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Use of Antimetastatic SOD3-Mimetic Albumin as a Primer in Triple Negative Breast Cancer

Use of Antimetastatic SOD3-Mimetic Albumin as a Primer in Triple Negative Breast Cancer Hindawi Journal of Oncology Volume 2019, Article ID 3253696, 11 pages https://doi.org/10.1155/2019/3253696 Research Article Use of Antimetastatic SOD3-Mimetic Albumin as a Primer in Triple Negative Breast Cancer 1 1 1 2 Shanta M. Messerli, Amanda M. Schaefer , Yongxian Zhuang , Bohdan J. Soltys, 1 1 3 2 1 Noah Keime , Jenny Jin , Li Ma, Carleton J. C. Hsia , and W. Keith Miskimins Cancer Biology and Immunotherapies, Sanford Research, Sioux Falls,  SD, USA AntiRadical erapeutics LLC, Sioux Falls,  SD, USA Department of Physics, Georgia Southern University, Statesboro, GA , USA Correspondence should be addressed to Carleton J. C. Hsia; cjchsia@yahoo.com and W. Keith Miskimins; keith.miskimins@sanfordhealth.org Received 8 September 2018; Accepted 30 December 2018; Published 28 February 2019 Guest Editor: Wei-Ting Chao Copyright © 2019 Shanta M. Messerli et al. is Th is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Of the deaths attributed to cancer, 90% are due to metastasis. Treatments that prevent or cure metastasis remain elusive. Low expression of extracellular superoxide dismutase (EcSOD or SOD3) has been associated with poor outcomes and increased metastatic potential in multiple types of cancer. Here, we characterize the antimetastatic therapeutic mechanisms of a macromolecular extracellular SOD3-mimetic polynitroxyl albumin (PNA, also known as VACNO). PNA is macromolecular human serum albumin conjugated with multiple nitroxide groups and acts as an SOD-mimetic. Here we show that PNA works as a SOD3- mimetic in a highly metastatic 4T1 mouse model of triple negative breast cancer (TNBC). In vitro, PNA dose dependently inhibited 4T1 proliferation, colony formation, and reactive oxygen species (ROS) formation. In vivo, PNA enhanced reperfusion time in the hypoxic cores of 4T1 tumors as measured by ultrasound imaging. Furthermore, PNA enhanced ultrasound signal intensity within the cores of the 4T1 tumors, indicating PNA can increase blood flow and blood volume within the hypoxic cores of tumors. Lung metastasis from 4T1 flank tumor was inhibited by PNA in the presence or absence of doxorubicin, a chemotherapy agent that produces superoxide and promotes metastasis. In a separate study, PNA increased the survival of mice with 4T1 flank tumors when used in conjunction with three standard chemotherapy drugs (paclitaxel, doxorubicin, and cyclophosphamide), as compared to treatment with chemotherapy alone. In this study, PNA-increased survival was also correlated with reduction of lung metastasis. These results support the hypothesis that PNA works through the inhibition of extracellular superoxide/ROS production leading to the conversion of 4T1 cells from a metastatic tumorigenic state to a cytostatic state. These findings support future clinical trials of PNA as an antimetastatic SOD3-mimetic drug to increase overall survival in TNBC patients. 1. Introduction reactive oxygen species (ROS) may have a major impact on survival of these patients [4, 5]. Due to the inadequacy of The development of antimetastatic drugs is urgently needed a single therapy to effectively eliminate tumors and prevent to reduce cancer-related deaths caused by metastasis [1]. In aggressive regrowth, alternative therapies that target the humans, metastasis accounts for up to 90% of all cancer- broader tumor microenvironment (TME) are also warranted. related deaths [2]. For breast cancer, triple negative breast In this study, we examine the efficacy of PNA, an extracellular cancer (TNBC) has the greatest chance of developing distant superoxide dismutase (EcSOD or SOD3) mimetic that was originally developed for ischemic stroke [6, 7]. In stroke, PNA metastases, despite existing systemic therapy with radiation, is expected to increase the Golden Hour for treatment via its chemotherapy, and immunotherapy [3]. As there are no currently available targeted therapies for this aggressive remarkable ability to treat both ischemic and hemorrhagic stroke safely without neuroimaging before thrombolytic ther- disease, we hypothesized that a new treatment which targets apy. PNA works by enhancing blood flow without oxidative the overproduction of extracellular superoxide and its related 2 Journal of Oncology example, increased extracellular superoxide radical has been PNA associated with increased prostate cancer cell invasion [14]. Therefore, we hypothesize that the SOD mimetic activity of the nitroxide groups on PNA will reduce the metastatic phenotype of the tumors, as well as decrease the formation of new cancer cells. The cores of tumors are oeft n chronically hypoxic, which is closely associated with a negative prognosis and a metastatic phenotype which is more resistant to cancer therapy [15, 16]. By targeting the hypoxic core of primary and metastatic sites, PNA may act to enhance blood flow and ROS Levels improve the therapeutic effects of standard chemotherapy, radiotherapy, and/or immunotherapy. Represents Recently, it was discovered that deficiency in EcSOD nitroxide group or SOD3 is involved in metastasis of multiple cancer types including TNBC and pancreatic cancer [4]. It is also estab- Figure 1: PNA is a human serum albumin that has been modified lished that, in the tumorigenic state, the levels of superoxide with multiple caged nitroxide groups. eTh nitroxide groups exhibit radicals in the TME are elevated [17–20]. Therefore, a SOD3 antioxidant activity and have the ability to catalytically remove superoxide in the vasculature and intracellular spaces. Tumors mimetic, like PNA, may be suitable as a targeted therapy display elevated levels of reactive oxygen species that promote to replace the deficient SOD3 in cancer cells. The data growth and metastasis. PNA is expected to reduce tumor ROS, reported here provides support for this hypothesis in a highly leading to inhibitory effects on tumor growth, metabolism, and metastatic mouse 4T1 TNBC model. metastasis. In this study we demonstrate that PNA inhibits metastasis leading to improved overall survival in a highly metastatic murine model of TNBC. The mechanisms involved in this stress and serving as a neuroprotectant [8]. PNA is human process are as follows: (1) removal of superoxide and its serum albumin (HSA) that has been modified with multiple related ROS of cancer cells;(2) improvement in blood flow to the tumor hypoxic core;(3) reversal of metastasis induced by caged nitric oxide groups (Figure 1). With albumin as the carrier, PNA is distributed in the intravascular and lymph the superoxide producing chemotherapeutic drug, e.g., dox- compartments to intercept metastasizing cancer cells. PNA orubicin;(4) extension of survival when used conjunctively with three standard conjunctive chemotherapeutic drugs targets the vasculature of the TME leading to the restoration of blood o fl w to the hypoxic tumor core, presumably through with concomitant inhibition of metastasis to the lung. the removal of superoxide in the intravascular space. This TME activity is facilitated by the nitroxide groups that exhibit 2. Materials and Methods superoxide mimetic activities [6, 7, 9]. us Th , PNA has been demonstrated to catalytically dismutate superoxide in the .. Cell Culture. The mouse mammary carcinoma line 4T1 vasculature and increase the bioavailability of endogenous was purchased from ATCC and used within 5 passages. nitric oxide (NO), leading to improved blood flow. Improved Cells were cultured in DMEM with 10% fetal bovine serum blood flow in tumors has been shown to reverse tumor (FBS), penicillin/streptomycin solution, and amphotericin hypoxia [10], leading to inhibitory effects on tumor growth, and incubated at 37 Cin5% CO2. For theSYTOX Green metabolism, and metastasis. (ThermoScientific) cell proliferation assay, 4T1 cells were The design of PNA, consisting of a novel combination plated in 96-well plates and allowed to attach overnight before of aHSA and nitroxide asametal-free superoxide dismu- being treated with HSA or PNA at indicated concentrations in tase (SOD3) mimetic, has advantages over small molecular triplicate. After 72 hr of treatment, cells were stained with 20 weight and membrane permeable nitroxide that mimics 𝜇 MSYTOX Green for 15 min. and dead cells’ u fl orescence SOD1, SOD2, and SOD3, in cancer therapy. Albumin has was measured in a Spectromax M5 plate reader (Molecular a number of characteristics that make it an attractive drug Devices) at excitation/emission 485/530 with a 515 emission vehicle or primer, including the ability of the protein to carry cutoff. Then the cells were permeabilized by addition of 6 % hydrophobic drugs, such as paclitaxel, through the blood and Triton-X for 30 min, and total cell uo fl rescence was measured deliver them directly to cancer cells [11]. With HSA as the at excitation/emission 485/530 with a 515-emission cutoff. carrier of the nitroxides, PNA is targeted to the vasculature Data is representative of three independent experiments. to increase blood flow and is in equilibrium with the lymph For the colony assays, 4T1 cells were plated at 250 cells per system [7, 9]. The SOD3 mimetic activity of PNA can break well in a Corning 6-well plate. After 24 hours, the cells were down ROS, such as superoxide, and alter the ROS balance treated in triplicate with the indicated concentration of PNA within cancer cells and the tumor microenvironment. ROS or HSA. After 5 days of treatment, cells were washed with have been shown to be involved in several cellular processes PBS and fixed with 70% ethanol for 5 minutes. Colonies were including proliferation, growth, migration, and apoptosis washed again with PBS and then stained with Coomassie Blue [12]. In cancer cells, ROS plays a pivotal role in many signaling [40% methanol, 12% glacial acetic acid, and 0.24% Coomassie pathways involved in carcinogenesis and metastasis [13]. For Blue]. This was followed by another wash, and then the plates Journal of Oncology 3 were imaged using an AlphaImager System. Colony number administered once a week, and PNA and has were delivered and sizewereanalyzed using AlphaImager Systemimage 3 times a week. analysis sow ft are (AlphaInnotech, Santa Clara, CA). Lung metastasis was quantified in 4T1 mice through injection of India ink into whole lungs via the trachea .. Flow Cytometry. The production of ROS was detected following sacrifice of the animals as described [22, 23]. The by using the dye 2’,7’-dichlorouo fl rescein diacetate (DCF- number of visible metastases was manually counted using a DA), a cell-permeant indicator for reactive oxygen species. dissecting microscope (Nikon SMZ1000). DCF is used as a qualitative marker for cellular oxidant stress Quantification of necrotic areas of primary tumor sec- rather than a marker for specific ROS [21]. 