Patent Application
20160158386
The present invention relates generally to a clinically relevant model of colorectal cancer (CRC) and methods of using the model to screen for compounds that inhibit tumorigenesis.
Colorectal cancer (CRC) is the third most common cancer worldwide and the fourth most common cause of death. CRC accounts for over 9% of all cancer incidences. In 2013, it is estimated that 142,820 new CRC cases will be diagnosed in the United States and that 50,830 people will succumb to the disease (American Cancer Society: Cancer Facts and Figures 2013, Atlanta, Ga., 2013). The poor ratio of survival to incidence of CRC is due, at least in part, to the high percentage of cases that are diagnosed at an advanced stage. The overall five-year relative survival of patients with advanced stage metastatic CRC stands at 12.5% (Howlader et al. SEER Cancer Statistics Review, 1975-2010. NCI. Bethesda, Md., 2013).
Although xenograft, chemical-induced, and genetically-engineered models (e.g., mouse models) of CRC have been developed to study CRC, tumors in these models fail to reproducibly metastasize to the regional intestinal lymph nodes and liver, the target organs relevant to human CRC. Thus, there is an unmet need in the field for the development of a model of CRC that is capable of recapitulating the pathogenesis of the human disease. Such a model of CRC would be a valuable tool for testing therapeutics and developing novel treatment strategies.
The present invention provides a model of colorectal cancer (CRC) that recapitulates the pathogenesis of the human disease, as well as methods for generating and using the model.
In a first aspect, the invention features a non-human mammal including a donor tumorigenic cell implant on the colonic mucosal surface, wherein implantation does not result in breach (e.g., opening, tear, rupture, or puncture) of the colon wall (i.e., the integrity of the deeper colon wall layers is maintained). In one embodiment, the donor tumorigenic cell implant is capable of invasive growth through the colon wall to the colonic serosal surface (e.g., growth resulting in penetration through the collagen IV-rich basement membrane of the muscularis externa to the serosal surface). In another embodiment, the invasive growth of the donor tumorigenic cell implant is characterized by metastases in common target organs (i.e., target metastatic organs or metastatic tissues) of CRC (e.g., human CRC), such as the intestinal lymph nodes, liver, or lungs. In another embodiment, the non-human mammal does not exhibit detectable tumor formation in the peritoneal cavity (e.g., peritoneal carcinomatosis) post-implantation. In another embodiment, the donor tumorigenic cell implant includes cells of a cancer cell line. The cancer cell line, in one embodiment, is a CRC cell line (e.g., HCT116). In another embodiment, the cancer cell line is a non-CRC cell line (e.g., a lung cancer cell line, a liver cancer cell line, a brain cancer cell line, a lymph node cancer cell line, a kidney cancer cell line, a stomach cancer cell line, a ovarian cancer cell line, a skin cancer cell line, a pancreatic cancer cell line, a thyroid cancer cell line, a prostate cancer cell line, or a breast cancer cell line, e.g., MDA-231). In another embodiment, the donor tumorigenic cell implant is an intact tumor, or fragment thereof. The intact tumor, or fragment thereof, in one embodiment, may be malignant (e.g., metastatic, regionally invasive, and/or distantly invasive). In another embodiment, the intact tumor, or fragment thereof, may be benign (e.g., non-metastatic and/or locally invasive). In some embodiments, the intact tumor, or fragment thereof, is an intact CRC tumor, or fragment thereof. In other embodiments, the intact tumor, or fragment thereof, is an intact non-CRC tumor, or fragment thereof (e.g., a breast cancer tumor, a lung cancer tumor, a liver cancer tumor, a brain cancer tumor, a lymph node cancer tumor, a kidney cancer tumor, a stomach cancer tumor, a ovarian cancer tumor, a skin cancer tumor, a pancreatic cancer tumor, a thyroid cancer tumor, or a prostate cancer tumor, or fragment thereof). In another embodiment, a subset (e.g., 5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more) of cells of the donor tumorigenic cell implant is capable of invasive growth. In other embodiments, growth of every (i.e., 100%) cell of the donor tumorigenic cell implant may be characterized as invasive growth. In another embodiment, the non-human mammal is a rodent, such as a mouse or a rat. In some embodiments, the rodent (e.g., mouse or rat) may be immunodeficient or immunocompromised. An immunodeficient mouse, in certain embodiments, may be a NOD/SCID mouse or a NOD/SCID interleukin-2 receptor gamma chain null (NSG) mouse. In other embodiments, the non-human mammal is wild-type and/or immune-competent (e.g., a wild-type or immune-competent rodent, e.g., a wild-type or immune-competent mouse or rat).
In a second aspect, the invention features a method for generating a non-human mammal (e.g., rodent, e.g., mouse or rat) of the first aspect (i.e., a non-human mammal (e.g., rodent) model for CRC), the method including exteriorizing the colonic mucosal surface of a host non-human mammal, implanting one or more tumorigenic cells onto the colonic mucosal surface, and re-inserting the exteriorized colon comprising the one or more implanted tumorigenic cells into the host non-human mammal.
In a third aspect, the invention features a method of screening for a compound that inhibits growth of tumorigenic cells (e.g., inhibits growth of tumorigenic cells by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater, e.g., compared to an untreated or control-treated group), the method including contacting the donor tumorigenic cell implant of a non-human mammal of the invention with a candidate compound and determining whether the candidate compound inhibits growth of the tumorigenic cells, thereby identifying the candidate compound as a compound that inhibits growth of tumorigenic cells.
In a fourth aspect, the invention features a method of screening for an adjuvant that inhibits growth of tumorigenic cells (e.g., inhibits growth of tumorigenic cells by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or greater, e.g., compared to an untreated or control-treated group), the method including: removing the donor tumorigenic cell implant from the colonic mucosal surface of a non-human mammal of the first aspect, administering to the non-human mammal a candidate compound, and determining whether the candidate compound inhibits growth of tumorigenic cells, thereby identifying the candidate compound as an adjuvant that inhibits growth of tumorigenic cells.
In one embodiment of the third or fourth aspect of the invention, the step of determining whether the candidate compound inhibits growth of tumorigenic cells includes evaluating the ability of the candidate compound to evoke at least one response (e.g., 1, 2, 3, 4, or 5 responses) selected from the group consisting of: reduction or stabilization in the number of tumorigenic cells; reduction or stabilization of tumor size; reduction or stabilization of tumor load; reduction or stabilization of tumorigenic cell invasiveness; and reduction or stabilization of tumor metastasis. In another embodiment of the third or fourth aspect, the candidate compound may be a small molecule, a peptide, a polypeptide, an antibody, an antibody fragment, or an immunoconjugate. In another embodiment of the third or fourth aspect, the donor tumorigenic cell implant may be capable of invasive growth through the colon wall to the colonic serosal surface (e.g., growth resulting in penetration through the collagen IV-rich basement membrane of the muscularis externa to the serosal surface). In other embodiments of the third or fourth aspect, invasive growth of the tumorigenic cells may be characterized by metastases in one or more (e.g., 1, 2, or 3 or more) common target organs (i.e., target metastatic organs or metastatic tissues) of CRC (e.g., human CRC), such as the intestinal lymph nodes, liver, or lungs. In other embodiments of the third or fourth aspect, the non-human mammal does not exhibit detectable tumor formation in the peritoneal cavity (e.g., peritoneal carcinomatosis) post-implantation. In another embodiment of the third or fourth aspect, the non-human mammal is a rodent, such as a mouse or rat.
