Murine Mammary Carcinoma Cells Research Articles

Abstract 1664: Alpha-tocopheryloxyacetic acid (aTEA) induced immune activation synergizes with radiation therapy to treat primary and metastatic murine mammary carcinoma

Abstract Immunotherapy has now emerged as a promising treatment for metastatic cancer. However, immunotherapy for advanced stage breast carcinoma remains an unmet need. Here we demonstrate that the novel immunomodulator alpha-tocopheryloxyacetic acid (aTEA) synergizes with radiation therapy (RT) in a metastatic murine mammary carcinoma model to improve tumor control more than each individual therapy, and we propose a mechanism based on the stimulation of type I interferons by aTEA. For in-vivo studies, C57BL/6 mice bearing 4T1 murine mammary carcinoma lesions were treated with dietary aTEA, 20 Gy x 2 fractions ionizing radiotherapy, or the combination of aTEA/RT. Immune activation in animals treated with dietary aTEA was determined by analyzing the percentage of proliferating CD4 and CD8 T cells using a Ki-67 assay. Pulmonary metastatic burden was determined using a clonogenic metastasis assay. For in-vitro studies, murine mammary carcinoma cell lines were treated with aTEA or LPS and whole cell lysates were analyzed by Western blot. Expression of interferon regulation factor 3 (IRF3) pathway proteins, including phospho-TANK binding protein kinase 1 (TBK1) and stimulator of interferon genes (STING) were determined. Our results indicate that treatment with the combination of aTEA and RT significantly improves tumor control compared to either treatment alone. Combination therapy resulted in complete and durable tumor regression in a subset of animals treated, an effect not observed with aTEA or RT alone. Dietary administration of aTEA stimulates proliferation of both CD4 and CD8 T cells and significantly reduces metastatic burden in 4T1 tumor bearing mice. This immune stimulatory effect of aTEA appears to be mediated by reactive oxygen species (ROS) production and release of type I interferons through a STING mediated pathway. These preliminary results suggest that the combination of aTEA and ionizing radiation may be a viable therapy for the treatment of metastatic breast carcinoma. This therapeutic combination may provide more durable response to radiotherapy while simultaneously reducing further metastatic spread. Citation Format: Joshua M. Walker, Diego M. Barragan, Melissa J. Kasiewicz, Charles R. Thomas, William L. Redmond. Alpha-tocopheryloxyacetic acid (aTEA) induced immune activation synergizes with radiation therapy to treat primary and metastatic murine mammary carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1664.

Abstract 3365: Provasculogenic effects of alcohol and estrogen: implications for breast cancer development

Abstract Over 200,000 cases of breast cancer are diagnosed within the United States, resulting in over 40,000 deaths annually. Of the many lifestyle habits that are associated with cancer development, various epidemiological observations have provided a strong link between increased alcohol consumption and breast cancer progression. Specifically, this observation was made in women on estrogen replacement therapy thus suggesting a possible synergistic link between alcohol and estrogen. Our laboratory has demonstrated that estrogen increases breast cancer neo-vascularization both in vivo and in vitro. Provided that alcohol and estrogen can potentially synergize to promote breast cancer progression, we hypothesize that this progression is attributed to an increase in tumor neo-vascularization. In the current study, Tg-1 murine mammary carcinoma cells were treated with varying concentrations of alcohol +/- estrogen and analyzed for angiogenesis markers, such as VEGF and eNOS, and the pro-proliferation marker, MEK. At 24 hours post treatment, VEGF and eNOS were upregulated in response to both estrogen and alcohol compared to untreated cells and cells treated with either alcohol or estrogen alone. MEK levels remained constant through the conditions with a marginal upregulation in presence of alcohol and estrogen when compared to untreated cells. To further characterize neo-vascularization, we performed a scratch wound assay using the murine endothelial cell line, SVEC4-10, which was cultured in Tg1-1 conditioned media. The scratch wound assay results indicated enhanced migration of endothelial cells in response to conditioned media from Tg1-1 cells cultured in the presence of both alcohol and estrogen. SVEC4-10, treated with Tg-1-1 conditioned media were processed by Western blot analyses for MEK expression. MEK was upregulated in SVEC4-10 cells cultured in the media obtained from cells cultured with both alcohol and estrogen, indicating enhanced proliferation. Our results indicate a pro-neovasculogenic effect elicited by alcohol and estrogen, as determined by enhanced proliferation and migration of endothelial as well as increased expression of the pro-vasculogenic markers, VEGF and eNOS. Future experiments will be aimed to characterize the expression of other essential pro-vasculogenic markers, such as basic fibroblast growth factor (bFGF) and angiopoietin (Ang). The identification of these cellular and metabolic markers will establish a biochemical link between estrogen activity and signal transduction induced cellular effects. Citation Format: Rachana R. Maniyar, Robert B. Bednarczyk, Ghada M. Ben Rahoma, Neha Tuli, Abraham Mittelman, Raj K. Tiwari, Robert Suriano. Provasculogenic effects of alcohol and estrogen: implications for breast cancer development. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3365.

