Germ layer specification and organotropism in lymphoma invasion

In this study, patients with diffuse large B-cell lymphoma (DLBCL) involving extranodal sites were stratified into three groups based on the germ-layer origin of the affected organs: ENI-ectoderm (central nervous system, breast, skin, and nasal cavity), ENI-endoderm (gastrointestinal tract, thyroid, lung, liver, and pancreas), and ENI-mesoderm (bone, bone marrow, testis, kidney/adrenal gland, and uterus/ovary). Patients with only lymph node involvement served as the control group. Following R-CHOP therapy, ENI-mesoderm group exhibited significantly poorer progression-free and overall survival compared to ENI-ectoderm or ENI-endoderm. Distinct oncogenic mutation profiles were identified across the three groups. ENI-ectoderm was characterized by mutations in MYD88, PIM1, and TBL1XR1; ENI-endoderm characterized by TP53 and TET2 mutation; and ENI-mesoderm characterized by mutations in MYD88, PIM1, TBL1XR1, and CD79B. To validate the direct role of these mutations in organotropism, the researchers established cell lines mimicking the mutational signatures and conducted xenotransplantation experiments in zebrafish. Cells carrying mutations common to the ENI-ectoderm and ENI-mesoderm groups (MYD88/PIM1/TBL1XR1) rapidly migrated to the brain (representative organ of ENI-ectoderm) and CHT (representative organ of ENI-mesoderm), while cells with CD79B mutations primarily targeted CHT. Cells harboring TP53 or TET2 mutations precisely migrated to the liver and gastrointestinal tract (representative organs of ENI-endoderm).

Notably, the temporal sequence of organotropic migration mirrored the developmental timing of germ layers. In zebrafish models, migration to the brain was observed within two days of injection, followed by migration to the gastrointestinal tract and liver by day four, with migration to hematopoietic tail tissue occurring last. Single-cell RNA sequencing further revealed that malignant B-cell evolution followed a trajectory mimicking germ-layer development, branching from a cell state enriched for NF-κB and T-cell activation signals into two divergent paths: one upregulating B-cell receptor signaling and the other maintaining sustained T-cell activation.

In contrast, T-cell differentiation in the tumor microenvironment did not recapitulate germ-layer developmental timing. Trajectory analysis showed that CD4⁺ and CD8⁺ T cells in the mesodermal group remained largely arrested in an undifferentiated naïve state, characterized by high enrichment of naïve T cells. From this starting point, T cells in the ectodermal and endodermal groups diverged toward distinct fates: ectodermal T cells primarily differentiated into effector proliferative phenotypes (e.g., CD8⁺ Prolif, CD4⁺ Tfh), exhibiting activation, proliferation, cytotoxicity, and antigen presentation capabilities, while endodermal T cells evolved toward immune tolerance and exhaustion phenotypes (e.g., CD4⁺ Treg, CD8⁺ Tex). These findings suggest that T-cell characteristics in the tumor microenvironment are shaped more by the specific local milieu of target organs than by germ-layer developmental timing.

Distinct immune checkpoint molecules were also identified across groups: LGALS9 in ENI-ectoderm, PD-L1 in ENI-endoderm, and B7-H3 in ENI-mesoderm. In vitro experiments confirmed that high expression of LGALS9 and PD-L1 activated effector T cells (such as CD8⁺ and Temra cells) and maintained tumor cell sensitivity to rituximab, significantly reducing tumor viability. Conversely, high B7-H3 expression kept T cells arrested in a naïve state and mediated resistance to rituximab. Crucially, knockdown of B7-H3 reversed this immune evasion, restoring effector T-cell populations and resensitizing tumor cells to rituximab.

This study provides the first evidence that extranodal invasion in lymphoma follows a germ-layer-dependent pattern of organotropism, with significant biological heterogeneity across mutational profiles, signaling pathways, migratory timing, and evolutionary trajectories. By offering a novel developmental biology perspective on the molecular basis of tumor extranodal infiltration, the findings lay important theoretical groundwork for understanding tumor invasion and metastasis. The discovery of germ-layer-dependent invasion patterns paves the way for developing precision intervention strategies, offering new therapeutic targets for personalized cancer treatment and holding significant promise for improving clinical outcomes and patient prognosis.

 

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