Title<br />“Molecular probe targeting breast cancer diagnosis with the help of nanoparticles upon conjugation with specific antibodies”<br />Project will be done in Dr. Joe Gray’s Lab under the guidance of Dr. Fanqing Chen at Lawrence Berkeley National Laboratory (LBNL), California.<br />Graduate Grant proposal<br />Submitted by:<br />Puja Patel<br />From: Biosciences department<br />Student id: xy5865<br />Presented to:<br />California State University, East Bay<br />Abstract<br />The main objective of this study is to create a translational technology platform for multi-mode breast cancer imaging that 1) detects breast cancer by MRI and is capable of detecting cancer cells with greater sensitivity than with existing technologies, and (2) targets the over expressed HER1 molecules that localize on the surface of a subclass of breast cancer cells and (3) Biopsy of breast cancer cell line by flow cytometry. <br />Specific GOALS<br />I propose to develop a novel nanoparticle-based molecular probe that can be targeted to HER1-overexpressing breast cancer for diagnosis.<br />Background & Significance<br />Cancer occurs as a result of mutations, or abnormal changes in the genes responsible for cell’s growth & regulations. An erratic, uncontrolled growth & proliferation of breast cells is defined as breast cancer. A rapidly dividing cell may form lump or mass of extra tissue which is known as tumor. It can either be malignant (cancerous) or benign (non-cancerous). Breast cancer is usually referred to as malignant tumor that has developed from the cells in the breast. One of the major challenges for breast cancers is the lack of sensitive and accurate imaging solutions and highly localized non-invasive image-guided treatments. Primary breast tumors measures less than about 2 mm in diameter. This is below the detection limit of most clinical imaging techniques. The clinical impact of inability to detect small tumors is that breast cancers are frequently not detected until they have progressed to a stage when the probability of curative treatment is significantly reduced. Rapid, sensitive, specific and noninvasive imaging techniques are urgently needed to detect cancer in its earliest stages to improve clinical outcomes, and to reduce the use of expensive biopsies. In addition, targeted, molecular imaging-guided intervention has not been evaluated adequately in breast cancer to allow highly localized, non-invasive therapeutic treatments.<br /> When regular cells transform into cancer cells, their protein expression profiles change dramatically. Certain proteins such as cancer cell-specific antigens & transmembrane-receptor tyrosine kinases are expressed on the surface of cancer cells and thus have a crucial role in proliferation, motility & metastasis of cancer cells. In recent years, several surface bound molecular markers for breast cancer have been identified. The epidermal growth factor (EGF)-like proteins stimulate cells to divide by activating members of the EGF receptor (EGFR) family, which consists of the EGFR itself (HER1) and the receptors known as HER2-4. Women have cell membrane surface-bound tyrosine receptor kinases called HER2-positive breast cancer. This gene is normally involved in the signal transduction pathways leading to cell growth and differentiation. HER2 (also known as ErbB-2) stands for Human Epidermal growth factor Receptor 2 and also a member of ErbB2 protein family. EGFR is a 170-kDa transmembrane glycoprotein encoded by the HER1 proto-oncogene & is expressed at high levels in at 20% of breast cancers. It is related to the pathogenesis of breast cancer, and is over expressed in many basal-like breast tumors ADDIN EN.CITE <EndNote><Cite><Author>Nielsen</Author><Year>2004</Year><RecNum>757</RecNum><record><rec-number>757</rec-number><ref-type name="
>17</ref-type><contributors><authors><author>Nielsen, T. O.</author><author>Hsu, F. D.</author><author>Jensen, K.</author><author>Cheang, M.</author><author>Karaca, G.</author><author>Hu, Z.</author><author>Hernandez-Boussard, T.</author><author>Livasy, C.</author><author>Cowan, D.</author><author>Dressler, L.</author><author>Akslen, L. A.</author><author>Ragaz, J.</author><author>Gown, A. M.</author><author>Gilks, C. B.</author><author>van de Rijn, M.</author><author>Perou, C. M.</author></authors></contributors><auth-address>Genetic Pathology Evaluation Centre, University of British Columbia, Vancouver Hospital & British Columbia Cancer Agency, Vancouver, British Columbia, Canada.