Journal: Molecular Pharmaceutics

Article Title: Hydrophilic Small Molecules That Harness Transthyretin To Enhance the Safety and Efficacy of Targeted Chemotherapeutic Agents

doi: 10.1021/acs.molpharmaceut.9b00432

Figure Lengend Snippet: In vivo fluorescence imaging and ex vivo biodistribution and tumor targeting analysis of TFM1 in a xenograft mouse models of human metastatic prostate cancer. (a) Representative in vivo images of male athymic nu/nu mice ( n = 3) bearing LNCaP (PSMA+) or DU145 (PSMA−) tumors, injected with vehicle, TFM1, or BFM1 at a dose of 17 nmol/kg via tail vein injection and scanned at 1, 4, 24, 48, and 72 h using LI-COR Odyssey CLx Imaging System at excitation and emission wavelength 685 and 800 nm, respectively. (b) 72 h postinjection, mice were sacrificed, and ex vivo tissue biodistribution analysis was performed by imaging of the excised tumors, liver, kidneys, heart, and blood samples. AFU/mg of excised organs and blood after 72 h. Bar graph showing the respective mean (±s.d.) ( n = 6 for tumors or n = 3 for other organs). (c) Representative ex vivo images of excised LNCaP and DU145 tumors after 72 h and bar graph showing the respective mean (±s.d.) AFU/mg of excised tumors ( n = 6). The significance of differences was measured by one-way ANOVA followed by Tukey’s multiple comparison test (ns, not significant; ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001; ∗∗∗∗ p ≤ 0.0001).

Article Snippet: Human prostate carcinoma cell lines LNCaP (PSMA+) (ATCC CRL-1740) and DU145 (PSMA−) (ATCC HTB81) cell lines were obtained from American Type Culture Collection (ATCC), Manassas, USA.

Techniques: In Vivo, Fluorescence, Imaging, Ex Vivo, Injection, Comparison

Journal: Molecular Pharmaceutics

Article Title: Hydrophilic Small Molecules That Harness Transthyretin To Enhance the Safety and Efficacy of Targeted Chemotherapeutic Agents

doi: 10.1021/acs.molpharmaceut.9b00432

Figure Lengend Snippet: TFMs efficiently release MMAE after cathepsin B cleavage and have selective cytotoxicity on LNCaP (PSMA+) versus DU145 (PSMA−) cells. (a) Valine–citrulline dipeptide cleavable linker TFM3 is efficiently cleaved (within 15 min in buffer) by cathepsin B hydrolysis (step a) and spontaneous fragmentation (step b) of the para-aminobenzylcarbamate intermediate. The formation of free MMAE and Fragment A after cleavage of TFM3 was confirmed by HPLC and LC–MS/MS analysis. Similar results were obtained for cathepsin B hydrolysis of TFM2 and BFM2 as shown in Figure S5 . The HPLC spectrum is a representative of triplicate experiments ( n = 3). (b–e) Selective uptake of BFM2, TFM2, and TFM3 by PSMA receptors and the effect of TTR on lowering the cytotoxicity of TFM2 and TFM3 on PSMA– cells. MTT cell proliferation assay was used to determine the cytotoxicity of MMAE, BFM2, TFM2, and TFM3 against LNCaP (PSMA+) and DU145 (PSMA−) cell lines in the absence and presence of TTR. (b) MMAE shows similar cytotoxicity against LNCaP and DU145 cell lines regardless of the absence and presence of TTR. (c) Selective cytotoxicity of BFM2 against LNCaP (PSMA+) compared to DU145 (PSMA−) cell lines. The activity of BFM2 on these cell lines was similar in the absence and presence of TTR. Selective cytotoxicity of (d) TFM2 and (e) TFM3 against LNCaP (PSMA+) compared to DU145 (PSMA−) cell lines. Both TFM2 and TFM3 were less toxic against DU145 (PSMA−) cell lines in the presence of TTR. Each time point is expressed as means ± s.d. ( n = 5).

Article Snippet: Human prostate carcinoma cell lines LNCaP (PSMA+) (ATCC CRL-1740) and DU145 (PSMA−) (ATCC HTB81) cell lines were obtained from American Type Culture Collection (ATCC), Manassas, USA.

Techniques: Liquid Chromatography with Mass Spectroscopy, MTT Cell Proliferation, Activity Assay

Journal: Molecular Pharmaceutics

Article Title: Hydrophilic Small Molecules That Harness Transthyretin To Enhance the Safety and Efficacy of Targeted Chemotherapeutic Agents

