# Patent

## Title
Method for identifying agents that inhibit cell migration, promote cell adhesion and prevent metastasis

## Abstract

Disclosed are methods for identification of agents that modulate cell attachment, cell migration and cell viability. Cancer and primary cells adhered to a matrix are treated with agent(s) that modulate ActRII signaling and cell adhesion. Agents are tested that modulate cell adhesion, detachment, invasion and viability. Agents that modulate the expression, phosphorylation, function and translocation of ActRII signaling pathway members also can predict agents that modulate cell adhesion, detachment, invasion and viability. The methods have utility in tissue engineering as well as identifying agents that prevent cancer cell metastasis, wound dehiscence, aortic dissection and aid retina attachment and skin replacement and fertility. Examples of tissue engineering include techniques that allow for the generation of biological substitutes that restore, maintain, or improve tissue function or a whole organ.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a Continuation in Part of U.S. patent application Ser. No. 13/709,685, now U.S. Pat. No. 8,716,466, filed Dec. 10, 2010, which is a divisional of U.S. patent application Ser. No. 13/366,692, now U.S. Pat. No. 8,329,891, filed Feb. 6, 2012, which is a divisional of Ser. No. 12/709,897, now U.S. Pat. No. 8,110,355, filed Feb. 22, 2010, which claims the benefit of provisional U.S. Patent Application filed Feb. 20, 2009, having a Ser. No. 61/154,251, the disclosures of which are hereby incorporated by reference in their entireties.


FIELD OF THE INVENTION
The invention relates to an assay that can identify agents that inhibit cell migration, promote cell adhesion and prevent metastasis.
BACKGROUND OF THE INVENTION
Metastasis of cells from a primary tumor in mammals leads to the spread of cancer to other tissues of the body (1-3). The spread of metastases may occur via the blood or the lymphatics or through both routes. Cancer cell metastasis commonly occur in lungs, liver, brain, and the bones. These secondary aggressive cancers lead to organ dysfunction and ultimately death (Metastatic Cancer: Questions and Answers”. National Cancer Institute). To date, there are few effective treatments for metastatic cancers.
Signaling pathways that promote cell detachment and/or metastasis are largely unknown. However, we have discovered signaling pathways that unexpectedly regulate cell adhesion to the extra-cellular matrix (ECM) are prime candidates for promoting metastasis (4). These pathways may be impacted upon by the many genetic and epigenetic changes as a cell transitions to malignancy, together with microenvironmental changes such as changes in hormone signaling.
Wound dehiscence is the premature “bursting” open of a wound along surgical suture. It is a surgical complication that results from poor wound healing. Risk factors are age, diabetes, obesity, poor knotting/grabbing of stitches and trauma to the wound after surgery. Agents that promote cell adhesion could speed healing. Retinal detachment is a disorder of the eye in which the retina peels away from its underlying layer of support tissue. Initial detachment may be localized, but without rapid treatment the entire retina may detach, leading to vision loss and blindness. Agents that promote cell adhesion could speed healing. Aortic dissection is a tear in the wall of the aorta that causes blood to flow between the layers of the wall of the aorta and force the layers apart. Aortic dissection is a medical emergency and can quickly lead to death, even with optimal treatment. If the dissection tears the aorta completely open (through all three layers), massive and rapid blood loss occurs. Agents that promote cell adhesion could speed healing. Major skin trauma (e.g. burns, amputation) can be treated with temporary, artificial or autologous skin replacement. Agents that promote cell adhesion could speed healing. Blastocyst attachment to the endometrium of the uterine wall is essential for pregnancy and is a cause of infertility. Agents that promote blastocyst attachment could prevent infertility. Tissue engineering is a technique that allows for the generation of biological substitutes that restore, maintain, or improve tissue function or a whole organ. This technology includes the use of stem cells grown in the presence of scaffolds and growth factors for the generation of new tissues for transplantation into recipients. Examples of this technology include but are not limited to the development of bioartificial windpipes for implantation into recipients, cartilage for repair of knee, skin for the repair of wounds, and other bioartificial organs for the repair of blood vessels, bladders, pancreas, foreskin, penis, bone, liver, and heart. Agents that promote stem cell attachment could speed the generation and function of these tissues for transplantation, as well as promote their attachment and integration into the organ or body that they are transplanted into. Agents that promote stem cell attachment also would enhance 3-D printing of stem cells for the creation of tissues. Edible artificial animal muscle tissue can be cultured in vitro as a meat source. Agents that enhance muscle and fat cell attachment to collagen or other matrices, or allow solid meat formation, would speed the production and improve the texture of in vitro meat. Psoriasis, eczema and dandruff are skin conditions that result in the shedding of skin cells and inflammation. Agents that prevent skin detachment and inflammation would prevent psoriasis, eczema and dandruff. Unfortunately, signaling pathways that regulate cell adhesion are largely unknown.
We have identified a pathway, the activin receptor type II (ActRII) signaling pathway, that regulates cell adhesion. Blocking ActRII signaling alters cellular morphology and increases cell detachment. Cell detachment correlates with an increase in the expression of ADAM-15, a disintegrin which cleaves integrin molecules (5) and cadherin (6). In this context, it has recently been demonstrated that the expression of ADAM-15 is strongly correlated with the metastatic potential of prostate, breast (7) and pancreatic cancers (8) and is highly up-regulated in aggressive prostate cancer (6). Furthermore, ADAM-15 has been shown to be involved in cell migration and invasion (9, 10). Signal transduction components of the activin signaling pathway are highly down-regulated in prostate cancer (11, 12). In vitro, inhibition of ADAM15 expression in PC-3 cells decreases cell migration and adhesion to specific extracellular matrix proteins, and is accompanied by a reduction in the cleavage of N-cadherin by ADAM15 at the cell surface (6). In patients with bone metastasis from prostate cancer, circulating levels of activin A are significantly higher (13). These findings indicate that ActRII mediates cell adhesion (and viability) via the regulation of ADAM-15 expression and function.
Modulation of ActRII signaling and associated disintegrins can be used to identify drugs that enhance ActRII signaling and promote cell attachment, or inhibit the expression and/or function of disintegrins associated with promoting cell detachment. Accordingly, it is desirable to provide a method, kit, and apparatus for utilizing ActRII signaling and associated disintegrins to identify compounds and conditions capable of modulating adhesion characteristics of cell.
SUMMARY OF THE INVENTION
The foregoing needs are met, to a great extent, by the present invention, wherein in one respect methods and kits for identifying agents that inhibit cell detachment and increase cell attachment, and to the use of such agents to develop methods of preventing, treating or alleviating and/or the symptoms of cancer, and other diseases and conditions is provided. Some examples of such diseases and conditions and other applications are described herein. More specifically, the present invention is directed to the identification of agents that could be used to treat cancer, or other conditions and diseases and other applications described herein where cell detachment or migration is detrimental, or where cell adhesion is beneficial. The methods may include detecting, either directly or indirectly, agents that increase ActRII signaling, and/or decrease disintegrin or metalloprotease expression and/or function, and prevent cell detachment and cell invasion. Conversely, methods may include detecting, either directly or indirectly, agents that decrease ActRII signaling, and/or increase disintegrin or metalloprotease expression and/or function, and increase cell attachment and cell invasion.
An embodiment of the present invention pertains to a method of identifying an agent to modify cell adhesion. In this method, cells from an animal are adhered to a matrix, cell detachment from a matrix is induced by suppressing ActRII signaling, the agent is introduced, and a change in cell detachment as a result of introducing the agent is measured.
Another embodiment of the present invention relates to a kit to identify an agent to modify cell adhesion. The kit includes a cell growth media, a container having a surface matrix for cell adhesion, and a cell detachment solution having a concentration of an ActRII signaling suppressor sufficient to induce cell detachment.
Yet another embodiment of the present invention pertains to a composition to induce cell detachment comprising a concentration of an ActRII signaling suppressor sufficient to induce cell detachment.
There has thus been outlined, rather broadly, certain embodiments of the invention in order that the detailed description thereof herein may be better understood, and in order that the present contribution to the art may be better appreciated. There are, of course, additional embodiments of the invention that will be described below and which will form the subject matter of the claims appended hereto.
In this respect, before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of embodiments in addition to those described and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein, as well as the abstract, are for the purpose of description and should not be regarded as limiting.
As such, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the ActRII signaling pathway linked to the expression and function of the disintegrin ADAM15.
FIGS. 2A-2F illustrates that ActRII blocking antibody suppression of ActRII signaling promotes morphological changes, increased detachment and decreased viability of prostate cancer cells.
FIGS. 3A-3C illustrates that anti-ActRII oligonucleotide (antisense-P) suppression of ActRII signaling promotes increased ADAM15 expression, increased detachment and decreased viability of prostate cancer cells.
FIGS. 4A-4D illustrates that suppressing ActRII signaling promotes morphological changes, increased detachment and decreased viability of prostate cancer cells.
FIG. 5 illustrates antisense oligonucleotide suppression of ActRII along with ADAM-15 suppression to prevent cell detachment.
FIG. 6 illustrates a linear increase in absorbance with increasing cell number.
FIG. 7A illustrates via cell density micrographs that conventional antisense does not induce detachment of PC-3 cancer cells.
FIG. 7B illustrates via % cell attachment that conventional antisense does not induce detachment of PC-3 cancer cells.
FIG. 8A illustrates via cell density micrographs that ActRIIB antisense-P does induce detachment of PC-3 cancer cells.
FIG. 8B illustrates via % cell attachment that ActRIIB antisense-P does induce detachment of PC-3 cancer cells.
FIG. 9A illustrates via cell density micrographs that ActRIIB siRNA does induce detachment of PC-3 cancer cells.
FIG. 9B illustrates via % cell attachment that ActRIIB siRNA does induce detachment of PC-3 cancer cells.
FIG. 10A illustrates via cell density micrographs that ActRIIB Stealth siRNA does induce detachment of PC-3 cancer cells.
FIG. 10B illustrates via % cell attachment that ActRIIB Stealth siRNA does induce detachment of PC-3 cancer cells.
FIG. 11 illustrates via cell count that Activin A does increase human fibroblast cell proliferation.



