Name: Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model (Video) | JoVE Journal
Uploaded: 2010-11-26T15:03:00.000+00:00
Duration: 8 min 6 s
Description: Learn to assess tumor-specific T-cell adoptive transfer immunotherapy efficacy using a mouse lung metastasis model. This method enables quantitative visualization of tumor nodules via India ink staining.

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5 minutes

Supported by grants from the National Institutes of Health (CA133085 to K.L.) and the American Cancer Society (RSG-09-209-01-TBG to K.L.).

1. Experimental Metastasis Mouse Model
<ol>
<li>On the day before the tumor cell injections, seed one T75 flask with up to 1 x 107 CMS4-Met cells in 10 mL of RPMI medium containing 10% serum to obtain fast-growing tumor cells. Incubate overnight at 37°C.</li>
<li>On the day of the injection, remove the medium and rinse the cells once with PBS, then harvest the tumor cells with 0.05% trypsin-EDTA at 37°C for 5 minutes. Stop the reaction with 10 mL of RPMI medium containing 10% serum. Transfer cells to a conical tube.</li>
<li>Spin down the cells at 1300 rpm in a Sorvall Legend RT centrifuge for 3 minutes at room temperature. Remove the supernatant. Resuspend the cells in 10 mL fresh 1X HBSS to wash, then spin down again and resuspend in the same manner.</li>
<li>Count the tumor cells on a hemocytometer. Dilute the cells in HBSS, so that the cells required for each injection are resuspended in a total volume of 100 μL. </li>
<li>Warm the 6-8 week old BALB/c mice in a beaker immersed in warm water to dilate the tail vein. Place the mouse on a tail vein restrainer on the bench. Use a microsyringe to inject 100 μL of tumor cells into the lateral tail vein. Use aseptic technique to avoid infection. After injection, apply slight pressure to the injection site until bleeding has stopped.</li>
<li>Return the mouse to the cage, and allow the tumor to grow to the desired stage.</li>
</ol>
2. Cytotoxic T-Lymphocyte (CTL) Adoptive Transfer Immunotherapy
<ol>
<li>On the day of the transfer, pipette up and down to resuspend purified cytotoxic T-lymphocytes, or CTLs, prepared as described in the text. Transfer all of the cells from one plate to a 15 mL conical tube, not letting the volume exceed more than 2/3 of the tube.</li>
<li>Insert a sterile Pasteur pipette into the conical tube and lay Lymphocyte Separation Medium, or LSM, under the cells until the total volume nears 14 mL.</li>
<li>Be careful not to disturb the layers. Centrifuge at 2000 rpm for 20 minutes at room temperature. Do not use the brake to slow the rotor after spinning.</li>
<li>Transfer the CTLs to a new 15 mL conical tube. Add HBSS to a volume of approximately 10 mL to wash.</li>
<li>Count the cells. Spin down at 1300 rpm for 5 minutes at room temperature. Resuspend in HBSS to the required cell density for injections, keeping the total volume per injection at 100 μL.</li>
<li>Use the same tail injection technique as seen earlier in this video to inject the CTLs into tumor-bearing mice.</li>
<li>Return the mouse to the cage, and allow the CTLs to interact with the tumor. Mice are usually sacrificed for analysis 14-21 days after CTL treatment.</li>
</ol>
3. Visualization of Lung Metastases 
<ol>
<li>To visualize lung metastases, place a sacrificed mouse on its back on a Styrofoam board. Pin the legs to ensure unobstructed access to the trachea. Spray the ventral side with 70% ethanol.</li>
<li>Starting from the mid-abdomen, use scissors to cut along the midline, through the ribcage, and up toward the salivary glands. Expose the trachea. Use forceps to remove tissues surrounding the trachea.</li>
<li>Thread a 200 μL pipette tip underneath the trachea. Holding the tip with one hand, gently lift the trachea up and away from the body.</li>
<li>Rotate the platform 180°. Use a 50 mL syringe to inject India Ink into the lungs via the trachea. Completely inflate the lungs with ink until you feel a strong resistance.</li>
<li>Use scissors to cut the trachea. Slide a pair of forceps under the lungs and lift them out of the mouse. Rinse the lungs briefly in a 1L beaker of water.</li>
<li>In a chemical fume hood, transfer the lungs to a glass scintillation vial containing 5 mL of Fekete's solution. The tumor tissue will emerge as white nodules on the black lungs after a few minutes. The tumors can now be counted and stored in Fekete's solution indefinitely.</li>
</ol>
4. Representative Metastases in Visualized Lungs
<ol>
<li>Here, lungs from mice that were injected with mammary carcinoma cell line 4T1 show white spots indicating tumors. Mice that were injected with 4T1 cells transfected with an IRF8 dominant-negative mutant K79E show the enhanced metastatic potential of tumor cells.</li>
<li>These images show a histological analysis of efficacy of CTL adoptive transfer immunotherapy. Tumor-bearing mice injected with saline showed multiple tumors 6 days later, indicated by yellow arrows (A). Mice injected with tumor-specific T cells, on the other hand, showed a reduction of tumors (B).</li>
<li>In the same experiment, India ink treatment gives a simpler way to measure the efficacy of CTL adoptive transfer. Here, the white tumor nodules on tumor-bearing lungs clearly distinguish them from lungs that have successfully undergone CTL adoptive transfer, and counting the nodules allows for a degree of quantitation not possible with histology.</li>
</ol>
5. Representative Results
 <img src="/files/ftp_upload/2077/2077fig1.jpg" alt="Tumor metastasis mouse model diagram; tumor cell transplantation, CTL transfer, lung metastasis analysis." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. Experimental scheme for experimental tumor metastasis and CTL adaptive transfer immunotherapy mouse model. Red dots indicate tumor cells and green dots indicate CTLs.
 <img src="/files/ftp_upload/2077/2077fig2.jpg" alt="Tumor growth comparison in mouse lungs; experimental results; cancer research; 4T1 cell lines." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Disruption of IRF8 function enhanced the metastatic potential of tumor cells. Mouse mammary carcinoma cell line 4T1 was stably transfected with vector (4T1.Vector) or vector expressing an IRF8 dominant-negative mutant K79E (4T1.IRF8K79E) (15, 16). Tumor cells were injected i.v. into mouse lateral tail veins. Tumor-bearing lungs were inflated with India ink to visualize tumor nodules. Tumor nodules are easily seen as white spots on the black lung tissue background.
 <img src="/files/ftp_upload/2077/2077fig3.jpg" alt="Histology comparison, lung tissue, microscopic image, cellular structure analysis, H&amp;E staining." data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. Histological analysis of efficacy of CTL adoptive transfer immunotherapy. Mouse sarcoma cell line CMS4-Met was injected i.v. into mouse lateral tail veins. Three days later, saline (A) or tumor-specific T cells (B) were injected into the tumor-bearing mice. Lungs were analyzed six days after CTL treatment by conventional H&amp;E histological staining. Tumor nodules are indicated by arrows.
 <img src="/files/ftp_upload/2077/2077fig4.jpg" alt="Control vs CTL tumor growth comparison, experiment results, tumor size change analysis." data-altupdatedat="1788235205000" data-alt="Figure 4"> 
Figure 4. Visualization of tumor nodules by India ink inflation. Mouse sarcoma cell line CMS4-Met was injected i.v. into mouse lateral tail veins. Three days later, saline (Control) or tumor-specific T cells (+CTL) were injected into the tumor-bearing mice. Lungs were analyzed fourteen days after CTL treatment. The white spots, which are tumor nodules, allow for easy quantification of the efficacy of CTL treatment.

