NMRI Nude Mice Research Articles

Visible light and/or UVA offer a strong amplification of the anti-tumor effect of curcumin

Curcumin, a dietary pigment from the plant Curcuma longa, inhibits cell proliferation and induces apoptosis in different cell lines. The therapeutic benefit is hampered by a very low absorption after trans-dermal or oral application. Therefore, great efforts were undertaken to enhance the effectiveness of curcumin. Recently, it was demonstrated that curcumin offers the described effects also at low concentrations (0.2–1 μg/ml) when applied in combination with UVA or visible light. The efficacy of this combination was shown in human epidermal keratinocytes and in a panel of other cell species in vitro as well as in a xenograft tumor model with A431 tumor cells injected subcutaneously in the flanks of NMRI nude mice in vivo. The treatment of keratinocytes with curcumin and light resulted in the inhibition of cell growth, and in the induction of apoptosis, whereas no toxic cell membrane damage was detectable. The treatment of tumor bearing nude mice with curcumin and visible light resulted in reduced tumor volumes, reduced proliferation rates, and the induction of apoptosis in the tumors. On the molecular level inhibition of extracellular regulated kinases 1/2 and epidermal growth factor receptor was observed which may aid to inhibition of proliferation and induction of apoptosis. This review covers the experiences of the new combination treatment of human tumors.

Abstract 68: Targeting STAT3 in vitro and in vivo reveals a novel therapeutic strategy to sensitize colorectal cancer cells to chemoradiotherapy.

Abstract Introductory sentence: Increased activity of signal transducer and activator of transcription 3 (STAT3) is common in human malignancies, including colorectal cancers. Recently, we reported that STAT3 expression correlated with resistance to 5-fluorouracil (5-FU) based chemoradiotherapy. This is of considerable clinical relevance, because a large proportion of rectal cancers are resistant to preoperative multimodal treatment. We therefore examined whether STAT3 contributes to resistance to chemoradiotherapy. Experimental procedures: STAT3 mRNA and protein expression levels were determined in 12 colorectal cancers cell lines. STAT3 was inhibited using two different siRNAs and a small-molecular inhibitor (STATTIC) in the cell lines SW480 and SW837. Successful RNAi-mediated silencing of STAT3 or inhibition of phosphoSTAT3(Tyr705) was detected by Western blot and reduction of transcription factor activity was measured by a luciferase reporter assay. Additionally, we established doxycycline-inducible stable shRNA single cell populations and a non-silencing shRNA (shNEG) in SW480. To test the influence of STAT3 knock down or inhibition, clonogenic survival assays were performed. Therefore, RNAi or inhibitor treated cells were exposed to chemoradiotherapy using 3μM 5-FU and X-ray-irradiation at 1, 2, 4, 6, and 8 Gy. Finally, we tested the effect of a chemoradiotherapy combined with STATTIC treatment in a SW837 xenograft model in NMRI nude mice. To verify the sensitizing effect of STATTIC, tumor growth was recorded and growth delay assays were performed. Data: STAT3 was overexpressed in resistant cells at mRNA and protein level. siRNA transfected SW480, SW837, and SW480shRNA single cell clones showed a significant reduction of STAT3 protein and transcription factor activity after 96 hours. STATTIC inhibition led to a decreased phosphorylation of STAT3 after 1 hour. The silencing/inhibition resulted in a significantly increased chemoradiosensitivity with dose-reduction factors of 1.3 to 2.5 at a surviving fraction of 0.37. In vivo, additional STAT3 inhibition during chemoradiotherapy led to a profound chemoradiosensitization effect and a significant tumor growth delay in STATTIC treated mice. Survival of these mice was also enhanced, if compared to the control group. Conclusions: STAT3 is highly overexpressed in resistant colorectal cancer cells, and silencing or inhibition of STAT3 leads to a significantly increased chemoradiosensitivity in vitro and in vivo. This highlights the potential relevance of STAT3 for mediating treatment resistance and provides a first proof of concept that STAT3 represents a novel molecular target in rectal cancer to sensitize a priori resistant colorectal tumor cells to chemoradiotherapy. Citation Format: Melanie Spitzner, Birte Roesler, Christian Bielfeld, Carolin Herzberg, Georg Emons, Jochen Gaedcke, Margret Rave-Fränk, Tim Beißbarth, Thomas Ried, B. Michael Ghadimi, Marian Grade. Targeting STAT3 in vitro and in vivo reveals a novel therapeutic strategy to sensitize colorectal cancer cells to chemoradiotherapy. [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 68. doi:10.1158/1538-7445.AM2013-68

Abstract 3817: The potential oncogenic significance of Wip1 in neuroblastoma.

