Silenced APP Protein Reveals Why Pediatric Brain Tumors Hide From the Immune System

Diffuse intrinsic pontine glioma, or DIPG, is among the most lethal cancers known to medicine. Arising in the brainstem of young children, it grows in a diffuse, infiltrative pattern that makes surgical resection impossible, and the blood-brain barrier shields it from most systemic therapies. Median overall survival remains under twelve months, and for decades the standard of care has been limited to palliative radiotherapy, with the FDA approval of ONC201 for H3K27M-mutant diffuse midline glioma marking a rare therapeutic advance. Now, an integrated multi-omics study published in Acta Neuropathologica has mapped the tumor microenvironment of this devastating disease in unprecedented detail, and in doing so has uncovered a surprising molecular culprit: the amyloid precursor protein, better known for its role in Alzheimer’s disease, emerges as a central regulator of whether the immune system inside these tumors fights back or stands down.

The research team, led by investigators at Nationwide Children’s Hospital and Cincinnati Children’s Hospital Medical Center, began with a resource that few groups possess: autopsy-derived tumor specimens from twenty-six DIPG patients, each paired with matched normal frontal lobe tissue obtained through the Pediatric Brain Tumor Repository under institutional review board-approved protocols with informed consent from families. Bulk RNA sequencing of these paired samples revealed a transcriptome in upheaval. More than two thousand genes were significantly upregulated in tumor tissue, and among them were signatures of immune modulatory factors, including checkpoint genes such as PDCD1 and LAG3 and myeloid-associated genes including CSF1, CD163, CD86, and CCL2. Gene Ontology analysis showed that five of the top enriched pathways involved MHC-mediated antigen presentation, indicating that despite their reputation as immunologically cold tumors, DIPG tissues retain active immunological interfaces.

The apparent paradox, in which antigen presentation machinery is broadly induced while cytotoxic T cells remain scarce, likely reflects a dysfunctional immune interface rather than a functional anti-tumor response. Indeed, when the researchers correlated the expression of macrophage lineage markers such as CD11b, CD14, and STAT6 with patient survival, higher expression of each was significantly associated with shorter survival duration. This finding reinforced a growing consensus that tumor-associated myeloid cells, rather than functioning as anti-tumor sentinels, actively promote disease progression in DIPG. Consistent with earlier work showing sparse lymphocyte infiltration in these tumors, the data painted a portrait of a microenvironment dominated by microglia and monocyte-derived macrophages that have been recruited and shaped by the tumor itself.

To probe how that recruitment happens, the team turned to laboratory experiments with four patient-derived DIPG cell lines and THP-1 human monocytes in transwell migration assays. The results were striking in their asymmetry. SU-DIPG-IV and SU-DIPG-XXXVI, both carrying the H3.1K27M mutation, markedly enhanced monocyte migration and drove the cells toward an immunosuppressive CD11b-positive CD163-positive phenotype, while CCHMC-DIPG-1 and CCHMC-DIPG-2 showed no such capacity. Multiplex cytokine assays revealed that the two potent lines secreted far higher levels of CCL2, IL-10, and TGF-beta1. Yet when the investigators analyzed RNA sequencing data from forty-six pediatric high-grade glioma cell lines in the Childhood Cancer Model Atlas, they found that chemokine expression did not track with histone mutation status at all.

Instead, the decisive variable was the tumor cell’s lineage state. Pediatric glioma cells oscillate among four transcriptional identities, resembling neural progenitor cells, astrocytes, oligodendrocyte precursors, or mesenchymal cells. Using single-sample gene set enrichment analysis, the researchers computed chemokine scores and lineage scores for every cell line and found a strong positive correlation between chemokine expression and the mesenchymal-like program, with a Pearson coefficient of 0.73 and a p-value of 6.5 times ten to the minus nine. Chemokine scores correlated negatively with both the neural progenitor-like and oligodendrocyte precursor-like scores and showed no significant relationship with the astrocyte-like state. Mesenchymal-like cell lines expressed significantly higher levels of CCL2, CCL5, and CCL7 than their counterparts. This suggests that the mesenchymal program, potentially driven by NF-kappaB and STAT3 signaling, is the engine of myeloid recruitment, and it hints at a self-reinforcing loop in which mesenchymal tumor cells summon macrophages that in turn stabilize the mesenchymal phenotype.

