Figure 6. Entrapped TAMs in hypoxic zones express immunosuppressive markers.

(A) UMAP plot of TAM subclusters in GL261.

(B) Violin plots of marker gene expression in TAM subcluster 3 (hypoxic) vs. other subclusters combined.

(C) Heatmap showing DEGs in hypoxic TAMs, with top upregulated DEGs labeled.

(D, E) Representative IF images from GL261 transplants (n=3) show expression of CD68 or CD206 in relation to hypoxia markers GLUT1 or Pimo.

(F) IF images show expression of immunotolerance markers Arginase-1 (Arg1) and CD206 in sequestered TAMs in hypoxic zones (asterisks). Quantification: n=10 randomly selected tumor areas from 3 mice per condition.

(G) Schematic of TAM patterning in hypoxic zones of GBM.

(H) IF image and quantifications of CD8⁺ CTLs or FOXP3⁺ Tregs in relation to hypoxic cores (asterisks) outlined by UnaG⁺ cells (dashed lines) of GL261 at 4 wks post-transplant. n=15 tumor areas from 3 mice per group.

(I) Multiplex 5-color IF imaging from n=3 tumor sections show localization of CTLs (CD8a⁺) and TAMs (CD68⁺) in relation to hypoxic zones (UnaG⁺) or blood vessels (PECAM1⁺).

(J, K) IF images of fresh frozen human GBM sections stained for immune cell markers for TAMs or T cells and GLUT1 (asterisks) in hypoxic or adjacent non-hypoxic zones. DAPI for nuclear staining. Enlarged images of boxed area in (J) are shown on right.

Unpaired t-test (F, H); *P<0.01, ***P<0.001.

See also Figures S5–S7.