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PURPOSE: To develop optoacoustic, spectrally distinct, actively targeted gold nanoparticle–based near-infrared probes (trastuzumab [TRA], TRA-Aurelia-1, and TRA-Aurelia-2) that can be individually identifiable at multispectral optoacoustic tomography (MSOT) of human epidermal growth factor receptor 2 (HER2)–positive breast tumors. MATERIALS AND METHODS: Gold nanoparticle–based near-infrared probes (Aurelia-1 and 2) that are optoacoustically active and spectrally distinct for simultaneous MSOT imaging were synthesized and conjugated to TRA to produce TRA-Aurelia-1 and 2. Freshly resected human HER2-positive (n = 6) and HER2-negative (n = 6) triple-negative breast cancer tumors were treated with TRA-Aurelia-1 and TRA-Aurelia-2 for 2 hours and imaged with MSOT. HER2-expressing DY36T2Q cells and HER2-negative MDA-MB-231 cells were implanted orthotopically into mice (n = 5). MSOT imaging was performed 6 hours following the injection, and the Friedman test was used for analysis. RESULTS: TRA-Aurelia-1 (absorption peak, 780 nm) and TRA-Aurelia-2 (absorption peak, 720 nm) were spectrally distinct. HER2-positive human breast tumors exhibited a significant increase in optoacoustic signal following TRA-Aurelia-1 (28.8-fold) or 2 (29.5-fold) (P = .002) treatment relative to HER2-negative tumors. Treatment with TRA-Aurelia-1 and 2 increased optoacoustic signals in DY36T2Q tumors relative to those in MDA-MB-231 controls (14.8-fold, P < .001; 20.8-fold, P < .001, respectively). CONCLUSION: The study demonstrates that TRA-Aurelia 1 and 2 nanoparticles operate as a spectrally distinct HER2 breast tumor–targeted in vivo optoacoustic agent. Keywords: Molecular Imaging, Nanoparticles, Photoacoustic Imaging, Breast Cancer Supplemental material is available for this article. © RSNA, 2023

Background: Triple negative breast cancer (TNBC), unlike other breast cancer subtypes, lacks a specific targetable receptor. As such tumor specific delivery, which can limit off-target effects of anti-neoplastic therapies, has been an unmet clinical need in treating this aggressive breast cancer subtype. To address this need, we exploited the tumor hallmark of an acidic microenvironment and developed a pH targeted nanoparticle by conjugation of the V3 pH specific peptide on a wormhole pore mesoporous silica nanoparticles (V3-RUBY) and assessed the ability of V3-RUBY to specifically release cargo in ex vivo patient samples and in orthotopically implanted TNBC tumors as detect by multispectral optoacoustic imaging technology (MSOT). Methods: The silica nanoparticles with wormhole pore architecture were synthesized by sol-gel chemistry and characterized by transmission electron microscopy (TEM), zeta potential, and dynamic light scattering (DLS). The surface base particle was crosslinked with a gatekeeper molecule, chitosan. Further targeting of the particle using V3 pHLIP (low insertion peptide), resulted in the V3-RUBY nanoparticle which allows for pH-sensitive cargo release. The particle was loaded with imaging dye to assess tumor specificity. Fresh ex vivo TNBC patient tumor tissues were resected and rapidly treated with V3-RUBY containing propodium iodide (PI) to evaluate tumor uptake of V3-RUBY and cargo release within the tumor cells, as measured by the red fluorescence of PI when bound to nucleic acids. (PI independently cannot cross cell membranes.) In vivo, female athymic mice were implanted with MDA-MB-468 breast cancer cells by the mammary fat pad injection. Once the tumor reached 3mm in size, athymic mice were intravenously injected with V3-RUBY nanoparticles carrying IR780 infrared imaging dye and were imaged with MSOT inVision 512TF. Results: The RUBY nanoparticle with wormhole pores was 27 nm diameter. The dual targeting approach of a nanoparticle with V3 targeting peptide and chitosan demonstrated pH specificity around tumor pH. In ex vivo patient TNBC samples, V3-RUBY demonstrated active targeting and dye release at pH 6.8, which approximated the pH measured at surgical tumor removal, in 10 fresh patient samples compared to pH 7.4 controls (p Conclusion: The tumor-specific release of payload by V3-RUBY suggests the potential of a pH specific target in TNBC, with such a nanoparticle holding promise to deliver both diagnostic and therapeutic cargo directly to the tumor and limit off-target toxicity. Future translation of these technologies could have promise in TNBC, as well as other high-grade breast cancer subtypes and expand treatment options in this challenging area of oncology. TablePatientBreast Cancer SubtypeTumor GradePathologic StagePI Uptake (a.u.) pH 6.8PI Uptake (a.u.) 7.41TNBC3ypT3N3a33.20.82TNBC3ypT2N038.41.53TNBC3ypT4bN1a36.81.74TNBC3T1bN022.40.85TNBC2T1cN021.80.86TNBC2ypT0N021.50.67TNBC3ypT1aN2a22.00.48TNBC3ypT4dN239.52.19TNBC3T2N2a31.52.210TNBC3T2N1a32.21.8 Citation Format: Alexandra Thomas, Akiko Chiba, Abhilash Samykutty, Molly W. McNally, Lacey R. McNally. Tumor specific cargo release in ex vivo patient samples and murine models of triple negative breast cancer by a pH-targeted nanoparticle: V3-RUBY [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-04.