</auth-address><titles><title>Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma</title><secondary-title>Clin Cancer Res</secondary-title></titles><periodical><full-title>Clin Cancer Res</full-title></periodical><pages>5367-74</pages><volume>10</volume><number>16</number><keywords><keyword>Breast Neoplasms/classification/genetics/mortality/*pathology</keyword><keyword>Female</keyword><keyword>Humans</keyword><keyword>Immunohistochemistry/methods</keyword><keyword>Keratin/analysis</keyword><keyword>Neoplasm Invasiveness</keyword><keyword>Neoplasms, Basal Cell/genetics/mortality/*pathology</keyword><keyword>Oligonucleotide Array Sequence Analysis</keyword><keyword>Receptor, erbB-2/analysis/genetics</keyword><keyword>Receptors, Estrogen/analysis/genetics</keyword><keyword>Research Support, U.S. Gov't, P.H.S.</keyword><keyword>Survival Analysis</keyword><keyword>Tumor Markers, Biological/analysis</keyword></keywords><dates><year>2004</year><pub-dates><date>Aug 15</date></pub-dates></dates><accession-num>15328174</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15328174 </url></related-urls></urls></record></Cite></EndNote>1, which have a worse outcome than other types of breast cancers ADDIN EN.CITE <EndNote><Cite><Author>Kurebayashi</Author><Year>2004</Year><RecNum>756</RecNum><record><rec-number>756</rec-number><ref-type name="
>17</ref-type><contributors><authors><author>Kurebayashi, J.</author><author>Okubo, S.</author><author>Yamamoto, Y.</author><author>Sonoo, H.</author></authors></contributors><auth-address>Department of Breast and Thyroid Surgery, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan.</auth-address><titles><title>Inhibition of HER1 signaling pathway enhances antitumor effect of endocrine therapy in breast cancer</title><secondary-title>Breast Cancer</secondary-title></titles><periodical><full-title>Breast Cancer</full-title></periodical><pages>38-41</pages><volume>11</volume><number>1</number><keywords><keyword>Antineoplastic Agents/*pharmacology</keyword><keyword>Antineoplastic Agents, Hormonal/pharmacology</keyword><keyword>Breast Neoplasms/*drug therapy</keyword><keyword>Cell Line, Tumor</keyword><keyword>Drug Synergism</keyword><keyword>Epidermal Growth Factor/antagonists & inhibitors</keyword><keyword>Estradiol/*analogs & derivatives/pharmacology</keyword><keyword>Estrogen Receptor Modulators/therapeutic use</keyword><keyword>Female</keyword><keyword>Gene Expression Regulation/genetics</keyword><keyword>Genes, erbB-1/*drug effects/genetics</keyword><keyword>Humans</keyword><keyword>Quinazolines/pharmacology</keyword><keyword>Research Support, Non-U.S. Gov't</keyword><keyword>Signal Transduction/*drug effects/genetics</keyword></keywords><dates><year>2004</year></dates><accession-num>14718791</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14718791 </url></related-urls></urls></record></Cite></EndNote>2. When cells have too many copies of HER2 gene, cells grow faster. Women with HER2-positive breast cancer have a more aggressive disease and a higher risk of recurrence than those who do not have this type. Due the high expression of EGFR/HER 1, it leads to poor prognosis in breast cancer patients.<br /> Statistics have shown that in 2009, an estimated 192,370 new cases of invasive breast cancer were expected to be diagnosed in women in the U.S. About 40,170 women in the U.S. were expected to die in 2009 from breast cancer, though death rates have been decreasing since 1990. These decreases are thought to be the result of treatment advances, earlier detection through screening, and increased awareness. HER2 protein over-expression affects approximately 20-30% of breast cancer patients. It is indeed necessary for the development of new, imaging-based, non-invasive tests that utilize breast cancer-specific markers, which could significantly reduce the need for biopsy, and the attendant cost and morbidity. <br />39909752657475 Answer to the above mentioned challenges is use of Quantum dot (QD) technology that has already made a remarkable impact on cancer imaging. QD are miniscule made up of semiconductors whose conducting characteristics are closely related to the size & shape of the individual crystal. These nanoparticles give us the ability to see cells and molecules that we otherwise cannot detect through conventional imaging. The ability to pick up what happens in the cell - to monitor therapeutic intervention and to see when a cancer cell is mortally wounded or is actually activated - is critical to the successful diagnosis and treatment of the disease. Qdot are novel class of flurophores which have a diameter of