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WO2009076548A1 - Methods of inhibiting tumor development using adipose-derived regenerative cells - Google Patents

Methods of inhibiting tumor development using adipose-derived regenerative cells

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Publication number
WO2009076548A1
WO2009076548A1 PCT/US2008/086470 US2008086470W WO2009076548A1 WO 2009076548 A1 WO2009076548 A1 WO 2009076548A1 US 2008086470 W US2008086470 W US 2008086470W WO 2009076548 A1 WO2009076548 A1 WO 2009076548A1
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adipose
tissue
tumor
cells
cancer
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PCT/US2008/086470
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French (fr)
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Mark H. Hedrick
John K. Fraser
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Cytori Therapeutics, Inc.
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/35Fat tissue; Adipocytes; Stromal cells; Connective tissues
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/04Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08Peptides having 5 to 11 amino acids
    • A61K38/09Luteinising hormone-releasing hormone [LHRH], i.e. Gonadotropin-releasing hormone [GnRH]; Related peptides

Abstract

Provided herein are compositions that include adipose-derived regenerative cells, and methods of making and using the same, that are useful for the inhibition of tumor development.

Description

METHODS OF INHIBITING TUMOR DEVELOPMENT USING ADIPOSE- DERIVED REGENERATIVE CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application Serial No. 61/013, 442, filed on December 13, 2007, by Hedrick et al. and entitled "METHODS OF INHIBITING TUMOR DEVELOPMENT USING ADIPOSE-DERIVED REGENERATIVE CELLS," which is hereby expressly incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of medicine, specifically, to the study of cancer and the effect of adipose tissue and its components on tumor development.

BACKGROUND OF THE INVENTION

[0003] Autologous fat transfer in combination with adipose-derived regenerative cells ("ADRCs") is a promising therapy for women who have undergone surgery for breast cancer. Studies have shown that implanting freshly isolated, uncultured, ADRCs in combination with adipose tissue in a fat transfer procedure results in longer-term graft retention. In this use, implanted ADRC and adipose tissue are potentially brought into close contact with residual cancer cells. Therefore, an effect of implanted ADRC and/or adipose tissue on tumor cell proliferation could indicate a role of adipose tissue and its components in cancer recurrence.

[0004] A number of cancers, notably breast cancers, are estrogen-receptor- positive (ER+). Estrogen is involved in functions that include the development and proliferation of milk glands in the breast. It has been reported that estrogen has an oncogenic action in breast cells, and also that because it strongly stimulates proliferation it can cause growth of cells that have oncogenic mutations (Patel, et al., 2007, "Optimizing the antihormonal treatment and prevention of breast cancer," Breast Cancer 14(2): 1 13-122, incorporated herein by reference). The estrogen receptor is thus a target in treatments for estrogen-receptor-positive (ER+) cancers. [0005] Certain cytostatic factors have been reported to reduce tumor cell proliferation. For example, investigators have reported that interleukin-6 has the ability to reduce proliferation of hepatocellular carcinoma (Moran, et al., 2005, "Interleukin-6 inhibits cell proliferation in a rat model of hepatocellular carcinoma," Liver Int. Apr;25(2):445-57, incorporated herein by reference). Cytokines, including IL-2, IL- 12, GM-CSF, TNF, and the T-cell costimulatory molecule CD80, have been postulated to cause a systemic immune reaction in tumor cells (Takahashi, et al., "Gene Therapy for Breast Cancer. - Review of Clinical Gene Therapy Trials for Breast Cancer and MDRl Gene Therapy Trial in Cancer Institute Hospital," Breast Cancer 13(1):8-15), incorporated herein by reference.