DEFINITIONS
The present invention is described herein using several definitions, as set forth below and throughout the application.
As used in this specification and the claims, the singular forms “a,” “an,” and “the” include plural forms unless the content clearly dictates otherwise.
As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.
As used herein, the term “including” has the same meaning as the term “comprising.”
As used herein, the term “cancer cell line” or “cancer cells” may be used interchangeably and refers to cells isolated from a tumor, metastasis or abnormal growth derived from an animal or human. These cells typically, but not always, grow rapidly in culture when supplemented with appropriate growth factors, often fetal animal serum. This term also refers to normal cells transformed into cells that display typical features of cancer cells, i.e. they divide in cell culture under trophic support and/or form tumors when administered to animals.
Cancer cell lines may include, but are not limited to, human prostate cancer cells (PC-3, LNCaP, DU-145), human mammary epithelial cells (e.g. MCF-7, MCF-10A, MDA-MB-438, MDA-231, MDA-468, T47D, SkBr3), human neuronal cells (M17, SHSY5Y, H4, U87), human acute myeloid leukemia cells (THP-1), human bone cancer cells (Saos-2 cells), human melanoma cells (721), human glioblastoma cells (A172), human head and neck carcinoma cells (A253), human skin epithelial cells (A431), human lung carcinoma epithelial cells (A-549), human peripheral blood mononuclear cell lymphoma (BCP-1), human pancreatic adenocarcinoma cells (BxPC3), human squamous cell carcinoma (Cal-27), human CML acute phase T cell leukemia cells (CML T1), human CML blast crisis Ph+ CML cells (EM2), human CML blast crisis Ph+ CML cells (EM3), human metastatic lymph node melanoma cells (FM3), human lung cancer cells (H1299), human hybridoma cells (HB54), human fibroblasts (HCA2), human kidney embryonic epithelial cell (HEK-293), human cervical cancer epithelial cell (HeLa), human myeloblast blood cells (HL-60), human mammary epithelial cells (HMEC), human colon epithelial adenocarcinoma cells (HT-29), human umbilical cord vein endothelial cells (HUVEC), human T-cell-leukemia white blood cells (Jurkat), human lymphoblastoid EBV immortalized B cells (JY cells), human lymphoblastoid CML blast crisis cells (K562 cells), human lymphoblastoid erythroleukemia cells (Ku812), human lymphoblastoid CML cells (KCL22), human lymphoblastoid AML cells (KG1), human lymphoblastoid CML cells (KY01), human melanoma cells (Ma-Mel 1, 2, 3 through 48), human WBC myeloid metaplasic AML cells (MONO-MAC 6), human T cell leukemia (Peer), human osteosarcoma cells (Saos-2), human T cell leukemia/B cell line hybridoma (T2), human colorectal carcinoma/lung metastasis epithelium cells (T84), human colorectal adenocarcinoma cells (HCT-15, HT-29), human monocyte AML cells (THP1), human glioblastoma-astrocytoma epithelial cells (U373), human glioblastoma-astrocytoma epithelial-like cells (U87), human leukemic monocytic lymphoma cells (U937), human lymphoblastoid cells (WT-49), human B-cell EBV transformed cells (YAR), human breast adenocarcinoma cells (NC1/ADR-RES, MDA-MB-231), human CNS glioblastoma cells (SF-268), human ovary adenocarcinoma cells (SK-OV-3), human lung carcinoma cells (NCIH460), human lung adenocarcinoma cells (A549), human liver carcinoma cells (Hep3B), human uterine sarcoma-drug sensitive cells (MES-SA), human uterine sarcoma-drug resistant cells (MES-SA/DX5), human skin primary melanoma cells (WM39), ape-kidney fibroblast cells (COS-7), African green monkey kidney epithelial cells (Vero cells), murine brain/cerebral cortex endothelial cells (bEnd.3), murine embryonic mesenchymal cells (C3H-10T1/2), murine T cell leukemia ECACC cells (EL4), murine embryonic fibroblasts (NIH-3T3), murine embryonic calvarial cells (MC3T3), murine hepatoma epithelial cell (Hepal cic7), murine adenocarcinoma cells (MC-38), murine epithelial cells (MTD-1A), murine endothelial cells (MyEnd), murine renal carcinoma cells (RenCa), murine melanoma cells (X63), murine lymphoma cells (YAC-1), murine T cell tumor cells (RMA/RMAS), murine breast adenocarcinoma cells (4T1), murine mammary normal epithelial cells (NmuMG), rat glioblastoma cells (9L), rat neuroblastoma cells (B35), canine mammary tumor cells (CMT), canine osteosarcoma cells (D17), canine histiocytosismonocyte/macrophages (DH82), rat pheochromocytoma cells (PC-12), rat pituitary tumor (GH3), canine kidney epithelial cells (MDCK II), murine B lymphoma B cells (lymphocyte A20), murine bone marrow stromal cells (ALC), murine melanoma cells (B16), murine colorectal carcinoma cells (CT26), baby hamster kidney fibroblasts (BHK-21), Asian tiger mosquito larval tissue (C6/36), insect-ovary cells (Sf-9), Chinese hamster ovary cells (CHO), onyvax prostate cancer cells (OPCN, OPCT), tobacco cells (BY-2), zebrafish cells (ZF4 and AB9), Madin-Darby Canine Kidney (MDCK) epithelial cells, Xenopus kidney epithelial cells (A6).
As used herein, the term “primary cells” refers to cells isolated from tissues of animals.
Primary cells include, but are not limited to, keratinizing epithelial cells, wet stratified barrier epithelial cells, exocrine secretory epithelial cells, holinone secreting cells, metabolism and storage cells, barrier function cells of lung, gut, exocrine glands and urogenital tract, kidney cells, epithelial cells lining closed internal body cavities, ciliated cells, extracellular matrix secretion cells, blood and immune system cells, cells of the nervous system, autonomic neuron cells, sense organ and peripheral neuron supporting cells, central nervous system neurons and glial cells, lens cells, pigment cells, germ cells, nurse cells, interstitial cells, stem cells and other cells (see Appendix 1 for list of cells).
A “cell array” refers to a substrate comprising a plurality of cancer or primary cell lines.
The present methods and kits may utilize cell arrays to test agents for cell adhesion, cell invasion and cell viability.
The term “oligonucleotide” or “siRNA” refers to a nucleic acid sequence or fragments or portions thereof, which may be single or double stranded, and represents the sense or antisense strand. A oligonucleotide may include DNA or RNA, and may be of natural or synthetic origin. For example, a oligonucleotide or siRNA may include cDNA or mRNA. Oligonucleotides may include nucleic acid that has been amplified (e.g., using polymerase chain reaction). The oligonucleotide may contain phosphorothioate bonds.
The term “oligonucleotide” is understood to be a molecule that has a sequence of bases on a backbone comprised mainly of identical monomer units at defined intervals. The bases are arranged on the backbone in such a way that they can enter into a bond with a nucleic acid having a sequence of bases that are complementary to the bases of the oligonucleotide. The most common oligonucleotides have a backbone of sugar phosphate units. Oligonucleotides of the method which function as antisense oligonucleotides are generally at least about 10-15 nucleotides long and more preferably at least about 15 to 25 nucleotides long, although shorter or longer oligonucleotides may be used in the method. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. An oligonucleotide (e.g., antisense sequence) that is specific for a target nucleic acid will “hybridize” to the target nucleic acid under suitable conditions. As used herein, “hybridization” or “hybridizing” refers to the process by which an oligonucleotide single strand anneals with a complementary strand through base pairing under defined hybridization conditions. Oligonucleotides used as antisense oligonucleotides for specifically inactivating gene transcription or translation are capable of specifically hybridizing to the target gene.
The present methods and kits may utilize oligonucleotides. The term “antisense” and “sense” are complimentary to “antisense oligonucleotide” and “sense oligonucleotide”, respectively, and refers to an oligonucleotide that hybridizes to a target nucleic acid and is capable of specifically hybridizing to the target nucleic acid. Antisense or sense may be fully complementary to a target nucleic acid sequence or partially complementary. The level of complementarity will depend on many factors based, in general, on the function of the antisense or sense oligonucleotide. An antisense oligonucleotide can be used, for example to prevent gene transcription and translation for the formation of proteins. A sense oligonucleotide does not prevent gene transcription and translation for the foil-nation of proteins. Antisense and sense oligonucleotides can be labeled or unlabeled, or modified in any of a number of ways well known in the art. An antisense and sense oligonucleotides may specifically hybridize to a target nucleic acid.
As used herein, a “target nucleic acid” refers to a nucleic acid molecule containing a sequence that has at least partial complementarity with a target DNA/RNA sequence. A target DNA/RNA may specifically hybridize to a target nucleic acid.
As used herein, an “inhibitor” refers to a molecule that blocks or decreases the function, phosphorylation or translocation of a protein or the binding of a protein to another.
An “amino acid sequence” refers to a polypeptide or protein sequence.
The term “blocking antibody” refers to an antibody that blocks normal signaling by that protein, usually by binding to a particular amino acid sequence of that protein.
As used herein, the term “agent,” which may be used interchangeably with the terms “chemical”, “inhibitor” or “drug,” refers to any compound that can be applied to cells.
As used herein, the term “chemical library” means a group of distinct drugs and drug classes maintained as a group that are tested individually or in combination for their ability to affect a cellular change, e.g. cell adhesion.
