JP2851288B2 - Autocrine motility factors in cancer diagnosis and management - Google Patents

Description

TECHNICAL FIELD The present invention relates generally to the field of cancer diagnosis and management. More particularly, the present invention relates to novel tumor motility factors and their use in devising new approaches to cancer diagnosis, prevention and treatment.

BACKGROUND ART Cell motility is necessary for tumor cells to traverse many stages in a complex cascade of invasion and metastasis.
Such steps include detachment and subsequent infiltration of cells from the primary tumor to adjacent tissues, migration of cells through the vessel wall into the circulation (intravascular foreign body entry), and extravasation of cells into secondary sites. ing. The movement of cells through biological barriers, such as the endothelial basement membrane of the vasculature, can occur by chemotactic mechanisms. Studies on the chemotaxis of some tumor cells in vitro have shown that various compounds, such as complement inducers, collagen peptides, formyl peptides, and certain connective tissue components, can act as chemoattractants. Todaro et al. (Proc. Natl. Acad. Sci USA,
77 : 5258-5262, 1980) report transformed autocrine growth factors for cells. Various other growth factors are also known. However, the existence and role of autocrine factors that control the chemotactic (directional) and chemokinetic (random) motility of tumor cells has not been previously known or described. It may be important to note here that cell motility is an aspect of cell behavior that differs from cell growth and proliferation.

DISCLOSURE OF THE INVENTION It is therefore an object of the present invention to provide an autocrine factor that regulates motility of tumor cells (such an autocrine factor is referred to herein as "AM
F ") is identified and provided.

Still another object of the present invention is to provide an antibody having a specific binding affinity for AMF or AMF receptor.

Yet another object of the present invention is to provide a kit for detecting, localizing and predicting metastasis and tumor angiogenesis in humans.

Yet another object of the present invention is to provide a method for predicting, preventing and / or treating metastatic invasion and cancer growth in humans.

It is an additional object of the present invention to provide a pharmaceutical composition comprising an effective amount of a neutralizing antibody against AMF in a pharmaceutically acceptable carrier to suppress tumor cell motility.

Various other objects and advantages of the invention will be apparent from the specific description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS These and other objects, features and many of the attendant advantages of this invention will be better understood when reading the following specific description when considered in conjunction with the accompanying drawings: FIG. 1 shows a schematic diagram of the Boyden test, and FIG. 2 shows (a) ¹²⁵ I-binding to suspended tumor cells.
Scatchard analysis of AMF; and (b) Dose response curves of cell motility to purified AMF.

BEST MODE FOR CARRYING OUT THE INVENTION The above objects and various other objects and advantages of the present invention are achieved by a polypeptide having the following properties: (a) secreting and producing productive cells of mammalian cells; Stimulates random migration, (b) has a molecular weight>30,000; and (c) is inhibited by pertussis toxin. The polypeptide of the invention may be at least partially or wholly NH 3
_{At the 2} terminus (single letter code) or at the NH ₂ terminus of the active fragment of the polypeptide, it was found to have the following amino acid sequence order: DKELRFRDCTKSLAEANKK Unless otherwise specified, all technical and scientific terms used herein are Has the same meaning as commonly understood by those skilled in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned below are
Incorporated here as a reference.

Materials and MethodsCell lines Human MDA231 and MDA435 breast cancer cell lines were obtained from
Cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with% fetal calf serum. Both of these estrogen-independent cell lines produce metastases in the lungs of 6-week-old NIH nude mice 6 weeks after injection of 5 × 10 ⁵ cells into the lateral tail vein.