[0006] Cells in adipose tissue express the estrogen receptor (see, e.g., Ijichi, et al., 2007, "Estrogen-related receptor alpha modulates the expression of adipogenesis-related genes during adipocyte differentiation," Biochem. Biophys. Res. Commun. 358(3):813-8). Adipose tissue also has been described as an important and highly active part of the immune system. As set forth in a review by Schaffler, et al., 2007, "Adipose tissue as an immunological organ: Toll-like receptors, Clq/TNFs and CTRPs," TRENDS in Immunology 28(9), doi:10.1016/j.it.2007.07.003, incorporated herein by reference, the following properties of adipocytes are evidence of the immunological importance of adipose tissue: adipocytes produce proinflammatory cytokines, e.g., IL-6, TNF, and chemokines, e.g., monocyte chemoattractant protein (MCP)-I; adipocytes produce adipokines, e.g., leptin, adiponectin, and resistin, that regulate monocyte and macrophage function and target various cells of the innate and adaptive immune system; preadipocytes can convert into macrophage- like cells, linking adipose tissue to the innate immune response; adipocytes produce molecules of the newly described Clq/TNF and ClqTNF-related protein (CTRP) superfamilies, which all belong to the innate immune system, and; preadipocytes and adipocytes express a broad spectrum of functional Toll-like receptors (TLRs). TLR3 and 4 expression on MSCs has been reported to potentially block the immunosuppressive activity of MSCs (Liotta, et al., 25 Oct 2007, "TLR3 and TLR4 are Expressed by Human Bone Marrow-Derived Mesenchymal Stem Cells And Can Inhibit Their T-cell Modulatory Activity by Impairing Notch Signalling," Stem Cells DOI: 10.1634/stemcells.2007-0454, incorporated herein by reference). [0007] Adipose-derived-regenerative cells (ADRCs) are a population of cells isolated from adipose tissue by methods that include eliminating mature adipocytes and free lipid. The ADRC cell fraction contains adult stem cells as well as other types of cells, including progenitor cells. Certain types of stem cells have been examined for their effect on cancer development. Many of the studies done to date report the stimulation of tumor growth in the presence of stem cells, based on research performed using a variety of experimental systems and cell types. For example, Karnoub, et al., 2007, "Mesenchymal stem cells within tumour stroma promote breast cancer metastasis," Nature 449:557-565; Ramasamy, et al., 2007, "Mesenchymal stem cells inhibit proliferation and apoptosis of tumor cells: impact on in vivo tumor growth," Leukemia 21 : 304-310, and; Djouad, et al., 2003, "Immunosuppressive effect of mesenchymal stem cells favors tumor growth in allogeneic animals," Blood 102:3837-3844, all report stimulation of tumor growth in the presence of mesenchymal stem cells (MSC). MSC inhibition of tumor growth has been reported by, e.g., Ohlsson, et al., 2003, "Mesenchymal progenitor cell-mediated inhibition of tumor growth in vivo and in vitro in gelatin matrix," Exp MoI Pathol 75:248-255, and Maestroni, et al., 1999, "Factor(s) from nonmacrophage bone marrow stromal cells inhibit Lewis lung carcinoma and B16 melanoma growth in mice," Cell MoI Life Sci 55:663-667, all incorporated herein by reference.

[0008] Although there is evidence that adipose tissue and its components are important in immunological and hormonal signaling, it has not been shown that implanting freshly isolated adipose tissue and/or ADRCs has an inhibitory effect on the growth or development of tumors.

SUMMARY OF THE INVENTION

[0009] The present invention relates to the unexpected finding that implanted adipose tissue and/or ADRCs inhibits the growth of tumor cells at the implant site. The invention provides methods for administration of adipose tissue and/or components of adipose tissue to treat a cancer patient, for example, a patient who has undergone surgery to remove cancerous tissue. In embodiments of the invention, adipose tissue and/or adipose- derived regenerative cells are administered to a breast cancer patient who has undergone surgery to remove cancerous tissue. In further embodiments, the adipose tissue and adipose- derived regenerative cells (ADRC) are administered in combination, to fill the defect generated by the surgery. The implant can inhibit the growth of neoplastic cells potentially remaining in the area, reducing the tumor regrowth or metastasis.

[0010] In an in vivo study, a striking reduction of tumor size in animals that received fat grafts or ADRC-supplemented fat grafts was observed. The study included three arms: (1) a baseline arm with fat grafting alone; (2) an arm in which the graft was supplemented with ADRC; and (3) an arm that received tumor implantation alone (no fat graft or ADRC). Surprisingly, tumor growth observed in either of the treatment groups (arms 1 or 2) was significantly reduced compared with tumor growth in the nontreatment group (arm 3).

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Analysis of Tumors Arising from MCF-7 Estrogen Receptor Positive Cells. The single bar shows the average tumor size observed when tumors were allowed to develop in the absence of ADRC and adipose tissue. The light bars in the paired sets show average tumor size observed when adipose tissue alone was implanted at the tumor site. The dark bars in the paired sets show the average tumor size observed when a combination of ADRC and adipose tissue was implanted. Each paired set represents implantation with ADRC and adipose tissue, or adipose tissue alone, from one of three human donors.

[0012] Figure 2 Analysis of Tumors Arising from MDA-MB-231 Estrogen Receptor Negative Cells. As described above with regard to the MCF-7 tumors, this graph shows the average tumor sizes that developed from estrogen-receptor-negative cells in the absence or presence of ADRC and adipose tissue, or in the presence of adipose tissue alone.