Nanoparticles are widely studied as theranostic vehicles for cancer, however, clinical translation has been limited due to poor tumor-specificity. Features that maximize tumor uptake remain controversial, particularly using clinically relevant, orthotopic models. Herein, we report a systematic study that assesses two major features for impact on tumor specificity, i.e., active vs. passive targeting and nanoparticle size, to evaluate relative influences for a pancreatic cancer model, in vivo. Active targeting via V7 peptide is superior to passive targeting for uptake by orthotopic pancreatic tumors, irrespective of nanoparticle size, observed through cell internalization and release mechanisms. However, size has a secondary effect on uptake for actively-targeted nanoparticles. Nanoparticle size had no significant effect on uptake of passively-targeted nanoparticles. Results highlight the superiority of active targeting over nanoparticle size for pancreatic tumor uptake. These findings suggest a framework for optimizing similar non-aggregate nanoparticles for diagnostic and therapeutic treatment of pancreatic and recalcitrant cancers.

While particle carriers have potential to revolutionize disease treatment, using these carriers requires knowledge of spatial and temporal biodistribution. The goal of this study was to track orally administered particle uptake and trafficking through the murine gastrointestinal (GI) tract using multispectral optoacoustic tomography (MSOT).Polylactic acid (PLA) particles encapsulating AlexaFluor 680 (AF680) dye conjugated to bovine serum albumin (BSA) were orally gavaged into mice. Particle uptake and trafficking were observed using MSOT imaging with subsequent confirmation of particle uptake via fluorescent microscopy. Mice treated with PLA-AF680-BSA particles exhibited MSOT signal within the small bowel wall at 1 and 6 h, colon wall at 6, 12, and 24 h, and mesenteric lymph node 24 and 48 h. Particle localization identified using MSOT correlated with fluorescence microscopy. Despite the potential of GI tract motion artifacts, MSOT allowed for teal-time tracking of particles within the GI tract in a non-invasive and real-time manner.Future use of MSOT in conjunction with particles containing both protein-conjugated fluorophores as well as therapeutic agents could allow for non-invasive, real time tracking of particle uptake and drug delivery Keywords: Multispectral optoacoustic tomography, Gastrointestinal tract, Colon, Oral gavage, Nanoparticle tracking, Diagnostic imaging

A library of 49 analogs of imidazo[1,2-a]pyridine with 2-halo, aryl, styryl and phenylethynyl-substitution at C-2 position and N-/O-/S-methyl linkage at C-3 position, have been synthesized and evaluated for their anti-proliferative activity against breast (MCF-7, MDA-MB-231), pancreatic (MiaPaca-2), lung (A549), prostate (PC-3) and colon (HCT-116) cancer cell lines and normal cells (HEK-293). Among the screened compounds, 5b exhibited best anticancer potential in all tested cancer cells with IC

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal disease and the leading cause of cancer death worldwide. The survival rate of patients with this form of cancer is about 8%. The physiological barrier of the tumor microenvironment composed of a dense stroma and disorganized blood vessels creates a barrier for early identification and treatment of this deadly disease. In recent years, nanoparticle-based controlled delivery systems were developed to exploit the pathophysiology of biological systems such as acidic tumor microenvironment or the altered tumor-specific enzymes to improve the diagnosis and treatment efficacy. Here, we demonstrate the collagenase IV-mediated tumor site-selective release of the IR-780 imaging probe from the M-Ge-SDC1 nanoparticles, revealing the feasibility of the collagenase IV (MMP-9) responsive target specificity for diagnosing pancreatic cancer by multispectral optoacoustic tomography (MSOT) imaging. Methods: Mesoporous silica nanoparticles (MSN) with wormhole pore topology were synthesized and were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The surface of MSN was conjugated with Gelatin-A to obtain M-Ge. The M-Ge particles were loaded with propidium Iodide (PI) or IR780 infrared imaging dye. The M-Ge surface was further conjugated with Syndecan-1 (SDC1) to improve the target specificity to release imaging cargo from the nanoparticles. Female athymic mice were orthotopically implanted with S2VP10 tumor cells. After a week of tumor implantation, mice were intravenously injected with M-Ge-SDC1 nanoparticles containing IR780 dye and were imaged with MSOT and AMI. Results: In the current study, Mesoporous silica nanoparticles with 27 nm diameter were synthesized. The Gelatin-A crosslinking on the surface of MSN particles as a gatekeeper was developed that could degrade upon contact with collagenase IV in the tumor microenvironment. The conjugation of SDC1 further improved the tumor specificity. The athymic mice orthotopically implanted with S2VP10 cells closely resemble human PDAC. Our results demonstrated that intravenous delivery of M-Ge-SDC1 nanoparticles could enzymatically degrade (MMP-9) and release IR780 at the tumor site and conjugation of SDC1 further improved the tumor specificity to detect the orthotopically implanted pancreatic tumors (p Conclusion: Due to the lack of effective screening tools, PDAC has the lowest survival rate and limited therapeutic efficacy for current FDA-approved drugs compared to other malignancies. Innovative technologies to develop engineered nanoparticles with active targeting moiety and dynamic imaging technology can overcome these limitations. Implementing such systems can enhance PDAC detection that can be translated into the clinic to improve health care. Citation Format: Abhilash Samykutty, Molly McNally, William M. MacCuaig, Jordan Hagood, Girish Mishra, Barish H. Edil, William E. Grizzle, Lacey R. McNally. Matrix metalloproteinase-9 responsive active targeted silica nanoparticles for pancreatic cancer detection by multispectral optoacoustic tomography [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 300.