As used herein, the term “assay” or “assaying” means qualitative or quantitative analysis or testing.
As used herein, the term “cell viability” refers to whether a cell is living or dead. Cell viability measurements may be used to evaluate the death or life of cells. Cell viability tests can be used to determine the effectiveness of agents to promote life, or induce death.
As used herein, the term “cell invasion” and “migrate” refers to the ability of a cell to move from its starting position to a new place, under the influence of chemotaxic agents or not.
As used herein, the term “cell attachment” and “cell adhesion” refers to cell binding to a substrate, including but not limited to plastic and other substrates referred to in [0049] and the like.
As used herein, the term “ADAM-15 expression and function” refers to the concentration of ADAM-15 protein, and the activity of that protein to act as a metalloproteinase to cleave ECM proteins. More particularly, the term ADAM-15 refers to a disintegrin and metalloproteinase domain-containing protein 15.
As used herein, the term “ActRI, Smad-2 and Smad-4 expression and phosphorylation” refers to the concentrations of ActRI, Smad-2 and Smad-4, and whether ActRI, Smad-2 and Smad-4 are phosphorylated.
As used herein, the term “ActRII binding to ALKs” refers to the binding of one member of the ActRII family of proteins to another member of the ALK family of proteins.
As used herein, the term “Smad-2:Smad-4 coupling” refers to the binding of Smad2 to Smad-4 as a complex.
DETAILED DESCRIPTION
In some embodiments, the methods include: (a) using cancer cell lines and primary cells; (b) attaching cells to a matrix; (c) inducing detachment of cancer cells or primary cells by blocking the ActRII signaling pathway using oligonucleotides, oligonucleotides with phosphorothioate bonds (antisense-P phospho-oligonucleotides), siRNA, blocking antibodies, or inhibitors of protein function, binding or translocation; (d) using agents from chemical libraries or other agents; (e) measuring cell attachment; (f) measuring cell invasion; (g) measuring cell viability; (h) measuring the expression and activity of disintegrins, (i) measuring ActRII, activin receptor-like kinases (ALKs) 1-7, Smad-2, Smad-3 and Smad-4 expression and their phosphorylation; (j) measuring ActRII binding to ALKs; (k) measuring Smad-2:Smad-4 coupling and translocation to the nucleus. In one method, cancer cell lines are induced to detach from a culture or microwell plate and agents are tested to determine their ability to prevent detachment and cell invasion.
In a preferred embodiment, the determination of the ability of the agent to inhibit cell detachment is made by treating cultured cancer cells with antisense-P phosphooligonucleotides and/or siRNA and then treating the cells with chemical agents and measuring cell detachment, invasion, morphology and viability.
In some embodiments, the methods may include using plastic culture plates, or plastic culture plates that are coated with different binding substrates, including but not limited to fibronectin, vitronectin, bovine serum albumin, gelatin, Matrigel, fibrous matrix proteins (such as collagen I, collagen IV), fibrinogen, non-collagenous components (such as laminin molecules; GTFALRGDNGDNGQ (SEQ ID NO 7)—portion of the laminin alpha-chain), proteoglycans (such as chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate and keratan sulfate, syndecans, perlecan), non-sulfated glycosaminoglycan (such as hyaluronan), entactin, core proteins (such as lumican, keratocan, mimecan, fibromodulin, PRELP, osteoadherin and aggrecan), dystroglycan glycoprotein complex and Lutheran blood group glycoprotein. Plates may consist of one or more wells (e.g. 6-, 12-, 24-, 48-, 96-, 384-well) for high throughput screening of agents.
In some embodiments, the methods may include using any cancer cell line that can adhere to the surface of the plate.
In some embodiments, the methods may include using any primary cells from animals, or human cells from biopsies, that can adhere to the surface of the plate.
In some embodiments, the methods may include using, but are not limited to oligonucleotides, oligonucleotides with phosphorothioate bonds, RNA interference (RNAi) or inhibitors for decreasing the expression or function of the activin signaling pathway including ActRIIA and B, activin receptor-like kinases (ALKs 1-7), Smad-2, Smad-3, Smad-4, activins (A, B, C, D and E). In another embodiment, the methods may include using blocking antibodies against members of the activin signaling pathway including ActRII, ActRI, Smad-2, Smad-4, activins (A, B, C, D and E), for decreasing protein function. In another embodiment, the methods may include using oligonucleotides, oligonucleotides with phosphorothioate bonds, RNAi or inhibitors to decrease phosphorylation of activin signaling pathway members. In another embodiment, the methods may include using oligonucleotides, oligonucleotides with phosphorothioate bonds, RNAi or inhibitors to inhibit ActRIIA and/or ActRIIB binding to ALKs 1-7. In another embodiment, the methods may include using oligonucleotides, oligonucleotides with phosphorothioate bonds, RNAi or inhibitors to decrease Smad-2:Smad-4 translocation to the nucleus. In another embodiment, the methods may include natural cellular inhibitors of the signal transduction pathway of ActRII signaling such as inhibin, follistatin, and myostatin.
The treatments may include single agents, combinations of agents or no agents. The treatments also may include solutions used to dissolve the agents.
Cell detachment and adhesion may be detected by any suitable method, which may include, but are not limited to, cell cytometry (e.g. trypan blue), fluorescent based cell detection assays (e.g. Calcein AM (Life Technologies, Inc.), and Mitotracker Red (Life Technologies, Inc.)), luminescent based detection assays (e.g. Cell-Titer glo (Promega, Inc.) and spectrophotometry based detection assays (e.g. crystal violet, MTS/MTT assays such as Promega CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay, Promega, Inc. and Chemicon Cell Adhesion Assays).
Cell invasion may be detected by any suitable method, which may include, but are not limited to, the scratch wound assay, cell invasion assays using fluorescent detection of cell invasion (e.g. activin, serum) (BD BioCoat matrigel invasion chambers, Fisher Scientific; EMD, Calbiochem; Chemicon International) with or without the use of a chemotaxic agent.
Cell viability may be detected by any suitable method, which may include, but are not limited to, cell cytometry (e.g. trypan blue), fluorescent based cell detection assays (e.g. calcein AM (Life Technologies, Inc.), and Mitotracker Red (Life Technologies, Inc.)), luminescent based detection assays (e.g. Cell-Titer glo (Promega, Inc.)) and spectrophotometry based detection assays (e.g. crystal violet, MTS/MTT assays such as Promega CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay, Promega, Inc.)
Cell morphology may be assessed by capturing images using a microscope and analyzing morphological changes using software such as MetaMorph (Molecular Devices, LLC).
The disclosed methods will determine which agents or combination of agents prevent cell detachment, prevent cell invasion, promote cell attachment, promote cell invasion, increase viability, decrease viability and alter morphology.
The disclosed methods may be utilized to identify agents that: 1) prevent metastasis or the other conditions/diseases such as those described herein; 2) detach cells from a matrix; and 3) promote cell adhesion to a matrix. In addition, the disclosed methods may be utilized to study cell surface markers during cell detachment and how agents affect the expression and activity of those markers. Also contemplated are kits for performing the disclosed methods. A kit may include one or more reagents for determining, either directly or indirectly, whether an agent(s) can prevent cell detachment, prevent cell invasion, promote cell attachment, promote cell invasion and alter morphology/viability.
The present methods may be performed to induce cell detachment from a matrix and to identify agents that prevent cell detachment. Cells are cultured to attain 80% confluence as known in the art. Typically cells are cultured under sterile conditions at 37° C., 5% CO2, in media supplemented with 1% penicillin-streptomycin, 2 mM glutamine, 0.4 mM sodium bicarbonate and 1-10% fetal bovine serum as determined by the cell type and as known in the art. Cells are then treated with antisense oligonucleotides with phosphorothioate bonds or RNAi to a final concentration of 0.01-10 μM. Oligonucleotides can be added directly, or added to media that has been preincubated with lipofectamine (4 ng/ml; Life Technologies, New York, N.Y.) for 5 min. at room temperature, and this mixture then incubated at room temperature for 20 min. prior to addition to cells. Cell attachment, invasion and viability are significantly altered by suppressing ActRII signaling via these treatments. Agent(s) are then added to cells at concentrations ranging from picomolar to millimolar concentrations. Fluids that the agent(s) are dissolved in are added to separate wells at the same concentration. Other control wells contain no agent(s). After 0-5 days of treatment, cell attachment, invasion, viability, and morphology are measured as described herein.
The ability of agents to prevent cell detachment is measured by the difference in cell attachment between those cells treated with and without agents. Alternatively, the percentage change in cell attachment by an agent can be measured by the level of cell attachment in the presence of the agent and oligonucleotides or blocking antibody divided by the difference between cells treated with and without oligonucleotides/or antibody alone as known in the art.
Cell detachment can be induced by, but is not limited to, the following oligonucleotides:









