David Shrayer

Histologic Progression of B16 F10 Metastatic Melanoma in C57BL/6 Mice Over a Six Week Time Period : Distant Metastases before Local Growth

B16F10 murine metastatic melanoma in the tails of C57BL/6 mice after subcutaneous injection is a well‐established model. However, the histologic progression from injected cells to established local growth of melanoma has not been studied systematically. We therefore have investigated the histologic changes and growth of B16F10 melanoma at the injection site over a six‐week time period. One million B16F10 melanoma cells were injected subcutaneously into the dorsal aspect of tails of C57/BL6 mice. Mice were sacrificed at zero, 12, 24, 48, 72 and 96 hours, and at one, two, three, four, five and six weeks. Sections were stained with Hematoxylin and Eosin and immunostained with antibodies to S100. Beginning at time zero, melanoma cells were detected between the dermis and the myofascial bundle of the tail. At week four, distant metastases were clinically evident in the inguinal region, though injection site tumors did not become evident until week six. Histological analysis showed melanoma cells at the injecion site at all time periods and no injection site tumor until week six. Indeed, the injection site tumors arose two weeks after distant metastases were clinically apparent. A progression of S100 positivity was also observed. S100 immunostaining was negative in all injection site of B16F10 cells until the cells underwent a morphologic change from small and monomorphic at the injection site, to large, pleomorphic cells at week six in the clinically evident injection site tumors. Inguinal metastases were also S100 positive at week four, though injection site cells were still S100 negative. We conclude that in this particular established model for melanoma, local growth at the injection site may occur after the development of regional metastases. This may prove to be a good model for investigation of local growth of tumor cells and their interaction with metastatic lesions.

Immunization of mice with melanoma cells transfected to secrete the superantigen, staphylococcal enterotoxin A

Abstract Immunization of mice with a melanoma vaccine coupled with staphylococcal enterotoxin A (SEA) inhibits the growth of primary melanoma tumors in mice. We have now successfully transfected B16 cells with the sea gene and have immunized C57BL/6 mice subcutaneously once per week for 4 weeks prior to tumor challenge with vaccines of irradiated B16 cells or, 4 weeks following tumor challenge of naïve mice with B16 cells, with irradiated B16 cells transfected with the sea gene. Primary tumor growth following both types of treatments was inhibited significantly. To characterize immune responses to these immunogens, we examined the production of antibodies to the B700 melanoma antigen, the stimulation of endogenous IL-2 production, the expression of CD4, CD8, Vβ and CD25 T cell markers, and the induction of NK activity. At 4 weeks following immunization of mice, there was a significant increase (P<0.05) in levels of interleukin-2 production by splenocytes from mice immunized with SEA-secreting B16 cells or with the parental B16 cells, compared to controls. Levels of antibodies to the B700 melanoma antigen were also significantly higher in mice immunized with the SEA-secreting B16 cells, as was expression of CD4, CD8, CD25 and Vβ T cell antigens, particularly CD4. Natural killer cell activity (at various E:T ratios) was tenfold higher in splenocytes of mice immunized with SEA-secreting B16 cells, and fivefold higher in mice immunized with the parental B16 cells, compared to controls. These data confirm the possibility of using irradiated murine melanoma cells transfected to secrete SEA in vaccines targeted at preventing the development and growth of melanoma.

Abstract B104: Liposomal C6 Ceramide appears to potentiate chemotoxicity of paclitaxel, gemcitabine and cetuximab against aggressive pancreatic cancer.

