Rhiana Menen

Tumor-specific fluorescence antibody imaging enables accurate staging laparoscopy in an orthotopic model of pancreatic cancer.

BACKGROUND/AIMS Laparoscopy is important in staging pancreatic cancer, but false negatives remain problematic. Making tumors fluorescent has the potential to improve the accuracy of staging laparoscopy. METHODOLOGY Orthotopic and carcinomatosis models of pancreatic cancer were established with BxPC-3 human pancreatic cancer cells in nude mice. Alexa488-antiCEA conjugates were injected via tail vein 24 hours prior to laparoscopy. Mice were examined under bright field laparoscopic (BL) and fluorescence laparoscopic (FL) modes. Outcomes measured included time to identification of primary tumor for the orthotopic model and number of metastases identified within 2 minutes for the carcinomatosis model. RESULTS FL enabled more rapid and accurate identification and localization of primary tumors and metastases than BL. Using BL took statistically significantly longer time than FL (p<0.0001, fold change and 95% CI for BL vs. FL: 8.12 (4.54,14.52)). More metastatic lesions were detected and localized under FL compared to BL and with greater accuracy, with sensitivities of 96% vs. 40%, respectively, when compared to control. FL was sensitive enough to detect metastatic lesions <1mm. CONCLUSIONS The use of fluorescence laparoscopy with tumors labeled with fluorophore-conjugated anti-CEA antibody permits rapid detection and accurate localization of primary and metastatic pancreatic cancer in an orthotopic model. The results of the present report demonstrate the future clinical potential of fluorescence laparoscopy.

Submillimeter-Resolution Fluorescence Laparoscopy of Pancreatic Cancer in a Carcinomatosis Mouse Model Visualizes Metastases Not Seen with Standard Laparoscopy

BACKGROUND Staging laparoscopy can visualize peritoneal and liver metastases in pancreatic cancer otherwise undetectable by preoperative imaging. However, false-negative rates may be as high as 18%-26%. The aim of the present study was to improve detection of metastatic pancreatic cancer with the use of fluorescence laparoscopy (FL) in a nude-mouse model with the tumors expressing green fluorescent protein (GFP). METHODS The carcinomatosis mouse model of human pancreatic cancer was established by intraperitoneal injections of green fluorescent protein-expressing MiaPaca-2 human pancreatic cancer cells into 6-week-old female athymic mice. Two weeks later, mice underwent diagnostic laparoscopy. Laparoscopy was performed first under standard brightfield lighting, followed by fluorescent lighting. The number of metastatic foci identified within the four quadrants of the peritoneal cavity was recorded. After laparoscopy, the animals were sacrificed, opened, and imaged with the OV-100 Small Animal Imaging system as a positive control to identify metastasis. Tumors were collected and processed for histologic review. RESULTS FL enabled visualization of pancreatic cancer metastatic foci not visualized with standard brightfield laparoscopy (BL). Under FL, in 1 representative mouse, 26 separate micrometastatic lesions were identified. In contrast, only very large tumors were seen using BL. Use of the OV-100 images, as positive controls, confirmed the presence of tumor foci. FL thus allowed identification and exact localization of submillimeter tumor foci. Such small-sized tumor foci were not distinguished from surrounding tissue under BL. All malignant lesions were histologically confirmed. CONCLUSIONS The use of FL enables the identification of tumor foci that cannot be seen with standard laparoscopy. The technology described in this report has important potential for the clinical development of FL.

The Tumor-Educated-Macrophage Increase of Malignancy of Human Pancreatic Cancer Is Prevented by Zoledronic Acid

We previously defined macrophages harvested from the peritoneal cavity of nude mice with subcutaneous human pancreatic tumors as “tumor-educated-macrophages” (Edu) and macrophages harvested from mice without tumors as “naïve-macrophages” (Naïve), and demonstrated that Edu-macrophages promoted tumor growth and metastasis. In this study, Edu- and Naïve-macrophages were compared for their ability to enhance pancreatic cancer malignancy at the cellular level in vitro and in vivo. The inhibitory efficacy of Zoledronic acid (ZA) on Edu-macrophage-enhanced metastasis was also determined. XPA1 human pancreatic cancer cells in Gelfoam co-cultured with Edu-macrophages proliferated to a greater extent compared to XPA1 cells cultured with Naïve-macrophages (P = 0.014). XPA1 cells exposed to conditioned medium harvested from Edu culture significantly increased proliferation (P = 0.016) and had more migration stimulation capability (P<0.001) compared to cultured cancer cells treated with the conditioned medium from Naïve. The mitotic index of the XPA1 cells, expressing GFP in the nucleus and RFP in the cytoplasm, significantly increased in vivo in the presence of Edu- compared to Naïve-macrophages (P = 0.001). Zoledronic acid (ZA) killed both Edu and Naïve in vitro. Edu promoted tumor growth and metastasis in an orthotopic mouse model of the XPA1 human pancreatic cancer cell line. ZA reduced primary tumor growth (P = 0.006) and prevented metastasis (P = 0.025) promoted by Edu-macrophages. These results indicate that ZA inhibits enhanced primary tumor growth and metastasis of human pancreatic cancer induced by Edu-macrophages.

