Tumor Growth In Rodent Models Research Articles

Abstract 5243: Averting tumor growth in rodent breast cancer models with a liquid ablation approach

Abstract In low-resource settings, surgery or radiation are inaccessible to 90% of cancer patients due to inadequate infrastructure and lack of trained personnel. Resource appropriate technologies must be developed to make cancer treatments accessible. Ablative techniques, such as radiofrequency ablation are often used as a minimally invasive alternative to surgery. However, energy-based ablation techniques require expensive machinery and a steady supply of electricity and expensive consumables. To address these shortcomings, we have developed a low-cost ablation technique that stabilizes tumor growth with a simple injection of ethanol in combination with a polymer, ethylcellulose (EC). The EC injected with ethanol creates a slow-release depot of ethanol at the injection site by undergoing a solvent induced phase-transition initiated by water in tissues. EC-ethanol improves upon traditional ethanol ablation by providing increased precision while remaining low-cost and simple to administer. We evaluated the precision of EC-ethanol as well as its vascular sclerosing capabilities as a strategy to avert tumor growth in rodent models of breast cancer. EC-ethanol injected at 1 mL/hr with 6% w/w EC was used as this demonstrated to be the optimal formulation and rate for causing tumor necrosis (Morhard et al., Scientific Reports, 2017) and enhanced the anti-tumor immunostimulatory response (Nief et al., 2019, Under Review). We demonstrate the precision delivery of EC-ethanol in orthotopic 67NR mammary tumor as with small-animal MRI. Further, ethanol devascularization is monitored with photoacoustic imaging 24 hours post ablation. Local tumor control was demonstrated both in 67NR mammary tumor and in 7,12-Dimethylbenz[a]anthracene (DMBA) induced rat mammary tumor models. Repeated ablations on significantly decrease primary tumor growth compared to untreated tumors in both the mouse and rat models (P<0.05). In rats weekly 200 µL EC-ethanol injections began when tumors reached 2 mL in volume (n=5), and in 30 days tumors decreased 20% ± 3% untreated controls grew an average of 721%. For mice (n=5), 50 µL was given daily for 6 days on day 3 after tumor implantation to account for small body weight and rapidly growing tumors. Mice treated with EC-ethanol lived significantly longer (P<0.05 log-rank test) than untreated mice and mice injected with the same volume of pure ethanol. EC-ethanol is a low-cost, non-invasive tumor destruction method that shows a clear control of tumor growth when applied as a fractionated dose. We utilized a mouse and rat tumor model to ensure that the EC-ethanol's success is not dependent on the model. Ongoing studies will examine the impact of varying injected volumes at well-defined fractionation schedules on ethanol distribution, necrosis, tumor growth, and survival. Citation Format: Corrine A. Nief, Erika Chelales, Robert Morhard, Maomao Chem, Jeffrey Everitt, Jenna Mueller, Junjie Yao, Mark W. Dewhirst, Nirmala Ramanujam. Averting tumor growth in rodent breast cancer models with a liquid ablation approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5243.

Ultrasound-mediated delivery of siESE complexed with microbubbles attenuates HER2+/- cell line proliferation and tumor growth in rodent models of breast cancer.

The highly tunable, noninvasive and spatially targeted nature of microbubble-enhanced, ultrasound-guided (MB+US) drug delivery makes it desirable for a wide variety of therapies. In breast cancer, both HER2+ and HER2- type neoplasms pose significant challenges to conventional therapeutics in greater than 40% of breast cancer patients, even with the widespread application of biologics such as trastuzumab. To address this therapeutic challenge, we examined the novel combination of tumor-injected microbubble-bound siRNA complexes and monodisperse size-isolated microbubbles (4-µm diameter) to attenuate tumor growth in vivo, as well as MB+US-facilitated shRNA and siRNA knockdown of ESE-1, an effector linked to dysregulated HER2 expression in HER2+/- cell line propagation. We first screened six variants of siESE and shESE for efficient knockdown of ESE in breast cancer cell lines. We demonstrated efficient reduction of BT-474 (PR+, ER+, HER2+; luminal B) and MDA-MB-468 (PR-, ER-, HER2-; triple-negative) clonogenicity and non-adherent growth after knockdown of ESE-1. A significant reduction in proliferative potential was seen for both cell lines using MB+US to deliver shESE and siESE. We then demonstrated significant attenuation of BT-474 xenograft tumor growth in Nod/SCID female mice using direct injection of microbubble-adsorbed siESE to the tumor and subsequent sonication. Our results suggest a positive effect on drug delivery from MB+US, and highlights the feasibility of using RNAi and MB+US for breast cancer pathologies. RNAi coupled with MB+US may also be an effective theranostic approach to treat other acoustically accessible tumors, such as melanoma, thyroid, parotid and skin cancer.