The application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawings will be provided by the Office upon request and payment of the necessary fee.
The present invention is based in part on the generation of a model of colorectal cancer that exhibits metastasis to clinically relevant sites.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al. Dictionary of Microbiology and Molecular Biology. 2nd Ed. J. Wiley & Sons (New York, N.Y. 1994). For purposes of the present invention, the following terms are defined below.
The term “antibody” herein is used in the broadest sense and refers to any immunoglobulin (Ig) molecule comprising two heavy chains and two light chains, and any fragment, mutant, variant or derivation thereof so long as they exhibit the desired biological activity (e.g., epitope binding activity). Examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies, and antibody fragments.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to theft specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Ohler et al., Nature. 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson al., Nature. 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991), for example.
An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody, such as the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, and multispecific antibodies formed from antibody fragment(s). in certain embodiments, the antibody fragment binds the same antigen to which the intact antibody binds.
The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, kidney cancer, renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma and various types of head and neck cancer.
By “metastasis” is meant the spread of cancer from its primary site to other places in the body. Cancer cells can break away from a primary tumor, penetrate into lymphatic and blood vessels, circulate through the bloodstream, and grow in a distant focus (metastasize) in normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis can be characterized as a sequential process, contingent on tumor cells breaking off from the primary tumor, traveling through the bloodstream or lymphatics, and stopping at a distant site. After the tumor cells come to rest at another site, they can re-penetrate through the blood vessels or lymphatic walls, continue to multiply, and eventually another tumor is formed. At the new site, the cells establish a blood supply and can grow to form a life-threatening mass. In certain embodiments, this new tumor is referred to as a metastatic (or secondary) tumor. In certain embodiments, the term metastatic tumor refers to a tumor that is capable of metastasizing, but has not yet metastasized to tissues or organs elsewhere in the body. In certain embodiments, the term metastatic tumor refers to a tumor that has metastasized to tissues or organs elsewhere in the body. In certain embodiments, metastatic tumors are comprised of metastatic tumor cells.
The “metastatic organ” or “metastatic tissue” is used in the broadest sense, refers to an organ or a tissue in which the cancer cells from a primary tumor or the cancer cells from another part of the body have spread. Examples of metastatic organ and metastatic tissue include, but are not limited to, lung, liver, brain, ovary, bone, bone marrow, and lymph node. With respect to colorectal cancer (CRC), predominant metastatic organ and metastatic tissue are the regional intestinal lymph nodes, liver, and lungs.
By “micrometastasis” is meant a small number of cells that have spread from the primary tumor to other parts of the body. Micrometastasis may or may not be detected in a screening or diagnostic test.
By “macrometastasis” is meant a number of cells that are detectable and have spread from the primary tumor site to other parts of the body.
By “non-metastatic” is meant a cancer that is benign or that remains at the primary site (e.g., a locally invasive cancer) and has not penetrated into the lymphatic or blood vessel system or to tissues other than the primary site. In certain embodiments, a non-metastatic cancer is any cancer that is a Stage 0, I, or II cancer,
“Invasiveness” or “invasive growth,” as used herein, refers to the ability of a cancer or tumor to leave the tissue site at which it originated and proceed to proliferate at a different site (e.g., nearby or distant site) of the body. In some embodiments, a cancer can be “locally invasive” and proceed to proliferate at a nearby site of the body, such as surrounding tissue. In other embodiments, a cancer can be “regionally invasive” or “distantly invasive” and proceed to proliferate at a regional or distant site of the body, respectively.
Reference to a cancer or tumor as a “Stage 0,” “Stage I,” “Stage II,” “Stage III,” or “Stage IV” indicates classification of the tumor or cancer using the Overall Stage Grouping or Roman Numeral Staging methods known in the art. Although the actual stage of the cancer is dependent on the type of cancer, in general, a Stage 0 cancer is an in situ lesion, a Stage I cancer is small localized tumor, a Stage II is a local advanced tumor, a Stage III cancer is a local advanced tumor that exhibits involvement of the local lymph nodes, and a Stage IV cancer represents metastatic cancer. The specific stage for each type of tumor is known to the skilled clinician.
“Tumor,” as used herein, refers to any neoplastic cell growth, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer”, “cancerous”, “cell proliferative disorder”, “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein. Tumors may be solid tumors, such as tumors of the colon (CRC tumor), or non-solid or soft tumors, such as leukemia. Examples of soft tissue tumors include leukemia (e.g., chronic myelogenous leukemia, acute myelogenous leukemia, adult acute lymphoblastic leukemia, acute myelogenous leukemia, mature B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphocytic leukemia, or hairy cell leukemia), or lymphoma (e.g., non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, or Hodgkin's disease). A solid tumor includes any cancer of body tissues other than blood, bone marrow, or the lymphatic system. Solid tumors can be further separated into those of epithelial cell origin and those of non-epithelial cell origin. Examples of solid tumors include tumors of colon, breast, prostate, lung, kidney, liver, pancreas, ovary, head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, gastrointestinal tract, anus, gall bladder, labium, nasopharynx, skin, uterus, male genital organ, urinary organs, bladder, and skin. Solid tumors of non-epithelial origin include sarcomas, brain tumors, and bone tumors. The term “tumor,” as used herein, is also meant to be inclusive of “polyps.”
By “primary tumor” or “primary cancer” is meant the original cancer and not a metastatic lesion located in another tissue, organ, or location in the subject's body. In certain embodiments, primary tumor is comprised of primary tumor cells.
By “benign tumor ” or “benign cancer” is meant a tumor that remains localized at the site of origin and does not have the capacity to infiltrate, invade, or metastasize to a distant site.
“Tumorigenic cells,” as used herein, refer to any cells (e.g., cancer cells, e.g., human cancer cells or non-human cancer cells) that exhibit an abnormal growth state or are capable of changing their normal growth state to an abnormal growth state in which they eventually form tumors. Tumorigenic cells are capable of forming tumors, which are generally the result of uncontrolled growth of the cells. Tumorigenic cells can be distinguished from non-tumorigenic cells on the basis of their tumor-forming phenotype (see, e.g., Al-Hajrj, et al. Proc Natl Acad Sci USA. 100: 3983-8, 2003; U.S. Pub. No. 2002/0119565; U.S. Pub. No. 2004/0037815; U.S. Pub. No. 2005/0232927; WO 05/005601; U.S. Pub. No. 2005/0089518; U.S. appl. Ser. No. 10/864,207; Al-Hajj et al. Oncogene. 23: 7274, 2004; and Clarke et al. Ann Ny Acad. Sci. 1044: 90, 2005, all of which are herein incorporated by reference in their entireties for all purposes). Tumorigenic cells include, without limitation, tumor cells, embryonic cells, cells engineered to have abnormal growth, cancer cell lines, as well as cell masses of any of these cell types.
