Integrated functional and mass spectrometry-based flow cytometric phenotyping to describe the immune microenvironment...

A hallmark of the development of cancer is its ability to avoid detection and elimination by the immune system. There are many identified mechanisms of this immune evasion that can be measured both phenotypically and functionally. Functional studies directly show the ability of the tumor microenvironment to suppress immune responses, typically measured as lymphocyte proliferation, cytokine production or killing ability. While a direct measurement of function is ideal, these assays require ex vivo activation which may not accurately mimic in vivo conditions. Phenotypic assays can directly measure the distribution and activation of immune cell types rapidly after isolation, preserving the conditions present in the patient. While conventional flow cytometry is a rapid and well established assay, it currently allows for measurement of only 12-14 parameters. Mass spectrometry-based flow cytometry, or CyTOF, offers the ability to measure 3-fold more parameters than conventional optical-based modalities providing an advantage in depth of analysis that can be crucial for precious human samples. The goal of this report is to describe the system our group has developed to measure both the phenotype and function of immune cells in the bone marrow of patients with acute myeloid leukemia. We hope to explain our system in the context of previous studies aimed at measuring immune status in tumors and to inform the reader as to some experimental approaches our group has found useful in developing the basic data required to rationally pursue immune-based therapies for patients with cancer.