Reduced Tumor Growth Rate Research Articles

Use of tumor growth rate (TGR) to provide useful clinical information in phase I trials and reveal clear drug-specific profiles.

3010 Background: The evaluation of treatment response by RECIST does not take into account the tumor growth kinetics along the treatment sequence and may induce misleading information regarding the evaluation of the drug activity. TGR incorporates the time between the tumor evaluations and allows for a dynamic and quantitative evaluation of the tumor kinetics. How TGR is modified along the introduction of experimental therapeutics and is associated with outcome in phase I patients remains unknown. Methods: Medical records from all patients (n=304) prospectively treated at Institut Gustave Roussy in 19 phase I trials between 2009 and 2011 were analyzed. TGR was computed both during the pre-treatment period (REFERENCE) and between baseline and first evaluation (EXPERIMENTAL). TGR is defined as: log 10 (Vt/V0)/dt and is represented as a percentage increase of tumor volume per month for clinical relevance. The associations between TGR, RECIST, the most commonly used prognostic scores (ECOG, RMH score, PFS ratio) and the outcome (OS, PFS) were computed (multivariate analysis). The effect of treatment, prognostic scores, histology, and the number of previous treatment lines on TGR were assessed. Results: Overall, we observed a reduction of TGR between the REFERENCE vs. EXPERIMENTAL periods (11.5 vs. 1.5, P<.0001). Although most of the patients were classified as stable disease (57%) or progressive disease (28%) by RECIST at the first evaluation, 88% and 69% of them exhibited a decrease in TGR, respectively. TGR reveals clear drug specific profiles along the 19 experimental regimens tested, allowing for an early evaluation of the drug activity. The TGR decrease was associated with both PFS (P=.004) and OS (P=.001) and remained significant even after adjustment to the other prognostic scores for phase I patients. Conclusions: Adding TGR assessment in phase I patients is simple and provides clinically relevant information: (i) It allows for an early and precise assessment of the tumor growth, (ii) It reveals drug-specific profiles, suggesting its potential use for the early assessment of drug activity, (iii) TGR is independently associated with prognosis in phase I patients.

Abstract 4573: STINGVAX - A novel tumor vaccine with cyclic dinucleotides - can induce potent anti-tumor responses in vivo.

Abstract Cyclic dinucleotides (CDN) are ubiquitous bacterial intracellular messengers, and they have recently been shown to also function as a pathogen associated molecular pattern (PAMP) molecules that are sensed by eukaryotic host Stimulator of Interferon Genes (STING) that can activate the TBK-1/IRF-3 signaling pathway to induce type I interferon and other co-regulated genes. CDN was found to induce STING dependent augmentation of T-cell priming in multiple vaccination models, potentially by activating dendritic cells. We formulated CDN with a GM-CSF secreting tumor cell vaccine (STINGVAX) to mobilize and as well as activate dendritic cells both in vitro and in vivo. STINGVAX was tested in a stringent B16 melanoma treatment model, and we demonstrated significant reduction of tumor growth rate in vivo. STINGVAX's anti-tumor response was correlated with increased tumor infiltrating CD8 T-cells as well as increased number of p15E tumor-specific cytotoxic T-cells. STINGVAX's in vivo anti-tumor response was T-cell dependent as well as STING dependent. When we combined STINGVAX with multiple TLR 4/7/8 agonists signaling through the MyD88/TRIF pathway that is distinct from CDN/STING/TBK-1 signaling treatment of palpable B16 tumor resulted in regression of 30-60% of these non-immunogenic tumors. When the STINGVAX treated tumor microenvironment was examined, both IFNγ+CD8+ and PD-L1 was upregulated, potentially demonstrating an adaptive immune resistance mechanism which would render STINGVAX an excellent candidate to be combined with anti-PD-1 blockade. Cumulatively, STINGVAX is a novel tumor vaccine with a high potential for translation in clinical oncology. Citation Format: Thomas W. Dubensky, Meredith L. Leong, David B. Kanne, Edward E. Lemmens, Ken Metchette, Weiqun Liu, Marcella Fasso, Juan Fu, Joshua J. Woodward, Drew Pardoll, Daniel A. Portnoy, Young J. Kim. STINGVAX - A novel tumor vaccine with cyclic dinucleotides - can induce potent anti-tumor responses 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 4573. doi:10.1158/1538-7445.AM2013-4573

