Fig 1: In vivo experiments: comparison of the effects assessed for GMI-1359, GMI-1271 and CTCE-9908 administered at the subcutaneous 22rv1 xenograft model. (A) Diagram of treatments: tumour bearing animals were randomized approximately 10 days or when tumors reached 80–100 mm3 of volume in four experimental groups as follows Group 1: mice receiving intraperitoneal (i.p.) injections of 100 µL PBS (vehicle); Group 2: mice receiving CTCE-9908 (25 mg/kg, i.p); Group 3: mice receiving GMI-1359 twice a day ip at 40 m for consecutive 14 days; Group 4: mice receiving GMI-1271 40 mg/Kg BID for consecutive 10 days; (B) 22rv1 tumour weights assessed after 35 days from cell injection and 28 from start of treatments. (C) Time To Progression analysis (median values and 95% CI); (D) Kaplan Meyer representation for the analyses of effects of GMI-1359, CTCE-9908 and GMI-1271; (E) hazard ratio values and relative significance for the different comparisons; (F) dot blots for CXCR4 performed on tissue extracts obtained from eight tumors/group. Two hundred µg of extracted proteins were spotted in a nitrocellulose paper. (G) CXCR4 densitometric units analyzed by Image J software as described in the Material and Methods section (H) ELISA determination for HECA-542 in the plasma of animal of control or treated withGMI-1359, CTCE-9908 and GMI-1271. Statistics for all comparisons are in the text and Supplementary Materials Figure S1. *p < 0.05 versus respective controls (vehicle).
Fig 2: CXCR4 expression and HECA-452 immune-reactivity after treatment with non-toxic DTX doses. (A) Western blotting analysis performed on PC3, DU145 and 22rv1 cells cultured with 20 nM DTX (time course experiment). (B) Fluorescence analyses on 22rv1, DU145 and PC3 cells cultured with DTX for 96 hr. (C) CXCR4 expression levels by FACS (fluorescence index) on parental and resistant PCa cells. (D) Western blotting analyses performed on parental (P) and DTX resistant (R) 22rv1, DU145 and PC3 cells. (E) HECA-452 immuno-reactivity in parental cell treated with DTX and (F) DTX-resistant cells. Western blots were loaded with 40 µg/lane of proteins. Western blots images are representative o three different gels/experiments. MFI values were calculated for each cell line as indicated in MM ± standard deviation calculated from individual three FACS analyses. * p < 0.05 versus respective controls.
Fig 3: CXCR4 inhibition increases the sensitivity to DTX in vitro. (A) IC50 values for DTX calculated in cells derived from bone metastases (PC3, PC3b, C4–2B and VCaP), primary (22rv1), lymph-nodes (LnCaP) and brain (DU145). (B) IC50 values calculated for DTX in presence of 10–100 ng/mL SDF1a. (C,D) GMI-1359, GMI-1271 and CTCE-9908 were co-administrated with different doses of DTX (0–200 µM) and IC50 values for DTX calculated in DTX sensitive (C) and resistant (D) cells. Not to work at too high concentrations, which could mask the combined effects, GMI-1359, GMI-1271 and CTCE-9908 were used at doses close to their IC20 values, so GMI-1359 was administered at 12.5 µM (C4–2B), 8.7 µM (PC3), 15 µM (PC3DTXR) and 10.3 µM (DU145DTXR). GMI-1271 was administered at 10.7 µM (PC3 and PC3DTXR) and 6.9 µM (DU145 and DU145DTXR). Finally, CTCE-9908 was added at 25 ng/mL (PC3), 45 ng/mL (C4–2B), 15 ng/mL (DU145DTXR) and 58 ng (PC3). Data are representative of three similar experiments performed in triplicate. * p < 0.05.
Fig 4: CXCR4 influence osteoclast proliferation: (A) RAW275.6 cells were grown in presence of conditioned media from PC3, 22rv1, LnCaP, VCaP and C42B and stained with crystal violet. (B) OD495 nm for solubilized RAW276.5 cultures and effects of GMI-1359, GMI-1271 and CTCE-9908 on C4-2B, PC3 and 22rv1 administration. (C) Representative staining for tartrate-resistant acid phosphatase (TRAP) in RAW276.5 plates treated with PC3, 22rv1, C4-2B, VCaP and LnCaP cells. (D) effects of 0.5 µM CTCE-9908, GMI-1359 and GMI-1271 on TRAP activity from RAW276.5 treated with PC3CM. (E) ALP activities (OD 405 nm) measured in RAW275.6 pre-treated with pharmacological doses of CTCE-9908, GMI-1359 and GMI-1271 and successively administered with CMs (diluted 1:4 in complete medium) derived from PC3, 22rv1 and C4–2B PCa cells treated. Statistics: *p < 0.05.
Fig 5: Immune-reactivity (IR) of CXCR4 and HECA-452 in prostate cancer cells. (A) Antigen quantification for both antibodies in seven prostate cancer cells (Mean Fluorescence Index, MFI ± Standard Deviation, SD from three separate analyses). (B) MFI values were grouped for bone metastatic and non-bone metastatic PCa cells. Box plots show median values of MFI and 95% of confidence. * p < 0.05 in the comparison between bone versus non bone metastatic sites. (C,D) Effects of CAF-CM (1:1 in complete medium) and exogenous (10 ng/mL) SDF1a on CXCR4 (C) and HECA-452 (D) immune-reactivity levels (MFI) in 22rv1 and PC3, used as models. (E, F) Effects of BMS-CM, Murine osteoblast-like MC3T3-E1 (OB) and RAW-CM cells on CXCR4 (E) and HECA-452 (F) levels by FACS assays in PC3 and 22rv1 cell models. Data represent the values of MFI calculated for each cell line as indicated in MM ± the values of standard deviation calculated from individual three FACS analyses. * p < 0.05 versus controls.
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