Halina Onishko

Tumor-targeted delivery of liposome-encapsulated doxorubicin by use of a peptide that selectively binds to irradiated tumors.

Tumor-targeted drug delivery improves anti-tumor efficacy and reduces systemic toxicity by limiting bioavailability of cytotoxic drugs to within tumors. Targeting reagents, such as peptides or antibodies recognizing molecular targets over-expressed within tumors, have been used to improve liposome-encapsulated drug accumulation within tumors and resulted in enhanced tumor growth control. In this report, we expand the scope of targeting reagents by showing that one peptide, HVGGSSV which was isolated from an in vivo screening of phage-displayed peptide library due to its selective binding within irradiated tumors, enabled highly selective tumor-targeted delivery of liposome-encapsulated doxorubicin and resulted in enhanced cytotoxicity within tumors. Targeting liposomes (TL) and non-targeting liposomes (nTL) were labeled with Alexa Fluor 750. Biodistribution of the liposomes within tumor-bearing mice was studied with near infrared (NIR) imaging. In the single dose pharmacokinetic study, the liposomal doxorubicin has an extended circulation half life as compared to the free doxorubicin. Targeting liposomes partitioned to the irradiated tumors and improved drug deposition and retention within tumors. The tumor-targeted delivery of doxorubicin improved tumor growth control as indicated with reduced tumor growth rate and tumor cell proliferation, enhanced tumor blood vessel destruction, and increased treatment-associated apoptosis and necrosis of tumor cells. Collectively, the results demonstrated the remarkable capability of the HVGGSSV peptide in radiation-guided drug delivery to tumors.

Noninvasive assessment of cancer response to therapy

Rapid assessment of cancer response to a therapeutic regimen can determine efficacy early in the course of treatment. Although biopsies of cancer can be used to rapidly assess pharmacodynamic response, certain disease sites are less accessible to repeated biopsies. Here, we simultaneously assess response in all sites of disease within days of starting therapy by use of peptide ligands selected for their ability to discern responding from nonresponding cancers. When conjugated to near-infrared imaging agents, the HVGGSSV peptide differentiates between these two types of cancer. Rapid, noninvasive assessment of the pharmacodynamic response within cancer promises to accelerate drug development and minimize the duration of treatment with ineffective regimens in cancer patients.

Radiation induces an antitumour immune response to mouse melanoma.

Purpose: Irradiation of cancer cells can cause immunogenic death. We used mouse models to determine whether irradiation of melanoma can enhance the host antitumour immune response and function as an effective vaccination strategy, and investigated the molecular mechanisms involved in this radiation-induced response. Materials and methods: For in vivo studies, C57BL6/J mice and the B16F0 melanoma cell line were used in a lung metastasis model, intratumoural host immune activation assays, and tumour growth delay studies. In vitro studies included a dendritic cell (DC) phagocytosis assay, detection of cell surface exposure of the protein calreticulin (CRT), and small interfering RNA (siRNA)-mediated depletion of CRT cellular levels. Results: Irradiation of cutaneous melanomas prior to their resection resulted in more than 20-fold reduction in lung metastases after systemic challenge with untreated melanoma cells. A syngeneic vaccine derived from irradiated melanoma cells also induced adaptive immune response markers in irradiated melanoma implants. Our data indicate a trend for radiation-induced increase in melanoma cell surface exposure of CRT, which is involved in the enhanced phagocytic activity of DC against irradiated melanoma cells (VIACUC). Conclusion: The present study suggests that neoadjuvant irradiation of cutaneous melanoma tumours prior to surgical resection can stimulate an endogenous anti-melanoma host immune response.

Inhibition of NF-kappa B activity in mammary epithelium increases tumor latency and decreases tumor burden.