Investigating New Therapeutic Strategies Targeting Hyperinsulinemia's Mitogenic Effects in a Female Mouse Breast Cancer Model

Epidemiological and experimental studies have identified hyperinsulinemia as an important risk factor for breast cancer induction and for the poor prognosis in breast cancer patients with obesity and type 2 diabetes. Recently it was demonstrated that both the insulin receptor (IR) and the IGF-IR mediate hyperinsulinemia's mitogenic effect in several breast cancer models. Although IGF-IR has been intensively investigated, and anti-IGF-IR therapies are now in advanced clinical trials, the role of the IR in mediating hyperinsulinemia's mitogenic effect remains to be clarified. Here we aimed to explore the potential of IR inhibition compared to dual IR/IGF-IR blockade on breast tumor growth. To initiate breast tumors, we inoculated the mammary carcinoma Mvt-1 cell line into the inguinal mammary fat pad of the hyperinsulinemic MKR female mice, and to study the role of IR, we treated the mice bearing tumors with the recently reported high-affinity IR antagonist-S961, in addition to the well-documented IGF-IR inhibitor picropodophyllin (PPP). Although reducing IR activation, with resultant severe hyperglycemia and hyperinsulinemia, S961-treated mice had significantly larger tumors compared to the vehicle-treated group. This effect maybe secondary to the severe hyperinsulinemia mediated via the IGF-1 receptor. In contrast, PPP by partially inhibiting both IR and IGF-IR activity reduced tumor growth rate with only mild metabolic consequences. We conclude that targeting (even partially) both IR and IGF-IRs impairs hyperinsulinemia's effects in breast tumor development while simultaneously sparing the metabolic abnormalities observed when targeting IR alone with virtual complete inhibition.

Study of immunomodulatory effects of arteether administrated intratumorally.

Recent studies have indicated the profound anti-tumor activity of artemisinin's compounds, among which; arteether is an oil-soluble derivative of artemisinin with an endoperoxide bridge that can induce apoptosis in tumor cells but not in the normal cells. An experiment was carried out on tumor-bearing Balb/c mice to estimate the effects of Arteether on tumor growth and antitumor immune responses. Briefly, 6mg/kg/day of Arteether and diluents were administered to two groups of mice. Tumor sizes were measured using digital verniercallipers. Mice were sacrificed and splenocytes were harvested for lymphocyte proliferation assay, the level of IL-4 and IFN-γ cytokines, and the percentage of splenic T regulatory cells were measured. According to the findings, there were no statistical differences between the groups with respect to the level of IFN-γ, IL-4 and proliferation assay; while our results showed that Arteether is effective in the reduction of tumor growth rate. In general, intra-tumoral injection of Arteether as an oil-soluble derivative of artemisinin brings to light some antitumor properties that may aid in development of more effective antitumor agents.

N2-Trimethylacetyl substituted and unsubstituted-N4-phenylsubstituted-6-(2-pyridin-2-ylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines: Design, cellular receptor tyrosine kinase inhibitory activities and in vivo evaluation as antiangiogenic, antimetastatic and antitumor agents

Six novel N4-phenylsubstituted-6-(2-pyridin-2-ylethyl)-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamines and their N2-trimethylacetyl substituted analogs were synthesized as receptor tyrosine kinase (RTK) inhibitors. A microwave-mediated Sonogashira reaction was used as a key step for the synthesis of these compounds. Biological evaluation, in whole cell assays, showed that some analogs had remarkable inhibitory activity against a variety of RTKs and in particular cytotoxic activity against A431 tumor cells in culture. The inhibitory data against RTKs in this study demonstrated that variation of the 4-anilino substituents of these analogs dictates both potency and specificity of inhibitory activity against various RTKs. The study also supported the hypothesis that interaction of substituents on the 2-amino group with hydrophobic site-II provides an increase in potency. Compound 8 of this series was selected for evaluation in vivo in a B16-F10 syngeneic mouse tumor model and exhibited significant reduction in tumor growth rate, in tumor vascular density and in metastases to the lung compared to the control.

