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. 2021 May 17;118(21):e2017925118. doi: 10.1073/pnas.2017925118

Fig. 4.

Fig. 4.

Biodistribution of tumor-cell transduction. (A–D) Representative confocal microscopy images of tumor slices from mice treated with a virus that delivers an antibody isotype control (Ad-D1.3) and a transcriptionally coupled eGFP reporter (green). Slices originating from corresponding locations within tumors are costained with fluorescently labeled antibodies for various cell markers to determine the biodistribution of Ad transduction. (A) Transduction of BT474 tumor cells is shown by eGFP colocalization to BT474-endogenous TdTomato expression (red) and/or positive HER2 staining by IHC (white). Cells positive for eGFP alone are classified as transduced stroma and further characterized in the panels below. (B) Transduction of the tumor vasculature is indicated by GFP colocalization to lectin staining (cyan) and/or positive vWF staining (magenta). (C) Transduction of stromal macrophages is indicated by GFP colocalization with F4/80 staining (magenta). (D) Transduction of stromal fibroblasts is indicated by GFP colocalization with vimentin staining (magenta). (E) Transduction rate of tumor cells (Td+Her2+, Td−Her2+, and Td+Her2−) versus stromal cells (Td−Her2−) in Ad-D1.3–treated tumors. Values from n = 3 individual mice are shown as black dots; error bars represent SE (SEM). (F–H) Subclassification of transduced nontumor stromal cells. Tumor tissue was sectioned and stained for various markers via IHC (anti-F4/80, anti-vimentin, or anti-vWF) and used to further characterize the TdTomato-negative subpopulations. Values from n = 3 individual mice are shown as black dots; error bars represent SE (SEM). (F) Transduction rate of vasculature-associated cells (vasc+vWF+, vasc+vWF−, and vasc−vWF+) versus other stromal cells (vasc−vWF−). (G) Transduction rate of macrophages (F4/80+) versus other stromal cells (F4/80−). (H) Transduction rate of fibroblasts (vim+) versus other stromal cells (vim−). (I) Based on quantifications from the other panels, transduction biodistribution could be determined for the entire tumor. For simplification, F4/80-, vimentin-, and vWF-positive cells have been classified generally as macrophages, fibroblasts, or vasculature-associated cells, respectively.