Abstract
In the past 20 years, the interest for the tumor microenvironment (TME) has exponentially increased. Indeed, it is now commonly admitted that the TME plays a crucial role in cancer development, maintenance, immune escape and resistance to therapy. This stands true for hematological malignancies as well. A considerable amount of newly developed therapies are directed against the cancer-supporting TME instead of targeting tumor cells themselves. However, the TME is often not clearly defined. In addition, the unique phenotype of each tumor and the variability among patients limit the success of such therapies. Recently, our group took advantage of the mass cytometry technology to unveil the specific TME in the context of chronic lymphocytic leukemia (CLL) in mice. We found the enrichment of LAG3 and PD1, two immune checkpoints. We tested an antibody-based immunotherapy, targeting these two molecules. This combination of antibodies was successful in the treatment of murine CLL. In this methods article, we provide a detailed protocol for the staining of CLL TME cells aiming at their characterization using mass cytometry. We include panel design and validation, sample preparation and acquisition, machine set-up, quality control, and analysis. Additionally, we discuss different advantages and pitfalls of this technique.
Highlights
In chronic lymphocytic leukemia (CLL), the microenvironment is crucial
Mass cytometry was developed in the late 2000s/early 2010s in order to push forward the number of cellular parameters that could be analyzed in parallel compared to conventional Flow Cytometry (FC) analysis
Data were reanalyzed for the purpose of this methods article, cluster numbers and number of associated cells differs from our original report [2]
Summary
In chronic lymphocytic leukemia (CLL), the microenvironment is crucial. CLL cells need to interact with their neighboring cells for their survival and proliferation. The development of such therapies is limited due to the lack of knowledge in the composition of the LME and to the singularity of this environment for each cancer type It is composed of a high variety of stromal and immune cells, which often display phenotypes exclusive to the LME, with the appearance of populations unique to this very specific environment. Whereas mass cytometry technology allows the analysis of up to 50 parameters in parallel, it is still associated with a relatively high throughput compared to conventional FC. This was made possible by combining FC platform with mass spectrometry analysis. It must be noted that novel flow cytometers (e.g., spectral flow cytometers) can detect a similar amount of parameters
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