Abstract Background: In neuroblastoma (NB) 17q+ is the most powerful genetic predictor of adverse clinical outcome. We found aberrations of chromosome 17 in 85% in a national study of NB with 17q+ in 46% and whole 17 gain in 39%. Gain of 17q correlated with poor survival in our population-based material. In NB gain of wild-type p53-induced phosphatase 1 (Wip1) at 17q23 is frequently detected. Wip1 is a serine/threonine phosphatase and was described as a gatekeeper in the Mdm2-p53 regulatory loop by promoting Mdm2-mediated proteolysis of p53. Methods: Comparative genomic hybridization (CGH) was used to analyze primary NB tumors and cell lines. NB cells were examined for Wip1 expression using immunoblotting, immunohistochemistry and qPCR. Stable Wip1 knockdown NB cells were generated by shRNA transfections. H2AX phosphorylation and clonogenicity of Wip1 knockdown were examined using flow cytometry and clonogenic assay, respectively. In vivo, Wip1 knocked- and non-silenced cells were injected in nude NMRI mice. Tumor formation was followed by daily evaluation by digital caliper. Results: Detailed CGH analysis revealed that Wip1 is present in at least one extra genomic copy in all 63 tumor samples containing 17q gain. Results from our wide range of NB cell lines demonstrate 17q aberrations in all samples. These findings have been confirmed by analyzing the expression pattern of Wip1 using different immunostaining techniques. Moreover, mRNA levels of Wip1 correlate to Wip1 protein expression. Transfection experiments with shRNA against Wip1 showed decreased cell viability, proliferation and colony formation in NB cells. In addition, substantial increase of gamma-H2AX expression after Wip1 knockdown compared to the non-silencing transfection was observed. Tumor xenograft development was significantly delayed showing median tumor development (0.10 mL) to be more than doubled (15 days median, vs. 33 days) after Wip1 downregulation compared to animals injected with cells transfected with the scrambled construct. Conclusions: Wip1 downregulation inhibits tumor development. Our results showed that Wip1 is present in at least one extra genomic copy in the majority of primary NB. We identified NB cell lines expressing high, moderate and low levels of Wip1 where Wip1 knocked cells exhibited decreased cell growth and clonogenic capacity compared to non-silenced cells. Moreover, knockdown of Wip1 induced phosphorylation of histone H2AX, indicating a significant role of Wip1 in the DNA damage response of these tumors. Our data suggest that Wip1 expression have significant oncogenic function in neuroblastoma development. Citation Format: Jelena Milosevic, Malin Wickström, Lotta Elfman, Ninib Baryawno, Baldur Sveinbjörnsson, Tommy Martinsson, John Inge Johnsen, Per Kogner. The potential oncogenic significance of Wip1 in neuroblastoma. [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 3817. doi:10.1158/1538-7445.AM2013-3817

Abstract 2069: Combination of mTOR targeting with cetuximab and conventional chemotherapy: a preclinical study on an orthotopic xenograft model of head and neck cancer.

Abstract Background: Preclinical and clinical studies on head and neck squamous cell carcinoma (HNSCC) demonstrated synergistic anti-tumoral effects when combining anti-EGFR agents with conventional chemotherapeutic drugs such as cisplatin (C) and 5-fluorouracil (F). In a previous experimental study, our group pointed out an enhancement of PIK3CA signaling (pAKT) in tumors re-growing after cetuximab-based treatment (Bozec et al. Br J Cancer 2007). This context justifies investigations on mTOR inhibitors combined with cetuximab (Cetux) in HNSCC. Methods: The effects on tumor growth of treatments combining an mTOR inhibitor (Temsirolimus (Tem)), an anti-EGFR (Cetux) and a clinically validated-chemotherapeutic regimen (C+F) were examined in an orthotopic (floor of the mouth) model of human HNSCC (CAL33 cell line, over-expressing EGFR, secreting VEGF-A, H1047R PIK3CA mutated). In parallel, in-vitro experiments were conducted in order to strengthen the data arising from the in-vivo study. On day 0, cells were injected in nude NMRI mice. Treatment was started on day 3 and consisted of 8 groups (10 mice/group) treated as follows: Control, Tem (5 mg/kg i.p. five times a week), Cetux (2.5 mg/kg i.p. once a week), cisplatin (6 mg/kg) + 5-fluorouracil (17 mg/kg) (i.p. once a week), concomitant treatment combining Tem with either Cetux, C+F, or Cetux+C+F. The tumor growth was recorded by IVIS imaging and on day 13 after animal euthanasia with a caliper ruler. Results: All tested treatments produced significant growth inhibition as compared to controls (p<0.01). The Tem-Cetux and Tem-Cetux+C+F regimens produced the strongest growth-inhibiting effects, however the addition of C+F to the Tem-Cetux regimen did not result in a significantly greater effect as compared to Tem-Cetux (p = 0.18). The growth-inhibiting effects of Tem-Cetux were significantly stronger than those of Tem or Cetux alone (p<0.004). Moreover, a significant synergism was demonstrated between Temsirolimus and Cetuximab effects (significant interaction: p = 0.001, ANOVA). Furthermore, in contrast to mice receiving the Tem-Cetux+C+F regimen, mice treated with Tem-Cetux had no clinical signs of secondary effects. Accordingly, the mice weight loss measured on day 12 was significantly lower in mice receiving Tem-Cetux regimens as compared to mice receiving the Tem-Cetux+C+F regimen (p=0.005). IHC analysis on tumor vasculature (VEGFR2), proliferation status (Ki67) and downstream signaling phospho-proteins (pAKT, p-P70/S6k, pERK) strengthen our data on tumor growth and proliferation. Conclusion: These data on the association of temsilorimus with cetuximab could serve as a strong basis for innovative treatments aiming at an optimal management of patients with recurrent/metastatic HNSCC. Citation Format: Alexandre Bozec, Nathalie Ebran, Martino Monteverde, Nicolas Toussan, Anne Cayre, Marie-Cristine Etienne-Grimaldi, Cristiana Lo Nigro, Anne Sudaka, Marco Merlano, Frederique Penault-Llorca, Gerard Milano. Combination of mTOR targeting with cetuximab and conventional chemotherapy: a preclinical study on an orthotopic xenograft model of head and neck 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 2069. doi:10.1158/1538-7445.AM2013-2069

Abstract 2751: Wnt/β-catenin regulates the expression of MGMT through TCF/LEF transcription: new strategy for DNA alkylators in cancer therapy.