ActRIIA antisense-P:



SEQ ID NO 1



5′-TCCAGTTCAGAGTCCCATTTC-3′



 



ActRIIA sense:



SEQ ID NO 2



5′-GAAATGGGACTCTGAACTGGA-3′



 



ActRIIB antisense-P:



SEQ ID NO 3



5′-TCTCCCGTTCACTCTGCCAC-3′



 



ActRIIB sense:



SEQ ID NO 4



5′-GTGGCAGAGTGAACGGGAGA-3′



 



ADAM-15 Antisense-P:



SEQ ID NO 5



5′-CGCACTCTTCCCTGGTAGCA-3′



 



ADAM-15 Sense:



SEQ ID NO 6



5′-TGCTACCAGGGAAGAGTGCG-3′






Cell detachment can be induced by, but is not limited to, the following antibodies and inhibitors: Anti-human ActRIIB affinity-purified mouse monoclonal antibody (A0856; US Biological, Swampscott, Mass.); Anti-human mouse activin antibody (US Biologicals, MA); SB 431542 (GlaxoSmithKline). Any oligonucleotide or antibody that decreases signaling via the ActRII signaling pathway can be used to induce cell detachment.
The present methods may be performed to identify agents that promote cell attachment and prevent cell detachment from a matrix. Cells cultured as described herein at 50% confluence are treated with and without agents and after 0-5 days cell attachment, invasion and viability are measured in any suitable manner. Specific examples of suitable methods of measuring cell attachment, invasion and viability include thus methods described herein.
The present methods may be used for identifying agents that prevent cell detachment and promote cell adhesion. These agents may be used to treat any suitable metastasis and other diseases and conditions and applications. Specific examples of suitable metastasis and other diseases conditions and applications include those described.
In a particular embodiment of the invention, a kit for identifying agent that prevent ActRII-mediated cell detachment is provided. The kit may include any suitable reagents for performing various assays capable of identifying agent that prevent ActRII-mediated cell detachment. In the following list of reagents, the term ‘antisense-P’ is used to designate antisense with phosphorothioate bonds. In a particular example, the kit includes:

    
    
        1) sense and antisense-P oligonucleotides to ActRII
        2) lipofectamine
        3) 96-well plate (coated with or without a particular matrix for different cell types). Plate would be either visible, fluorescent of luminescent light compatible. Cells would be dependent upon each researchers requirements as would the media that they would put the lipofectamine/oligonucleotides into for treatment of cells.
        4) PBS buffer (for washing wells)
        5) cell detection reagents
    
    


In addition, depending upon the cell number detection method, the kit may include various additional reagents for performing particular assays such as, for example, fluorescent detection of cell number, luminescent detection of cell number, spectrophotometric detection of cell number, and the like. For fluorescent detection of cell number, the kit may include reagents for calcein AM (e.g., fluorescent compatible 96-well plate). For luminescent detection of cell number, the kit may include reagents for CellTiter-Glo® (e.g., luminescent compatible 96-well plate). For spectrophotometric detection of cell number, the kit may include reagents for crystal violet assay such as:

    
    
        1) PBS buffer (for washing wells)
        2) crystal violet solution (0.5%; staining cells)
        3) 33% (v/v) acetic acid (digesting cells)
        4) (visible light compatible 96-well plate)
    
    


To perform the spectrophotometric detection of cell number: Media is removed from cells cultured in 96-well plates, the cells washed with D-PBS (Gibco, Carlsbad, Calif., USA), and 50 μL of crystal violet added to each well at room temperature for 10 min. Following this, 200 uL of 33% (v/v) acetic acid is added to the wells, the plate shaken for 2 min. and then read at 570 nm on a plate reading spectrophotometer.
METHODS AND RESULTS
EXAMPLE 1
ActRII Oligonucleotide-Induced Detachment of Cancer Cells in a Microwell Format for the High Throughput Screening of Small Molecules to Inhibit Cell Detachment
Prostate cancer cell lines (e.g. PC-3 cells) are cultured in 96-well opaque plates (luminescence compatible) at 37° C. in 100 μL of F-12 Nutrient Mixture (Ham; Gibco, Life Technologies, New York, N.Y.) supplemented with 1% penicillin-streptomycin (P/S; Life Technologies), 2 mM glutamine (Life Technologies), 0.4 mM sodium bicarbonate (Sigma, St. Louis, Mo.), and 5% fetal bovine serum (FBS, #26400-036; Gibco, Life Technologies). When cells reach 80% confluence (at least 24 h after plating to allow suitable attachment), cells are treated every day for 1-3 days with:

    
    
        1) treatment medium+0.4 μM ActRIIB sense-P oligonucleotide (SEQ ID NO 4); or
        2) treatment medium+0.4 μM ActRIIB antisense-P oligonucleotide (SEQ ID NO 3); or
        3) treatment medium+0.4 μM ActRIIB antisense-P oligonucleotide (SEQ ID NO 3)+small molecule (0-10 μM).
        4) treatment medium+0.4 μM ActRIIB sense-P oligonucleotide (SEQ ID NO 4)+small molecule (0-10 μM).
    
