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, and transplanted into mice of the same background and sex. If cells were provided from other research groups and the exact background remains unknown, we recommend the use of the F1 generation of a cross between C57BL/6 and 129S mice as transplant recipients to guarantee maximal tolerance.
1. Cell Preparation
<ol>
	<li>Seed KP cells 24 h before transplantation at approximately 50% confluency in RPMI supplemented with 10% fetal calf serum (FCS), glutamine, and 100 U/mL penicillin and 100 &#956;g/mL streptomycin (hereafter referred to as standard culture medium). Incubate the cell cultures at 37&#176; C, 5% CO2, and around 95% relative humidity.</li>
	<li>On the next day, harvest cells using 1 mL of trypsin-EDTA (0.05% in phosphate-buffered saline [PBS]) for 5 min per 10 cm plate and, subsequently, resuspend detached cells with 9 mL of standard culture medium.</li>
	<li>Count the cells in a hemocytometer and transfer the number of cells needed for the experiments in a 50-mL conical centrifuge tube. 
	NOTE: We recommend transplanting between 2.5 x 105 and 1 x 106 KP cells per mouse, but this might be adapted based on the researcher&#39;s needs.</li>
	<li>Subsequently, centrifuge the cells for 5 min at 300 x g, aspirate the supernatant, and, using a pipette, resuspend the cells at a density of 2 x 107/mL (for the inhalation of 1 x 106 KP cells per mouse) in serum and antibiotic-free RPMI, supplemented with 0.01 M ethylenediaminetetraacetic acid (EDTA).</li>
	<li>Keep the cells on ice until transplantation.</li>
</ol>
2. Orthotopic Transplantation via Intratracheal Delivery
<ol>
	<li>Sedate a mouse (8 - 12 weeks of age) by a subcutaneous injection of a mixture of ketamine (100 mg/kg of body weight) and xylazine (10 mg/kg of body weight).</li>
	<li>While the anesthesia sets in, prepare the catheter for intubation. Therefore, blunt the needle of a catheter by simply cutting the end with scissors. Afterward, push the catheter completely over the end of the needle.</li>
	<li>Confirm the appropriate level of anesthesia by pedal reflex via firm toe pinching and apply ophthalmic ointment to the eyes.</li>
	<li>Fix the mouse on the intubation platform (Figure 1A) by hooking its upper incisors over a suture and confirm that the chest is vertical underneath the suture.</li>
	<li>Place a fiber optic cable in between the front legs to illuminate the chest (Figure 1B).</li>
	<li>Carefully open the mouth of the mouse and pull out the tongue using disinfected flat forceps. Look for the emission of white light to locate the larynx and visualize the epiglottis and arytenoid cartilages (Figure 1C).</li>
	<li>Once the opening of the trachea is clearly visible, gently slide the catheter into the trachea (Figure 1D). The length of the catheter to be inserted depends on the age and size of the animal, since it should not go below the bifurcation to guarantee an even distribution of the lung adenocarcinoma cells within the lung. Quickly remove the needle from the catheter.</li>
	<li>The proper placement of the catheter in the trachea is indicated by the white light shining through the catheter (Figure 1E). In order to confirm the placement of the catheter in the trachea, attach a 1-mL syringe containing water to the catheter. The water in the syringe will rapidly move up and down in accordance with the breathing (Figure 1F). 
	NOTE: This step can be omitted by experienced researchers.</li>
	<li>Warm up the cell suspension by holding the tube in hand and, subsequently, pipette 50 &#181;L of the suspension containing 1 x 106 cells (the number of cells may be variable) into the center of the catheter hub. The suspension will be aspirated immediately. Subsequently, attach a 1 mL syringe and dispense 300 &#181;L of air to assure a consistent distribution within the lungs.</li>
	<li>Gently remove the catheter, remove the mouse from the intubation platform, and put it on a heat pad until it recovers from the anesthesia.</li>
</ol>
3. Lung Preparation for Flow Cytometry
<ol>
	<li>At the desired experimental endpoint, sedate the mouse by a subcutaneous injection of a mixture of ketamine (100 mg/kg of body weight) and xylazine (10 mg/kg of body weight) and euthanize it by cervical dislocation.</li>
	<li>Soak the carcass in 70% ethanol and secure the mouse on a dissection board using tape.</li>
	<li>Make a ventral midline incision and gently invert the skin to expose the thoracic wall muscles and the abdominal organs. Puncture the diaphragm and cut the ribs with scissors to expose the thoracic cavity.</li>
	<li>Perfuse the lungs 3x with 6 - 8 mL of ice-cold PBS through the right ventricle using a 27 G needle after cutting a small opening in the left ventricle to allow blood to leave. The lungs should be cleared of blood and turn completely white.</li>
	<li>Take out the lungs and mince the lobes into small pieces using scissors. Transfer the lung pieces to a 2 mL microcentrifuge tube and incubate it in 1.5 mL of lung digestion buffer (RPMI, 5% FCS, 150 U/mL collagenase I, and 50 U/mL DNase I).</li>
	<li>Incubate the lung pieces 30 - 60 min at 37 &#176;C and with constant shaking.</li>
	<li>Transfer the lung cell suspension through a 70 &#181;m cell strainer into a 50 mL tube. Clear the strainer with the back of a sterile 10-mL syringe and rinse the strainer with 15 mL of PBS with 2% FCS.</li>
	<li>Centrifuge the cells at 300 x g for 5 min at 4 &#176;C and aspirate the supernatant. Resuspend the cells in 1 mL of ammonium-chloride-potassium (ACK) lysing buffer and incubate them for 5 min at room temperature for the lysis of residual erythrocytes.</li>
	<li>Centrifuge the cells at 300 x g for 5 min at 4 &#176;C and resuspend the cells in 1 mL of PBS with 2% FCS and proceed with the desired staining protocol for flow cytometry23. 
	NOTE: Alternatively, the cells may be resuspended in RPMI containing 30% FCS and 10% DMSO and frozen using a freezing container for later analysis.</li>
</ol>