Abstract Background: O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that protects DNA from the biological effects of alkylating agents. MGMT has been implicated in resistance to alkylating drugs such as temozolomide used in the clinic against brain tumors. Temozolomide has been shown to be ineffective in tumors expressing elevated levels of MGMT. Finding agents that induce MGMT deficiency and increase the susceptibility to alkylating chemotherapeutic agents is highly warranted. Methods: The correlation between Wnt activity and MGMT expression was investigated in primary medulloblastomas, gliomas and colon cancer using gene expression cohorts. Cell lines from medulloblastomas, gliomas, colon cancer and neuroblastomas were examined for Wnt/β-catenin activity and MGMT expression using immunoblotting and real-time RT-PCR. siRNA against β-catenin, forced overexpression of β-catenin, and MGMT promotor reporter plasmids were used to study β-catenin mediated regulation on MGMT. Cell cytotoxicity and clonogenicity of chemotherapeutic drugs in combination with celecoxib were examined in cell lines using fluorometric microculture cytotoxicity assay and clonogenic assay, respectively. In vivo, nude NMRI mice carrying xenografts were treated with celecoxib in combination with temozolomide. Results: Wnt signaling activity was shown to be correlated to increased MGMT expression levels both in primary tumors and cell lines. Transfection experiments revealed that β-catenin regulates MGMT expression. The clinically available COX-2 inhibitor celecoxib augmented the cytotoxic effect of temozolomide in cells with elevated MGMT expression through a reduction of Wnt/β-catenin activity and a subsequent reduction in MGMT expression. Conclusions: This study demonstrates that celecoxib reduces the transcription of the MGMT gene through a prostaglandin E2-Wnt/β-catenin route and thus increases the susceptibility to temozolomide. These finding are the first reported that Wnt/β-catenin pathway regulates the expression of MGMT and thus provides a rational approach for improved efficacy of temozolomide therapy in the treatment of medulloblastoma and other cancers. Citation Format: Malin Wickström, Ninib Baryawno, Cecilia Dyberg, Jelena Milosevic, Baldur Sveinbjörnsson, Paul A. Northcott, Michael Taylor, Marcel Kool, Per Kogner, John I. Johnsen. Wnt/β-catenin regulates the expression of MGMT through TCF/LEF transcription: new strategy for DNA alkylators in cancer therapy. [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 2751. doi:10.1158/1538-7445.AM2013-2751

Importance of Attenuation Correction (AC) for Small Animal PET Imaging.

The purpose of this study was to investigate whether a correction for annihilation photon attenuation in small objects such as mice is necessary. The attenuation recovery for specific organs and subcutaneous tumors was investigated. A comparison between different attenuation correction methods was performed. Methods: Ten NMRI nude mice with subcutaneous implantation of human breast cancer cells (MCF-7) were scanned consecutively in small animal PET and CT scanners (MicroPETTM Focus 120 and ImTek’s MicroCATTM II). CT-based AC, PET-based AC and uniform AC methods were compared. Results: The activity concentration in the same organ with and without AC revealed an overall attenuation recovery of 9–21% for MAP reconstructed images, i.e., SUV without AC could underestimate the true activity at this level. For subcutaneous tumors, the attenuation was 13 ± 4% (9–17%), for kidneys 20 ± 1% (19–21%), and for bladder 18 ± 3% (15–21%). The FBP reconstructed images showed almost the same attenuation levels as the MAP reconstructed images for all organs. Conclusions: The annihilation photons are suffering attenuation even in small subjects. Both PET-based and CT-based are adequate as AC methods. The amplitude of the AC recovery could be overestimated using the uniform map. Therefore, application of a global attenuation factor on PET data might not be accurate for attenuation correction.

Abstract B113: Establishment of a clinically relevant orthotopic xenograft model for human liver cancer.

Abstract In order to allow a better understanding of liver carcinoma, the establishment of functional and reproducible in vivo models is widely pursued. Of available model systems, orthotopically growing xenografts in immunodeficient mice reflect well the clinical situation. In the present study, the growth behaviour and sensitivity towards the multikinase inhibitor sorafenib of human liver carcinoma cells implanted orthotopically into NMRI nude mice was investigated. Liver carcinoma cell line SKHEP1 was injected orthotopically into nude mice. Three days after inoculation of 5 Mio. tumor cells, mice were subdivided into two groups, and treatment was started either with vehicle (control) or sorafenib (100 mg/kg/d, po, days 3–36). Tumor growth was monitored via a) fluorescence-based in vivo imaging (IVI, using Alexa750 labeled anti-human CD10 antibody) once weekly, b) macroscopic examination at the end of the experiment, and c) histological examination of murine liver tissue. IVI pictures were merged with x-ray for an optimal localization of the fluorescent tissue. Antitumoral and antimetastatic capacity was determined by comparing the fluorescent intensity as well as the fluorescent area for primary tumors and metastasized organs over the time. Seven days after implantation, SKHEP1-derived tumors could be detected in the liver in all animals via IVI. Weekly follow-up analyses confirmed, that liver engraftment was prominent and observed in all animals (16/16). Lymphnode metastases (6/9 control mice and 3/7 Sorafenib-treated mice) could be determined 14 days after tumor cell inoculation. Beyond that bone marrow metastases (4/9) could be detected exclusively in the untreated control group. The detection limit of the investigated model was about 5×104 cells within one region of interest. Signal to noise ratio was excellent due to a highly specific antibody, the use of a near-infrared fluorophore combined with the absence of auto-fluorescense of murine hair. IVI produced very bright and reproducible pictures optimal for exact quantification of the tumor load. Sorafenib showed a strong antitumoral and antimetastastatic activity, inducing optimal T/C values as low as 2.7% for the primary tumor and 40% for the lymphnode metastases on day 56. In addition, the total number of metastasized animals was reduced in comparison to the untreated animals. The occurrence of bone marrow metastases was almost completely reduced by sorafenib treatment. Tolerability of the test compound was very high as no marked body weight losses or drug related toxicities could be observed. Our xenografts show a close resemblance to the liver cancer disease regarding the growth behaviour of primary tumor and metastatic involvement of other organs. Collection of whole-body IVI data proved to be a time- and animal-saving analysis that allows to closely monitor hepato-carcinoma growth. Quantification and localization of tumor load was determined in a very exact and prompt manner. Thus, orthotopically implanted liver xenografts monitored by a fluorescent-based in vivo imaging system will be a useful tool in the development of new therapies against human liver carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B113.

