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783: Modification of topoisomerases in cancer stem cells derived from MCF7 breast cancer cell line - clinical implications for cancer treatment
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EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
that rapidly releases DOX in regions where local tissue temperatures are elevated. Material and Method: 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine and 1,2 - distearoyl - sn - glycero - 3 - phosphoethanolamine - N - (amino(polyethylene glycol)-2000) were purchased from Avanti Polar Lipids. Results and Discussion: In vitro drug-release profiles, the amounts of DOX released from the ThermoDox and ammonium bicarbonate (ABC) liposomes at 37ºC were relatively low; nevertheless, when the environmental temperature was heated to 42ºC, significant amounts of their encapsulated DOX were released instantly. The lipid bilayer of ThermoDox enhances significantly the permeability of liposomal membranes at their phase transition temperature. Conversely, upon heating to 40ºC, ABC decomposes and generates CO2 bubbles, subsequently creating permeable defects in the lipid bilayer and ultimately facilitating the swift release of DOX. Finally, the ability of different liposomal formulations to suppress tumor growth was examined. The body weight of test mice was also evaluated since it reflects the general toxicity of different delivery formulations. The mice receiving the Lysolipid liposomes had a significantly higher toxicity than those treated with ABC liposomes. Analytical results demonstrate that the ABC liposomes, with their greater kinetic stability, delivered DOX to the tumor more effectively than the Lysolipid formulation, thereby reducing the toxicity caused by the nonspecificity of DOX. Conclusion: This study demonstrates the feasibility of using the proposed bubble-generating ABC liposomal system to release drugs locally under mild hyperthermia. The highly thermoresponsive properties of the ABC liposomes contribute to efforts to establish effective tumor-selective chemotherapy. No conflict of interest.
dissemination. Nanotechnology-based 3D scaffolds decorated with exosomes purified from ascitic fluid from ovarian cancer patients were used for fabrication of M-Trap device. In vitro dynamic cell-adhesion assays and in vivo bioluminescence imaging systems were used to evaluate the effect of M-Trap technology in peritoneal metastasis. Results: We here describe a novel approach in oncology trying to hamper the process of metastasis by interfering with this intense dialogue, and a probe of concept in a model of ovarian cancer intraperitoneal dissemination: (i) we characterized exosomes as components within the ascitic fluid of ovarian cancer patients owning the ability to dialogue with tumor cells and modulate their localization; (ii) we generated a nanotechnology-based capture device by embedding exosomes onto a 3D-scaffold (M-Trap) where metastatic tumor cells preferentially home; (iii) we demonstrated that M-Trap completely remodeled the peritoneal pattern of metastasis in an ovarian cancer model; (iv) we finally evaluated the impact of M-Trap on the outcome of the animal model with a benefit on survival. Conclusions: We present exosomes as key drivers of the crosstalk between metastatic tumor cells and their environment during massive peritoneal dissemination in ovarian cancer. We then created M-Trap device as an artificial pre-metastatic niche based on exosomes that might compete with natural niches for the capture of tumor cells metastasizing in the peritoneal cavity. In clinical setting, this would represent the capacity to modulate the pattern of metastatic dissemination, the alteration of the process of metastasis by preventing the generation of new foci, and the possibility to transform a systemic disease into a focalized disease where the therapeutic approaches would present again a reasonable effectiveness. No conflict of interest.
