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

The Flemish mutation is an A692G substitution in the gene encoding the amyloid precursor protein, or APP. When APP is processed, the change corresponds to an A21G substitution within amyloid-β, the short peptide that aggregates into the plaques characteristic of Alzheimer’s disease. Carriers of this mutation develop a rare, early-onset form of the disease with two unusual pathological hallmarks: pronounced cerebral amyloid angiopathy, in which amyloid accumulates in the walls of cerebral blood vessels, and senile plaque cores that are unusually large. Affected individuals suffer from both progressive dementia and cerebral hemorrhages, a combination that distinguishes the Flemish variant from more common presentations of familial Alzheimer’s disease. Until now, the molecular reason for this vascular tropism remained obscure.

To understand the structural consequences of the mutation, the team turned to cryo-electron microscopy, a technique that images flash-frozen biological molecules at near-atomic resolution and has in recent years transformed the study of amyloid diseases. Filaments were extracted from parietal lobe tissue of the two affected individuals and their structures determined. Although tau paired helical filaments, the tangles that accompany most forms of Alzheimer’s disease, were present in the samples, the predominant filaments were built not from tau but from the amyloid-β peptide itself, specifically the Aβ40 species carrying the A21G substitution. The Flemish fold, it turned out, is written directly into the peptide sequence.

Structurally, the filaments are composed of two identical protofilaments, each spanning residues D1 through V40 of the amyloid-β peptide, packed together with two-start helical symmetry. This means that two protofilaments run in parallel along the length of the filament, twisting around each other in a helical arrangement. Comparative analysis revealed a striking continuity with previously known amyloid structures: the region of the peptide preceding the mutation site, residues Y10 through F19, adopts essentially the same substructure in both the Flemish fold and in wild-type Aβ42 filaments. The mutation’s effect is therefore not a wholesale remodeling of the peptide but a subtle yet consequential local change. The glycine substitution removes a single methyl group from residue 21, and the loss of that small hydrophobic moiety is enough to redirect the packing of the entire filament.

What emerges downstream of residue 21 is a completely different arrangement from all previously characterized Aβ folds. Since the first amyloid structures were determined, structural biologists have catalogued a growing repertoire of filament folds in Alzheimer’s disease and related disorders, each associated with different clinical presentations. The Flemish fold now joins this catalogue as a distinct entry, defined by its own hydrophobic interface, the interdigitating surface through which the two protofilaments and successive peptide layers stabilize one another. That an alteration of a single methyl group can generate a fold with no known counterpart underscores the exquisite sensitivity of amyloid assembly to sequence chemistry, and suggests that the growing library of familial APP mutations may each encode their own structural signatures.

The structural work alone could not explain why the Flemish fold associates with blood vessels. To address that question, the researchers employed a cell-based assay, and their results provide the study’s most intriguing mechanistic clue. The distinctive Flemish fold was found to be associated with the vascular tropism characteristic of this variant. In other words, the particular architecture of the filament, dictated by the A21G substitution, correlates with the propensity of amyloid to deposit in vessel walls rather than, or in addition to, the brain parenchyma. Cerebral amyloid angiopathy arises when amyloid-β, particularly the Aβ40 species, accumulates within the smooth muscle and basement membrane of leptomeningeal and cortical arteries, weakening them until they rupture. The Flemish findings now provide a structural vocabulary for that process: a fold that appears optimized, in some sense, for the vascular environment.

The implications extend beyond a single family. Dominantly inherited mutations in APP and in the presenilin genes have long served as guideposts for understanding Alzheimer’s disease more broadly, on the logic that if a mutation is sufficient to cause the disease, the pathway it disrupts must be central to the illness. The identification of a mutation-specific filament fold strengthens an increasingly influential idea in the field: that amyloid polymorphism, the existence of distinct filament structures, may correspond to distinct disease phenotypes. Previous cryo-EM studies have shown that different folds correlate with different dementias, including variants of Alzheimer’s disease and cerebral amyloid angiopathy with differing clinical courses. The Flemish fold, being tied to a specific pedigree and a specific clinical syndrome, offers one of the cleanest demonstrations yet that structure and phenotype are mechanistically linked.

The rarity of the Flemish pedigrees makes the achievement particularly notable. Only two such families are known worldwide, and obtaining postmortem brain tissue from affected members is exceptionally difficult. The fact that filaments from two independent individuals yielded consistent structures, with Aβ40-A21G as the predominant component, provides confidence that the Flemish fold is a reproducible biological entity rather than an artifact of a single brain. The presence of tau paired helical filaments alongside the amyloid filaments also confirms that the Flemish variant produces a bona fide Alzheimer-type pathology, complete with both plaques and tangles, even as its vascular features set it apart.

For the molecular understanding of Flemish-type dementia and cerebral hemorrhage, the study delivers what its authors describe as the definition of a familial Alzheimer-disease-associated amyloid fold. This framing matters because it establishes the Flemish fold as a structural unit of analysis in its own right. Future work can now ask mechanistic questions at atomic resolution: how the hydrophobic interface of the Flemish fold interacts with vascular basement membranes, whether the fold nucleates more readily on vascular surfaces, whether the faster clearance of Aβ40 from brain tissue funnels the peptide into vessels, and whether small molecules could be designed to destabilize the Flemish fold specifically. The loss of a single methyl group at residue 21, and the filament architecture it produces, has gone from a genetic curiosity to a defined structural target.

More broadly, the work exemplifies the maturing relationship between cryo-EM and neurodegenerative disease research. In the space of a decade, amyloid structures have progressed from generic models to fold-specific structures that distinguish diseases, mutations, and even individual patients. Each new fold added to the catalogue is a potential biomarker, a phenotypic explanation, and a drug-discovery template all at once. The Flemish fold demonstrates that even in a disease as intensively studied as Alzheimer’s, the structural landscape remains incompletely mapped, and that rare families, however small in number, can illuminate molecular mechanisms invisible in the common sporadic form of the illness. For the members of the two Flemish pedigrees and for the broader effort to understand cerebral hemorrhage in Alzheimer’s disease, the structure now provides a foundation on which diagnostic, mechanistic, and therapeutic advances can be built, one methyl group at a time.