4T1 cells were tions was performed using stereology methodology. At the treated with PNA or HSA at concentrations ranging from 30 time of sacrifice, primary tumors were harvested, xfi ed in 10% to 120𝜇 M for 16-18 hours and then stained with DCF-DA (10 bueff red formalin, and paraffin embedded (FFPE). Tumors 𝜇 M). DCF u fl orescence was measured using BD C sampler were sectioned to 5𝜇 m. Hematoxylin and Eosin (H & E) software on the Accuri C6 Flow Cytometer (BD Biosciences). staining was performed, and analysis of live and dead tissue on tumor sections was performed using Stereo Investigator . . Polynitroxyl Albumin. Polynitroxylated albumin (PNA, software (version 10, Micro Bright Field; Williston, VT) by aka Vascular Albumin with Caged Nitric Oxide or VACNO) outlining areas of karyolysis indicated by reduced number of is a drug product prepared as a sterile, nonpyrogenic nuclei and increased eosin staining. Briefly, the tumor area preparation in saline solution. Each 100 mL PNA con- was outlined, and a grid of counting frames was applied to tains 20 g of injectable-grade human serum albumin systematically quantify across the entire tumor section. The (HSA) and 2.5 g of 4-(2-acetamido)-2,2,6,6-tetramethyl-1- area of live tumor tissue versus nonviable was calculated using piperidinyloxy (AcTPO) covalently attached to the albumin the area fraction fractionator probe. Counting frames of 200 molecules with added stabilizers, including 0.08 moles of 𝜇 mby200𝜇 mwere placedoverthe tumorsection with an sodium caprylate and 0.08 moles of sodium acetyltrypto- average of 50 counting frames per tumor. Tumor tissue was phanate per gram of albumin. defined as viable or necrotic and the corresponding area of each was calculated using the sow ft are package. .. Mouse Studies. Balb/c mice (Jackson Laboratory) be- tween 4 and 6 weeks of age were maintained in a barrier facil- .. Ultrasound Measurement of Tumor Perfusion. The day prior to ultrasound imaging, a depilatory cream (Nair) was ity on high efficiency particulate air (HEPA)-filtered racks. The animals were fed with autoclaved laboratory rodent diet applied to skin on the tumor and surrounding regions to pre- (Envigo diet 2918). All animal studies were approved by the vent interference with the ultrasound transducer. On the day Sanford Institutional Animal Care and Use Committee and of imaging, animals were anesthetized with 1.5% isou fl rane conducted in accordance with the principles and procedures in oxygen, placed on a heated stage, and restrained using outlined in the NIH Guide for the Care and Use of Animals. surgical tape. Anesthesia was maintained during imaging 4T1 cells (5 X 10 ) were injected subcutaneously into using 1.5% isou fl rane in oxygen administered via nose cone. the hind limb of female Balb/c mice. In the rfi st in vivo Warmed ultrasound gel (Parker Laboratories) was applied to the depilated skin and ultrasound images were captured experiment examining how PNA affects metastasis, 60 mice were randomly divided into the following groups: PNA (12.5 (Vevo2100; Visual Sonics). ml/kg), control HSA (10%), doxorubicin (4 mg/kg, Sigma To measure tumor vascularity, Color Doppler imaging was performed on 4T1 tumors 14 days after injection. Tumors Aldrich), and doxorubicin (4 mg/kg) plus PNA (12.5 ml/kg). When tumors became palpable (4 days), intraperitoneal (i.p.) had been treated starting 4 days after initiation with PNA injections with PNA, control HSA, doxorubicin, or PNA plus or control HSA 3 times per week as described above. Color doxorubicin were performed. PNA and HSA treatments were Doppler imaging was performed across the entire tumor performed three times a week. using serial scans. After image-acquisition, the tumor was In the second in vivo 4T1 experiment, 50,000 4T1 outlined on the serial scans and the volume was calculated cells were subcutaneously injected into Balb/c mice, and and percent of vascularity within the outlined tumor was 45 mice were divided into the following groups: (1) con- calculated by manufacturer’s software (Visual Sonics). To calculate tumor perfusion time following adminis- trol chemotherapy group (15 mice),(2) chemotherapy plus PNA delivered intraperitoneally (i.p.) (15 mice), and (3) tration of PNA, a microbubble contrast agent (Vevo Micro- chemotherapy plus PNA delivered intravenously (i.v.) (15 Marker; Visual Sonics) was used. Tumors had been treated starting 4 days aeft r initiation with PNA or control HSA mice). When tumors became palpable (4 days), the groups received the following treatments: in weeks 1-2, control 3 times per week as described above. Tumor perfusion group received HSA combined with paclitaxel (60 mg/kg), studies were performed 16 days aer ft tumor initiation. The paclitaxel (60 mg/kg) plus PNA delivered i.p., and paclitaxel following procedures were performed in addition to those plus PNA delivered i.v. and in weeks 3-4, the control group mentioned above: a 27-gauge butteryfl needle connected to a received HSA plus doxorubicin (4mg/kg) plus cyclophos- catheter was inserted via tail vein for intravenous injection of phamide (100mg/kg). The experimental groups received dox- the microbubbles contrast agent. The microbubble contrast orubicin, cyclophosphamide (100 mg/kg) and PNA delivered agent was prepared according to manufacturer instructions and a bolus injection of 50𝜇 L was delivered via tail vein i.p. and doxorubicin, cyclophosphamide, and PNA delivered i.v. The paclitaxel, doxorubicin, and cyclophosphamide were catheter u fl shed with 20-30 𝜇 L of saline. Perfusion of the 4 Journal of Oncology 6.00 ∗∗ allowing the restoration of endogenous nitric oxide in the ∗∗ 5.00 vasculature. We have recently reported that the restora- 4.00 tion of blood flow by PNA in acute hypoxia arising in 3.00 ischemia stroke also restores blood flow in chronic hypoxia 2.00 in solid tumors [22]. PNA was shown to promote blood flow 1.00 and drug delivery to hypoxic flank tumors in a xenograft 0.00 tumor model using electron paramagnetic resonance (EPR) No HSA PNA HSA PNA HSA PNA Treatment measurements [22]. To confirm that PNA is also effective Control 30 M 60 M 120 M in opening up the 4T1 TNBC flank hypoxic tumor core, Treatment we tested whether PNA increased tumor blood flow using ultrasound bursting and imaging techniques. Figure 3(a) Figure 2: Treatment with PNA reduces intracellular reactive oxygen shows ultrasound measurements demonstrating that PNA species. 4T1 cells were treated with PNA or corresponding concen- tration of control HSA for 16-18 hours and then stained with 2’,7’- increased the perfusion rate of the hypoxic core of 4T1 flank dichlorou fl orescein diacetate and measured via flow cytometry. In tumors compared to the tail vein infusion of HSA. Figure 3(b) thepresenceof reactive oxygen species (ROS), thedye is converted shows the reperfusion kinetics of the microbubble burst by to the uo fl rescent molecule 2’,7’-dichlorou fl orescein (DCF) (n=3 for the ultrasound. Figures 3(c) and 3(d) illustrate the difference each group; data is representative of three independent experiments; of the perfusion volume of the an fl k tumor detected by the ∗ =p< 0.05,∗∗ =p< 0.01 versus HSA). microbubble technique from tail vein infusion of HSA versus PNA, respectively. These ultrasound measurements together with the EPR measurements independently validated that the tumor microvasculature was calculated using manufacturer SOD3-mimetic activity PNA improves blood flow within the software (VevoCQ; Visual Sonics). A baseline perfusion prior hypoxic core of two types of solid tumors. to administration of PNA or control HSA was obtained. Following baseline acquisition, PNA (12.5 mL/kg) or control . . PNA Is Cytotoxic and Inhibits Cancer Cell Proliferation HSA (10%) were delivered via tail vein injection. Thirty in TNBC. It is well known that elevated levels of ROS are minutes aeft r drug injection, a second dose of microbubbles protumorigenic through activation of signaling pathways was administered and tumor perfusion was calculated as that promote cell proliferation, cell survival, and oncogenic described above. Tumor perfusion following drug delivery transcriptional programs [12–14]. Since PNA reduces cancer was calculated by taking the 30-minute perfusion time minus cell ROS in 4T1 cells (Figure 2), we predicted that PNA would the baseline perfusion time. also reduce proliferation of 4T1 cells. In order to examine this Statistics. Data is expressed as means ±SEM. P values hypothesis, PNA was tested in vitro in 4T1 mouse TNBC cell were determined by paired student’s t-tests. cultures. In this dose response study, cells were exposed for 72 hr to 30𝜇 M, 60𝜇 M, and 120𝜇 Mof PNA or corresponding 3. Results concentrations of control HSA. Live and dead cell numbers were determined using a SYTOX Green assay, and live cell .. PNA Reduces Reactive Oxygen Species. The key property number was then extrapolated from this data (Figure 4). of PNA is its antioxidant activity, which is mediated by PNA compared to HSA signicfi antly reduced the number of catalytic dismutation of superoxide and related ROS. We live cells in a dose dependent manner (n=3 for each group, hypothesized that PNA treatment of tumor cells would ∗∗=p<0.001;∗∗∗=p<0.0001). reduce cellular ROS levels, leading to changes in growth and The effect of PNA on cell proliferation and survival was survival. To test this, cultured 4T1 cells were treated with further examined in colony formation assays. 