DETAILED DESCRIPTION OF THE INVENTION

[0013] Cancers treated using the methods of the invention include, but are not limited to, cancers that result in estrogen-receptor-positive (ER+) tumors. For example, treatment of cancers of the breast, uterus, endometrium, ovary, fallopian tube, cervix, vagina, liver, pituitary, central nervous system, hypothalamus, bone, skin, kidney, urethra, pancreas, and prostate, as well as melanoma, colorectal tumors, and desmoid tumors are contemplated. [0014] Other cancers which may be treated include, but are not limited to, fibrosarcoma, medullablastoma, and astrocytoma. It is to be understood, however, that the scope of the present invention is not to be limited to any specific type of cancer.

[0015] As described herein, growth of tumors arising from ER+ cell line MCF-7 is reduced in the presence of ADRCs and adipose tissue. In embodiments of the invention, expression of the estrogen receptor by implanted adipose tissue and/or ADRCs results in disruption of hormone-signaling pathways in hormone-dependent tumors, and reduced tumor growth. For example, expression of the estrogen receptor can result in sequestration of the estrogen in the adipose tissue. Lowering the availability of the hormone, e.g., progesterone or estrogen, to tumor cells, in turn can lower hormone-stimulated cell proliferation. In certain embodiments of the invention, administration of adipose tissue and/or ADRCs to patient with an ER+ cancer to reduce tumor growth is contemplated. In further embodiments, the treatment is provided in combination with a chemotherapeutic agent that disrupts hormone signaling in ER+ tumors.

[0016] Adipose-derived stem cells ("ADSCs") have also been reported to have inflammatory-modulating properties. (See, e.g., Mclntosh, et al., 2006, "The Immunogenicity of Human Adipose-Derived Cells: Temporal Changes In Vitro," Stem Cells 24; 1246- 1253.) Modulation of inflammation by ADSCs within a population of ADRCs can potentially inhibit tumor growth. Furthermore, factors including those described above are known to be expressed in ADRCs. For example, CD34+ isolated by flow cytometry from fresh ADRCs were found to express TLR3 at higher levels than CD45+ cells isolated from the same population (data not shown). In embodiments of the invention, adipose tissue and/or ADRCs express such factors that reduce tumor growth or metastasis in a cancer patient.

[0017] The invention contemplates treatment of patients including human patients. The term patient as used in the present application refers to all different types of mammals including humans and the present invention is effective with respect to all such mammals.

[0018] Citation of documents herein is not intended as an admission that any of the documents cited herein is pertinent prior art, or an admission that the cited documents are considered material to the patentability of the claims of the present application. All statements as to the date or representations as to the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates or contents of these documents.

Modes of Carrying out the Invention

[0019] It is to be understood that this invention is not limited to particular formulations or process parameters, as these may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting. Further, it is understood that a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.

Preparation of ADRCs and Adipose Tissue

[0020] In practicing the methods disclosed herein, the cells that are used to generate an adipose tissue-containing graft may be obtained from adipose tissue. Adipose tissue can be obtained by any method known to a person of ordinary skill in the art. For example, adipose tissue may be removed from a patient by suction-assisted lipoplasty, ultrasound-assisted lipoplasty, or excisional lipectomy. In addition, the procedures may include a combination of such procedures, such as a combination of excisional lipectomy and suction-assisted lipoplasty. Tissue may be obtained while the donor is living or dead, provided that the adipogenic cells remain viable. The tissue extraction should be performed in a sterile or aseptic manner to minimize contamination. Suction-assisted lipoplasty may be desirable to remove the adipose tissue from a human patient as it provides a minimally invasive method of collecting tissue with minimal potential for cell damage that may be associated with other techniques, such as ultrasound-assisted lipoplasty.

[0021] Means for obtaining adipogenic cells from adipose tissue have been described in the art. Most methods apply enzymatic digestion of washed adipose tissue fragments followed by centrifugation to separate buoyant adipocytes and debris from the non-buoyant cell fraction.

[0022] Adipose tissue processing can be performed by methods described in the literature and known to those of skill in the art, e.g., in U.S. App. Ser. No. 10/316,127 (U.S. Pub. No. 2003/0161816), entitled SYSTEMS AND METHODS FOR TREATING PATIENTS WITH PROCESSED LIPOASPIRATE CELLS, filed December 9, 2002, and U.S. App. Ser. No. 10/877,822 (U.S. Pub. No. 2005/0084961), entitled SYSTEMS AND METHODS FOR SEPARATING AND CONCENTRATING REGENERATIVE CELLS FROM TISSUE, filed June 25, 2004. The contents of both publications are expressly incorporated herein by reference.