Abstract LB-303: Identification of a mammalian sterile-20 like kinase 4 (MST4) inhibitor to target pituitary tumorigenesis

Abstract Pituitary tumors of the gonadotrope lineage are often large and invasive, resulting in hypopituitarism. There are no medical treatments currently available. Using a combined genetic and genomic screen of human pituitary tumor samples, we identified the Mammalian sterile-20 like kinase 4 (MST4) as a pro-tumorigenic effector, driving increased pituitary cell proliferation and survival in response to a hypoxic microenvironment. MST4 is a member of the germinal center kinase III subfamily of Ste-20 kinases with limited data available concerning its role in tumorigenesis. To identify selective inhibitors of the MST4 kinase, computational-based virtual library screening was employed to dock the commercially available SelleckChem kinase inhibitor library into the ATP binding site of the MST4 crystal structure. Several candidate MST4 kinase inhibitors were identified with the potential to bind with high affinity. Using a newly developed TRFRET in vitro kinase assay, one of the small molecule compounds exhibited a potent inhibition of the MST4 kinase (IC50 = 6.18 nM +/- 2.2 nM). To determine the ability of this inhibitor to antagonize MST4 actions, a hypoxic LβT2 gonadotrope pituitary cell model was used. Under chronic hypoxia (5% O2), cells stably overexpressing MST4 showed an increased rate of proliferation (at 7 days, 3.16-fold, p = 0.006) compared to vector control cells as assessed by BrdU staining. These effects were completely blocked by the inhibitor at 50nM (-2.61-fold, p = 0.01, NS from controls). Similarly, MST4 protection from hypoxia induced apoptosis under 1% O2 for 17h was also inhibited at the same dose as assessed by TUNEL staining (-2.72-fold, p<0.001, NS from controls). Analysis of transfectants expressing wildtype and Mst4-mutants (K53E-kinase dead, T178A-loss of autophosphorylation, and C-terminus deletion) confirmed the inhibitory effect was dependent on the kinase sequence. Together, these data identify a potent inhibitor of the MST4 kinase with the capacity to selectively block pituitary cell growth under a hypoxic microenvironment and support future studies of its ability to modulate pituitary tumor growth in rodent models. Citation Format: Weipeng Xiong, Chris Matheson, Donald S. Backos, Mei Xu, Katja Kiseljak-Vassiliades, Smita Salian-Mehta, Philip Reigan, Margaret E. Wierman. Identification of a mammalian sterile-20 like kinase 4 (MST4) inhibitor to target pituitary tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-303. doi:10.1158/1538-7445.AM2015-LB-303

Targeted prodrug approaches for hormone refractory prostate cancer.

Due to the propensity of relapse and resistance with prolonged androgen deprivation therapy (ADT), there is a growing interest in developing non-hormonal therapeutic approaches as alternative treatment modalities for hormone refractory prostate cancer (HRPC). Although the standard treatment for HRPC consists of a combination of ADT with taxanes and anthracyclines, the clinical use of chemotherapeutics is limited by systemic toxicity stemming from nondiscriminatory drug exposure to normal tissues. In order to improve the tumor selectivity of chemotherapeutics, various targeted prodrug approaches have been explored. Antibody-directed enzyme prodrug therapy (ADEPT) and gene-directed enzyme prodrug therapy (GDEPT) strategies leverage tumor-specific antigens and transcription factors for the specific delivery of cytotoxic anticancer agents using various prodrug-activating enzymes. In prostate cancer, overexpression of tumor-specific proteases such as prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) is being exploited for selective activation of anticancer prodrugs designed to be activated through proteolysis by these prostate cancer-specific enzymes. PSMA- and PSA-activated prodrugs typically comprise an engineered high-specificity protease peptide substrate coupled to a potent cytotoxic agent via a linker for rapid release of cytotoxic species in the vicinity of prostate cancer cells following proteolytic cleavage. Over the past two decades, various such prodrugs have been developed and they were effective at inhibiting prostate tumor growth in rodent models; several of these prodrug approaches have been advanced to clinical trials and may be developed into effective therapies for HRPC.