4T1 cells were PNA (30-120𝜇 M) for 24 hours. Control cultures received studied for their ability to form colonies within 5 days in the the same concentrations of HSA, the protein component of presence of varying doses of PNA and HSA as in Figure 4. PNA. Cellular ROS levels were estimated by staining cells In Figure 5, colonies were stained and imaged, and the with DCF-DA. At each concentration, 4T1 cells treated with number and size of the colonies were quantified. There was a PNA had significantly reduced ROS as compared to the significant dose dependent decrease in the number (Figures corresponding control HSA treatment (Figure 2). u Th s, PNA 5(a) and 5(b), left panel) and size of colonies (Figures 5(a) and potentially shifts 4T1 cancer cells from a tumorigenic to a 5(b), right panel) in response to PNA compared to control cytostatic state (see Figure 1). HSA at concentrations ranging from 30 to 120𝜇 M(p<0.001). Results from Figures 4 and 5 suggest that PNA inhibited .. PNA Treatment of Tumor-Bearing Animals Shows Reduced proliferation and colony formation of 4T1 cells by reducing Tumor Perfusion Time and Increased Relative Blood Volume the superoxide and ROS levels. Compared to Control in a Breast Cancer Mouse Model. Our previous studies show that PNA promotes blood flow in .. Small Molecule, Membrane Permeable Nitroxide Com- animal models of cerebral ischemia, leading to significant pound Affects T Growth and Survival Differently Compared reductions in infarct size [6, 7]. Similar findings have to PNA. Macromolecular PNA is primarily extracellular been found in cardiac ischemia reperfusion models [24]. and, therefore, is an EcSOD/SOD3 mimetic. TEMPOL (4- hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), on the other This activity is mediated by dismutation of superoxide in blood vessels, preventing the formation of peroxynitrite and hand, is a small molecule nitroxide that is membrane Mean DCF  Journal of Oncology 5 Perfusion Kinetics 30 minutes post-injection Control 0 0 0 5 10 15 20 25 Time (sec) Treatment (a) (b) -13 rBV [dB] Gain: 17 DR: 20 (c) -10 rBV [dB] Gain: 14 DR: 20 (d) Figure 3: PNA treated tumors have reduced tumor perfusion time and increased relative blood volume in the metastatic TNBC T model. Ultrasound imaging was used to visualize tumor microvasculature. eTh contrast agent was administered intravenously, images were acquired, and perfusion time was calculated with manufacturer software. PNA or HSA control was administered intravenously. And after 30 minutes, a second dose of contrast agent was administered, and perfusion time was calculated. (a) PNA treated tumors have reduced tumor perfusion time compared to HSA control. Tumor perfusion time was calculated by taking the 30-minute perfusion time minus the baseline perfusion time. (b) The kinetics of perfusion as measured by echo power versus time indicate PNA treated tumors have higher echo power at peak perfusion time compared to HSA control, indicating increased blood flow within the tumor. (c) Tumors were outlined in ultrasound B-mode (left panel). Parametric imaging of relative blood volume is shown 30 minutes aer ft drug injection at the time of peak perfusion (right panel). HSA treated tumors show little to no signal intensity in the core of the tumor. (d) PNA treated tumors show greater relative blood volume within the tumor core. permeable, reaching all cellular compartments. A dose The difference between control and PNA treated groups was response study showed that TEMPOL significantly inhibits approaching signicfi ance (p= 0.0705) but suggested that PNA the 4T1 colony size, at concentrations ranging from 18 to is having a direct effect on primary tumors as well as on 180 𝜇 M, suggesting cytostatic effects on the cancer cells metastasis. (Figure 6(a)). However, the TEMPOL did not signicfi antly reduce colony number (Figure 6(b)), suggesting a lack of .. PNA Reduces Lung Metastasis from T Primary Tumors. cytotoxic effects on the cancer cells. These results indicate that Subcutaneous or orthotopic 4T1 tumors rapidly metastasize PNA and small molecule, membrane permeable nitroxides to distant tissues in a pattern similar to human TNBC, have differences in their specificities and therapeutic indices. including metastasizing to the lung [25, 26]. We found that Eec ff ts of PNA on 4T1 tumors in vivo. In vivo, 4T1 cells PNA reduced lung metastasis as compared to control HSA establish rapidly growing tumors that metastasize to lung, treatment but not to a level of significance (Figure 7(b), liver, bone, and brain [25]. u Th s, they are a useful model p=0.056). However, we found that there is a dramatic increase of human TNBC. Subcutaneous 4T1 tumors were developed in metastasis to the lung induced by a superoxide producing in Balb/c mice and treated with PNA or HSA as a control. chemotherapy drug doxorubicin (Figure 7(c)). Remarkably, Tumors from PNA treated mice were not significantly dif- in the presence of PNA and doxorubicin the lung metastasis ferent in size from those of HSA treated animals. However, was nearly abolished (Figure 7(c), paired two-tailed t test, histological examination of tumors from both groups showed p=0.038). Thisprovidesstrong rationale forcombining PNA that PNA treated tumors had less live tissue and more necrotic with other drugs used in adjuvant or neoadjuvant therapy. (dead) tissue than the HSA treated controls (Figure 7(a)). In this regard, mice bearing 4T1 tumors were subjected to Control (N=3) PNA (N=4) Seconds Echo power (a.u.) 6 Journal of Oncology ∗∗∗ capacity of cells. PNA also reduced the size of the colonies, ∗∗ ∗∗∗ indicating reduced ability of the cancer cells to proliferate. More importantly, we have demonstrated that PNA inhibits metastasis, particularly that induced by chemotherapeutic drugs such as doxorubicin, leading to enhanced survival 1000 (Figures 7 and 8). We discuss below the significance of the current results for future preclinical and clinical studies that can guide the development of PNA as a primer used No HSA PNA HSA PNA HSA PNA treatment in conjunction with standard cancer therapies. us, Th our control preclinical studies may be relevant to neoadjuvant treatment 30 M 60 M 120 M and to clinical trials such as the I-SPY2 trials [27]. Treatment Two key aspects of PNA facilitate its biological activities. Figure 4: PNA treatment significantly reduces live cell number. 4T1 First, it catalyzes the dismutation of superoxide by virtue of cells were plated and treated the following day with PNA (red bars) the multiple covalently attached nitroxide groups. Second, it or HAS (blue bars), and control (black bar) was media only. Aeft r is based on a macromolecule carrier, HSA, which is capable 72 hr, cells were stained with SYTOX Green and uo fl rescence of targeting extracellular compartments. These properties of was measured. Percent of dead cells and percent of live cells PNA are expected to create an essentially superoxide-free were calculated using uo fl rescence values. Live cells’ uo fl rescence is milieu within the vasculature and within the tumor microen- shown. Results indicate PNA, compared to HSA, reduces live cell vironment. In the vasculature, elimination of superoxide numbers at concentrations ranging from 30 to 120𝜇 MPNA (n=3 prevents the formation of peroxynitrite through its reaction for each group;∗∗=p<0.01,∗∗∗=p<0.001). with NO. This allows the restoration of endogenous NO to promote blood flow to the metastasized solid tumor. This is supported by our published studies that demonstrate PNA a chemotherapy regimen including paclitaxel, doxorubicin, mediation of increased blood flow and prevention of ischemic and cyclophosphamide, with or without PNA (Figure 8(a)). reperfusion injury in models of stroke, myocardial infarction, This regimen is similar to that in the I-SPY 2 clinical trials and sickle cell disease [6, 29]. for breast cancer [27]. In this study, PNA again significantly Enhanced blood o fl w, as shown here and in our previous decreased the number of lung metastases (paired t test with study [28], is likely to have a number of eeff cts on solid p<0.01). Furthermore, PNA enhanced overall survival of mice tumors. It is expected to increase oxygen delivery to tumor against chemotherapy-mediated toxicity in the 4T1 mouse cells, which would likely reverse hypoxia leading to inactiva- model (Figure 8(b)). Statistical analysis using the Log-Rank tion of hypoxia inducible factor 1𝛼 . This in turn would alter (Mantel-Cox) test indicates a statistical difference between tumor metabolism and growth, as well as several aspects of PNA and chemotherapy treated mice compared to HSA and metastasis including angiogenesis, migration, invasion, and chemotherapy treated mice, with p<0.0013. Together, these intravasation [30]. data demonstrate that PNA reduces metastasis in this triple PNA reduces cellular ROS in 4T1 breast cancer cell negative breast cancer model and that this correlates with cultures and we have shown that this corresponds to reduced longer survival times. u Th s, this indicates PNA conjunctive cell proliferation and survival. Because PNA is based on HSA, therapy with standard chemotherapeutic agents improves it is expected to target extracellular spaces within the tumor survival of 4T1 tumor bearing mice corresponding to a microenvironment. We did not observe reduced 4T1 tumor reduction in toxicity of the chemotherapeutic agent and a sizes in mice treated with PNA. However, the tumors did have reductioninlung metastasis. reduced amount oflivetissue(Figure 7(a)) and increased areas of noncellular or necrotic tissue (not shown), indicating that the drug was directly acting on the tumor. PNA in the 4. Discussion extravascular spaces within the tumor microenvironment is Our findings demonstrate that the SOD3-mimetic activity expected to reduce tumor superoxide and ROS levels and of an extracellular albumin-based macromolecular nitroxide, this may directly lead to interference with metastasis. For example, ROS are able to directly activate matrix metallo- PNA, converts cancer cells from a tumorigenic state to a cytostatic state by reducing superoxide and ROS levels (see proteinases (MMPs) [31]. MMPs are key eeff ctors of tumor Figure 1). Earlier we reported that PNA works as an SOD3 invasion and metastasis through degradation of extracellular matrix components. Therefore, PNA may directly interfere mimetic to alter the hypoxic state of a solid tumor through blood ow fl enhancement [28]. In this report we established with metastasis by blocking MMP activation by eliminating that the same therapeutic mechanism for PNA also works superoxide within the tumor microenvironment. This pro- posed mechanism may also relate to the effects of doxoru- in the 4T1 TNBC model. We