The transcription factor nuclear factor kappa B (NF-κB) is activated in human breast cancer tissues and cell lines. However, it is unclear whether NF-κB activation is a consequence of tumor formation or a contributor to tumor development. We developed a doxycycline (dox)-inducible mouse model, termed DNMP, to inhibit NF-κB activity specifically within the mammary epithelium during tumor development in the polyoma middle T oncogene (PyVT) mouse mammary tumor model. DNMP females and PyVT littermate controls were treated with dox from 4 to 12 weeks of age. We observed an increase in tumor latency and a decrease in final tumor burden in DNMP mice compared with PyVT controls. A similar effect with treatment from 8 to 12 weeks indicates that outcome is independent of effects on postnatal virgin ductal development. In both cases, DNMP mice were less likely to develop lung metastases than controls. Treatment from 8 to 9 weeks was sufficient to impact primary tumor formation. Inhibition of NF-κB increases apoptosis in hyperplastic stages of tumor development and decreases proliferation at least in part by reducing Cyclin D1 expression. To test the therapeutic potential of NF-κB inhibition, we generated palpable tumors by orthotopic injection of PyVT cells and then treated systemically with the NF-κB inhibitor thymoquinone (TQ). TQ treatment resulted in a reduction in tumor volume and weight as compared with vehicle-treated control. These data indicate that epithelial NF-κB is an active contributor to tumor progression and demonstrate that inhibition of NF-κB could have a significant therapeutic impact even at later stages of mammary tumor progression.

NF-kappaB activation within macrophages leads to an anti-tumor phenotype in a mammary tumor lung metastasis model.

IntroductionMetastasis from primary tumor to the lungs is a major cause of the mortality associated with breast cancer. Both immune and inflammatory responses impact whether circulating mammary tumor cells successfully colonize the lungs leading to established metastases. Nuclear factor -kappaB (NF-κB) transcription factors regulate both immune and inflammatory responses mediated in part by the activities of macrophages. Therefore, NF-κB activity specifically within macrophages may be a critical determinant of whether circulating tumor cells successfully colonize the lungs.MethodsTo investigate NF-κB signaling within macrophages during metastasis, we developed novel inducible transgenic models which target expression of the reverse tetracycline transactivator (rtTA) to macrophages using the cfms promoter in combination with inducible transgenics that express either an activator (cIKK2) or an inhibitor (IκBα-DN). Doxycyline treatment led to activation or inhibition of NF-κB within macrophages. We used a tail vein metastasis model with mammary tumor cell lines established from MMTV-Polyoma Middle T-Antigen-derived tumors to investigate the effects of modulating NF-κB in macrophages during different temporal windows of the metastatic process.ResultsWe found that activation of NF-κB in macrophages during seeding leads to a reduction in lung metastases. The mechanism involved expression of inflammatory cytokines and reactive oxygen species, leading to apoptosis of tumor cells and preventing seeding in the lung. Activation of NF-κB within macrophages after the seeding phase has no significant impact on establishment of metastases.ConclusionsOur results have identified a brief, defined window in which activation of NF-κB has significant anti-metastatic effects and inhibition of NF-κB results in a worse outcome.

Aberrant activation of NF-κB signaling in mammary epithelium leads to abnormal growth and ductal carcinoma in situ

BackgroundApproximately 1 in 5 women diagnosed with breast cancer are considered to have in situ disease, most often termed ductal carcinoma in situ (DCIS). Though recognized as a risk factor for the development of more invasive cancer, it remains unclear what factors contribute to DCIS development. It has been shown that inflammation contributes to the progression of a variety of tumor types, and nuclear factor kappa B (NF-κB) is recognized as a master-regulator of inflammatory signaling. However, the contributions of NF-κB signaling to tumor initiation are less well understood. Aberrant up-regulation of NF-κB activity, either systemically or locally within the breast, could occur due to a variety of commonly experienced stimuli such as acute infection, obesity, or psychological stress. In this study, we seek to determine if activation of NF-κB in mammary epithelium could play a role in the formation of hyperplastic ductal lesions.MethodsOur studies utilize a doxycycline-inducible transgenic mouse model in which constitutively active IKKβ is expressed specifically in mammary epithelium. All previously published models of NF-κB modulation in the virgin mammary gland have been constitutive models, with transgene or knock-out present throughout the life and development of the animal. For the first time, we will induce activation at later time points after normal ducts have formed, thus being able to determine if NF-κB activation can promote pre-malignant changes in previously normal mammary epithelium.ResultsWe found that even a short pulse of NF-κB activation could induce profound remodeling of mammary ductal structures. Short-term activation created hyperproliferative, enlarged ducts with filled lumens. Increased expression of inflammatory markers was concurrent with the down-regulation of hormone receptors and markers of epithelial differentiation. Furthermore, the oncoprotein mucin 1, known to be up-regulated in human and mouse DCIS, was over-expressed and mislocalized in the activated ductal tissue.ConclusionsThese results indicate that aberrant NF-κB activation within mammary epithelium can lead to molecular and morphological changes consistent with the earliest stages of breast cancer. Thus, inhibition of NF-κB signaling following acute inflammation or the initial signs of hyperplastic ductal growth could represent an important opportunity for breast cancer prevention.

