Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Summary

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Abstract

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

Protocol

1. Experimental Metastasis Mouse Model

1. On the day before the tumor cell injections, seed one T75 flask with up to 1 x 10⁷ CMS4-Met cells in 10 mL of RPMI medium containing 10% serum to obtain fast-growing tumor cells. Incubate overnight at 37°C.
2. On the day of the injection, remove the medium and rinse the cells once with PBS, then harvest the tumor cells with 0.05% trypsin-EDTA at 37°C for 5 minutes. Stop the reaction with 10 mL of RPMI medium containing 10% serum. Transfer cells to a conical tube.
3. Spin down the cells at 1300 rpm in a Sorvall Legend RT centrifuge for 3 minutes at room temperature. Remove the supernatant. R....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Solutions:
India Ink Solution (17):
Pour 150 ml of distilled water into a 250 ml flask. Add 4 drops ammonium hydroxide to the distilled water. Add 30 ml India Ink stock (i.e. Sanford Black Magic Waterproof Drawing Ink 4465 Item 44011) to the ammonia and water mixture. Top off with distilled water to a volume of 200 ml. Solution is ready for injection.
Fekete's Solution (17):
Fekete's solution is used to bleach India ink-inflated tumor-bearing lungs to distinguish white tumor nodules from the black background of normal tissues.
Add 350ml 95% EtOH to 1L glass bottle. Add 150ml distilled water Add 50ml formaldehyde Add 25ml glacial acidic acid