were able to confirm that this action of PNA occurs within 30 minutes of i.v. infusion with bicin and other chemotherapy reagents on metastasis. For example, we found that doxorubicin increases the number two independent biophysical methods (EPR and ultrasound). Our data suggest that PNA is cytotoxic to murine 4T1 of lung metastases compared to control mice, and this was TNBC cells as well as inhibiting their proliferation. In reversed by PNA. This is consistent with other studies show- ing doxorubicin-induced increases in metastasis in mouse colony forming assays, PNA reduced the number of colonies, suggesting an effect of PNA on the clonogenic survival models and human patients [32, 33]. Many chemotherapy Relative Fluorescence Unites Journal of Oncology 7 HSA 30M Control PNA 30M Control HSA 120M HSA 60M PNA 120M PNA 60M (a) ∗∗∗ ∗∗ ∗∗ ∗∗ 20 5000 HSA PNA HSA PNA HSA PNA HSA PNA HSA PNA HSA PNA 30M 60M 120M 30M 60M 120M (b) Figure 5: PNA treatment significantly reduces TNBC cancer cell proliferation. Colony forming assays were performed in PNA treated 4T1 cell cultures. Cells were plated at low density and then treated with the indicated concentration of PNA (red bars) or HSA (blue bars) as a control. PNA significantly reduces number of colonies compared to HSA treatments (a) and size of colonies (b). n=3 per group; data is representative of three independent experiments (∗ =p< 0.05,∗∗ =p< 0.01, and∗∗∗ =p< 0.001 versus HSA). Colony Number Colony Size 180 8000 ∗∗ ∗∗ 0 0 0 1.8 18 180 0 1.8 18 180 Tempol Concentration (M) Tempol Concentration (M) (a) (b) Figure 6: TEMPOL produces significant decreases in colony size but not colony number. 4T1 cells were plated at low cell number and treated the following day with TEMPOL at varying concentrations, and the control was DMEM alone. Aeft r 5 days, cells were fixed, stained, and imaged using Alpha Imager sow ft are. (a) Colony number was counted and (b) total colony size was measured using Alpha Imager software. Data shown suggests that TEMPOL alone inhibits cellular proliferation at concentrations ranging from 18 to 180𝜇 M, according to a two-tailed t test, with p<0.001, but is not significantly cytotoxic at the concentrations shown. Colony Number Number of Colonies Colony Size (pixels) Size of colonies (pixels) 8 Journal of Oncology   1.5×10  8  1.0×10  7  5.0×10 Con PNA Con PNA Dox Dox+PNA (a) (b) (c) Figure 7: Live tumor area and number of metastatic lung nodules decreases with PNA treatment while survival increases with PNA treatment. (a) Subcutaneous 4T1 tumors are treated with HSA (control) or PNA. Tumors sections were stained by H&E and live tissue area was determined by stereology. (b) Tumor bearing mice were treated with or without PNA and aer ft 3 weeks lungs were harvested for counting lung metastatic nodules. (c) 4T1 tumor bearing mice were treated with doxorubicin in the presence or absence of PNA. PNA combined with doxorubicin compared to doxorubicin alone significantly reduces lung metastasis, according to a paired two-tailed t test, p <0.05 (∗). ∗∗ 3 ∗∗ 2 50      Control Chemotherapy Chemotherapy plus PNA Days elapsed Control Chemotherapy PNA plus Chemotherapy (a) (b) Figure 8: PNA treatment combined with chemotherapy reduces lung metastasis and enhances survival of mice carrying T tumors compared to chemotherapy treatment alone. (a) Tumor bearing mice were treated with a chemotherapy regimen, with and without PNA, and aer ft 3 weeks lungs were harvested for counting metastatic lung nodules. PNA in the presence of chemotherapy significantly reduced lung metastases compared to chemotherapy alone, according to a paired two-tailed t test, p>0.01. (b) PNA combined with chemotherapy significantly increased overall survival of mice compared to chemotherapy alone, according to the Log-Rank (Mantel-Cox) test, with p value =.0013 (∗∗). agents, including doxorubicin, increase tumor superoxide albumin is mainly synthesized in the liver, enters the circu- and ROS, which is associated with the process of metastasis. lation, and occurs predominantly in the extravascular space in tissues such as skin, gut, muscle, and other u fl ids such as We show that PNA eliminates the increase in metastasis associated with chemotherapy. This is most likely related to cerebrospinal uid. fl A substantial amount of albumin is also the ability of PNA to eliminate superoxide and ROS in the found within the intravascular space, but only a very small tumor microenvironment. amount is thought to exist intracellularly [34–36]. In our Since our experimental design has been to inject PNA studies, after injection into the intraperitoneal space, PNA into the intraperitoneal cavity, the subsequent biodistribution is expected to enter the circulation via the lymphatic system of PNA warrants consideration. The overall biodistribution and be distributed similarly to endogenous albumin. Imaging of PNA in our studies is expected to be similar to endoge- studies confirm that uo fl rescent albumin injected into the nous serum albumin since PNA consists of macromolecular intraperitoneal space distributes maximally to the spleen, human serum albumin conjugated with multiple nitroxide livers, lungs, and other organs within 3 hrs of injection; groups to provide the SOD mimetic activity. Endogenous levels subsequently decline [37]. Endogenous albumin cycles Mean number of lung metastases Area (m ) Number of lung mets Percent survival Number of lung mets Journal of Oncology 9 through the lymphatic system approximately 28 times during inhibits metastasis, as well as its effectiveness in other types of its lifetime (Peters, 1996; Evans, 2002) and injected PNA solid tumors. As a novel SOD mimetic drug, PNA would be is expected to behave similarly. More importantly, PNA is ideally suited for evaluation in trials like the I-SPY 2 platform [27] and FDA breakthrough therapy designation (BTD) to not expected to accumulate significantly intracellularly in vivo, similar to endogenous albumin. u Th s, the activity of bring PNA from benchtobedside in 3-5 years. PNA is most similar to ecSOD/SOD3, which is also localized extracellularly. 5. Conclusions The intraperitoneal administration of PNA may have two effects. First, PNA would remove extracellular superoxide PNA reduces ROS in TNBC, is cytotoxic, and is cytostatic to in all bodily compartments working as EcSOD/SOD3 with mouse TNBC breast cancer cells in a dose dependent manner. minimal eeff ct on the intracellular superoxide levels con- PNA combined with doxorubicin, compared to doxorubicin trolled by SOD1 and SOD2. In contrast a low molecular alone, significantly reduces lung metastasis in a mouse model weight nitroxide chemical like TEMPOL would dismutate of breast cancer. Furthermore, PNA reduces metastasis and both intracellular and extracellular superoxide because it enhances survival of mice against chemotherapy-mediated readily crosses membranes and accumulates within cells toxicity in a 4T1 mouse breast cancer model. These in vitro (Figure 6). Second, it has been known for over four decades and in vivo experimental n fi dings suggest PNA is a promising that the lymphatics are the rfi st site of metastasis for most cancer therapeutic. The antimetastatic mechanism of the solid cancers and there has been a concerted effort in SOD3-mimetic PNA on a murine TNBC 4T1 mouse model trying to target cancer therapeutics to the lymphatic system has been established. If the same antimetastatic mechanisms (e.g., see [38]). Accumulation of PNA in the lymph nodes can be established with a human TNBC mouse model, these and surrounding lymphatics is a possibility and may affect preclinical efficacy data will guide a Phase 1b clinical protocol immune cells and metastasis. The possible immunodulatory design in recurrent TNBC patients. In addition, PNA could effects on metastasis by PNA will be evaluated in future be tested in trials like the I-SPY2 fast track clinical trial studies. protocol to select and graduate new drug candidates through As a macromolecule with multiple nitroxide groups, PNA FDA breakthrough therapy designation (BTD). acts as an SOD3 mimetic. SOD3 (aka EcSOD) is secreted from cells and is found either free in the extracellular space or Data Availability adhered to the surface of cells. SOD3 expression is frequently diminished in breast and other types of cancer, which The data used to support the findings of this study are contributes to metastasis [4]. Furthermore, low expression included within the article. of SOD3 in breast cancer is associated with reduced relapse free survival in all types of breast cancer [4]. Overexpression of SOD3 in a human breast cancer xenograft or in the 4T1 Conflicts of Interest syngeneic model led to reduced lung metastasis and longer Shanta M.Messerli,Amanda M.Schaefer,Yongxian Zhuang, survival [39, 40]. Noah Keime, Jenny Jin, Li Ma, Bohdan J. Soltys, and W. Keith Taken together, PNA appears to be capable of acting as Miskimins declare no conflicts of interest. Carleton J. C. Hsia an SOD3 mimetic to alter the oxidative state within solid is sole proprietor with AntiRadical Therapeutics. tumors, inhibiting their ability to metastasize (see Figure 1). We speculate that elimination of superoxide within the tumor microenvironment directly reverses ROS stimulated metas- Acknowledgments tasis by preventing activation of MMPs, reducing invasion, This research was supported by the National Institutes and inhibiting cancer cell proliferation. PNA enhancement of of Cancer (1R01CA180033-04; W. Keith Miskimins), the blood flow is expected to reverse solid tumor hypoxia leading National Institute of General Medical Sciences (CoBRE to changes in tumor metabolism and hypoxia-dependent Grant P20GM103548;W.Keith Miskimins), the COBRE metastatic events. Another potential outcome of increased P20GM103548P20 Pilot grant (Project lead-Shanta M. blood ofl w in tumors is to act as a primer to improve Messerli), Sanford Research, and project seed funds from the drug delivery to tumors to enhance the therapeutic index of state of South Dakota. chemotherapy and immunotherapy. An additional advantage of PNA treatment of cancer may be attributable to its ability to protect normal tissues from oxidative stress and References inflammatory injuries leading to better quality of life. We have previously demonstrated the ability of PNA to prevent [1] M. 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Use of Antimetastatic SOD3-Mimetic Albumin as a Primer in Triple Negative Breast Cancer

