In Situ Spatial mRNA Analysis Implicates Increased TGFB1 and PDGFA1 Expression As Potential Drivers of Fibrosis in Pediatric Acute Lymphoblastic Leukemia

Growing evidence suggests that changes in the hematopoietic microenvironment (HME) can influence treatment response in leukemia. Among HME disturbances, bone marrow (BM) fibrosis stands out as the most severe and recognizable form, characterized by excessive deposition of reticulin fibers. In acute lymphoblastic leukemia (ALL), BM fibrosis has been linked to unfavorable outcome and high reticulin fiber density at diagnosis has been associated with elevated levels of therapy-surviving leukemia cells. However, the mechanisms that cause fibrosis in ALL have not been thoroughly investigated thus far. In contrast, transforming growth factor beta 1 (TGFB1) and platelet-derived growth factor alpha 1 (PDGFA1) have been identified to play major roles in classical primary myelofibrosis (PMF).

We therefore aimed to investigate potential fibrosis mechanisms in ALL by comparing the cellular composition and spatial expression of TGFB1 and PDGFA1 in ALL bone marrows compared with classical primary myelofibrosis (PMF).

We analyzed formalin-fixed paraffin-embedded BM biopsies from pediatric ALL patients (n=9) compared with PMF samples (n=3) and hematologically normal controls (n=3). Sequential immunofluorescence (IF) staining with CD45, CD271, CD31, and CD34 antibodies and DAPI as nuclear stain was realized by repetitive rounds of staining, scanning, bleaching and restaining resulting in 5-color images (Bräunig et al., Cytometry A. 2023 (10):763-776). Slides were scanned with an Olympus VS120 slide scanner. The chosen markers were sufficient to identify hematopoietic stem and progenitor cells (HSPCs) (CD271-/ CD31-/CD34/45+), megakaryocytes (MKs) (CD45-/low/ CD271-/ CD34low/+/ CD31+), mesenchymal stromal cells (MSCs) (CD45 -/low/ CD271+/ CD31/34-), and endothelial cells (ECs) (CD45-/ CD271-/ CD34low/+/ CD31+). CD271+ mesenchymal stromal cell (MSC) density was assessed by volumetric analysis using the image analysis platform Arivis. Combined RNAscope® and IF staining was established for spatial analysis of TGFB1 and PDGFA1 mRNA expression.

As expected, ALL bone marrows showed high cellularities and prominent populations of blast cells expressing typical surface markers. The density of MSCs in ALL was seemingly increased, especially around vessels, with increasing degrees of fibrosis in comparison to the control samples. CD271+ MSC densities as calculated by normalized CD271+ volumes were significantly higher in ALL with grade 2 fibrosis compared to non-fibrotic ALL bone marrows and controls. Megakaryocyte (MK) numbers in ALL samples were generally reduced. Interestingly, however, TGFB1 and PDGFA1 expression was considerably increased in ALL MKs compared to MKs from PMF patients and normal controls. This was unexpected as MKs in ALL do not belong to the malignant clone. Furthermore, TGFB1 and PDGFA1 expression intensities in MKs in fibrotic ALL correlated with fibrosis grade and fibrotic pathway activity. In addition, TGFB1 and PDGFA1 were also expressed in leukemic blasts, however at lower intensities compared to ALL MKs. Expression of TGFB1 and PDGFA1 in ALL blasts was furthermore confirmed by reanalysis of publically available single-cell RNA sequencing data (Witkowski et al., Cancer Cell, 2020 37(6): 867-882).

Taken together, simultaneous in-situ RNA expression and IF staining identified – for the first time – increased TGFB1 and PDGFA1 expression in pediatric ALL, thus suggesting a role of these driver cytokines in ALL fibrosis. Hence, our findings provide novel insights into fibrosis pathomechanisms in ALL that point to potential therapeutic targets to improve treatment outcomes in fibrotic ALL.