Detection of Colon Cancer Metastases With Fluorescence Laparoscopy in Orthotopic Nude Mouse Models

OBJECTIVE To improve detection of colon cancer metastases using fluorescence laparoscopy (FL). DESIGN An orthotopic mouse model of human colon cancer was established by intracecal injection of HCT-116 human colon cancer cells expressing green fluorescent protein into 12 mice. One group modeled early disease and the second modeled late metastatic disease. For the early-disease model, 2 weeks after implantation, 6 mice underwent 2 modalities of laparoscopy: bright field laparoscopy (BL) and FL. The number of metastases identified within each of the 4 abdominal quadrants was recorded with both laparoscopy modalities. This process was repeated in the late-metastatic disease group 4 weeks after implantation. All animals were then humanely sacrificed and imaged using open fluorescence laparoscopy (OL) as a positive control to identify metastases. SETTING Basic science laboratory. PARTICIPANTS Twelve female, 6-week-old nude mice. INTERVENTIONS Detection of tumor foci by FL compared with BL. MAIN OUTCOME MEASURES Number of tumors identified in each quadrant. RESULTS Fluorescence laparoscopy enabled superior visualization of colon cancer metastases compared with BL in the early (P = .03) and late (P = .002) models of colon cancer. Compared with OL, BL was significantly inferior in the early (P = .04) and late (P < .001) groups. Fluorescence laparoscopy was not significantly different from OL in the early (P = .85) or late (P = .46) group. Thus, FL allowed identification of micrometastases that could not be distinguished from surrounding tissue using BL. CONCLUSIONS The use of FL enables identification of metastases that could not be visualized using standard laparoscopy. This report illustrates the important clinical potential for FL in the surgical treatment of cancer.

Abstract 2265: Sub-fractions of conditioned medium, from hypoxia-induced cells with multipotent potential, exhibit a significant anti-oncogenic activity

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We have previously shown that a human soluble cell conditioned media (CCM) from hypoxia-induced, potentially multipotent cells, grown in a perfusion bioreactor system inhibits tumor cell growth in vitro and in two animal models. Here we present data that indicate that a low molecular weight sub-fraction from the CCM has an increased anti-oncogenic activity. This activity was demonstrated in orthotopic pancreatic models, nRAS melanoma assays and over 10 additional human cancer cell lines in vitro. In an orthotopic BxPC3 pancreatic cancer model, there was a statistically significant improvement in survival following weekly intravenous injections of 0.1ml of sub fractioned CCM, as well as a significant improvement with 0.5ml intraperitoneal injections post-resection, as measured by Kaplan Meier analysis (p<0.05) (figures below). Uveal and nRAS melanoma cancer cell growth was inhibited by greater than 90% in in-vitro proliferation assays. The mechanism of action of the CCM is the induction of apoptosis through the upregulation of Caspase 3 and 9, as demonstrated by immunostaining of Annexin V and immunoblot ayalysis. In a Miapaca nude mouse daily dose study, tumor mass and metastatic extent was substantially reduced in treated mouse versus the control. Further, in a BXPC3 nude mouse daily dose study, 60% (p<0.05) of treated mice were tumor free after four weeks of treatment, while only 10% were tumor free in the control group . Little or no apparent drug toxicity was observed. These results indicate that the CCM sub-fraction could be a useful raw material for a treatment for a large range of neoplastic diseases. Citation Format: Emmett Pinney, Mayra Montes-Camacho, Kayler Brintle, Christian Posch, Rhiana Menen, David Easter, Michael Bouvet, Robert Hoffman, Gail Naughton. Sub-fractions of conditioned medium, from hypoxia-induced cells with multipotent potential, exhibit a significant anti-oncogenic activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2265. doi:10.1158/1538-7445.AM2014-2265

Abstract 3130: Zoledronic acid inhibits proliferation and metastasis of human pancreatic cancer in the patient-derived orthotopic xenograft (PDOX) model by targeting tumor-educated macrophages

