Figure 1: Folate receptor beta is only expressed on tumor resident MDSCs and TAMs.

(A) qPCR analysis of tumor and spleen isolated MDSCs subsets, CD11b+ Ly-6G+ (G-MDSCs) and CD11b+ Ly-6C+ (M-MDSCs), from 7-day IP RM-1 tumor bearing animals. folr1, folr2, and folr4 were analyzed. Data from 5 mice pooled. (B) Prevalence of FRβ expression in MDSCs and TAMs isolated from various tumor cell lines. Solid tumor digests were stained in vitro with OTL38 (50 nM) to determine the proportion of the tumor comprised of FRβ expressing MDSCs (CD11b+ GR-1+) and TAMs (CD11b+ Ly-6C− F4/80+). Data are represented as percent FRβ+ MDSCs and TAMs within the total tumor mass.(n=2–5 mice per group) (C) Analysis of OTL38 delivery to a solid tumor. 4T1 tumors were grown to approximately 600 mm³ in size. OTL38 (50 nM) was injected IV with or without 200x folic acid (mixed with OTL38) to compete binding. Mice were then imaged after 2 hrs. to analyze uptake. (D) MDSCs and TAMs subset analysis from mice bearing solid 800 mm³ RM-1 tumors and treated by IV delivery of OTL38 (50 nM). OTL38 was delivered as described in panel C. Tumor-derived and spleen MDSCs subsets and TAMs were isolated and analyzed for uptake of OTL38. Red histogram is tumor OTL38 uptake, blue histogram is PBS injected control, and green histogram is spleen OTL38 uptake. (n=5 mice per group) (E) Quantification of flow data shown in panel D with percent uptake indicating percentage M-MDSCs and TAMs that labeled positively for OTL38. (F) Expression of FRβ in CD14+ cells in both cancerous lung tissue and patient matched peripheral blood samples. Mann-Whitney non-parametric test was used for quantification. (G) FRβ expression in renal carcinoma tissue. Determination of FRβ expression in human cancer. Formalin fixed, paraffin embedded tissue sections were processed for antigen retrieval and stained with anti-FRβ antibody (m909). Bladder cancer sections were stained with anti-CD11b antibody (brown stain, black arrows) and co-stained with anti-FRβ antibody (red stain, white arrows).