Introduction: Pancreatic adenocarcinoma a highly lethal malignancy (5 yr survival ∼ 5%) is poorly responsive to all chemotherapy, in part related to presence of chemo resistant pathways: prosurvival (AKT/PI3K/mTOR and mutant KRAS. We have previously demonstrated reversal of these resistant pathways by C6 Ceramide and now are pursuing optimum intracellular delivery using selected liposomal preparations ofC6Ceramide. Methods: C6 Ceramide housed in 4 different liposomal vesicle formulations were tested against pancreatic cell lines L3.6, PANC1 and MIA, using combination therapy with chemotherapy agents Gemcitabine, Paclitaxel and Cetuximab with determination of the MTT anti tumor response. Liposomal preparations included : (#1)18:1 PC(1,2-dioleoyl-sn-glycero-3-phosphocholine; (#3),18:0 PC (1,2-distearoyl-sn-glycero-3-phosphocholine;(#5) DPGG (1,2-dipalmitoyl-sn-glycero-3-galloyl); and (#7) 18:0 PEG2 PE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N [methoxy(polyethylene glycol)-2000. (#1)18:1 PC- has low transition temp and is fluid at bodyT*(# 3). 18:0 PC- has high transition Temperature(T*) is rigid at body T*;#5 DPGG- avoids RE System (is fluid at bodyT*, induces long circulations)#7. 18:0 PEG 2 PE - industry standard (also avoids RE system) Biologic effects of the 4 lipid formulations with/without C6 Ceramide were initially assessed invitro. Results: Dose response MTT assay demonstrated no distinctive response of C6 combined liposomes vesicles # 1 & 3, but confirmed high activity in preparations #5 & 7. ![Figure][1] Conclusion: Liposomal C6 preparations show high activity in potentiating chemo toxicity by Gemcitabine, Paclitaxel and Cetuximab against Pancreatic Cancer. Confirmatory in vivo studies are in process. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B104. Citation Format: Harold J. Wanebo, Cong Cao, Shaun Lu, David Shrayer, Yinsheng Wan, Wayne Bowen, Wayne Bowen. Liposomal C6 Ceramide appears to potentiate chemotoxicity of paclitaxel, gemcitabine and cetuximab against aggressive pancreatic cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B104. [1]: pending:yes

Abstract 600: Liposomal C6 ceramide appears to potentiate enhanced chemotoxicity of pancreatic cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Introduction: Pancreatic adenocarcinoma a highly lethal malignancy (5 yr survival ∼ 5%) is poorly responsive to all chemotherapy, in part related to presence of chemo resistant pathways: prosurvival (AKT/PI3K/mTOR and mutant KRAS. We previously demonstrated reversal of these resistant pathways by C6 Ceramide and now pursue optimum intracellular delivery using selected liposomal preparations of C6 Ceramide. Methods: C6 Ceramide(C6Cer) in 4 different liposomal vesicle formulations were tested by MTT assay against pancreatic cell lines L3.6, PANC1 and MIA, combined with chemotherapy: Gemcitabine, Paclitaxel and Cetuximab.Liposomal preparations included : (#1)18:1 PC (1,2-dioleoyl-sn-glycero-3-phosphocholine; (#3),18:0 PC (1,2-distearoyl-sn-glycero-3-phosphocholine;(#5) DPGG (1,2-dipalmitoyl-sn-glycero-3-galloyl); and (#7) 18:0 PEG2 PE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy(polyethylene glycol)-2000. (#1)18:1 PC- has low transition temp.(T*) and is fluid at body T*(# 3). 18:0 PC-is rigid with high transition (T*) . #5 DPGG- avoids RE System (is fluid at body T*, induces long circulations)#7. 18:0 PEG 2 PE - industry standard (also avoids RE system).Biologic effects of the 4 lipid formulations with/without C6 Ceramide were initially assessed invitro. Results: Dose response MTT assay demonstrated no distinctive response of C6 combined liposomes vesicles # 1 & 3, but confirmed high activity in preparations #5 & 7. View this table: MTT Response Conclude: Liposomal C6 preparations show high activity in potentiating chemo toxicity by Gemcitabine, Paclitaxel and Cetuximab against Pancreatic Cancer. Confirmatory in vivo studies are in process. Citation Format: Harold J. Wanebo, Cao Cong, Li Shu, David Shrayer, Yeushung Wan, Wayne Bowen. Liposomal C6 ceramide appears to potentiate enhanced chemotoxicity of pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 600. doi:10.1158/1538-7445.AM2013-600