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Macrophages harvested from the peritoneal cavity of nude mice with subcutaneous human pancreatic tumors were defined as “tumor-educated macrophages” (EMφ) and macrophages harvested from mice without tumors were defined as “naive macrophages” (NMφ). EMφ promote tumor growth and metastasis. The aim of the present study was to determine the efficacy of Zoledronic acid (ZA) on EMφ in a pancreatic cancer patient derived orthotopic xenograft (PDOX) nude mouse model. In this study, EMφ and NMφ were compared for their ability to enhance tumor progression. We initially demonstrated that the cancer cells exposed to the conditioned medium harvested from EMφ culture significantly increased proliferation (p = 0.016) and had more migration stimulation capability (p < 0.001) compared to cultured cancer cells treated with the conditioned medium from NMφ. Next, we examined the efficacy of ZA on EMφ, and NMφ and found that ZA had the ability to kill both EMφ and NMφ in vitro. We then demonstrated that EMφ promoted tumor growth and metastasis in an orthotopic mouse model of a human pancreatic cancer cell line, ZA reduced the tumor growth (p = 0.006) and metastasis (p = 0.025) promoted by EMφ. Finally, we examined the efficacy of ZA for pancreatic cancer in the PDOX model, and found that the combination of gemcitabine (GEM) and ZA reduced tumor weight (p = 0.016) and tumor growth (p = 0.005) compared to GEM alone. ZA alone reduced metastasis (p = 0.009). These results suggest that ZA inhibits the proliferation and the metastasis of human pancreatic cancer by targeting EMφ. Citation Format: Yukihiko Hiroshima, Mohamed K. Hassenein, Rhiana Menen, Matthew H.g. Katz, Jason B. Fleming, Sho Sato, Takashi Murakami, Mako Yamamoto, Fuminari Uehara, Shinji Miwa, Shuya Yano, Masashi Momiyama, Ali Maawy, Takashi Chishima, Kuniya Tanaka, Michael Bouvet, Itaru Endo, Robert M. Hoffman. Zoledronic acid inhibits proliferation and metastasis of human pancreatic cancer in the patient-derived orthotopic xenograft (PDOX) model by targeting tumor-educated macrophages. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3130. doi:10.1158/1538-7445.AM2014-3130

Abstract 1539: Tumor-educated macrophages promote proliferation of human pancreatic cancer cells in vitro and in vivo.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Macrophages promote tumor growth by stimulating tumor-associated angiogenesis, cancer-cell invasion, migration, and intravasation, as well as suppression of antitumor immune responses. We previously defined the macrophages which were harvested from the peritoneal cavity of nude mice with subcutaneous human pancreatic tumors as “tumor-educated macrophages (EMϕ)” and demonstrated EMϕ promoted tumor growth and metastasis in orthotopic mouse models. The aim of this study is to elucidate the mechanism of tumor promotion by EMϕ. Transgenic nude mice ubiquitously-expressing GFP were injected subcutaneously with the human pancreatic cancer cell line XPA1 stably expressing RFP in the cytoplasm and GFP in the nucleus. GFP-expressing macrophages from the GFP mice with the dual-color pancreatic tumor were harvested and defined as EMϕ. Macrophages were also harvested from transgenic GFP mice without tumors and identified as “naive macrophages (NMϕ)”. EMϕ and NMϕ were compared for their ability to enhance cancer-cell proliferation. Using a skin-flap-window imaging model, the mitotic index of the cancer cells significantly increased after injection of EMϕ compared to NMϕ (p = 0.001). Next, we cultured cancer cells in 3-D on Gelfoam® and found that the cancer cells cultured with EMϕ proliferated to a greater extent compared to those cultured with NMϕ (p = 0.014). We also found that the cancer cells cultured in the conditioned medium harvested from EMϕ had enhanced proliferated compared to cancer cells cultured in the conditioned medium from NMϕ (p = 0.016). These results suggest that EMϕ secrete factors which increase cancer cell growth and can serve as a novel therapeutic target for pancreatic cancer. Citation Format: Yukihiko Hiroshima, Masashi Momiyama, Mohamed Hassanein, Ali Maawy, Rhiana Menen, Takashi Chishima, Kuniya Tanaka, Michael Bouvet, Itaru Endo, Robert M. Hoffman. Tumor-educated macrophages promote proliferation of human pancreatic cancer cells in vitro and in vivo . [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 1539. doi:10.1158/1538-7445.AM2013-1539

Abstract 1690: Tumor-educated macrophages promote tumor growth and peritoneal metastasis in an orthotopic nude mouse model of human pancreatic cancer

