Cell culture media and method for culturing breast cancer cells

Abstract

A cell culture medium comprising adenosine triphosphate; a carrier protein; cholesterol, linoleic acid, and lipoic acid; glutathione; at least one nucleotide salvage pathway precursor base; phosphoethanolamine; selenium; transferrin; triiodothyronine; all-trans-retinoic acid (ATRA) and vitamin C; zinc, magnesium, and copper; an agent that increases intracellular cAMP; epidermal growth factor (EGF); hydrocortisone; insulin; and charcoal stripped fetal bovine serum, wherein said cell culture medium is substantially free, if not entirely free, of vitamin D, androgenic hormones, androgenic ligands, estrogenic hormones, estrogenic ligands, and/or androgenic receptors.

Claims

1. A cell culture medium comprising: (a) adenosine triphosphate; (b) a carrier protein; (c) cholesterol, linoleic acid, and lipoic acid; (d) glutathione; (e) at least one nucleotide salvage pathway precursor base; (f) phosphoethanolamine; (g) selenium; (h) transferrin; (i) triiodothyronine; (j) all-trans-retinoic acid (ATRA) and vitamin C; (k) zinc, magnesium, and copper; (l) an agent that increases intracellular cAMP; (m) epidermal growth factor (EGF); (n) hydrocortisone; (o) insulin; and (p) charcoal-stripped fetal bovine serum; and wherein said cell culture medium is substantially free of estrogenic hormones and estrogenic ligands.

2. The cell culture medium of claim 1, wherein said culture medium is substantially free of vitamin D.

3. The cell culture medium of claim 2, wherein said culture medium is substantially free of androgenic hormones and androgenic ligands.

4. A method of culturing breast cancer cells comprising, obtaining a sample of breast cancer cells from cancerous breast tissue, adding an amount of the cell culture medium of claim 3 to the sample of breast cancer cells, and maintaining the sample of breast cancer cells in the cell culture medium of claim 3 at conditions appropriate for cell growth.

5. A method of culturing breast cancer cells comprising, obtaining a sample of breast cancer cells from cancerous breast tissue, adding an amount of the cell culture medium of claim 2 to the sample of breast cancer cells, and maintaining the sample of breast cancer cells in the cell culture medium of claim 2 at conditions appropriate for cell growth.

6. The cell culture medium of claim 1, wherein said culture medium is substantially free of androgenic hormones and androgenic ligands.

7. A method of culturing breast cancer cells comprising, obtaining a sample of breast cancer cells from cancerous breast tissue, adding an amount of the cell culture medium of claim 6 to the sample of breast cancer cells, and maintaining the sample of breast cancer cells in the cell culture medium of claim 6 at conditions appropriate for cell growth.

8. A method of culturing breast cancer cells comprising, obtaining a sample of breast cancer cells from cancerous breast tissue, adding an amount of the cell culture medium of claim 1 to the sample of breast cancer cells, and maintaining the sample of breast cancer cells in the cell culture medium of claim 1 at conditions appropriate for cell growth.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) For a fuller understanding of the nature of the present invention, reference should be had to the following detailed description taken in connection with the accompanying drawings in which:

(3) FIGS. 1A and 1B show immunofluorescence images of normal breast tissue sections, demonstrating that the vitamin D receptor (VDR) is not expressed in proliferating breast cancer cells. Normal human breast tissue sections were stained with primary antibodies for Ki67, a marker for proliferating cells, and VDR and/or estrogen receptor (ER). In FIG. 1A, the cells that only express Ki67 are green and marks the proliferating cells. The cells that only express VDR are red. The overlap of these two fluorescent probes in the same cell would produce a yellow signal which is not detected, which indicates that notably VDR does not co-localize with Ki67. FIG. 1B illustrates an example of overlapping markers as a positive control in which some breast cells express both ER (green) and VDR (red), wherein the co-localized expression is are in yellow. Images were taken at approximately 200× magnification.

(4) FIGS. 2A and 2B show immunofluorescence images of normal breast tissue sections, demonstrating that the androgen receptor (AR) is not expressed in proliferating breast cells. Normal human breast tissue sections were stained with primary antibodies for Ki67, a marker for proliferating cells, and VDR and/or androgen receptor (AR). In FIG. 2A, the cells that only express Ki67 are red and marks the proliferating cells. The cells that only express AR are green. The overlap of these two fluorescent probes in the same cell would produce a yellow signal which is not detected, which indicates that notably AR does not co-localize with Ki67. FIG. 2B illustrates an example of overlapping markers as a positive control in which some breast cells express both AR (green) and VDR (red), wherein the co-localized expression is are in yellow. Images were taken at approximately 200× magnification.

