AIM To investigate near-infrared photoimmunotherapeutic effect mediated by an anti-tissue factor(TF) antibody conjugated to indocyanine green(ICG) in a pancreatic cancer model.METHODS Near-infrared photoimmunotherapy(...AIM To investigate near-infrared photoimmunotherapeutic effect mediated by an anti-tissue factor(TF) antibody conjugated to indocyanine green(ICG) in a pancreatic cancer model.METHODS Near-infrared photoimmunotherapy(NIR-PIT) is a highly selective tumor treatment that utilizes an antibody-photosensitizer conjugate administration, followed by NIR light exposure. Anti-TF antibody 1849-ICG conjugate was synthesized by labeling of rat IgG2 b anti-TF monoclonal antibody 1849(anti-TF 1849) to a NIR photosensitizer,ICG. The expression levels of TF in two human pancreatic cancer cell lines were examined by western blotting. Specific binding of the 1849-ICG to TF-expressing BxPC-3 cells was examined by fluorescence microscopy. NIR-PITinduced cell death was determined by cell viability imaging assay. In vivo longitudinal fluorescence imaging was used to explore the accumulation of 1849-ICG conjugate in xenograft tumors. To examine the effect of NIRPIT, tumor-bearing mice were separated into 5 groups:(1) 100 μg of 1849-ICG i.v. administration followed by NIR light exposure(50 J/cm2) on two consecutive days(Days 1 and 2);(2) NIR light exposure(50 J/cm2) only on two consecutive days(Days 1 and 2);(3) 100 μg of 1849-ICG i.v. administration;(4) 100 μg of unlabeled antiTF 1849 i.v. administration; and(5) the untreated control. Semiweekly tumor volume measurements, accompanied with histological and immunohistochemical(IHC) analyses of tumors, were performed 3 d after the 2nd irradiation with NIR light to monitor the effect of treatments. RESULTS High TF expression in BxPC-3 cells was observed via western blot analysis, concordant with the observed preferential binding with intracellular localization of 1849-ICG via fluorescence microscopy. NIR-PIT-induced cell death was observed by performing cell viability imaging assay. In contrast to the other test groups, tumor growth was significantly inhibited by NIR-PIT with a statistically significant difference in relative tumor volumes for 27 d after the treatment start date [2.83 ± 0.38(NIR-PIT) vs 5.42 ± 1.61(Untreated), vs 4.90 ± 0.87(NIR), vs 4.28 ±1.87(1849-ICG), vs 4.35 ± 1.42(anti-TF 1849), at Day 27, P < 0.05]. Tumors that received NIR-PIT showed evidence of necrotic cell death-associated features upon hematoxylin-eosin staining accompanied by a decrease in Ki-67-positive cells(a cell proliferation marker) by IHC examination.CONCLUSION The TF-targeted NIR-PIT with the 1849-ICG conjugate can potentially open a new platform for treatment of TF-expressing pancreatic cancer.展开更多
AIM To investigate the therapeutic effect of combined integrinα6β4-targeted radioimmunotherapy(RIT)and PI3 K/m TOR inhibitor BEZ235 in a pancreatic cancer model.METHODS Phosphorylation of Akt,m TOR,the downstream ef...AIM To investigate the therapeutic effect of combined integrinα6β4-targeted radioimmunotherapy(RIT)and PI3 K/m TOR inhibitor BEZ235 in a pancreatic cancer model.METHODS Phosphorylation of Akt,m TOR,the downstream effectors eukaryotic initiation factor 4 E binding protein 1(4 EBP1)and S6 ribosomal protein(S6)were evaluated in Bx PC-3 human pancreatic cancer cells treated with Yttrium-^(90)(^(90)Y)labeled anti-integrinα6β4 antibody(ITGA6 B4)and BEZ235 by western blotting.The cytotoxic effect of BEZ235 was investigated using a colony formation assay.Therapeutic efficacy enhancement by oral BEZ235 administration was assessed using mice bearing Bx PC-3 xenograft tumors.Tumor volume measurements and immunohistochemical analyses(cell proliferation marker Ki-67,DNA damage marker p-H2 AX and p-4 EBP1 staining)of tumors were performed for evaluation of combined treatment with^(90)Y-ITGA6 B4 plus BEZ235,or each arm alone.RESULTS We found that phosphorylation of