Identification of AREG and PLK1 pathway modulation as a potential key of the response of intracranial 9L tumor to microbeam radiation therapy.

Synchrotron microbeam radiation therapy (MRT) relies on the spatial fractionation of a synchrotron beam into parallel micron-wide beams allowing deposition of hectogray doses. MRT controls the intracranial tumor growth in rodent models while sparing normal brain tissues. Our aim was to identify the early biological processes underlying the differential effect of MRT on tumor and normal brain tissues. The expression of 28,000 transcripts was tested by microarray 6 hr after unidirectional MRT (400 Gy, 50 µm-wide microbeams, 200 µm spacing). The specific response of tumor tissues to MRT consisted in the significant transcriptomic modulation of 431 probesets (316 genes). Among them, 30 were not detected in normal brain tissues, neither before nor after MRT. Areg, Trib3 and Nppb were down-regulated, whereas all others were up-regulated. Twenty-two had similar expression profiles during the 2 weeks observed after MRT, including Ccnb1, Cdc20, Pttg1 and Plk1 related to the mitotic role of the Polo-like kinase (Plk) pathway. The up-regulation of Areg expression may indicate the emergence of survival processes in tumor cells triggered by the irradiation; while the modulation of the "mitotic role of Plk1" pathway, which relates to cytokinetic features of the tumor observed histologically after MRT, may partially explain the control of tumor growth by MRT. The identification of these tumor-specific responses permit to consider new strategies that might potentiate the antitumoral effect of MRT.

Evaluating the cancer therapeutic potential of cardiac glycosides.

Cardiac glycosides, also known as cardiotonic steroids, are a group of natural products that share a steroid-like structure with an unsaturated lactone ring and the ability to induce cardiotonic effects mediated by a selective inhibition of the Na+/K+-ATPase. Cardiac glycosides have been used for many years in the treatment of cardiac congestion and some types of cardiac arrhythmias. Recent data suggest that cardiac glycosides may also be useful in the treatment of cancer. These compounds typically inhibit cancer cell proliferation at nanomolar concentrations, and recent high-throughput screenings of drug libraries have therefore identified cardiac glycosides as potent inhibitors of cancer cell growth. Cardiac glycosides can also block tumor growth in rodent models, which further supports the idea that they have potential for cancer therapy. Evidence also suggests, however, that cardiac glycosides may not inhibit cancer cell proliferation selectively and the potent inhibition of tumor growth induced by cardiac glycosides in mice xenografted with human cancer cells is probably an experimental artifact caused by their ability to selectively kill human cells versus rodent cells. This paper reviews such evidence and discusses experimental approaches that could be used to reveal the cancer therapeutic potential of cardiac glycosides in preclinical studies.

Chronic Social Isolation Is Associated with Metabolic Gene Expression Changes Specific to Mammary Adipose Tissue

Chronic social isolation is linked to increased mammary tumor growth in rodent models of breast cancer. In the C3(1)/SV40 T-antigen FVB/N (TAg) mouse model of "triple-negative" breast cancer, the heightened stress response elicited by social isolation has been associated with increased expression of metabolic genes in the mammary gland before invasive tumors develop (i.e., during the in situ carcinoma stage). To further understand the mechanisms underlying how accelerated mammary tumor growth is associated with social isolation, we separated the mammary gland adipose tissue from adjacent ductal epithelial cells and analyzed individual cell types for changes in metabolic gene expression. Specifically, increased expression of the key metabolic genes Acaca, Hk2, and Acly was found in the adipocyte, rather than the epithelial fraction. Surprisingly, metabolic gene expression was not significantly increased in visceral adipose depots of socially isolated female mice. As expected, increased metabolic gene expression in the mammary adipocytes of socially isolated mice coincided with increased glucose metabolism, lipid synthesis, and leptin secretion from this adipose depot. Furthermore, application of media that had been cultured with isolated mouse mammary adipose tissue (conditioned media) resulted in increased proliferation of mammary cancer cells relative to group-housed-conditioned media. These results suggest that exposure to a chronic stressor (social isolation) results in specific metabolic reprogramming in mammary gland adipocytes that in turn contributes to increased proliferation of adjacent preinvasive malignant epithelial cells. Metabolites and/or tumor growth-promoting proteins secreted from adipose tissue could identify biomarkers and/or targets for preventive intervention in breast cancer.

