Figures and data
3BP sensitivity of pancreatic ductal adenocarcinoma cell lines.
(A) Response of six different PDAC cell lines to 3- BP. The indicated cell lines were exposed to increasing doses of 3BP for 72 h and evaluated with a SYBR green growth assay. Data are represented as the mean +/- SD of three technical replicates and are normalized to untreated controls. (B) Expression levels of MCT-1, MCT-4 and GLUT-1 (TPM) and corresponding IC50s of 3BP. (C) Immunohistochemistry performed on PDAC cell lines with monoclonal mouse antibody against MCT1.
MCT-1 mediates 3BP activity.
(A) Strategy for CRISPR based knockout of SLC16A1 in MiaPaCa-2 cells. (B) Table of Knockout clones. (C) IHC performed on representative KO clones with monoclonal mouse antibody against MCT1 (1:2000 dilution). (D) IHC performed with MCT1 antibody on pooled monoclonal MCT1 KO cells used to test the MCT1 specific activity of 3BP and ME3BP-7. (E and F) Comparison of cell growth over time of MiaPaCa-2 and MiaPaCa-2 MCT1-KO in the absence and presence of 3BP (50 µM) normalized to time point 0 h. Data are represented as the mean +/- SD of two technical replicates. (G) Dose-response curves of MiaPaCa-2 cells and MiaPaCa-2 MCT1-KO at 36 h. Cell viability normalized to the number of cells at 0 h.
Data represent the mean +/- SD of two technical replicates.
New formulations and serum stability of 3BP in cyclodextrin complexes.
(A) HPLC: Evaluation of different microencapsulated β-cyclodextrin complexes using size-exclusion chromatography (SEC).The agents examined were i. 3BP (1 mg/mL), ii. succinyl–β-CD (20 mg/mL), iii. a mixture of 10 µL of 3BP and 10 µL succinyl–β-CD, and iv. ME3BP-7 (10 mg/mL). Samples were monitored at 220 nm as shown in A-D. (B) Serum stability assay using DLD-1 cells. (C) ME3BP-7 specificity assessed with MIA PaCa-2 parental and MIA PaCa-2 MCT-1 KO cells.
Comparison of MCT-1 specific cytotoxicity of 3BP, ME3BP-7 and current standard of care agents for PDAC upon short exposures.
Viability of MCT-1 isogenic panel after (A) Drug exposure for 30 minutes at 200 uM. (B) Drug exposure for 2 hours at 200 uM (C) Drug exposure for 30 minutes at 100 uM (D) Drug exposure for 2 hours at 100 uM
ME3BP-7 inhibits tumor growth of orthotopically implanted pancreatic cancer cell line Panc 02.13 with high MCT1 expression.
(A) Timeline and design of in vivo tumor experiments. (B) Bioluminescence images of nude mice bearing orthotopic Panc 02.13 tumors. (C) Mean fold change in radiance from day of treatment initiation (** P < 0.01, *** P<0.001 1 way ANOVA. (D) Weights of residual tumors harvested upon termination of therapy (** P < 0.01 Mann-Whitney U test).
ME3BP-7 reduces tumor burden in orthotopically implanted human patient derived xenograft TM01212 with diffuse expression of MCT1.
A) Timeline and design of in vivo therapeutic study. B) IHC of orthotopic PDx TM01212 showing diffuse but uniform expression of MCT1. (C) Representative ultrasound image of orthotopically implanted tumors in NSG mice. (D) Mean fold change in tumor volume (n = 10) from day of treatment initiation. (E) Weights of res dual tumors harvested upon termination of therapy. (F and G) H&E of lung and liver with metastases from untreated animals. (H) Number of metastatic lesions harvested from control and treated mice upon termination of therapy. (** P < 0.01 Mann-Whitney U test)
Immunohistochemistry performed on a PDAC tissue microarray with MCT1 antibody.
(A) Overview (1x) of IHC performed on tissue microarray (HPanA150CS03, BioMax U.S) of pancreatic carcinoma cases with MCT1 antibody. (B) IHC of normal pancreas (10x) from the same microarray with MCT1 antibody. (C) Representative examples of uniform high, uniform moderate and focal high expression of MCT1 in human PDAC samples from the array (10x).
