1. Establish a new drug discovery and development system to screen compounds for their capability to prevent cancer metastasis in vitro and in vivo
The current criteria for screening and identifying a cancer drug for further development do not fit clinical reality. Traditional in vitro and in vivo screening for cancer drugs has been depending on, for decades, its potency to inhibit proliferation of cancer cell lines and growth of tumors implanted in the immune-deficient nude mice. However, in reality, when patients are diagnosed as a cancer, they will ask for a surgery or radiotherapy to remove or kill the primary tumor as early and quickly as possible to prevent any potential metastases. Patients die of cancer metastases, not of the primary tumor. And no one wants to risk the cytotoxic chemotherapy to see if his tumor will be shrunk by the hardly-tolerable chemotherapy drugs that themselves may have potential of teratogenicity, carcinogenicity and mutagenicity.
The chemotherapy drugs used today in clinics are inherited from the thinking and designs made four decades ago, that is to kill and /or inhibit rapidly-dividing cancer cells. The potency of those chemotherapy agents on tumor cell lines is well demonstrated in the in vitro setting. However, these drugs cannot specifically target cancer cells in the body. Due to their non-specific effects on normal cells and organs, these drugs cause toxicity and deteriorate the quality of patient’s life, weaken the host immunosurveillance system, and may result in an irreversible damage to human’s own recovery power. Many clinical trials have proven that chemotherapy drugs cannot prevent primary cancer from metastasis. At 2009 American Society of Clinical Oncology (ASCO) conference, clinical oncologists demonstrated again that early chemo (including the anti-angiogenesis Bevacizumab, or Avastin) failed to prevent cancer recurrence. In addition, common carcinomas (lung, colon) are generally refractory to current chemotherapy.
Sadly, metastasis research seems to be ignored by the majority of cancer researchers. Of nearly 8,900 NCI grant proposals awarded in 2003, 92% did not even mention the word “metastasis”. No one has been focusing on development of a therapy to prevent cancer metastases from occurring after a cancer surgery or radiotherapy. With today’s understanding of cancer genome, metastasis, and circulating tumor cells (CTCs), it is the time to develop various cancer metastasis alert and prevention strategies.
1.1. CTC enrichment
We will establish a system that can be used to enrich and detect CTCs (Ficoll-paque, ISET, MEMS and cellular immunochemistry assays, and/ or antibodies such as A45-B/B3, AE1/AE3 and others) in order to determine the effectiveness of the potential metastasis-preventing agents.There are many approaches to enriching or sorting CTCs from peripheral blood, including flow cytometrythat sorts cells by size and surface antigen expression; high-throughput optical-imaging systems and fiber optic array scanningthat rely on Fiber-Optic Array Scanning Technology (FAST) that identify CTCs based on fluorescent labels; microchips designed to capture CTCs as blood flows past EpCAM-coated microposts; filters with pore size designed to retain CTCs but permits smaller cells to pass; immunomagnetic-bead purification and immunomagnetic cell separator; negative enrichment that eliminates all cells from blood samples, except CTCs. This enrichment technique requires expression of EpCAM by CTCs. An appropriate method will be selected from the above, and developed and validated for future use.
1.2. Enhance activity of metastatic suppression genes and proteins
We will screen and test old and new molecules that can enhance activity of metastatic suppression genes and proteins. Although many metastatic suppression genes and proteins are identified, we will focus on colon-cancer related genes and proteins to test the proof-of- concept. These molecules may be those that were previously abandoned due to their lack of cytotoxic activity.
1.3. Keep disseminating tumor cells in bone marrow under dormancy
We will screen and test molecules that can maintain or enhance bone marrow’s capacity to keep disseminating tumor cells (DTCs) under dormancy. BM is the site of blood cell formation and a common homing organ for dormant tumor cells. The mechanism of delicate balance between BM and DTCs has not been understood. The possibility to scavenge the DTCs within BM or to keep DTCs dormant is a new area to exploit.
1.4. Drugs that enhance host’s immunosurveillance
We will screen and test drugs for their ability to enhance host’s immunosurveillance, a very important force in fighting cancer metastasis but is ignored by most of cancer drug developers. In the 2009 annual ASCO, clinicians showed treatment vaccines that boost the immune system’s response to tumors had benefits for patients with various types of cancer.
1.5. Drugs that inhibit CTCs adhesion and intravasation
We will screen and test molecules that can inhibit CTC adhesion and intravasation to blood and lymphatic vessel walls. This is a new idea towards metastasis prevention.
1.6. Drugs that inhibit tumor cell motility and metastasis
We will screen and test molecules that inhibit tumor cell motility and metastasis (KGF receptor, tyrosine kinase inhibitors). We will screen and test molecules that inhibit the transition from disseminated occult tumor cells to dormant micrometastasis.