Abstract A15: SMC2 role in regulating tumor angiogenesis via FGF signaling

Abstract Fibroblast growth factors (FGFs) play a crucial role during embryonic development, tissue homeostasis, wound healing and angiogenesis. FGF-binding protein 1 (FGFBP1) is a secreted protein that binds FGFs stored in the extracellular matrix and transports them to their receptor, therefore they can modulate FGF signaling. FGFBP1 contributes to the progression of many cancers including colon, pancreas and squamous cell cancer. It acts as an angiogenic switch during tumor development. E0771 cells, a murine mammary carcinoma cell line, was subcutaneously injected into syngeneic C57BL/6 mice. While these cells form aggressive tumors within two weeks and metastasize to the lungs in wild type FGFBP1 (FGFBP1+/+) mice, there was no tumor growth or metastasis in FGFBP1 knock out (FGFBP1-/-) mice. A genome-wide shRNA library screen revealed a novel role for structural maintenance of the chromosome 2 (SMC2) in compensating for the loss of FGFBP1 in the stroma of FGFBP1-/- mice. E0771 cells with SMC2 knock down (KD) or CRISPR targeting SMC2 formed aggressive tumors in FGFBP1-/- mice and metastasized to the lungs. Surprisingly, FGFBP1 expression level was 32 folds lower in SMC2 KD E0771 cells yet FGF2 was 16 folds up in the tumors from SMC2 KD E0771 cells. Tumor histology shows impaired and leaky angiogenesis in FGFBP1-/- compared to FGFBP1+/+ mice. Furthermore, higher levels of GM-CSF and G-CSF were detected in the conditioned media (CM) of SMC2 KD E0771 cells compared to control cells suggesting differential immune cell recruitment. Also, an endothelial monolayer exposed to CM from SMC2 KD E0771 cells showed delayed wound closure and loose junctions suggesting a tendency for endothelium disintegration and initiation of blood vessel formation. Interestingly, the CM effect was blocked using PD173074, a pan FGFR inhibitor. In conclusion, SMC2 demonstrate a role in regulating FGF signaling which affects the course of angiogenesis during tumor development. Note: This abstract was not presented at the conference. Citation Format: Ghada M. Sharif, Marcel O. Schmidt, Casey Shuptrine, Weiner M. Louis, Anna T. Riegel, Anton Wellstein. SMC2 role in regulating tumor angiogenesis via FGF signaling. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A15.

Secretion of N- and O-linked Glycoproteins from 4T1 Murine Mammary Carcinoma Cells.

Breast cancer is one of the most common cancers that affect women globally and accounts for ~23% of all cancers diagnosed in women. Breast cancer is also one of the leading causes of death primarily due to late stage diagnoses and a lack of effective treatments. Therefore, discovering protein expression biomarkers is mandatory for early detection and thus, critical for successful therapy. Two-dimensional electrophoresis (2D-E) coupled with lectin-based analysis followed by mass spectrometry were applied to identify potential biomarkers in the secretions of a murine mammary carcinoma cell line. Comparisons of the protein profiles of the murine 4T1 mammary carcinoma cell line and a normal murine MM3MG mammary cell line indicated that cadherin-1 (CDH), collagenase 3 (MMP-13), Viral envelope protein G7e (VEP), Gag protein (GAG) and Hypothetical protein LOC433182 (LOC) were uniquely expressed by the 4T1 cells, and pigment epithelium-derived factor (PEDF) was exclusively secreted by the MM3MG cells. Further analysis by a lectin-based study revealed that aberrant O-glycosylated CDH, N-glycosylated MMP-13 and LOC were present in the 4T1 medium. These differentially expressed N- and O-linked glycoprotein candidates, which were identified by combining lectin-based analysis with 2D-E, could serve as potential diagnostic and prognostic markers for breast cancer.

Antitumor activity of a novel small molecule TLR7 agonist via immune response induction and tumor microenvironment modulation.

Immunotherapy is emerging as a powerful and active tumor-specific approach against cancer via triggering the immune system. Toll-like receptors (TLRs) are fundamental elements of the immune system, which facilitate our understanding of the innate and adaptive immune pathways. TLR agonists used as single agents can effectively eradicate tumors due to their potent stimulation of innate and adaptive immunity. We examined the effects of a novel adenine type of TLR7 agonists on both innate and adaptive immune activation in vitro and in vivo. We established the local and distant tumor‑bearing mice derived from murine mammary carcinoma cell line (4T1) to model metastatic disease. Our data demonstrated that SZU101 was able to stimulate innate immune cells to release cytokines at the very high level compared with LPS at the same or lower concentration. Locally intratumoral SZU101 injection can elicit a systemic antitumor effect on murine breast tumor model. SZU101 affected the frequency of intratumoral immune cell infiltration, including the percentage of CD4+ and CD8+ increase, and the ratio of Tregs decrease. Our data reveal that the antitumor effect of SZU101 is associated with multiple mechanisms, inducing tumor‑specific immune response, activation of innate immune cells and modulation of the tumor microenvironment.