The term “implant,” and variations thereof, refers to transplanted cells, for example, tumorigenic cells (e.g., an intact tumor, or fragment thereof) which are introduced into a recipient host and which remain substantially stably established at the site of transplantation in the recipient.
By “donor” cell, tumor, or tumorigenic cell is meant a cell, tumor, or tumorigenic cell that is not derived from the recipient host organism, but may be syngeneic (where the donor and recipient are genetically identical), allogeneic (where the donor and recipient are of different genetic origins but of the same species), or xenogeneic (where the donor and recipient are from different species). For example, the donor cell, tumor, or tumorigenic cell may be derived from a human. A “donor tumorigenic cell implant” refers to transplanted tumorigenic cells, as used herein, which are derived from a source other than the recipient organism.
By “tumor load” is meant the amount of cancer in the body. Tumor load is also referred to as tumor burden, and may be a function of tumor number and tumor size.
“Adjuvant therapy” herein refers to therapy given after surgery, where no evidence of residual disease can be detected, so as to reduce the risk of disease recurrence. The goal of adjuvant therapy is to prevent recurrence of the cancer, and therefore to reduce the chance of cancer-related death.
A “small molecule” is defined herein to have a molecular weight below about 500 Daltons.
By “immunoconjugate” is meant an antibody conjugated to one or more heterologous molecule(s) (e.g., an antibody-drug conjugate (ADC)), including but not limited to a cytotoxic agent.
By “reduce” or “inhibit” is meant the ability to cause an overall decrease, for example, of 20% or greater, of 50% or greater, or of 75%, 85%, 90%, 95%, or greater. In certain embodiments, reduce or inhibit can refer to the growth of tumorigenic cells or a tumor, which can be measured by a reduction or inhibition in the number of tumorigenic cells, size of tumors, tumor load, tumorigenic cell or tumor invasiveness, and/or tumor metastasis.
The term “non-human animal” refers to all animals, except humans, and includes, without limitation, birds, farm animals (e.g., cows), sport animals (e.g., horses), fish, reptiles, and non-human mammals (e.g., cats, dogs, and rodents).
The term “non-human mammal” refers to all members of the class Mammalia, except humans.
The term “rodent” refers to all members of the order Rodentia, including rats, mice, rabbits, hamsters, and guinea pigs.
Colorectal cancer (CRC) initially manifests as benign polyps on the mucosal surface of the large intestine. If left unresected, these polyps can progress to invasive adenocarcinomas that penetrate through the submucosal and muscularis externa layers of the colorectal wall to reach the serosal side. Eventual regional spread to the intestinal lymph nodes and distant spread to the liver results in the outgrowth of gross metastases that are the major cause of CRC mortality. Deciphering the routes of CRC metastasis to these sites therefore has the potential to uncover therapeutic opportunities that may impact mortality rates. Investigations into metastatic routes, however, have been hampered by the lack of availability of relevant in vivo metastatic models of CRC. Indeed, despite the wide availability of xenograft, chemical-induced, and genetically-engineered models (e.g., mouse models) of CRC (Heijstek et al. Dig. Surg. 22: 16-25, 2005. Epub 2005 Apr. 14; Kobaek-Larsen et al. Comp. Med. 50 (1): 16-26, 2000; Rosenberg et al. Carcinogenesis. 30 (2): 183-196, 2009. Epub 2008 Nov. 26; Taketo et al. Gastroenterology. 136 (3): 780-798, 2009), tumors in these models fail to reproducibly metastasize to the regional intestinal lymph nodes and liver, the target organs relevant to human CRC.
Lumen Implantation Model of Colorectal Cancer
The present invention is based, at least in part, on the development of a clinically relevant model of colorectal cancer (CRC). In contrast to known models of CRC, the model of CRC of the invention is generated by a novel lumen implantation technique, and, importantly, is capable of recapitulating the etiology of human CRC.
A non-human animal (e.g., a non-human mammal) of any species, subspecies, genetic variant, tissue variant, or combination thereof, can be used in the generation of the lumen implantation model (LIM) of CRC. The non-human mammal may, for example, be a rodent. Examples of rodent species include, without limitation, rat, mouse, hamster, rabbit, guinea pig, and gerbil. The non-human mammal can be male or female. The non-human mammal can be any age, provided that the lumen implantation technique can be successfully executed. Accordingly, the non-human mammal can be, for example, less than one week old, from about one week to about five years old, from about one week to about three years old, from about two weeks to about two years old, from about three weeks to about one year old, from about four weeks to about six months old, from about six weeks to about three months old, from about eight weeks to about twelve weeks old, older than three years old, or older than five years old.
The non-human mammal can be wild-type (e.g., immune-competent) or immunodeficient. For example, when the lumen of the recipient host non-human mammal is implanted with a donor cell, tumor, or tumorigenic cell that is xenogeneic (e.g., human), the host non-human mammal is immunodeficient, When the lumen of the recipient host non-human mammal is implanted with a donor cell, tumor, or tumorigenic cell that is syngeneic, however, the host non-human mammal can be non-immunodeficient (e.g., wild-type).
In certain instances, the non-human mammal is a mouse. The mouse can be a nude mouse. The mouse can be a severely combined immunodeficient (SCID) mouse, for example, a NOD/SCID interleukin-2 receptor gamma chain null (NSG) mouse. The NSG mouse is described in Pearson et al. Curr. Top. Microbiol. Immunol. 324:25-51, 2008; Shultz et al. Curr Top Microbiol Immunol. 324:25-51 2005; Strom et al. Methods Mol. Biol. 640:491-509, 2010; McDermott et al. Blood. 116 (2): 193-200, 2010; Lepus et al. Hum. Immunol. 70 (10):790-802, 2009; Brehm et al. Clin Immunol. 135 (I):84-98, 2010. Any suitable immunodeficient non-human mammal can be used. Suitable non-human mammals include rodents, which can be obtained from such sources as The Jackson Laboratory of Bar Harbor, Me., Charles River Laboratories International, Inc. of Wilmington, Mass., and Harlan Laboratories of Indianapolis. Ind.
The lumen implantation technique involves the implantation of one or more (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 102, 103, 104, 105, 106, 107, 108, 109, or 1010 or more) donor tumorigenic cells that are capable of being implanted on the mucosal surface (luminal side) of the colon without breaching the colon wall. The donor tumorigenic cell(s) can be syngeneic (where the donor and recipient are genetically identical), allogeneic (where the donor and recipient are of different genetic origins but of the same species), or xenogeneic (where the donor and recipient are from different species, e.g., human) with respect to the recipient non-human mammal host. The donor tumorigenic cell(s) may be invasive or non-invasive, benign or malignant, metastatic or non-metastatic. The tumorigenic cells may be tumor cells, or alternatively, may be, for example, embryonic cells, cells engineered to have abnormal growth, cancer cell lines, as well as cell masses of any of these cell types.