781 Transfection of chitosan/KillerRed/gPGA complex to intrinsically generate photosensitizer for photodynamic therapy
783 Modification of topoisomerases in cancer stem cells derived from MCF7 breast cancer cell line − clinical implications for cancer treatment E. Priel1 , R. Peleg1 . 1 Ben-Gurion University of the Negev, The Shraga Segal Department of Microbiology Immunology & Genetics, Beer-Sheva, Israel
C.C. Huang1 , Z.X. Liao1 , Y.C. Li1 , H.M. Lu1 , H.W. Sung1 . 1 National Tsing Hua University, Department of Chemical Engineering, Hsinchu, Taiwan Background: Photodynamic therapy (PDT) has received considerable attention as a therapeutic treatment for cancer; however, it is frequently accompanied by prolonged phototoxic reaction of the skin due to slow clearance of synthetic photosensitizers (PSs) administered externally. KillerRed, a genetically encoded red fluorescent protein, can efficiently produce reactive oxygen species (ROS) that can kill cells when exposed to appropriate light excitation. This study was designed to investigate the genetic use of pKillerRed-mem, delivered using complexes of chitosan (CS) and poly(g-glutamic acid) (gPGA), to intracellularly express a membrane-targeted KillerRed protein that can be used as a potential PS for PDT. Material and Methods: The pKillerRed-mem was obtained from Evrogen JSC (Moscow, Russia). Test complexes containing pKillerRed-mem (CS/pKillerRed/gPGA complexes) were prepared via an ionic-gelation method in an aqueous environment, using an N (amine groups of CS):P (phosphate groups of plasmid DNA):C (carboxyl groups of gPGA) molar ratio of 10:1:4. Results: Following transfection with CS/pKillerRed/gPGA complexes, a red fluorescence protein of KillerRed was clearly seen at the cellular membranes. When exposed to green-light irradiation, the KillerRed-positive cells produced an excessive amount of ROS in a time-dependent manner. Data from viability assays indicate that ROS have an important role in mediating KillerRedinduced cytotoxicity, apoptosis, and anti-proliferation, suggesting that KillerRed can be used as an intrinsically generated PS for PDT treatments. Conclusions: As delivered by ternary CS/pKillerRed/gPGA complexes, a genetically encoded red fluorescence KillerRed protein with a membranetargeted property can be expressed efficiently within cells. Upon photoactivation, KillerRed-positive cells produce excessive amounts of ROS, markedly decreasing their viability and proliferation by triggering cell apoptosis. With the targets of its PDT action, this intrinsically generated PS KillerRed potentially mediates precise light-induced cell apoptosis in disease cells. No conflict of interest. 782 M-Trap: Exosome-based capture of tumor cells as a new therapeutic technology in peritoneal metastasis M. Abal1 , A. De la Fuente1 , L. Alonso-Alconada1 , J. Cueva1 , R. LopezLopez1 . 1 Hospital Univ. Santiago De Compostela, Translational Medical Oncology (IDIS), Santiago de Compostela, Spain Background: The molecular and cellular bases that determine the stepwise process of metastasis suggest an intense dialogue of the primary tumor with the environment. Tissue specific metastasis and pre-metastatic niches are concepts that are beginning to illustrate an active role of carcinomas in the determination of the most adequate sites to colonize: signals emitted and sensed both from the tumor and from the environment govern the remodeling of targeted tissues for a favored reception of prone tumor cells disseminated from primary lesions. Material and Methods: Human SKOV3 ovarian tumor cells intraperitoneally injected in SCID mice were used as model system for ovarian peritoneal
Introduction: Accumulating evidence suggests that tumors are driven by a small population of cells, termed ‘cancer stem cells’ (CSCs), which may be resistant to current therapeutic approaches. In breast carcinoma, the CSCs have been identified as a CD44+ /CD24− cell population. These rare cells are able to grow as non-adherent sphere-like structures, ‘mammospheres’, which enables their isolation and expansion in culture. To design therapeutic strategies for the eradication of CSC it is important to investigate the modifications of essential enzymes like topoisomerases that are the target of several anti-cancer drugs. Method: The MCF7 human breast cancer cell line were grown on lowattachment dishes in the presence of 10% FCS or in a serum-free medium containing EGF, bFGF, and insulin and allowed to form spheres. Isolated mammospheres show enriched CD44+ /CD24− cell population, as verified by immunostaining and flow cytometry analyses. Topoisomerases activities were examined using specific methods. Results: Topoisomerase I (topo I) activity is decreased and topo II activity is increased in mammospheres compared to the adherent MCF7 cell line. No changes were observed in the relative levels of the enzyme proteins, suggesting possible posttranslational modifications that regulate their activities. Indeed, topo I activity in mammospheres is regulated, at least in part, by PARP-1, as observed by the recovery of topo I activity after treatment with PARP-1 inhibitor 3-Aminobenzamide. In accordance with the activity of topoisomerases, mammosphere-derived cells show reduced sensitivity to topo I inhibitors, CPT or TPT, and increased sensitivity to topo II inhibitor etoposide. However, intact mammospheres show increased resistance to both drugs. A combined treatment of intact mammospheres with either CPT and gefitinib, or etoposide and erlotinib, increased the anti-cancer effect of both drugs. These treatment mechanisms remain to be elucidated. Conclusions: In this study we demonstrate for the first time that in mammospheres, nuclear essential enzymes, such as topoisomerases, differ in their activity and regulation, compared to the non-CSC population, and therefore provide an explanation for the resistance/sensitivity of CSC to topoisomerase antagonists. In addition, the results of this study suggest that a combination of TK inhibitors with topoisomerase inhibitors might be effective treatments for breast cancer, providing a CSC eradication approach that might ensure long-term cancer-free survival. No conflict of interest.