In vitro and in vivo efficacy of edelfosine-loaded lipid nanoparticles against glioma

Edelfosine is the prototype molecule of a family of anticancer drugs collectively known as synthetic alkyl-lysophospholipids. This drug holds promise as a selective antitumor agent, and a number of preclinical assays are in progress. In this study, we observe the accumulation of edelfosine in brain tissue after its oral administration in Compritol® and Precirol® lipid nanoparticles (LN). The high accumulation of edelfosine in brain was due to the inhibition of P-glycoprotein by Tween® 80, as verified using a P-glycoprotein drug interaction assay. Moreover, these LN were tested in vitro against the C6 glioma cell line, which was later employed to establish an in vivo xenograft mouse model of glioma. In vitro studies revealed that edelfosine-loaded LN induced an antiproliferative effect in C6 glioma cell line. In addition, in vivo oral administration of drug-loaded LN in NMRI nude mice bearing a C6 glioma xenograft tumor induced a highly significant reduction in tumor growth ( p < 0.01) 14 days after the beginning of the treatment. Our results showed that Tween® 80 coated Compritol® and Precirol® LN can effectively inhibit the growth of C6 glioma cells in vitro and suggest that edelfosine-loaded LN represent an attractive option for the enhancement of antitumor activity on brain tumors in vivo.

Abstract 1609: In vivo and ex vivo comparison of two human AML bone marrow engraftment mouse models using fire fly luciferase expressing MOLM-13 and MV4-11 cells

Abstract Acute myeloid leukemia (AML) is the most common leukemia affecting adults. In 70-90% of all AML patients FLT3, a receptor tyrosine kinase which plays an important role in normal hematopoiesis, is highly expressed. Furthermore, in about 30% of all AML patients activating internal tandem duplications (ITDs) or point mutations within the FLT3 gene have been identified. FLT3, therefore, is one of the most promising drug targets for the treatment of AML. Most often novel FLT3 protein kinase inhibitors are tested in subcutaneous xenograft mouse models using various human AML cell lines. To generate more relevant, orthotopic AML in vivo models, we stably transduced the human AML cell lines MOLM-13 and MV4-11 with fire fly luciferase (MOLM-13-Luci and MV4-11-Luci) suitable for in vivo bioluminescence imaging. MOLM-13 carries a heterozygous and MV4-11 a homozygous FLT3 internal tandem duplication (ITD) mutation. In vivo growth of MOLM-13-Luci and MV4-11-Luci cells after subcutaneous implantation in NMRI nude mice proofed that stable expression of luciferase did not alter growth characteristics of these cells. To initiate orthotopic growth, both AML cell lines were implanted intravenously via the tail vein into NOD SCID mice after Cyclophosphamide treatment. Cyclophosphamide facilitates engraftment of the human cells by reducing the endogenous bone marrow cell population. Approximately two (MOLM-13-Luci), or three (MV4-11-Luci) weeks after implantation, luciferase activity was detectable in the pelvic region of the mice indicating growth of both tumor cell lines. Within the next weeks luciferase activity was also seen in additional parts of the mouse at different levels. After necropsy a set of different organs and tissues was analysed ex vivo for luciferase activity. The pattern of metastasising tumor cells was compared in both models. Furthermore, detection of human Ki-67 protein using appropriate human specific antibodies allowed verification of growth of MOLM-13-Luci and MV4-11-Luci cells in different tissues. Sutent, a small-molecule kinase inhibitor, totally abolished tumor growth of both orthotopically growing tumors, indicating that both in vivo models are highly suitable for the characterisation of novel drugs directed against AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1609. doi:10.1158/1538-7445.AM2011-1609

Abstract LB-209: Expression of O6-methylguanine DNA-methyltransferase is regulated by Wnt/β-catenin signalling in cancer cells: A new strategy for DNA alkylators in cancer therapy