In instances of implantation of more than one donor tumorigenic cell, the implanted cells may be an intact tumor, or fragment thereof. The intact tumor or fragment thereof, can be an intact malignant tumor, or fragment thereof, such as a Stage III CRC tumor, which has given rise to regional metastases (e.g., in the intestinal lymph node) in the donor organism, or a Stage IV CRC tumor, which has given rise to distant metastases (e.g., in the liver or lungs) in the donor organism. Alternatively, the intact tumor or fragment thereof, can be an intact benign or locally invasive tumor, or fragment thereof, such as a Stage 0, Stage I, or Stage II CRC tumor, or fragment thereof, which is confined to the site or tissue of primary origin.
The intact tumor, or fragment thereof, is not limited to a CRC tumor, or fragment thereof. For example, the intact tumor, or fragment thereof, can be an intact a non-CRC tumor, or fragment thereof, such as, without limitation, a breast cancer tumor, lung cancer tumor, liver cancer tumor, brain cancer tumor, lymph node cancer tumor, kidney cancer tumor, stomach cancer tumor, ovarian cancer tumor, skin cancer tumor, pancreatic cancer tumor, thyroid cancer tumor, or prostate cancer tumor, or fragment thereof.
The intact tumor, or fragment thereof, implanted on the colonic mucosal surface of the recipient non-human mammal host can be a solid tumor (e.g., a colon/CRC tumor, breast cancer tumor, lung cancer tumor, or liver cancer tumor), or fragment thereof. The intact tumor, or fragment thereof, can be derived from any suitable donor organism, such as a human or mouse. The intact tumor, or fragment thereof, can be from a particular cell line, such as a CRC cell line (e.g., HCT116,LS174T, or LoVo primary human CRC-derived cell line) or a breast cancer cell line (e.g., MDA-231 human breast cancer cell line).
The implanted one or more donor tumorigenic cells can be of any collective size. In instances when an intact tumor, or fragment thereof, is implanted, the intact tumor, or fragment thereof, is around 0.1-100 mm3 in size, e.g., around 1-100 mm3 in size, e.g., around 10 mm3 in size.
The implantation site can be along any region of the mucosal surface of the colon of the non-human mammal. In certain embodiments, the implantation site is located nearby the anus of the recipient non-human mammal in order to allow for the option of removal of the implanted tumor from the implantation site. In mice, for example, a tumor implantation distance of about 1-20 mm (e.g., about 5-15 mm, e.g., about 11-12.5 mm) away from the anus of the host mouse is preferable.
In general, a mouse LIM of CRC can be created by anesthetizing the mouse (e.g., by isoflurane inhalation), placing the mouse in a supine position with extremities secured and inserting a blunt-ended hemostat (Micro-Mosquito, No. 13010-12, Fine Science Tools) or other suitable tool around 1 cm into the anus, clasping a single mucosal fold (e.g., by closing the hemostat to the first notch), retracting and cleaning exteriorized mucosa (e.g, with povidone/iodine), rinsing (e.g., with lactated ringers solution), and blotting dry. One or more donor tumorigenic cells (e.g., a donor tumor fragment or intact polyp of ˜10 mm3) can be then be sutured onto the mucosa (e.g., using absorbable 4-0 vicryl sutures (Ethicon)), ensuring that the suture only penetrates the superficial mucosal layer. After rehydrating the mucosa with PBS, the exteriorized colon can be re-inserted together with the sutured tumor, thus reversing the rectal prolapsed. To minimize tumor dislodgement during defecation, mice can be housed on cage floor inserts and fed a 100% rodent liquid diet (AlN-76A, Casein Hydrolysate without Fiber; BioServe) from around 3 days pre-surgery to around 7 days post-surgery.
The generated non-human mammal LIMs of CRC have numerous advantages over established CRC models, as demonstrated in the Examples section below. These advantages of the LIM include, without limitation, the implantation of tumors onto the mucosal surface, compared to tumor implantation onto the serosal surface in the existing cecum implantation model, resulting in: (i) the potential to give rise to distant metastases in clinically relevant sites, compared to the widespread tumor dissemination throughout the peritoneal cavity due to tumor cell shedding rather than actual metastasis in the existing cecum implantation model; (ii) the ability to implant intact tumor fragments into a host mouse instead of cell suspensions that are unable to maintain tumor structure as in existing cell suspension injection models; and (iii) the maintained integrity of the colon wall compared to the likelihood of puncturing the colon wall as in existing cell suspension injection models.
Screening of Candidate Compounds that Inhibit Tumorigenesis
The LIM finds utility, for example, in the screening of candidate compounds that possess anti-cancer activity (e.g., compounds that inhibit growth of tumorigenic cells). Anti-cancer activity can include activity in directly or indirectly mediating any effect in preventing, delaying, reducing or inhibiting tumor growth and/or development, which may provide for a beneficial effect to the host. Anti-cancer activity of a candidate compound could therefore be reflected by, without limitation, the ability of the candidate compound to, directly or indirectly, reduce or stabilize: the number of tumorigenic cells (e.g., reduce the number of tumorigenic cells by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more), tumor size (e.g., reduce the size of a tumor by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more), tumor load or burden (e.g., reduce tumor load or burden by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more), tumorigenic cell invasiveness (e.g., reduce invasiveness nearby tissue by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more), and/or tumor metastasis (e.g., reduce the number of metastases and/or metastatic organs or tissues by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more). Accordingly, the anti-cancer activity of a candidate compound can be assessed by determining the presence or absence of, for example, one or more of the above effects related to the inhibition of growth of tumorigenic cells in the LIM, wherein the presence of one or more effects on tumorigenic cell growth is indicative of the candidate compound possessing anti-cancer activity. For example, with respect to determining an effect on tumor size and/or number, the determining step can include measuring tumor size and/or number at a first time point and a second time point, comparing tumor size and/or number measured at the first time point relative to that measured at the first time point. In some instances, with respect to tumor invasiveness and metastasis, the determining step can include detecting the presence or absence of tumor invasion (e.g., invasive tumor growth through the colon wall to the colonic serosal surface) or metastases (e.g., metastases in the intestinal lymph nodes, liver, and/or lungs) by gross visual analysis (e.g., when detecting macrometastases) and/or by histological or cell counting analyses.
The candidate compounds that can be screened for anti-cancer activity using a LIM of the present invention include, without limitation, synthetic, naturally occurring, or recombinantly produced molecules, including small molecules, polynucleotides, peptides, polypeptides, antibodies, and immunoconjugates. Candidate compounds can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, and amidification, to produce structural analogs.
The candidate compounds may be formulated, dosed, and administered in any manner desired and/or appropriate in a fashion consistent with good medical practice and in order to examine anti-cancer activity. The candidate compounds may be prepared in therapeutic formulations using standard methods known in the art by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (20th edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.). Acceptable carriers, include saline, or buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagines, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, PLURONICS™, or PEG.