Single-cell RNA sequencing of eight DIPG patients, integrated with public datasets spanning H3K27M-mutant, G34R/V-mutant, and wildtype pediatric high-grade gliomas, then allowed the team to reconstruct cellular communication networks across more than twenty-two thousand cells. CellChat analysis revealed that tumor-associated macrophages were present in every histone subtype examined and were the sole recipients of several incoming signals, including TGF-beta, semaphorin-3, and, most intriguingly, the amyloid precursor protein signaling pathway transmitted through the CD74 receptor. Immunofluorescence staining of tumor sections confirmed that APP and CD74-expressing cells occupy spatial proximity within the tumor, consistent with a real ligand-receptor interaction occurring in the microenvironment. APP was broadly expressed across most cell populations, whereas CD74 was restricted to tumor-associated macrophages, defining a one-way channel of communication from tumor to immune cell.

The most consequential observation, however, was what was missing. APP expression was significantly reduced in DIPG tumor tissue compared with matched normal brain at both the RNA and protein levels, a result confirmed by western blotting and mirrored by decreased expression of the related family members APLP1 and APLP2. To understand what this loss means for macrophage behavior, the researchers treated THP-1-derived macrophages with recombinant human APP protein. The stimulation triggered a robust proinflammatory transformation: interferon-stimulated genes such as IFIT1 and IFIT2 were induced, inflammatory cytokines including IL1B, CCL2, and CXCL10 rose, and immunosuppressive markers such as CD163, CD206, and ARG2 were suppressed. Gene set enrichment analysis confirmed strong activation of interferon alpha, interferon gamma, and TNF alpha signaling via NF-kappaB, and multiplex cytokine assays showed time-dependent increases in secreted IFN-gamma, TNF-alpha, IL-1beta, and IL-6.

These functional data support a compelling hypothesis: when APP levels fall in the tumor, macrophages lose a signal that would otherwise push them toward an inflammatory, potentially anti-tumor state, and instead settle into the non-inflammatory, immunosuppressive phenotype that characterizes DIPG. The authors caution that the causal relationship remains to be established, and they note an apparent discrepancy with a recent glioblastoma study in which APP appeared to suppress macrophage phagocytosis via CD74, suggesting that context and the specific cellular readout matter. To lay groundwork for therapeutic intervention, the team went further, building three-dimensional structural models of membrane-bound APP and the CD74 trimer using I-TASSER-MTD, refining them in GROMACS, and performing protein-protein docking with HADDOCK. Membrane-restrained Gaussian network modeling then identified a mechanically rigid binding interface in which APP residues 19 through 24 engage a hydrophobic pocket in CD74 centered on residues 118 through 122.

That structurally defined interface offers an actionable target. Peptides engineered to engage the CD74 hydrophobic pocket could, in principle, mimic APP signaling and reprogram tumor-associated macrophages toward proinflammatory phenotypes with anti-tumor capacity, complementing emerging CAR-T cell therapies directed against GD2, B7-H3, and IL13-Ralpha2. The authors acknowledge important limitations: the sequencing work relied on post-mortem tissue from patients who had received varied treatments, the THP-1 model does not fully recapitulate the complexity of primary human macrophages, and the computational docking will require validation through site-directed mutagenesis and co-immunoprecipitation. Still, the study represents a substantial advance in understanding how lineage identity governs immune recruitment in pediatric high-grade glioma and how a protein famous in neurodegeneration may hold the key to thawing one of childhood cancer’s coldest tumors. Future studies using biopsy specimens, primary human systems, and syngeneic preclinical models will determine whether restoring APP-CD74 signaling can genuinely convert the immunosuppressive microenvironment of DIPG into one that fights back.

Subject of Research: Tumor-associated macrophage recruitment and the APP-CD74 signaling axis in pediatric high-grade glioma

Article Title: Integrated multi-omics identifies lineage-dependent myeloid cells recruitment and the APP-CD74 axis as an immunoregulatory target in pediatric high-grade glioma

Article References: Wang, Z., Kumar, A., Umaru, B., Iyer, A. M., Khan, K., Pang, H.-H., Fouladi, M., & Drissi, R. (2026). Integrated multi-omics identifies lineage-dependent myeloid cells recruitment and the APP-CD74 axis as an immunoregulatory target in pediatric high-grade glioma. Acta Neuropathologica, 152(1), Article 39. https://doi.org/10.1007/s00401-026-03089-0

Image Credits: AI Generated

DOI: 10.1007/s00401-026-03089-0

Keywords: diffuse intrinsic pontine glioma, pediatric high-grade glioma, tumor-associated macrophages, APP-CD74 axis, amyloid precursor protein, CD74, tumor microenvironment, single-cell RNA sequencing, mesenchymal-like lineage state, chemokines, immunotherapy, multi-omics