Purpose: While nanoparticles are widely studied as potential theranostic treatments for cancer, weak tumor specificity has hindered clinical translation. Features that contribute to tumor specificity are historically controversial, particularly when using clinically relevant models. Aggressive cancers, such as pancreatic ductal adenocarcinoma (PDAC), stand to benefit from development of highly specific nanoparticles as a theranostic drug delivery system. This work evaluates active targeting and nanoparticle size, in a silica-based nanoparticle for specific accumulation and release of contrast agent within an orthotopically implanted tumor. Methods: Mesoporous silica nanoparticles (MSNs) were synthesized with wormhole-like pores using a silica precursor to coat a surfactant scaffold. Chitosan was attached to MSN surface as a pH-responsive gatekeeper for encapsulated agents. A series of acidification and basification procedures resulted in loading of photoacoustic contrast agent IR780 within MSN pores. MSNs were further functionalized for attachment of V7 peptide to target aggressive and acidic pancreatic cancer. pH-sensitivity and tumor specificity/uptake was validated using an in vitro PDAC cell model (S2VP10L) prior to implantation and assessment in an animal model. Functionalized MSNs were intravenously injected into athymic mice with orthotopically implanted PDAC tumors. Near infrared fluorescence and optoacoustic imaging were used to evaluate the biodistribution of MSNs subsequent to treatment. Results: Zeta potential, DLS, and TEM were utilized to show three differently sized MSNs of 26, 45, and 73 nm and confirm conjugation of chitosan and V7 peptide. Dye-release assays indicated significantly increased agent release from MSNs in acidic pH (~90%) compared to biological pH (~15%) (p=0.001). Treatment of PDAC cell line with MSNs showed highest uptake and specificity with actively targeted 26nm particles and that all actively targeted MSNs exhibited greater specificity than all passively targeted MSNs (p Conclusion: Active targeting outperforms nanoparticle size for facilitation of tumor-specific uptake in an acidic PDAC murine model. Active targeting was necessary for high accumulation of MSNs and contrast agent in the tumor. Nanoparticle size had a secondary, but notable influence on tumor uptake in which smaller sized MSNs resulted in higher tumor specificity. Citation Format: William M. MacCuaig, Abhilash Samykutty, Molly McNally, Ajay Jain, William E. Grizzle, Lacey R. McNally. Comparing influences of active targeting and nanoparticle size on tumor specificity in pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2458.

The antibody-drug conjugates (ADCs) are one the fastest growing biotherapeutics in oncology and are still in their infancy in gastrointestinal (GI) cancer for clinical applications to improve patient survival. The ADC based approach is developed with tumor specific antigen, antibody carrying cytotoxic agents to precisely target and deliver chemotherapeutics at the tumor site. To date, 11 ADCs have been approved by US-FDA, and more than 80 are in the clinical development phase for different oncological indications. However, The ADCs based therapies in GI cancers are still far from having high-efficient clinical outcomes. The limited success of these ADCs and lessons learned from the past are now being used to develop a newer generation of ADC against GI cancers. In this review, we did a comprehensive assessment of the key components of ADCs, including tumor marker, antibody, cytotoxic payload, and linkage strategy, with a focus on technical improvement and some future trends in the pipeline for clinical translation. The various preclinical and clinical ADCs used in gastrointestinal malignancies, their target, composition and bioconjugation, along with preclinical and clinical outcomes, are discussed. The emphasis is also given to new generation ADCs employing novel mAb, payload, linker, and bioconjugation methods are also included.

Pancreatic cancer remains a recalcitrant neoplasm associated with chemoresistance and high fatality. Because it is frequently resistant to apoptosis, exploiting autophagic cell death could offer a new treatment approach. We repurpose echinomycin, an antibiotic encapsulated within a syndecan-1 actively targeted nanoparticle, for treatment of pancreatic cancer. Tumor-specific uptake, biodistribution, efficacy of nanodelivered echinomycin, and mechanism of cell death were assessed in aggressive, metastatic models of pancreatic cancer. In these autophagic-dependent pancreatic cancer models, echinomycin treatment resulted in autophagic cell death noted by high levels of LC3 among other autophagy markers, but without hallmarks of apoptosis, e.g., caspase activation and chromatin fragmentation, or necrosis, e.g., plasma membrane degradation and chromatin condensation/degrading. In vivo, biodistribution of syndecan-1-targeted nanoparticles indicated preferential S2VP10 or S2CP9 tumor uptake compared to the liver and kidney (S2VP10 p = 0.0016, p = 0.00004 and S2CP9 p = 0.0009, p = 0.0001). Actively targeted nanodelivered echinomycin resulted in significant survival increases compared to Gemzar (S2VP10 p = 0.0003, S2CP9 p = 0.0017) or echinomycin only (S2VP10 p = 0.0096, S2CP9 p = 0.0073). We demonstrate that actively targeted nanodelivery of echinomycin results in autophagic cell death in pancreatic and potentially other high-autophagy, apoptosis-resistant tumors. Collectively, these findings support syndecan-1-targeted delivery of echinomycin and dysregulation of autophagy to induce cell death in pancreatic cancer.