nanometer and possesses high quantum yield tunable emission wavelength. As small crystals, they can be mixed into liquid solution, making them ideal for fluorescent tagging in biological applications. Qdot technology offers several advantages over organic dyes traditionally used for cellular imaging. One advantage is that the Qdots can be fine-tuned to emit different wavelengths simply by altering the size of the core nanostructure. Another advantage is that either a single wavelength or broadband source can be used to excite the Qdots. <br />Fig 1: Schematic of the overall structure of a Qdot nanocrystal <br />conjugate. The layers represent the distinct structural elements,<br />and are drawn roughly to the scale.<br />OBJECTIVES<br />A nanoparticles targeting platform based on breast-specific anti-EGFR single chain Fv (scFv) antibody developed by Jim Marks in the Breast Cancer SPORE in UCSF will be developed and tested in selected xenograft mouse models of breast cancer. A Gd3+-DOTA- and 64Cu-DOTA- coated CdSe nanocrystal will be used for MRI imaging. The project is highly collaborative and leverages the expertise of several labs in the UCSF Breast Cancer SPORE and UC Berkeley, such as scFv expertise of the Marks lab, xenograft mouse model in the Hann lab, and nuclear imaging capability at UCSF and LBNL, the nanofabrication expertise of the Chen lab and the Molecular Foundry at LBNL. Once the proof of principle has been established by targeting Her1/EGFR with scFv and the killing of breast cancer cell lines in the pilot phase, this technology can be applied to other surface protein molecules associated with breast cancer, using other targeting mechanisms such as specific peptides and small molecules. To achieve these goals the following specific aims will be accomplished:<br />Specific Aim 1. Develop multi-modality MRI contrast agent imaging nanoparticles. The particles will be highly luminescent CdSe nanocrystal densely coated with Gd-DOTA and 64Cu-DOTA. <br />Aim 1. The toxicity of the nanoparticles will be studied along with the consideration for developing robust, efficient and versatile protocols for the preparation of the nanoparticles, which will be composed of CdSe nanocrystals functionalized with DOTA, a chelator for Gd3+ and 64Cu2+ for MRI imaging.<br />Specific Aim 2: Develop multi-modality imaging nanoparticles using EGFR as targeted biomarkers of breast cancer via the incorporation of single chain antibody-based targeting mechanism. <br />Aim 2 a: Develop a nanoparticle functionalized with scFv antibody designed specifically to bind to HER1 that have been identified by the Marks’ lab. <br />Aim 2 b: The association of the nanoparticle with the purified protein & cultured breast cancer cells will be studies. <br />Aim 2 c: Establish the biostability and toxicity of the nanoparticle in animals and develop MIR imaging of xenografted breast tumor in nude mice. The targeted nanoparticles will be applied to imaging studies in xenograft mouse models (carried out in collaboration with the Nuclear Medicine Dept. in LBNL and with Byron Hann at UCSF).<br />Aim 3: FACS analysis done by imaging breast cancer cells clustered with quantum dots & antibodies. <br />Experimental design & METHODs<br />Aim 1. Construction of the Gd and 64Cu-charged DOTA-CdSe nanocrystal<br />We have constructed silica-coated CdSe nanocrystals with different surface groups, such as amine, thiol, and carboxyl groups. Using EDC-NHS chemistry, we have been able to conjugate DOTA on the surface of CdSe nanocrystals at high density. Gd3+ and 64Cu2+ will be charged into the CdSe nanocrystal-linked DOTA. We will perform MIR imaging in collaboration with LBNL. The carboxyl group on DOTA will be EDC-activated and conjugated to NHS, and NHS will react with amine groups on CdSe nanocrystal.<br />Multiphoton excitation of CdSe nanocrystals avoids interference from absorption of light by tissue. Recently, nanoparticles have also been shown to absorb and emit light directly in the visible region as a multiple photon process. This involves the simultaneous excitation by two or more lower energy photons to reach the same excited energy state that can also be reached by a single higher energy photon. The ability for a chromophore to absorb two photons is dependent on the parameters shown in eqn. 1, where “” is the two photon cross section and “I” is the intensity of light. The cross sectional area is the two-photon equivalent of the single photon absorption extinction coefficient, and is a quantitative measure of the ability of the chromophore to absorb two photons simultaneously. CdSe nanocrystal has been shown to be an ideal multiphoton fluorophore because of its large cross-section.