Method for the Analysis of the Tumor Microenvironment by Mass Cytometry: Application to Chronic Lymphocytic Leukemia

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 antibodybased 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 setup , quality control, and analysis. Additionally, we discuss different advantages and pitfalls of this technique.

Advanced Flow Cytometry Analysis Algorithms for Optimizing the Detection of “Different From Normal” Immunophenotypes in Acute Myeloid Blasts

“…According to previously published data ( Byford et al, 2018 ; Kimball et al, 2018 ; Gonder et al, 2020 ), viSNE was used as a non-linear technique to reduce dimensionality, but also to identify the phenotypically related cell cluster as assigned by the parameters tSNE1 and tSNE2.…”

The Tumor Microenvironment-Dependent Transcription Factors AHR and HIF-1α Are Dispensable for Leukemogenesis in the Eµ-TCL1 Mouse Model of Chronic Lymphocytic Leukemia

“…Control C57BL/6 and diseased Eµ-TCL1, or Eµ-TCL1 CD19 Cre/WT Hif1α fl/fl , Eµ-TCL1 CD19 Cre/WT Ahr fl/fl , Eµ-TCL1 CD19 Cre/WT Hif1a fl/fl Ahr fl/fl , and the corresponding control mice Eµ-TCL1 CD19 Cre/WT Hif1α WT/WT Ahr WT/WT were euthanized at humane endpoint by CO 2 inhalation or cervical dislocation. Spleens were collected, and single cell suspensions were prepared as previously described [29,30]. CD19+ B cells or CD5+CD19+ CLL cells were sorted with a BD FACSAria III at 4 • C. Then, 1-5 × 10 6 sorted cells were centrifuged and resuspended in 500 µL of Nucleozol reagent.…”

CyTOF® for the Masses

“…Therefore, starting with 800,000 - 1 million cells per sample is advisable. Typically, only 50-70% of the sample can be recovered in the data; the remainder is loss due to aggregation on the walls of the spray chamber and injector ( 28 , 57 ). Of note, these numbers are based on the CyTOF Helios ® instrument (Fluidigm, San Francisco, CA).…”

“…As in conventional flow cytometry, panel design is key to mass cytometry experiment success ( 36 , 57 , 67 ). The initial marker selection relies heavily on the scientist’s combined biological knowledge and familiarity with statistical testing methods, varying depending on the sample type, cell type, and overall experimental objectives.…”