Reconstituted tumor microenvironment for anticancer drug development

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Extracellular matrix bioscaffolds capable of supporting the formation and growth of tumors from tumor cells introduced thereto containing tumor associated macrophages and carcinoma-associated fibroblast-like cells cultured under conditions effective to provide a cellular matrix capable of supporting the formation and growth of tumors from tumor cells introduced to the matrix. Bioscaffold kits and methods for using the bioscaffolds for testing, identifying and drug development of known or novel anticancer therapeutics are also disclosed.

The instant application claims 35 U.S.C. §119(e) priority to U.S. Provisional Patent Application Serial No. 61/212,794 filed April 15, 2009, the disclosure of which is incorporated herein by reference.

The present invention relates, generally, to an extracellular matrix bioscaffold that includes carcinoma-associated fibroblast-like cells and tumor associated macrophages supporting tumor cell growth and tumor formation, which may be used for the testing, identification and development of known or novel anticancer therapeutics.

Despite numerous advances and continuing research efforts, cancer is still one of the leading causes of human death worldwide. Understanding the environment surrounding cancer cell growth and motility is one avenue being explored and could provide useful information for the development of novel therapeutics. Current research suggests that cancer cell growth and invasion may be driven, at least in part, by the interactions between cells and a specific extracellular matrix (ECM). In the case of many epithelial-derived carcinomas, for example, a specialized ECM or basement membrane surrounds the primary tumor and is required for cell growth. With this in mind, many researchers have attempted to develop reconstituted basement membrane matrices that support cell growth in order to study the milieu of such carcinoma cells, as well as identify potential treatment target sites.

One such reconstituted basement membrane is set forth in U.S. Patent Nos. 4,829,000 and 5,158,874. The membrane, or "matrigel," set forth in these patents is rich in the extracellular matrix proteins laminin, collagen IV, heparan sulfate proteoglycans, entactin, and nidogen. It is formed by first extracting these components from Engelbreth Holm-Swarm (EHS) mouse sacrcomas, then heating and polymerized the extract to form a three dimensional matrix. Such a matrix is embodied within the biological membrane and cell culture reagent BD Matrigel™, available from BD Biosciences. Indeed, this product is widely used by researchers for studying carcinoma cell interaction and identifying putative chemo therapeutic agents.

Many current methods for novel therapeutic evaluation rely on the detection of a change in carcinoma cell growth and motility within or through such a matrix. In particular, methods of assaying the effects of such therapeutics involve measuring a decrease in the number of cells (or the absence of cells) that grow within or penetrate through the matrix upon application of the agent. The use of such models, however, have certain drawbacks. In a large majority of instances, for example, in vitro results using such biological membranes do not correlate well with follow-up tests in vivo. Some researchers surmise that this may be because the murine- based Matrigel™ does not adequately mimic the actual cancer cell microenvironment. Thus, the myriad of cell types, growth factors, chemokines, and other relevant proteins and communication molecules that are involved in tumor cell growth and invasion are almost entirely ignored in the artificial environment. Such a drastic change in the tumor mileu from in vitro to in vivo test conditions could be a major reason why there is such a high incidence of drug failure. Accordingly, there remains a need in the art for more accurately replicating the tumor milieu or microenvironment, both in vitro and in vivo, which would lead to improved testing methodologies for chemo therapeutic compounds.

Beyond just drug screening assays, however, the increased understanding in the genetic variability of cancer cells makes specific and targeted treatment of a particular carcinoma genotype and/or phenotype increasingly possible and more desirable. Again, one current limitation in doing so is the inability to culture cells within an environment that substantially mimics the microenvironment within the patient. To this end, a matrix is also desirable that would replicate such conditions outside of the patient for the purpose of testing and identifying the effects of one or more known or novel agents on those cells. Such a matrix would, in certain instances, be adaptable to current in vitro or in vivo testing methodologies and would ultimately contribute to a personalized therapeutic strategy in treating carcinoma growth and invasion.

The instant invention through its embodiments and examples addresses these needs.

In one embodiment, the instant invention provides an extracellular matrix bioscaffold in which . tumor associated macrophages and carcinoma-associated fibroblast-like cells are cultured under conditions effective to provide a cellular matrix capable of supporting the formation and g &r-1owth of tumors from tumor cells introduced to the matrix.

Carcinoma-associated fibroblast-like cells may include mesenchymal stem cells that are differentiated on a tumor conditioned medium. In a non-limiting embodiment, mesenchymal stem cells are differentiated on tumor conditioned medium for about 1 to 30 days. Resulting cells express stromal-derived factor- 1 or may otherwise be positive for one or more biological markers selected from α-smooth muscle actin, vimentin, and fibroblast surface protein.

Tumor associated macrophages of the instant invention refer to a population of leukocytes exhibiting a macrophage phenotype that promotes tumor cell proliferation, metastasis and/or angiogenesis, or otherwise promotes chemotaxis of MSCs. In one non-limiting embodiment the tumor associated macrophages are phorbol ester differentiated leukocytes, such as HL-60 cells or U937 cells. In further embodiments, the HL-60 cells or U937 cells are differentiated in the presence of 3 nM of TPA for 96 hours.

Tumors capable of being grown in the extracellular matrix of the invention include any tumor cell line provided herein or otherwise known, which may be formed by incubating the tumor cells on the matrix for about one to about four days. In one embodiment, the tumor cells include one or more biological reporter genes. Such biological reporter genes may encode a reporter selected from a green fluorescence protein, luciferase, and combinations thereof. To this end, the biological reporter genes may be provided on one or more expression vectors that are transfected or otherwise expressed within the tumor cells.

In a further embodiment of the instant invention, methods for assaying the efficacy of a chemotherapeutic compound against a tumor cell line are provided, wherein the compound is administered to an extracellular matrix bioscaffold according to the present invention within which tumors are grown from the tumor cell line, and the size of the tumors are measured after.