222 Lurbinectedin (PM01183): Pharmacokinetics/Pharmacodynamic (PK/PD) Properties in Pancreas, Ovarian and NSCLC Xenografts

breast and platinum-resistant/refractory ovarian) clinical evaluation. In vitro studies demonstrate that lurbinectedin causes delayed progression of S phase and cycle arrest in G2M. Also, lurbinectedin was 3−4 time less potent in NER deficient cells whereas it was 100 time more potent in HR deficient cells. The in vivo antitumor activity of lurbinectedin was evaluated in human ovarian carcinoma models, growing subcutaneously (OCX) or in the peritoenal cavity (IPX). Material and Methods: The in vivo pharmacological behavior of lurbinectedin was evaluated in M5076 (murine reticulosarcoma), MN-MCA1 (murine fibrosarcoma), 2 human OCX (HOC 18 and MNB-PTX-1) and 2 IPX (HOC8 and HOC22). Lurbinectedin was given i.v. at 0.2 mg/kg/day on days 0, 7, 14 (q7d×3). The antitumor effect was calculated either by T/C (as a percentage of the change in tumor size for treated [T] and placebo [C]) or by the increase of like span (ILS) (as 100·[(T − C)/C], where ‘T’ and ‘C’ are the median survival time of the treated and placebo); treatment-related anti-metastic activity was also evaluated. Results: In M5076 lurbinectedin was only marginally active (T/C, 73.4%) against the primary tumor whereas in MN-MCA1 demonstrated a good antitumor activity (best T/C 15%). In both tumor models lurbinectedin produced a striking antimetastatic effect (p < 0.001). Lurbinectedin treatment resulted in no antitumor activity in the MNB-PTX-1 model. In animals bearing OCX HOC18 tumors, lurbinectedin treatment induced a strong and long-lasting antitumor effect (minimal T/C, 13% Day 16). Tumor volume were reduced (P < 0.01) compared to placebo on days 10−17. Also, lurbinectedin treatment reduced tumor growth rate: all animals were sacrificed due to tumor volume on Day 63, later than placebo (Day 15). Lurbinectedin treatment of HOC8 IPX xenografts resulted in (P < 0.05) antitumor activity compared to untreated animals (ILS % of 140.0). Eight out of 9 of mice bearing HOC22 IPX tumors and treated with lurbinectedin survived 4 months after the death of the last placebo-treated animal and, then considered as tumor-free. Conclusions: The in vivo antitumor effect of lurbinectedin was demonstrated in a panel of human and murine experimental models, characterized by different behaviors and drug sensitivity.

223 Lurbinectedin (PM01183) Synergizes with Gemcitabine in NSCLC, Ovarian and Pancreas Tumor Xenografts

breast and platinum-resistant/refractory ovarian) clinical evaluation. In vitro studies demonstrate that lurbinectedin causes delayed progression of S phase and cycle arrest in G2M. Also, lurbinectedin was 3−4 time less potent in NER deficient cells whereas it was 100 time more potent in HR deficient cells. The in vivo antitumor activity of lurbinectedin was evaluated in human ovarian carcinoma models, growing subcutaneously (OCX) or in the peritoenal cavity (IPX). Material and Methods: The in vivo pharmacological behavior of lurbinectedin was evaluated in M5076 (murine reticulosarcoma), MN-MCA1 (murine fibrosarcoma), 2 human OCX (HOC 18 and MNB-PTX-1) and 2 IPX (HOC8 and HOC22). Lurbinectedin was given i.v. at 0.2 mg/kg/day on days 0, 7, 14 (q7d×3). The antitumor effect was calculated either by T/C (as a percentage of the change in tumor size for treated [T] and placebo [C]) or by the increase of like span (ILS) (as 100·[(T − C)/C], where ‘T’ and ‘C’ are the median survival time of the treated and placebo); treatment-related anti-metastic activity was also evaluated. Results: In M5076 lurbinectedin was only marginally active (T/C, 73.4%) against the primary tumor whereas in MN-MCA1 demonstrated a good antitumor activity (best T/C 15%). In both tumor models lurbinectedin produced a striking antimetastatic effect (p < 0.001). Lurbinectedin treatment resulted in no antitumor activity in the MNB-PTX-1 model. In animals bearing OCX HOC18 tumors, lurbinectedin treatment induced a strong and long-lasting antitumor effect (minimal T/C, 13% Day 16). Tumor volume were reduced (P < 0.01) compared to placebo on days 10−17. Also, lurbinectedin treatment reduced tumor growth rate: all animals were sacrificed due to tumor volume on Day 63, later than placebo (Day 15). Lurbinectedin treatment of HOC8 IPX xenografts resulted in (P < 0.05) antitumor activity compared to untreated animals (ILS % of 140.0). Eight out of 9 of mice bearing HOC22 IPX tumors and treated with lurbinectedin survived 4 months after the death of the last placebo-treated animal and, then considered as tumor-free. Conclusions: The in vivo antitumor effect of lurbinectedin was demonstrated in a panel of human and murine experimental models, characterized by different behaviors and drug sensitivity.