<ol><li>Ryan, M. H., Bristol, J. A., McDuffie, E., Abrams, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regression+of+extensive+pulmonary+metastases+in+mice+by+adoptive+transfer+of+antigen-specific+CD8%28%2B%29+CTL+reactive+against+tumor+cells+expressing+a+naturally+occurring+rejection+epitope%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Regression of extensive pulmonary metastases in mice by adoptive transfer of antigen-specific CD8(+) CTL reactive against tumor cells expressing a naturally occurring rejection epitope.</a> J Immunol. 167 (8), 4286-4292 (2001).</li>
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<li>Galon, J., Costes, A., Sanchez-Cabo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Type%2C+density%2C+and+location+of+immune+cells+within+human+colorectal+tumors+predict+clinical+outcome%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Type, density, and location of immune cells within human colorectal tumors predict clinical outcome.</a> Science. 313 (5795), 1960-194 (2006).</li>
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<li>Camus, M., Tosolini, M., Mlecnik, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coordination+of+intratumoral+immune+reaction+and+human+colorectal+cancer+recurrence%5BTitle%5D)+AND+%22Cancer+research%22%5BJournal%5D)">Coordination of intratumoral immune reaction and human colorectal cancer recurrence.</a> Cancer research. 69 (6), 2685-2693 (2009).</li>
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<li>Srivastava, M. K., Sinha, P., Clements, V. K., Rodriguez, P., Ostrand-Rosenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Myeloid-derived+suppressor+cells+inhibit+T-cell+activation+by+depleting+cystine+and+cysteine.+Cancer+research%5BTitle%5D)+AND+%22%22%5BJournal%5D)">Myeloid-derived suppressor cells inhibit T-cell activation by depleting cystine and cysteine. Cancer research.</a> 70 (1), 68-77 (2010).</li>
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<li>Heijstek, M. W., Kranenburg, O., Rinkes, B. orel, H, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+models+of+colorectal+cancer+and+liver+metastases%5BTitle%5D)+AND+%22Digestive+surgery%22%5BJournal%5D)">Mouse models of colorectal cancer and liver metastases.</a> Digestive surgery. 22 (1-2), 1-2 (2005).</li>
<li>Yang, D., Ud Din, N., Browning, D. D., Abrams, S. I., Liu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeting+lymphotoxin+beta+receptor+with+tumor-specific+T+lymphocytes+for+tumor+regression%5BTitle%5D)+AND+%22Clin+Cancer+Res%22%5BJournal%5D)">Targeting lymphotoxin beta receptor with tumor-specific T lymphocytes for tumor regression.</a> Clin Cancer Res. 13 (17), 5202-5210 (2007).</li>
<li>Yang, D., Thangaraju, M., Browning, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((IFN+Regulatory+Factor+8+Mediates+Apoptosis+in+Nonhemopoietic+Tumor+Cells+via+Regulation+of+Fas+Expression%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">IFN Regulatory Factor 8 Mediates Apoptosis in Nonhemopoietic Tumor Cells via Regulation of Fas Expression.</a> J Immunol. 179 (7), 4775-4782 (2007).</li>
<li>Yang, D., Thangaraju, M., Greeneltch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Repression+of+IFN+regulatory+factor+8+by+DNA+methylation+is+a+molecular+determinant+of+apoptotic+resistance+and+metastatic+phenotype+in+metastatic+tumor+cells%5BTitle%5D)+AND+%22Cancer+research%22%5BJournal%5D)">Repression of IFN regulatory factor 8 by DNA methylation is a molecular determinant of apoptotic resistance and metastatic phenotype in metastatic tumor cells.</a> Cancer research. 67 (7), 3301-3309 (2007).</li>
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Mice were purchased from the National Cancer Institute (Friderick, MD) and housed in the Medical College of Georgia animal facility. Experiments and care/welfare were in agreement with federal regulations and an approved protocol by the Medical College of Georgia IACUC committee.