Isolation and Purification of Autocrine Motility Factor MDA231 and MDA435 human breast cancer cells are grown to 60% confluency in DMEM in the absence of added protein.
The medium was lyophilized and the residue is dissolved in distilled H ₂ O to about 2 ml. This solution is applied to a PD-10 (Sephadex G25 medium) column. Discard the first 2.5 ml and collect the next 4 ml. The effluent contains AMF separated from low molecular weight substances. The collected fraction is adjusted to pH with 10 × phosphate buffered saline (PBS).
Apply 7.4 0.02M formate buffered saline and apply to a Sephacryl S-300 column in PBS (column source). Elution with PBS yields an active fraction corresponding to a material having a molecular weight of about 54 KDa. This fraction is dialyzed and concentrated 25-fold. This substance was converted to 50 mM Tris-acetate having a pH of 8.0.
Apply to an anion exchange column (source) and elute with a linear salt gradient (0-1 M NaCl) with the following modifications: NaCl concentration 0.2
When 5M is reached, hold this concentration for 10 minutes before taking the gradient. AMF is eluted in the 0.3M-0.4M NaCl fraction. The active fraction is dialyzed and concentrated to a small volume (about 0.5 ml). The concentrate is made 0.02M phosphate in normal saline at pH 7.4. This is applied to a heparin column in PBS.
The column elutes AMF between 0.35 M and 0.4 salt gradient with NaC
Elute with a 1 linear gradient (0.15M to 1M). After each purification step,
The column fractions (dialyzed to remove salts) are assayed for motility stimulatory activity by the modified Boyden Chamber method.

Cell Motility Assays Motility assays are based on modified Boyden (Jigmund et al., J. Ex.
p. Med. 137: 387-410, 1973) achieved by use of a chamber. It was separated horizontally by a microporous polycarbonate filter having a pore size of about 8μ.
FIG. 1 shows an apparatus comprising one well (FIG. 1). A motility stimulant (or scientific attractant) is placed in the lower well and brought into contact with the filter. Add a suspension of cells (eg, A2085 melanoma cells) at a concentration of about 10 ⁶ cells / ml to the upper well. The chamber is then placed in a humidified incubator at 37 ° C. in an atmosphere of air and about 5% CO ₂ for about 4 hours. At this time, the cells are deposited on the upper side of the filter by gravity. However, some cells (about 5-10%) migrate below the filter in response to motility stimulants. Since the average diameter of the cells is larger than the pore size, energy expenditure must occur during migration. At the end of the culture period, the filters are removed and subjected to a fixed staining operation. This means that, first, the filter is
Immersion in an eosin solution for 2 minutes and then in a hematoxylin solution for 3 minutes. The filter is then washed in water and placed on a glass slide, top side up. The button on the upper side of the stained cells is completely removed with a small piece of dry tissue paper. Then, the stained cells that migrated through the filter were
It becomes clear. These are counted microscopically at approximately 500 × magnification. Five different high power fields are visualized with a grid in one eyepiece, the cells in the five fields are counted and the average is calculated. Positive control / negative control ratio ≧ 5 indicates a positive response of the cells to the motility stimulant.

Measurement of random and directed (chemotactic) motility Measurement of random motility is achieved by exposing cells to a fixed concentration of stimulant and measuring migration as described above. This involves adding equally increasing concentrations of attractant to both upper and lower wells prior to assay culture. The random migration of cells as a function of the amount of attractant is then measured. Directed migration occurs if cells migrate better on a positive gradient (higher concentration of attractant in the lower well compared to the upper well) than a negative gradient (higher concentration in the upper well than in the lower well). The result of such a test is called a “checkerboard”
It is shown in the table (Table 1). Random motility responds to AMF A
It turns out to be quite significant in the case of 2058 melanoma cells.

"Diagonal" indicates random movement of cells. The lower triangle indicates the directed migration of cells on a positive gradient of the motility stimulant.

Assay of cellular pathways involved in AMF-induced motility Materials: DMEM supplement with L-glutamine (2 μg), penicillin and streptomycin, with or without 10% heat-inactive fetal calf serum, commercially available sources such as And purchased from Melroy Laboratories, Inc. (Springfield, VA). Pertussis toxin and cholera toxin were obtained from List Biological Laboratories, Inc. (Campbell, CA). Phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate (PDD), calcium ionophore A23187, diltiazem, nifedipine, verapamil, trifluoperazine,
Leupeptin, forskolin and 8-Br cAMP are
All purchased from Sigma Chemical Company (St. Louis, MO). 1-Oleoyl-2-acetylglycerol was purchased from Molecular Probes (Eugen, OR). Nucleopore membranes (without polyvinylpyrrolidone) as well as 48-well chemotaxis chambers were purchased from Neuro Probes Inc. (Cabin Joan, MD).