Abstract 1845: Sensitivity of PC9 cells to erlotinib is affected by extracellular matrix

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Tyrosine kinase inhibitors of epidermal growth factor receptor, such as erlotinib and gefinitib, have been effective in the initial treatment of non-small cell lung cancer. With time, however, initially responsive tumors almost invariably rebound and proliferate under therapy. Of general interest is the impact of tumor cell heterogeneity on rebound time. Previously, we isolated 89 single-cell derived discrete sublines (DS) from the commonly studied PC9 cell line by monitoring the distribution of cell fates (division, quiescence, and cell death) and the drug-induced proliferation (DIP) rate under erlotinib treatment. In this work we examine the role of the extracellular matrix (ECM) in determining proliferation behavior and the distribution of cell fates. Cells suspended in 3D matrices show increased sensitivity to erlotinb with the rate of cell death depending on the ECM composition. DS sublines that were highly resilient in 2D culture (i.e., positive DIP rate and <10% cell death under treatment) asymptote to about 40% cell death in Matrigel after ten days in 1 µM erlotinb. Both the rate of cell death and the total percentage of the cell population that dies increased in collagen I and in collagen I coated hydrogel. At asymptotic levels, the cell population contained both dividing and quiescent cells. In contrast, DS sublines with negative DIP rates in 2D culture reached 90-99% cell death in each ECM considered. We conclude that the pathways conferring resilience in these cells are sensitive to ECM conditions. Intriguingly, we find that resilient DS sublines tend to have diversity in cluster morphologies in the matrices, suggesting a possible correlation between ECM engagement and/or adhesion and resilience. Molecular studies should clarify mechanistic relationships between ECM and sensitivity to targeted therapy, and whether such relationship may extend to clinical tumors. Citation Format: Halina M. Onishko, Jie Zhao, Katherine L. Jameson, Peter L. Frick, Darren R. Tyson, Vito Quaranta, Thomas E. Yankeelov, Erin C. Rericha. Sensitivity of PC9 cells to erlotinib is affected by extracellular matrix. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1845. doi:10.1158/1538-7445.AM2014-1845

Abstract 3010: The role of NF-kappaB in mammary tumor initiation

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Nuclear factor kappa B signaling is accepted as a major component of pro-survival signaling within breast cancer cells. However, less is known about how NF-kappaB could contribute to breast tumor initiation. Inflammatory signaling can be activated within the breast by such common factors as obesity, involution following lactation, aging, and psychological stress. Thus, understanding how activated NF-kappaB could lead to the initiation of breast tumors would provide opportunities to develop prevention strategies. We generated a doxycycline-inducible mouse model to study the effects of constitutively active IKK2 on ductal morphogenesis. In this model, Tet-o-cIKK2 mice are crossed with MMTV-rtTA transgenics to target inducible expression specifically to the mammary epithelium. Expression of cIKK2 in transplanted mammary tissue results in enlarged terminal end-buds, increased lateral branching, and intraductal hyperplasia. Transgene induction for only 3 days in a 6 week old female induces filling of ductal lumens, hypercellularity and loss of strict basal/luminal structure. Quantitative PCR of mammary tissue reveals up-regulated markers of macrophage infiltration such as CCL2 and F4/80 and markers of inflammation including Cox-2, TNF-alpha, and CXCL1. Interestingly, constitutive IKK2 activation also results in a decrease in hormone receptor levels (ERα and PR). Immunofluorescent staining shows that activation of classical NF-kappaB increases nuclear p65, as expected. However, nuclear p52 is also increased in transgene positive cells, suggesting that a subset of the effects resulting from activation of classical NF-kappaB are mediated by cross talk with the alternative pathway. Taken together, we find that activation of NF-kappaB in mammary epithelium is sufficient to induce hyperplastic growth and loss of ductal structure. Ongoing studies seek to define what additional factors, such as the right hormonal context, will cause the lesions to progress to malignancy. Citation Format: Whitney Barham, Oleg Tikhomirov, Lianyi Chen, Ryan Ortega, Halina Onishko, Linda Connelly, Fiona E. Yull. The role of NF-kappaB in mammary tumor initiation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3010. doi:10.1158/1538-7445.AM2014-3010