<table><tbody><tr><td colspan="4">&lt;p class=&quot;jove_title&quot;&gt;Solutions:&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;India Ink Solution (17):&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;ol&gt; &lt;li&gt; Pour 150 ml of distilled water into a 250 ml flask.&lt;/li&gt; &lt;li&gt; Add 4 drops ammonium hydroxide to the distilled water.&lt;/li&gt; &lt;li&gt; Add 30 ml India Ink stock (i.e. Sanford Black Magic Waterproof Drawing Ink 4465 Item 44011) to the ammonia and water mixture.&lt;/li&gt; &lt;li&gt; Top off with distilled water to a volume of 200 ml. Solution is ready for injection.&lt;/li&gt; &lt;/ol&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_step&quot;&gt;Fekete&#039;s Solution (17):&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;Fekete&#039;s solution is used to bleach India ink-inflated tumor-bearing lungs to distinguish white tumor nodules from the black background of normal tissues.&lt;/p&gt;</td></tr><tr><td colspan="4">&lt;ol&gt; &lt;li&gt; Add 350ml 95% EtOH to 1L glass bottle.&lt;/li&gt; &lt;li&gt; Add 150ml distilled water&lt;/li&gt; &lt;li&gt; Add 50ml formaldehyde&lt;/li&gt; &lt;li&gt; Add 25ml glacial acidic acid&lt;/li&gt; &lt;/ol&gt;</td></tr></tbody></table>