(5) FIGS. 3A-3D show immunofluorescence images of breast cancer tissue sections, demonstrating that the Vitamin D receptor (VDR) is not expressed in proliferating cells. FIG. 3A shows a merged image of staining with VDR (in red), proliferative marker Ki67 (in green) and counterstaining with DAPI (in blue) to mark nuclei. FIG. 3B shows Ki67 alone, FIG. 3C shows VDR alone, and FIG. 3D shows the VDR and Ki67 merge without nuclear stain. Images were taken at approximately 100× magnification. As is clear from FIGS. 3A and 3D, there is no co-localization of VDR and Ki67, indicating the proliferating cells do not express AR.

(6) FIGS. 4A-4D shows immunofluorescence images of breast cancer tissue sections, demonstrating that the androgen receptor (AR) is not expressed in proliferating cells. FIG. 4A shows a merged image of staining with AR (in red), proliferative marker Ki67 (in green) and counterstaining with DAPI (in blue) to mark nuclei. FIG. 4B shows Ki67 alone, FIG. 4C shows AR alone, and FIG. 4D shows the AR and Ki67 merge without nuclear stain. Images were taken at approximately 100× magnification. As is clear from FIGS. 4A and 4D, there is no co-localization of AR and Ki67, indicating the proliferating cells do not express AR.

(7) FIG. 5 shows an immunofluorescence image of a normal breast tissue section. An antibody to recognize any of ER, AR, and VDR was used (denoted herein as HR, shown in green), and was co-stained with Ki67 (in red) and DAPI (in blue). Images were taken at approximately 200× magnification. Once again, there is no co-expression of any of ER, AR, or VDR and Ki67, indicating these receptors are not present in proliferating breast cancer cells.

(8) FIGS. 6A-6D show breast cancer cells under various conditions as imaged by DIC. FIG. 6A shows human breast cancer cells cultured in regular WIT-OCE medium that contains Vit-D and (0.1 mg/L) 2% fetal bovine serum that contains androgenic hormones in 5% O.sub.2. In this medium there are significantly fewer cells that are large with abundant cytoplasm compared to FIGS. 6B-6D, consistent with growth arrested cells and inhibition of cell proliferation by FBS and vitamin D. FIG. 6B shows human breast cancer cells cultured in WIT-BC that is free of Vitamin D and 2% charcoal stripped fetal bovine serum that is free of androgenic hormones at 5% O.sub.2. Compared to FIG. 6A there is a significant increase in cell numbers and the small cell size indicates minimal growth arrest. The growth conditions in FIGS. 6A and 6C were identical, except Vitamin D inhibitor (DLAMP2, 100 nM) was added to the culture medium in FIG. 6C, which partially reversed the growth inhibition observed in FIG. 6A. Routine cell culture is carried out at ambient air O.sub.2 which is 18-21%. We discovered that breast cancer cells are best cultured in lower O.sub.2 levels; less than 18% O.sub.2, but greater that 2.5% O.sub.2 (FIG. 6D). Compared to FIG. 6C there are fewer tumor cells in FIG. 6D, indicating that 5% 0.sub.2 levels are better than 2.5% O.sub.2. The optimal level was approximately 5-10% O.sub.2.

(9) FIG. 7 shows the inhibitory effect of VDR agonist on breast cancer cell proliferation. In this experiment six different breast cancer cell lines that represent the three major breast cancer subtypes were tested, including ER+ Breast Cancer (MCF7, T47D), HER2+ Breast Cancer (BT474, MDA-MB-453), and Triple Negative Breast Cancer (TNBC) (MDA-MB-468, SUM159). These cell lines were cultured both with and without VDR agonist calcitriol. The breast cancer cell lines were allowed to attach overnight and Calcitriol (100 nM) was added to the media the next morning. Media was refreshed every two days and cells were stained with 0.1% trypan blue and counted using a Cellometer for proliferation assays after 4 days.

(10) FIG. 8 shows the inhibitory effect of AR agonist on breast cancer cell proliferation. Four different breast cancer cell lines (T47D, ZR751, MCF7 and BT474) were cultured both with and without AR agonist R18811. The breast cancer cell lines were allowed to attach overnight in phenol-red free DMEM with 5% charcoal stripped fetal bovine serum. After 3 days, an AR agonist R1881 (10 nM) and 17 beta-Estradiol (10 nM) were added to the media. Cells were maintained in this medium for 4 days after which they were trypsinized, stained with 0.1% trypan blue and counted using a Cellometer. All the conditions were carried out in triplicate and the experiments were repeated at least twice.

(11) FIG. 9 shows immunofluorescence images of normal breast tissue sections, demonstrating that the estrogen receptor (ER) is not expressed in proliferating cells. The merged image shows staining with ER (in green) and the proliferative marker Ki67 (in red). Images were taken at approximately 100× magnification. As is clear there is no co-localization of ER and Ki67 which would produce a yellow signal, indicating the proliferating cells do not express ER.