Optionally, the formulation contains a pharmaceutically acceptable salt (e.g., sodium chloride) at about physiological concentrations. Optionally, the formulations of the invention can contain a pharmaceutically acceptable preservative. In some embodiments the preservative concentration ranges from 0.1 to 2.0%, typically v/v. Suitable preservatives include those known in the pharmaceutical arts. Benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben are preferred preservatives. Optionally, the formulations of the invention can include a pharmaceutically acceptable surfactant at a concentration of 0.005 to 0.02%.
The candidate compounds can be administered singly or can be combined in combinations of two or more (e.g., 3, 4, or 5 or more candidate compounds), especially where administration of a combination of compounds may result in a synergistic effect.
Adjuvant Model of Colorectal Cancer and Screening of Adjuvants
The LIM can also be utilized to generate an adjuvant model of CRC to be subsequently used, for example, in the screening of adjuvants that possess anti-cancer activity (e.g., compounds that inhibit of growth of tumorigenic cells). To this end, the implanted primary tumor is surgically removed after implantation, and adjuvant screening with candidate compounds can then be performed on the non-human mammal (e.g., rodent, e.g., mouse or rat) adjuvant model in a manner analogous to the screening of compounds that inhibit growth of tumorigenesis, discussed above.
In this adjuvant model of colorectal cancer, the surgical removal of the implanted primary tumor can be performed at various time points post-implantation, corresponding to different stages of CRC disease progression (e.g., Stage 0, I, II, III, or IV). The same or different candidate compounds can then be tested for efficacy as an adjuvant in the treatment of different stages of CRC.
A candidate compound that inhibits growth/re-growth of tumorigenic cells in an adjuvant setting compared to a counterpart untreated or control-treated adjuvant model identifies a candidate compound as an adjuvant.
The duration of adjuvant therapy trials, as well as the formulation, dosage, and administration route of an adjuvant candidate or identified adjuvant can be altered as necessary in any manner desired and/or appropriate in a fashion consistent with good medical practice, similar to candidate compounds for primary therapy, as described above.
The present invention is illustrated by the following Examples, which are in no way intended to be limiting of the invention.
One skilled in the art will recognize many materials and methods similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the materials and methods described below.
Mice
Wild-type NOD/SCID female mice (8-12 weeks old) were purchased from Charles River Laboratories. Wild-type NSG female mice (8-12 weeks old; stock number 00 55 57), ApcMin/+ mice (stock number 002020), and 12.4 KbVilCre mice (stock number 004586; referred to as Villin-Cre) were purchased from the Jackson Laboratory. KrasLSLG12D/+ mice were licensed from Tyler Jacks from the Massachusetts Institute of Technology. Apc/Kras compound mutant mice from colony number 4028 were bred with CAG-mRFP1 mice (stock number 005884) purchased from the Jackson Laboratory. Apc/Kras compound mutant mice from colony number 4700 were bred with Rosa26-CAG-LSL-tdTomato mice (stock number 007909) purchased from the Jackson Laboratory. All experiments were approved by the Animal Research Ethics and Protocol Review Committee of Genentech.
Cell Culture and Gene Transfer
HCT116,LS174T, and LoVo primary human colorectal cancer-derived cell lines were purchased from ATCC and maintained in complete RPMI medium (RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin) at 37° C. and 5% CO2. Cells were transduced with a TZV-CMV-Discosoma red fluorescent protein (DsRed) lentiviral vector (Open Biosystems) at a multiplicity-of-infection (MOI) of 10 in complete RPMI medium supplemented with 8 mg ml−1 polybrene for 6 hr at 37° C. and 5% CO2. After 4 passages in culture, DsRed-positive cells were isolated by fluorescence-activated cell sorting on a FACSAria (BO Biosciences). Sorted DsRed-positive cells were expanded for 2-3 passages, and then stored in liquid nitrogen. Early passage cells were used for all in vivo experiments.
Lumen Implantation Technique
Mice were anesthetized by isoflurane inhalation, placed in a supine position, and the extremities secured to a gauze-covered platform with tape. A blunt-ended hemostat (Micro-Mosquito. No. 13010-12, Fine Science Tools) was inserted ˜1 cm into the anus, and the hemostat angled towards the mucosa and opened slightly such that a single mucosal fold could be clasped by closing the hemostat to the first notch. The hemostat was retracted from the anus, and the clasped exteriorized mucosa cleansed with povidone/iodine, rinsed with lactated ringers solution and blotted dry. A donor tumor fragment or intact polyp of ˜10 mm3 was sutured onto the mucosa using absorbable 4-0 vicryl sutures (Ethicon), ensuring that the suture only penetrated the superficial mucosal layer. After rehydrating the mucosa with PBS, the hemostat was released and a blunt gavage needle used to re-insert the exteriorized colon together with the sutured tumor, thus reversing the rectal prolapse. The average tumor implantation distance was 11.8±0.5 mm away from the anus (n=18). Mortality post-surgery was less than 1%, with morbidity at 2-4 wpi attributable to reversible rectal prolapse in less than 5% of mice and morbidity at 7 wpi attributable to weight loss due to increased tumor burden. To minimize tumor dislodgement during defecation, mice were housed on cage floor inserts and fed a 100% rodent liquid diet (AlN-76A, Casein Hydrolysate without Fiber; BioServe) from 3 days pre-surgery until 7 days post-surgery.
Subcutaneous Tumor Generation
Cell lines were harvested via trypsinization, counted with trypan blue to assess viability, and resuspended in cold complete RPMI medium at a concentration of 100×106 cells/ml. Cold Matrigel (BD Biosciences) was added to the cell suspension at a 1:1 ratio to achieve a final cell concentration of 50×106 cells/ml. NOD/SCID mice were injected with 5×106 cells in a volume of 100 μl subcutaneously in the left flank. Tumor dimensions were measured using calipers and tumor volume was calculated as 0.523×length×width×width. For subcutaneous tumors used as donors for the lumen implantation technique, tumors were harvested between 1000-2000 mm3, necrotic tissue grossly dissected away under a microscope, and the remaining viable tissue divided into 10 mm3 fragments and placed on ice in complete RPMI medium.
Endoscopy
Prior to endoscopic imaging, mice were anesthetized by isoflurane inhalation, placed in a supine position, and their colons evacuated of stool using a gavage needle. Endoscopic imaging equipment consisted of a Hopkins II 0° straight forward 1.9 mm outer diameter telescope encompassed by an examination and protection sheath, an Image-I high definition three-chip digital camera attached to a Mikata Point Setter telescope holding system, a fiber optic light guide cable connected to a D Light System xenon light source, an electronic CO2 insufflator to maintain colon insufflation during imaging, and an AIDA Connect high definition documentation system connected to a high definition color monitor (Karl Storz). Endoscopic videos were reviewed using VLC Media Player (VideoLAN Team) and still images were captured from these videos.