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Copyright © 2019 Shanta M. Messerli et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi Journal of Oncology Volume 2019, Article ID 3253696, 11 pages https://doi.org/10.1155/2019/3253696 Research Article Use of Antimetastatic SOD3-Mimetic Albumin as a Primer in Triple Negative Breast Cancer 1 1 1 2 Shanta M. Messerli, Amanda M. Schaefer , Yongxian Zhuang , Bohdan J. Soltys, 1 1 3 2 1 Noah Keime , Jenny Jin , Li Ma, Carleton J. C. Hsia , and W. Keith Miskimins Cancer Biology and Immunotherapies, Sanford Research, Sioux Falls,  SD, USA AntiRadical erapeutics LLC, Sioux Falls,  SD, USA Department of Physics, Georgia Southern University, Statesboro, GA , USA Correspondence should be addressed to Carleton J. C. Hsia; cjchsia@yahoo.com and W. Keith Miskimins; keith.miskimins@sanfordhealth.org Received 8 September 2018; Accepted 30 December 2018; Published 28 February 2019 Guest Editor: Wei-Ting Chao Copyright © 2019 Shanta M. Messerli et al. is Th is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Of the deaths attributed to cancer, 90% are due to metastasis. Treatments that prevent or cure metastasis remain elusive. Low expression of extracellular superoxide dismutase (EcSOD or SOD3) has been associated with poor outcomes and increased metastatic potential in multiple types of cancer. Here, we characterize the antimetastatic therapeutic mechanisms of a macromolecular extracellular SOD3-mimetic polynitroxyl albumin (PNA, also known as VACNO). PNA is macromolecular human serum albumin conjugated with multiple nitroxide groups and acts as an SOD-mimetic. Here we show that PNA works as a SOD3- mimetic in a highly metastatic 4T1 mouse model of triple negative breast cancer (TNBC). In vitro, PNA dose dependently inhibited 4T1 proliferation, colony formation, and reactive oxygen species (ROS) formation. In vivo, PNA enhanced reperfusion time in the hypoxic cores of 4T1 tumors as measured by ultrasound imaging. Furthermore, PNA enhanced ultrasound signal intensity within the cores of the 4T1 tumors, indicating PNA can increase blood flow and blood volume within the hypoxic cores of tumors. Lung metastasis from 4T1 flank tumor was inhibited by PNA in the presence or absence of doxorubicin, a chemotherapy agent that produces superoxide and promotes metastasis. In a separate study, PNA increased the survival of mice with 4T1 flank tumors when used in conjunction with three standard chemotherapy drugs (paclitaxel, doxorubicin, and cyclophosphamide), as compared to treatment with chemotherapy alone. In this study, PNA-increased survival was also correlated with reduction of lung metastasis. These results support the hypothesis that PNA works through the inhibition of extracellular superoxide/ROS production leading to the conversion of 4T1 cells from a metastatic tumorigenic state to a cytostatic state. These findings support future clinical trials of PNA as an antimetastatic SOD3-mimetic drug to increase overall survival in TNBC patients. 1. Introduction reactive oxygen species (ROS) may have a major impact on survival of these patients [4, 5]. Due to the inadequacy of The development of antimetastatic drugs is urgently needed a single therapy to effectively eliminate tumors and prevent to reduce cancer-related deaths caused by metastasis [1]. In aggressive regrowth, alternative therapies that target the humans, metastasis accounts for up to 90% of all cancer- broader tumor microenvironment (TME) are also warranted. related deaths [2]. For breast cancer, triple negative breast In this study, we examine the efficacy of PNA, an extracellular cancer (TNBC) has the greatest chance of developing distant superoxide dismutase (EcSOD or SOD3) mimetic that was originally developed for ischemic stroke [6, 7]. In stroke, PNA metastases, despite existing systemic therapy with radiation, is expected to increase the Golden Hour for treatment via its chemotherapy, and immunotherapy [3]. As there are no currently available targeted therapies for this aggressive remarkable ability to treat both ischemic and hemorrhagic stroke safely without neuroimaging before thrombolytic ther- disease, we hypothesized that a new treatment which targets apy. PNA works by enhancing blood flow without oxidative the overproduction of extracellular superoxide and its related 2 Journal of Oncology example, increased extracellular superoxide radical has been PNA associated with increased prostate cancer cell invasion [14]. Therefore, we hypothesize that the SOD mimetic activity of the nitroxide groups on PNA will reduce the metastatic phenotype of the tumors, as well as decrease the formation of new cancer cells. The cores of tumors are oeft n chronically hypoxic, which is closely associated with a negative prognosis and a metastatic phenotype which is more resistant to cancer therapy [15, 16]. By targeting the hypoxic core of primary and metastatic sites, PNA may act to enhance blood flow and ROS Levels improve the therapeutic effects of standard chemotherapy, radiotherapy, and/or immunotherapy. Represents Recently, it was discovered that deficiency in EcSOD nitroxide group or SOD3 is involved in metastasis of multiple cancer types including TNBC and pancreatic cancer [4]. It is also estab- Figure 1: PNA is a human serum albumin that has been modified lished that, in the tumorigenic state, the levels of superoxide with multiple caged nitroxide groups. eTh nitroxide groups exhibit radicals in the TME are elevated [17–20]. Therefore, a SOD3 antioxidant activity and have the ability to catalytically remove superoxide in the vasculature and intracellular spaces. Tumors mimetic, like PNA, may be suitable as a targeted therapy display elevated levels of reactive oxygen species that promote to replace the deficient SOD3 in cancer cells. The data growth and metastasis. PNA is expected to reduce tumor ROS, reported here provides support for this hypothesis in a highly leading to inhibitory effects on tumor growth, metabolism, and metastatic mouse 4T1 TNBC model. metastasis. In this study we demonstrate that PNA inhibits metastasis leading to improved overall survival in a highly metastatic murine model of TNBC. The mechanisms involved in this stress and serving as a neuroprotectant [8]. PNA is human process are as follows: (1) removal of superoxide and its serum albumin (HSA) that has been modified with multiple related ROS of cancer cells;(2) improvement in blood flow to the tumor hypoxic core;(3) reversal of metastasis induced by caged nitric oxide groups (Figure 1). With albumin as the carrier, PNA is distributed in the intravascular and lymph the superoxide producing chemotherapeutic drug, e.g., dox- compartments to intercept metastasizing cancer cells. PNA orubicin;(4) extension of survival when used conjunctively with three standard conjunctive chemotherapeutic drugs targets the vasculature of the TME leading to the restoration of blood o fl w to the hypoxic tumor core, presumably through with concomitant inhibition of metastasis to the lung. the removal of superoxide in the intravascular space. This TME activity is facilitated by the nitroxide groups that exhibit 2. Materials and Methods superoxide mimetic activities [6, 7, 9]. us Th , PNA has been demonstrated to catalytically dismutate superoxide in the .. Cell Culture. The mouse mammary carcinoma line 4T1 vasculature and increase the bioavailability of endogenous was purchased from ATCC and used within 5 passages. nitric oxide (NO), leading to improved blood flow. Improved Cells were cultured in DMEM with 10% fetal bovine serum blood flow in tumors has been shown to reverse tumor (FBS), penicillin/streptomycin solution, and amphotericin hypoxia [10], leading to inhibitory effects on tumor growth, and incubated at 37 Cin5% CO2. For theSYTOX Green metabolism, and metastasis. (ThermoScientific) cell proliferation assay, 4T1 cells were The design of PNA, consisting of a novel combination plated in 96-well plates and allowed to attach overnight before of aHSA and nitroxide asametal-free superoxide dismu- being treated with HSA or PNA at indicated concentrations in tase (SOD3) mimetic, has advantages over small molecular triplicate. After 72 hr of treatment, cells were stained with 20 weight and membrane permeable nitroxide that mimics 𝜇 MSYTOX Green for 15 min. and dead cells’ u fl orescence SOD1, SOD2, and SOD3, in cancer therapy. Albumin has was measured in a Spectromax M5 plate reader (Molecular a number of characteristics that make it an attractive drug Devices) at excitation/emission 485/530 with a 515 emission vehicle or primer, including the ability of the protein to carry cutoff. Then the cells were permeabilized by addition of 6 % hydrophobic drugs, such as paclitaxel, through the blood and Triton-X for 30 min, and total cell uo fl rescence was measured deliver them directly to cancer cells [11]. With HSA as the at excitation/emission 485/530 with a 515-emission cutoff. carrier of the nitroxides, PNA is targeted to the vasculature Data is representative of three independent experiments. to increase blood flow and is in equilibrium with the lymph For the colony assays, 4T1 cells were plated at 250 cells per system [7, 9]. The SOD3 mimetic activity of PNA can break well in a Corning 6-well plate. After 24 hours, the cells were down ROS, such as superoxide, and alter the ROS balance treated in triplicate with the indicated concentration of PNA within cancer cells and the tumor microenvironment. ROS or HSA. After 5 days of treatment, cells were washed with have been shown to be involved in several cellular processes PBS and fixed with 70% ethanol for 5 minutes. Colonies were including proliferation, growth, migration, and apoptosis washed again with PBS and then stained with Coomassie Blue [12]. In cancer cells, ROS plays a pivotal role in many signaling [40% methanol, 12% glacial acetic acid, and 0.24% Coomassie pathways involved in carcinogenesis and metastasis [13]. For Blue]. This was followed by another wash, and then the plates Journal of Oncology 3 were imaged using an AlphaImager System. Colony number administered once a week, and PNA and has were delivered and sizewereanalyzed using AlphaImager Systemimage 3 times a week. analysis sow ft are (AlphaInnotech, Santa Clara, CA). Lung metastasis was quantified in 4T1 mice through injection of India ink into whole lungs via the trachea .. Flow Cytometry. The production of ROS was detected following sacrifice of the animals as described [22, 23]. The by using the dye 2’,7’-dichlorouo fl rescein diacetate (DCF- number of visible metastases was manually counted using a DA), a cell-permeant indicator for reactive oxygen species. dissecting microscope (Nikon SMZ1000). DCF is used as a qualitative marker for cellular oxidant stress Quantification of necrotic areas of primary tumor sec- rather than a marker for specific ROS [21]. 