Cell culture: The human melanoma cell line A2058 was maintained as described by Todaro et al., Supra.

Production of autocrine motility factors: In a modification of the technique (Liotta et al., Proc. Natl. Acad. Sci. USA 83: 3302-3306,
1986), A2058 cells were inoculated into DMEM without protein supplementation for 48 hours. The media was concentrated using a centricon ultrafiltration assembly with a molecular weight cutoff of 30,000 daltons.

Chemotaxis assay: Assays used to measure cell motility include those described by Harvas et al.
Year) was a modification of the technology described. This technology A2
According to 058, melanoma cells (approximately 75-90% of the total surface) were harvested with trypsin-EDTA and collected at room temperature for at least 1 hour in DMEM supplemented with 10% fetal calf serum. Then, 2 × 10 ⁶ ml cells in DMEM with bovine serum albumin 1mg / ml
And resuspended. Assays were performed in a 48-well microchemotaxis chamber with an 8 μm Nuclepore membrane coated with type IV collagen (Herbas et al., 1980, supra). The chamber was incubated at 37 ° C. for 4-5 hours and then developed using Diff Quick Stain (American Scientific). The stained membrane was placed on a glass slide with the original cell side up so that the cell pellet was blown off the surface. Cells that migrated through the pore were trapped between the glass and the membrane and could be easily counted by light microscopy at high power fields (500X). Unstimulated random movement was <20% of directed movement.

Prior to or during the chemotaxis assay, chemicals could be co-cultured with the cells to alter cell metabolism or stimulate a chemokinetic response. At the beginning of the assay, chemicals could also demonstrate chemotactic potential in addition to the lower chamber.

Production of murine antibodies against AMF Purified AMF protein (10 μg) was emulsified with Freund's complete adjuvant and injected into the toes of 3C3H mice. Two weeks later, mice were co-injected with 5 μg of AMF in PBS injected intravenously into the tail vein in a volume of 0.1 ml. One month later, mice were bled and sera were tested for their ability to suppress tumor cell motility. In this assay, mouse sera were pre-cultured with AMF in the Boyden Chamber transfer assay. 1/100
At a dilution of 0, the mouse serum had a 90% inhibition of tumor cell motility compared to the pooled mouse serum control
Occurred. Purified AMF protein (10 μg) was emulsified in Freund's complete adjuvant and injected subcutaneously on the back of a New Zealand white rabbit. 5 μg of the booster injection was applied at 6 and 12 weeks. At three and four months, the rabbits were bled and the sera were tested for motility inhibitory activity. At a dose of 1/1000, the immune serum was
Motility was destroyed compared to control pre-immune sera. Serum was heat inactivated at 56 ° C. for 30 minutes.

Determination of AMF Purity The purity of the isolated AMF was determined by the following criteria: (a) Single 54 kDA band was analyzed by one- and two-dimensional polyacrylamide gel electrophoresis performed by standard methods well known in the art. Was found. The protein was identified with a silver stain.

(B) The protein band cleaved from the gel retains motility stimulating activity.

(C) NH ₂ terminal amino acid sequence order (1 to 19) shows one type of amino acid residues in each cycle.

(D) Murine and rabbit anti-AMF antibodies block the motility stimulating activity of human tumor AMF.

Based on the above criteria, the isolated AMF of the present invention was found to be substantially pure. The term "substantially" as used herein means as pure as possible to obtain by standard techniques.

Amino acid sequence sequencing Edman degradation of purified AMF is performed on an Applied Biosystems (Foster City, CA) model 470A gas phase sequencer using trifluoroacetic acid chemistry provided by the manufacturer. Phenylthiodantoin amino acids were identified and quantified by using Parking-Elmer series 3B HPLC and UV detection.