Whole Organ Imaging and Macrometastasis Evaluation
Colons were harvested intact, flushed with PBS, opened longitudinally, pinned down on thin cardboard pieces, and imaged both mucosally and serosally. Livers and lungs were harvested, washed in PBS, and imaged. All organs were imaged using a DFC295 color digital camera (Leica) attached to a M80 stereomicroscope (Leica). Macroscopic metastasis formation was assessed visually using a S4 stereomicroscope (Leica). For the intestinal lymph nodes, the entire intestinal tract from the anus to the stomach was examined for evidence of lymph node involvement, and the number of macrometastases quantified. For the liver and lungs, the entire external surface of whole organs was examined and the number of macrometastases quantified. Following macro metastasis quantification, organs were fixed in 4% paraformaldehyde in PBS overnight. Prior to overnight fixation in 4% paraformaldehyde, lungs were perfused with 4% paraformaldehyde in PBS. Primary colorectal tumor dimensions were determined using a reference measurement scale and tumor volume was calculated as 0.523×length×width×width.
Tissue Digestion and Flow Cytometry
Entire tissues were processed on a GentieMACS dissociator (Miltenyi Biotec), digested in complete RPMI medium supplemented with 1 mg/ml collagenase/dispase for 30 min at 37° C. with agitation at 210 rpm, and filtered through a 70-μm strainer. Following red blood cell lysis and centrifugation, cells were resuspended in PBS supplemented with 2% fetal bovine serum, 20 mM HEPES and 5 μg/ml propidium iodide, filtered into FACS tubes and analyzed on a FACSAria flow cytometer (BO Biosystems). For FACS controls, normal tissues from non-tumor-bearing mice were used, and DsRed-positive analysis gates were established such that zero DsRed-positive events were detectable in control tissue specimens. An average of 5×106 viable events were analyzed per specimen. Data were expressed as the number of DsRed-positive cells per 1×106 viable events.
Circulating Tumor Cells
Mice were euthanized by CO2 inhalation. Immediately after breathing subsided, the rib cage was splayed open to expose the heart. A syringe fitted with a 27 gauge needle was inserted into the right chamber of the heart, and ˜50 μl of blood was withdrawn. Blood was immediately transferred to EOTA-coated Microtainer tubes (BD Biosciences). Following red blood cell lysis, blood samples were resuspended in PBS supplemented with 2% fetal bovine serum, 20 mM HEPES, and 5 μg/ml propidium iodide, and analyzed by flow cytometry. For FACS controls, blood from non-tumor-bearing mice was used, and DsRed-positive analysis gates were established such that zero DsRed-positive events were detectable in control blood specimens. An average of 5×106 viable events were analyzed per specimen. Data were expressed as the number of DsRed-positive cells per 1×106 viable events.
Human Colorectal Cancer Clinical Specimens
Freshly resected human colorectal cancer specimens were obtained from Bio-options Inc., from consenting patients in accordance with federal and state guidelines. Specimens were shipped overnight at 4° C. in DMEM high glucose medium supplemented with 10% fetal bovine serum, glutamine, vancomycin, metronidazole, cefotaxime, amphotericin B, penicillin, streptomycin, and protease inhibitor cocktail. Specimens were cut into ˜2 mm3 tumor fragments, and individual fragments implanted under the kidney capsule of athymic nu/nu male mice (6-8 weeks old) purchased from Harlan Sprague Dawley. Six months post-implantation, tumors that grew under the kidney capsule were used as donor tumors, and ˜2 mm3 donor tumor fragments were implanted into the colonic lumens of NOD/SCID mice.
Anti-VEGF-A and Anti-VEGF-C Antibodies
The anti-VEGF-A monoclonal antibody G6-31 has been described previously (U.S. Pat. No. 7,758,859; Liang et al. J. Biol. Chem. 281 (2): 951-961, 2006. Epub 2005 Nov. 7). The anti-VEGF-C monoclonal antibody VC4.5 was isolated from synthetic phage antibody libraries built on a single framework (Lee et al. J. Mol. Biol. 340: 1073-1093, 2004) by selection against a matured form of human VEGF-C (R&D Systems). One positive clone VC4 as full-length IgG was verified to block the interaction between human VEGF-C and human VEGFR3, inhibit VEGF-C induced cell activity and cross-bind murine VEGF-C. VC4 was further affinity improved to VC4.5 with phage display selection, as previously described (Lee et al. Blood. 108: 3103-3111, 2006. Epub 2006 Jul. 13) and shown to improve the potency of blocking VEGF-C from receptor binding and cell signaling. VC4.5 exhibits similar affinity towards human and murine VEGF-C (Kd=0.3-1 nM) as determined by surface plasmon resonance measurement using BIAcore instruments by immobilizing either VC4.5 IgG or VEGF-C on the chip. If desired, other anti-VEGF-A or anti-VEGF-C antibodies may be utilized.
Vascular Targeting
One day prior to lumen implantation, NOD/SCID mice were treated with the function-blocking monoclonal antibodies anti-VEGF-A (G6-31; 5 mg/kg in PBS) and/or anti-VEGF-C (VC4.5; 40 mg/kg in PBS) by intraperitoneal injection. On day 0, HCT116-DsRed tumor fragments were implanted onto the colonic mucosa. Antibodies were administered once per week.
Histopathology and Immunostaining
Tissues were fixed in 4% paraformaldehyde in PBS overnight, rinsed in PBS, cryoprotected in 30% sucrose in PBS overnight at 4° C., embedded in Optimal cutting temperature (OCT) compound and frozen at −80° C., and sectioned at 8 μm. For histopathological analyses, tissue sections were stained with haematoxylin and eosin (H&E) using a Jung Autostainer XL (Leica), and whole tissue section scans were acquired using a NanoZoomer (Hamamatsu). For immunohistochemical analyses, tissue sections were incubated with primary antibody overnight at 4° C. and secondary antibody for 30 min at room temperature. Primary antibodies used were rabbit anti-collagen IV (polyclonal ab6586; Abcam; 1:100 dilution), goat anti-DsRed (polyclonal sc-33354; Santa Cruz Biotechnology; 1:100 dilution), and rat anti-pan endothelial cell marker (clone MECA-32; Pharmingen; 2 μg/ml). Secondary antibodies used were conjugated to Alexa Fluor 488 or 594 (Invitrogen). Images were acquired on an Axioplan 2 imaging microscope (Zeiss) with an ORCA-ER digital camera (Hamamatsu). Vascular density was expressed as a ratio of the MECA-32-positive vascular area over the total DAPI-positive viable tumor area multiplied by 100. Histology specimens were reviewed by a trained pathologist with CRC disease expertise.
Statistical Analyses
Group differences were evaluated by two-tailed Student's t test. Correlations were evaluated by Pearson correlation coefficients. Contingency analyses were evaluated by two-sided chi-squared test, using actual mouse numbers as input data. For Kaplan-Meier survival analyses, P values were computed using the Log-rank test, and hazard ratios were computed using Apcmin/+; Villin-Cre mice as the comparator. P values less than 0.05 were considered significant.
