Authors: Sarah Reppert, Katerina Andreev, Sandra Wittmann & Susetta Finotto
Lung cancer is one of the most frequently occurring cancer types. Successful lung cancer therapies in patients require preliminary investigations of promising therapeutic reagents in animal models. This protocol describes a method to induce lung tumours in mice and to deliver an immunoregulatory molecule directly to the lung by intranasal application. Here we describe the usage of murine cell lines L1C2 and B16F10 for the induction of lung adenocarcinoma or metastatic melanoma respectively. In this model the tumour cells are injected intravenously in the tail vein of the mice. To apply therapeutic reagents locally in the lung during tumour growth, the mice are anaesthetized and the therapeutic molecule is applied drop by drop into the nose of the mouse at different time points after tumour induction. To monitor the outcome of the therapy tumour size can be defined by analyzing Haematoxilin/Eosin stained slices of the lung.
Advancements in novel therapies for lung cancer in patients require an understanding and monitoring of the immune response mechanisms in humans as well as improvements of the experimental model of the disease. To investigate the immune responses in lung cancer, mouse models are a helpful tool. In our laboratory we standardized a method to induce lung cancer and to apply therapeutic molecules to the lungs of mice. The technique of lung tumour induction facilitates the analysis of the effects of different therapeutical molecules in the anti-tumour immune response. Here we show two models of lung tumours, the B16-F10 metastatic melanoma model and the L1C2 adenocarcinoma model. Previous studies have been performed in our group both in wild type and in knockout mice in the melanoma model (1) as well as in the L1C2 adenocarcinoma model (2). Tumour development can be analyzed at different stages after intravenous tumour cell injection. It can be evaluated by quantifying the lung areas which are invaded with tumour cells on the lung surface as well as by analyzing lung sections stained with Haematoxilin/Eosin. Our laboratory showed that intranasal application of particular neutralizing antibodies as well as other biological active molecules ensures a localized effect in the lung (3-5). Using this protocol we repeatedly achieved a reduction of lung tumour growth in the L1C2 adenocarcinoma mouse model after intranasal application of a neutralizing anti-IL17A antibody (6). In contrast to other methods like intraperitoneal or intravenous injection of therapeutical molecules, the intranasal protocol provides the possibility to deliver the therapy directly to the affected lung.
Tumour cell induction
Tumour cell induction
For induction of L1C2 adenocarcinoma we recommend the compatible Balbc/J mice, for the induction of B16-F10 melanoma C57BL/6 mice are required. We suggest using 2×10e5 tumour cells per mouse for intravenous injection in the tail vein. The experimental procedure takes approximately 21 days. In case of using other mouse strains than wild type mice, the number of cells for injection and the time duration of the experiment needs to be tested and established first. Intranasal application of antibodies can be performed at different time points as well as in different dosages after tumour cell injection. The amount of antibody depends on the amount of protein you intend to neutralize in the lung. The data sheet of the antibody or previous publications might give a hint. As a possible control animals can be treated with PBS or the corresponding isotype antibody instead of the specific blocking antibody. We suggest the application of the antibody at two time points after tumour cell injection (Figure 1). To analyze the tumour growth, lung tissue can be embedded in paraffin cut into 4 micrometers thick sections which will be then stained with Hemtaoxilin/Eosin (H&E) in accordance to standardized histological laboratory protocols. Afterwards the tumour-bearing area can be analyzed. For this purpose, stained sections can be displayed on a computer monitor with a computer linked Nikon Coolscan V ED scanner using the Program SF launcher thereby determining the ratio of the area of the lung section occupied by tumour to the lung section tumour-free area. To classify the tumour type the affected areas the slide must be further analyzed with a light microscope (e.g. Zeiss Axio Observer.D1; Axio Vision 4.7 software) using different magnifications.
Prepare the cell culture media DMEM +10% FCS, +1% penicillin/streptomycin for B16F10 cells and RPMI +10% FCS, +1% penicillin/streptomycin for L1C2 cells and store it at 4°C.
Ketamin can be stored at 4°C for a few weeks. Avertin can be stored at 4°C for a few days.
CAUTION! All experiments should be performed according to national and institutional guidelines for animal care and use.