[0023] For suction-assisted lipoplastic procedures, adipose tissue can be collected by insertion of a cannula into or near an adipose tissue depot present in the patient followed by aspiration of the adipose into a suction device. In one embodiment, a small cannula may be coupled to a syringe, and the adipose tissue may be aspirated using manual force. Using a syringe or other similar device may be desirable to harvest relatively moderate amounts of adipose tissue (e.g., from 0.1 ml to several hundred milliliters of adipose tissue). Procedures employing these relatively small devices have the advantage that the procedures can be performed with only local anesthesia, as opposed to general anesthesia. Larger volumes of adipose tissue above this range (e.g., greater than several hundred milliliters) may require general anesthesia at the discretion of the donor and the person performing the collection procedure. When larger volumes of adipose tissue are desired to be removed, relatively larger cannulas and automated suction devices may be employed in the procedure.

[0024] Excisional lipectomy procedures include, and are not limited to, procedures in which adipose tissue-containing tissue (e.g., skin) is removed as an incidental part of the procedure; that is, where the primary purpose of the surgery is the removal of tissue (e.g., skin in bariatric or cosmetic surgery) and in which adipose tissue can be removed along with the tissue of primary interest (e.g., extraction of perirenal or omental adipose during abdominal surgery). Subcutaneous adipose tissue may also be extracted by excisional lipectomy in which the adipose tissue is excised from the subcutaneous space without concomitant removal of skin. Harvesting adipose tissue via excisional lipectomy of the inguinal fat depot is contemplated when using adipose tissue from mice.

[0025] The adipose tissue that is removed from a patient or animal can be collected into a device for further processing.

[0026] The amount of tissue collected will be dependent on a number of variables including, but not limited to, the body mass index of the donor, the availability of accessible adipose tissue harvest sites, concomitant and pre-existing medications and conditions (such as anticoagulant therapy), and, in the case of research animals, the number of donors selected.

[0027] In some embodiments, the ADRCs or adipose tissue are autologous, and in some embodiments the ADRCs or adipose tissue are nonautologous, or a mixture of autologous and nonautologous ADRCs and/or adipose tissue. For transplantation of nonautologous tissue or cells, if desired, the patients can be treated with drugs to suppress the immune system. Appropriate methods and drugs are known to those of skill in the art.

[0028] If the ADRCs are to be mixed with adipose tissue for implanting, the ADRCs can be prepared from a volume of adipose tissue that is equal to the volume of the adipose tissue with which the ADRCs will be mixed for implantation i.e., a 1 :1 ratio. In general, this ratio, of the volume or weight of a first portion of adipose tissue to that of the second portion of adipose tissue (from which cells to be mixed with the first portion are obtained) can range from about 1 :5 to 5: 1, or any ratio in between, e.g., 1 :4, 1 :3, 1 :2, 1 :1, 2: 1, 3: 1, 4: 1 as preferred by the physician. In some embodiments, the adipose tissue can be mixed with about 1 x 104, 5 x 104, 1 x 105, 2 x 105, 3 x 105, 5 x 10s, 1 x 106, or more ADRCs or ADSCs.

[0029] It is understood by those of skill in the art that the amount of adipose tissue used for processing can depend, in part, on the amount of tissue available from the patient or donor.

Administration

[0030] Adipose tissue and ADRCs can be administered together or separately to a patient using methods known by those of skill in the art and described in the literature. (See, e.g., US 2005/025755, "Methods of using adipose tissue-derived cells in augmenting autologous fat transfer," incorporated herein by reference in its entirety.) In these methods, ADRCs and fat can be mixed and co-implanted. ADRCs alone can be provided locally or systemically, e.g., by injection. When provided systemically, the cells can be altered to incorporate homing/targeting strategies known in the art.

Combination Treatment [0031] Using the treatments of the present invention with other chemotherapeutic agents can allow administration of reduced dosages of the chemotherapeutic agents. A reduced dosage of an agent refers to a dosage lower than that typically administered for the single agent, wherein benefits of reducing the dosage are observed. Potential benefits include, e.g., a reduction in an undesirable side effect, e.g., nausea and vomiting, hair loss, osteoporosis, musculoskeletal pain, anemia, blood clots, occurrence of cancers, e.g., endometrial cancer, arising other sites, etc. Another benefit of reducing a drug dosage could be a delay in the time before a cancer becomes resistant to the drug or chemotherapeutic agent (e.g., tamoxifen). Other side effects of chemotherapeutic agents have been described extensively in the literature and are known to those of skill in the art. For example, potential side effects of antihormonal treatments are described by Patel, et al., 2007, incorporated herein by reference.