The most widely utilized genetically-engineered mouse model of intestinal cancer is the ApcMin/+ mouse, which harbors a dominant nonsense mutation in one Apc allele (Su at al. Science. 256 (5057): 668-670, 1992). ApcMin/+ mice develop numerous adenomas within the intestinal tract; however, these adenomas rarely, if ever, progress to invasive or metastatic adenocarcinomas (Moser et al. Science. 247 (4940): 322-324, 1990). Moreover, these adenomas primarily localize to the small intestine, with relatively few adenomas manifesting in the colon (Moser at al. Science. 247 (4940): 322-324, 1990). Introduction of oncogenic Kras to the mutant Apc background promotes intestinal adenoma multiplicity (Janssen at al. Gastroenterology. 131 (4): 1096-1109, 2006. Epub 2006 Aug. 16; Luo et al. Int. J. Exp. Pathol. 90 (5): 558-574, 2009) and accelerates progression to invasiveness (Janssen et al. Gastroenterology. 131 (4): 1096-1109, 2006. Epub 2006 Aug. 16; Haigis et al. Nat. Genet. 40 (5): 600-608, 2008. Epub 2008 Mar. 30; Sansom et al. Proc. Natl. Acad. Sci. USA. 103 (38): 14122-14127, 2006. Epub 2006 Sep. 7), with a marked enhancement of tumor development in the relevant anatomical location of the colon (Luo et al. Int. J. Exp. Pathol. 90 (5): 558-574, 2009). Development of intestinal lymph node metastases has not been observed in Apc/Kras compound mutant mice, with distant liver metastases only detected in 20-27% of Apc/Kras compound mutant mice by transgene RT-PCR (Janssen at al. Gastroenterology. 131 (4): 1096-1109, 2006. Epub 2006 Aug. 16) or gross observation (Hung et al. Proc. Natl. Acad. Sci. USA. 107: 1565-1570, 2010). We generated ApcMin/+; KrasLSLG12D/+; Villin-Cre compound mutant mice (mice carrying a Cre-dependent activated allele of Kras (KrasLSLG12D) on the ApeMin/+ background, crossed with mice carrying a Villin-Cre transgene that directs expression of Cre recombinase throughout the intestine), and confirmed an enhancement of tumor development in the colon compared to ApcMin/+; Villin-Cre control mice (
Given that Apc/Kras compound mutant tumors exhibit features of early stage malignant progression (Janssen et al. Gastroenterology. 131 (4): 1096-1109, 2006. Epub 2006 Aug. 16; Haigis et al. Nat. Genet. 40 (5): 600-608, 2008. Epub 2008 Mar. 30; Sansom et al. Proc. Natl. Acad. Sci. USA. 103 (38): 14122-14127, 2006. Epub 2006 Sep. 7), we postulated that maintaining a compound mutant tumor in vivo beyond the shortened lifespan of an Apc/Kras mutant mouse might enable a realization of metastatic potential. We therefore aimed to transplant a single intact ApeMin/+; KrasLSLG12D/+; Villin-Cre donor tumor within the mucosal layer of a host wild-type C57BL/6 mouse colon, as this would faithfully represent an orthotopic primary colorectal tumor at clinical stage 0 with the potential to progress to stage IV disease. Several colon orthotopic transplantation techniques have been described to date, including the injection of cancer cell suspensions directly into the rectal mucosa (Donigan et al. Surg. Endosc. 24 (3): 642-647, 2010. Epub 2009 Aug. 18) or serosal wall of the cecum (Cespedes et al. Am. J. Pathol. 170 (3): 1077-1085, 2007), the instillation of tumor cell suspensions into the colonic lumen following electrocoagulation of the mucosa to promote tumor cell uptake (Bhullar et al. J. Am. Call. Surg. 213 (1): 54-60; discussion 60-61, 2011. Epub 2011 Mar. 31), and the surgical implantation of intact tumor fragments onto the serosal side of the cecal wall (Fu et al. Natl. Acad. Sci. USA. 88 (20): 9345-9349, 1991; Jin et al. Tumour. Biol. 32 (2): 391-397, 2011. Epub 2010 Nov. 19). One drawback of the cecum implant procedure is the fact that tumors are implanted on the serosal side of the cecal wall, thus bypassing the requirement of primary tumor invasion through the mucosa to the serosa for metastasis to occur. Importantly, a major caveat of all of these established techniques is the potential for inadvertent seeding of tumor cells into the peritoneal space, whether from a breach of the colon wall during tumor cell injection into the mucosa, or from the shedding of tumor cells from the cecal implant following return to the peritoneal cavity. Indeed, although these techniques have been utilized to generate mouse models of colorectal cancer (CRC) that reportedly develop metastases in the intestinal lymph nodes, liver and lungs, these techniques also give rise to widespread peritoneal carcinomatosis (Bhullar et al. J. Am. Call. Surg. 213 (1): 54-60; discussion 60-61, 2011. Epub 2011 Mar. 31; Cespedes et al. Am. J. Pathol. 170 (3): 1077-1085, 2007; Fu et al. Natl. Acad. Sci. USA. 88 (20): 9345-9349, 1991; Jin et al. Tumour. Biol. 32 (2): 391-397, 2011. Epub 2010 Nov. 19). It is thus plausible that tumor formation in these secondary sites may not be true metastases, but rather a result of peritoneal seeding. That peritoneal carcinomatosis is not a prominent feature of human metastatic CRC (Klaver et al. World. J. Gastroenterl. 18 (39): 5489-5494, 2012) further suggests a limited utility of these models for investigating routes of metastatic dissemination.
To circumvent these issues, we have developed a rectal prolapse induction technique that exteriorizes the lumen of the host colon, thus rendering the colonic mucosal surface amenable to surgical manipulation (
In an effort to shorten the time frame of malignant progression in our model so that routes of metastasis could be interrogated, we applied our lumen implantation technique to the poorly-differentiated HCT116 human CRC-derived cell line. HCT116 cells were transduced with the gene encoding the red fluorescent protein, DsRed, and implanted subcutaneously in a mouse to generate donor xenograft tumors. Following surgical implantation of donor tumor fragments of ˜10 mm3 onto the mucosal surface of host NOD/SCID mouse colons, ex vivo gross imaging (
Stage 0 polyps invariably progressed to stage I tumors, which breached the submucosal/muscularis externa layers, such that by 2-3 wpi invasive stage II adenocarcinomas that penetrated through the collagen IV-rich basement membrane of the muscularis externa (Vreemann et al. Biol. Chem. 390: 481-492, 2009) to reach the serosal side of the colon wall were evident (
Having developed lumen implantation as a viable technique for generating stage 0 colorectal tumors that progressed to stage II adenocarcinomas, we next assessed tumor-bearing mice for evidence of regional and/or distant metastatic progression corresponding to stage III/IIV disease. At 6-7 wpi, regional intestinal lymph node metastases were detectable as macroscopic tumor nodules located adjacent to the serosa within the draining lymphatic network that ran parallel to the colon wall (
We next determined if the lumen implantation procedure would yield similar progression and metastasis profiles if colorectal tumors of different origin were used as donors. To this end, we used the well-differentiated primary human CRC-derived LS174T cell line. We transduced LS174T cells with a lentivirus encoding DsRed, generated LS174T-DsRed subcutaneous tumors in a donor mouse, and transplanted donor tumor fragments onto the mucosal surface of host mouse colons. Similar to lumen-implanted HCT116 tumors, lumen-implanted LS174T tumors initially grew within the mucosal layer and eventually invaded through the colon wall, such that the bulk of the primary tumor burden was situated on the serosal side of the wall at the 8 wpi harvest endpoint (
Table 1 summarizes the tumor take rates following lumen implantation of colorectal donor tumors of various types and sources into host mice of various strains using the lumen implantation model (LIM) of CRC. Take rate is defined as the total number of host mice that have undergone successful transplantation of a donor tumor divided by the total number of host mice in which surgical transplantation was attempted, expressed as a percentage. These findings highlight the clinical relevance of disease progression in the LIM.