Akt(p-Akt),4 EBP1(p-4 EBP1)and S6(p-S6)was inhibited by BEZ235.Colony formation in Bx PC-3 cells was additively suppressed by the combination of^(90)Y-ITGA6 B4 and BEZ235.Pretreatment with BEZ235 before^(90)Y-ITGA6 B4 exposure resulted in significant reduction of cells plating efficiency(PE)(0.54±0.11 vs 2.81±0.14 with 185 k Bq/m L^(90)Y-ITGA6 B4 exposure,P<0.01;0.39±0.08 vs 1.88±0.09 with 370 k Bq/m L^(90)Y-ITGA6 B4 exposure,P<0.01)when 5×10~3 cells per dish were plated.In vivo,the combined treatment with^(90)Y-ITGA6 B4 plus BEZ235 enhanced the inhibition of tumor growth and statistically significant differences of relative tumor volume were observed for 27 d after the treatment start date when compared with the^(90)Y-ITGA6 B4 single injection treatment(1.03±0.38 vs 1.5±0.15 at Day 27,P<0.05),and for 41 d when compared with the BEZ235 treatment alone(1.8±0.7 vs 3.14±1.19 at Day 41,P<0.05).Tumors from treatment groups showed reduction in volumes,decreased Ki-67-positive cells,increased p-H2 AX-positive cells and decreased p-4 EBP1 expression.CONCLUSION The therapeutic efficacy of^(90)Y-ITGA6 B4-RIT can be improved by combining with dual PI3 K and m TOR inhibitor,BEZ235,in a pancreatic cancer model suggesting potential clinical application.展开更多
基金Supported by a Grant-in-Aid for Scientific Research(C)from the Ministry of Education,Culture,Sports,Science,and Technology,Japan,No.17K10460(to Aung W)
文摘AIM To investigate near-infrared photoimmunotherapeutic effect mediated by an anti-tissue factor(TF) antibody conjugated to indocyanine green(ICG) in a pancreatic cancer model.METHODS Near-infrared photoimmunotherapy(NIR-PIT) is a highly selective tumor treatment that utilizes an antibody-photosensitizer conjugate administration, followed by NIR light exposure. Anti-TF antibody 1849-ICG conjugate was synthesized by labeling of rat IgG2 b anti-TF monoclonal antibody 1849(anti-TF 1849) to a NIR photosensitizer,ICG. The expression levels of TF in two human pancreatic cancer cell lines were examined by western blotting. Specific binding of the 1849-ICG to TF-expressing BxPC-3 cells was examined by fluorescence microscopy. NIR-PITinduced cell death was determined by cell viability imaging assay. In vivo longitudinal fluorescence imaging was used to explore the accumulation of 1849-ICG conjugate in xenograft tumors. To examine the effect of NIRPIT, tumor-bearing mice were separated into 5 groups:(1) 100 μg of 1849-ICG i.v. administration followed by NIR light exposure(50 J/cm2) on two consecutive days(Days 1 and 2);(2) NIR light exposure(50 J/cm2) only on two consecutive days(Days 1 and 2);(3) 100 μg of 1849-ICG i.v. administration;(4) 100 μg of unlabeled antiTF 1849 i.v. administration; and(5) the untreated control. Semiweekly tumor volume measurements, accompanied with histological and immunohistochemical(IHC) analyses of tumors, were performed 3 d after the 2nd irradiation with NIR light to monitor the effect of treatments. RESULTS High TF expression in BxPC-3 cells was observed via western blot analysis, concordant with the observed preferential binding with intracellular localization of 1849-ICG via fluorescence microscopy. NIR-PIT-induced cell death was observed by performing cell viability imaging assay. In contrast to the other test groups, tumor growth was significantly inhibited by NIR-PIT with a statistically significant difference in relative tumor volumes for 27 d after the treatment start date [2.83 ± 0.38(NIR-PIT) vs 5.42 ± 1.61(Untreated), vs 4.90 ± 0.87(NIR), vs 4.28 ±1.87(1849-ICG), vs 4.35 ± 1.42(anti-TF 1849), at Day 27, P < 0.05]. Tumors that received NIR-PIT showed evidence of necrotic cell death-associated features upon hematoxylin-eosin staining accompanied by a decrease in Ki-67-positive cells(a cell proliferation marker) by IHC examination.CONCLUSION The TF-targeted NIR-PIT with the 1849-ICG conjugate can potentially open a new platform for treatment of TF-expressing pancreatic cancer.