[0032] Methods for experimentally determining therapeutically-effective dosages of chemotherapeutic drugs and other agents for use in combination treatment regimens are described in the literature. For example, the use of metronomic chemotherapy dosing, i.e., providing more frequent, lower doses in order to minimize toxic side effects, has been described extensively in the literature. A combination treatment regimen encompasses treatment regimens in which administration of a chemotherapeutic agent is initiated prior to, during, or after treatment with the second agent, e.g., an antibody, and continues until any time during treatment with the other agent or after termination of treatment with the other agent. It also includes treatments in which the agents being used in combination are administered simultaneously or at different times and/or at decreasing or increasing intervals during the treatment period.

[0033] Combination treatment includes periodic treatments that start and stop at various times to assist with the clinical management of the patient. For example, an agent in the combination can be administered weekly at the onset of treatment, decreasing to biweekly, and decreasing further as appropriate.

[0034] Commonly used chemotherapeutic agents, and the preparation of such agents, are described, e.g., in U.S. Pat. No. 6,858,598, hereby incorporated by reference in its entirety. Chemotherapeutic agents useful in the methods of the present invention include, e.g., selective estrogen-receptor modulators (SERMS), including tamoxifen and raloxifene, that block the estrogen receptor. Other useful agents include aromatase inhibitors, which block the synthesis of estrogen, e.g., anastrazole, letrozole, and exeraestane. Use of estrogen receptor down-regulators, e.g., fulvestrant, is also contemplated. Goserelin, a long acting gonadotropin releasing hormone, can also be used with the therapies described herein. Agents used for treating ER+ breast cancers, and methods for administering and determining dosages of these agents, are known by those of skill in the art and described in the literature, e.g., by Patel, et al., 2007.

[0035] Other agents contemplated for use in the methods of the present invention include taxanes (i.e., taxol, docetaxel, paclitaxel), campthothecin (i.e., irinotecan, or CPT- 1 1), dacarbazine (DTIC), adriamycin, bleomycin, gemcitabine, cyclophosphamide, oxaliplatin, fludarabine, cisplatin and carboplatin. The phrase "taxane" includes a family of diterpene alkaloids all of which contain a particular eight-member "taxane" ring structure.

[0036] Gemcitabine is a pyrimidine antimetabolite with antineoplastic activity against a wide range of solid tumors including metastatic pancreatic carcinoma, non-small cell lung cancer, ovarian and breast cancer. It is sold under the trademark, GEMZAR™ by Eli Lilly and Company, Indianapolis, Ind. Its use and production are known in the art and are described, e.g., in U.S. Pat. Nos. 5,464,826, 5,912,366, and 6,001,994, incorporated herein by reference in their entirety, as well as in Kaye, J. Clin. Oncol. 12, 1527 (1994), and in Plunkett et al., Nucleosides Nucleotides 8, 775 (1989).

[0037] Irinotecan, a derivative of camptothecin, is also called CPT-I l . It is widely used as a first-line therapy for colorectal cancer. Methods for preparing and administering irinotecan are described in the art, e.g., in U.S. Pat. No. 4,604,463.

[0038] Methods for experimentally determining chemotherapeutic dosages in animal models prior to administering the drug to humans are known in the art and described in the literature. Chemotherapy doses for animals are normally expressed as mg/kg and those for humans in mg/m2. An approximate conversion factor for these units is 1 mg/kg to 35 mg/ mg/m2.

[0039] Single-therapy dosages in humans are described, e.g., in Cancer Management: A Multidisciplinary Approach (Medical, Surgical, and Radiation Oncology), eds. Pazdur R, Coia L R, Hoskins W J, Wagman L D (2000), Publisher PRR, Melville, N.Y., pp 984-988. For example, breast cancer in humans can be treated using taxol (or paclitaxel) at a dosage of 175 mg/m2 (5 mg/kg) repeated every 34 weeks, or 80-100 mg/m2 (2.3-2.9 mg/kg) per week. Paclitaxel is typically administered in a 15420 mg/m2 dose over a 6 to 24 hour infusion. For renal cell carcinoma, squamous carcinoma of head and neck, carcinoma of esophagus, small and non-small cell lung cancer, and breast cancer, paclitaxel is typically administered as a 250 mg/m2 24 hour infusion every 3 weeks. For refractory ovarian cancer paclitaxel is typically dose-escalated starting at 1 10 mg/m2. Docetaxel is typically administered in a 60-100 mg/m2 i. v. over 1 hour, every three weeks.

