The authors would like to thank Safia Zahma for her help with the preparation of tissue sections.

All experimental protocols as outlined below follow ethical guidelines and were approved by the Austrian Federal Ministry of Science, Research and Economy.
NOTE: The protocol here describes an orthotopic transplantation model of mouse lung adenocarcinoma cells into syngeneic recipients. Cells may be isolated from tumor-bearing lungs of KrasLSL-G12D:p53fl/fl (KP) mice7,18, if available in-house, an...

<ol>
	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2018.">Cancer statistics, 2018.</a> CA: A Cancer Journal for Clinicians. 68 (1), 7-30 (2018).</li><li>Zappa, C., Mousa, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-small+cell+lung+cancer:+current+treatment+and+future+advances.">Non-small cell lung cancer: current treatment and future advances.</a> Translational Lung Cancer Research. 5 (3), 288-300 (2016).</li><li>Dolly, S. O., Collins, D. C., Sundar, R., Popat, S., Yap, T. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+Therapies+for+Lung+Cancer.">Targeted Therapies for Lung Cancer.</a> Cancer Treatment Research. 170, 165-182 (2016).</li><li>Brody, 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=PD-L1+expression+in+advanced+NSCLC:+Insights+into+risk+stratification+and+treatment+selection+from+a+systematic+literature+review.">PD-L1 expression in advanced NSCLC: Insights into risk stratification and treatment selection from a systematic literature review.</a> Lung Cancer. 112, 200-215 (2017).</li><li>Safari, R., Meuwissen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+use+of+advanced+mouse+models+for+lung+cancer.">Practical use of advanced mouse models for lung cancer.</a> Methods in Molecular Biology. 1267, 93-124 (2015).</li><li>DuPage, M., Dooley, A. L., Jacks, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+mouse+lung+cancer+models+using+adenoviral+or+lentiviral+delivery+of+Cre+recombinase.">Conditional mouse lung cancer models using adenoviral or lentiviral delivery of Cre recombinase.</a> Nature Protocols. 4 (7), 1064-1072 (2009).</li><li>Kwon, M. 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H., 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=Orthotopic+reconstitution+of+human+small-cell+lung+carcinoma+after+intravenous+transplantation+in+SCID+mice.">Orthotopic reconstitution of human small-cell lung carcinoma after intravenous transplantation in SCID mice.</a> Anticancer Research. 12 (5), 1407-1410 (1992).</li><li>Li, 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=A+novel+bioluminescence+orthotopic+mouse+model+for+advanced+lung+cancer.">A novel bioluminescence orthotopic mouse model for advanced lung cancer.</a> Radiation Research. 176 (4), 486-493 (2011).</li><li>Mase, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrabronchial+orthotopic+propagation+of+human+lung+adenocarcinoma--characterizations+of+tumorigenicity,+invasion+and+metastasis.">Intrabronchial orthotopic propagation of human lung adenocarcinoma--characterizations of tumorigenicity, invasion and metastasis.</a> Lung Cancer. 36 (3), 271-276 (2002).</li><li>McLemore, T. L., 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=Novel+intrapulmonary+model+for+orthotopic+propagation+of+human+lung+cancers+in+athymic+nude+mice.">Novel intrapulmonary model for orthotopic propagation of human lung cancers in athymic nude mice.</a> Cancer Research. 47 (19), 5132-5140 (1987).</li><li>Tsai, L. H., 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=The+MZF1/c-MYC+axis+mediates+lung+adenocarcinoma+progression+caused+by+wild-type+lkb1+loss.">The MZF1/c-MYC axis mediates lung adenocarcinoma progression caused by wild-type lkb1 loss.</a> Oncogene. 34 (13), 1641-1649 (2015).</li><li>Winslow, M. 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To study lung physiologic and pathologic events in the lung, invasive and non-invasive intratracheal intubation methods for the instillation of various reagents are widely used26,27,28,29,30,31,32. In the cancer field, researchers use the intratracheal (and intranasal) instillation of Cre-re...