Investigation of In vitro PDT Activities and In vivo Biopotential of Zinc Phthalocyanines Using 131I Radioisotope

Novel octylthio-containing asymmetrically substituted Zn(II) phthalocyanine (Zn(II)Pc1) and a symmetric derivative (Zn(II)Pc2) have been prepared to investigate the biological potential and ability to photosensitize singlet oxygen for photodynamic therapy applications. In this study, the singlet oxygen generation potential and in vitro photodynamic activities of these compounds have been tested. Both ZnPcs reveal to be very efficient singlet oxygen generators and promising PSs for PDT applications. In vitro PDT activities of the compounds were evaluated in EMT-6 murine mammary carcinoma and HeLa human cervix carcinoma cell lines. Moreover, Zn(II)Pc1 displayed the phototoxic effects in the mammary cancer cell line (6.25 μm concentration at 30 J/cm(2) light dose and 12.5 μm concentration at 20 J/cm(2) light dose), while Zn(II)Pc2 did not show any phototoxic effects both in two cell lines. Zn(II)Pcs were radiolabeled with (131) I in high yields. Biodistribution studies revealed that the radiolabeled Zn(II)Pc1 showed significant uptake in l. intestine, pancreas, brain, and ovary, while Zn(II)Pc2 has significant uptake in ovary and pancreas in normal rats. Hence, these Pcs derivatives could be promising candidate for tumor nuclear imaging.

Abstract 5015: Tumor dependence on HER2 signaling as a player in immune infiltration required for trastuzumab activity

Abstract The identification of a robust clinical or molecular predictor of trastuzumab benefit has proven challenging. Recent clinical studies have shown that either tumor dependence on HER2 signaling or the presence of high levels of tumor-infiltrating lymphocytes is predictive of trastuzumab sensitivity. Preclinical data suggest that only a fraction of the HER2+ breast carcinoma (BC) cells are truly addicted to the oncogene and that innate and adaptive immune cells contribute substantially to the therapeutic effect of trastuzumab. Based on these data, we aimed to determine whether tumor cells addicted to HER2 guide the development of tumor immune cell infiltration required for trastuzumab cytotoxic activity. Gene expression profile analysis of 53 HER2+ BC patients treated with adjuvant trastuzumab and chemotherapy identified a 41-gene classifier able to predict tumor relapse risk in both adjuvant and neoadjuvant settings. Tumors classified as low-risk presented high HER2 mRNA levels and were mainly HER2-enriched based on PAM50 signature (p<0.0001); these tumors showed significantly enhanced immune cell pathways and T cell infiltration as compared with high-risk samples. Gene-set enrichment analysis identified chemokine and cytokine ligands and receptor pathways enriched in low-risk patients (ES = 0.63, p<0.0001, FDR<0.0001), with CXCL9, CXCL10, CXCL11 and CX3CL1 also significantly (p<0.05) associated with low relapse risk. The high CXCL9 expression in low-risk tumors was validated by immunohistochemistry on FFPE tumor slides. PD-L1 expression levels were also significantly higher in low- than in high-risk tumors (p = 0.0014). Mean expression levels of relevant chemokines in human HER2+ BC cell lines as analyzed by qRT-PCR were 3- to 30-fold higher in HER2-addicted lines HER2 (ZR75.30, SKBr3 and BT474) than in non-addicted lines (MDAMB361 and MDAMB453). Similarly, murine CXCL10 and CX3CL1 levels were 2- and 10-times higher, respectively, in murine MI6 mammary carcinoma cells than in wild-type (WT) HER2 cells derived from mice transgenically expressing human delta16-HER2, which develop HER2-addicted tumors, or human WT-HER2, which develop non-addicted mammary tumors. Analysis of PBMC migration induced by tumor cells showed a higher recruitment of immune cells by HER2-addicted than non-addicted cells in vitro. Overall, our results support the notion that the activated HER2 oncogene induces production of chemokines/cytokines that recruit the immune infiltrate favorable to trastuzumab activity. Moreover, it seems likely that tumor cells themselves exert a PD-L1-dependent immunosuppressive activity that trastuzumab reverts to anti-tumor immunity. Supported by AIRC and Fondazione Pezcoller. Citation Format: Tiziana Triulzi, Viola Regondi, Loris De Cecco, Gaia Ghedini, Maria Luisa Carcangiu, Serenella M. Pupa, Elda Tagliabue. Tumor dependence on HER2 signaling as a player in immune infiltration required for trastuzumab activity. [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 5015. doi:10.1158/1538-7445.AM2015-5015

Abstract 1096: Murine mammary carcinoma produce more CCL2 in response to TLR agonist treatment than dendritic cells because of differences in NF-kB levels

Abstract Previously we reported that murine mammary carcinoma and dendritic cells elicit distinct responses to Toll-like receptor (TLR) agonists. Here we used a combination of computational biology and molecular analysis to identify the reason for the distinct responses. For this purpose we created a Netlogo model of the TLR4-Myd88 signaling pathway from TLR4 through nuclear translocation of NF-kB culminating in CCL2 transcription. Assessing CCL2 allowed us to validate our model by comparing how much CCL2 was generated by the model versus how much CCL2 was produced in our lab experiments. CCL2 was also of interest because previous studies have shown that CCL2 expression correlates with breast cancer progression. The 4T1 murine mammary carcinoma was used for these studies because it represents a model for stage IV breast cancer and also produces CCL2. Bone marrow derived CD11c+ dendritic cells (DC), which elicit a prototypical TLR agonist response, were used as a control. The model was developed by incorporating qRT-PCR data of the relative mRNA concentrations from each cell type. The experimental values were then used as the starting concentration of proteins in the model. The model of the TLR4-Myd88 signaling cascade allowed us to make predictions by running computational experiments that generate CCL2 expression data over a set amount of time. For instance, the model could be used to predict what happens in response to a TLR agonist if any of the TLR signaling proteins are up- or down-regulated. So far, the model predicted that the tumor cells would produce significantly more CCL2 than DC, results that were validated with experimental data. These results were surprising because most of the TLR signaling proteins were expressed at greater levels by the DC. Since only Tab3 and NF-kB were expressed at greater levels by the tumor cells we used the model to address whether the increased amount of NF-kB in the tumor cells was responsible for the greater level of CCL2 production by the tumor cells. The model showed that when the level of NF-kB in the tumor cells was reduced to the level found in the DC the tumor cells produced lower levels of CCL2. These data suggest that the level of NF-kB in the tumor cells was responsible for the greater production of CCL2 by the tumor cells relative to the DC. Thus, our computational model successfully predicted the experimental data and revealed that NF-kB may be a critical protein responsible for tumor-derived CCL2 expression. Collectively, a more thorough understanding of the differences in TLR signaling between the tumor cells and DC may reveal how targeting the TLR4 signaling cascade can be used to slow tumor progression. Citation Format: Tiffany Phuong, Carli B. Jones, Robert A. Kurt, Chun Wai Liew. Murine mammary carcinoma produce more CCL2 in response to TLR agonist treatment than dendritic cells because of differences in NF-kB levels. [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 1096. doi:10.1158/1538-7445.AM2015-1096

