Imaging assessment of photosensitizer emission induced by radionuclide-derived Cherenkov radiation using charge-coupled device optical imaging and long-pass filters

BACKGROUND Radionuclides produce Cherenkov radiation(CR),which can potentially activate photosensitizers(PSs)in phototherapy.Several groups have studied Cherenkov energy transfer to PSs using optical imaging;however,cost-effectively identifying whether PSs are excited by radionuclide-derived CR and detecting fluorescence emission from excited PSs remain a challenge.Many laboratories face the need for expensive dedicated equipment.AIM To cost-effectively confirm whether PSs are excited by radionuclide-derived CR and distinguish fluorescence emission from excited PSs.METHODS The absorbance and fluorescence spectra of PSs were measured using a microplate reader and fluorescence spectrometer to examine the photo-physical properties of PSs.To mitigate the need for expensive dedicated equipment and achieve the aim of the study,we developed a method that utilizes a chargecoupled device optical imaging system and appropriate long-pass filters of different wavelengths(manual sequential application of long-pass filters of 515,580,645,700,750,and 800 nm).Tetrakis(4-carboxyphenyl)porphyrin(TCPP)was utilized as a model PS.Different doses of copper-64(^(64)CuCl_(2))(4,2,and 1 mCi)were used as CR-producing radionuclides.Imaging and data acquisition were performed 0.5 h after sample preparation.Differential image analysis was conducted by using ImageJ software(National Institutes of Health)to visually evaluate TCPP fluorescence.RESULTS The maximum absorbance of TCPP was at 390-430 nm,and the emission peak was at 670 nm.The CR and CRinduced TCPP emissions were observed using the optical imaging system and the high-transmittance long-pass filters described above.The emission spectra of TCPP with a peak in the 645-700 nm window were obtained by calculation and subtraction based on the serial signal intensity(total flux)difference between^(64)CuCl_(2)+TCPP and^(64)CuCl_(2).Moreover,the differential fluorescence images of TCPP were obtained by subtracting the^(64)CuCl_(2)image from the^(64)CuCl_(2)+TCPP image.The experimental results considering different^(64)CuCl_(2)doses showed a dosedependent trend.These results demonstrate that a bioluminescence imaging device coupled with different longpass filters and subtraction image processing can confirm the emission spectra and differential fluorescence images of CR-induced TCPP.CONCLUSION This simple method identifies the PS fluorescence emission generated by radionuclide-derived CR and can contribute to accelerating the development of Cherenkov energy transfer imaging and the discovery of new PSs.

Combined treatment of pancreatic cancer xenograft with 90Y-ITGA6B4-mediated radioimmunotherapy and PI3K/m TOR inhibitor

AIM To investigate the therapeutic effect of combined integrin α6β4-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-integrin α6β4 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.

Near-infrared photoimmunotherapy of pancreatic cancer using an indocyanine green-labeled anti-tissue factor antibody

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.

Overproduction of reactive oxygen species–obligatory or not for induction of apoptosis by anticancer drugs

Many studies demonstrate that conventional anticancer drugs elevate intracellular level of reactive oxygen species （ROS） and alter redox-homeostasis of cancer cells. It is widely accepted that anticancer effect of these chemotherapeutics is due to induction of oxidative stress and ROS-mediated apoptosis in cancer. On the other hand, the harmful side effects of conventional anticancer chemotherapy are also due to increased production of ROS and disruption of redox-homeostasis of normal cells and tissues. This article describes the mechanisms for triggering and modulation of apoptosis through ROS-dependent and ROS^independent pathways. We try to answer the question： ＂Is it possible to induce highly specific apoptosis only in cancer cells, without overproduction of ROS, as well as without harmful effects on normal cells and tissues？＂ The review also suggests a new therapeutic strategy for selective killing of cancer cells, without significant impact on viability of normal cells and tissues, by combining anticancer drugs with redox-modulators, affecting specific signaling pathways and avoiding oxidative stress.

Folate receptor-targeted near-infrared photodynamic therapy for folate receptor-overexpressing tumors

BACKGROUND Photodynamic therapy(PDT)is a minimally invasive form of cancer therapy,and the development of a novel photosensitizer(PS)with optimal properties is important for enhancing PDT efficacy.Folate receptor(FR)membrane protein is frequently overexpressed in 40%of human cancer and a good candidate for tumor-specific targeting.Specific active targeting of PS to FR can be achieved by conjugation with the folate moiety.A folate-linked,near-infrared(NIR)-sensitive probe,folate-Si-rhodamine-1(FolateSiR-1),was previously developed and is expected to be applicable to NIR-PDT.AIM To investigate the therapeutic efficacy of NIR-PDT induced by FolateSiR-1,a FRtargeted PS,in preclinical cancer models.METHODS FolateSiR-1 was developed by conjugating a folate moiety to the Si-rhodamine derivative through a negatively charged tripeptide linker.FR expression in the designated cell lines was examined by western blotting(WB).The selective binding of FolateSiR-1 to FR was confirmed in FR overexpressing KB cells(FR+)and tumors by fluorescence microscopy and in vivo fluorescence imaging.Low FR expressing OVCAR-3 and A4 cell lines were used as negative controls(FR-).The NIR light(635±3 nm)-induced phototoxic effect of FolateSiR-1 was evaluated by cell viability imaging assays.The time-dependent distribution of FolateSiR-1 and its specific accumulation in KB tumors was determined using in vivo longitudinal fluorescence imaging.The PDT effect of FolateSiR-1 was evaluated in KB tumor bearing mice divided into four experimental groups:(1)FolateSiR-1(100μmol/L)alone;(2)FolateSiR-1(100μmol/L)followed by NIR irradiation(50 J/cm2);(3)NIR irradiation(50 J/cm2)alone;and(4)no treatment.Tumor volume measurement and immunohistochemical(IHC)and histological examinations of the tumors were performed to analyze the effect of PDT.RESULTS High FR expression was observed in the KB cells by WB,but not in the OVCAR-3 and A4 cells.Substantial FR-specific binding of FolateSiR-1 was observed by in vitro and in vivo fluorescence imaging.Cell viability imaging assays showed that NIR-PDT induced cell death in KB cells.In vivo longitudinal fluorescence imaging showed rapid peak accumulation of FolateSiR-1 in the KB tumors 2 h after injection.In vivo PDT conducted at this time point caused tumor growth delay.The relative tumor volumes in the PDT group were significantly reduced compared to those in the other groups[5.81±1.74(NIR-PDT)vs 12.24±2.48(Folate-SiR-1),vs 11.84±3.67(IR),vs 12.98±2.78(Untreated),at Day 16,P<0.05].IHC analysis revealed reduced proliferation marker Ki-67-positive cells in the PDT treated tumors,and hematoxylin-eosin staining revealed features of necrotic-and apoptotic cell death.CONCLUSION FolateSiR-1 has potential for use in PDT,and FR-targeted NIR-PDT may open a new effective strategy for the treatment of FR-overexpressing tumors.