Fig 2: The modifications of CK146 via the prodrug strategy and drug delivery strategy. (A) Synthesis of CK146 (4); reaction conditions: (i) 1,14-bromotetradecane, 2-butanone, 110 °C, 3 h; (ii) Di-tert-butyl decarbonate, DMAP, DCM, RT, 16 h; (iii) MeCN, 110 °C, 72 h; (iv) TFA, MeCN, RT, 16 h; (B) Synthesis of CK145 (5) and CK147 (8); reaction conditions: (v) α-Boc-Lys (ε-Ac)-OH, EDC HCl, HOBt, DIEA, DMF, RT, 24 h; (vi) Dithiodiglycolic acid, EDC HCl, HOBt, DIPEA, DMF, RT, 72 h; (vii) TCEP, Tris-HCl, NaOH, PSMA-Maleimide (9), DMF, N2, RT, 24 h; (viii) TFA, DCM, RT, 16 h.

Fig 3: Representative IHC staining results to show PSMA expression in tumor-associated vasculature of HCC. (a, c, and e) Representative pictures to show different PSMA expression levels in HCC. (a) No PSMA expression (score = 0); (c) positive PSMA expression in ≤50% of tumor-associated vasculature (score = 1); (e) positive PSMA expression in >50% of tumor-associated vasculature (score = 2) (scale bar = 100 μm). (b, d, and f) Representative pictures to show CD31 expression in serial sections; arrowheads indicate the microvascular structure (scale bar = 100 μm). HCC, hepatocellular carcinoma; IHC, immunohistochemistry; PSMA, prostate-specific membrane antigen.

Fig 4: Cancer stem cell markers are co-expressed with NANOGP8 in CRPC (castration-resistant prostate cancer) tumors. Surgery and chemical castration induced mouse implanted tumors to form CRPC. (A-C) Immunofluorescence staining showed that NANOG antibody (recognizing NANOGP8 in tumor samples) could specifically bind to Region 1 in the pathological section of the implanted tumor; Using laser micro-dissection to isolate Region 1 and Region 2 as indicated in the section. (D) Immunoblotting indicated that the cellular phenotype of Region 1 (NANOGP8+) is PSA– PSMA–/ALDH+ CD44+ α2β1+ ABCG2+. Scale bars, 25 µm.

Fig 5: Antiproliferative activity characterization of ICL-CCIC-0019, CK146 and CK147. (a) Immunoblotting analysis of PSMA and CHKA in 22Rv1, C4-2B, LNCap, PC3, PNT1A, R1-AD1 and R1-D567 cell lines (n = 2); (b) Growth inhibitory activity comparison among ICL-CCIC-0019, CK146 and CK147 in 22Rv1, C4-2B, LNCap, PC3, PNT1A, R1-AD1, R1-D567 and HCT-116 cell lines (n = 6, mean ± SD).