Abstract Background: O6-Methylguanine (O6-MG)-DNA-methyltransferase (MGMT) encodes the DNA-repair protein O6-alkylguanine (O6-AG) DNA alkyltransferase (AGT) that repair DNA adducts caused by alkylating agents. MGMT has important implications in cancer treatment since its expression induces resistance to methylating and chloroethylating anticancer drugs which are effective treatment options in tumours lacking MGMT expression. The demand to find agents that induce MGMT deficiency and increase the susceptibility to alkylating chemotherapeutic agents is therefore highly warranted. Methods: The correlation between Wnt activity and MGMT expression was investigated in primary medulloblastomas, gliomas and colon cancer using gene expression cohorts. Cell lines from medulloblastomas, gliomas, colon cancer and neuroblastomas were examined for Wnt/β-catenin activity and MGMT expression using immunoblotting and Real-Time quantitative PCR (Q-PCR). Small interfering RNA (siRNA) against β-catenin, forced overexpression of β-catenin, and MGMT promotor reporter plasmids were used to study β-catenin mediated regulation on MGMT. Cell cytotoxicity and clonogenicity of chemotherapeutic drugs in combination with celecoxib were examined in cell lines using fluorometric microculture cytotoxicity assay and clonogenic assay, respectively. In vivo, nude NMRI mice carrying D283 MED xenografts were treated with celecoxib in combination with temozolomide. Results: Wnt signaling activity was shown to be correlated to increased MGMT expression levels both in primary tumors and cell lines of different origins. Transfection experiments revealed that β-catenin regulates MGMT expression. Proinflammatory prostaglandin E2 activates the Wnt/β-catenin cascade and increase MGMT expression. The clinically available COX-2 inhibitor celecoxib augmented the cytotoxic effect of the DNA alkylating drug, temozolomide in cells with elevated MGMT expression both in vitro and in established medulloblastoma xenografts in nude mice through inhibition of Wnt/β-catenin activity and subsequent reduction in MGMT expression. Conclusions: This study demonstrates that celecoxib reduces the transcription of the MGMT gene through a prostaglandin E2-Wnt/β-catenin route and thus increases the susceptibility to temozolomide. These finding are the first reported that Wnt/β-catenin pathway regulates the expression of MGMT and thus provides a rational approach for improved efficacy of chemotherapeutic drugs inducing DNA alkylation in cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-209. doi:10.1158/1538-7445.AM2011-LB-209

Abstract 4168: Comprehensive characterisation of a newly established patient-derived pancreatic adenocarcinoma xenograft collection

Abstract There is a high medical need to identify new treatments for patients with pancreatic cancer, since in many cases only palliative treatment is possible. The standard 1st line chemotherapy in inoperable, locally advanced (stage II and III) and metastatic (stage IV) adenocarcinoma of the pancreas is Gemcitabine as a single agent with a median survival of about 6 month. In addition, Gemcitabine is indicated as adjuvant chemotherapy after surgery. Although the anti-metabolite 5-Flourouracil (5-FU) and the EGF-R inhibitor Tarceva (Erlotinib) have been approved for 2nd line treatment, new and more efficient drugs are urgently needed. In the present study, more than 60 samples of pancreatic carcinomas were transplanted subcutaneously (s.c.) into NMRI nude mice directly after tumor resection. In most cases, tumor material from chemonaive patients with defined histology and staging was used for implantation. Up to now, 17 tumor models were passaged in nude mice and characterized comprehensively. In general, the histology of the primary tumor was comparable to that of the established xenograft. Chemosensitivity in vivo was evaluated by treatment of tumor bearing nude mice with 5-FU (100 or 75mg/kg, q7dx3, i.p.), Gemcitabine (240mg/kg, q7dx3, i.v.) and Erlotinib (25 and 50mg/kg, qdx21, p.o.). In general, tumor growth was not inhibited with best T/C values &amp;gt;50% highlighting the general chemoresistance of pancreatic cancer. Only for two models (PAXF 1872 and PAXF 1998), a high sensitivity towards Gemcitabine was evident with best T/C values of 8% and 3.8%, respectively. There was no correlation to the transcriptional levels of proteins involved in transportation and metabolism of Gemcitabine. Concerning Erlotinib, best T/C values were ranging from 89.6% (PAXF 1876) to 38.5% (PAXF 1982) with no correlation to EGFR expression status. A more broad chemosensitivity profile was established with the ex-vivo clonogenic assay. Interestingly, several tumors responded strongly to treatment with Rapamycin (IC50 ≤ 10nM). Based on this data, in vivo treatment experiments with RAD001 (Everolimus) were done and data are presented. Mutational analysis of p53 (exons 4 to 10) revealed only 4 out of 13 models as p53 wild-type. Nevertheless, the p53 pathway is dysregulated in all tumor models and for selected tumors, an aneuploid cell population was identified by ex vivo cell cycle analysis. In summary, a unique collection of patient-derived pancreatic xenograft models of high clinical relevance has been established. These models are available for translational research studies including in-vivo efficacy testing of new investigational drugs. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4168.

Abstract 4354: Celecoxib reduces MGMT expression and potentiates the effect of temozolomide in childhood medulloblastoma

Abstract Background: O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that protects DNA from the biological effects of alkylating agents. MGMT has been implicated in resistance to alkylating drugs such as temozolomide used in the clinic against brain tumors. Temozolomide has been shown to be ineffective in tumors expressing MGMT and in medulloblastoma high expression of MGMT is correlated with poor prognosis. Therefore, the demand to find agents that deplete MGMT is warranted. Methods: medulloblastoma cell cytotoxicity of chemotherapeutic drugs in combination with Celecoxib was examined in MGMT positive cell lines using fluorometric microculture cytotoxicity assay. In vivo, nude NMRI mice carrying MGMT positive D283 MED xenografts were treated with celecoxib in combination with temozolomide. For mechanistic studies, western blot and quantitative PCR were used. Results: the clinically available COX-2 inhibitor Celecoxib induced a synergistic or an additive cytotoxic effect in combination with Temozolomide, Cyclophosphamide, Doxorubicin, Irinotecan, Vincristine, Rapamycin or CCI-779 in vitro. In vivo, Celecoxib synergistically potentiates the effect of Temozolomide through downregulation of MGMT expression. Conclusions: this study shows that Celecoxib reduces the DNA repair capacity of MGMT in medulloblastoma and increases the susceptibility to temozolomide. These findings support the use of pharmacological MGMT depletion as a rational approach for intensification of temozolomide therapy in the treatment of medulloblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4354.