1.7. Metastasis models
There are several mouse tumor models to be selected, including transplantable murine tumors grown in syngeneic hosts, xenografts of human tumors growth in immunodeficient murine (NCR-nu/nu; SCID; RH-Foxn1rnu), genetically engineered mouse (GEM), and orthotopic metastatic models of human tumors (OMMHU). I will establish and validate new models that should mimic patient-like orthotopic metastasis more closely to screen and test anti-metastasis drugs. Mice will also be treated with candidate drugs starting before (preventive protocols) or after (treatment protocols) administration of test drugs. Intravenous injection with appropriate cancer cells that express green fluorescent protein and luciferase may apply.
2. Nanotechnologyfor cancer metastasis prevention
The study has two goals: 1. to re-engineer nanomaterials to capture the circulating tumor cells (CTCs); 2. specifically deliver nanomaterials conjugated with targeting ligands and drugs to tumors. To achieve the goals, a nanomaterial must be coated with a targeting ligand and a drug.
2.1. Coating strategy for polymers
We are interested in re-engineering polymers and dendrimers with therapeutic drugs. Different kinds of dendrimers (1- 15 nm) are commercially available (e.g., polyamidoamine, PAMAM; Superfect; poly propylene imine, PPI). Dendrimers are of particular interest because these highly branched polymeric nano-carriers are globular in shape, monodispersive in nature, and have unique organization and highly-controlled architecture to allow them carry many molecules (high payload) such as targeting agents, therapeutics, imaging contrast agents and reporter molecules (to detect if an anticancer drug is working). Self-organization, or self-assembly (or bottom-up) is an approach to re-engineering dendrimers with targeting ligands and drugs, or imaging agents. Central to many of these approaches is the use of amphiphilic substances, which have hydrophobic and hydrophilic regions that cause them to spontaneously form nanostructures. Drugs, targeting ligands, or imaging agents can be conjugated to dendrimers by encapsulating drugs within the dendritic structure, or by attaching drugs to their terminal functional groups via electrostatic or covalent bonds (e.g., an amide bond or by ester bonds using either L-lactic acid or diethylene glycol as a linker). The covalent bonding results in a dendrimer-drug conjugate that is controlled by hydrolysis, while a linker can achieve controlled drug release but formulation of the delivery system is not easy.
2.2. Coating strategy for nanocarbon
The surface of carbon nanotubes (CNTs) is highly hydrophobic. For biomedical utilization, CNTs’ surface needs to be modified either covalently or noncovalently: covalent functionalization forms bonds on CNTs sidewalls, whereas noncovalent functionalization build interactions between the hydrophobic domain of an amphiphilic molecule and the CNT surface to afford aqueous CNTs wrapped by surfactant. Noncovalent functionalization of CNTs by coating their surface with amphiphilic surfactants or polymers essentially preserves the physical properties (e.g., π-network of CNTs) of CNTs. We will pursue non-covalent functionalization coatingto make CNTs 1) biocompatible and nontoxic; 2) sufficiently stable to resist detachment from the nanotubes surface in serum; 3) having functional groups for conjugation with antibodies or other molecules. Aromatic molecules such as pyrene can bind to the polyaromatic graphitic surface of the CNTs via π-π interaction. Therefore, amine-reactive pyrene derivative, or glycodendrimers-conjugated pyrene can be anchored on to the CNT surface via π-π stacking. We will select various amphiphiles to suspend CNTs in aqueous solutions with hydrophobic domains attached to the CNTs surface via van der Waals forces and hydrophobic effects, and polar heads for water solubility. Surfactants such as Tween-20, pluronic tri-block, sodium dodecyl sulfate (SDS), TritonX-100 copolymer may be tried to noncovalently functionalize nanotubes surfaces to reduce the non-specific binding of proteins and solubilize CNTs in water. The amounts of surfactants will be carefully calculated and tested to stabilize the CNTs while avoiding to lyse cell membranes and denature proteins.
2.3.Drugs to be conjugated
Drugs to be conjugated to the nanomaterials will be selected based on the drug’s physico-chemical properties that best fit to the nanomaterials to be used. In addition, the drug selection will also be based on our disease focus and degree of our understanding of the disease. At present, we may try those drugs without the IP issue one by one: alkylating agents nitrosoureas (Lomustine, CCNU; or methyl-CCNU); antimetabolites pyrimidine analog 5-fluorouracil; folic acid analog methotrexate; cisplatin or carboplatin, prednisone and diethylstilbestrol.