<br />Rate = ½ () <I2> (1)<br />MRI imaging with DOTA. Magnetic resonance imaging (MRI) is widely used clinically because it provides high spatial resolution images, particularly through the application of contrast agents which are currently employed in approximately 35% of all clinical MRI examinations. These are derived from either iron particles or paramagnetic complexes (predominantly Gd, complexes). ADDIN EN.CITE <EndNote><Cite><Author>Artemov</Author><Year>2003</Year><RecNum>221</RecNum><record><rec-number>221</rec-number><ref-type name="
>17</ref-type><contributors><authors><author>Artemov, D.</author></authors></contributors><titles><title>Molecular magnetic resonance imaging with targeted contrast agents</title><secondary-title>J .Cellular Biochem.</secondary-title></titles><periodical><full-title>J .Cellular Biochem.</full-title></periodical><pages>518-524</pages><volume>90</volume><dates><year>2003</year></dates><urls></urls></record></Cite><Cite><Author>Aime</Author><Year>2003</Year><RecNum>210</RecNum><record><rec-number>210</rec-number><ref-type name="
>17</ref-type><contributors><authors><author>Aime, S.</author><author>Dastru, W.</author><author>Crich, S. G.</author><author>Gianolio, E.</author><author>Mainero, V.</author></authors></contributors><titles><title>Innovative magnetic resonance imaging diagnostic agents based on paramagnetic Gd(III) complexes</title><secondary-title>Biopolymers</secondary-title></titles><periodical><full-title>Biopolymers</full-title></periodical><pages>419-428</pages><volume>66</volume><dates><year>2003</year></dates><urls></urls></record></Cite><Cite><Author>Caravan</Author><Year>1999</Year><RecNum>255</RecNum><record><rec-number>255</rec-number><ref-type name="
>17</ref-type><contributors><authors><author>Caravan, P.</author><author>Ellison, J. J.</author><author>McMurry, T. J.</author><author>Lauffer, R. B.</author></authors></contributors><titles><title>Gadolinium(III) chelates as MRI contrast agents: Structure, dynamics, and applications</title><secondary-title>Chem. Rev.</secondary-title></titles><periodical><full-title>Chem. Rev.</full-title></periodical><pages>2293-2352</pages><volume>99</volume><dates><year>1999</year></dates><urls></urls></record></Cite></EndNote>23-25 One of the clinically approved, and commonly used contrast agents are Gd-DOTA (Fig 1. DOTA = 1,4,7,10-tetrakis (carboxymethyl)-1,4,7,10-tetraazacyclododecane), which show low toxicity and patient discomfort. Clinical safety results from its low osmolality, low viscosity, low chemo-toxicity, high solubility, and high in vivo stability for the macrocylic complex. The vast majority of MRI applications depend on the bulk biodistribution of the contrast agent rather than molecular targeting methods. As a small molecule, Gd agents get into the microvasculature around tumors, which is at a much higher density than normal tissue. This increased concentration of Gd in highly vascularized tissue around tumors is the basis for the MRI contrast mechanism. Thus, specifically targeted contrast agents would be extremely useful for improving the ability of MRI to localize cancer. In addition to MRI, DOTA can also be used to chelate short-lived radioactive tracer, such as 64Cu2+<br />Aim 2. Conjugation of targeting antibody onto the nanoparticle<br />Fig. 2. Antibody conjugated nanocrystal (Qdots) are internalized by cancer cells. A. Left, antibody -nanocrystal conjugates, Right, nanocrystal only. B. only antibody-nanocrystal conjugates are internalized by tumor cells. C. live cell image of internalized nanocrystal. D. FACS analysis of the anti-EGFR scFv antibody.Single chain antibodies against EGFR/HER1 will be obtained from the Jim Marks lab. The antibodies will be crosslinked to SMCC, a heterobifunctional crosslink that can react with thiol group on that CdSe nanocrystal and amine groups on the protein. The antibody-conjugated CdSe nanocrystal will be tested for binding activity to EGFR/HER1 by BiaCore and uptake by cultured breast cancer cell lines (Fig.5. antibody targeted CdSe nanocrystal). <br />Molecular Imaging and targeting. Recent efforts have been directed at developing “molecular probes” that can locate and image cancer cells by engineering the imaging probe to recognize a marker of a disease state, on the cell surface. For example, it has been well established that the incorporation of a specificity element (such as an antibody) helps to