Moritz L. Blume

Supplementary Fig. 5 from Tumor Vessel Normalization via PFKFB3 Inhibition Alleviates Hypoxia and Increases Tumor Necrosis in Rectal Cancer upon Radiotherapy

Supplementary Fig. 6 from Tumor Vessel Normalization via PFKFB3 Inhibition Alleviates Hypoxia and Increases Tumor Necrosis in Rectal Cancer upon Radiotherapy

Supplementary Fig. 2 from Tumor Vessel Normalization via PFKFB3 Inhibition Alleviates Hypoxia and Increases Tumor Necrosis in Rectal Cancer upon Radiotherapy

Supplementary Fig. 3 from Tumor Vessel Normalization via PFKFB3 Inhibition Alleviates Hypoxia and Increases Tumor Necrosis in Rectal Cancer upon Radiotherapy

Supplementary Fig.1 from Tumor Vessel Normalization via PFKFB3 Inhibition Alleviates Hypoxia and Increases Tumor Necrosis in Rectal Cancer upon Radiotherapy

Supplementary Fig. 4 from Tumor Vessel Normalization via PFKFB3 Inhibition Alleviates Hypoxia and Increases Tumor Necrosis in Rectal Cancer upon Radiotherapy

Supplementary Fig. 7 from Tumor Vessel Normalization via PFKFB3 Inhibition Alleviates Hypoxia and Increases Tumor Necrosis in Rectal Cancer upon Radiotherapy

3PO reduces colorectal cancer cell invasion and endothelial cell sprouting capabilities. A, Invasion assay performed with HCT-116, HT-29, SW-1463, and SW-837 cancer cells. Cells were plated in a Boyden multi-well chamber and treated with 10 µmol/L 3PO for a period of 96 hours. Data are displayed as means SD, n = 3. ∗∗, P < 0.01; ∗∗∗∗, P < 0.0001; Student t test. B, Representative pictures of migration assay performed with SW-837 treated with 10 and 25 µmol/L 3PO; 0 and 30 hours after removing culture inserts. C, Cell migration assay quantification of SW-837 treated with different concentrations of 3PO for 48 hours. Data are displayed as means ± SD, n = 3. D, Relative mRNA expression levels of PAXILLIN, CDC42, and WASF-1 in SW-837 cells treated with different concentrations of 3PO after 72 hours. Data are displayed as means SD, ∗, P < 0.05; ∗∗, P < 0.01; one-way ANOVA. E, Representative pictures of cell sprouting assay performed with HUVECs treated with 10 and 20 µmol/L 3PO after 0 and 24 hours. F and G, Sprouting assay quantification of HUVECs upon PFKFB3 inhibition. Sprouting length, vessel number, and EC proliferation analyzed upon treatment with 10 and 20 µmol/L 3PO for a period of 24 hours. H, Sprouting length quantification upon 24 hours of 3PO treatment on already established spheroids. Data are displayed as means SD, n = 3. ∗∗∗, P < 0,001; ∗∗∗∗, P < 00,001; one-way ANOVA.

In vivo 3PO administration in combination with radiotherapy induces TVN and alleviates hypoxia in RC. A, Representative images of CD31+αSMA+ co-stained sections of PDX tumors upon treatment with vehicle (comtrol), 3PO, RT, and 3PO + RT. Scale bars, 100 μm. B–D, Quantification of CD31+ αSMA+ area (%), vessel lumen size (µm2), and tumor vessel number in PDX tumors from control, 3PO, RT, and 3PO + RT-treated mice. Data are displayed as means SD. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001, ∗∗∗∗, P < 0.0001; one-way ANOVA. E, Representative images of hypoxic zones stained with pimonidazole (brown staining) from sections of PDX tumors upon treatment with vehicle (control), 3PO, RT, and 3PO + RT. Scale bars, 100 µm. F, Quantification of PIMO+ area (%) in PDX tumors from control, 3PO, RT, and 3PO + RT-treated mice. Data are displayed as means SD. ∗, P < 0.05; ∗∗∗, P < 0.001; one-way ANOVA. G, Representative micrographs of γH2AX-stained sections of PDX tumors from control, 3PO, RT, and 3PO + RT-treated mice. Scale bar, 20 μm. H, DNA damage levels were assessed using γH2AX staining, and treated PDX tumors were immunostained and quantified for γH2AX. Data are displayed as means SD. ∗∗∗∗, P < 0.0001; one-way ANOVA.

Gene set enrichment analysis of 3PO-treated colon organoids. A, Representative pictures of patient-derived tumor organoids treated with 30 µmol/L 3PO for 24 hours. Scale bars, 200 μm. B, Patient-derived tumor organoid size quantification. Data are displayed as means ± SD. ∗, P < 0.05; one-way ANOVA. C, GSEA with gene sets derived from the GO Biological Process ontology. For the enrichment plot (score curves), refer to Supplementary Fig. S2. FDR, false discovery rate. D and E, Protein expression levels of NDUFB6, ACAD9, and LDHA in colorectal cancer organoids (PT-73, PT-70, and PT-79) and colorectal cancer cell lines treated with 3PO for 24 hours. ACTIN and HSC70 were used as loading control. Uncropped Western blot pictures at Supplementary Fig. S5. In all cases, densitometry analysis is performed using loading control as references. F, Seahorse analysis for extracellular acidification rate performed with patient-derived tumor organoids treated with 10 µmol/L 3PO for 24 hours. Data is displayed as SD. ∗∗∗, P < 0.001; ∗∗∗∗, P < 0.0001.