Vo, D. N. | Search bioRxiv

bioRxiv2025

Combined multi-color immunofluorescence staining and spatial in situ mRNA 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 multi-color immunofluorescence staining with in-situ RNA expression analysis (RNAscope(R)) investigate the spatial expression of putative fibrotic drivers in ALL bone marrows. We analyzed standard BM biopsies from pediatric ALL patients. Sequential 5-color immunofluorescence (IF) staining with CD45, CD271, CD31, CD34 and DAPI was used to identify different BM cell types. Combined RNAscope(R) and IF staining was established for spatial mRNA 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 bone marrows showed high cellularities and prominent populations of blast cells. CD271+ MSC 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 to PMF patients 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 to 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 bone marrow pathophysiology.

cell biology↗

bioRxiv2024

NK cells control the progression of myelodysplastic syndrome but become initial disease target in NUP98-HOXD13 mouse model

Studies in NUP98/HOXD13 mouse model (NHD13tg), progressing from myelodysplastic syndrome (MDS) to different forms of leukemia, demonstrated that T cells had a limited anti-leukemia effect, suggesting the involvement of other immune cells. Natural killer (NK) cells control viral infection and cancer. In MDS and acute myeloid leukemia (AML), patients often acquire disease-induced NK cell dysfunctions. Here, we report that NK cells from NHD13tg mice were reduced before the MDS-onset and specific NK cell depletion accelerated the disease progression and severity. NK cells from NHD13tg mice showed perturbed differentiation and impaired IL-15/IL-2 responses. These defects were cell-intrinsic and mainly affected the KLRG1+ mature NK cells. The expression of Nfil3, Klf2 and Id2 genes, crucial for NK cell development, homeostasis and IL-15 responsiveness, was altered in immature NK cells from NHD13tg mice. Interestingly, these genes were changed in MDS and AML bone marrow patient-samples compared to healthy donors. Our findings highlight a critical role for NK cells in controlling MDS progression and identify new genetic markers for MDS and AML.

immunology↗