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
that rapidly releases DOX in regions where local tissue temperatures are elevated. Material and Method: 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine and 1,2 - distearoyl - sn - glycero - 3 - phosphoethanolamine - N - (amino(polyethylene glycol)-2000) were purchased from Avanti Polar Lipids. Results and Discussion: In vitro drug-release profiles, the amounts of DOX released from the ThermoDox and ammonium bicarbonate (ABC) liposomes at 37ºC were relatively low; nevertheless, when the environmental temperature was heated to 42ºC, significant amounts of their encapsulated DOX were released instantly. The lipid bilayer of ThermoDox enhances significantly the permeability of liposomal membranes at their phase transition temperature. Conversely, upon heating to 40ºC, ABC decomposes and generates CO2 bubbles, subsequently creating permeable defects in the lipid bilayer and ultimately facilitating the swift release of DOX. Finally, the ability of different liposomal formulations to suppress tumor growth was examined. The body weight of test mice was also evaluated since it reflects the general toxicity of different delivery formulations. The mice receiving the Lysolipid liposomes had a significantly higher toxicity than those treated with ABC liposomes. Analytical results demonstrate that the ABC liposomes, with their greater kinetic stability, delivered DOX to the tumor more effectively than the Lysolipid formulation, thereby reducing the toxicity caused by the nonspecificity of DOX. Conclusion: This study demonstrates the feasibility of using the proposed bubble-generating ABC liposomal system to release drugs locally under mild hyperthermia. The highly thermoresponsive properties of the ABC liposomes contribute to efforts to establish effective tumor-selective chemotherapy. No conflict of interest.
dissemination. Nanotechnology-based 3D scaffolds decorated with exosomes purified from ascitic fluid from ovarian cancer patients were used for fabrication of M-Trap device. In vitro dynamic cell-adhesion assays and in vivo bioluminescence imaging systems were used to evaluate the effect of M-Trap technology in peritoneal metastasis. Results: We here describe a novel approach in oncology trying to hamper the process of metastasis by interfering with this intense dialogue, and a probe of concept in a model of ovarian cancer intraperitoneal dissemination: (i) we characterized exosomes as components within the ascitic fluid of ovarian cancer patients owning the ability to dialogue with tumor cells and modulate their localization; (ii) we generated a nanotechnology-based capture device by embedding exosomes onto a 3D-scaffold (M-Trap) where metastatic tumor cells preferentially home; (iii) we demonstrated that M-Trap completely remodeled the peritoneal pattern of metastasis in an ovarian cancer model; (iv) we finally evaluated the impact of M-Trap on the outcome of the animal model with a benefit on survival. Conclusions: We present exosomes as key drivers of the crosstalk between metastatic tumor cells and their environment during massive peritoneal dissemination in ovarian cancer. We then created M-Trap device as an artificial pre-metastatic niche based on exosomes that might compete with natural niches for the capture of tumor cells metastasizing in the peritoneal cavity. In clinical setting, this would represent the capacity to modulate the pattern of metastatic dissemination, the alteration of the process of metastasis by preventing the generation of new foci, and the possibility to transform a systemic disease into a focalized disease where the therapeutic approaches would present again a reasonable effectiveness. No conflict of interest.