Abstract 1545: Education of macrophages through modulation of NF-kappaB: an opportunity for targeted therapy.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Macrophages are a plastic cell type, capable of adapting to numerous signals within their environment. As part of the innate immune system, macrophages were traditionally considered anti-tumor (M1), but it has been well established that macrophages can also help to create a pro-tumor, pro-metastatic tumor niche (M2). NF-κB transcription factors can regulate both pro- (MMPs, VEGF) and anti-tumor (iNOS) downstream targets within macrophages, suggesting that modulation of NF-κB may play a role in the two different macrophage phenotypes. However, our understanding of NF-κB signaling specifically within macrophages during tumor progression is limited. To this end, we have developed murine transgenic models that enable us to induce expression of an activator or dominant inhibitor of NF-κB in macrophages by adding doxycycline to the drinking water of mice. We have combined these novel transgenics with the polyoma model of mammary cancer for our studies. We have recently shown that activation of NF-κB in macrophages significantly limits metastasis in a tail vein model of tumor progression. In this model, constitutive IKK2 activity within macrophages leads to an anti-tumor immune response including altered immune cell populations within the lung microenvironment, changes in chemokine and cytokine expression and rapid killing of tumor cells during the seeding phase mediated by reactive oxygen species. Our current work has extended these findings to an orthotopic mammary tumor model. Again, we find that activation of NF-κB in macrophages results in decreased primary tumor growth and decreased tumor seeding into the blood. To model this activation in vitro, we have utilized immortalized bone marrow derived macrophages from IκBα knock-out mice compared to a wild type line. IκBα KO macrophages display changes in morphology and adhesion relative to wild type macrophages. This correlates with increased cytotoxic behavior in co-culture with polyoma tumor cells, mirroring the in vivo phenotype of the cIKK2 expressing macrophages. Given these findings, we believe that targeted activation of NF-κB signaling in macrophages could be harnessed to overcome the education of macrophages by tumor cells, and could be exploited as a novel targeted therapy. Citation Format: Whitney Barham, Oleg Tikhomirov, Lianyi Chen, Ryan Ortega, Linda Gleaves, Halina Onishko, Taylor Sherrill, Linda Connelly, Timothy S. Blackwell, Fiona E. Yull. Education of macrophages through modulation of NF-kappaB: an opportunity for targeted 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 1545. doi:10.1158/1538-7445.AM2013-1545

Abstract 2894: Environmentally responsive nanoparticles for the intracellular delivery of RNAi therapeutics into tumor-associated macrophages

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Tumor-associated macrophages (TAMs) represent a potentially promising therapeutic target in cancer because they have been shown to facilitate tumor growth, invasiveness, and metastasis. However, methods to specifically target therapies to TAMs are lacking. To address this problem, we designed and synthesized mannosylated micellar nanoparticles (ManNPs), composed of tri-block co-polymers. The three polymer blocks include (1) an azido-displaying block for functionalization with biomolecules via azide-alkyne ‘click’ chemistry, (2) a cationic block for the condensation of polyanions such as siRNA, and (3) a pH-responsive terpolymer block that facilitates endosomal disruption. This terpolymer is hydrophobic at pH 7.4, allowing these polymers to self-assemble into 25 nm micellar nanoparticles under physiologic conditions. However, they become protonated at lower pH ranges representative of endosomal compartments (5.8 - 6.2), leading to disassembly of the nanoparticles, and increased ability to disrupt endosomal membranes and enable cytoplasmic delivery. This pH-dependent behavior has been validated using a red blood cell hemolysis assay. This environmentally-responsive behavior facilitates improved cytoplasmic delivery of siRNA, access to the intracellular silencing machinery, and consequently, knockdown of target gene expression. Further, we demonstrate that mannosylation of these nanoparticles via ‘click’ chemistry significantly enhances their ability to deliver siRNA into murine bone marrow-derived primary macrophages (BMDMs), relative to control, untargeted nanoparticles. Targeted nanoparticle uptake is mediated specifically through the macrophage mannose receptor (CD206), as validated through competition experiments with free mannose, or by pre-incubating BMDMs with lipopolysaccharide to downregulate CD206 expression. This is particularly important for cancer applications because CD206 is upregulated in tumor-suppressed and non-activated macrophages, enabling more specific targeting of TAMs versus healthy macrophages in other tissues. The ManNPs described here present new opportunities to target TAMs in various cancers, providing an enabling technology for the modification of the immunosuppressive tumor environment by targeting TAM activity. Studies are pending to demonstrate this behavior in an in vivo murine model of metastatic breast 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 2894. doi:1538-7445.AM2012-2894