1 Prepare the antibody suspension as described above and store it on ice. - Anaesthetize the mice. This step can be performed using options A, B or C. - A) inject 200µl avertin intraperitonally (i.p) into the mice or - B) inject 60-70µl ketamin i.p or - C) use the isoflurane system - c1. prepare the isoflurane-equipment according to the manufacturer´s instructions - c2. put the mice in the plastic chamber and anaesthetize the mice by flooding the box with isolflurane/oxygen CAUTION! Increase the doses of isoflurane slowly - By the time the mouse is immobilized and the heart beat is getting slower, clamp the mouse in the neck and tilt the head back. Pipet the solution carefully, drop-wise into the nose of the mouse, so that the mouse inhales the solution. - Wait until the mouse wakes up. After Avertin and Ketamin treatment the mouse needs approximately 30min to get awake. Anaesthesia duration after isoflurane treatment is about 5min. Use an infrared lamp to warm the mouse until it is awake. - CAUTION! If you use ketamin or avertin for anaesthesia wait 24h to anaesthetize the mice again. Repeated use might be harmful for the mouse. - TIMING: 10-15min.
Tumour cells (Figure 2 a, b) which are intravenously injected in the mouse tail vein (Figure 2d) reach the lung and begin to grow. They form colonies and expand. The lung surface is afflicted by tumour cell colonies which can differ in size and number. In case of the B16-F10 metastatic melanoma model the cell colonies can be recognized as black areas on the lung surface (Figure 3a). Usually the whole lung surface is affected at day 28-33 after tumour cell injection with B16-F10 cells causing the death of the mice (1). In case of the L1C2 adenocarcinoma model 10-20% of the lung area is occupied by the tumour 21 days after tumour cell injection. In addition to the tumour in the lung, L1C2-treated mice often develop metastases in the thorax. On H&E stained slices the growing colonizing tumour cells are observable as high dense, purple, round regions (Figure 3b, c)
Intranasal application of antibodies allows the evaluation of new experimental therapeutical strategies for the local treatment of lung diseases such as lung cancer. The antibody can directly target cells or proteins in the lung, without covering long distances as after intraperitoneal or intravenous applications. The antibody reaches the nasal cavity as the mouse inhales the solution drop-wise (Figure 4). Using the isoflurane system anaesthesia only lasts for few minutes.
We thank Professor Rainer Wiewrodt for providing us with the L1C2 cell line.
Table 1: Troubleshooting tumor cell injection
Table 2: intranasal application
Figure 1: Experimental design for tumor cell injection.
Injection of the tumour cell line on day 0 and application of the antibody at different time points after tumour cell induction. CAUTION! All experiments should be performed according to national and institutional guidelines for animal care and use.
Figure 2: Tumour cell injection procedure
(a+b) Microscopic picture of the L1C2 adenocarcinoma (a) and B16F10 melanoma (b) cell line in culture at 400 magnifications. (c) Neubauer chamber for cell counting. (d) Injection of the tumour cells into the tail vein of the mouse. (e) Bulge in the tail (arrow) indicates that tumour cells were injected in the tail tissue not in the vein. CAUTION! All experiments should be performed according to national and institutional guidelines for animal care and use.
Figure 3: Tumour growth analysis
(a) Lungs of naïve (left) and B16-F10 melanoma bearing (right) C57BL/6 mice injected with 2105 tumour cells. (b) Analysis of Hematoxilin/Eosin stained lung sections of C57-BL/6 B16-F10 melanoma-bearing mice at day 14 after injection of 2×10e5 cells. (c) Hematoxilin/Eosin stained histological lung sections of BALBc/J L1C2 adenocarcinoma-bearing mice analyzed at day 21 after injection of 2×10e5 tumour cells. Arrows indicate the tumour area. CAUTION! All experiments should be performed according to national and institutional guidelines for animal care and use*.
Figure 4: Intranasal application
The mouse was anesthezised and held tightly in the neck. Then the antibody solution was pipetted dropwise on the nose of the mouse.
Sarah Reppert, Katerina Andreev, Sandra Wittmann & Susetta Finotto, Molekulare Pneumologie, AG Finotto, Universitätsklinikum Erlangen
Correspondence to: Susetta Finotto ([email protected])
Source: Protocol Exchange (2012) doi:10.1038/protex.2012.037. Originally published online 18 July 2012.