    


The oligomers with phosphorothioate bonds (sense and antisense-P; Integrated DNA Technology, Coralville, Iowa) are added to media that has been preincubated with lipofectamine (4 ng/μl; Life Technologies, New York, N.Y.) for 5 min. at room temperature. This mixture is incubated at room temperature for 20 min. prior to addition to cells. Replicates=3-6. As a modification to the above protocol, antisense oligonucleotides can be replaced with a specific binding antibody to ActRII such as anti-human ActRIIB affinity-purified mouse monoclonal antibody (A0856; US Biological, Swampscott, Mass.).
The number of viable cells attached to the plate is determined using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega). Briefly, the CellTiter-Glo® Reagent is added directly to the wells of the plate (100 μL per well for 96-well plates) and the plate mixed on an orbital shaker for 2 min. Following this, the plate is incubated at room temperature for 10 min. and the luminescence recorded (typical integration time of 0.25-1 second per well) on a plate-reading luminometer. Note: Control wells are prepared containing medium without cells to obtain a value for background luminescence, and is subtracted from the values obtained for cells treated in 1, 2 and 3 above.
The % of cell detachment rescued by the small molecules is determined as the luminescence of (3−2)/(1−2)×100.
EXAMPLE 2
Antisense Oligonucleotide Suppression of ActRII Along with ADAM-15 Suppression Prevents Cell Detachment
To test that cell detachment induced by suppressing ActRII signaling was mediated via the metalloprotease ADAM-15, we treated PC-3 prostatic cancer cells with antisense-P against ActRII and then treated these cells with antisense-P against ADAM-15. A significant decrease in cell attachment was detected after 3 days of treatment with antisense-P against ActRII, and this was reversed by treatment with antisense-P against ADAM-15 (p<0.01, n=5). As shown in FIG. 5, neither sense against ActRII nor together with sense against ADAM-15 altered cell attachment. These results illustrate that cell detachment induced by suppressing ActRII signaling is mediated via the metalloprotease ADAM-15, and further indicate that antisense-P against ADAM-15 as a chemical that can prevent prostate cancer cell detachment.
Androgen-insensitive PC3 cells derived from a grade IV human prostate adenocarcinoma of epithelial origin (examples described herein) were maintained at 37° C. in F-12 Nutrient Mixture (Ham; Gibco, Life Technologies, New York, N.Y.) supplemented with 1% penicillin-streptomycin (P/S; Gibco, Life Technologies, New York, N.Y.), 2 mM glutamine (Life Technologies, New York, N.Y.), 0.4 mM sodium bicarbonate (Sigma, St. Louis, Mo.), and 5% fetal bovine serum (FBS, #26400-036; Gibco, Life Technologies, New York, N.Y.). For the experiment, PC-3 cells were plated in 6 well plates with 10% serum at 2×105 cells/well for 24 h, after which cells treated every day for 3 days with:

    
    
        1) media containing 10% serum+lipofectamine+sense-P oligonucleotide to ActRIIB (sActRIIB) (SEQ ID NO 4);
        2) media containing 10% serum+lipofectamine+antisense-P oligonucleotide to ActRIIB (aActRIIB) (SEQ ID NO 3);
        3) media containing 10% serum+lipofectamine+sense-P oligonucleotide to ActRIIB (sActRIIB) (SEQ ID NO 4)+sense-P oligonucleotides to ADAM-15 (SEQ ID NO 6)
        4) media containing 10% serum+lipofectamine+aActRIIB (SEQ ID NO 3)+antisense-P oligonucleotides to ADAM-15 (SEQ ID NO 5).
    
    


The oligomers with phosphorothioate bonds (Sense and antisense-P; Integrated DNA Technology, Coralville, Iowa) were added to media (240 μl) that had been preincubated with lipofectamine (4 ng/μl; Life Technologies, New York, N.Y.) for 5 min. at room temperature. This mixture was then incubated at room temperature for 20 min. prior to addition to cells. Antisense-P was used at a final concentration of 0.4 μM. The number of viable PC-3 cells attached to the plate was measured after trypsinization followed by cell counting using the trypan blue staining assay at the end of 72 h. Results are presented as cell attachment (% of sense control; mean±SEM, n=5). Statistical differences are denoted by an *p<0.01 as shown in FIG. 5. In addition to the methods and results presented herein, methods and results described in “Simon D, Vadakkadath Meethal S, Wilson A C, et al. Activin receptor signaling regulates prostatic epithelial cell adhesion and viability. Neoplasia 11:365-376” the disclosure of which is incorporated herein in its entirety.
EXAMPLE 3
Demonstration of Effectiveness of Antisense ActRIIB, siRNA, and Stealth siRNA in Comparison to Conventional ActRII Antisense for Inducing Cell Detachment
Cell Culture: PC-3 cells are androgen-insensitive cells derived from a grade 4 human prostate adenocarcinoma of epithelial origin (Kaighn, et al. 1978; Kaighn, et al. 1979). Cells were maintained at 37° C. in F-12 Nutrient Mixture (Ham; Gibco, Life Technologies, New York, N.Y.) supplemented with 1% penicillin-streptomycin with 2 mM glutamine (Gibco, Life Technologies, New York, N.Y.), 0.4 mM sodium bicarbonate (Sigma Chemical Co., St Louis, Mo.), and 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, Ga.). The cells were plated with 10% fetal bovine serum for 24 h prior to treatment for 72 h in serum-free medium containing respective oligonucleotides. For the crystal violet assay, cytology and cell counting, cells were cultured in sterile 48-well plates (Corning Inc. Corning, N.Y.).
For experiments using oligomers with phosphorothioate bonds (antisense-P; Integrated DNA Technology, Coralville, Iowa), oligomers of ActRII Antisense and Sense Oligonucleotides were added to the medium (total volume 400 μl) that had been preincubated with Lipofectamine 2000 (4 ng/μl; Life Technologies, New York, N.Y.) for 5 minutes at room temperature. This mixture was then incubated at room temperature for 20 minutes before adding to the cells. Antisense-P was used at a final concentration of 0.4 μM. Liu antisense and sense (Liu et al., 2000) were synthesized by Integrated DNA Technology (Coralville, Iowa) and used at a final concentration of 0.2-0.4 μM. See attached for sequence information.
For experiments using siRNAs, the siRNAs were generated by feeding the sequence of ActRIIA and ActRIIB into the algorithm supplied by Integrated DNA Technology. The sequences common to human, rat and mouse were synthesized (Integrated DNA Technology, Coralville, Iowa). siRNA against ActRIIA and ActRIIB, and the negative control oligonucleotide (DS NC1) were used at a final concentration of 10-320 nM. See attached for sequence information.
Stealth RNAi against ActRIIA (Cat# ACVR2AVHS41227), ActRIIB (Cat# ACVR2BVHS41230), negative control RNAi (Cat#12935300) and Lipofectamine@RNAiMAX (Cat#13778075) were purchased from Life Technologies (Grand Island, N.Y.). Incubation and treatment conditions were as described for antisense except Stealth ActRIIA/B and negative control RNAi were used at 50-100 nM final concentration. Experiments were performed in serum-free media without penicillin/streptomycin. See attached for sequence information.
Cell number was assessed using the trypan blue (Life Technologies, New York, N.Y.) staining technique. Briefly, the cells were incubated with trypsin (2.5% trypsin without phenol red (Cat #15090-046; Life Technologies, New York, N.Y.; (1 mL 2.5% trypsin/25 cm2 flasks or 50 μL trypsin/well for 48-well format) for one minute, cells were dislodged by tapping, the trypsin activity stopped by incubation with fetal bovine serum containing medium (generally 5 ml/25 cm2 flask; 100 μl/well) and cells then collected into Eppendorf tubes. Cells (50 μl) containing medium is then mixed with an equal volume of trypan blue, vortexed, and cell number counted using a hemocytometer.
Crystal violet assay is a standard assay used for quantitating the relative density of cells adhering to multi-well plates. Crystal violet physically binds the peptidoglycan layer of cell membranes which correlates with cell number. The amount of crystal violet taken up by a monolayer of cells after solubilization is quantified at 570 nm using a plate reader (SPECTRAmax M3, Molecular Devices, Sunnyvale, Calif.). Briefly, the culture medium was carefully removed and plates with a monolayer of cells were washed gently with PBS (0.5 ml/well; warmed to room temperature). After removing the PBS, 100 μl of crystal violet solution (crystal violet solution: 100 ml—crystal violet 0.2%, ethanol 2% and water 98 ml) was added gently to each well and incubated for 10 min. at room temperature. After 10 min., the plate was washed twice in tap water by gently immersing in a large beaker. The plate was then drained upside down on paper towels and warm SDS (1%; 300 μl/well) added to solubilize the cellular material and stain. The plate was then agitated for 5 min. on a shaker until the color was uniform. The plate was centrifuged at 4500 rpm for 2 min. and the absorbance measured at 570 nm in the plate reader. The average absorbance from each treatment was calculated and normalized to controls and expressed as a percentage of control.
The cells were examined cytographically using a Zeiss Axiovert inverted microscope connected to a Fluo Arc light source, and images were captured with an Axio CamMRC-5 camera using Axio Vision 4.0 software (Zeiss Axiophot, Thornwood, N.Y.).
To validate the reliability of the crystal violet assay as a method to assess the number of attached cells after oligonucleotide treatment, we plated increasing cell numbers (0, 20000, 40000, 80000, 100000, 140000 and 180000 cells/well) into 48-well plates in culture media containing FBS as described above. 24 h post-plating, wells were treated with media containing no FBS and cell number was measured using both the trypan blue and crystal violet assays, and then again 72 h later (96 h timepoint).
In determining the statistical analysis, the absorbance of blank wells was subtracted from the absorbance of treated wells. The absorbance of treated wells was then normalized to control untreated wells to determine percentage of cells attached. The average and standard error from the mean were then calculated and plotted (see figures). The statistical analysis were performed using R online statistical software program. A single-factor Analysis of Variance (ANOVA) test was used to reveal significant differences among treatment means, and Tukey's HSD multiple comparison procedure was used to test whether the treatments were significantly different from one another.
In FIG. 6, it is shown that there is a linear increase in absorbance with increasing cell number. PC-3 prostate cancer cells were plated into 48-well plates at increasing concentrations and incubated in serum-containing media for 24 or 96 h. Cell density was determined at these time points using the colorimetric crystal violet assay. A linear relationship was observed between the number of plated cells and absorbance (570 nm) at both 24 h and 72 h.
In FIG. 7, it is shown that conventional ActRII antisense disclosed by Liu et al., 200019 does not induce detachment of PC-3 cancer cells. PC-3 prostate cancer cells were plated in media containing 10% bovine serum albumin in 48-well plates (140,000 cells/well) for 24 h. Cells were then cultured in serum-free media containing 1) control sense oligonucleotides (0.2 or 0.4 μM); or 2) antisense oligonucleotides (0.2 or 0.4 μM). Cells were treated each day. After 72 h cell density was determined using the crystal violet assay. (A) Cell density, and (B) Cells attached (%), normalized to control sense oligonucleotides. Results are presented as a percentage change from the control treated cells (mean±SEM; n=4, ns).
The conventional ActRII antisense oligonucleotides include the following oligonucleotides:









ActRII antisense:



SEQ ID NO 8



5′-TGCAGCAGCTCCCAT-3′



 



ActRII sense:



SEQ ID NO 9



5′-ATGGGAGCTGCTGCA-3′






In FIG. 8, it is shown that ActRII antisense-P induces detachment of PC-3 cancer cells. PC-3 prostate cancer cells were plated in media containing 10% bovine serum albumin in 48-well plates (100,000 cells/well) for 24 h. Cells were then cultured in serum-free media containing 1) ActRIIB-P sense oligonucleotides (0.4 μM); or 2) ActRIIB antisense oligonucleotides (0.4 μM). After 72 h cell density was determined using the crystal violet assay. (A) Cell density, and (B) Cells attached (%), normalized to control sense oligonucleotides. Results are presented as a percentage change from the control treated cells (mean±SEM; n=6, P<0.05; different letters indicate significant differences between treatments).
Cell detachment can be induced by, but is not limited to, the following oligonucleotides:









ActRIIA antisense-P:



SEQ ID NO 1



5′-TCCAGTTCAGAGTCCCATTTC-3′



 



ActRIIA sense:



SEQ ID NO 2



5′-GAAATGGGACTCTGAACTGGA-3′



 



ActRIIB antisense-P:



SEQ ID NO 10



5′-TCTCCCGTTCACTCTGCC-3′



 



ActRIIB sense:



SEQ ID NO 11



5′-GTGGCAGAGTGAACGGGA-3′






In FIG. 9, it is shown that ActRII siRNA induces detachment of PC-3 cancer cells. PC-3 prostate cancer cells were plated in media containing 10% bovine serum albumin in 48-well plates (140,000 cells/well) for 24 h. Cells were then cultured in serum-free media with penicillin/streptomycin containing 1) control siRNA (DS NC1; 200 nM); 2) ActRIIA siRNA (200 nM); 3) ActRIIB siRNA (200 nM); or ActRIIA and ActRIIB siRNA (100 nM+100 nM). Cells were treated each day. After 72 h cell density was determined using the crystal violet assay. (A) Cell density, and (B) Cells attached (%), normalized to control (DS NC1). Results are presented as a percentage change from the control treated cells (mean±SEM; n=4, P<0.001; different letters indicate significant differences between treatments).
Cell detachment can be induced by, but is not limited to, the following siRNA oligonucleotides:









ActRIIA



SEQ ID NO 12



5′-UGCUAUAAACUUCCAAAGG-3′



 



ActRIIA



SEQ ID NO 13



3′-ACGAUAUUUGAAGGUUUCC-5′



 



ActRIIB



SEQ ID NO 14



5′-UGGGACCAUGAUGCAGAGG-3′



 



ActRIIB



SEQ ID NO 15



3′-ACCCUGGUACUACGUCUCC-5′






In FIG. 10 it is shown that ActRII Stealth siRNA induces detachment of PC-3 cancer cells. PC-3 prostate cancer cells were plated in media containing 10% bovine serum albumin in 48-well plates (140,000 cells/well) for 24 h. Cells were then cultured in serum-free media without penicillin/streptomycin containing 1) control Stealth siRNA (100 nM); 2) Stealth ActRIIA siRNA (100 nM); 3) Stealth ActRIIB siRNA (100 nM); or Stealth ActRIIA and Stealth ActRIIB siRNA (50 nM+50 nM). Cells were treated each day. After 72 h cell density was determined using the crystal violet assay. (A) Cell density, and (B) Cells attached (%), normalized to control Stealth siRNA. Results are presented as a percentage change from the control treated cells (mean±SEM; n=4, P<0.05; different letters indicate significant differences between treatments).
Cell detachment can be induced by, but is not limited to, the following Stealth siRNA oligonucleotides:









ActRIIA



SEQ ID NO 16



5′-UAAUUCUUUCACCUACACAUCCAGC-3′



 



ActRIIA



SEQ ID NO 17



3′-AUUAAGAAAGUGGAUGUGUAGGUCG-5′



 



ActRIIB



SEQ ID NO 18



5′-AAUGAACUGUAGCAGGUUCUCGUGC-3′



 



ActRIIB



SEQ ID NO 19



3′-UUACUUGACAUCGUCCAAGAGCACG-5′






In FIG. 11 it is shown via cell count that Activin A does increase human fibroblast cell proliferation. Human fibroblasts were maintained at 37° C. in MEM (Gibco, Carlsbad, Calif., USA) media containing 10% FBS and 1% penicillin/streptomycin (100 U/ml penicillin). For experiment, the cells were plated in 6 well plates at a density of approximately 1.0×105 cells/well for 48 h. The cells were then cultured in the above media without serum but containing 1% insulin/transferrin/sodium selenite (ITS) liquid media supplement (Sigma, St. Louis, Mo.) for 24 h prior to treatment with or without activin A (50 ng/mL; Cell Guidance Systems; Cambridge, United Kingdom; n=3 each) for 72 h. The cells were then counted using the trypan blue method.
As a result of the activity shown in FIG. 11, activin A may be used in a variety of ways to aid in the detection methods described herein. For example, activin A may be utilized to provide a positive control in the method and/or kit for inducing cell detachment. More particularly, activin A may be included with the kit for use in serum-free conditions and added to the control wells that are not treated with siRNA and/or Stealth siRNA. It is generally known that serum-free conditions lead to cell detachment and therefore the use of activin A would act to counter this cell detachment and promote attachment and proliferation in such serum-free conditions. In a second example, activin A may be utilized to promote attachment of any suitable mammalian cell. Examples of suitable mammalian cells include immortalized cell lines and primary cells. Activin A could be included in media to promote cell attachment under serum free conditions. In a particular example, activin A could be included with the 1% insulin/transferrin/sodium selenite (ITS) liquid media supplement (Sigma, St. Louis, Mo.) as a new supplement for the culture of cells under serum-free conditions.
The many features and advantages of the invention are apparent from the detailed specification, and thus, it is intended by the appended claims to cover all such features and advantages of the invention which fall within the true spirit and scope of the invention. Further, since numerous modifications and variations will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation illustrated and described, and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the invention.