In the clinic, certain tumor types specifically metastasize to certain organs (Fidler et al. Nat. Rev. Cancer. 3 (6): 453-458, 2003). Indeed, CRC predominantly metastasizes to the regional intestinal lymph nodes, liver, and lungs, whereas other organs are largely spared (Chambers et al. Nat. Rev. Cancer. 2 (8): 563-572, 2002). To determine whether the LIM of CRC would accurately recapitulate the preferential target organ specificity observed in humans, we assessed metastatic tumor burden at 6-7 wpi in various internal organs by both macroscopic examination and DsRed-positive tumor cell flow cytometry. NOD/SCID mice bearing lumen-implanted HCT116-DsRed tumors preferentially developed metastases in the liver, lungs, and intestinal lymph nodes, with minimal metastatic burden detectable in the adrenal gland, kidney, spleen, brain, and bone marrow (
To determine if HCT116 cells were capable of metastasis regardless of implantation site, we implanted HCT116-DsRed cells subcutaneously in both NOD/SCID and NSG mice and assessed metastatic burden. Subcutaneously-implanted tumors did not readily metastasize compared to their lumen-implanted counterparts, in both NOD/SCID (
To date, no in vivo model of CRC has demonstrated predictable and reproducible distant metastatic outgrowth within relevant target organs from an orthotopically-established primary colorectal tumor (Heijstek et al. Dig. Surg. 22: 16-25, 2005. Epub 2005 Apr. 14; Kobaek-Larsen et al. Comp. Med. 50 (1): 16-26, 2000; Rosenberg et al. Carcinogenesis. 30 (2): 183-196, 2009. Epub 2008 Nov. 26; Taketo et al. Gastroenterology. 136 (3): 780-798, 2009). The lack of available models has precluded investigations into the route(s) of metastatic spread to distant organs. In the clinic, it is unknown whether colorectal metastases in the liver arise secondary to an initial colonization of the regional intestinal lymph nodes, or whether these liver metastases arise via direct hematogenous spread from the primary tumor, independent of lymph node metastatic growth (Bacac at al. Annu. Rev. Pathol. 3: 221-247, 2008). The first hypothesis is supported by the observations that (i) staging criteria are based on the degree to which the cancer has spread; clinical presentation of intestinal lymph node metastases alone is indicative of stage III disease while the presence of liver metastases are cause for the more advanced stage IV diagnosis (Schwartz et al. Am. J. Health. Syst. Pharm. 65 (11): S8-14, S22-24, 2008), (ii) high co-incidence has been reported for distant liver metastases and intestinal lymph node metastases (Derwinger et al. World. J. Surg. Oncol. 6: 127, 2008), (iii) primary tumor lymphatic vessel density correlates with metastasis to both the lymph nodes and liver (Saad et al. Mod. Pathol. 19 (10): 1317-1323. Epub 2006 Jun. 23), and (iv) tumor cell entry into the lymphatics is presumably easier than entry into the hematogenous vasculature due to a discontinuous basement membrane and a lack of pericyte coverage (Saharinen et al. Trends. Immunol. 25 (7): 387-395, 2004). Several observations support the second hypothesis, including (i) that some CRC patients present with liver metastases in the absence of lymph node involvement (Derwinger et al. World. J. Surg. Oncol. 6: 127, 2008), (ii) that surgical removal of an increased number of draining intestinal lymph nodes in stage III CRC patients may not improve overall survival (Prandi et al. Ann. Surg. 235 (4): 458-463, 2002; Tsikitis et al. J. Am. Coll. Surg. 208 (1): 42-47, 2009; Wong et al. JAMA. 298 (18): 2149-2154, 2007), and (iii) that venous invasion of the primary tumor is an independent prognostic indicator of distant liver metastasis development in CRC (Suzuki et al. Am. J. Surg. Pathol. 33 (11): 1601-1607, 2009). Having developed a LIM as a clinically-relevant model that recapitulates the metastatic tropism of human CRC, we were uniquely positioned to interrogate dissemination routes. In patients a correlation exists between the presence/absence of lymph node metastases and the presence/absence of liver metastases (Derwinger et al. World. J. Surg. Oncol. 6: 127, 2008). In both our NOD/SCID and NSG lumen implantation models, lymph node metastatic burden (both total number of involved lymph nodes and total DsRed-positive tumor cell burden within the lymph nodes) did not correlate with liver metastatic burden (
Given that vascular endothelial growth factors (VEGFs) play critical roles in primary tumor vascularization (Carmeliet et al. Nature. 473 (7347): 298-307, 201 1), with VEGF-A and VEGF-C primarily functioning to promote hematogenous and lymphatic vascularization, respectively (Adams et al. Nat. Rev. Mol. Cell. Biol. 8: 464-478, 2007; Oh et al. Dev. Biol. 188: 96-109, 1997), we assessed the effects of function-blocking antibodies against these factors on metastatic dissemination in our model. To this end, we utilized a neutralizing anti-VEGF-A antibody (Liang et al. J. Biol. Chem. 281 (2): 951-961, 2006. Epub 2005 Nov. 7) and generated an anti-VEGF-C antibody. NOD/SCID host mice were treated with antibodies once per week beginning one day prior to HCT116-DsRed or LS174T-DsRed tumor implantation, and metastasis formation was assessed at 6-7 wpi or 8 wpi, respectively. Anti-VEGF-A inhibited macroscopic metastasis formation in the liver (
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.
All patents, patent applications, patent application publications, and other publications cited or referred to in this specification are herein incorporated by reference to the same extent as if each independent patent, patent application, patent application publication or publication was specifically and individually indicated to be incorporated by reference. Such patent applications specifically include U.S. Provisional Patent Application Nos. 61/857,638, filed Jul. 23, 2013, and 61/954,788, filed Mar. 18, 2014, from which this application claims benefit.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US14/47860 | 7/23/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61857638 | Jul 2013 | US | |
| 61954788 | Mar 2014 | US |