221 Lurbinectedin (PM01183) in Combination with Gemcitabine in Patient-Derived, Pancreatic Ductal Adenocarcinoma (PDA) Xenografts

breast and platinum-resistant/refractory ovarian) clinical evaluation. In vitro studies demonstrate that lurbinectedin causes delayed progression of S phase and cycle arrest in G2M. Also, lurbinectedin was 3−4 time less potent in NER deficient cells whereas it was 100 time more potent in HR deficient cells. The in vivo antitumor activity of lurbinectedin was evaluated in human ovarian carcinoma models, growing subcutaneously (OCX) or in the peritoenal cavity (IPX). Material and Methods: The in vivo pharmacological behavior of lurbinectedin was evaluated in M5076 (murine reticulosarcoma), MN-MCA1 (murine fibrosarcoma), 2 human OCX (HOC 18 and MNB-PTX-1) and 2 IPX (HOC8 and HOC22). Lurbinectedin was given i.v. at 0.2 mg/kg/day on days 0, 7, 14 (q7d×3). The antitumor effect was calculated either by T/C (as a percentage of the change in tumor size for treated [T] and placebo [C]) or by the increase of like span (ILS) (as 100·[(T − C)/C], where ‘T’ and ‘C’ are the median survival time of the treated and placebo); treatment-related anti-metastic activity was also evaluated. Results: In M5076 lurbinectedin was only marginally active (T/C, 73.4%) against the primary tumor whereas in MN-MCA1 demonstrated a good antitumor activity (best T/C 15%). In both tumor models lurbinectedin produced a striking antimetastatic effect (p < 0.001). Lurbinectedin treatment resulted in no antitumor activity in the MNB-PTX-1 model. In animals bearing OCX HOC18 tumors, lurbinectedin treatment induced a strong and long-lasting antitumor effect (minimal T/C, 13% Day 16). Tumor volume were reduced (P < 0.01) compared to placebo on days 10−17. Also, lurbinectedin treatment reduced tumor growth rate: all animals were sacrificed due to tumor volume on Day 63, later than placebo (Day 15). Lurbinectedin treatment of HOC8 IPX xenografts resulted in (P < 0.05) antitumor activity compared to untreated animals (ILS % of 140.0). Eight out of 9 of mice bearing HOC22 IPX tumors and treated with lurbinectedin survived 4 months after the death of the last placebo-treated animal and, then considered as tumor-free. Conclusions: The in vivo antitumor effect of lurbinectedin was demonstrated in a panel of human and murine experimental models, characterized by different behaviors and drug sensitivity.

Transition from Colitis to Cancer: High Wnt Activity Sustains the Tumor-Initiating Potential of Colon Cancer Stem Cell Precursors