Abstract 1354: Chronic social stress in a mouse model of triple-negative breast cancer is associated with oncogenic changes in mammary fat.

Abstract Chronic social isolation is linked to increased mammary tumor growth in rodent models of breast cancer. In the C3(1)/SV40 T-antigen (Tag) FVB/N mouse model of “triple-negative” breast cancer, the heightened stress response elicited by social isolation has been associated with increased expression of metabolic genes in the mammary gland before invasive tumors develop (i.e. during the in situ carcinoma stage). To further understand how underlying accelerated mammary tumor growth is associated with social isolation, we separated the mammary gland adipose tissue from adjacent ductal epithelial cells and analyzed individual cell types for changes in metabolic gene expression. Specifically, increased expression of the key metabolic genes Acaca, Hk2 and Acly was found primarily in the adipocyte, rather than epithelial fraction. Increased metabolic gene expression in the mammary adipocytes of socially isolated mice coincided with increased glucose metabolism, lipid synthesis, and leptin expression. Furthermore, culture media from isolated mouse mammary adipose tissue resulted in relatively increased proliferation and decreased apoptosis of mammary epithelial cancer cells derived from the same mouse strain. Additional gene expression analyses identified significant upregulation of the novel carbohydrate response element binding protein beta isoform (CHREBP-β) in socially isolated mammary fat. We hypothesize that the response to social isolation induces metabolic changes in mammary fat that are coordinated through CHREBP-β and are pro-oncogenic. We are currently performing global lipidomic and metabolomic analyses of mammary adipose tissue and secreted culture media to identify bioactive molecules involved in tumor growth. Citation Format: Paul A. Volden, Erin Wonder, Maxwell Skor, Feenalie Patel, Martha McClintock, Matthew Brady, Suzanne Conzen. Chronic social stress in a mouse model of triple-negative breast cancer is associated with oncogenic changes in mammary fat. [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 1354. doi:10.1158/1538-7445.AM2013-1354

In Vivo Imaging and Quantification of Iron Oxide Nanoparticle Uptake and Biodistribution.

Recent advances in nanotechnology have allowed for the effective use of iron oxide nanoparticles (IONPs) for cancer imaging and therapy. When activated by an alternating magnetic field (AMF), intra-tumoral IONPs have been effective at controlling tumor growth in rodent models. To accurately plan and assess IONP-based therapies in clinical patients, noninvasive and quantitative imaging technique for the assessment of IONP uptake and biodistribution will be necessary. Proven techniques such as confocal, light and electron microscopy, histochemical iron staining, ICP-MS, fluorescent labeled mNPs and magnetic spectroscopy of Brownian motion (MSB), are being used to assess and quantify IONPs in vitro and in ex vivo tissues. However, a proven noninvasive in vivo IONP imaging technique has not yet been developed. In this study we have demonstrated the shortcomings of computed tomography (CT) and magnetic resonance imaging (MRI) for effectively observing and quantifying iron/IONP concentrations in the clinical setting. Despite the poor outcomes of CT and standard MR sequences in the therapeutic concentration range, ultra-short T2 MRI methods such as, Sweep Imaging With Fourier Transformation (SWIFT), provide a positive iron contrast enhancement and a reduced signal to noise ratio. Ongoing software development and phantom and in vivo studies, will further optimize this technique, providing accurate, clinically-relevant IONP biodistribution information.

Macronutrient Intake and Cancer: How Does Dietary Restriction Influence Tumor Growth and Why Should We Care?

This perspective on the report by Rogozina and colleagues (beginning on page 712 in this issue of the journal) reviews mechanisms that may underlie inhibitory effects of dietary restriction on tumor growth in the mouse mammary tumor virus transforming growth factor alpha (MMTV-TGF-alpha) breast cancer model and comments on the potential clinical relevance of these mechanisms. The inhibitory effect of caloric restriction on carcinogenesis and tumor growth in rodent models is a classic finding that is receiving increasing attention. In some experimental models, the magnitude of the effect is significant, rivaling what can be achieved by pharmacologic approaches to cancer prevention or treatment. Major challenges include defining the underlying mechanisms and determining the clinical relevance of laboratory models of caloric restriction.