[0040] It should be noted that specific dose regimen depends upon dosing considerations based upon a variety of factors including the type of neoplasia; the stage of the neoplasm; the age, weight, sex, and medical condition of the patient; the route of administration; the renal and hepatic function of the patient; and the particular agents and combination employed, etc. Accordingly, in some embodiments, the ADRCs or ADSCs described herein are administered prior to, simultaneously with, or following treatment with a chemotherapeutic agent. For example, the ADRCs or ADSCs can be administered about 30 days, about 15 days, about 10 days, about 7 days, about 3 days, about 1 day, about 14 hours, about 7 sours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 30 min, prior to of following administration of a chemotherapeutic agent.

Tumor Development

[0041] Tumor development can be evaluated using a number of methods known to those of skill in the art and described in the literature, including methods described herein. For example, tumor size, metastatic potential, and effect on activation of factors important in regulation of tumor growth or invasion can be determined.

[0042] Tumor size is calculated using measurements of tumor dimensions. As described in the examples herein, a calculation can be made as follows: longest dimension x shortest dimension2 x 0.5 = tumor volume.

[0043] Metastases can be detected by imaging techniques known in the art, including X-ray, CT scan, or MRI. Methods for evaluating the effects of therapeutic agents at individual stages of metastasis have been described, e.g., by Glaves, et al., 1984, "Selective therapy of metastasis. I. Quantitation of tumorigenic circulating and covert cancer cells disseminated from metastatic and nonmetastatic tumors," Cancer Drug Delivery l(4):293-302. Glaves, et al., report quantitative studies on the total numbers and potential tumorigenicity of cancer cells in the circulation and also secondarily arrested in the lungs of mice following their spontaneous dissemination from the primary tumor.

[0044] Other parameters that can be used to evaluate tumor development include the activation of factors important in regulation of tumor growth or invasion, e.g., matrix metalloproteinases.

[0045] The contents of all cited references, including literature references, issued patents, published patent applications, and co-pending patent applications, cited throughout this application are hereby expressly incorporated by reference in their entirety.

Examples

[0046] The present invention is further illustrated by the following examples, which should not be construed as limiting in any way.

Example I: The Effect of Implanting Adipose-Derived Regenerative Cells Combined with ADRCs and Adipose Tissue, or Adipose Tissue Alone, on In Vivo Tumor Growth from ER+ Breast Cancer Cells

[0047] The in vivo growth of tumors arising from ER+ MCF-7 cells was reduced when animals were implanted with either adipose-derived regenerative cells combined with adipose tissue, or adipose tissue alone, at the site of the tumor. This finding suggests that adipose-derived regenerative cells and adipose tissue inhibit the development OfER+ tumors.

[0048] MCF-7 tumor cells (American Type Culture Collection) were grown in DMEM/F12 medium (a 50:50 mix of DMEM and Hams F-12 medium) supplemented with 10% fetal calf serum and antibiotic/anti-mycotic solution using the cell culture approach. They were harvested from tissue culture in log phase growth (<80% of confluence).

[0049] On the day of MCF-7 cell implantation the animals were anesthetized and the lower dorsal area was shaved and swabbed with alcohol. An incision of approximately 0.5-lcm was made in the midline skin and a 17β-estradiol pellet inserted into the dorsal abdominal subcutaneous space. The incision was closed with surgical adhesive and the animals were removed from the inhalation nose cone and monitored for recovery prior to being returned to their cage. [0050] To form the tumors, approximately 1x106 tumor cells were implanted in the right lateral mammary fat pad of immunodeficient mice (six to twelve week old female Fox Chase SCID mice). An incision of approximately 0.5- lcm was made in the skin over the right mammary fat pad. The fat pad was exposed by blunt dissection and tumor cells (approximately 1x106 MCF-7 cells or approximately 1x106 MDA-MB-231 cells) were injected in 0.05-0.1 mL PBS. The incision was then closed by use of a surgical adhesive and the animal was removed from the inhalation nose cone and monitored while it recovered prior to returning it to its cage.

[0051] Human adipose tissue was obtained from three individuals undergoing elective liposuction following informed consent. A portion of the tissue was washed with Lactated Ringers solution to remove the majority of free lipid and blood. The washed tissue was then placed in a sterile container inside a tissue culture hood and inspected to remove large fragments that might clog the injection syringe. Prepared tissues were then stored at room temperature until implantation. The remainder of the tissue was used to generate ADRC using methods described elsewhere herein.