Sequence Listing

















ActRIIA antisense-P:
5′-TCCAGTTCAGAGTCCCATTTC-3′
SEQ ID NO 1


 


ActRIIA sense:
5′-GAAATGGGACTCTGAACTGGA-3′
SEQ ID NO 2


 


ActRIIB antisense-P:
5′-TCTCCCGTTCACTCTGCCAC-3′
SEQ ID NO 3


 


ActRIIB sense:
5′-GTGGCAGAGTGAACGGGAGA-3′
SEQ ID NO 4


 


ADAM-15 Antisense-P:
5′-CGCACTCTTCCCTGGTAGCA-3′
SEQ ID NO 5


 


ADAM-15 Sense:
5′-TGCTACCAGGGAAGAGTGCG-3′
SEQ ID NO 6


 


laminin molecules
GTFALRGDNGDNGQ
SEQ ID NO 7


 


ActRII antisense:
5′-TGCAGCAGCTCCCAT-3′
SEQ ID NO 8


 


ActRII sense:
5′-ATGGGAGCTGCTGCA-3′
SEQ ID NO 9


 


ActRIIB antisense-P:
5′-TCTCCCGTTCACTCTGCC-3′
SEQ ID NO 10


 


ActRIIB sense:
5′-GTGGCAGAGTGAACGGGA-3′
SEQ ID NO 11


 


ActRIIA
5′-UGCUAUAAACUUCCAAAGG-3′
SEQ ID NO 12


 


ActRIIA
3′-ACGAUAUUUGAAGGUUUCC-5′
SEQ ID NO 13


 


ActRIIB
5′-UGGGACCAUGAUGCAGAGG-3′
SEQ ID NO 14


 


ActRIIB
3′-ACCCUGGUACUACGUCUCC-5′
SEQ ID NO 15


 


ActRIIA
5′-UAAUUCUUUCACCUACACAUCCAGC-3′
SEQ ID NO 16


 


ActRIIA
3′-AUUAAGAAAGUGGAUGUGUAGGUCG-5′
SEQ ID NO 17


 


ActRIIB
5′-AAUGAACUGUAGCAGGUUCUCGUGC-3′
SEQ ID NO 18


 


ActRIIB
3′-UUACUUGACAUCGUCCAAGAGCACG-5′
SEQ ID NO 19









REFERENCES


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Appendix 1



Keratinizing epithelial cells such as epidermal keratinocyte (differentiating epidermal cell), epidermal basal cell (stem cell), keratinocyte of fingernails and toenails, nail bed basal cell (stem cell), medullary hair shaft cell, cortical hair shaft cell, cuticular hair shaft cell, cuticular hair root sheath cell, hair root sheath cell of Huxley's layer, hair root sheath cell of Henle's layer, external hair root sheath cell, hair matrix cell (stem cell).
Wet stratified barrier epithelial cells such as surface epithelial cell of stratified squamous epithelium of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, basal cell (stem cell) of epithelia of cornea, tongue, oral cavity, esophagus, anal canal, distal urethra and vagina, urinary epithelium cell (lining urinary bladder and urinary ducts).
Exocrine secretory epithelial cells such as salivary gland mucous cell (polysaccharide-rich secretion), salivary gland serous cell (glycoprotein enzyme-rich secretion), von Ebner's gland cell in tongue (washes taste buds), mammary gland cell (milk secretion), lacrimal gland cell (tear secretion), ceruminous gland cell in ear (wax secretion), eccrine sweat gland dark cell (glycoprotein secretion), eccrine sweat gland clear cell (small molecule secretion), apocrine sweat gland cell (odoriferous secretion, sex-hormone sensitive), gland of Moll cell in eyelid (specialized sweat gland), sebaceous gland cell (lipid-rich sebum secretion), Bowman's gland cell in nose (washes olfactory epithelium), Brunner's gland cell in duodenum (enzymes and alkaline mucus), seminal vesicle cell (secretes seminal fluid components, including fructose for swimming sperm), prostate gland cell (secretes seminal fluid components), bulbourethral gland cell (mucus secretion), Bartholin's gland cell (vaginal lubricant secretion), gland of Littre cell (mucus secretion), uterus endometrium cell (carbohydrate secretion), isolated goblet cell of respiratory and digestive tracts (mucus secretion), stomach lining mucous cell (mucus secretion), gastric gland zymogenic cell (pepsinogen secretion), gastric gland oxyntic cell (hydrochloric acid secretion), pancreatic acinar cell (bicarbonate and digestive enzyme secretion), Paneth cell of small intestine (lysozyme secretion), type II pneumocyte of lung (surfactant secretion), Clara cell of lung.
Hormone secreting cells such as anterior pituitary cells, somatotropes, lactotropes, thyrotropes, gonadotropes, corticotropes, intermediate pituitary cell, secreting melanocyte-stimulating hormone, magnocellular neurosecretory cells, cells secreting oxytocin, secreting vasopressin, gut and respiratory tract cells, secreting serotonin, secreting endorphin, secreting somatostatin, secreting gastrin, secreting secretin, secreting cholecystokinin, secreting insulin, secreting glucagon, secreting bombesin, thyroid gland cells, thyroid epithelial cell, parafollicular cell, parathyroid gland cells, parathyroid chief cell, oxyphil cell, adrenal gland cells, chromaffin cells, secreting steroid hormones (mineralcorticoids and gluco corticoids), leydig cell of testes secreting testosterone, theca interna cell of ovarian follicle secreting estrogen, corpus luteum cell of ruptured ovarian follicle secreting progesterone, granulosa lutein cells, theca lutein cells, juxtaglomerular cell (renin secretion), macula densa cell of kidney, peripolar cell of kidney, mesangial cell of kidney. Metabolism and storage cells such as hepatocyte (liver cell), white fat cell, brown fat cell, liver lipocyte.
Barrier function cells of lung, gut, exocrine glands and urogenital tract.
Kidney cells such as kidney glomerulus parietal cell, kidney glomerulus podocyte, kidney proximal tubule brush border cell, Loop of Henle thin segment cell, kidney distal tubule cell, kidney collecting duct cell.
Epithelial cells lining closed internal body cavities such as blood vessel and lymphatic vascular endothelial fenestrated cell, blood vessel and lymphatic vascular endothelial continuous cell, blood vessel and lymphatic vascular endothelial splenic cell, synovial cell (lining joint cavities, hyaluronic acid secretion), serosal cell (lining peritoneal, pleural, and pericardial cavities), squamous cell (lining perilymphatic space of ear), squamous cell (lining endolymphatic space of ear), columnar cell of endolymphatic sac with microvilli (lining endolymphatic space of ear), columnar cell of endolymphatic sac without microvilli (lining endolymphatic space of ear), dark cell (lining endolymphatic space of ear), vestibular membrane cell (lining endolymphatic space of ear), stria vascularis basal cell (lining endolymphatic space of ear), stria vascularis marginal cell (lining endolymphatic space of ear), cell of Claudius (lining endolymphatic space of ear), cell of Boettcher (lining endolymphatic space of ear), choroid plexus cell (cerebrospinal fluid secretion), pia-arachnoid squamous cell, pigmented ciliary epithelium cell of eye, nonpigmented ciliary epithelium cell of eye, corneal endothelial cell.
Ciliated cells with propulsive function such as respiratory tract ciliated cell, oviduct ciliated cell (in female), uterine endometrial ciliated cell (in female), rete testis ciliated cell (in male), ductulus efferens ciliated cell (in male), ciliated ependymal cell of central nervous system (lining brain cavities).
Extracellular matrix secretion cells such as ameloblast epithelial cell (tooth enamel secretion), planum semilunatum epithelial cell of vestibular apparatus of ear (proteoglycan secretion), organ of Corti interdental epithelial cell (secreting tectorial membrane covering hair cells), loose connective tissue fibroblasts, corneal fibroblasts, tendon fibroblasts, bone marrow reticular tissue fibroblasts, other nonepithelial fibroblasts such as pericyte, nucleus pulposus cell of intervertebral disc, cementoblast/cementocyte (tooth root bonelike cementum secretion), odontoblast/odontocyte (tooth dentin secretion), hyaline cartilage chondrocyte, fibrocartilage chondrocyte, elastic cartilage chondrocyte, osteoblast/osteocyte, osteoprogenitor cell (stem cell of osteoblasts), hyalocyte of vitreous body of eye, stellate cell of perilymphatic space of ear contractile cells such as skeletal muscle cells, red skeletal muscle cell (slow), white skeletal muscle cell (fast), intermediate skeletal muscle cell, nuclear bag cell of muscle spindle, nuclear chain cell of muscle spindle, satellite cell (stem cell), heart muscle cells such as ordinary heart muscle cell, nodal heart muscle cell, purkinje fiber cell, smooth muscle cell (various types), myoepithelial cell of iris, myoepithelial cell of exocrine glands.
Blood and immune system cells such as erythrocyte (red blood cell), megakaryocyte (platelet precursor), monocyte, connective tissue macrophage (various types), epidermal Langerhans cell, osteoclast (in bone), dendritic cell (in lymphoid tissues), microglial cell (in central nervous system), neutrophil granulocyte, eosinophil granulocyte, basophil granulocyte, mast cell, helper T cell, suppressor T cell, cytotoxic T cell, natural Killer T cell, B cell, natural killer cell, reticulocytes, stem cells and committed progenitors for the blood and immune system (various types).
Cells of the nervous system such as sensory transducer cells which include auditory inner hair cell of organ of Corti, auditory outer hair cell of organ of Corti, basal cell of olfactory epithelium (stem cell for olfactory neurons), cold-sensitive primary sensory neurons, heat-sensitive primary sensory neurons, merkel cell of epidermis (touch sensor), olfactory receptor neuron, pain-sensitive primary sensory neurons (various types), photoreceptor cells of retina in eye (Photoreceptor rod cells, photoreceptor blue-sensitive cone cell of eye, photoreceptor green-sensitive cone cell of eye, photoreceptor red-sensitive cone cell of eye), proprioceptive primary sensory neurons (various types), touch-sensitive primary sensory neurons (various types), type I carotid body cell (blood pH sensor), type II carotid body cell (blood pH sensor), type I hair cell of vestibular apparatus of ear (acceleration and gravity), type II hair cell of vestibular apparatus of ear (acceleration and gravity), type I taste bud cell.
Autonomic neuron cells such as cholinergic neural cell (various types), adrenergic neural cell (various types), peptidergic neural cell (various types).
Sense organ and peripheral neuron supporting cells such as inner pillar cell of organ of Corti, outer pillar cell of organ of Corti, inner phalangeal cell of organ of Corti, outer phalangeal cell of organ of Corti, border cell of organ of Corti, Hensen cell of organ of Corti, vestibular apparatus supporting cell, type I taste bud supporting cell, olfactory epithelium supporting cell, Schwann cell, satellite cell (encapsulating peripheral nerve cell bodies), enteric glial cell.
Central nervous system neurons and glial cells such as astrocyte (various types), neuron cells (large variety of types, still poorly classified), oligodendrocyte, spindle neuron.
Lens cells such as anterior lens epithelial cell, crystallin-containing lens fiber cell.
Pigment cells such as melanocyte, retinal pigmented epithelial cell.
Germ cells such as oogonium/oocyte, spermatid, spermatocyte, spermatogonium cell (stem cell for spermatocyte), spermatozoon.
Nurse cells such as ovarian follicle cell, sertoli cell (in testis), thymus epithelial cell.
Interstitial cells like interstitial kidney cells.
Stem cells such as embryonic stem cells, mesenchymal stem cells, subcutaneous preadipocytes, visceral preadipocytes, osteoclast precursors, neural progenitors, bone marrow mononuclear cells, cord blood mononuclear cells, bone marrow progenitors, cord blood progenitors, cord blood erythroid progenitors, fetal liver progenitors, bone marrow stromal cells.
Other cells such as type I pneumocyte (lining air space of lung), pancreatic duct cell (centroacinar cell), nonstriated duct cell (of sweat gland, salivary gland, mammary gland, etc.), principal cell, intercalated cell, duct cell (of seminal vesicle, prostate gland, etc.), intestinal brush border cell (with microvilli), exocrine gland striated duct cell, gall bladder epithelial cell, ductulus efferens nonciliated cell, epididymal principal cell, epididymal basal cell.