Abstract 4121: Autophagy influences the development of the pre-metastatic niche

Abstract Background: Autophagy is a lysosomal degradation process that is characterized by cellular self-consumption. Autophagy allows for the recycling and removal of damaged cytoplasmic material. Many types of cancer have elevated autophagic activity in order to sustain a high rate of metabolism and circumvent stress-induced cell death. However, little is known of autophagy's role in metastasis. Therefore, we tested the effect of autophagy modulation in different mouse models and cell based assays that reflect different points along the metastatic cascade. Methods: Pharmacologic autophagy inhibition in the murine mammary carcinoma cell line 4T1 was achieved with 10uM chloroquine (CQ) or 1nM bafilomycin A1 (Baf A1). Knockdown of autophagy critical genes Becn1 or Atg7 by lentiviral delivery of shRNA was also used as a means of inhibition. Proliferation, invasion, migration, and anchorage independent growth of cells were assessed in the presence or absence of active autophagy. For an experimental model of metastasis, Balb/c mice were challenged with 4T1 cells expressing luciferase (4T1-luc) injected via the tail vein. Mice were pre-treated with CQ (60 mg/kg, qd, i.p.) and monitored until the development of luciferase positive metastases. Mice were also challenged with 4T1 Becn1 knockdowns or cells pre-treated with CQ for 2h to assess effect of cellular autophagy impairment. An orthotopic model was also utilized by implanting 4T1-luc cells into the mammary fat pad, with tumors resected at 100-200 mm3 and mice followed until metastasis development. CQ was given either adjuvantly, 24h after surgery, or neoadjuvantly, 24h after implantation. Autophagy stimulation, 2% Trehalose in drinking water, was also given as a neoadjuvant therapy. Bone marrow derived cells (BMDCs), mediators of pre-metastatic niche formation, were measured by flow cytometry in the lungs and blood of treated mice prior to the arrival of tumor cells. Results: Autophagy inhibition was able to reduce cell proliferation, but did not significantly delay metastasis in the experimentally induced model. Similarly, CQ was not effective as an adjuvant therapy. Yet, as a neoadjuvant therapy both CQ and Becn1 knockdown were able to significantly delay metastases. Autophagy stimulation actually sped up metastasis development. Autophagy modulation had no effect on invasion, migration, or anchorage independent growth. Conversely, altering autophagy did impact the number of BMDCs present in the lung and blood. Trehalose treated mice had more than either vehicle or CQ. In the blood, CQ treated mice had even less BMDCs present than vehicle treated mice. Conclusions: Autophagy does not seem to be required for metastatic colonization and survival. Nor does it seem to affect the metastatic capability of cells. Rather, autophagy may be influencing the pre-metastatic microenvironment by impacting BMDCs. Thus autophagy appears to be most essential before the arrival of tumor cells to the site of metastasis. Citation Format: Rebecca A. Barnard, Daniel Regan, Ryan J. Hansen, Paola Maycotte, Andrew Thorburn, Daniel L. Gustafson. Autophagy influences the development of the pre-metastatic niche. [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 4121. doi:10.1158/1538-7445.AM2015-4121

Localized experimental bone metastasis drives osteolysis and sensory hypersensitivity at distant non-tumor-bearing sites.

Patients with breast cancer metastasis to bone suffer from inadequate pain relief. Animal models provide increased understanding of cancer-induced bone and sensory alterations. The objective of this study was to investigate the measures of pain at distant non-tumor-bearing sites in animals with localized bone metastasis. Immunocompetent BALB/c mice are injected intra-tibially with murine mammary carcinoma cells (4T1) or saline, and the sensitivity to mechanical and thermal stimuli in the contralateral paw was examined. In addition to previously demonstrated development of osteolysis and hypersensitivity to mechanical and thermal stimuli in the cancer-injected tibia, these animals exhibited an increase in sensory hypersensitivity in the contralateral limb. No bone lesions were evident on radiographs of the contralateral limbs. Histomorphometry detected decreased bone volume per tissue volume and increased osteoclast number in the contralateral tibia and vertebral bones of cancer-bearing animals. Neuroplasticity was examined by immunofluorescence for calcitonin gene-related peptide (CGRP) in sensory neurons and glial fibrillary acidic protein (GFAP) in lumbar spinal cords. CGRP-immunoreactivity and GFAP-immunoreactivity were significantly elevated both ipsilateral and contralateral in tumor-bearing animals. The anti-inflammatory and osteolysis-targeting drug rapamycin reduced hypersensitivity to mechanical and cold stimuli, attenuated GFAP over-expression, and lowered osteoclast number. The osteoclast-targeting drug pamidronate reduced sensitivity to cold and protected against bone loss. Localized bone cancer drives hypersensitivity, bone remodeling, and sensory neuron plasticity at sites distant from the primary tumor area. Drugs targeting these mechanisms may be useful in the treatment of pain distant from the primary tumor site.