experimental metastasis and ctl adoptive transfer immunotherapy mouse model

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

The overall goal of this procedure is to examine the efficacy of tumor specific T-cell adoptive transfer immunotherapy in vivo. This is accomplished first by transplanting tumor cells via lateral tail vein injection to the mice in order to establish lung metastases. The second step of the procedure is to treat the tumor bearing mice with tumor specific cytotoxic T lymphocytes CTLs.The final step is to use India ink as a means to visualize and quantify tumor nodules. Ultimately, results can be obtained that show the CIE of different treatments through measuring tumor size and quantifying the number of tumor nodules. Hi, my name is Mary Zimmerman and I'm a member of Dr.Kevin Lu's lab at the Medical College of Georgia.Hello, my name is Shelene Hu.I'm second year student and I'm also working Dr.Lou's lab. The main advantage of this technique over traditional methods such as survival curves is that this procedure is both qualitative and Quantitative. The implication of the techniques can extend toward cancer therapy because it mimics T-cell adoptive immunotherapy in human cancer patients.So let's get Started On the day before the tumor cell injections seed one T 75 flask with up to one times 10 to the seventh. CMS four met cells in 10 milliliters of RPMI medium containing 10%serum. To obtain fast-growing tumor cells incubate overnight at 37 degrees Celsius on the day of the injection.Remove the medium and rinse the cells once with PBS, then harvest the tumor cells with 0.05%trypsin EDTA at 37 degrees Celsius for five minutes. Stop the reaction with 10 milliliters of RPMI medium containing 10%serum. Transfer the cells to a conical tube.Spin down the cells at 1300 RPM in a val legend RT centrifuge for three minutes. At room temperature, remove the snat, resuspend the cells in 10 milliliters of fresh one XHB Ss to wash. Then spin down again and resuspend in the same manner.Count the tumor cells on a hemo.Cytometer. Dilute the cells in HBSS so that the cells required for each injection are resuspended. In a total volume of 100 microliters warm the six to eight week old B SEA mice in a beaker, immersed in warm water.To dilate the tail vein, place the mouse in a tail vein restrainer on the bench. Use a micros syringe to inject 100 microliters of tumor cells into the lateral tail vein. Use a septic technique to avoid infection after injection.Use slight pressure to the injection site until bleeding has stopped. Return the mouse to the cage and allow the tumor to grow to the desired stage. On the day of the transfer, pipette up and down to resuspend purified cytotoxic T lymphocytes or CTLs prepared.As described in the text, transfer all the cells from one plate to a 15 milliliter conical tube, not letting the volume exceed more than two thirds of the tube. Insert a sterile Pasteur pipette into the conical tube and layer lymphocyte separation. Medium or LSM under the cells until the total volume nears 14 milliliters.Be careful not to disturb the layers. Centrifuge a 2000 RPM for 20 minutes at room temperature. Do not use the brake to slow the rotor after spinning, transfer the CTLs to a new 15 milliliter conical tube.Add HBSS to a volume of approximately 10 milliliters. To wash. Count the cells spin down at 1300 RPM for five minutes At room temperature resuspend an HBSS to the required cell density for injections.Keeping the total volume per injection at 100 microliters. Use the same tail injection technique as seen earlier in this video. To inject the CTLs into tumor bearing mice, return the mouse to the cage and allow the CTLs to interact with the tumor.Mice are usually sacrificed for analysis 14 to 21 days after CTL treatment. To visualize lung metastases, place a sacrificed mouse on its back on a styrofoam board. Pin the legs to ensure unobstructed access to the trachea.Spray the ventral side with 70%ethanol starting from the mid abdomen. Use scissors to cut along the midline through the rib cage and up toward the salivary glands. Expose the trachea.Use forceps to remove tissues surrounding the trachea. Thread a 200 microliter pipette tip underneath the trachea holding the tip with one hand. Gently lift the trachea up and away from the body.Rotate the platform 180 degrees. Use a 50 milliliter syringe to inject India ink into the lungs via the trachea. Completely inflate the lungs with ink until you feel a strong resistance.Use scissors to cut the trachea. Slide a pair of forceps under the lungs and lift them out of the mouse. Rinse the lungs briefly in a one liter beaker of water in a chemical fume hood.Transfer the lungs to a glass instalation bile containing five milliliters of FTI solution. The tumor tissue will emerge as white nodules on the black lungs. After a few minutes, the tumors can now be counted and start in FTI solution indefinitely.Here.Lungs for mice transfected with mammary carcinoma. Cell line four T one show white spots indicating tumors. Mice transfected with an IRF eight dominant negative immune to K 79 E show the enhanced metastatic potential of tumor cells.These images show a histological analysis of efficacy of CTL adoptive transfer immunotherapy. Tumor bearing mice injected with saline, showed multiple tumors six days later indicated by arrows. Mice injected with tumor specific T cells, on the other hand, showed a reduction of tumors in the same experiment.India ink treatment gives a simpler way to measure the efficacy of CTL adoptive transfer. Here the white tumor nodules on tumor bearing lungs clearly distinguish them from lungs that have successfully undergone CTL adoptive transfer and counting the nodules allows for a degree of quantification not possible with histology. While attempting this procedure, it is important to remember to warm up the mice for easier and more accurate tail vein injections.After watching this video, you should have a good understanding of how to assess the efficacy of the treatment through this simple procedure to quantify tumor size and numbers. Thanks for watching and good luck with your experiments.

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Learn to assess tumor-specific T-cell adoptive transfer immunotherapy efficacy using a mouse lung metastasis model. This method enables quantitative visualization of tumor nodules via India ink staining.

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

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