At the intersection of the newly emerging fields of optoacoustic imaging and theranostic nanomedicine, promising clinical progress can be made in dismal prognosis of ovarian cancer. An acidic pH targeted wormhole mesoporous silica nanoparticle (V7-RUBY) was developed to serve as a novel tumor specific theranostic nanoparticle detectable using multispectral optoacoustic tomographic (MSOT) imaging. We report the synthesis of a small, < 40 nm, biocompatible asymmetric wormhole pore mesoporous silica core particle that has both large loading capacity and favorable release kinetics combined with tumor-specific targeting and gatekeeping. V7-RUBY exploits the acidic tumor microenvironment for tumor-specific targeting and tumor-specific release. In vitro, treatment with V7-RUBY containing either paclitaxel or carboplatin resulted in increased cell death at pH 6.6 in comparison to drug alone (p10X higher than in the kidney using both multispectral optoacoustic tomography (MSOT) imaging with secondary confirmation using near infrared fluorescence imaging (p

Improved detection of breast cancer using highly sensitive, tumor-specific imaging would facilitate diagnosis, surveillance and assessment of response to treatment. We conjugated osteopontin peptide to an infrared fluorescent dye to serve as a contrast agent for detection of breast cancer by multispectral optoacoustic tomography (MSOT). Selective binding of the osteopontin-based probe was identified using flow cytometry and near infrared fluorescent imaging in triple negative and HER2 positive breast cancer cell lines in vitro. Osteopontin-750 accumulation was evaluated in vivo using MSOT with secondary confirmation of signal accumulation using near infrared fluorescent imaging. The osteopontin-based probe demonstrated binding to breast cancer cells in vitro. Similarly, after intravenous administration of the osteopontin-750 probe, it accumulated preferentially in the subcutaneous breast tumor in nude mice (557 MSOT a.u. compared to untargeted organs such as kidney (53.7 MSOT a.u.) and liver (32.1 MSOT a.u.). At 2.5 h post-injection, signal intensity within the tumor was 9.7 and 17 times greater in the tumor bed than in the kidney or liver, respectively. Fluorescence imaging ex vivo comparing tumor signal to that of nontarget organs confirmed the results in vivo. MSOT imaging demonstrated selective accumulation of the fluorescent osteopontin targeting probe to tumor sites both in vitro and in vivo, and provided high-resolution images. Further development of this tool is promising for advanced diagnostic imaging, disease surveillance and therapeutic models that limit nontarget toxicity.

Examining the dynamics of an agent in the tumor microenvironment can offer critical insights to the influx rate and accumulation of the agent. Intratumoral kinetic characterization in the in vivo setting can further elicudate distribution patterns and tumor microenvironment.Dynamic contrast-enhanced Multispectral Optoacoustic Tomographic imaging (DCE-MSOT) acquires serial MSOT images with the administration of an exogenous contrast agent over time. We tracked the dynamics of a tumor-targeted contrast agent, HypoxiSense 680 (HS680), in breast xenograft mouse models using MSOT. Arterial input function (AIF) approach with MSOT imaging allowed for tracking HS680 dynamics within the mouse. The optoacoustic signal for HS680 was quantified using the ROI function in the ViewMSOT software. A two-compartment pharmacokinetics (PK) model constructed in MATLAB to fit rate parameters. The contrast influx (kin) and outflux (kout) rate constants predicted are kin = 1.96 × 10−2 s-1 and kout = 9.5 × 10-3 s-1 (R = 0.9945). Keywords: Pharmacokinetic modeling, Targeted contrast agent, Intratumoral kinetics, Tumor microenvironment, Hypoxia, Multispectral optoacoustic imaging

Acquired resistance to chemotherapy remains a major stumbling block in cancer treatment. Chronic inflammation has a crucial role in induction of chemoresistance and results, in part, from the induction and expansion of inflammatory cells that include myeloid-derived suppressor cells (MDSCs) and IL-13+ Th2 cells. The mechanisms that lead to induction of activated MDSCs and IL-13+ Th2 cells have not yet been identified. Here we demonstrated that doxorubicin (DOX) treatment of 4T1 breast tumor-bearing mice led to the induction of IL-13R+miR-126a+ MDSCs (DOX-MDSC). DOX-MDSC promote breast tumor lung metastasis through MDSC miR-126a+ exosomal-mediated induction of IL-13+ Th2 cells and tumor angiogenesis. The induction of DOX-MDSC is regulated in a paracrine manner. DOX treatment not only increases interleukin (IL)-33 released from breast tumor cells, which is crucial for the induction of IL-13+ Th2 cells, but it also participates in the induction of IL-13 receptors and miR-126a expressed on/in the MDSCs. IL-13 released from IL-13+Th2 cells then promotes the production of DOX-MDSC and MDSC miR-126a+ exosomes via MDSC IL-13R. MDSC miR-126a+ exosomes further induce IL13+ Th2 cells in a positive feed-back loop manner. We also showed that MDSC miR-126a rescues DOX-induced MDSC death in a S100A8/A9-dependent manner and promotes tumor angiogenesis. Our findings provide insight into the MDSC exosomal-mediated chemoresistance mechanism, which will be useful for the design of inhibitors targeting the blocking of induction of miR-126a+ MDSCs.