Dose response and effect on time course and motility of pertussis toxin: Pertussis toxin (PT) for overnight culture in flask, for preculture for various periods before assay,
Alternatively, they were added to A2058 at different times after the start of the assay. The PT dose tested was about 10 ng / ml to 1.5 μg / ml. Cell viability at any of the doses tested was comparable to viability in untreated controls (> 90%). The treated and untreated cells were then tested for motility response to A2058 conditioned media. Cell motility in response to DMEM alone was included as a negative control for each treatment group of cells.

Overnight culture of cells at any of the test PT doses resulted in significant suppression of cell motility (Table 2). Preculture for 30 minutes to 2 hours at a dose of 0.5-1.5 μg / ml also resulted in greater than 50% suppression. There was a gradual decrease in the inhibitory effect when pertussis toxin was added at the beginning or after the start of the assay. One to two hours after starting the assay, PT had a minimal effect on the observed motility.

Dose response of the PT is consistent with the inhibition of PT in the case of G _i and G ₀ proteins. The time course showed much reduced inhibition when PT was added at an inappropriate dose or for insufficient time to saturate the G protein site. Thus, the data obtained in this study was consistent with the hypothesis that AMF stimulates cell motility through receptors that require a G protein to activate the cells.

Cholera toxin dose response and time course: pertussis toxin as opposed to cholera toxin (CT) is believed to act on the G _s proteins that stimulate adenylate cyclase to produce second messenger, the cAMP. Cholera toxin was added to A2058 cells for overnight culture in flasks or for a variable period of pre-culture before the start of the chemotaxis assay. Test doses of cholera toxin were approximately 0.1-50 μg / ml. At all doses tested, cell viability was comparable to that of untreated cells (> 90%). The treated and untreated cells were then tested for a chemotactic response to A2058 conditioned media.

The suppression effect is not perfect (suppression rate 30-60%)
However, overnight treatment with CT reduced the response to A2058 conditioned media. If the cells are exposed to cholera toxin for a short period of pre-culture just prior to the start of the chemotaxis assay, suppression is
It was minimal (<5%).

Other agents effective encompassed adenylate cyclase system for cell motility: cholera toxin, ADP of G _s protein activity arrangement that can stimulate adenylate Ikuraze
It is thought to work by ribosylation. Because the effect of cholera toxin on A2058 cell motility was minimal, additional tests were performed to determine if other drugs acting on the cAMP pathway were inhibitory. Forskolin
Stimulates adenylate cyclase directly without acting through the mediator G protein. The cAMP analog, 8-Br cAMF, can enter native cells. Both chemicals
A2058 cells were added for overnight culture in flasks or for a 2 hour pre-culture prior to the onset of chemotaxis. Both showed only a partial inhibition of cell motility which was essentially identical to the inhibition of cholera toxin for comparable times.

Since these cells respond in a dose-dependent manner to various concentrations of conditioned medium obtained by culturing confluent cells in serum-free medium, the motility factor is:
It was concluded that it was derived from cells. The results obtained in the modified Boyden chamber experiment show that the autocrine factor of the present invention has chemotaxis (directivity) and chemokinesis (random motility).
It also demonstrates having both. Since the random stimuli were found to be about three times greater than directed motility, it was concluded that the cells responded to the motility factor gradient as well as to a high uniform concentration of attractant.

When measured by gel filtration and gel electrophoresis, the migration stimulator of the present invention was found to have a molecular weight of about 54 kilodaltons. This form may be a precursor of the active agent. Cellular or serum components are capable of activating or suppressing the action of motility factors. Motility factors are inactivated by exposure to streptococcal proteases, but active chymotrypsin-derived fragments can be produced (data not shown). Activity is destroyed by boiling, but is stable upon exposure to 56 ° C.
Additionally, the activity is stable in the pH range 4-11 (data not shown). These properties indicate that the autocrine substances (AM
F) shows that it differs from various known growth factors and chemoattractants. Also, known growth factors such as PDG
F, αTGF, βTGF, EGF, IGF, transferrin or F
GF was not found to displace or block AMF (data not shown). Amino acid analysis revealed a unique sequence order of the 19 amino-terminal amino acids of AMF. The slightly smaller form of the active substance was also found to have a unique amino-terminal sequence order. Protein database searches failed to show other polypeptides with such sequence order.