2.4. Targeting ligands to be conjugated
Since CTCs are characterized by their signatures such as Ki-67, CD44+/ CD24-, EpCAM, HER2, Twist, we will coat dendrimers, nanocarbon, or even gold-nanoparticles with chemically functional antibodies against these signatures along with drugs mentioned above to harness CTCs. Active targeting for early cancer metastasis involves conjugating targeting molecules to the surface of nanocarriers. Examples of targeting molecules include antibodies, ligands, peptides (Arginine-Glycine-Aspartate triad, RGD) nucleic acids, and other molecules that bind directly to a receptor overexpressed on a tumor-cell surface such as alphaV beta3 integrin (up-regulated in both invasive cancer cells and angiogenic endothelial cells). Aptamers, or small fragments of RNA or DNA, have been used as a targeting molecule due to their small size and lack of immune response. Aptamers fold into shapes that induce high binding specificity to their target molecules. We will also try to conjugate Aptamers to the nanomaterials.
2.5. Synthesis and purification of conjugates
After mixture and stirring reaction of conjugation for 1-2 days at (usually) room temperature, the conjugates are subjected to purification by dialysis or membrane filtration to get rid of salts and un-conjugated molecules, or by size exclusion (gel filtration) chromatography. The products will be dried by using anhydrous Na2SO4, or under vacuum.
2.6. Physico-chemical stability and biostability
Biostability tests will be conducted after mixing the conjugates with buffers at different pH: 0.02 M phosphate buffer for pH 7.4, 0.06 M HCl for pH1.2, and 0.05 M borate buffer for pH 8.5. At appropriate intervals, samples will be withdrawn and quenched by adding appropriate quenching solvent, and samples are quantitatively analyzed by using the following methods (4.7.) to determine degradation. Biostability of conjugates will be tested in human serum at 370C and at appropriate concentrations. Serum proteins will be precipitated by using methanol, or perchloric acid, or separated from the conjugates by using a solid-phase extraction column plate at different time points to determine the amount of entire conjugates by using appropriate analytical methods on a case-by-case basis. For comparison, identical amounts of test drugs and test target ligands may be quantitatively determined at the same time under the same conditions.
2.7. Characterization of conjugates
Conjugates can be characterized by using infrared spectroscopy, 13C and 1H NMR spectroscopy, HPLC analysis (retention time and peak area of a conjugate), transmission electron microscopy and atomic force microscopy. Size and zeta potential of conjugates will be measured by a ZetaPALS instrument.
2.8. CTC enrichment and binding
The CTCs can be enriched and sorted from cancer patients following appropriate IRB approval. Demonstration of the specific binding and deactivating CTCs could open a new opportunity for novel cancer treatment.
2.9. Nano-drugs specifically targeting at αVβ3-integrin or MMPs on tumors
αVβ3-integrin is the overexpressed receptor on a tumor-cell surface of both invasive cancer cells and angiogenic endothelial cells. Matrix metalloproteinases (MMPs) are extracellular proteolytic, zinc-dependent proteinases involved in tumor progression, angiogenesis, and metastasis. MMPs are capable of degrading most of the multiple components of the extracellular matrix (ECM) to facilitate growth, migration, and invasion of tumor cells. In the tumor microenvironment, host and tumor derived MMPs are often misregulated leading to uncontrolled degradation of the ECM. Of the 24 identified human MMP gene products, MMP7 is notably produced by cells of epithelial origin and contributes to tumor formation in a number of epithelial-derived adenocarcinomas. αVβ3-integrin, MMP7 (or MMP2 and MMP9) are interesting targets that provide the necessary starting point for testing my hypothesis that nano-drugs derived from nanoparticles, nanoparticle-based conjugates, or polymer-based nano-substrates may specifically inhibit tumor growth in vivo by inhibiting αVβ3-integrin or MMPs.
EXPECTED RESULTS AND IMPACTS Conjugation reaction may be easier to conduct than the stabilization and optimization reactions after conjugates are formed. We will try different conjugation reactions, and select 1-2 successful reactions, and repeat the reactions for reproducibility. If successful, we will optimize the reaction conditions, test physico-chemical stability and biostability, and further determine the % binding between the drug conjugates and selected CTCs after obtaining CTCs. If the CTCs can be specifically captured in vitro, this research will open a door to the next in vivo study. The entire research plans and lessons we learn from the studies will lead us to manipulating CTCs and preventing cancer metastasis.
3. natural products as a source of Cancer metastasis prevention agents
To characterize natural products (as an extract or isolated compound) as an alternative cancer treatment, identify medical herbs, extract active ingredients from a medical herb, and test them. Currently, we are working on two Traditional Chinese Medicine Plants, i.e., CMAPC-Tu and CMAPC-Jiu for the purposes.
