Extended Data Fig. 3: Pharmacological inhibition of PIKfyve blocks pancreatic cancer progression and tumor growth.
From: Targeting PIKfyve-driven lipid metabolism in pancreatic cancer
a-c. Relative abundance of PI3P (a.), PI4P (b.), and PI(4,5)P2 (c.) in PANC1 cells upon treatment with apilimod (1000 nM) or ESK981 (1000 nM) for the indicated times. Bars are +/-SD. d. Representative H&E staining of whole pancreatic tissue from vehicle and ESK981 treated mice (left). Scalebar = 5 mm for the whole-pancreas images, 100 μm for the zoomed-in images. Quantification of histologically normal pancreatic tissue in vehicle or ESK981 treated mice (right). Box represents 25th and 75th percentiles; whiskers represent the range. (Unpaired two-tailed t-test.). e. Representative CK19 IHC staining of whole pancreatic tissue from vehicle or ESK981 treated mice (left). Scalebar = 5 mm for the whole-pancreas images, 100 μm for the zoomed-in images. f. Schematic of in vivo efficacy studies utilizing cell-derived xenograft (CDX) or allograft models. ESK981 was dosed at 30 mg/kg per day for 5 days/week (PO) in all studies. Mouse cartoons were adapted from Adobe Stock Image (Asset #304271210). g. Relative body weight of C57BL/6 mice used in Fig. 2g–i measured on the indicated treatment day. Bars are +/-SEM. h. Endpoint raw pancreas + tumor weight of CB17 SCID mice bearing UM19 orthotopic tumors. Pancreata of 5 age-matched non-tumor bearing CB17 SCID mice were used as references. Box represents 25th and 75th percentiles; whiskers represent the range. (One-way ANOVA with Tukey’s.). i. Tumor volumes of subcutaneous allograft model using KPC-derived KPC-1344 cells in response to vehicle or ESK981 in C57BL/6 mice. Bars are +SEM. (Two-way ANOVA.). j. Tumor weights (left) and images (right) of KPC-1344 model tumors at study endpoint. Box represents 25th and 75th percentiles; whiskers represent the range. (Unpaired two-tailed t-test.). k. Tumor volumes of subcutaneous CDX model using MIA PaCa-2 cells in response to vehicle or ESK981 in SCID mice. Bars are +SEM. (Two-way ANOVA.). l. Waterfall plot displaying changes in tumor volume comparing endpoint to baseline in response to vehicle or ESK981 treatment in MIA PaCa-2 CDX model. m. Tumor volumes of subcutaneous CDX model derived from BxPC-3 cells in response to vehicle or ESK981 in SCID mice. Data plotted are mean tumor volumes +SEM. (Two-way ANOVA.). n. Individual weights (left) and images (right) of tumors from CDX model derived from BxPC-3 cells at endpoint. Box represents 25th and 75th percentiles; whiskers represent the range. (Unpaired two-tailed t-test.). o. Kaplan–Meier estimates of time to tumor tripling of BxPC-3 CDX tumors after vehicle or ESK981 treatment. (Two-sided log-rank test.). p. Representative images of H&E and Ki67 IHC staining in MIA-PaCa-2 (left) and BxPC-3 (right) CDX models post vehicle or ESK981 treatment. q. Representative image of TUNEL staining from MDA-PaCa-2 CDX tumors after 5 days of treatment of vehicle or ESK981. r. Left panel, representative images of TUNEL staining from primary UM-2 CDX tumors after 5 days of treatment of vehicle or ESK981. Scalebar = 50 μm. Right panel, quantification of TUNEL positivity in indicated groups. Box represents 25th and 75th percentiles; whiskers represent the range. (Unpaired two-tailed t-test). s. Immunoblot analysis of primary UM2 CDX tumors after 5 days treatment of vehicle or ESK981 showing changes in apoptosis marker cleaved PARP (c-PARP). Vinculin was used as a loading control. t. Individual tumor volumes of a PDAC primary CDX UM-2 model before and after 5 days treatment of vehicle or ESK981. Statistics and reproducibility: a-c. One biological replicate of each condition was analyzed together, in two independent experiments for a total of n = 2 for each group. d. n = 11 individual animals for both groups. e. This image is representative of n = 11 individual animals for both groups. g. n = 8 individual animals for each group. h. vehicle n = 9, ESK981 n = 9, No Tumor n = 5 individual animals. i. n = 8 from 4 mice for each cohort, P-value: 4.4e-6. j. n = 8 tumors from 4 mice for each group. k. n = 16 from 8 mice for vehicle, n = 14 from 7 mice for ESK981. P-value: 5.8e-157. n = 14 from 7 mice for each group as 1 mouse in the vehicle reached humane endpoint prior to endpoint analysis. m. n = 12 tumors from 6 individual animals for vehicle, n = 10 tumors from 5 individual animals for ESK981. P-value: 5.7e-32. n. n = 10 tumors from 5 animals for each cohort as 1 mouse in the vehicle cohort reached humane endpoint prior to endpoint analysis. o. n = 12 tumors from 6 individual animals for vehicle, n = 10 tumors from 5 individual animals for ESK981. P-value = 1.7e-28. p. These images are representative of images from n = 4 tumors from individual animals for MIA PaCa-2 vehicle and ESK981 groups, n = 4 for BxPC-3 vehicle group, and n = 6 BxPC-3 ESK981 group. q. These images are representative of three. r. n = 4 (vehicle) or n = 6 (ESK981) individual tumors (from n = 2 or n = 3 animals, respectively) and each represent the mean of 5 representative images per tumor. s. This experiment was performed once. t. Vehicle n = 4 tumors from 2 animals; ESK981 n = 6 tumors from 3 animals.