4T1 cells were tions was performed using stereology methodology. At the treated with PNA or HSA at concentrations ranging from 30 time of sacrifice, primary tumors were harvested, xfi ed in 10% to 120𝜇 M for 16-18 hours and then stained with DCF-DA (10 bueff red formalin, and paraffin embedded (FFPE). Tumors 𝜇 M). DCF u fl orescence was measured using BD C sampler were sectioned to 5𝜇 m. Hematoxylin and Eosin (H & E) software on the Accuri C6 Flow Cytometer (BD Biosciences). staining was performed, and analysis of live and dead tissue on tumor sections was performed using Stereo Investigator . . Polynitroxyl Albumin. Polynitroxylated albumin (PNA, software (version 10, Micro Bright Field; Williston, VT) by aka Vascular Albumin with Caged Nitric Oxide or VACNO) outlining areas of karyolysis indicated by reduced number of is a drug product prepared as a sterile, nonpyrogenic nuclei and increased eosin staining. Briefly, the tumor area preparation in saline solution. Each 100 mL PNA con- was outlined, and a grid of counting frames was applied to tains 20 g of injectable-grade human serum albumin systematically quantify across the entire tumor section. The (HSA) and 2.5 g of 4-(2-acetamido)-2,2,6,6-tetramethyl-1- area of live tumor tissue versus nonviable was calculated using piperidinyloxy (AcTPO) covalently attached to the albumin the area fraction fractionator probe. Counting frames of 200 molecules with added stabilizers, including 0.08 moles of 𝜇 mby200𝜇 mwere placedoverthe tumorsection with an sodium caprylate and 0.08 moles of sodium acetyltrypto- average of 50 counting frames per tumor. Tumor tissue was phanate per gram of albumin. defined as viable or necrotic and the corresponding area of each was calculated using the sow ft are package. .. Mouse Studies. Balb/c mice (Jackson Laboratory) be- tween 4 and 6 weeks of age were maintained in a barrier facil- .. Ultrasound Measurement of Tumor Perfusion. The day prior to ultrasound imaging, a depilatory cream (Nair) was ity on high efficiency particulate air (HEPA)-filtered racks. The animals were fed with autoclaved laboratory rodent diet applied to skin on the tumor and surrounding regions to pre- (Envigo diet 2918). All animal studies were approved by the vent interference with the ultrasound transducer. On the day Sanford Institutional Animal Care and Use Committee and of imaging, animals were anesthetized with 1.5% isou fl rane conducted in accordance with the principles and procedures in oxygen, placed on a heated stage, and restrained using outlined in the NIH Guide for the Care and Use of Animals. surgical tape. Anesthesia was maintained during imaging 4T1 cells (5 X 10 ) were injected subcutaneously into using 1.5% isou fl rane in oxygen administered via nose cone. the hind limb of female Balb/c mice. In the rfi st in vivo Warmed ultrasound gel (Parker Laboratories) was applied to the depilated skin and ultrasound images were captured experiment examining how PNA affects metastasis, 60 mice were randomly divided into the following groups: PNA (12.5 (Vevo2100; Visual Sonics). ml/kg), control HSA (10%), doxorubicin (4 mg/kg, Sigma To measure tumor vascularity, Color Doppler imaging was performed on 4T1 tumors 14 days after injection. Tumors Aldrich), and doxorubicin (4 mg/kg) plus PNA (12.5 ml/kg). When tumors became palpable (4 days), intraperitoneal (i.p.) had been treated starting 4 days after initiation with PNA injections with PNA, control HSA, doxorubicin, or PNA plus or control HSA 3 times per week as described above. Color doxorubicin were performed. PNA and HSA treatments were Doppler imaging was performed across the entire tumor performed three times a week. using serial scans. After image-acquisition, the tumor was In the second in vivo 4T1 experiment, 50,000 4T1 outlined on the serial scans and the volume was calculated cells were subcutaneously injected into Balb/c mice, and and percent of vascularity within the outlined tumor was 45 mice were divided into the following groups: (1) con- calculated by manufacturer’s software (Visual Sonics). To calculate tumor perfusion time following adminis- trol chemotherapy group (15 mice),(2) chemotherapy plus PNA delivered intraperitoneally (i.p.) (15 mice), and (3) tration of PNA, a microbubble contrast agent (Vevo Micro- chemotherapy plus PNA delivered intravenously (i.v.) (15 Marker; Visual Sonics) was used. Tumors had been treated starting 4 days aeft r initiation with PNA or control HSA mice). When tumors became palpable (4 days), the groups received the following treatments: in weeks 1-2, control 3 times per week as described above. Tumor perfusion group received HSA combined with paclitaxel (60 mg/kg), studies were performed 16 days aer ft tumor initiation. The paclitaxel (60 mg/kg) plus PNA delivered i.p., and paclitaxel following procedures were performed in addition to those plus PNA delivered i.v. and in weeks 3-4, the control group mentioned above: a 27-gauge butteryfl needle connected to a received HSA plus doxorubicin (4mg/kg) plus cyclophos- catheter was inserted via tail vein for intravenous injection of phamide (100mg/kg). The experimental groups received dox- the microbubbles contrast agent. The microbubble contrast orubicin, cyclophosphamide (100 mg/kg) and PNA delivered agent was prepared according to manufacturer instructions and a bolus injection of 50𝜇 L was delivered via tail vein i.p. and doxorubicin, cyclophosphamide, and PNA delivered i.v. The paclitaxel, doxorubicin, and cyclophosphamide were catheter u fl shed with 20-30 𝜇 L of saline. Perfusion of the 4 Journal of Oncology 6.00 ∗∗ allowing the restoration of endogenous nitric oxide in the ∗∗ 5.00 vasculature. We have recently reported that the restora- 4.00 tion of blood flow by PNA in acute hypoxia arising in 3.00 ischemia stroke also restores blood flow in chronic hypoxia 2.00 in solid tumors [22]. PNA was shown to promote blood flow 1.00 and drug delivery to hypoxic flank tumors in a xenograft 0.00 tumor model using electron paramagnetic resonance (EPR) No HSA PNA HSA PNA HSA PNA Treatment measurements [22]. To confirm that PNA is also effective Control 30 M 60 M 120 M in opening up the 4T1 TNBC flank hypoxic tumor core, Treatment we tested whether PNA increased tumor blood flow using ultrasound bursting and imaging techniques. Figure 3(a) Figure 2: Treatment with PNA reduces intracellular reactive oxygen shows ultrasound measurements demonstrating that PNA species. 4T1 cells were treated with PNA or corresponding concen- tration of control HSA for 16-18 hours and then stained with 2’,7’- increased the perfusion rate of the hypoxic core of 4T1 flank dichlorou fl orescein diacetate and measured via flow cytometry. In tumors compared to the tail vein infusion of HSA. Figure 3(b) thepresenceof reactive oxygen species (ROS), thedye is converted shows the reperfusion kinetics of the microbubble burst by to the uo fl rescent molecule 2’,7’-dichlorou fl orescein (DCF) (n=3 for the ultrasound. Figures 3(c) and 3(d) illustrate the difference each group; data is representative of three independent experiments; of the perfusion volume of the an fl k tumor detected by the ∗ =p< 0.05,∗∗ =p< 0.01 versus HSA). microbubble technique from tail vein infusion of HSA versus PNA, respectively. These ultrasound measurements together with the EPR measurements independently validated that the tumor microvasculature was calculated using manufacturer SOD3-mimetic activity PNA improves blood flow within the software (VevoCQ; Visual Sonics). A baseline perfusion prior hypoxic core of two types of solid tumors. to administration of PNA or control HSA was obtained. Following baseline acquisition, PNA (12.5 mL/kg) or control . . PNA Is Cytotoxic and Inhibits Cancer Cell Proliferation HSA (10%) were delivered via tail vein injection. Thirty in TNBC. It is well known that elevated levels of ROS are minutes aeft r drug injection, a second dose of microbubbles protumorigenic through activation of signaling pathways was administered and tumor perfusion was calculated as that promote cell proliferation, cell survival, and oncogenic described above. Tumor perfusion following drug delivery transcriptional programs [12–14]. Since PNA reduces cancer was calculated by taking the 30-minute perfusion time minus cell ROS in 4T1 cells (Figure 2), we predicted that PNA would the baseline perfusion time. also reduce proliferation of 4T1 cells. In order to examine this Statistics. Data is expressed as means ±SEM. P values hypothesis, PNA was tested in vitro in 4T1 mouse TNBC cell were determined by paired student’s t-tests. cultures. In this dose response study, cells were exposed for 72 hr to 30𝜇 M, 60𝜇 M, and 120𝜇 Mof PNA or corresponding 3. Results concentrations of control HSA. Live and dead cell numbers were determined using a SYTOX Green assay, and live cell .. PNA Reduces Reactive Oxygen Species. The key property number was then extrapolated from this data (Figure 4). of PNA is its antioxidant activity, which is mediated by PNA compared to HSA signicfi antly reduced the number of catalytic dismutation of superoxide and related ROS. We live cells in a dose dependent manner (n=3 for each group, hypothesized that PNA treatment of tumor cells would ∗∗=p<0.001;∗∗∗=p<0.0001). reduce cellular ROS levels, leading to changes in growth and The effect of PNA on cell proliferation and survival was survival. To test this, cultured 4T1 cells were treated with further examined in colony formation assays. 