Fig 2: Functional analysis of hPSC‐derived PTEC (a‐b) SGLT2 localization and Glucose uptake of d16 PTEC. (A) Immunofluorescence staining for SGLT2 (green), Laminin (red); Scale bar = 20µm; (B) Flow cytometry analysis of 400µM 2‐NBDG uptake in absence or presence of 1µM Dapagliflozin of d16 PTEC; (C) Flow cytometry analysis for 1mg/ml FITC‐Albumin‐endocytosis of d16 PTEC; (D) Immunofluorescence staining for Megalin/LRP2 and FITC‐Albumin of d16 PTEC; Scale bar = 50µm

Fig 3: Assessment of hPSC‐derived PTEC. hPSC were seeded, expanded and differentiated in suspension on Matrigel‐coated alginate beads for scalable automated manufacture of PTEC (A) Flow cytometry analysis of cells on d16 post‐differentiation induction for AQP1, Na⁺/K⁺‐ATPase, SGLT2, Megalin/LRP2. Starting cells were the hiPSC lines BCRTi005‐A, WISCi004‐A and the ESC‐line WAe001‐A, respectively; (B) Flow cytometry analysis of d16 cells showing LTL and (C) Fluorescence microscopy for Phalloidin (yellow); DAPI (blue) shows nuclei. Scale bar = 50µm; (D) Immunofluorescence analysis of d20 cells showed expression of AQP1, Na⁺/K⁺‐ATPase, Megalin/LRP2 (all in red); SGLT2, E‐Cadherin (CDH1) (green). Scale bar = 100µm; (E) Transmission electron microscope images of d16 cells showed apical‐basal polarization (A). Basal side oriented toward Matrigel beads. Microvilli on apical side (MV). (A‐C) Tight junction (TJ); nuclei (N), mitochondria (M) and lysosomes (L)

Fig 4: GlucoGlo binds models of human NSCLC in an SGLT2-dependent manner. (a) Representative flow cytometry tracings of cell lines after staining with SGLT2 antibody. Mean fluorescence intensity (MFI) of stained cells corresponds to the red histogram; unstained cells were used as a baseline (blue histogram). (b) Representative images of cell lines co-cultured with GlucoGlo (red pseudocoloration) and counterstained with DAPI (blue pseudocoloration) and examined by fluorescence microscopy, shown at 20 × magnification. (c) Fluorescence intensity (FI) and (d) signal to background ratios (SBR) of cells incubated with GlucoGlo were significantly higher in SGLT2-expressing cell lines compared to control lines measured by ANOVA (FI: p = 0.0065; SBR: p < 0.0001).

Fig 5: B. (a) Chemical structure of GlucoGlo. A modified SGLT2-inhibitor was covalently bound to a modified indocyanine green molecule to create GlucoGlo. The absorption peak and profile (b) and normalized emission intensity (c) for GlucoGlo were in the near-infrared range and were similar to the gold standard of NIR imaging, indocyanine green.