781 Transfection of chitosan/KillerRed/gPGA complex to intrinsically generate photosensitizer for photodynamic therapy
783 Modification of topoisomerases in cancer stem cells derived from MCF7 breast cancer cell line − clinical implications for cancer treatment E. Priel1 , R. Peleg1 . 1 Ben-Gurion University of the Negev, The Shraga Segal Department of Microbiology Immunology & Genetics, Beer-Sheva, Israel
C.C. Huang1 , Z.X. Liao1 , Y.C. Li1 , H.M. Lu1 , H.W. Sung1 . 1 National Tsing Hua University, Department of Chemical Engineering, Hsinchu, Taiwan Background: Photodynamic therapy (PDT) has received considerable attention as a therapeutic treatment for cancer; however, it is frequently accompanied by prolonged phototoxic reaction of the skin due to slow clearance of synthetic photosensitizers (PSs) administered externally. KillerRed, a genetically encoded red fluorescent protein, can efficiently produce reactive oxygen species (ROS) that can kill cells when exposed to appropriate light excitation. This study was designed to investigate the genetic use of pKillerRed-mem, delivered using complexes of chitosan (CS) and poly(g-glutamic acid) (gPGA), to intracellularly express a membrane-targeted KillerRed protein that can be used as a potential PS for PDT. Material and Methods: The pKillerRed-mem was obtained from Evrogen JSC (Moscow, Russia). Test complexes containing pKillerRed-mem (CS/pKillerRed/gPGA complexes) were prepared via an ionic-gelation method in an aqueous environment, using an N (amine groups of CS):P (phosphate groups of plasmid DNA):C (carboxyl groups of gPGA) molar ratio of 10:1:4. Results: Following transfection with CS/pKillerRed/gPGA complexes, a red fluorescence protein of KillerRed was clearly seen at the cellular membranes. When exposed to green-light irradiation, the KillerRed-positive cells produced an excessive amount of ROS in a time-dependent manner. Data from viability assays indicate that ROS have an important role in mediating KillerRedinduced cytotoxicity, apoptosis, and anti-proliferation, suggesting that KillerRed can be used as an intrinsically generated PS for PDT treatments. Conclusions: As delivered by ternary CS/pKillerRed/gPGA complexes, a genetically encoded red fluorescence KillerRed protein with a membranetargeted property can be expressed efficiently within cells. Upon photoactivation, KillerRed-positive cells produce excessive amounts of ROS, markedly decreasing their viability and proliferation by triggering cell apoptosis. With the targets of its PDT action, this intrinsically generated PS KillerRed potentially mediates precise light-induced cell apoptosis in disease cells. No conflict of interest. 782 M-Trap: Exosome-based capture of tumor cells as a new therapeutic technology in peritoneal metastasis M. Abal1 , A. De la Fuente1 , L. Alonso-Alconada1 , J. Cueva1 , R. LopezLopez1 . 1 Hospital Univ. Santiago De Compostela, Translational Medical Oncology (IDIS), Santiago de Compostela, Spain Background: The molecular and cellular bases that determine the stepwise process of metastasis suggest an intense dialogue of the primary tumor with the environment. Tissue specific metastasis and pre-metastatic niches are concepts that are beginning to illustrate an active role of carcinomas in the determination of the most adequate sites to colonize: signals emitted and sensed both from the tumor and from the environment govern the remodeling of targeted tissues for a favored reception of prone tumor cells disseminated from primary lesions. Material and Methods: Human SKOV3 ovarian tumor cells intraperitoneally injected in SCID mice were used as model system for ovarian peritoneal
Introduction: Accumulating evidence suggests that tumors are driven by a small population of cells, termed ‘cancer stem cells’ (CSCs), which may be resistant to current therapeutic approaches. In breast carcinoma, the CSCs have been identified as a CD44+ /CD24− cell population. These rare cells are able to grow as non-adherent sphere-like structures, ‘mammospheres’, which enables their isolation and expansion in culture. To design therapeutic strategies for the eradication of CSC it is important to investigate the modifications of essential enzymes like topoisomerases that are the target of several anti-cancer drugs. Method: The MCF7 human breast cancer cell line were grown on lowattachment dishes in the presence of 10% FCS or in a serum-free medium containing EGF, bFGF, and insulin and allowed to form spheres. Isolated mammospheres show enriched CD44+ /CD24− cell population, as verified by immunostaining and flow cytometry analyses. Topoisomerases activities were examined using specific methods. Results: Topoisomerase I (topo I) activity is decreased and topo II activity is increased in mammospheres compared to the adherent MCF7 cell line. No changes were observed in the relative levels of the enzyme proteins, suggesting possible posttranslational modifications that regulate their activities. Indeed, topo I activity in mammospheres is regulated, at least in part, by PARP-1, as observed by the recovery of topo I activity after treatment with PARP-1 inhibitor 3-Aminobenzamide. In accordance with the activity of topoisomerases, mammosphere-derived cells show reduced sensitivity to topo I inhibitors, CPT or TPT, and increased sensitivity to topo II inhibitor etoposide. However, intact mammospheres show increased resistance to both drugs. A combined treatment of intact mammospheres with either CPT and gefitinib, or etoposide and erlotinib, increased the anti-cancer effect of both drugs. These treatment mechanisms remain to be elucidated. Conclusions: In this study we demonstrate for the first time that in mammospheres, nuclear essential enzymes, such as topoisomerases, differ in their activity and regulation, compared to the non-CSC population, and therefore provide an explanation for the resistance/sensitivity of CSC to topoisomerase antagonists. In addition, the results of this study suggest that a combination of TK inhibitors with topoisomerase inhibitors might be effective treatments for breast cancer, providing a CSC eradication approach that might ensure long-term cancer-free survival. No conflict of interest.