// Huang-Ge Zhang 1, 2 , Pengxiao Cao 2 , Yun Teng 2 , Xin Hu 4, 5 , Qilong Wang 2, 8 , Ashish S. Yeri 6 , Xiaoying Zhuang 2 , Abhilash Samykutty 2 , Jingyao Mu 2 , Zhong-Bin Deng 3 , Lifeng Zhang 2 , James A. Mobley 7 , Jun Yan 3 , Kendall Van Keuren-Jensen 6 , Donald Miller 3 1 Louisville Veterans Administration Medical Center, Louisville, KY 40206, USA 2 James Brown Cancer Center, Department of Microbiology and Immunology, University of Louisville, KY 40202, USA 3 Department of Medicine, University of Louisville, KY 40202, USA 4 Program in Biostatistics, Bioinformatics and Systems Biology, The University of Texas Graduate School of Biomedical Sciences at Houston, TX 77030, USA 5 Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 6 Translational Genomics Research Institute, Phoenix, AZ 85004, USA 7 Mass Spectrometry/Proteomics Shared Facility, University of Alabama at Birmingham, Birmingham, AL 35294, USA 8 Department of Clinical Oncology, Huai’an First People’s Hospital, Nanjing Medical University, Huai’an, 223300, China Correspondence to: Huang-Ge Zhang, email: H0Zhan17@louisville.edu Keywords: isolation and identification extracellular microvesicles, in vivo predominately population, HG-NV, exosomes Received: November 12, 2015 Accepted: April 16, 2016 Published: May 12, 2016 ABSTRACT Extracellular microvesicles (EVs) have been recognized for many potential clinical applications including biomarkers for disease diagnosis. In this study, we identified a major population of EVs by simply screening fluid samples with a nanosizer. Unlike other EVs, this extracellular nanovesicle (named HG-NV, HG-NV stands for HomoGenous nanovesicle as well as for Huang-Ge- nanovesicle) can be detected with a nanosizer with minimal in vitro manipulation and are much more homogenous in size (8–12 nm) than other EVs. A simple filtration platform is capable of separating HG-NVs from peripheral blood or cell culture supernatants. In comparison with corresponding exosome profiles, HG-NVs released from both mouse and human breast tumor cells are enriched with RNAs. Tumor derived HG-NVs are more potent in promoting tumor progression than exosomes. In summary, we identified a major subset of EVs as a previously unrecognized nanovesicle. Tumor cell derived HG-NVs promote tumor progression. Molecules predominantly present in breast tumor HG-NVs have been identified and characterized. This discovery may have implications in advancing both microvesicle biology research and clinical management including potential used as a biomarker.

Liver metastasis accounts for many of the cancer deaths in patients. Effective treatment for metastatic liver tumors is not available. Here, we provide evidence for the role of miR-18a in the induction of liver M1 (F4/80+interferon gamma (IFNγ)+IL-12+) macrophages. We found that miR-18a encapsulated in grapefruit-derived nanovector (GNV) mediated inhibition of liver metastasis that is dependent upon the induction of M1 (F4/80+IFNγ+IL-12+) macrophages; depletion of macrophages eliminated its anti-metastasis effect. Furthermore, the miR-18a mediated induction of macrophage IFNγ by targeting IRF2 is required for subsequent induction of IL-12. IL-12 then activates natural killer (NK) and natural killer T (NKT) cells for inhibition of liver metastasis of colon cancer. This conclusion is supported by the fact that knockout of IFNγ eliminates miR-18a mediated induction of IL-12, miR-18a treatment has an anti-metastatic effects in T cell deficient mice but there is no anti-metastatic effect on NK and NKT deficient mice. Co-delivery of miR-18a and siRNA IL-12 to macrophages did not result in activation of co-cultured NK and NKT cells. Taken together our results indicate that miR-18a can act as an inhibitor for liver metastasis through induction of M1 macrophages.

The lack of access to the brain is a major obstacle for central nervous system drug development. In this study, we demonstrate the capability of a grapefruit-derived nanovector (GNV) to carry miR17 for therapeutic treatment of mouse brain tumor. We show that GNVs coated with folic acid (FA-GNVs) are enhanced for targeting the GNVs to a folate receptor-positive GL-26 brain tumor. Additionally, FA-GNV-coated polyethylenimine (FA-pGNVs) not only enhance the capacity to carry RNA, but the toxicity of the polyethylenimine is eliminated by the GNVs. Intranasal administration of miR17 carried by FA-pGNVs led to rapid delivery of miR17 to the brain that was selectively taken up by GL-26 tumor cells. Mice treated intranasally with FA-pGNV/miR17 had delayed brain tumor growth. Our results demonstrate that this strategy may provide a noninvasive therapeutic approach for treating brain-related disease through intranasal delivery.