TGFβ modulates inflammatory cytokines and growth factors to create premetastatic microenvironment and stimulate lung metastasis.

The formation of tumor-promoting premetastatic microenvironment plays a pivotal role on metastatic progression. Understanding how the primary tumor can promote the formation of premetastatic microenvironment in the lung will aid discovery of a final cure for metastatic breast cancer. The murine 4T1 mammary carcinoma cells were injected into the mammary fat pads of the BALB/c mice. Days 0-14 were considered the premetastatic phase. Lung tissues were examined using hematoxylin-eosin staining and transmission electron microscopy. After intravenous injection of TGFβ1 pretreated 4T1 cells, the relative pulmonary vascular permeability was quantified, the extravasation, survival, and proliferation of tumor cells in premetastatic lungs were evaluated, and the levels of S100A8, S100A9, VEGF, and Angpt2 were detected in tumor-bearing mice. The results showed that during the premetastatic phase, an inflammatory response and inflammation-induced vascular hyperpermeability were established, leading to an abnormal pulmonary microenvironment, which facilitated extravasation of circulating tumor cells, and subsequent survival and proliferation of metastatic tumor cells in a TGFβ-dependent manner. Moreover, the expressions of S100A8, S100A9, VEGF, and Angpt2 were increased, and an induction of these genes by TGFβ was further observed in premetastatic lungs. Thus, this study demonstrated that TGFβ promoted the creation of premetastatic microenvironment by modulating certain crucial inflammatory cytokines and growth factors, and finally enhanced the ability of circulating cells to seed the lung.

Inhibitory effect of iron withdrawal by chelation on the growth of human and murine mammary carcinoma and fibrosarcoma cells

Since iron uptake is essential for cell growth, rapidly dividing cancer cells are sensitive to iron depletion. To explore the effect of iron withdrawal on cancer cell growth, mouse and human mammary carcinoma cells (4T1 and MDA-MB-468, respectively) and mouse and human fibrosarcoma cells (L929 and HT1080, respectively) were cultured in the absence or presence of DIBI, a novel iron-chelating polymer containing hydroxypyridinone iron-ligand functionality. Cell growth was measured by a colorimetric assay for cell metabolic activity. DIBI-treated 4T1, MDA-MB-468, L929 and HT1080 cells, as well as their normal counterparts, showed a dose- and time-dependent reduction in growth that was selective for human cancer cells and mouse fibrosarcoma cells. The inhibitory effect of DIBI on fibrosarcoma and mammary carcinoma cell growth was reversed by addition of exogenous iron in the form of iron (III) citrate, confirming the iron selectivity of DIBI and that its inhibitory activity was iron-related. Fibrosarcoma and mammary carcinoma cell growth inhibition by DIBI was associated with S-phase cell cycle arrest and low to moderate levels of cell death by apoptosis. Consistent with apoptosis induction following DIBI-mediated iron withdrawal, fibrosarcoma and mammary carcinoma cells exhibited mitochondrial membrane permeabilization. A comparison of DIBI to other iron chelators showed that DIBI was superior to deferiprone and similar to or better than deferoxamine for inhibition of fibrosarcoma and mammary carcinoma cell growth. These findings suggest that iron withdrawal from the tumor microenvironment with a selective and potent iron chelator such as DIBI may prevent or inhibit tumor progression.

4T1 Murine Mammary Carcinoma Cells Enhance Macrophage-Mediated Innate Inflammatory Responses.

Tumor progression and the immune response are intricately linked. While it is known that cancers alter macrophage inflammatory responses to promote tumor progression, little is known regarding how cancers affect macrophage-dependent innate host defense. In this study, murine bone-marrow-derived macrophages (BMDM) were exposed to murine carcinoma-conditioned media prior to assessment of the macrophage inflammatory response. BMDMs exposed to 4T1 mammary carcinoma-conditioned medium demonstrated enhanced production of pro-inflammatory cytokines tumor necrosis factor α, interleukin-6, and CCL2 in response to lipopolysaccharide (LPS) while production of interleukin-10 remained unchanged. The increased LPS-induced production of pro-inflammatory cytokines was transient and correlated with enhanced cytokine production in response to other Toll-like receptor agonists, including peptidoglycan and flagellin. In addition, 4T1-conditioned BMDMs exhibited strengthened LPS-induced nitric oxide production and enhanced phagocytosis of Escherichia coli. 4T1-mediated augmentation of macrophage responses to LPS was partially dependent on the NFκB pathway, macrophage-colony stimulating factor, and actin polymerization, as well as the presence of 4T1-secreted extracellular vesicles. Furthermore, peritoneal macrophages obtained from 4T1 tumor-bearing mice displayed enhanced pro-inflammatory cytokine production in response to LPS. These results suggest that uptake of 4T1-secreted factors and actin-mediated ingestion of 4T1-secreted exosomes by macrophages cause a transient enhancement of innate inflammatory responses. Mammary carcinoma-mediated regulation of innate immunity may have significant implications for our understanding of host defense and cancer progression.