Ulcerative colitis (UC) increases the risk of colorectal cancer (CRC), but the mechanisms involved in colitis-to-cancer transition (CCT) are not well understood. CCT may involve a inflammation-dysplasia-carcinoma progression sequence compared with the better characterized adenoma-carcinoma progression sequence associated with sporadic CRC. One common thread may be activating mutations in components of the Wnt/β-catenin signaling pathway, which occur commonly as early events in sporadic CRC. To examine this hypothesis, we evaluated possible associations between Wnt/β-catenin signaling and CCT based on the cancer stem cell (CSC) model. Wnt/β-catenin immunostaining indicated that UC patients have a level of Wnt-pathway-active cells that is intermediate between normal colon and CRC. These UC cells exhibiting activation of the Wnt pathway constituted a major subpopulation (52% + 7.21) of the colonic epithelial cells positive for aldehyde dehydrogenase (ALDH), a putative marker of precursor colon CSC (pCCSC). We further fractionated this subpopulation of pCCSC using a Wnt pathway reporter assay. Over successive passages, pCCSCs with the highest Wnt activity exhibited higher clonogenic and tumorigenic potential than pCCSCs with the lowest Wnt activity, thereby establishing the key role of Wnt activity in driving CSC-like properties in these cells. Notably, 5/20 single cell injections of high-Wnt pCCSC resulted in tumor formation, suggesting a correlation with CCT. Attenuation of Wnt/β-catenin in high-Wnt pCCSC by shRNA-mediated downregulation or pharmacological inhibition significantly reduced tumor growth rates. Overall, the results of our study indicates (i) that early activation of Wnt/β-catenin signaling is critical for CCT and (ii) that high levels of Wnt/β-catenin signaling can further demarcate high-ALDH tumor-initiating cells in the nondysplastic epithelium of UC patients. As such, our findings offer plausible diagnostic markers and therapeutic target in the Wnt signaling pathway for early intervention in CCT.

Pharmacological targeting of mammalian target of rapamycin inhibits ovarian granulosa cell tumor growth

Few targeted therapies have been developed for ovarian granulosa cell tumor (GCT), even though it represents 5% of all malignant ovarian tumors in women. As misregulation of PI3K/AKT signaling has been implicated in GCT development, we hypothesized that the AKT signaling effector mammalian target of rapamycin (mTOR) may play a role in the pathogenesis of GCT and could represent a therapeutic target. Analyses of human GCT samples showed an increase in protein levels of mTOR and its downstream effectors RPS6KB1, RPS6, eIF4B and PPARG relative to normal granulosa cells, suggestive of an increase in mTOR pathway activity and increased translational activity and/or protein stability. We next sought to evaluate mTOR as a GCT therapeutic target using the Pten (tm1Hwu/tmiHwu);Ctnnb1 (tm1Mmt/+);Amhr2 (tm3(cre)Bhr/+) (PCA) mouse model, in which mTOR, RPS6KB1, eIF4B and PPARG are upregulated in tumor cells in a manner similar to human GCT. Treatment of PCA mice with the mTOR-specific inhibitor everolimus reduced tumor growth rate (1.5-fold; P < 0.05) and also reduced total tumor burden (4.7-fold; P < 0.05) and increased survival rate (78 versus 44% in the vehicle group) in a PCA surgical model of GCT peritoneal carcinomatosis. Everolimus decreased tumor cell proliferation and tumor cell volume relative to controls (P < 0.05), whereas apoptosis was unaffected. Phosphorylation of RPS6KB1 and RPS6 were decreased (P < 0.05) by everolimus, but RPS6KB1, RPS6, eIF4B and PPARG expressions were not affected. These results suggest that mTOR is a valid and clinically useful pharmacological target for the treatment of GCT, although its inhibition does not reverse all consequences of aberrant PI3K/AKT signaling in the PCA model.

Carbonic Anhydrase IX Promotes Tumor Growth and NecrosisIn Vivoand Inhibition Enhances Anti-VEGF Therapy

Bevacizumab, an anti-VEGFA antibody, inhibits the developing vasculature of tumors, but resistance is common. Antiangiogenic therapy induces hypoxia and we observed increased expression of hypoxia-regulated genes, including carbonic anhydrase IX (CAIX), in response to bevacizumab treatment in xenografts. CAIX expression correlates with poor prognosis in most tumor types and with worse outcome in bevacizumab-treated patients with metastatic colorectal cancer, malignant astrocytoma, and recurrent malignant glioma. We knocked down CAIX expression by short hairpin RNA in a colon cancer (HT29) and a glioblastoma (U87) cell line which have high hypoxic induction of CAIX and overexpressed CAIX in HCT116 cells which has low CAIX. We investigated the effect on growth rate in three-dimensional (3D) culture and in vivo, and examined the effect of CAIX knockdown in combination with bevacizumab. CAIX expression was associated with increased growth rate in spheroids and in vivo. Surprisingly, CAIX expression was associated with increased necrosis and apoptosis in vivo and in vitro. We found that acidity inhibits CAIX activity over the pH range found in tumors (pK = 6.84), and this may be the mechanism whereby excess acid self-limits the build-up of extracellular acid. Expression of another hypoxia inducible CA isoform, CAXII, was upregulated in 3D but not two-dimensional culture in response to CAIX knockdown. CAIX knockdown enhanced the effect of bevacizumab treatment, reducing tumor growth rate in vivo. This work provides evidence that inhibition of the hypoxic adaptation to antiangiogenic therapy enhances bevacizumab treatment and highlights the value of developing small molecules or antibodies which inhibit CAIX for combination therapy.