[0052] Three days (Donor 1) or four days (Donors 2 and 3) following tumor implantation, human adipose tissue (approximately 0.2 ml) was implanted by creating a skin incision of approximately 0.5 cm adjacent to the incision sites use for tumor implantation. Blunt dissection was used to create a small subcutaneous pocket over the mammary fat pad and a blunt needle was extended into this pocket for delivery of vehicle control, adipose tissue alone, or adipose tissue plus cells. The animals designated to receive tumor only were injected with 0.2 ml saline. The animals designated to the adipose implantation arms received 0.2 ml of washed human adipose tissue. The animals designated to the adipose tissue plus ADRC implantation arms received 0.2 ml of washed human adipose tissue supplemented with 2-5 xlO5 cells ADRC. When a combination of ADRC and adipose tissue was implanted, both the ADRC and adipose tissue were obtained from the same donor. Adipose tissue and ADRC were implanted on the same day that the tissue was received in the laboratory.

[0053] At 8 weeks following tumor inoculation, animals were euthanized by exposure to carbon dioxide and subjected to necropsy. A midline incision was made through the ventral skin to expose the fat pads. The longest and shortest dimension of each tumor was measured using a caliper. Tumor volume was calculated according to the formula: longest dimension x shortest dimension2 x 0.5

[0054] The tumor sizes were averaged and are shown graphed in Fig. 1. The first bar, labeled on the x-axis as "Tumor Only," represents the size of the tumors that developed when no ADRC or adipose tissue was implanted at the tumor site. The light bars represent the tumor sizes measured when adipose tissue alone was implanted at the tumor site, and the dark bars show the tumor sizes measured when ADRC combined with adipose tissue were implanted. Animals deemed to have an inadequate implantation of tumor (due, for example, to observed leakage of cells from the mammary fat pad at the time of implantation) were euthanized and not included in the analysis.

[0055] The tumor volumes observed were significantly less in the treated implants as compared with the untreated implants. This result indicates that treatment of the tumors with ADRCs and adipose tissue, or adipose tissue alone, have an inhibitory effect on tumor cell proliferation.

[0056] The thoracic cavity was dissected and the heart and lungs removed and dissected to evaluate the presence of macroscopic metastasis in the heart and lungs. Resection of the lungs and heart was performed by resection of the ventral ribcage to expose the thoracic cavity. The heart and lungs were resected and each of these organs was subjected to gross evaluation for the presence of observable tumor masses. Each organ was sliced into sections of 2-5mm in thickness. Each slice of each organ was subjected to gross evaluation for the presence of observable tumor masses. The presence of metastases in each organ was recorded. There was no evidence of an increase in the frequency of detection of metastases in the treatment groups receiving ADRCs and adipose tissue or adipose tissue alone.

Example II: The Effect of Implanting Adipose-Derived Regenerative Cells Combined with ADRCs and Adipose Tissue, or Adipose Tissue Alone, on In Vivo Tumor Growth from ER" Breast Cancer Cells

[0057] In a similar experiment, the in vivo growth, of tumors arising from estrogen receptor-negative MDA-MB-231 cells, was shown to potentially be inhibited by adipose-derived regenerative cells combined with adipose tissue, or adipose tissue alone.

[0058] Using the same three tissue donors described in Example I, the effect of ADRC combined with adipose tissue, or adipose tissue alone, on the in vivo growth of tumors arising from estrogen receptor-negative human breast cancer cell line MDA-MB-231 was evaluated.

[0059] MDA-MB-231 tumor cells (American Type Culture Collection) were grown in DMEM/F12 medium (a 50:50 mix of DMEM and Hams F-12 medium) supplemented with 10% fetal calf serum and antibiotic/anti-mycotic solution using the cell culture approach. They were harvested from tissue culture in log phase growth (<80% of confluence).

[0060] As described in Example 1 with respect to the MCF-7 cell line, approximately 1x106 MDA-MB-231 tumor cells were implanted in the mammary fat pad of immunodeficient mice. Three to four days after implantation of the tumor cells, adipose (0.2 ml) plus ADRC (2-5x105 cells), or adipose alone, were implanted at the same site. The treatment plan is shown in Table 2.

[0061] The tumors were dissected and analyzed as described in Example 1, and the tumor sizes were averaged and graphed as shown in Fig. 2. As in Fig. 1, the bar labeled on the x-axis as "Tumor Only" represents the size of the tumors that developed when no ADRC or adipose tissue was implanted at the tumor site. The light bars represent the tumor sizes measured when adipose tissue alone was implanted at the tumor site, and the dark bars show the tumor sizes measured when ADRC combined with adipose tissue were implanted.

[0062] The tumor volumes observed were significantly less in the implants treated with Donor samples 2 and 3 as compared with the untreated implants. The sizes of the tumors arising from the Donor 1 treated implants were not as clearly affected by treatment.

