Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

Chris J. Weir; Amanda L. Hudson; Lyndsay Peters; Viive M. Howell

doi:10.3791/53019

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Method Article

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

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DOI:

10.3791/53019

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October 2nd, 2015

Chris J. Weir¹ , Amanda L. Hudson¹ , Lyndsay Peters² , Viive M. Howell¹

¹Bill Walsh Translational Cancer Research Laboratory, Kolling Institute of Medical Research, Royal North Shore Hospital, University of Sydney, ²Northern Blood Research Laboratory, Kolling Institute of Medical Research, Royal North Shore Hospital, University of Sydney

Summary

The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 µl blood sample is also described.

Abstract

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 µl whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.

Introduction

Malignant mesothelioma (MM) is an aggressive malignancy which arises from transformed cells in the membrane (mesothelium) that lines the lung and abdominal cavities, heart and internal reproductive organs, and is the most common primary tumor of the lung cavity or pleura^1,2. Exposure to asbestos fibres accounts for 80% of all MM, and while bans on asbestos use were introduced decades ago in most western countries, its widespread use in the community has left a deadly legacy. The World Health Organization has estimated that 107,000 people worldwide die each year from asbestos related diseases, with mortality rates continuing to increase. A new non-occupation....