Analyzing the Pivotal Trial That Compared Sunitinib and IFN-α in Renal Cell Carcinoma, Using a Method That Assesses Tumor Regression and Growth

We applied a method that analyzes tumor response, quantifying the rates of tumor growth (g) and regression (d), using tumor measurements obtained while patients receive therapy. We used data from the phase III trial comparing sunitinib and IFN-α in metastatic renal cell carcinoma (mRCC) patients. The analysis used an equation that extracts d and g. For sunitinib, overall survival (OS) was strongly correlated with log g (Rsq = 0.44, P < 0.0001); much less with log d (Rsq = 0.04; P = 0.0002). The median g of tumors in these patients (0.00082 per days; log g = -3.09) was about half that (P < 0.001) of tumors in patients receiving IFN-α (0.0015 per day; log g = -2.81). With IFN-α, the OS/log g correlation (Rsq = 0.14) was weaker. Values of g from measurements obtained by study investigators or central review were highly correlated (Rsq = 0.80). No advantage resulted in including data from central review in regressions. Furthermore, g can be estimated accurately four months before treatment discontinuation. Extrapolating g in a model that incorporates survival generates the hypothesis that g increased after discontinuation of sunitinib but did not accelerate. In patients with mRCC, sunitinib reduced tumor growth rate, g, more than did IFN-α. Correlating g with OS confirms earlier analyses suggesting g may be an important clinical trial endpoint, to be explored prospectively and in individual patients.

Abstract LB-356: Micelle-encapsulated thiostrepton: an effective nanomedicine for inhibiting tumor growth

Abstract The thiazole antiobiotic, thiostrepton, has been found to suppress the expression of FOXM1, the oncogenic forkhead transcription factor overexpressed in cancer cells. Thiostrepton has also been shown to induce cell death in cancer cells through a proteasome inhibitory mechanism. As thiostrepton is highly hydrophobic, encapsulation into nano-sized delivery vehicles not only aides its solubilization but also improves its targeting for tumors in vivo. This is achieved by encapsulating thiostrepton into the interior of an amphiphilic micelle, composed from an assembly of lipid-PEG (polyethylene glycol) conjugates. In this study, we examined the anti-tumor effects of micelle-thiostrepton in both subcutaneous xenograft models (HepG2 liver cancer, Capan-2 pancreatic cancer and MDA-MB-231 breast cancer) and endogenous liver tumor models (DEN/PB-induced adenocarcinomas). In the case of the xenograft tumor-bearing mice, biodistribution studies showed micelle-thiostrepton to accumulate into both the liver (30% of injected dose) and the xenograft tumors (60% of injected dose) from 4 hours post-administration. In the endogenous liver cancer models, micelle-thiostrepton accumulated into the liver only, allowing a high concentration of tumor-associated thiostrepton drug. Continuous administration of micelle-thiostrepton at doses of 40mg/kg, 3 times a week for 30 days retarded the growth rate of all subcutaneous tumors and reduced the occurrence of adenocarinomas in endogenous liver cancer models. Immunoblotting and immunohistochemical staining revealed that treatment with micelle-thiostrepton inhibited the expression of the oncogenic transcription factor FOXM1 in tumors, a reduction of which generally coincides with suppressed cell proliferation and increased cell death. Furthermore, it was found that the reduced tumor growth rate of thiostrepton-treated tumors was indeed related to increased apoptosis, as indicated by the elevated expression of cleaved caspase-3 in such tumors samples. Our data suggest that the thiazole antibiotic/proteasome inhibitor thiostrepton, when formulated into nanoparticles, may be highly suited as a nanomedicine for treating cancer, particularly that of liver 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 LB-356. doi:1538-7445.AM2012-LB-356

