy on tumor growth and survival can be analyzed by measuring changes in the bioluminescent images (BLI) produced by the tumors in vivo. This BLI data combined with positronic-emission tomography/computational tomography (PET/CT) analysis using the metabolic marker 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) is used to monitor changes in tumor metabolism over time. These imaging platforms allow for multiple noninvasive measurements within the tumor/BM microenvironment.

Here we use bioluminescent, X-ray, and positron-emission tomography/computed tomography imaging to study how inhibiting mTOR activity impacts bone marrow-engrafted myeloma tumors in a xenograft model. This allows for physiologically relevant, non-invasive, and multimodal analyses of the anti-myeloma effect of therapies targeting bone marrow-engrafted myeloma tumors in vivo.

Multiple myeloma (MM) tumors engraft in the bone marrow (BM) and their survival and progression are dependent upon complex molecular and cellular ...

Cancer Research

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

Biology

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology. 
Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority of the practical and technical details are commonly omitted in published manuscripts which, therefore, makes it challenging to reproduce the models, particularly when it involves surgical techniques. Bioluminescence technology allows for the evaluation of small amounts of cancer cells even when a tumor is not palpable. Utilizing luciferase-expressing cancer cells, we establish a breast cancer orthotopic inoculation technique with a high tumorigenesis rate. Lung metastasis is assessed utilizing an ex vivo technique. We, then, establish a mastectomy model with a low local recurrence rate to assess the metastatic tumor burden. Herein, we describe, in detail, the surgical techniques of orthotopic implantation and mastectomy for breast cancer with a high tumorigenesis rate and low local recurrence rates, respectively, to improve breast cancer model efficiency.

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority ...

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

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