Lung cancer is still by far the biggest cancer-related killer in both men and women1. Indeed, according to the American Cancer Society, every year more people die of lung cancer than of breast, prostate, and colon cancer together1. Until recently, the majority of patients suffering from non-small cell lung cancer (NSCLC), which is the most abundant subtype of lung cancer, were treated with platinum-based chemotherapy in a first-line setting, mostly with the addition of angiogenesis inhibitors2. Only a subset of patients harbors oncogenic mutations in the epidermal growth factor receptor (EGFR), in anaplastic lymphoma kinase (ALK), or in ROS1, and can be treated with available targeting drugs3,4. With the advent of immune checkpoint inhibitors, new hope for lung cancer patients has arisen, although until now, only 20&#8211;40% of patients respond to immune therapy5. Hence, further research is required to improve this outcome by fine-tuning immune checkpoint therapy and investigating combinatory treatment options.
To study lung cancer, a vast array of preclinical models are available, including spontaneous models triggered by chemicals and carcinogens and genetically engineered mouse models (GEMM) where autochthonous tumors arise following the conditional activation of oncogenes and/or the inactivation of tumor suppressor genes6,7,8. These models are of particular value to investigate fundamental processes in lung tumor development, but they also require extensive mice breeding, and experiments are time-consuming. Therefore, many studies evaluating potential inhibitors take advantage of subcutaneous (patient-derived) xenograft models where human lung cancer cell lines are subcutaneously injected into immunodeficient mice9.
In these models, the micromilieu of tumors is not represented accordingly; hence, researchers also use orthotopic transplantation models, where tumor cells are injected intravenously, intrabronchially, or directly into the lung parenchyma10,11,12,13,14,15,16,17,18,19,20. Some of these methods are technically challenging, difficult to be reproduced, and require intensive training of the researchers.21 Here we adapted a non-invasive orthotopic, intratracheal transplantation method in immunocompetent mice, where tumors develop within 3-5 weeks and exhibit significant similarities to human tumors, to induce the expression of the T-cell suppressor Programmed death-ligand 1 (PD-L1) on tumor cells.11,12,20 The use of mouse tumor cells derived from GEMM models and syngeneic recipient mice allows proper studying of the tumor microenvironment including immune cells. Furthermore, gene editing tools like CRISPR/Cas9 technology22 can be used in vitro before transplantation which facilitates the investigation of the impact of genetic factors in lung tumorigenesis.

<table border="0" cellpadding="0" cellspacing="0" style="width:663px;" width="664">
	<tbody>
		<tr height="23">
			<td height="23" style="height:23px;width:255px;">mouse lung adenocarcinoma cell line</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">isolated in house</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:255px;">C57Bl/6 mice</td>
			<td rowspan="2" style="width:123px;"></td>
			<td rowspan="2" style="width:119px;"></td>
			<td rowspan="2" style="width:167px;">F1 of the cross of the two backgrounds may be used (8-12 weeks)</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:255px;">129S mice</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640 Medium</td>
			<td>Life Technologies</td>
			<td>11544446</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Fetal Calf Serum</td>
			<td>Life Technologies</td>
			<td>11573397</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicillin/Streptomycin Solution</td>
			<td>Life Technologies</td>
			<td>11548876</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-Glutamine</td>
			<td>Life Technologies</td>
			<td>11539876</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin, 0.25% (1X) with EDTA</td>
			<td>Life Technologies</td>
			<td>11560626</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">UltraPure 0.5M EDTA, pH 8.0</td>
			<td style="width:123px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15575020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ketasol (100 mg/ml Ketamine)</td>
			<td style="width:123px;">Ogris Pharma</td>
			<td style="width:119px;">8-00173</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Xylasol (20 mg/ml Xylazine)</td>
			<td style="width:123px;">Ogris Pharma</td>
			<td style="width:119px;">8-00178</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">BD Insyste (22GA 1.00 IN)</td>
			<td style="width:123px;">BD</td>
			<td style="width:119px;">381223</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Blunt forceps</td>
			<td style="width:123px;">Roboz</td>
			<td style="width:119px;">RS8260</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Leica CLS150 LED</td>
			<td style="width:123px;">Leica</td>
			<td style="width:119px;">30250004</td>
			<td>Fibre Light Illuminator</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Student Iris Scissors</td>
			<td style="width:123px;">Fine Science Tools</td>
			<td style="width:119px;">91460-11</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNase I (RNase-Free)</td>
			<td>New England Biolabs</td>
			<td>M0303S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">Collagenase Type I</td>
			<td>Life Technologies</td>
			<td>17100017</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:255px;">ACK Lysing Buffer</td>
			<td style="width:123px;">Lonza</td>
			<td>10-548E</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">CD274 (PD-L1, B7-H1) Monoclonal Antibody (MIH5), PE-Cyanine7</td>
			<td style="width:123px;">eBioscience</td>
			<td style="width:119px;">25-5982-82</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:255px;">Rat IgG2a kappa Isotype Control, PE-Cyanine7</td>
			<td style="width:123px;">eBioscience</td>
			<td style="width:119px;">25-4321-82</td>
			<td></td>
		</tr>
	</tbody>
</table>

orthotopic transplantation of syngeneic lung adenocarcinoma cells to study pd-l1 expression

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.

We used the orthotopic transplantation model via intratracheal tumor cell delivery to test whether the tumor microenvironment stimulates PD-L1 expression. Therefore, we isolated mouse lung AC cells from the autochthonous KP model (KP cells), 10 weeks following tumor induction via Cre-recombinase-expressing adenovirus (Ad.Cre) delivery24. Subsequently, we labeled the lung AC cells using a green fluorescent protein (GFP)-expressing lentivirus25</sup...