Effect of chronic fish oil supplementation on renal function parameters of healthy and cachectic rats

Fish oil (FO) supplementation, rich in n‐3 PUFA, has a positive effect on various diseases, including cancer and kidney diseases. The objective of this study was evaluate renal function of healthy and tumor‐bearing Walker 256 rats chronically supplemented with fish oil. Weanling male rats (aged 21 days) were divided in two groups, one received a standard commercial chow (C) and the other were supplemented orally for 70 days with FO, 1g/kg body weight (FO). At 90 days of age half the animals of each group were injected into the right flank with a sterile suspension of 2 × 107 Walker 256 tumor cells (W and FOW group). Renal function parameters, urinary excretion of TBX2 and immunolocalization of ED‐1 macrophage, were measured in all groups. Tumor‐bearing group present a significant reduction of total plasma protein concentration which reflects cachectic proteolysis, and FO supplementation reversed this fact. Tumor growth rate was also markedly reduced in FOW group. Glomerular filtration (inulin clearance) did not present significant changes in the FO or W groups if compared to C, but increased significantly in WFO group. Renal plasma flow (p‐aminohippuric acid clearance) increased significantly in all supplemented groups, 54% in FO and 91% in FOW. W group had low plasma osmolality compared to C, an alteration reverted in supplemented animals. The FENa+ of FO rats were similar to C. In contrast, FENa+ was slightly decreased in the W compared to C, a tendency which it is reverted in WFO group. Proximal Na+ reabsorption, evaluated by lithium clearance, was similar among the groups. Urinary excretion of TBX2 was significantly decreased after FO supplementation in C and W. The number of macrophages in renal tissue was significantly higher in W, a value that was reduced to control levels by FO supplementation. In resume, FO supplementation reduced cachexia, with a reduction in tumor growth rate, and had a renoprotector effect, with a rise in renal plasma flow and a preservation of glomerular filtration.

Recombinant Lysyl Oxidase Propeptide Protein Inhibits Growth and Promotes Apoptosis of Pre-Existing Murine Breast Cancer Xenografts

Lysyl oxidase propeptide (LOX-PP) ectopic overexpression inhibits the growth of cancer xenografts. Here the ability and mode of action of purified recombinant LOX-PP (rLOX-PP) protein to inhibit the growth of pre-existing xenografts was determined. Experimental approaches employed were direct intratumoral injection (i.t.) of rLOX-PP protein into murine breast cancer NF639 xenografts, and application of a slow release formulation of rLOX-PP implanted adjacent to tumors in NCR nu/nu mice (n = 10). Tumors were monitored for growth, and after sacrifice were subjected to immunohistochemical and Western blot analyses for several markers of proliferation, apoptosis, and for rLOX-PP itself. Direct i.t. injection of rLOX-PP significantly reduced tumor volume on days 20, 22 and 25 and tumor weight at harvest on day 25 by 30% compared to control. Implantation of beads preloaded with 35 micrograms rLOX-PP (n = 10) in vivo reduced tumor volume and weight at sacrifice when compared to empty beads (p<0.05). A 30% reduction of tumor volume on days 22 and 25 (p<0.05) and final tumor weight on day 25 (p<0.05) were observed with a reduced tumor growth rate of 60% after implantation. rLOX-PP significantly reduced the expression of proliferation markers and Erk1/2 MAP kinase activation, while prominent increases in apoptosis markers were observed. rLOX-PP was detected by immunohistochemistry in harvested rLOX-PP tumors, but not in controls. Data provide pre-clinical findings that support proof of principle for the therapeutic anti-cancer potential of rLOX-PP protein formulations.

Combination with bortezomib enhances the antitumor effects of nanoparticle-encapsulated thiostrepton

Bortezomib is well-known for inducing cell death in cancer cells, specifically through the mechanism of proteasome inhibition. Thiostrepton, a thiazole antibiotic, has also been described for its proteasome inhibitory action, although differing slightly to bortezomib in the proteasomal site to which it is active. Previously we had shown the synergic effect of bortezomib and thiostrepton in breast cancer cells in vitro, where sub-apoptotic concentrations of both proteasome inhibitors resulted in synergic increase in cell death when combined as a treatment. Here, we administered such a combination to MDA-MB-231 xenograft tumors in vivo, and found that the effect of complementary proteasome inhibitors reduced tumor growth rates more efficiently than compared with when administered alone. Increased induction of apoptotic activity in tumors was found be associated with the growth inhibitory activity of combination treatment. Further examination additionally revealed that combination-treated tumors exhibited reduced proteasome activity, compared with non-treated and single drug-treated tumors. These data suggest that this drug combination may be useful as a therapy for solid tumors.