## Claims

1. A composition to induce cell detachment, wherein the activin receptor II signaling suppressor includes the siRNA having at least one of the sequences SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, and SEQ ID NO 15 comprising a concentration of an siRNA form of activin receptor II signaling suppressor sufficient to induce cell detachment.

2. A composition to induce cell detachment, wherein the activin receptor II signaling suppressor includes the Stealth siRNA having at least one of the sequences SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, and SEQ ID NO 19 comprising a concentration of a Stealth siRNA form of activin receptor II signaling suppressor sufficient to induce cell detachment.

3. A kit to identify an agent to modify cell adhesion, the kit comprising:
a cell growth media;
a container having a surface matrix for cell adhesion; and
a cell detachment solution having a concentration of an activin receptor II signaling suppressor including an siRNA sufficient to induce cell detachment, the siRNA having at least one of the sequences SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, and SEQ ID NO 15.

4. The kit according to claim 3, further comprising:
lipofectamine.

5. The kit according to claim 3, further comprising:
a 96-well microtiter plate for growing the cells.

6. The kit according to claim 3, further comprising:
a cell detection reagent.

7. The kit according to claim 3, further comprising:
a fluorescent cell detection reagent.

8. The kit according to claim 3, further comprising:
a luminescent cell detection reagent.

9. The kit according to claim 3, further comprising:
an activin A to promote cell attachment in a positive control.

10. A method of identifying an agent to modify cell adhesion comprising the steps of:
adhering cells from an animal to a matrix;
introducing a cell detachment solution having a concentration of an activin receptor II signaling suppressor including an siRNA sufficient to induce cell detachment, wherein the cell detachment solution includes the siRNA having at least one of the sequences SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, and SEQ ID NO 15
introducing the agent; and
measuring a change in cell detachment as a result of introducing the agent, wherein the agent is identified as modifying cell adhesion in response to a measured change in cell detachment.

11. The method according to claim 10, further comprising:
generating a positive control by introducing an activin A to promote cell attachment.

12. A method of identifying an agent to modify cell adhesion comprising the steps of: adhering cells from an animal to a matrix; introducing a cell detachment solution having a concentration of an activin receptor II signaling suppressor including a Stealth siRNA sufficient to induce cell detachment, wherein the cell detachment solution includes the Stealth siRNA having at least one of the sequences SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, and SEQ ID NO 19;
introducing the agent; and measuring a change in cell detachment as a result of introducing the agent, wherein the agent is identified as modifying cell adhesion in response to a measured change in cell detachment.

13. The method according to claim 12, further comprising:
generating a positive control by introducing an activin A to promote cell attachment.

14. A kit to identify an agent to modify cell adhesion, the kit comprising: a cell growth media; a container having a surface matrix for cell adhesion; and a cell detachment solution having a concentration of an activin receptor II signaling suppressor including a Stealth siRNA sufficient to induce cell detachment, the Stealth siRNA having at least one of the sequences SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, and SEQ ID NO 19.

15. The kit according to claim 14, further comprising: lipofectamine.

16. The kit according to claim 14, further comprising: a 96-well microtiter plate for growing the cells.

17. The kit according to claim 14, further comprising: a cell detection reagent.

18. The kit according to claim 14, further comprising: a fluorescent cell detection reagent.

19. The kit according to claim 14, further comprising: a luminescent cell detection reagent.

20. The kit according to claim 14, further comprising: an activin A to promote cell attachment in a positive control.