Type III Collagen Directs Stromal Organization and Limits Metastasis in a Murine Model of Breast Cancer

Breast cancer metastasis is the leading cause of cancer-related deaths in women worldwide. Collagen in the tumor microenvironment plays a crucial role in regulating tumor progression. We have shown that type III collagen (Col3), a component of tumor stroma, regulates myofibroblast differentiation and scar formation after cutaneous injury. During the course of these wound-healing studies, we noted that tumors developed at a higher frequency in Col3(+/-) mice compared to wild-type littermate controls. We, therefore, examined the effect of Col3 deficiency on tumor behavior, using the murine mammary carcinoma cell line 4T1. Notably, tumor volume and pulmonary metastatic burden after orthotopic injection of 4T1 cells were increased in Col3(+/-) mice compared to Col3(+/+) littermates. By using murine (4T1) and human (MDA-MB-231) breast cancer cells grown in Col3-poor and Col3-enriched microenvironments in vitro, we found that several major events of the metastatic process were suppressed by Col3, including adhesion, invasion, and migration. In addition, Col3 deficiency increased proliferation and decreased apoptosis of 4T1 cells both in vitro and in primary tumors in vivo. Mechanistically, Col3 suppresses the procarcinogenic microenvironment by regulating stromal organization, including density and alignment of fibrillar collagen and myofibroblasts. We propose that Col3 plays an important role in the tumor microenvironment by suppressing metastasis-promoting characteristics of the tumor-associated stroma.

Abstract 5032: Synergy between an engineered cytokine, NKTR-214, and CTLA-4 blockade in murine colon and breast tumor models

Abstract Background: Immunotherapy offers the potential for durable responses in a growing list of cancer indications. NKTR-214 is comprised of the cytokine IL2 conjugated o multiple PEG units. Upon in vivo administration some of these PEG molecules are released to leave activated IL2-conjugates that bind selectively at the IL2 receptor beta subunit (IL2Rβ). The active IL-2-conjugates favor proliferation of tumor-killing CD8+ memory T cells (CD8T) over immunosuppressive regulatory T cells (Treg) in the tumor microenvironment (Charych, JCO 2013, 31:15, Suppl. 1).We have also shown that NKTR-214 results in sustained exposure of drug in the tumor and robust immune system activation that translates between rodents and monkeys at safe doses, (AACR; Mol Cancer Ther 2013;12 (11 Suppl): Abstract B296). The receptor selectivity and improved pharmacokinetic profile leads to substantially improved single-agent efficacy and safety over the original IL-2 cytokine. Combinations of different checkpoint blockade antibodies have shown great promise clinically and in preclinical tumor models, highlighting the importance of targeting multiple immune activation pathways. Here we examine the combination of NKTR-214 with anti-CTLA4 antibody in murine breast and colon tumor models to explore the interaction of two mechanisms of action: checkpoint inhibition and direct CD8T cell activation via the IL-2 receptor beta. Methods: Female BALB/c mice bearing established tumors implanted with CT-26 (Murine Colon Carcinoma) or EMT6 (Murine Mammary Carcinoma) cells were treated with single agent NKTR-214, murine anti-CTLA-4 antibody or the two agents in combination. NKTR-214 was administered at 0.8 mg/kg i.v. and anti-CTLA-4 at 100 μg/mouse i.p. Results: In both the EMT6 breast and the CT-26 colon models, the combination of NKTR-214 with anti-CTLA-4 yielded significantly better tumor growth inhibition compared to either agent dosed alone. In the EMT6 model 10/12 animals were tumor free by day 20 in the combination arm and stayed tumor free until the end of study (60 days). Similarly in the CT-26 model, 8/12 animals were tumor free by day 25 in the combination arm and stayed tumor free until the end of study (day 60). Single agent anti CTLA-4 or NKTR-214 did not show significant tumor inhibition in either model. No toxicities were observed when NKTR-214 was combined with anti CTLA-4 antibody. Conclusions: NKTR-214 is a highly differentiated immunotherapy with a new mechanism of action that shows efficacy both as a single agent (as previously shown in a mouse melanoma model) and in combination. Combining NKTR-214 mediated T cell activation with CTLA-4 blockade is highly synergistic in murine models of cancer and holds the promise for durable responses in patients. Citation Format: Steve Lee, Murali Addepalli, Ute Hoch, Rhoneil Pena, Payal Shirsat, Yolanda Kirksey, Stephen K. Doberstein, Deborah Charych, Seema Kantak. Synergy between an engineered cytokine, NKTR-214, and CTLA-4 blockade in murine colon and breast tumor models. [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 5032. doi:10.1158/1538-7445.AM2014-5032

