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

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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, w....

Results

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

Discussion

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.......

Materials

List of materials used in this article

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