[0063] As described in Example 1, metastasis to the heart and lungs was evaluated. There was no evidence of an increase in the frequency of detection of metastases in the treatment groups receiving ADRCs and adipose tissue or adipose tissue alone.

Example HI: The Effect of Implanting Adipose-Derived Regenerative Cells on In Vivo Tumor Growth from ER+ Breast Cancer Cells

[0064] In an experiment similar to that described in Example 1 , ER+ MCF-7 cells are implanted in immunodeficient animals and subsequently implanted with 2-5 x 106 adipose-derived regenerative cells at the site of the tumor. A finding of reduction in tumor size or metastasis in the treated animals relative to the untreated animals indicates that adipose-derived regenerative cells inhibit the development of ER+ tumors. Example IV: The Effect of Implanting Adipose-Derived Regenerative Cells on In Vivo Tumor Growth in a Patient following Breast Cancer Surgery

[0065] A patient with ER+ breast cancer undergoes surgery in which tumors and other suspected cancerous tissue are removed. Adipose tissue is removed from the patient's abdomen and half of this tissue is processed to yield autologous adipose-derived regenerative cells using methods described herein and known in the art. The regenerative cells are mixed with the remaining adipose tissue, and implanted during a fat transplant procedure at the site of surgery according to methods known and practiced by those of skill in the art.

[0066] Following implantation, the patient is treated with a chemotherapeutic agent at a reduced dosage that decreases the occurrence of negative side effects typically produced by the agent.

[0067] At three, six, nine, and twelve months following implantation, the patient is evaluated by for regrowth or metastasis of the tumor.

Claims

WHAT IS CLAIMED IS:
1. A method for inhibiting tumor growth in a patient, comprising administering at or near the site of the tumor a composition comprising adipose tissue, adipose-derived regenerative cells, or both, wherein tumor growth at or near the site of implantation, or tumor metastasis, is reduced.
2. The method of claim 1, wherein said cells or adipose tissue were obtained from the patient.
3. The method of claim 1, wherein said patient is treated with a chemotherapeutic agent or radiation therapy.
4. The method of claim 3, wherein said chemotherapeutic agent is administered at a reduced dosage.
5. The method of claim 1, wherein said patient has undergone surgery to remove cancerous tissue at the tumor site.
6. The method of claim 5, wherein said adipose-derived regenerative cells or adipose tissue were obtained from the patient.
7. The method of claim 5, wherein said patient is treated with a chemotherapeutic agent or radiation therapy.
8. The method of claim 7, wherein said chemotherapeutic agent is administered at a reduced dosage.
9. The method of claim 1 or 7, wherein the tumor is estrogen-receptor positive.
10. The method of claim 9, wherein said chemotherapeutic agent is a selective estrogen receptor modulator, an aromatase inhibitor, an estrogen-receptor down-regulator, or gonadotropin releasing hormone.
1 1. A method for fat transfer, in a patient with a tumor, comprising implanting at or near the site of the tumor a composition comprising adipose tissue, adipose-derived regenerative cells, or both, wherein recurrence of tumor growth at or near the site of implantation, or tumor metastasis, is inhibited.
12. The method of claim 11, wherein said cells or adipose tissue were obtained from the patient.
13. The method of claim 1 1 , wherein said patient is treated with a chemotherapeutic agent or radiation therapy.
14. The method of claim 13, wherein said chemotherapeutic agent is administered at a reduced dosage.
15. The method of claim 1 1, wherein said patient has undergone surgery to remove cancerous tissue at the tumor site.
16. The method of claim 15, wherein said adipose-derived regenerative cells or adipose tissue were obtained from the patient.
17. The method of claim 15, wherein said patient is treated with a chemotherapeutic agent or radiation therapy.
18. The method of claim 17, wherein said chemotherapeutic agent is administered at a reduced dosage.
19. The method of claim 11, wherein the tumor is estrogen-receptor positive.
20. The method of claim 19, wherein said chemotherapeutic agent is a selective estrogen receptor modulator, an aromatase inhibitor, an estrogen-receptor down-regulator, or gonadotropin releasing hormone.
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US9849149B2 (en) 2001-12-07 2017-12-26 Cytori Therapeutics, Inc. Methods of using regenerative cells in the treatment of erectile dysfunction
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US9597395B2 (en) 2001-12-07 2017-03-21 Cytori Therapeutics, Inc. Methods of using adipose tissue-derived cells in the treatment of cardiovascular conditions
US9872877B2 (en) 2001-12-07 2018-01-23 Cytori Therapeutics, Inc. Methods of using regenerative cells to promote epithelialization or neodermis formation
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