Abstract 2637: Role of d16HER2 splice variant in HER2-positive breast cancer

Abstract Almost 90% of human primary breast cancers (BCs) express 4-9% of total wild-type (WT) HER2 as a splice variant lacking exon 16 (d16HER2). Consequent in-frame activating deletion of 2 cysteine residues causes an imbalanced conformation resulting in receptor constitutive homodimerization, enhanced signaling activity, transformation and altered trastuzumab (T) binding. We recently produced a human d16HER2 transgenic (tg) mouse characterized by a rapid multifocal mammary tumors onset and the expression of active d16HER2 dimers on tumor cells, whose downstream signaling was found coupled to multiple activated nodes that include Src kinase. In order to dissect d16HER2 role in the aggressiveness and in the susceptibility to anti-HER2 therapy, we focused on the generation of stable d16HER2-expressing mammary tumor cell lines to be used in different bioassays. An immunomagnetic purification procedure was applied to generate primary homogeneously d16HER2-expressing cell cultures. These purified tumor cell lines were analyzed by flow cytometry and immunofluorescence and their migration/invasion ability was assessed through Boyden chamber test. d16HER2 downstream signaling was evaluated by western blot and T, Lapatinib (L) and Dasatinib (D) drugs sensitivity was measured with WST-1 and soft-agar bioassays. As controls, we compared in vitro d16HER2-models oncogenic features to those of the human BC BT474, which also co-expresses a low amount of d16HER2 transcript (4%), and to a murine mammary carcinoma cell line (wtHER2), derived from a primary mammary tumor developed in human WT HER2 tg mice. We found that d16HER2 in vitro models expressed high levels of stable homodimers combined with the recruitment of activated Src, STAT3, MAPK and Akt, as in vivo primary mammary tumors. Both in bidimensional (2D) and matrigel-cultured tumor cells, we confirmed the T lower binding capability for d16HER2 than other anti-HER2 MAbs directed to different extracellular domain epitopes. d16HER2 tumor cells had an enhanced migratory and invasive capacity compared to wtHER2 and BT474 cells and, notably, were completely resistant to T treatment and less responsive to L. In virtue of a highly activated Src kinase expression in d16HER2-positive models, we tested in 2D the therapeutic effects of D and observed that d16HER2-cells were significantly more sensitive than wtHER2 and BT474 cells. Preliminary 3D-drug susceptibility assays showed that the sensitivity of d16HER2 cells increased for all the tested drugs, if assessed in a suitable environment such as soft-agar. Our findings further indicate that the constitutive expression of d16HER2 variant identifies an aggressive tumor phenotype and confirm, at least in vitro in 2D conditions, that this isoform is resistant to T and L, whereas is sensitive to D. Further analyses are ongoing to analyze in vivo drug sensitivity of d16 in comparison to WT HER2 model. Supported by AIRC and Ministry of Health Citation Format: Gaia C. Ghedini, Arianna Palladini, Valentina Ciravolo, Lorenzo Castagnoli, Giulia Marzano, Roberta Zappasodi, Guido Santilli, Augusto Amici, Alessia Lamolinara, Manuela Iezzi, Patrizia Nanni, Elda Tagliabue, Serenella M. Pupa. Role of d16HER2 splice variant in HER2-positive breast cancer. [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 2637. doi:10.1158/1538-7445.AM2014-2637

Abstract 2012: GPNMB cooperates with Neuropilin-1 and Integrin a5b1 to promote breast cancer tumorigenesis and metastasis

Abstract Deaths attributed to breast cancer primarily occur following metastasis to distant organs such as bone, lung, liver and brain. In a screen for metastasis modulators, our lab has identified GPNMB as a gene whose overexpression in weakly metastatic breast cancer cell lines enhances primary tumor growth, promotes endothelial cell recruitment, drives lung and bone metastasis in vivo and induces the acquisition of an invasive phenotype in vitro. We have shown that elevated GPNMB levels in the breast tumor epithelium strongly correlate with decreased disease-free and overall survival, even within the basal/triple-negative breast cancer subset, associated with a high grade and poor prognosis. Based on our research findings, CDX-011, an antibody-drug conjugate that selectively targets GPNMB, was investigated in a multi-centre Phase II clinical trial for patients with metastatic breast cancer and showed promising activity in TNBCs, where treatment options are limited. Gene expression profiling of 66cl4 murine mammary carcinoma cells and BT549 basal breast cancer cells revealed that Neuropilin-1 (Nrp1) levels are increased in response to GPNMB overexpression. Using a panel of GPNMB-domain mutants (GPNMB ΔCYT and GPNMB RGD), we show that GPNMB forms a complex with Nrp1, and that this association requires the cytoplasmic tail of GPNMB and the PDZ-binding motif of Nrp1. The presence of a GPNMB/Nrp-1 complex potentiates the VEGF signaling cascade in a manner that depends on GPNMB association with Nrp1. We demonstrate that GPNMB enhances adhesion of breast cancer cells to fibronectin, increases the expression of α5β1 and associates with this receptor through its RGD integrin-binding domain. The recruitment of GPNMB into integrin complexes activates downstream Src and Fak signaling pathways and, reciprocally, induces phosphorylation of GPNMB on its half-ITAM motif. Using a spontaneous model of breast cancer tumor formation, we show that both the RGD domain and the cytoplasmic tail of GPNMB are required to increase primary tumor formation. However, only the GPNMB RGD mutant showed a decrease in metastatic burden when compared to WT GPNMB, indicating that GPNMB association with α5β1, but not Neuropilin-1 is required to promote lung metastasis. A survey of RNAseq datasets reveals that GPNMB is strongly correlated with Nrp1 and α5 in breast cancer and emphasizes the clinical relevance of our data. These findings outline the importance of novel GPNMB/α5β1 and GPNMB/Nrp1 complexes in potentiating breast cancer tumorigenesis and metastasis. Citation Format: Gordana Maric, Matthew Annis, April Rose, Dru Perkins, Patricia Macdonald, Peter Siegel. GPNMB cooperates with Neuropilin-1 and Integrin a5b1 to promote breast cancer tumorigenesis and metastasis. [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 2012. doi:10.1158/1538-7445.AM2014-2012