(12) FIGS. 10A-10D shows immunofluorescence images of breast cancer tissue sections, demonstrating that the estrogen receptor (ER) is not expressed in proliferating cells. FIG. 10A shows a merged image of staining with ER (in green), proliferative marker Ki67 (in red) and counterstaining with DAPI (in blue) to mark nuclei. FIG. 10B shows ER alone, FIG. 10C shows Ki67 alone, and FIG. 10D shows the ER and Ki67 images merged without nuclear stain. Images were taken at approximately 100× magnification. As is clear from FIGS. 10A and 10D, there is no co-localization of ER and Ki67, indicating the proliferating cells do not express ER in this tumor. However, in some cases ER expressing cells and Ki67 cells overlapped in tumors (not shown); thus, depending of the tumor type we sometimes add estrogens in the medium and sometimes exclude it from the medium in ER+ tumors.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(13) The present invention is directed to a cell culture medium for use in culturing any type of breast cancer cell. Specifically, the cell culture medium includes a number of proteins, vitamins, minerals, compounds, and molecules to provide nutritive value to cells and promote growth. Notably, and unique to the instant invention, the cell culture medium utilizes charcoal stripped fetal bovine serum and is free of vitamin D. In another embodiment of the present invention the cell culture medium may be substantially free of vitamin D. These are the aspects that optimize the cell culture medium for breast cancer cell culture.

(14) More in particular, it has been discovered that the proliferating cells (Ki67+ cells) in human breast tumors were mutually exclusive with cells that expressed vitamin D receptor (VDR) and androgen receptor (AR) by immuno-staining of normal human breast tissue and human breast tumor tissues. Based on this information it was deduced that vitamin D and androgenic hormones might be growth inhibitory for human breast cancers and may be counter-productive for the in vitro culture of these cells. Based on the foregoing, the formulation of the WIT medium previously develop for human ovarian carcinomas has been modified in order to optimize it for human breast carcinomas.

(15) In brief, FIGS. 1A through 5 and FIGS. 9 through 10D illustrate the expression patterns of vitamin D receptor (VDR), androgen receptor (AR), and estrogen receptor (ER) in normal and malignant human breast tissue. FIGS. 6A through 8 illustrate the effect of the ligands for these receptors on normal and malignant breast cell proliferation in culture.

(16) These figures show that the proliferating Ki67 cells are mutually exclusive with vitamin D receptor (VDR) positive cells, both in normal breast (FIGS. 1A-1B) and breast cancer tissue (FIGS. 3A-3D). The Ki67 cells are also mutually exclusive with androgen receptor (AR) positive cells, both in normal breast (FIGS. 2A-2B) and breast cancer tissue (FIGS. 4A-4D). In addition, cells that are double AR/VDR positive were also mutually exclusive with proliferating cells (FIG. 5). The Ki67 cells are also mutually exclusive with estrogen receptor (ER) positive cells, both in normal breast tissue (FIG. 9) and breast cancer tissue (FIGS. 10A-10B).

(17) In at least some embodiments, the cell culture medium is substantially free of one or more components, and in at least one embodiment, “substantially free” refers to a low amount of the component that has no statistically significant effect on cell growth. In at least some embodiments, “substantially free” means less than 1%, 0.1%, 0.01%, 0.001%, or 0.0001% v/v of a liquid or w/v of a solute. In at least some other embodiments, “substantially free” means a concentration of less than 0.01, 0.001, 0.0001, 0.00001, 0.000001, or 0.0000001 mg/L, and in yet other embodiments, “substantially free” means a concentration of less than 10 nM, 1 nM, 100 pM, or 1 pM.

(18) To begin, the present cell culture medium may contain many ingredients already found in commercially available cell culture media. For example, in at least one embodiment the cell culture medium includes adenosine triphosphate (ATP) as an energy source for the cells.

(19) In at least one embodiment, the cell culture medium includes at least one carrier protein, certain hormones such as hydrocortisone and/or one or more growth factors such as insulin and EGF (epidermal growth factor).

(20) In at least one further embodiment, the cell culture medium includes one or more lipid synthesis precursors, such as cholesterol, linoleic acid, lipoic acid, and/or O-phosphoryl ethanolamine.

(21) In another embodiment, the cell culture medium also includes one or more antioxidant, such as, but not limited to, glutathione, glutathione (reduced), and/or vitamin C (ascorbic acid). In one further embodiment, the cell culture medium comprises one or more trace metals to supplement the antioxidant(s), such as, but not limited to zinc, selenium, chromium, copper, manganese and/or magnesium.

(22) At least one embodiment of the cell culture medium comprises at least one nucleotide salvage pathway precursor base including but not limited to hypoxanthine, xanthine, adenine, guanine and/or thymidine.

(23) Yet one further embodiment of the cell culture medium of the present invention comprises an amount of phosphoethanolamine.