Motility induction by AMF was also found not to be blocked or replaced by known growth or serum factors. At concentrations below 1 nM, AMF significantly stimulates random and directed motility of breast cancer cells,
Failing to induce motility in leukocytes. The factor is
It stimulated random pseudopodia production by breast cancer cells and melanoma cells. After transfection with an active ras oncogene, AMF and its receptor are enhanced more than 100-fold in certain cells. Human breast cancer cells (as opposed to normal breast epithelium) produce large amounts of AMF. Antibodies that recognize AMF
It destroys human tumor cell motility in vitro without altering tumor cell viability.

The availability of an isolated and purified autocrine polypeptide tumor motility factor makes it possible to obtain anti-AMF antibodies with specific binding affinity for said motility factor. Such antibodies can be either polyclonal or monoclonal and are produced by standard techniques routes well known in the art. Also, such antibodies can be labeled with a suitable radioisotope or fluorophore and other markers or ligands and used for the detection, quantification and / or localization of AMF in human tissues or body fluids. In addition, radiolabeled AMF along with unlabeled AMF can be used in label competition assays to measure AMF receptor levels. Such a binding assay to determine the amount of receptor is performed as follows.

AMF binding assay: Purified AM using standard Bio-Rad enzyme bead method
F is isolated. Increasing amounts of labeled AMF are cultured in a volume of 1 ml with 100,000 A2058 melanoma cells in the presence or absence of a 100-fold excess of cold competitor. The cultivation is performed at 37 ° C. for 40 minutes and the cell-bound radioactivity is separated by centrifugation. AMF
Binding shows 80% specific binding and saturation at approximately 30,000 receptors / cell. Scatchard analysis according to the standard method shows a linear relationship between the specifically bound / release ratio and the specifically bound AMF with an estimated kd in the range of about 0.5 nM.

Also, the detection of cancer in humans is enabled by the present invention and now uses human urine samples from bladder cancer patients to illustrate the testing of human body samples for this purpose.

Urine samples from bladder cancer patients are collected and processed on a centrifugal microconcentrator (AMFICON) with a 10 kilodalton exclusion filter. Treated urine is reconstituted at 10-fold concentration with sterile phosphate buffered saline at pH 7.5 and stored at -20 ° C until use. Tumor grade is determined by a pathologist using a scale of 1-3. Grade 1 tumors show the largest differentiation and Grade 3 tumors show the smallest differentiation. American Joint Committee TN for bladder tumor
Grade according to the N Classification.

Urine Sample Assay: Although any cell line that responds to AMF can be used, the preferred cell line is human MDA435 cells (ATCC). The concentrated urine sample is applied to a microwell transfer chamber assay as described above. Each sample is tested at a series of dilutions with and without the addition of antibodies directed against human tumor AMF. AMF units are recorded as the percentage of tumor cells stimulated to migrate by the sample suppressed by the antibody. Generally, 80
% Stimulation migration is suppressed by an antibody concentration of about 10 μg / ml.

As shown in Table 3, control urine with non-neoplastic disorders such as kidney stones failed to contain significant amounts of motility factors. All cases of bladder migrating cell carcinoma showed a positive motor response in urine. The highest amount of motility factor production was found in the urine of patients with high-grade or stage D (metastatic) tumors. Table 3 Urine sample AMF unit SE 1 Control ks75 5 0.5 2 Control ks76 9 23 Tumor in situ 32 5 4 Papillary TCC 64 85 TCC77 44 36 TCC69 98 147 TCC73 123 328 Recur TCC79 130 22 9 TCC II485 169 14 10 TCC II491 105 8 11 TCC II554 41 12 12 TCC III457 72 6 13 TCCstgD584 234 25 TCC = moving cell carcinoma of the bladder Recur TCC = Recurrent TCC TCC II = Grade II TCC III = Grade III TCCstgD = Metastatic TCC KS = Nephrite SE = standard error AMF unit is the total number of migrating cells per filter.
It refers to the number obtained by subtracting the number of pre-migrating cells around the filter in the presence of anti-AMF serum.