Despite Pancreatic cancer being expected to contribute to only 3% of new cancer cases in both males and females in 2019, it is expected to cause an estimated 45,750 deaths making it the third highest contributor to cancer deaths. This is due to the low survivability associated with pancreatic cancer which has a 5-year survival rate of only 9%. In order to address the poor prognosis common with pancreatic cancer, biomarkers are being used to create therapies targeted to specific cancer cells. The S100A8/A9 complex is a heterodimer, known for modulating the inflammatory response, and it is upregulated in a variety of cancers including pancreatic cancers. For this reason, we selected it as a targeting agent to deliver Rapamycin to pancreatic cancer cells via a mesoporous silica-encased gold nanorod (MS-GNR). The use of this nanoparticle allows an anticancer molecule to be encapsulated within the mesoporous silica matrix by further encapsulation of the nanoparticle with the pH sensitive polymer chitosan. A low pH tumor microenvironment induces relaxation of the chitosan surrounding this chitosan-capped mesoporous silica gold nanorod (CMG) allowing for the anticancer molecule to be released only in cancer cells. The gold nanorod at the core of this enables these nanoparticles to be imaged in real time via Multispectral Optoacoustic Tomography (MSOT). Using these tools, we linked S100A9 to a mesoporous silica-encased gold nanorod which contained Rapamycin held in via the chitosan polymer (S100A9-Rap-CMG). MSOT imaging revealed a strong uptake in S2013Q and MiaPaca pancreatic cancer cells. Further tests showed that at a pH 6.8 S100A9-Rap-CMG treatment resulted in an increase in cell death by 69% compared to treatment at physiologic pH of 7.4 by the S100A9-Rap-CMG's which did not induce cell death. This data indicates the feasibility of S100A9 as a targeting agent for pancreatic cancer cells and the potential benefits of nano-drug delivery of Rapamycin to treat pancreatic adenocarcinoma. The ultimate benefit of the nano-drug delivery includes both increased tumor targeting and mitigating premature drug release and offsite delivery. Citation Format: Karl Thomas, Molly McNally, Abhilash Samykutty, Lacey R. McNally. Evaluation of S100A9 targeted nanoparticles containing rapamycin to treat pancreatic adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2790.

Background: Approximately two-thirds of operative breast cancer cases are suitable for breast conserving surgery (BCS). In order for BCS to be successful, negative surgical margins should be obtained. Despite improvement in imaging techniques, the ability to achieve negative margins for BCS remains variable with positive margin rates ranging from 21 to 50%. Due to this high re-excision rate, there is an unmet need for a reliable technology to localize the tumor and assess excision margins in real time. Multispectral Optoacoustic Tomography (MSOT) is an emerging imaging modality capable of real time imaging of numerous contrast agents with enhanced spatial resolution of 75µm at depths of 5 cm. This imaging modality has the ability to visualize tumor tissue in a standard operating room setting allowing for precise surgical excision with the potential of increasing rate of negative margins. We developed gold nanorods targeting Human epidermal growth factor receptor-2 (HER2) as a contrast agent to visualize the breast tumor tissue in cell line as well as in ex vivo patient samples using MSOT. Methods: Gold nanorods were created using hydrogen peroxide (GNR-H2O2) as a reducing agent to create an MSOT detectable contrast agent; the nanorods were stabilized via encapsulation with mesoporous silica together with subsequent chitosan capping. HER2+ breast cancer cells were specifically targeted by conjugating the mesoporous silica-coated chitosan capped gold nanorods (CMGs) to Trastuzumab resulting in TRA-CMG particles. The TRA-CMG particles were evaluated in HER2+ and HER2- breast cancer cell lines as well as fresh HER2+ (N=6) and HER2- freshly resected patient tumor tissues (N=6). All HER2- tumor samples were triple negative breast cancer (TNBC) subtype. Tumor uptake was evaluated in tissue mimicking phantoms using MSOT. In vivo, TRA-CMG were IV injected into female mice with DY36T2Q tumors and imaged using MSOT imaging 6 hours post injection (N=5). Results: TRA-CMG particles were 8nm wide and 98nm in length. Treatment of HER2+ breast cancer cell lines, DY36T2Q and SKBR3, with TRA-CMG resulted in 2.5x and 3.1x enhanced signal, respectively, as compared to HER2- MDA-MD468 cells (p Conclusion: The significant uptake of TRA-CMG particles in HER2+ tumors suggests the potential of this particle to be used for diagnostic imaging as well as with intraoperative imaging using MSOT. Future clinical applications include improving the rate of negative margins for patients undergoing breast conservation. Patient tumor chracteristics and TRC-CMG uptakePatientBreast Cancer SubtypeTumor GradePathologic StageTRA-CMG Uptake (a.u.)1TNBC3ypT3N3a1.22TNBC3ypT2N00.33TNBC3ypT4bN1a0.54ER+PR+HER2+2pT1cN213.35ER+PR-HER2+3pT1cN017.96TNBC2ypT0N00.47ER-PR-HER2+3pT2N019.48ER+PR-HER2+3pT2N015.69ER-PR-HER2+3pT1cN016.610TNBC3pT2N1a0.911ER+PR+HER2+3ypTN018.712TNBC2pT1cN00.7 Citation Format: Akiko Chiba, Alexandra Thomas, Karl N. Thomas, Abhilash Samykutty, Molly McNally, Lacey McNally. Uptake of trastuzumab targeted mesoporous silica-coated chitosan capped gold nanorods in breast cancer cell lines and ex vivo patient samples [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-01-04.

Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), found in cooked meat, is a known food carcinogen that causes several types of cancer, including breast cancer, as PhIP metabolites produce DNA adduct and DNA strand breaks. Curcumin, obtained from the rhizome of Curcuma longa, has potent anticancer activity. To date, no study has examined the interaction of PhIP with curcumin in breast epithelial cells. The present study demonstrates the mechanisms by which curcumin inhibits PhIP-induced cytotoxicity in normal breast epithelial cells (MCF-10A). Curcumin significantly inhibited PhIP-induced DNA adduct formation and DNA double stand breaks with a concomitant decrease in reactive oxygen species (ROS) production. The expression of Nrf2, FOXO targets; DNA repair genes BRCA-1, H2AFX and PARP-1; and tumor suppressor P16 was studied to evaluate the influence on these core signaling pathways. PhIP induced the expression of various antioxidant and DNA repair genes. However, co-treatment with curcumin inhibited this expression. PhIP suppressed the expression of the tumor suppressor P16 gene, whereas curcumin co-treatment increased its expression. Caspase-3 and -9 were slightly suppressed by curcumin with a consequent inhibition of cell death. These results suggest that curcumin appears to be an effective anti-PhIP food additive likely acting through multiple molecular targets.