4T1 cells were PNA (30-120𝜇 M) for 24 hours. Control cultures received studied for their ability to form colonies within 5 days in the the same concentrations of HSA, the protein component of presence of varying doses of PNA and HSA as in Figure 4. PNA. Cellular ROS levels were estimated by staining cells In Figure 5, colonies were stained and imaged, and the with DCF-DA. At each concentration, 4T1 cells treated with number and size of the colonies were quantified. There was a PNA had significantly reduced ROS as compared to the significant dose dependent decrease in the number (Figures corresponding control HSA treatment (Figure 2). u Th s, PNA 5(a) and 5(b), left panel) and size of colonies (Figures 5(a) and potentially shifts 4T1 cancer cells from a tumorigenic to a 5(b), right panel) in response to PNA compared to control cytostatic state (see Figure 1). HSA at concentrations ranging from 30 to 120𝜇 M(p<0.001). Results from Figures 4 and 5 suggest that PNA inhibited .. PNA Treatment of Tumor-Bearing Animals Shows Reduced proliferation and colony formation of 4T1 cells by reducing Tumor Perfusion Time and Increased Relative Blood Volume the superoxide and ROS levels. Compared to Control in a Breast Cancer Mouse Model. Our previous studies show that PNA promotes blood flow in .. Small Molecule, Membrane Permeable Nitroxide Com- animal models of cerebral ischemia, leading to significant pound Affects T Growth and Survival Differently Compared reductions in infarct size [6, 7]. Similar findings have to PNA. Macromolecular PNA is primarily extracellular been found in cardiac ischemia reperfusion models [24]. and, therefore, is an EcSOD/SOD3 mimetic. TEMPOL (4- hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), on the other This activity is mediated by dismutation of superoxide in blood vessels, preventing the formation of peroxynitrite and hand, is a small molecule nitroxide that is membrane Mean DCF  Journal of Oncology 5 Perfusion Kinetics 30 minutes post-injection Control 0 0 0 5 10 15 20 25 Time (sec) Treatment (a) (b) -13 rBV [dB] Gain: 17 DR: 20 (c) -10 rBV [dB] Gain: 14 DR: 20 (d) Figure 3: PNA treated tumors have reduced tumor perfusion time and increased relative blood volume in the metastatic TNBC T model. Ultrasound imaging was used to visualize tumor microvasculature. eTh contrast agent was administered intravenously, images were acquired, and perfusion time was calculated with manufacturer software. PNA or HSA control was administered intravenously. And after 30 minutes, a second dose of contrast agent was administered, and perfusion time was calculated. (a) PNA treated tumors have reduced tumor perfusion time compared to HSA control. Tumor perfusion time was calculated by taking the 30-minute perfusion time minus the baseline perfusion time. (b) The kinetics of perfusion as measured by echo power versus time indicate PNA treated tumors have higher echo power at peak perfusion time compared to HSA control, indicating increased blood flow within the tumor. (c) Tumors were outlined in ultrasound B-mode (left panel). Parametric imaging of relative blood volume is shown 30 minutes aer ft drug injection at the time of peak perfusion (right panel). HSA treated tumors show little to no signal intensity in the core of the tumor. (d) PNA treated tumors show greater relative blood volume within the tumor core. permeable, reaching all cellular compartments. A dose The difference between control and PNA treated groups was response study showed that TEMPOL significantly inhibits approaching signicfi ance (p= 0.0705) but suggested that PNA the 4T1 colony size, at concentrations ranging from 18 to is having a direct effect on primary tumors as well as on 180 𝜇 M, suggesting cytostatic effects on the cancer cells metastasis. (Figure 6(a)). However, the TEMPOL did not signicfi antly reduce colony number (Figure 6(b)), suggesting a lack of .. PNA Reduces Lung Metastasis from T Primary Tumors. cytotoxic effects on the cancer cells. These results indicate that Subcutaneous or orthotopic 4T1 tumors rapidly metastasize PNA and small molecule, membrane permeable nitroxides to distant tissues in a pattern similar to human TNBC, have differences in their specificities and therapeutic indices. including metastasizing to the lung [25, 26]. We found that Eec ff ts of PNA on 4T1 tumors in vivo. In vivo, 4T1 cells PNA reduced lung metastasis as compared to control HSA establish rapidly growing tumors that metastasize to lung, treatment but not to a level of significance (Figure 7(b), liver, bone, and brain [25]. u Th s, they are a useful model p=0.056). However, we found that there is a dramatic increase of human TNBC. Subcutaneous 4T1 tumors were developed in metastasis to the lung induced by a superoxide producing in Balb/c mice and treated with PNA or HSA as a control. chemotherapy drug doxorubicin (Figure 7(c)). Remarkably, Tumors from PNA treated mice were not significantly dif- in the presence of PNA and doxorubicin the lung metastasis ferent in size from those of HSA treated animals. However, was nearly abolished (Figure 7(c), paired two-tailed t test, histological examination of tumors from both groups showed p=0.038). Thisprovidesstrong rationale forcombining PNA that PNA treated tumors had less live tissue and more necrotic with other drugs used in adjuvant or neoadjuvant therapy. (dead) tissue than the HSA treated controls (Figure 7(a)). In this regard, mice bearing 4T1 tumors were subjected to Control (N=3) PNA (N=4) Seconds Echo power (a.u.) 6 Journal of Oncology ∗∗∗ capacity of cells. PNA also reduced the size of the colonies, ∗∗ ∗∗∗ indicating reduced ability of the cancer cells to proliferate. More importantly, we have demonstrated that PNA inhibits metastasis, particularly that induced by chemotherapeutic drugs such as doxorubicin, leading to enhanced survival 1000 (Figures 7 and 8). We discuss below the significance of the current results for future preclinical and clinical studies that can guide the development of PNA as a primer used No HSA PNA HSA PNA HSA PNA treatment in conjunction with standard cancer therapies. us, Th our control preclinical studies may be relevant to neoadjuvant treatment 30 M 60 M 120 M and to clinical trials such as the I-SPY2 trials [27]. Treatment Two key aspects of PNA facilitate its biological activities. Figure 4: PNA treatment significantly reduces live cell number. 4T1 First, it catalyzes the dismutation of superoxide by virtue of cells were plated and treated the following day with PNA (red bars) the multiple covalently attached nitroxide groups. Second, it or HAS (blue bars), and control (black bar) was media only. Aeft r is based on a macromolecule carrier, HSA, which is capable 72 hr, cells were stained with SYTOX Green and uo fl rescence of targeting extracellular compartments. These properties of was measured. Percent of dead cells and percent of live cells PNA are expected to create an essentially superoxide-free were calculated using uo fl rescence values. Live cells’ uo fl rescence is milieu within the vasculature and within the tumor microen- shown. Results indicate PNA, compared to HSA, reduces live cell vironment. In the vasculature, elimination of superoxide numbers at concentrations ranging from 30 to 120𝜇 MPNA (n=3 prevents the formation of peroxynitrite through its reaction for each group;∗∗=p<0.01,∗∗∗=p<0.001). with NO. This allows the restoration of endogenous NO to promote blood flow to the metastasized solid tumor. This is supported by our published studies that demonstrate PNA a chemotherapy regimen including paclitaxel, doxorubicin, mediation of increased blood flow and prevention of ischemic and cyclophosphamide, with or without PNA (Figure 8(a)). reperfusion injury in models of stroke, myocardial infarction, This regimen is similar to that in the I-SPY 2 clinical trials and sickle cell disease [6, 29]. for breast cancer [27]. In this study, PNA again significantly Enhanced blood o fl w, as shown here and in our previous decreased the number of lung metastases (paired t test with study [28], is likely to have a number of eeff cts on solid p<0.01). Furthermore, PNA enhanced overall survival of mice tumors. It is expected to increase oxygen delivery to tumor against chemotherapy-mediated toxicity in the 4T1 mouse cells, which would likely reverse hypoxia leading to inactiva- model (Figure 8(b)). Statistical analysis using the Log-Rank tion of hypoxia inducible factor 1𝛼 . This in turn would alter (Mantel-Cox) test indicates a statistical difference between tumor metabolism and growth, as well as several aspects of PNA and chemotherapy treated mice compared to HSA and metastasis including angiogenesis, migration, invasion, and chemotherapy treated mice, with p<0.0013. Together, these intravasation [30]. data demonstrate that PNA reduces metastasis in this triple PNA reduces cellular ROS in 4T1 breast cancer cell negative breast cancer model and that this correlates with cultures and we have shown that this corresponds to reduced longer survival times. u Th s, this indicates PNA conjunctive cell proliferation and survival. Because PNA is based on HSA, therapy with standard chemotherapeutic agents improves it is expected to target extracellular spaces within the tumor survival of 4T1 tumor bearing mice corresponding to a microenvironment. We did not observe reduced 4T1 tumor reduction in toxicity of the chemotherapeutic agent and a sizes in mice treated with PNA. However, the tumors did have reductioninlung metastasis. reduced amount oflivetissue(Figure 7(a)) and increased areas of noncellular or necrotic tissue (not shown), indicating that the drug was directly acting on the tumor. PNA in the 4. Discussion extravascular spaces within the tumor microenvironment is Our findings demonstrate that the SOD3-mimetic activity expected to reduce tumor superoxide and ROS levels and of an extracellular albumin-based macromolecular nitroxide, this may directly lead to interference with metastasis. For example, ROS are able to directly activate matrix metallo- PNA, converts cancer cells from a tumorigenic state to a cytostatic state by reducing superoxide and ROS levels (see proteinases (MMPs) [31]. MMPs are key eeff ctors of tumor Figure 1). Earlier we reported that PNA works as an SOD3 invasion and metastasis through degradation of extracellular matrix components. Therefore, PNA may directly interfere mimetic to alter the hypoxic state of a solid tumor through blood ow fl enhancement [28]. In this report we established with metastasis by blocking MMP activation by eliminating that the same therapeutic mechanism for PNA also works superoxide within the tumor microenvironment. This pro- posed mechanism may also relate to the effects of doxoru- in the 4T1 TNBC model. We