WE‐E‐BRE‐06: High‐Dose Microbeam Radiation Induces Different Responses in Tumor Microenvironment Compared to Conventional Seamless Radiation in Window Chamber Tumor Models

Purpose:Microbeam radiation therapy and GRID therapy are different forms of Spatially‐Fractioned Radiation Therapy (SFRT) that is fundamentally different from the conventional seamless and temporally fractionated radiation therapy. SFRT is characterized by a ultra‐high dose (10s –100s Gy) dose single treatment with drastic inhomogeneity pattern of given spatial frequencies. Preclinical and limited clinical studies have shown that the SFRT treatments may offer significant improvements in reducing treatment toxicity, especially for those patients who have not benefited from the state‐of‐the‐art radiation therapy approaches. This preliminary study aims to elucidate the underlying working mechanisms of SFRT, which currently remains poorly understood.Methods:A genetically engineered 4T1 murine mammary carcinoma cell line and nude mice skin fold window chamber were used. A nanotechnology‐based 160kV x‐ray irradiator delivered 50Gy (entrance dose) single treatments of microbeam or seamless radiation. Animals were in 3 groups: mock, seamless radiation, and 300μm microbeam radiation. The windows were imaged using a hyperspectral system to capture total hemoglobin/saturation, GFP fluorescence emission, RFP fluorescence emission, and vessel density at 9 time points up to 7 days post radiation.Results:We found unique physiologic changes in different tumor/normal tissue regions and differential effects between seamless and microbeam treatments. They include 1) compared to microbeam and mock radiation seamless radiation damaged more microvasculature in tumor‐surrounding normal tissue, 2) a pronounced angiogenic effect was observed with vascular proliferation in the microbeam irradiated portion of the tumor days post treatment (no such effect observed in seamless and mock groups), and 3) a notable change in tumor vascular orientation was observed where vessels initially oriented parallel to the beam length were replaced by vessels running perpendicular to the irradiation portion of the tumor.Conclusion:Our preliminary study indicated that microbeam radiation modified tumor microenvironment in ways significantly different than of the conventional seamless radiation.

Aluminum–phthalocyanine chloride associated to poly(methyl vinyl ether-co-maleic anhydride) nanoparticles as a new third-generation photosensitizer for anticancer photodynamic therapy

Photodynamic therapy is generally considered to be safer than conventional anticancer therapies, and it is effective against different kinds of cancer. However, its clinical application has been significantly limited by the hydrophobicity of photosensitizers. In this work, a system composed of the hydrophobic photosensitizer aluminum–phthalocyanine chloride (AlPc) associated with water dispersible poly(methyl vinyl ether-co-maleic anhydride) nanoparticles is described. AlPc was associated with nanoparticles produced by a method of solvent displacement. This system was analyzed for its physicochemical characteristics, and for its photodynamic activity in vitro in cancerous (murine mammary carcinoma cell lineage 4T1, and human mammary adenocarcinoma cells MCF-7) and noncancerous (murine fibroblast cell lineage NIH/3T3, and human mammary epithelial cell lineage MCF-10A) cell lines. Cell viability and the elicited mechanisms of cell death were evaluated after the application of photodynamic therapy. This system showed improved photophysical and photochemical properties in aqueous media in comparison to the free photosensitizer, and it was effective against cancerous cells in vitro.

Integrin α3β1 Can Function to Promote Spontaneous Metastasis and Lung Colonization of Invasive Breast Carcinoma

Significant evidence implicates α3β1 integrin in promoting breast cancer tumorigenesis and metastasis-associated cell behaviors in vitro and in vivo. However, the extent to which α3β1 is actually required for breast cancer metastasis remains to be determined. We used RNA interference to silence α3 integrin expression by approximately 70% in 4T1 murine mammary carcinoma cells, a model of aggressive, metastatic breast cancer. Loss of α3 integrin reduced adhesion, spreading, and proliferation on laminin isoforms, and modestly reduced the growth of orthotopically implanted cells. However, spontaneous metastasis to lung was strikingly curtailed. Experimental lung colonization after tail vein injection revealed a similar loss of metastatic capacity for the α3-silenced (α3si) cells, suggesting that critical, α3-dependent events at the metastatic site could account for much of α3β1's contribution to metastasis in this model. Reexpressing α3 in the α3si cells reversed the loss of metastatic capacity, and silencing another target, the small GTPase RhoC, had no effect, supporting the specificity of the effect of silencing α3. Parental, α3si, and α3-rescued cells, all secreted abundant laminin α5 (LAMA5), an α3β1 integrin ligand, suggesting that loss of α3 integrin might disrupt an autocrine loop that could function to sustain metastatic growth. Analysis of human breast cancer cases revealed reduced survival in cases where α3 integrin and LAMA5 are both overexpressed. α3 integrin or downstream effectors may be potential therapeutic targets in disseminated breast cancers, especially when laminin α5 or other α3 integrin ligands are also over-expressed.