Watch this Scientific Journal Video about Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression at JoVE.com

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

Here we describe a minimally invasive syngeneic orthotopic transplantation model of mouse lung adenocarcinoma cells as a time- and cost-reducing model to study non-small cell lung cancer.

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, ...

The orthotopic transplantation of mouse lung adenocarcinoma cells into syngeneic graft recipients allows the study of tumorigenesis in the presence of a fully active immune system under physiological conditions. Of course, the tumors develop into an immunocompetence mice and this is much more physiological that if you perform a transplant in immunodeficient mice. Gene editing of the tumor cells before a transplantation makes this model a direct and time-saving approach for studying the impact of genetic factors on tumor growth and gene expression profiles.Compared to inducible autochthonous lung tumor models, this method avoids the extensive breeding of mice, and therefore represents a refinement of previous lung cancer mouse models. Before beginning the procedure, use scissors to remove the end of a catheter needle and push the catheter completely over the end of the needle. After confirming a lack of response to toe pinch, apply ointment to the eyes of an anesthetized, synergetic eight to 12 week old mouse and hook the upper incisors over a suture string across an intubation platform with the chest perpendicular to the suture.Place a fiber optic cable between the front limbs to illuminate the chest and carefully open the mouth using disinfected flat forceps to extract the tongue. Look for the emission of white light to locate the larynx, epiglottis, and arytenoid cartilages. Once the opening of the trachea is clearly visible, gently slide the catheter into the trachea, up to but not beyond the bifurcation, to guarantee an even distribution of the lung adenocarcinoma cells within the lungs.Take care that the insertion of the catheter is very smooth. If you feel any resistance, remove the catheter and expose the trachea again to avoid injuring the mouse. Quickly remove the needle from the catheter and check whether the white light can be observed shining through the catheter.To confirm a proper placement of the catheter within the trachea, attach a one milliliter syringe of water to the catheter. The water in the syringe should move rapidly up and down in time with the breathing. Before delivering the cell suspension, warm the cells by hand and load 50 microliters of cells into the catheter.As soon as the suspension is aspirated, attach an empty one milliliter syringe to the catheter and dispense 300 microliters of air into the catheter to ensure a complete distribution of the tumor cells within the lungs. Then gently remove the catheter and place the mouse on a heat pad with monitoring until full recumbency. After the appropriate experimental endpoint, soak the carcass in 70%ethanol and secure the animal to a dissection board.Make a ventral midline incision and gently invert the skin to expose the thoracic wall muscles and the abdominal organs. Use scissors to puncture the diaphragm and to cut the ribs, exposing the thoracic cavity. Cut a small opening in the left ventricle and use a 27 gauge needle to perfuse the lung three times through the right ventricle with six to eight milliliters of ice cold PVS per infusion.After the last perfusion, the lungs should be completely clear of blood and appear white. After harvesting, use scissors to mince the lung tissue into small pieces. Transfer the lung fragments into a two milliliter microcentrifuge tube containing 1.5 milliliters of lung digestion buffer for a 30 to 60 minute incubation at 37 degrees Celsius, with shaking.At the end of the digestion, transfer the cell suspension through a 70 micron cell strainer into a 50 milliliter tube and use the end of a sterile 10-milliliter syringe plunger to press any remaining tissue fragments through the filter. Rinse the strainer with 15 milliliters of PVS, supplemented with 2%FCS. And collect the cells by centrifugation.Re-suspend the pellet in one milliliter of ammonium chloride potassium lysis buffer for a five minute incubation at room temperature and centrifuge the cells again. Then re-suspend the white blood cell pellet in one milliliter of PVS supplemented with 2%FCS and stain the cells for flow cytometric analysis according to the experimental protocol. As expected, the survival of the recipient mice is correlated to the number of engrafted cells.And the lungs harvested at the time of death demonstrate an even distribution of tumor nodules throughout all of the lobes. When comparing sections from lungs harboring autochthonous tumors, with tumors following orthotopically transplanted lung tumor cells, no morphological differences are observed. Flow cytometric analysis of PD-L1 expression of lung cells isolated three weeks after orthotopic tumor cell transplantation into immunocompetent mice, as just demonstrated, reveals up-regulation in PD-L1 cell surface marker expression, compared to in-vitro cultured lung tumor cells.Following orthotopic transplantation, other analytical methods can be applied, including immunohistochemical analysis, and mRNA profiling to address additional experimental questions.

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JoVE Journal

Cancer Research

16.3K vistas.  Medical University of Vienna. El trasplante ortotópico de células de adenocarcinoma pulmonar de ratón en receptores de injertos singéneos permite el estudio de la tumorigenesis en presencia de un sistema inmunitario completamente activo en condiciones fisiológicas. Por supuesto, los tumores se convierten en ratones de inmunocompetencia y esto es mucho más fisiológico que si realizas un trasplante en ratones inmunodeficientes. La edición génica de las células tumorales antes de un trasplante hace de este modelo u...