(24) One other embodiment of the cell culture medium of the present invention comprises transferrin.

(25) At least one further embodiment of the cell culture medium includes triiodothyronine, and another embodiment comprises an amount of vitamin A.

(26) Another embodiment of the present cell culture medium comprises at least one agent that increases intracellular cAMP, and in one further embodiment, the agent that increases intercellular cAMP comprises cholera toxin.

(27) One embodiment of the cell culture medium of the present invention further comprises at least one epidermal growth factor (EGF).

(28) A further embodiment of the present cell culture medium includes an amount of hydrocortisone, and yet another embodiment comprises insulin.

(29) At least one embodiment of the cell culture medium in accordance with the present invention comprises fetal bovine serum (FBS), and in one preferred embodiment, the cell culture medium of the present invention comprises fetal bovine serum that has been filtered through activated charcoal, referenced herein as charcoal stripped fetal bovine serum (csFBS).

(30) The cell culture medium in accordance with the present comprises components which are substantially free of vitamin D, and in at least one embodiment, the cell culture medium of the present invention is itself substantially free of vitamin D.

(31) In at least one further embodiment, the cell culture medium of the present invention is substantially free of vitamin A, as Retinol, and in yet one further embodiment, the cell culture medium in accordance with the present invention comprises an amount of all-trans-retinoic acid (ATRA).

(32) The methods and compositions herein described and the related kits are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting. It will be appreciated that variations in proportions and alternatives in elements of the components shown will be apparent to those skilled in the art and are within the scope of embodiments of the present invention. Theoretical aspects are presented with the understanding that Applicants do not seek to be bound by the theory presented. All parts or amounts, unless otherwise specified, are by weight.

EXAMPLES

Example 1

(33) The base WIT cell culture medium contains 0.1 mg/L Calciferol (vitamin D2) and this medium is supplemented with 2-5% fetal bovine serum (FBS) to culture human ovarian carcinoma cells (WIT-OC medium). It was hypothesized that both of these components should be removed from the cell culture medium for the successful culture of human breast cancer cells because FBS contains vitamins and hormones such as vitamin D and androgens. Hence, FBS would inhibit the proliferation of breast tumor cells. Thus, charcoal stripped fetal bovine serum (csFBS) was utilized to culture breast tumor cells because activated carbon removes non-polar material such as lipophilic (lipid-related) materials (virus, certain growth factors, hormones and cytokines) regardless of molecular weight but has little effect on salts, glucose, amino acids, etc. The WIT-OC medium also contained vitamin A acetate (Retinol) which is a weaker form of vitamin A, compared to all-trans-retinoic acid (ATRA). In preliminary experiments, it was determined that this stronger form of vitamin A (ATRA) was promoting the growth of the breast tumor cells.

(34) Thus, in order to formulate a cell culture medium optimized to culture human breast tumor cells, as opposed to ovarian tumors, and other tumors, a WIT cell culture medium was prepared without any vitamin D, vitamin A, or androgens, and this medium was supplemented 100 nM ATRA, and 0.5-2% charcoal stripped fetal bovine serum (csFBS). We refer to this medium as WIT-BC, or WIT-BCe when estrogen is added.

(35) Interestingly, while complete serum does not inhibit proliferation of ovarian tumor cells, it did significantly reduce the proliferation of breast tumor cells. In addition, while a weak form of vitamin A (Retinol) was adequate to culture ovarian tumors, a stronger form of vitamin A (ATRA) was needed for the optimum culture if human breast tumor cells.

(36) Tables

(37) Table 1 presents the results obtained from tests conducted in accordance with Example 1.

(38) TABLE-US-00001 TABLE 1 WIT Ovarian Tumors Breast Tumors (base ER− ER+ ER− ER+ medium) WIT-OC WIT-OCe WIT-BC WIT-BCe Vitamin D + + + − − Vitamin A + + + − − (Retinol) All-trans − − − + + Retinoic Acid Fetal Bovine − + + − − Serum Charcoal − − − + + Striped Fetal Bovine Serum Estrogen − − + − +/−

(39) Since many modifications, variations and changes in detail can be made to the described preferred embodiment of the invention, it is intended that all matters in the foregoing description and shown in the accompanying drawings be interpreted as illustrative and not in a limiting sense. Thus, the scope of the invention should be determined by the appended claims and their legal equivalents.

REFERENCES

(40) Ochi, Y., Shiomi, K., Hachiya, T., Yoshimura, M., and Miyakai, T., Dextran-coated charcoal technique to make the hormone-free serum as a diluent for standard curve of radioimmunoassay. Endocrinol. Japan, 1971, 20(1), 1˜7.

INCORPORATION BY REFERENCE

(41) All publications, patents, and patent publications referenced herein are hereby incorporated by reference in their entirety as if each individual publication, patent, and patent publication was specifically and individually indicated to be incorporated by reference.