There is a strong association between poor survival and increased macrophage density in many cancers. In the current study, we determined whether macrophages from tumor-bearing mice (tumor-educated macrophages) had greater tumor-promoting capability than macrophages from non-tumor-bearing mice. Ten transgenic nude mice ubiquitously-expressing GFP were injected subcutaneously with the human pancreatic cancer cell line, BXPC3 stably expressing RFP. GFP-expressing macrophages from tumor-bearing transgenic GFP mice were harvested and defined as “tumor-educated macrophages”. Macrophages were also harvested from transgenic GFP mice (n=10) without subcutaneous tumors and identified as “naive macrophages.” Three groups of mice were studied: 1) A control group without addition of macrophages (n=10); 2) A naive group, with weekly intraperitoneal (ip) injection of 106 naive macrophages per mouse (n=10); and 3) A tumor-educated group, with weekly ip injection of 106 tumor-educated macrophages per mouse (n=10). The study ended with termination of the tumor-educated group after 8 weeks due to a pre-morbid state identified in all mice. At this time, all mice in each of the three study arms were terminated, imaging was performed, and total tumor weight was obtained. In the control group, the average primary tumor weighed 668 mg; only three mice (30%) developed peritoneal metastases with an average weight of 241 mg. The naive-macrophage group had an average tumor weight of 823 mg (p=0.51); 50% developed peritoneal metastases with an average weight of 975 mg (p=0.029). The tumor-educated-macrophage group had an average primary tumor weight of 2095 mg (p=0.001); 75% of mice developed peritoneal metastases with an average weight of 2135 mg (p=0.008). When comparing naive- to tumor-educated- groups, primary tumor weight was significantly greater in the tumor-educated group (p=0.003), and the average weight of metastasis was 2.2 times greater in the tumor educated group, however this did not reach statistical significance (p = 0.17). When comparing naive- to tumor-educated-groups, there was no statistically significant difference with regard to body weight (p=0.87), or weight of metastasis (p=0.68); however primary tumor weight was significantly greater in the tumor-educated group (p=0.013). Tumor-educated-macrophages specifically promote tumor growth progression in an orthotopic nude mouse model of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1690. doi:1538-7445.AM2012-1690

Abstract 2689: Inhibition of metastasis of circulating tumor cells by extracellular matrix secreted by human embryonic cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL We have previously demonstrated the increased metastatic potential of human prostate cancer PC-3 CTCs compared to their parental counterparts in both chick embryo and mouse models. In the present study, we tested the effects of human embryonic extracellular matrix (ECM), secreted by human embryonic cells including conditioned medium (CM) and a semi-solid form (hECM), on metastasis of PC-3 CTC in the chick embryo model. The chorioallentoic membrane (CAM) of 18 chicken embryos were inoculated with either 106 PC-3 human prostate cancer cells or PC-3 CTCs, both stably expressing GFP. Twelve hours later, embryos were divided into 6 groups each containing three embryos: PC-3 parental control; PC-3 + CM; PC-3 + hECM; CTC control; CTC + CM; and CTC+ hECM. Twelve hours following inoculation of the cells, a single dose of 100 μL of either treatment was given to the appropriate group. Embryo brains were removed on day 8 post-inoculations, and processed for cryo-sectioning, generating 3 slides of brain tissue per embryo taken at various depths. Imaging was performed using the IV-100 scanning laser microscope (Olympus Corp, Tokyo, Japan) in order to count metastatic foci. PC-3 controls had an average of 11.1 metastatic foci compared to 2.55 in the PC-3 + hECM group (p< 0.0001) and 2.76 in the PC-3 + CM group (p< 0.0001) The treatment showed even greater response on the CTC cells which an average of 30.9 metastatic foci in the CTC controls compared to 4.38 in the CTC + hECM group (p< 0.0001) and 4.18 in the CTC + CM group (p< 0.0001). Thus, human embryonic secreted ECM compound drastically decreased the metastatic potential of human prostate cancer CTCs in the chick embryo model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2689. doi:1538-7445.AM2012-2689

Abstract 2676: A secreted tissue engineered extracellular matrix (shECM) prevents recurrence in post-resection pancreatic cancer models

Pancreatic cancer is a highly treatment-resistant cancer. Gemcitabine, which is first-line therapy, results in toxicity without meaningful survival benefit. We report here that a extracellular matrix (ECM) secreted by human embryone fibroblasts in culture inhibit human pancreatic cancer growth and post-surgical recurrence in an orthotopic nude mouse models. Post-resection, all untreated control mice had recurrent disease. In contrast, only 2 of 8 mice (p=0.004) in the treatment group had palpable masses after 6 weekly intravenous treatments. In a second study, targeting the one surgical site with ECM treatment followed by 8 weekly i.p. treatments of ECM, 7 of 8 treated mice survived beyond 10 weeks while none of the untreated control mice survival 8 weeks. No measurable toxicity was observed in the treatment group. These results demonstrate the potential of ECM treatment of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2676. doi:1538-7445.AM2012-2676