Feasibility study: phosphospecific flow cytometry enabling rapid functional analysis of bone marrow samples from patients with multiple myeloma.
BACKGROUND
Multiple myeloma (MM) is an incurable cancer accounting for about 2% of cancer deaths. Its diagnosis is based on a combination of criteria, which are not always easily measurable. Flow cytometry now allows multiplex analysis of intracellular signaling at the single cell level. We investigated the feasibility of using intracellular protein phosphorylation analysis by flow cytometry on primary plasma cells from bone marrow and its usefulness in MM diagnosis.


METHODS
Cells from frozen bone marrow of five MM patients and four normal donors were stimulated with LPS, IL-6, IL-21, IFNα and TNFα. Cells were stained by fluorescent cell barcoding to allow multiplex analysis. Staining with antibodies against phosphorylated NFkB-p65, Stat1, Stat3, and p38 were used to identify cellular responses following stimulation.


RESULTS
Activation profiles of MM and normal plasma cells have been established. MM cells showed heterogeneous response profiles while normal cells responses were homogeneous between donors. We also noticed that many MM samples seemed to show elevated basal level of Stat3 phosphorylation. These results suggest that different response profiles in primary MM cells might correspond to different subtypes of the disease. Thus, we provide an example of how these results may be used as a criterion for MM subtypes classification.


CONCLUSIONS
We demonstrate that flow cytometry can be used to study signaling pathways in primary MM cells. The heterogeneity observed in MM cells from different patients can prove valuable for MM characterization and represents an interesting avenue for future research in MM diagnosis.
Héma-Québec, Ingénierie cellulaire, Recherche et Développement, 1070 avenue des Sciences-de-la-vie, Québec, G1V 5C3, Canada.
Carl
C
Simard
Marc
M
Cloutier
Sonia
S
Néron
D001853:Bone Marrow / Q000473:pathology*; D005240:Feasibility Studies; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D009101:Multiple Myeloma / Q000175:diagnosis* / Q000378:metabolism* / Q000473:pathology; D010766:Phosphorylation; D050796:STAT3 Transcription Factor / Q000378:metabolism; D015398:Signal Transduction
D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human
Stat-1; Stat-3; flow cytometry; fluorescent cell barcoding; multiple myeloma; phosphorylation; plasma cells phenotyping