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The authors have nothing to disclose.

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>
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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, ...

orthotopic-transplantation-syngeneic-lung-adenocarcinoma-cells-to

Research

JoVE Journal

Cancer Research

16.5K Views.  Medical University of Vienna. 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.

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

Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure

Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to overcome this challenge. Here, a 2-step dose-escalation method for establishing dual-resistance to an epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), gefitinib, and a MET-TKI, PHA665752, is described. This method is based on simple stepwise dose-escalation of inhibitors for inducing acquired resistance in cell lines. The alternate method for generating resistant sublines involves exposing the cells to high concentrations of the inhibitor in one step. The stepwise dose-escalation method has a higher possibility of successfully inducing acquired resistance than this method. Activating EGFR mutations are biomarkers of a response to treatment with EGFR-TKI, which is an applied first-line treatment for non-small cell lung cancers (NSCLC) that harbor these mutations. However, despite reports of effective responses, the use of EGFR-TKI is limited because tumors inevitably acquire resistance. The major mechanisms behind EGFR-TKI resistance include a secondary mutation at the gatekeeper site, T790M in exon 20 of EGFR, and a bypass signal of MET. Thus, a potential solution for this issue would be a combination of EGFR-TKI and MET-TKI. This combined treatment has been shown to be effective in an in vitro study model. Acquired gefitinib-resistance was established through MET-amplification by stepwise dose-escalation of gefitinib for 12 months, and a cell line named PC-9MET1000 was generated in a previous study. To further investigate the mechanisms of acquired MET-TKI and EGFR-TKI resistance, a MET-TKI, PHA665752, was administered to these cells with stepwise dose-escalation in the presence of gefitinib for 12 months. This protocol has also been successfully applied for a number of combination therapies to establish acquired resistance to other inhibitor molecules.

Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure

An in vitro method for establishing dual resistance to an EGFR-TKI and a MET-TKI in cancer cells is described. This method is useful for developing treatments for patients with EGFR-mutations, who exhibit disease progression despite EGFR-TKI treatment with MET-amplification. It can also be modified for inhibitors targeting other molecules.

Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to ...

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

A Syngeneic Pancreatic Cancer Mouse Model to Study the Effects of Irreversible Electroporation

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches including the promising immunotherapeutic strategies. Irreversible electroporation (IRE) is a non-thermal ablation technique that induces tumor cell death without destruction of adjacent collagenous structures, thus enabling the procedure to be performed in tumors very close to blood vessels. Unlike thermal ablation techniques, IRE results in gradual apoptotic cell death, along with immediate ablation induced necrosis, and is currently in clinical use for selected patients with locally advanced PC. An ablative, non-target specific procedure like IRE can induce a myriad of responses in the tumor microenvironment. A few studies have addressed the effects of IRE on tumor growth in other tumor types, but none have focused on PC. We have developed a syngeneic mouse model of PC in which subcutaneous (SQ) and orthotopic tumors can be successfully treated with IRE in a highly controlled setting, facilitating various longitudinal studies post procedure. This animal model serves as a robust system to study the effects of IRE and ways to improve the clinical efficacy of IRE.

