Combined Multicolor Immunofluorescence Staining and Spatial In Situ messenger RNA Expression Analysis Identifies...

Area of Science:

• Hematology and Oncology
• Cancer Biology
• Molecular Pathology

Background:

• Bone marrow (BM) fibrosis is linked to poor outcomes in pediatric acute lymphoblastic leukemia (ALL), but its underlying mechanisms remain unclear.
• Understanding fibrosis drivers in ALL is crucial for developing targeted therapies and improving patient prognosis.

Purpose of the Study:

• To investigate the spatial expression and cellular origin of potential fibrotic drivers in pediatric ALL bone marrow.
• To establish and validate an advanced analysis method combining immunofluorescence (IF) and RNAscope for in situ molecular analysis.

Main Methods:

• Developed a novel method combining multicolor immunofluorescence (IF) staining with in situ RNA expression analysis (RNAscope).
• Analyzed pediatric ALL BM biopsies, alongside primary myelofibrosis (PMF) and normal BM controls.
• Quantified spatial expression of transforming growth factor beta 1 (TGFB1) and platelet-derived growth factor alpha 1 (PDGFA1) in relation to specific cell markers (CD45, CD271, CD31, CD34).

Main Results:

• Increased CD271+ mesenchymal stromal cell density was observed in ALL, correlating with fibrosis.
• Significantly elevated TGFB1 and PDGFA1 messenger RNA expression was found in ALL megakaryocytes (MKs) compared to controls.
• TGFB1 and PDGFA1 expression in ALL MKs and blasts correlated with the grade of BM fibrosis.

Conclusions:

• The study identifies TGFB1 and PDGFA1 as key fibrotic drivers in pediatric ALL, originating from both leukemic blasts and megakaryocytes.
• The developed combined IF and RNAscope technique is a powerful tool for elucidating bone marrow pathophysiology at a single-cell level.
• These findings suggest potential therapeutic targets for mitigating bone marrow fibrosis in ALL.