doi: 10.1021/acs.molpharmaceut.9b00432

Figure Lengend Snippet: Antitumor efficacy of TFM3 and BFM2 in LNCaP (PSMA+) and DU145 (PSMA−) xenograft mouse models of metastatic PCa. (a) Male athymic nu/nu mice ( n = 6) with LNCaP (PSMA+) tumors received TFM3, BFM2, MMAE, or vehicle at a dose of 300 nmol/kg via i.p. injection as indicated by black (for TFM3, BFM2, or vehicle) or green (for MMAE) arrows. Each point represents mean (±s.d.) tumor volume (mm 3 ). (b) Mean (±s.d.) % body weight changes from the beginning of treatment in the same mice with LNCaP tumors. (c) Mean (±s.d.) weight of excised LNCaP tumors from each treatment group after 14 days. (d) Male athymic nu/nu ( n = 6) mice with DU145 (PSMA−) tumors received TFM3, BFM2, MMAE, or vehicle at a dose of 300 nmol/kg via i.p. injection as indicated by black or green arrows. Each point represents mean (±s.d.) tumor volume (mm 3 ). (e) Mean (±s.d.) % body weight changes from the beginning of treatment in the same mice with DU145 tumors. (f) Mean (±s.d.) weight of excised DU145 tumors from each treatment group after 14 days. The significance of differences was measured by one-way ANOVA followed by Tukey’s multiple comparison test (ns, not significant; ∗ p ≤ 0.05; ∗∗ p ≤ 0.01; ∗∗∗ p ≤ 0.001; ∗∗∗∗ p ≤ 0.0001).

Article Snippet: Human prostate carcinoma cell lines LNCaP (PSMA+) (ATCC CRL-1740) and DU145 (PSMA−) (ATCC HTB81) cell lines were obtained from American Type Culture Collection (ATCC), Manassas, USA.

Techniques: Injection, Comparison

Journal: Cells

Article Title: Engineering of Humanized PSMA-Directed CAR T Cells for Potent and Specific Elimination of Prostate Cancer Cells

doi: 10.3390/cells14171333

Figure Lengend Snippet: CAR T cell activation profiles: CAR T cells are co-cultured with either C4-2 (PSMA+) or DU145 (PSMA-) target cells. Activation is assessed by monitoring the expression of CD25. ( A ) Percentage of CD25-positive cells. ( B ) Mean fluorescent intensity (MFI) of CD25 expression. Patterned bars: Co-culture with DU145 cells. Solid bars: Co-culture with C4-2 cells. Abbreviations: UT—untreated T cells; mD7—murine D7 CAR; hD7—humanized D7 CAR; mA5—murine A5 CAR; and hA5—humanized A5 CAR. Illustrated are means +/− SEM of three biological replicates performed in technical triplicates. The statistical analysis is performed in GraphPad Prism 7 using a two-way ANOVA with Sidak’s multiple comparisons test; **; ***; and **** indicate p < 0.01, p < 0.001, p < 0.0001, respectively.

Article Snippet: The PSMA-positive cell lines C4-2 (ATCC, CRL-3314, Manassas, VA, USA), LNCaP (ATCC, CRL-1740) and PC3-PSMA (kindly provided by Prof. Paloma H. Giangrande, University of Iowa) [ ], and the PSMA-negative cell line DU145 (DSMZ, ACC-261, Braunschweig, Germany) were maintained in RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) with supplemental penicillin (100 U/mL), streptomycin (100 mg/L) (Sigma-Aldrich, Munich, Germany) and 10% fetal calf serum (FCS) (PAN-Biotech GmbH, Aidenbach, Germany).

Techniques: Activation Assay, Cell Culture, Expressing, Co-Culture Assay

Journal: Cells

Article Title: Engineering of Humanized PSMA-Directed CAR T Cells for Potent and Specific Elimination of Prostate Cancer Cells

doi: 10.3390/cells14171333

Figure Lengend Snippet: Cytotoxicity of the PSMA-targeted CAR T cells on prostate cancer cells. Untreated (UT) T cells and CAR T cells are co-cultured with different PSMA+ ( A – C ) and PSMA- ( D ) target cell lines. Depicted is the mean and standard error of the mean (SEM) of three biological replicates, each performed in technical triplicates. Comparison of mD7 vs. hD7 or mA5 vs. h is non-significant in all co-cultures. Significance in A: mD7 vs. hA5 p = 0.0008; hD7 vs. mA5 p = 0.0392; and hD7 vs. hA5 p = 0.0006. Significance in B: mD7 vs. hA5 p = 0.007; hD7 vs. hA5 p = 0.0104. Significance in C: mD7 vs. hA5 p = 0.0125. To reduce visual clutter no significance bars comparing UT with CAR T cell conditions are shown. In all PSMA+ co-cultures (C4-2, LNCaP, PC3-PSMA, all CAR T cell conditions mediated statistically significant higher lysis when compared to UT T cells. The statistical analysis was performed in GraphPad Prism 7 using a two-way ANOVA with Tukey’s multiple comparisons test. *; **; and *** indicate p < 0.05, p < 0.01, p < 0.001, respectively. Abbreviations: UT—untreated T cells; mD7—murine D7 CAR; hD7—humanized D7 CAR; mA5—murine A5 CAR; and hA5—humanized A5 CAR.

Article Snippet: The PSMA-positive cell lines C4-2 (ATCC, CRL-3314, Manassas, VA, USA), LNCaP (ATCC, CRL-1740) and PC3-PSMA (kindly provided by Prof. Paloma H. Giangrande, University of Iowa) [ ], and the PSMA-negative cell line DU145 (DSMZ, ACC-261, Braunschweig, Germany) were maintained in RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) with supplemental penicillin (100 U/mL), streptomycin (100 mg/L) (Sigma-Aldrich, Munich, Germany) and 10% fetal calf serum (FCS) (PAN-Biotech GmbH, Aidenbach, Germany).

Techniques: Cell Culture, Comparison, Lysis