Combined Multicolor Immunofluorescence Staining and Spatial In Situ messenger RNA Expression Analysis Identifies Potential Fibrosis Drivers in Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most prevalent childhood cancer. Bone marrow (BM) fibrosis in ALL has been associated with adverse outcomes; however, little is known about the mechanisms that cause fibrosis in ALL. Therefore, we established a novel and advanced analysis method by combining multicolor immunofluorescence (IF) staining with in situ RNA expression analysis (RNAscope) to investigate the spatial expression of putative fibrotic drivers in ALL BMs. We analyzed standard BM biopsies from pediatric patients with ALL. Sequential 5-color IF staining with CD45, CD271, CD31, CD34, and DAPI was used to identify different BM cell types. Combined RNAscope and IF staining was established for spatial messenger RNA expression analysis of transforming growth factor beta 1 (TGFB1) and platelet-derived growth factor alpha 1 (PDGFA1), which are known to play major roles in primary myelofibrosis (PMF). PMF and normal BM samples served as controls. As expected, ALL BMs showed high cellularities and prominent populations of blast cells. CD271⁺ mesenchymal stromal cell density was increased in ALL and was associated with fibrosis in a similar manner as observed for PMF. TGFB1 and PDGFA1 expression was considerably increased in ALL megakaryocytes (MKs) compared with patients with PMF and normal controls. Furthermore, MK TGFB1 and PDGFA1 expression intensities in fibrotic ALL correlated with fibrosis grade. TGFB1 and PDGFA1 were also expressed in leukemic blasts, however, at lower intensities compared with ALL MKs. Taken together, advanced in situ RNA and IF staining not only revealed increased expression of TGFB1 and PDGFA1 in fibrotic pediatric ALL but also identified ALL blasts and MKs as their cellular origin at the single-cell level. These novel data strongly suggest a role of these cytokines as potential fibrosis drivers in ALL. More broadly, our findings demonstrate that combined RNA and surface marker analysis is a powerful tool to provide new and valuable insights into BM pathophysiology.