selectively direct a tag to the desired cells. As a non-invasive technique, these highly specific and sensitive molecular tags can be used in animal models to study the progress of a disease. Molecular imaging will have a theoretical advantage over biopsy procedures because it is not invasive, and safer. The long-term potential of these tags lies beyond diagnostics, for example, a real-time image of the tumor can also be used during surgery as a method to delineate the tumor margins. The molecular targeting mechanism also allows delivery of therapeutics to the tumor and imaging-guided intervention. Initially we will use HER1 as a molecular target, using a high quality internalizing scFv antibody from Marks lab as the targeting mechanism. EGFR is present on at least 20% of breast tumor cells and on cells that line the microvasculature of breast cancer tumors. The antibody-DOTA-CdSe nanocrystal will be injected through tail vein into nude mice carrying human breast cancer xenograft (Fig. 6). The mice will be imaged with both MRI. <br />Fig 3: Xenograft mouse model (center and right), all p53 +/- 6.5 mo post 4Gy IR Left flank tumor on center mouse (mouse 46). Signals are from luciferase reporter in the tumor.<br />Aim 3: Biopsy of Breast cancer cell lines such as SKBR 3 & MDAMB 468 using Flow cytometry.<br />Upon attachment with the surface of the cell culture plates, the cells are expected to express the protein HER 1, which is then treated with primary antibody such as mouse erbB2 monoclonal antibody taken from Invitrogen Cat No. 28-0003Z. Concentration of this would be ~75 mg/ml with a dilution factor of 1:200. For secondary antibody, Q11001MP Qdot 605 Goat F (ab’) 2 Anti Mouse IgG Conjugate (H+L) and the nanoplex 605 from Invitrogen will be used. The concentration will be ~75 mg/ml with a dilution of 1:200. The fluorescence emission will then be measured at 605nm using FL2 channel (Ex 488 nm / Em 585nm) in the flow cytometer. The samples would be in triplicates & will be further analyzed using Cell Quest Pro software from BD Biosciences. <br /><ul><li>Innovation</li></ul>We will construct a nanoparticle as illustrated in Fig.1. DOTA, anti-EGFR/HER1 antibody, and Pc4 will be conjugated to the CdSe/ZnS nanocrystal, respectively. The nanocomposite will have modalities of MRI, PET, NIR imaging, and antibody-based targeting. This nanoconstruct will offer sensitive and molecular targeted imaging and imaging-guided intervention. This is a novel nanotherapeutics with multifunctional capabilities. A molecular probe with MRI, PET, NIR, molecular and PDT modalities has never been attempted before. The presence of all these allows precise spatial resolution (MRI), quantitative imaging (PET), real-time tumor margin definition during biopsy and surgery (NIR), and in situ treatment (PDT). The molecular targeting brings added specificity. Future developments will include the conjugation of enhancer to the photodynamic chemicals, scFv antibodies targeting other breast cancer antigens identified in the SPORE, and specific peptides/inhibitors against breast cancer surface antigens.<br /><ul><li>Literature Review</li></ul>Biju V, Mundayoor S, Omkumar RV, Anas A, Ishikawa M. (2009) Bioconjugated quantum dots for cancer research: Present status, prospects and remaining issues. Biotechnology Advances. 2009 Dec 5<br />Bailey VJ, Puleo CM, Ho YP, Yeh HC, Wang TH. (2009) Quantum dots in molecular detection of disease. Conf Proc IEEE Eng Med Biol Soc. 2009; 1:4089-92<br />Mahmoud W, Sukhanova A, Oleinikov V, Rakovich YP, Donegan JF, Pluot M, Cohen JH, Volkov Y, Nabiev I. (2009) Emerging applications of fluorescent nanocrystal quantum dots for micro metastases detection. Proteomics & clinical application 9999-999A<br />Michalet, X. et al. (2001) Properties of fluorescent semiconductor nanocrystal and their application to biological labeling. Single Mol. 2, 261-276<br />Nielsen, T.O. et al. (2004) Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res 10, 5367-5374<br />Mattoussi, H. et al. (2000) Self-Assembly of CdSe-ZnS Quantum Dot Bioconjugates Using an Engineered Recombinant Protein. J. Am. Chem. Soc. 122, 12142-12150<br />Artemov, D. (2003) Molecular magnetic resonance imaging with targeted contrast agents. J.Cellular Biochem. 90, 518-524. <br />Ntziachristos, V., Bremer, C. & Weissleder, R. (2003) Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging. Eur. Radiol. 13, 195-208 <br />Jaiswal, J. & Simon, S. (2004) Potentials and pitfalls of fluorescent quantum dots for biological imaging. Trends Cell Bio. 14, 497-500 <br />