were able to confirm that this action of PNA occurs within 30 minutes of i.v. infusion with bicin and other chemotherapy reagents on metastasis. For example, we found that doxorubicin increases the number two independent biophysical methods (EPR and ultrasound). Our data suggest that PNA is cytotoxic to murine 4T1 of lung metastases compared to control mice, and this was TNBC cells as well as inhibiting their proliferation. In reversed by PNA. This is consistent with other studies show- ing doxorubicin-induced increases in metastasis in mouse colony forming assays, PNA reduced the number of colonies, suggesting an effect of PNA on the clonogenic survival models and human patients [32, 33]. Many chemotherapy Relative Fluorescence Unites Journal of Oncology 7 HSA 30M Control PNA 30M Control HSA 120M HSA 60M PNA 120M PNA 60M (a) ∗∗∗ ∗∗ ∗∗ ∗∗ 20 5000 HSA PNA HSA PNA HSA PNA HSA PNA HSA PNA HSA PNA 30M 60M 120M 30M 60M 120M (b) Figure 5: PNA treatment significantly reduces TNBC cancer cell proliferation. Colony forming assays were performed in PNA treated 4T1 cell cultures. Cells were plated at low density and then treated with the indicated concentration of PNA (red bars) or HSA (blue bars) as a control. PNA significantly reduces number of colonies compared to HSA treatments (a) and size of colonies (b). n=3 per group; data is representative of three independent experiments (∗ =p< 0.05,∗∗ =p< 0.01, and∗∗∗ =p< 0.001 versus HSA). Colony Number Colony Size 180 8000 ∗∗ ∗∗ 0 0 0 1.8 18 180 0 1.8 18 180 Tempol Concentration (M) Tempol Concentration (M) (a) (b) Figure 6: TEMPOL produces significant decreases in colony size but not colony number. 4T1 cells were plated at low cell number and treated the following day with TEMPOL at varying concentrations, and the control was DMEM alone. Aeft r 5 days, cells were fixed, stained, and imaged using Alpha Imager sow ft are. (a) Colony number was counted and (b) total colony size was measured using Alpha Imager software. Data shown suggests that TEMPOL alone inhibits cellular proliferation at concentrations ranging from 18 to 180𝜇 M, according to a two-tailed t test, with p<0.001, but is not significantly cytotoxic at the concentrations shown. Colony Number Number of Colonies Colony Size (pixels) Size of colonies (pixels) 8 Journal of Oncology   1.5×10  8  1.0×10  7  5.0×10 Con PNA Con PNA Dox Dox+PNA (a) (b) (c) Figure 7: Live tumor area and number of metastatic lung nodules decreases with PNA treatment while survival increases with PNA treatment. (a) Subcutaneous 4T1 tumors are treated with HSA (control) or PNA. Tumors sections were stained by H&E and live tissue area was determined by stereology. (b) Tumor bearing mice were treated with or without PNA and aer ft 3 weeks lungs were harvested for counting lung metastatic nodules. (c) 4T1 tumor bearing mice were treated with doxorubicin in the presence or absence of PNA. PNA combined with doxorubicin compared to doxorubicin alone significantly reduces lung metastasis, according to a paired two-tailed t test, p <0.05 (∗). ∗∗ 3 ∗∗ 2 50      Control Chemotherapy Chemotherapy plus PNA Days elapsed Control Chemotherapy PNA plus Chemotherapy (a) (b) Figure 8: PNA treatment combined with chemotherapy reduces lung metastasis and enhances survival of mice carrying T tumors compared to chemotherapy treatment alone. (a) Tumor bearing mice were treated with a chemotherapy regimen, with and without PNA, and aer ft 3 weeks lungs were harvested for counting metastatic lung nodules. PNA in the presence of chemotherapy significantly reduced lung metastases compared to chemotherapy alone, according to a paired two-tailed t test, p>0.01. (b) PNA combined with chemotherapy significantly increased overall survival of mice compared to chemotherapy alone, according to the Log-Rank (Mantel-Cox) test, with p value =.0013 (∗∗). agents, including doxorubicin, increase tumor superoxide albumin is mainly synthesized in the liver, enters the circu- and ROS, which is associated with the process of metastasis. lation, and occurs predominantly in the extravascular space in tissues such as skin, gut, muscle, and other u fl ids such as We show that PNA eliminates the increase in metastasis associated with chemotherapy. This is most likely related to cerebrospinal uid. fl A substantial amount of albumin is also the ability of PNA to eliminate superoxide and ROS in the found within the intravascular space, but only a very small tumor microenvironment. amount is thought to exist intracellularly [34–36]. In our Since our experimental design has been to inject PNA studies, after injection into the intraperitoneal space, PNA into the intraperitoneal cavity, the subsequent biodistribution is expected to enter the circulation via the lymphatic system of PNA warrants consideration. The overall biodistribution and be distributed similarly to endogenous albumin. Imaging of PNA in our studies is expected to be similar to endoge- studies confirm that uo fl rescent albumin injected into the nous serum albumin since PNA consists of macromolecular intraperitoneal space distributes maximally to the spleen, human serum albumin conjugated with multiple nitroxide livers, lungs, and other organs within 3 hrs of injection; groups to provide the SOD mimetic activity. Endogenous levels subsequently decline [37]. Endogenous albumin cycles Mean number of lung metastases Area (m ) Number of lung mets Percent survival Number of lung mets Journal of Oncology 9 through the lymphatic system approximately 28 times during inhibits metastasis, as well as its effectiveness in other types of its lifetime (Peters, 1996; Evans, 2002) and injected PNA solid tumors. As a novel SOD mimetic drug, PNA would be is expected to behave similarly. More importantly, PNA is ideally suited for evaluation in trials like the I-SPY 2 platform [27] and FDA breakthrough therapy designation (BTD) to not expected to accumulate significantly intracellularly in vivo, similar to endogenous albumin. u Th s, the activity of bring PNA from benchtobedside in 3-5 years. PNA is most similar to ecSOD/SOD3, which is also localized extracellularly. 5. Conclusions The intraperitoneal administration of PNA may have two effects. First, PNA would remove extracellular superoxide PNA reduces ROS in TNBC, is cytotoxic, and is cytostatic to in all bodily compartments working as EcSOD/SOD3 with mouse TNBC breast cancer cells in a dose dependent manner. minimal eeff ct on the intracellular superoxide levels con- PNA combined with doxorubicin, compared to doxorubicin trolled by SOD1 and SOD2. In contrast a low molecular alone, significantly reduces lung metastasis in a mouse model weight nitroxide chemical like TEMPOL would dismutate of breast cancer. Furthermore, PNA reduces metastasis and both intracellular and extracellular superoxide because it enhances survival of mice against chemotherapy-mediated readily crosses membranes and accumulates within cells toxicity in a 4T1 mouse breast cancer model. These in vitro (Figure 6). Second, it has been known for over four decades and in vivo experimental n fi dings suggest PNA is a promising that the lymphatics are the rfi st site of metastasis for most cancer therapeutic. The antimetastatic mechanism of the solid cancers and there has been a concerted effort in SOD3-mimetic PNA on a murine TNBC 4T1 mouse model trying to target cancer therapeutics to the lymphatic system has been established. If the same antimetastatic mechanisms (e.g., see [38]). Accumulation of PNA in the lymph nodes can be established with a human TNBC mouse model, these and surrounding lymphatics is a possibility and may affect preclinical efficacy data will guide a Phase 1b clinical protocol immune cells and metastasis. The possible immunodulatory design in recurrent TNBC patients. In addition, PNA could effects on metastasis by PNA will be evaluated in future be tested in trials like the I-SPY2 fast track clinical trial studies. protocol to select and graduate new drug candidates through As a macromolecule with multiple nitroxide groups, PNA FDA breakthrough therapy designation (BTD). acts as an SOD3 mimetic. SOD3 (aka EcSOD) is secreted from cells and is found either free in the extracellular space or Data Availability adhered to the surface of cells. SOD3 expression is frequently diminished in breast and other types of cancer, which The data used to support the findings of this study are contributes to metastasis [4]. Furthermore, low expression included within the article. of SOD3 in breast cancer is associated with reduced relapse free survival in all types of breast cancer [4]. Overexpression of SOD3 in a human breast cancer xenograft or in the 4T1 Conflicts of Interest syngeneic model led to reduced lung metastasis and longer Shanta M.Messerli,Amanda M.Schaefer,Yongxian Zhuang, survival [39, 40]. Noah Keime, Jenny Jin, Li Ma, Bohdan J. Soltys, and W. Keith Taken together, PNA appears to be capable of acting as Miskimins declare no conflicts of interest. Carleton J. C. Hsia an SOD3 mimetic to alter the oxidative state within solid is sole proprietor with AntiRadical Therapeutics. tumors, inhibiting their ability to metastasize (see Figure 1). We speculate that elimination of superoxide within the tumor microenvironment directly reverses ROS stimulated metas- Acknowledgments tasis by preventing activation of MMPs, reducing invasion, This research was supported by the National Institutes and inhibiting cancer cell proliferation. PNA enhancement of of Cancer (1R01CA180033-04; W. Keith Miskimins), the blood flow is expected to reverse solid tumor hypoxia leading National Institute of General Medical Sciences (CoBRE to changes in tumor metabolism and hypoxia-dependent Grant P20GM103548;W.Keith Miskimins), the COBRE metastatic events. Another potential outcome of increased P20GM103548P20 Pilot grant (Project lead-Shanta M. blood ofl w in tumors is to act as a primer to improve Messerli), Sanford Research, and project seed funds from the drug delivery to tumors to enhance the therapeutic index of state of South Dakota. chemotherapy and immunotherapy. An additional advantage of PNA treatment of cancer may be attributable to its ability to protect normal tissues from oxidative stress and References inflammatory injuries leading to better quality of life. We have previously demonstrated the ability of PNA to prevent [1] M. 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