Of course, antibodies to AMF can be used to block or suppress AMF activity, thereby preventing tumor invasion or metastatic growth dependent on tumor cell motility. Also, the availability of such neutralizing antibodies is improved by administering an effective amount of AMF-antibody to those afflicted with such conditions as breast cancer, melanoma, etc., to prevent these conditions from progressing. And to treat such conditions. Pharmaceutical compositions for treating cancer and metastases simply comprise an effective amount of a neutralizing antibody against AMF and a pharmaceutically acceptable carrier such as saline, non-toxic buffers, etc., to inhibit tumor cell motility. Prepared by blending.

Means for detecting tumor aggressiveness and / or metastatic activity include (a) an antibody having a specific binding affinity for AMF; (b) labeled AMF; and (c) AM in a body sample.
This is now enabled by a kit consisting of educational material and a separate container containing unlabeled AMF to perform the test utilizing the antibody and AMF provided in the kit for measuring F and / or receptor activity. Accessories such as microtiter plates, micropipettes, and devices for reading antibody titers are typically found in such kits and may be conveniently incorporated into kits of the invention.

In summary, the present invention provides new tools for understanding the mechanisms that control tumor cell invasion and opens new strategies for cancer diagnosis and treatment. Epithelial cells usually do not show invasive behavior. The motility factors described herein do not affect normal leukocyte migration. Therefore, therapeutics aimed at suppressing the factors described in the present invention should have low toxicity to normal resting tissues. The pharmacological formulation obtained according to the present invention, which suppresses tumor cell invasion and prevents metastasis from in situ to invasive cancer, can be a potent cancer inhibitor. Also, inhibitors of tumor invasion can prevent the growth of established metastases, as metastases may need to enter locally as they grow. Further, such agents may inhibit tumor angiogenesis. Antibodies to motility factors or their receptors can be applied by histoimmunohistology, radiosinography, or serum immunoassay to localize metastases and predict cancer aggressiveness in individual patients. As a gene product, autocrine motility factors or their receptors define a new class of oncogenes. The expression levels of these genes in a patient's tumor can provide important diagnostic information by monitoring the amount of AMF in a body sample.

Of course, invasion and metastasis are a major cause of cancer treatment failure. The present invention provides (a) to detect pre-invasive lesions and prevent their progression; (b) to accurately predict the aggressiveness of a patient's tumor; and (c) to identify and eradicate micrometastases To provide new clinical strategies. One of the at least understood aspects of tumor invasion is tumor cell migration. The present invention allows for the determination of the role of tumor cell motility factors.

The examples and embodiments described herein are for illustrative purposes, and various modifications or alterations thereto will be suggested to those skilled in the art and the spirit and authority of the present application and the scope of the appended claims It is understood that they should be included within.

Continued on the front page (72) Inventor Schiffman, Elliott Maryland, USA, Chevy, Chase, Pickwick, Rain, 3207 (56) References Proc. Natl. Acad. Sc i. , U.S. S. A, 83 (1986), p. 3302-3306

Claims (6)

(57) [Claims] Contacting a body sample taken from a patient suspected of having cancer with an autocrine tumor motility factor (AMF) sensitive cell line to determine the motility of the AMF sensitive cell line; and (2) Contacting an antiserum containing an AMF antibody with a body sample with an AMF-sensitive cell line to measure the motility of the AMF-sensitive cell line, including detecting the presence of cancer in a human patient. Wherein the difference in motility measured in (1) and (2) indicates that cancer is present in the patient. 2. The method according to claim 1, wherein the body sample is urine. 3. The method according to claim 1, wherein the cancer is urinary tract cancer. 4. The method according to claim 1, wherein the cancer is bladder cancer. 5. The method according to claim 1, wherein the cancer is a mobile epithelial cancer. 6. The method of claim 1, wherein the AMF-sensitive cell line is a human MDA435 cell.