Irreversible electroporation (IRE) is a non-thermal ablation technique used for the treatment of locally advanced pancreatic cancer. Being a relatively new technique, the effects of IRE on the tumor growth are poorly understood. We have developed a syngeneic mouse model that facilitates studying the effects of IRE on pancreatic cancer.

Pancreatic cancer (PC), a disease which kills approximately 40,000 patients each year in the US, has successfully evaded several therapeutic approaches ...

A Bioluminescent and Fluorescent Orthotopic Syngeneic Murine Model of Androgen-dependent and Castration-resistant Prostate Cancer

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and transgenic genetically engineered mouse models. Unlike subcutaneous tumors, orthotopic tumors contain more clinically accurate vasculature, tumor microenvironment, and responses to multiple therapies. In contrast to genetically engineered mouse models, orthotopic models can be performed with lower cost and in less time, involve the use of highly complex and heterogeneous mouse or human cancer cell lines, rather that single genetic alterations, and these cell lines can be genetically modified, such as to express imaging agents. Here, we present a protocol to surgically injecting a luciferase- and mCherry-expressing murine prostate cancer cell line into the anterior prostate lobe of mice. These mice developed orthotopic tumors that were non-invasively monitored in vivo and further analyzed for tumor volume, weight, mouse survival, and immune infiltration. Further, orthotopic tumor-bearing mice were surgically castrated, leading to immediate tumor regression and subsequent recurrence, representing castration-resistant prostate cancer. Although technical skill is required to carry out this procedure, this syngeneic orthotopic model of both androgen-dependent and castration-resistant prostate cancer is of great use to all investigators in the field.

The goal of this protocol is to demonstrate the intra-prostatic injection of prostate cancer cells, with subsequent castration. Orthotopic pre-clinical models of androgen-dependent and castration-resistant prostate cancer are critical to study the disease in the context of a clinically relevant tumor microenvironment and an immunocompetent host.

Orthotopic tumor modeling is a valuable tool for pre-clinical prostate cancer research, as it has multiple advantages over both subcutaneous and ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer (NSCLC). Testing for PD-L1 expression with the PD-L1 immunohistochemistry (IHC) 22C3 companion diagnostic assay, which gives a tumor proportion score (TPS), has been validated on tumor tissue. We developed an optimized laboratory-developed test (LDT) that uses the 22C3 antibody (Ab) concentrate on a widely available IHC autostainer for biopsy and cytology specimens. The PD-L1 TPS was evaluated with 120 paired whole-tumor tissue sections and biopsy samples and with 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30). The 22C3 Ab concentrate-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to PD-L1 IHC expression determined using the PD-L1 IHC 22C3 companion assay at both TPS cut points (&#8805;1%, &#8805;50%). The optimized LDT presented here, using the 22C3 Ab concentrate to determine the PD-L1 expression in both tumor tissue and in cytology specimens, will expand the ability of laboratories worldwide to assess the eligibility of patients with NSCLC for treatment with pembrolizumab monotherapy in a reliable and reproducible manner.

To expand the ability of laboratories worldwide to assess the eligibility of patients with lung cancer for treatment with pembrolizumab, in a reliable and reproducible manner, we developed an assay that uses the 22C3 antibody concentrate on a widely available immunohistochemical autostainer, for both biopsy and cytology specimens.

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone. To address this problem, the aim of the study is to refine a lesser-known model of OS in rats with a comprehensive histologic, imaging, biologic, implantation, and amputation surgical approach that prolongs survival. We used an immunocompetent, outbred Sprague-Dawley (SD), syngeneic rat model with implanted UMR106 OS cell line (originating from a SD rat) with orthotopic tibial tumor implants into 3-week-old male and female rats to model pediatric OS. We found that rats develop reproducible primary and metastatic pulmonary tumors, and that limb amputations at 3 weeks post implantation significantly reduce the incidence of pulmonary metastasis and prevent unexpected deaths. Histologically, the primary and metastatic OSs in rats were very similar to human OS. Using immunohistochemistry methods, the study shows that rat OS are infiltrated with macrophages and T cells. A protein expression survey of OS cells reveals that these tumors express ErbB family kinases. Since these kinases are also highly expressed in most human OSs, this rat model could be used to test ErbB pathway inhibitors for therapy.

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival ...