Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

Jared M. Newton; Aurelie Hanoteau; Andrew G. Sikora

doi:10.3791/57685

A subscription to JoVE is required to view this content. Sign in or start your free trial.

Method Article

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

10K views

⸱

DOI:

10.3791/57685

⸱

June 7th, 2018

Jared M. Newton^*¹^,² , Aurelie Hanoteau^*¹ , Andrew G. Sikora¹

¹Department of Otolaryngology-Head and Neck Surgery, Baylor College of Medicine, ²Interdepartmental Graduate Program in Translational Biology and Molecular Medicine, Baylor College of Medicine

^* These authors contributed equally

Summary

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

Abstract

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Introduction

The suppressive TIME has recently been recognized as a hallmark of cancer, and is known to play a significant role in the development, progression, and protection of solid tumor cancers as well as mitigate their susceptibility to immunotherapy¹. The TIME is composed of numerous cellular subsets and phenotypes, all of which provide critical insight into the immunologic state of the tumor. These immunologic subsets can be further stratified into cells of lymphocyte or myeloid origin, which together constitute the majority of the innate and adaptive immune responses²^,³. Recent advances in ....