Abstract A23: A collection of patient-derived pancreatic adenocarcinoma xenograft models: Chemosensitivity in vitro and in vivo and molecular characterization

Abstract Pancreatic cancer is a devasting disease with an incidence of 0.1% and - in most cases - only palliative treatment options. The standard 1st line chemotherapy in inoperable, locally advanced (stage II and III) and metastatic (stage V) pancreas adenocarcinoma is Gemcitabine as a single agent with a median survival of about 6 month. In addition, Gemcitabine is indicated as adjuvant chemotherapy after surgery. Although the antimetabolite 5-Flourouracil (5-FU) and the EGF-R inhibitor Tarceva (Erlotinib) have been approved for 2nd line treatment, new and more efficacious treatments are urgently needed. In the present study, more than 60 primary pancreatic carcinoma samples from patients were transplanted subcutaneously (s.c.) into NMRI nude mice. In most cases, tumor material from chemonaive patients with defined histology and staging was used for implantation. Up to now, 14 pancreatic carcinoma models named PAXF and passaged in nude mice have been established and characterized comprehensively. Tumor bearing animals were treated in-vivo with 5-FU (100 or 75mg/kg, q7dx3, i.p.), Gemcitabine (240mg/kg, q7dx3, i.v.) and Tarceva (25 and 50mg/kg, qdx21, p.o.). In most studies, tumor growth was inhibited with best T/C values &amp;gt; 50%, highlighting the general chemoresistance of pancreatic cancer. Only for PAXF1872 and PAXF1998, a high sensitivity towards Gemcitabine was evident with best T/C values of 8% and 3.8%, respectively. In general, there was no correlation of response to Tarceva with EGFR expression status and best T/C values ranging from 89.6% (PAXF1876) to 38.5% (PAXF1982). A more broad chemosensitivity profile was established with Gemcitabine, 5-FU, Tarceva, Taxol, Cisplatin, Rapamycin, Sorafenib and Sunitinib in the ex-vivo clonogenic assay. Interestingly, 2 out of 12 pancreatic cancer models responded with high sensitivity towards treatment with Rapamycin (IC50 &amp;lt;= 10nM) with a mean IC50 = 0.18 µM over all models. Finally, the established tumor models were characterized by using Affymetrix HG U133 plus2.0 gene expression arrays, exon sequencing (Ki-ras, B-raf, p53) and the Sequenom MassARRAY® System - OncoCarta(TM) panel. In all tumors analysed so far, an activating Ki-ras (H-ras, B-raf) mutation was identified. Mutational analysis of p53 (exons 4 to 10) revealed only 4 out of 13 models as p53 wild-type. For the PAXF1998 tumor model with animals cured by Gemcitabine therapy, mutations in Ki-ras (G12V, heterozygous) and p53 (V274A, homozygous) have been identified. In summary, a unique collection of patient-derived xenograft models of high clinical relevance has been established. These pancreatic carcinoma models are available for translational research studies including in-vivo efficacy testing of new investigational drugs. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A23.

Abstract C187: Antitumoral efficacy of the novel thrombin inhibitor Revasc©/Desirudin in an orthotopic metastatic AsPC-1 pancreas tumor model

Abstract Background: Thrombo-embolism is one of the most common secondary diseases in cancer patients and is the second leading cause of death of these patients. In pancreatic cancer patients, thrombosis occurs more frequently than in other cancer patients. Thus anticoagulants are widely used in cancer therapies mainly to prevent the development of thrombo-embolisms. However there is increasing evidence that anticoagulants have also antitumoral and anti-angiogenic effects. The aim of this study was to evaluate the anti-tumoral, anti-metastatic and anti-angiogenic efficacy of the novel anticoagulant Revasc© a recombinant hirudin and direct thrombin inhibitor, in an orthotopic mouse model of pancreatic cancer. Material and Methods: To evaluate the anti-tumoral, anti-metastatic and anti-angogenic efficacy of Revasc©, luciferase labeled pancreatic AsPC-1-tumor cells were orthotopically implanted into the pancreas of NMRI nude mice. After onset of tumor growth, mice were randomized into six groups. Group 1 received vehicle control (0.9% NaCl) twice daily, Group 2 received Gemcitabine (240 mg/kg) once weekly; Group 3 received Revasc at a concentration of 1 mg/kg twice daily, Group 4 received Revasc© at a concentration of 3 mg/kg twice daily, Group 5 received Revasc© at a concentration of 10 mg/kg daily and Group 6 received a combination therapy of twice daily 10 mg/kg Revasc© plus a weekly dose of 240 mg/kg Gemcitabine. At the end of the study, tumor weight and volume were determined and luciferase activity was monitored in the primary tumor and selected organs (spleen, liver, stomach, intestine, lung and inguinal lymph nodes). In addition, microvessel densities and vessel maturation was determined by immunohistochemisty. Results: In general, Revasc© was very well tolerated. The primary tumor weight was significantly reduced in the groups receiving Revasc© at a concentration of 3 mg/kg and 10 mg/kg as well as in the control group treated with Gemcitabine. The combination of Gemcitabine and Revasc©, however, did not show any additive effect. No significant reduction of metastasis could be observed in the Revasc© Groups, whereas Gemcitabine efficiently inhibited metastasis into the lung. However, Revasc© showed a significant anti-angiogenic potential at the lowest concentration of 1mg/kg, corroborating findings of other groups showing that thrombin inhibition acts anti-angiogenic. Conclusion: Taken together the study showed that Revasc© has an anti-tumoral and a mild anti-angiogenic effect. The data suggest that Revasc© has a potential to be used for the treatment of patients with pancreatic cancers; primarily to prevent thromboembolism but secondarly also to inhibit tumor growth. It remains to be shown if Revasc will lead to a solid anti-tumoral effect in patients and if the relatively short mean elimination half life of Revasc© (2–3 h) will allow to identify a convenient dosing scheme. Alternatively a pegylated hirudin derivative (PEG-hirudin) which has a longer half life and which has proven to have a very efficient antitumoral activity could be of high therapeutic interest. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C187.