Micelle-Encapsulated Thiostrepton as an Effective Nanomedicine for Inhibiting Tumor Growth and for Suppressing FOXM1 in Human Xenografts

The thiazole antiobiotic, thiostrepton, has been found to induce cell death in cancer cells through proteasome inhibition. As a proteasome inhibitor, thiostrepton has also been shown to suppress the expression of FOXM1, the oncogenic forkhead transcription factor overexpressed in cancer cells. In this study, we explored the potential in vivo anticancer properties of thiostrepton, delivered through nanoparticle encapsulation to xenograft models of breast and liver cancer. We encapsulated thiostrepton into micelles assembled from amphiphilic lipid-PEG (polyethylene glycol) molecules, where thiostrepton is solubilized within the inner lipid compartment of the micelle. Upon assembly, hydrophobic thiostrepton molecules are solubilized into the lipid component of the micelle shell, formed through the self-assembly of amphipilic lipid-PEG molecules. Maximum accumulation of micelle-thiostrepton nanoparticles (100 nm in diameter, -16 mV in zeta potential) into tumors was found at 4 hours postadministration and was retained for at least 24 hours. Upon continuous treatment, we found that nanoparticle-encapsulated thiostrepton reduced tumor growth rates of MDA-MB-231 and HepG2 cancer xenografts. Furthermore, we show for the first time the in vivo suppression of the oncogenic FOXM1 after treatment with proteasome inhibitors. Immunoblotting and immunohistochemical staining also showed increased apoptosis in the treated tumors, as indicated by cleaved caspase-3 expression. Our data suggest that the thiazole antibiotic/proteasome inhibitor thiostrepton, when formulated into nanoparticles, may be highly suited as a nanomedicine for treating human cancer.

The targeted delivery of IL17 to the mouse tumor neo-vasculature enhances angiogenesis but does not reduce tumor growth rate

There has been a long controversy as to whether interleukin-17 (IL17) has an impact on tumor growth. In order to assess whether IL17 may affect tumor growth, it would be convenient to achieve high levels of this pro-inflammatory cytokine at the tumor neo-vasculature, since IL17 is known to promote angiogenesis. Here, we have generated and tested in vivo a fusion protein, consisting of the F8 antibody (specific to the alternatively spliced EDA domain of fibronectin, a marker of angiogenesis) and of murine IL17 (mIL17). The resulting immunocytokine (termed F8-mIL17) was shown to selectively localize at the tumor neo-vasculature and to vigorously promote tumor angiogenesis, without however reducing or enhancing tumor growth rate both in immunocompetent and in immunodeficient mice.

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Effects of nanoparticle coatings on the activity of oncolytic adenovirus–magnetic nanoparticle complexes

Limitations to adenovirus infectivity can be overcome by association with magnetic nanoparticles and enforced infection by magnetic field influence. Here we examined three core-shell-type iron oxide magnetic nanoparticles differing in their surface coatings, particle sizes and magnetic properties for their ability to enhance the oncolytic potency of adenovirus Ad520 and to stabilize it against the inhibitory effects of serum or a neutralizing antibody. It was found that the physicochemical properties of magnetic nanoparticles are critical determinants of the properties which govern the oncolytic productivities of their complexes with Ad520. Although high serum concentration during infection or a neutralizing antibody had strong inhibitory influence on the uptake or oncolytic productivity of the naked virus, one particle type was identified which conferred high protection against both inhibitory factors while enhancing the oncolytic productivity of the internalized virus. This particle type equipped with a silica coating and adsorbed polyethylenimine, displaying a high magnetic moment and high saturation magnetization, mediated a 50% reduction of tumor growth rate versus control upon intratumoral injection of its complex with Ad520 and magnetic field influence, whereas Ad520 alone was inefficient. The correlations between physical properties of the magnetic particles or virus complexes and oncolytic potency are described herein.