Exosomes are emerging mediators of intercellular communication; whether the release of exosomes has an effect on the exosome donor cells in addition to the recipient cells has not been investigated to any extent. Here, we examine different exosomal miRNA expression profiles in primary mouse colon tumour, liver metastasis of colon cancer and naive colon tissues. In more advanced disease, higher levels of tumour suppressor miRNAs are encapsulated in the exosomes. miR-193a interacts with major vault protein (MVP). Knockout of MVP leads to miR-193a accumulation in the exosomal donor cells instead of exosomes, inhibiting tumour progression. Furthermore, miR-193a causes cell cycle G1 arrest and cell proliferation repression through targeting of Caprin1, which upregulates Ccnd2 and c-Myc. Human colon cancer patients with more advanced disease show higher levels of circulating exosomal miR-193a. In summary, our data demonstrate that MVP-mediated selective sorting of tumour suppressor miRNA into exosomes promotes tumour progression., Exosomes are involved in the development of metastasis but how their composition is regulated is not well known. Here the authors propose that major vault protein-dependent loading of miR-193a into exosomes could be a general mechanism by which cancer cells get rid of oncosuppressor miRNAs.

Purpose: Due to inadequate early detection and inability to operate at advanced stages, pancreatic ductal adenocarcinoma (PDAC) has remained one of the most difficult types of cancer to treat. A small range of non-specific symptoms coupled with quick metastasis rate result in a poor 5-year survival rate; 14% for those diagnosed within stage IA, and as low as 1% for those diagnosed during stage IV. Nanoparticles have recently emerged as a potential delivery agent for diagnostic and therapeutic agents, and although clinical success has not been ample due to targeting accuracy issues. This work shows a nanoparticle that has been functionalized with a pancreatic cancer-specific targeting ligand and exhibits specific particle release in pancreatic malignant environment (pH 6.6) as compared to non-malignant environments (pH 7.4). Methods: Wormhole-pored mesoporous silica nanoparticles were formed at 80°C using Tetrapropyl orthosilicate (TPOS) and a scaffold of hexadecyltrimethyl-ammonium bromide (CTAB). Acetic acid and ethanol were used in dialysis procedures to remove the CTAB scaffold and create worm-like pores. Chitosan was added to coat the silica particles and serve as a gatekeeper. IR-780 was added before the solution was acidified to load the dye into the worm-hole particles created. Shortly after, the solution pH was raised back to physiological levels (pH=7.4) to trap the dye within the particle. The particle surfaces were functionalized to attach a targeting ligand pH-low insertion peptide (V7) to conjugate the dye-loaded nanoparticles. Pancreatic adenocarcinoma cells (S2VP10 line) were plated in pH-7.4,6.8, and 6.6 PBS solutions with the loaded particles to assess uptake via near-infrared fluorescence and multispectral optoacoustic imaging. Results: Zeta potential and dynamic light scattering were used to ensure the 63nm size nanoparticle and proper coating. Near-infrared fluorescence imaging showed ~10X increased signal at pH 6.6 as compared to pH=7.4. MSOT imaging ~5X increased signal in the malignant microenvironment environment that is acidic as compared to the non-malignant environment at pH 7.4. Conclusion: The functionalized wormhole mesoporous silica nanoparticles coated with chitosan demonstrated pH-sensitivity in terms of cellular uptake via NIR fluorescence and MSOT imaging. Citation Format: William MacCuaig, Abhilash Samykutty, Molly McNally, Kylie Nairon, Surya Banks, William Grizzle, Lacey R. McNally. pH-responsive tumor-targeted mesoporous silica nanoparticle for the identification of pancreatic cancer using optoacoustic tomography [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1937.