Combined treatment with lexatumumab and irradiation leads to strongly increased long term tumour control under normoxic and hypoxic conditions

PurposeThe combination of ionizing radiation with the pro-apoptotic TRAIL receptor antibody lexatumumab has been shown to exert considerable synergistic apoptotic effects in vitro and in short term growth delay assays. To clarify the relevance of these effects on local tumour control long-term experiments using a colorectal xenograft model were conducted.Materials and methodsColo205-xenograft bearing NMRI (nu/nu) nude mice were treated with fractionated irradiation (5× 3 Gy, d1-5) and lexatumumab (0.75 mg/kg, d1, 4 and 8). The tumour bearing hind limbs were irradiated with graded single top up doses at d8 under normoxic (ambient) and acute hypoxic (clamped) conditions. Experimental animals were observed for 270 days. Growth delay and local tumour control were end points of the study. Statistical analysis of the experiments included evaluation of tumour regrowth and local tumour control.ResultsCombined treatment with irradiation and lexatumumab led to a pronounced tumour regrowth-delay when compared to irradiation alone. The here presented long-term experiments revealed a highly significant rise of local tumour control for normoxic (ambient) (p = 0. 000006) and hypoxic treatment (p = 0. 000030).ConclusionOur data show that a combination of the pro-apoptotic antibody lexatumumab with irradiation reduces tumour regrowth and leads to a highly increased local tumour control in a nude mouse model. This substantial effect was observed under ambient and more pronounced under hypoxic conditions.

Combination of the Pro-Apoptotic TRAIL-Receptor Antibody Mapatumumab With Ionizing Radiation Strongly Increases Long-Term Tumor Control Under Ambient and Hypoxic Conditions

Mapatumumab, an agonistic tumor necrosis factor-related apoptosis inducing ligand-receptor antibody, exerts highly synergistic apoptotic effects in vitro and in short-term growth delay assays when combined with irradiation. Because it remained unclear in how far these effects influence local tumor control, long-term experiments using a colorectal xenograft model were undertaken. Experiments were performed with irradiation (5 x 3 Gy, d1-5) and mapatumumab (10 mg/kg) in Colo205-xenograft-bearing NMRI (nu/nu) nude mice. Graded top up doses were delivered on the tumor-bearing hind leg under ambient and hypoxic conditions; follow-up was 270 days. Growth delay and local tumor control were end points of the study. Statistical analysis of the experiments included calculation of tumor regrowth and local tumor control. After combined treatment, a pronounced tumor regrowth-delay was observed when compared with irradiation alone. Long-term experiments revealed a highly significant increase in local tumor control for ambient (p = 0.00076) and hypoxic treatment (p = 0.000069). The present data demonstrate for the first time that combination of a pro-apoptotic antibody with irradiation results in evidently reduced tumor regrowth times and subsequently highly increased local tumor control under normoxic and hypoxic conditions in a xenograft mouse model.

Tumor volume in subcutaneous mouse xenografts measured by microCT is more accurate and reproducible than determined by 18F-FDG-microPET or external caliper

BackgroundIn animal studies tumor size is used to assess responses to anticancer therapy. Current standard for volumetric measurement of xenografted tumors is by external caliper, a method often affected by error. The aim of the present study was to evaluate if microCT gives more accurate and reproducible measures of tumor size in mice compared with caliper measurements. Furthermore, we evaluated the accuracy of tumor volume determined from 18F-fluorodeoxyglucose (18F-FDG) PET.MethodsSubcutaneously implanted human breast adenocarcinoma cells in NMRI nude mice served as tumor model. Tumor volume (n = 20) was determined in vivo by external caliper, microCT and 18F-FDG-PET and subsequently reference volume was determined ex vivo. Intra-observer reproducibility of the microCT and caliper methods were determined by acquiring 10 repeated volume measurements. Volumes of a group of tumors (n = 10) were determined independently by two observers to assess inter-observer variation.ResultsTumor volume measured by microCT, PET and caliper all correlated with reference volume. No significant bias of microCT measurements compared with the reference was found, whereas both PET and caliper had systematic bias compared to reference volume. Coefficients of variation for intra-observer variation were 7% and 14% for microCT and caliper measurements, respectively. Regression coefficients between observers were 0.97 for microCT and 0.91 for caliper measurements.ConclusionMicroCT was more accurate than both caliper and 18F-FDG-PET for in vivo volumetric measurements of subcutaneous tumors in mice.18F-FDG-PET was considered unsuitable for determination of tumor size. External caliper were inaccurate and encumbered with a significant and size dependent bias. MicroCT was also the most reproducible of the methods.