基金Supported by(partially)a Grant-in-Aid for Scientific Research(C)from the Ministry of Education,Culture,Sports,Science and Technology,Japan,No.17K10460 to Aung W
文摘AIM To investigate the therapeutic effect of combined integrinα6β4-targeted radioimmunotherapy(RIT)and PI3 K/m TOR inhibitor BEZ235 in a pancreatic cancer model.METHODS Phosphorylation of Akt,m TOR,the downstream effectors eukaryotic initiation factor 4 E binding protein 1(4 EBP1)and S6 ribosomal protein(S6)were evaluated in Bx PC-3 human pancreatic cancer cells treated with Yttrium-^(90)(^(90)Y)labeled anti-integrinα6β4 antibody(ITGA6 B4)and BEZ235 by western blotting.The cytotoxic effect of BEZ235 was investigated using a colony formation assay.Therapeutic efficacy enhancement by oral BEZ235 administration was assessed using mice bearing Bx PC-3 xenograft tumors.Tumor volume measurements and immunohistochemical analyses(cell proliferation marker Ki-67,DNA damage marker p-H2 AX and p-4 EBP1 staining)of tumors were performed for evaluation of combined treatment with^(90)Y-ITGA6 B4 plus BEZ235,or each arm alone.RESULTS We found that phosphorylation of Akt(p-Akt),4 EBP1(p-4 EBP1)and S6(p-S6)was inhibited by BEZ235.Colony formation in Bx PC-3 cells was additively suppressed by the combination of^(90)Y-ITGA6 B4 and BEZ235.Pretreatment with BEZ235 before^(90)Y-ITGA6 B4 exposure resulted in significant reduction of cells plating efficiency(PE)(0.54±0.11 vs 2.81±0.14 with 185 k Bq/m L^(90)Y-ITGA6 B4 exposure,P<0.01;0.39±0.08 vs 1.88±0.09 with 370 k Bq/m L^(90)Y-ITGA6 B4 exposure,P<0.01)when 5×10~3 cells per dish were plated.In vivo,the combined treatment with^(90)Y-ITGA6 B4 plus BEZ235 enhanced the inhibition of tumor growth and statistically significant differences of relative tumor volume were observed for 27 d after the treatment start date when compared with the^(90)Y-ITGA6 B4 single injection treatment(1.03±0.38 vs 1.5±0.15 at Day 27,P<0.05),and for 41 d when compared with the BEZ235 treatment alone(1.8±0.7 vs 3.14±1.19 at Day 41,P<0.05).Tumors from treatment groups showed reduction in volumes,decreased Ki-67-positive cells,increased p-H2 AX-positive cells and decreased p-4 EBP1 expression.CONCLUSION The therapeutic efficacy of^(90)Y-ITGA6 B4-RIT can be improved by combining with dual PI3 K and m TOR inhibitor,BEZ235,in a pancreatic cancer model suggesting potential clinical application.