Purpose: Recent advances in the nanotechnology and molecular imaging provides an excellent opportunity for development of delivery vehicles and imaging probes to improve real-time assessment and early detection of breast cancer progression.The absence of estrogen receptor (ER), progesterone receptor (PR), or HER-2 genes, represents a major clinical challenge for triple negative breast cancer (TNBC). Because of the absence of reliable markers, there is an unmet clinical need for developing efficient methods to identify TNBC. We have developed an acidic pH targeted Rosella nanoparticles that can actively release IR780 dye into orthotopically implanted TNBC tumors to improve tumor detection using multispectral optoacoustic imaging technology (MSOT). Methods: The Rosella nanoparticles are a mesoporous silica base with wormhole pore architecture containing a chitosan gatekeeper and V3 pHLIP targeting peptide. The particles were synthesized using the sol-gel method and characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The Rosella particles were loaded with propidium Iodide (PI) or IR780 infrared imaging dye to evaluate pH-sensitive cargo release. Female athymic mice were implanted with MDA-MB-468 breast cancer cells by the mammary fat pad injection. Once the tumor reaches 3mm in size, athymic mice were intravenously injected with ROSELLA nanoparticles carrying IR780 dye and were imaged with MSOT. Results: We have synthesized the Rosella nanoparticle with wormhole pores that is 27 nm diameter and can encapsulate the IR780 imaging probes for the detection of the TNBC. The Rosella particle contains a chitosan gatekeeper which can degrade upon contact with acidic pH tumor to prevent off-target release and is further sensitized to pH using V3 peptide. The Rosella nanoparticles can detect acidic tumor microenvironment and can penetrate inside the tumor cells. As a model of TNBC, we have injected MDA-MB-468 cells into the mammary fat pad of the female athymic mice to develop breast tumors. Once the tumor reached 3mm in size, we have intravenously delivered ROSELLA nanoparticles to the athymic mice with TNBC tumors. Our results were demonstrated that the intravenous injection of the ROSELLA particles could detect the orthotopically implanted TNBC tumors (p Conclusion: most aggressive subtypes of breast cancer, TNBC has a poor response for majority of the FDA-approved breast cancer drugs. To overcome these limitations, the distinct nanoformulations with potent imaging technology will enable to develop tumor-penetrating nanoparticles to more effectively deliver chemotherapeutics or imaging agents with least off-target effects. Future translation of these technologies has a high clinical impact concerning our current treatment options for TNBC patients. Citation Format: Abhilash Samykutty, Molly McNally, Alexandra Thomas, William Grizzle, Lacey R. McNally. Tumor microenvironment targeted Rosella nanoparticle for the detection of triple negative breast cancer by multispectral optoacoustic tomography [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1952.

While particle carriers have potential to revolutionize disease treatment, using these carriers requires knowledge of spatial and temporal biodistribution. The goal of this study was to track orally administered particle uptake and trafficking through the murine gastrointestinal (GI) tract using multispectral optoacoustic tomography (MSOT). Polylactic acid (PLA) particles encapsulating AlexaFluor 680 (AF680) dye conjugated to bovine serum albumin (BSA) were orally gavaged into mice. Particle uptake and trafficking were observed using MSOT imaging with subsequent confirmation of particle uptake via fluorescent microscopy. Mice treated with PLA-AF680-BSA particles exhibited MSOT signal within the small bowel wall at 1 and 6 h, colon wall at 6, 12, and 24 h, and mesenteric lymph node 24 and 48h. Particle localization identified using MSOT correlated with fluorescence microscopy. Despite the potential of GI tract motion artifacts, MSOT allowed for teal-time tracking of particles within the GI tract in a non-invasive and real-time manner. Future use of MSOT in conjunction with particles containing both protein-conjugated fluorophores as well as therapeutic agents could allow for non-invasive, real time tracking of particle uptake and drug delivery. Citation Format: Neal Bhutiani, Abhilash Samykutty, Kelly McMasters, Nejat Egilmez, Lacey R. McNally. Tracking of orally-administered particles within the gastrointestinal tract of murine models using multispectral optoacoustic tomography [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1954.

Traditional cancer imaging devices are limited in their ability to screen for multiple contrast agents simultaneously in real time. Multispectral Optoacoustic Tomography (MSOT) is an emerging imaging modality capable of real-time imaging of numerous contrast agents with enhanced spatial resolution of 75µm at depths of 5 cm. The use of exogenous contrast agents in MSOT remains largely unexplored, so we developed two species of spectrally distinct gold nanorod as contrast agents for use in MSOT. Our goal was to evaluate the potential of MSOT to spectrally differentiate two exogenous contrast agents simultaneously. Two gold nanorod species were created using hydrogen peroxide (GNR-H2O2) or ascorbic (GNR-ASC) acid as reducing agents to modify the length of each species to create nanorods with individual light absorbance spectra in the IR range (680-900 nm). These gold nanorods were highly stabilized via encapsulation with mesoporous silica along with a subsequent chitosan capping. Human epidermal growth factor receptor 2 positive (HER2+) cells were specifically targeted by conjugating these mesoporous silica-coated chitosan capped gold nanorods (CMGs) to Trastuzumab resulting in TRA-CMG particles. Both TRA-CMG-ASC and TRA-CMG-H2O2 resulted in optoacoustic spectrally distinct signals when imaged in tissue phantoms both individually as well as mixed within the same well after multispectral processing using linear regression. Treatment of HER2+ breast cancer cell lines, DY36T2Q and SKBR3, with TRA-CMG-H2O2 resulted in 2.5x and 3.1x enhanced signal, respectively, as compared to HER2- MDA-MD468 cells. Treatment of DY36T2Q and SKBR3 cells with TRA-CMG-ASC demonstrated 3.7x and 6.9x, respectively, compared to MDA-MD468. In all three cell lines treated with a combination of TRA-CMG-H2O2’s and TRA-CMG-ASC’s clear and distinct signals were observed for each particle, demonstrating that each TRA-CMG possessed and maintained a detectibly distinct optoacoustic spectrum, in the IR range, allowing them to be detectable as separate contrast agents in MSOT while proximate to other targeted contrast agents. Both particles have demonstrated that they can be simultaneously administered and targeted at HER2+ cell while also maintaining distinct photoacoustic signals in MSOT upon consolidation. Each particle species, targeted to the same cells, were capable of being monitored individually in the presence of the other gold nanorod contrast agent. Citation Format: Karl N. Thomas, Abhilash Samykutty, Molly McNally, Lacey R. McNally. Development of spectrally distinct silica coated gold nanorods for detection of cancer using MSOT [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1934.