Growth of human gastric cancer cells in nude mice is delayed by a ketogenic diet supplemented with omega-3 fatty acids and medium-chain triglycerides

BackgroundAmong the most prominent metabolic alterations in cancer cells are the increase in glucose consumption and the conversion of glucose to lactic acid via the reduction of pyruvate even in the presence of oxygen. This phenomenon, known as aerobic glycolysis or the Warburg effect, may provide a rationale for therapeutic strategies that inhibit tumour growth by administration of a ketogenic diet with average protein but low in carbohydrates and high in fat enriched with omega-3 fatty acids and medium-chain triglycerides (MCT).MethodsTwenty-four female NMRI nude mice were injected subcutaneously with tumour cells of the gastric adenocarcinoma cell line 23132/87. The animals were then randomly split into two feeding groups and fed either a ketogenic diet (KD group; n = 12) or a standard diet (SD group; n = 12) ad libitum. Experiments were ended upon attainment of the target tumor volume of 600 mm3 to 700 mm3. The two diets were compared based on tumour growth and survival time (interval between tumour cell injection and attainment of target tumour volume).ResultsThe ketogenic diet was well accepted by the KD mice. The tumour growth in the KD group was significantly delayed compared to that in the SD group. Tumours in the KD group reached the target tumour volume at 34.2 ± 8.5 days versus only 23.3 ± 3.9 days in the SD group. After day 20, tumours in the KD group grew faster although the differences in mean tumour growth continued significantly. Importantly, they revealed significantly larger necrotic areas than tumours of the SD group and the areas with vital tumour cells appear to have had fewer vessels than tumours of the SD group. Viable tumour cells in the border zone surrounding the necrotic areas of tumours of both groups exhibited a glycolytic phenotype with expression of glucose transporter-1 and transketolase-like 1 enzyme.ConclusionApplication of an unrestricted ketogenic diet enriched with omega-3 fatty acids and MCT delayed tumour growth in a mouse xenograft model. Further studies are needed to address the impact of this diet on other tumour-relevant functions such as invasive growth and metastasis.

Five Stabilized111In-Labeled Neurotensin Analogs in Nude Mice Bearing HT29 Tumors

Neurotensin (NT) receptors are overexpressed in different human tumors, such as human ductal pancreatic adenocarcinoma. New stable neurotensin analogs with high receptor affinity have been synthesized by replacing arginine residues with lysine and arginine derivatives. The aim of this study was to explore the biodistribution, tumor uptake, kidney localization, and stability characteristics of these new analogs in order to develop new diagnostic tools for exocrine pancreatic cancer. Four (111)In-labeled DTPA-chelated NT analogs and one (111)In-labeled DOTA-chelated NT analog were evaluated in NMRI nude mice bearing NT receptor-positive HT29 tumors. Experiments with a coinjection of unlabeled NT or lysine were performed to investigate receptor-mediated uptake and kidney protection, respectively. In addition, the in vivo serum stability of the most promising analog was analyzed. In the biodistribution study in mice, at 4 hours postinjection, a low percentage of the injected dose per gram (%ID/g) of tissue for all compounds was found in NT receptor-negative organs, such as the blood, spleen, pancreas, liver, muscle, and femur. A high uptake was found in the colon, intestine, kidneys, and in implanted HT29 tumors. The coinjection of excess unlabeled neurotensin significantly reduced tumor uptake, showing tumor uptake to be receptor-mediated. To a lesser extent, this was also observed for the colon, but not for other tissues. We concluded that DTPA-(Pip)Gly-Pro-(PipAm)Gly-Arg-Pro-Tyr-tBuGly-Leu-OH and the DOTA-linked counterpart have the most favorable biodistribution properties regarding tumor uptake.

Pre-treatment number of clonogenic cells and their radiosensitivity are major determinants of local tumour control after fractionated irradiation

Objective The response of tumours to fractionated radiotherapy is determined by many factors including repopulation, reoxygenation, the number of clonogenic cells, and their intrinsic radiosensitivity. However, after single radiation doses given under conditions of clamp hypoxia, the dose to control a tumour locally is dependent only on the number of clonogenic cells and their cellular radiosensitivity. Therefore, these parameters were investigated using local control after single doses given under hypoxia, to predict the outcome of fractionated irradiation. Materials and methods Ten hSCC cell lines (FaDu, UT-SCC-15, UT-SCC-14, XF354, UT-SCC-5, UT-SCC-45, SAS, CAL-33, UT-SCC-8, and HSC-4) were transplanted subcutaneously into the right hind-leg of NMRI nude mice. At 7 mm in diameter, tumours were irradiated either with graded single doses under clamp blood flow conditions ( n = 873) or with 30 graded fractions within 6 weeks ( n = 905) under ambient conditions. Local tumour control was determined 120 days after irradiation. Radiation response was quantified in terms of TCD 50, i.e. the dose required to control 50% of tumours locally. Results Ten tumour lines investigated showed a pronounced heterogeneity in both TCD 50(30fx/6w) after fractionated irradiation and TCD 50(SDclamp) after single dose irradiation. TCD 50(30fx/6w) varied between 45 Gy for UT-SCC-45 and 127 Gy for SAS; TCD 50(SDclamp) varied between 42 Gy for UT-SCC-14 and 66 Gy for CAL-33. Two tumours were excluded from further analysis due to immunogenicity or non-defined TCD 50. Linear regression analysis revealed a significant positive correlation between TCD 50(SDclamp) and TCD 50(30fx/6w) ( R 2 = 0.82, p = 0.002). Conclusions Significant association between TCD 50(SDclamp) and TCD 50(30fx/6w) suggests that the pre-treatment number of clonogenic tumour cells and their cellular radiosensitivity have a major impact on local control after fractionated radiotherapy.