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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 drugs^3,4. With the advent of immune checkpoint inhibitors, new hope for lung cancer patients has arisen, although until now, only 20–40% 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 genes^6,7,8. 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 parenchyma^{10,11,12,13,14,15,16,17,18,19,20}. 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.

Protokół

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 Kras^LSL-G12D:p53^fl/fl (KP) mice^7,18, 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

1. 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 μg/mL streptomycin (hereafter referred to as standard culture medium). Incubate the cell cultures at 37° C, 5% CO₂, and around 95% relative humidity.
2. 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.
3. 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's needs.
4. 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).
5. Keep the cells on ice until transplantation.

2. Orthotopic Transplantation via Intratracheal Delivery

1. 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).
2. 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.
3. Confirm the appropriate level of anesthesia by pedal reflex via firm toe pinching and apply ophthalmic ointment to the eyes.
4. 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.
5. Place a fiber optic cable in between the front legs to illuminate the chest (Figure 1B).
6. 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).
7. 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.
8. 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.
9. Warm up the cell suspension by holding the tube in hand and, subsequently, pipette 50 µL 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 µL of air to assure a consistent distribution within the lungs.
10. Gently remove the catheter, remove the mouse from the intubation platform, and put it on a heat pad until it recovers from the anesthesia.

3. Lung Preparation for Flow Cytometry

1. 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.
2. Soak the carcass in 70% ethanol and secure the mouse on a dissection board using tape.
3. 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.
4. 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.
5. 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).
6. Incubate the lung pieces 30 - 60 min at 37 °C and with constant shaking.
7. Transfer the lung cell suspension through a 70 µm 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.
8. Centrifuge the cells at 300 x g for 5 min at 4 °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.
9. Centrifuge the cells at 300 x g for 5 min at 4 °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.

Wyniki

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

Dyskusje

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 used^{26,27,28,29,30,31,32}. In the cancer field, researchers use the intratracheal (and intranasal) instillation of Cre-re...

Materiały

Name	Company	Catalog Number	Comments
mouse lung adenocarcinoma cell line			isolated in house
C57Bl/6 mice			F1 of the cross of the two backgrounds may be used (8-12 weeks)
129S mice
RPMI 1640 Medium	Life Technologies	11544446
Fetal Calf Serum	Life Technologies	11573397
Penicillin/Streptomycin Solution	Life Technologies	11548876
L-Glutamine	Life Technologies	11539876
Trypsin, 0.25% (1x) with EDTA	Life Technologies	11560626
UltraPure 0.5 M EDTA, pH 8.0	Thermo Fisher Scientific	15575020
Ketasol (100 mg/mL Ketamine)	Ogris Pharma	8-00173
Xylasol (20 mg/mL Xylazine)	Ogris Pharma	8-00178
BD Insyste (22 GA 1.00 IN)	BD	381223
Blunt forceps	Roboz	RS8260
Leica CLS150 LED	Leica	30250004	Fibre Light Illuminator
Student Iris Scissors	Fine Science Tools	91460-11
DNase I (RNase-Free)	New England Biolabs	M0303S
Collagenase Type I	Life Technologies	17100017
ACK Lysing Buffer	Lonza	10-548E
CD274 (PD-L1, B7-H1) Monoclonal Antibody (MIH5), PE-Cyanine7	eBioscience	25-5982-82
Rat IgG2a kappa Isotype Control, PE-Cyanine7	eBioscience	25-4321-82