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

Bräunig, Sandro LU ; Dencker, Carl LU ; Vo, Dang Nghiem LU ; Fan, Rong LU ; Sierras, Alba Lillo LU ; Enoksson, Jens ; Hultquist, Anne ; Li, Hongzhe LU and Scheding, Stefan LU (2025) In Laboratory Investigation 105(12).
Abstract

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... (More)

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.

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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
acute lymphoblastic leukemia, bone marrow fibrosis, bone marrow stromal cells, multicolor immunofluorescence staining, RNA scope, spatial RNA expression analysis
in
Laboratory Investigation
volume
105
issue
12
article number
104241
publisher
Nature Publishing Group
external identifiers
  • scopus:105018886657
  • pmid:40975399
ISSN
0023-6837
DOI
10.1016/j.labinv.2025.104241
language
English
LU publication?
yes
id
ad5c7956-6444-4b43-85c2-3308e92307c9
date added to LUP
2025-12-11 14:15:59
date last changed
2025-12-11 14:16:56
@article{ad5c7956-6444-4b43-85c2-3308e92307c9,
  abstract     = {{<p>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<sup>+</sup> 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.</p>}},
  author       = {{Bräunig, Sandro and Dencker, Carl and Vo, Dang Nghiem and Fan, Rong and Sierras, Alba Lillo and Enoksson, Jens and Hultquist, Anne and Li, Hongzhe and Scheding, Stefan}},
  issn         = {{0023-6837}},
  keywords     = {{acute lymphoblastic leukemia; bone marrow fibrosis; bone marrow stromal cells; multicolor immunofluorescence staining; RNA scope; spatial RNA expression analysis}},
  language     = {{eng}},
  number       = {{12}},
  publisher    = {{Nature Publishing Group}},
  series       = {{Laboratory Investigation}},
  title        = {{Combined Multicolor Immunofluorescence Staining and Spatial In Situ messenger RNA Expression Analysis Identifies Potential Fibrosis Drivers in Acute Lymphoblastic Leukemia}},
  url          = {{http://dx.doi.org/10.1016/j.labinv.2025.104241}},
  doi          = {{10.1016/j.labinv.2025.104241}},
  volume       = {{105}},
  year         = {{2025}},
}