U.S. patent application number 11/650782 was filed with the patent office on 2007-05-17 for composition of restricted cancer cells which produce cancer cell proliferation suppressive materials, and uses thereof.
Invention is credited to Shirin Asina, Kanti Jain, Albert L. Rubin, Barry Smith, Kurt Stenzel.
Application Number | 20070110774 11/650782 |
Document ID | / |
Family ID | 34139535 |
Filed Date | 2007-05-17 |
United States Patent
Application |
20070110774 |
Kind Code |
A1 |
Asina; Shirin ; et
al. |
May 17, 2007 |
Composition of restricted cancer cells which produce cancer cell
proliferation suppressive materials, and uses thereof
Abstract
Compositions of matter are described which contain restricted
cancer cells. When so restricted, the cells produce an unexpectedly
high amount of material which suppresses cancer cell proliferation.
The phenomenon crosses cancer type and species lines. Processes for
making these compositions, and their use, are also described.
Inventors: |
Asina; Shirin; (New York,
NY) ; Jain; Kanti; (New York, NY) ; Rubin;
Albert L.; (Englewood, NJ) ; Smith; Barry;
(New York, NY) ; Stenzel; Kurt; (New York,
NY) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
666 FIFTH AVE
NEW YORK
NY
10103-3198
US
|
Family ID: |
34139535 |
Appl. No.: |
11/650782 |
Filed: |
January 8, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10919767 |
Aug 16, 2004 |
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11650782 |
Jan 8, 2007 |
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10336313 |
Jan 3, 2003 |
6818230 |
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10919767 |
Aug 16, 2004 |
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08745063 |
Nov 7, 1996 |
5888497 |
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10336313 |
Jan 3, 2003 |
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08625595 |
Apr 3, 1996 |
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08745063 |
Nov 7, 1996 |
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Current U.S.
Class: |
424/277.1 |
Current CPC
Class: |
A61K 2035/126 20130101;
C12N 11/04 20130101; A61K 2035/128 20130101; A61K 35/22 20130101;
C12N 5/0693 20130101 |
Class at
Publication: |
424/277.1 |
International
Class: |
A61K 39/00 20060101
A61K039/00 |
Claims
1-53. (canceled)
54. A conditioned culture medium which has a cancer cell
proliferation-inhibiting effect produced by a process comprising
the steps of entrapping cancer cells in a biocompatible,
selectively-permeable structure comprising agarose, culturing the
entrapped cancer cells in culture medium to restrict proliferation
of said entrapped cancer cells, wherein growth of said cancer cells
is restricted by entrapment in said structure so that they produce
a material that suppresses the proliferation of cancer cells which
permeates through said structure into said culture medium to
produce conditioned culture medium, and collecting the conditioned
culture medium.
55. The conditioned culture medium of claim 54, wherein said
biocompatible, selectively-permeable structure is a bead.
56. The conditioned culture medium of claim 55, wherein said bead
is a solid, agarose coated, agarose containing bead.
57. The conditioned culture medium of claim 54, wherein said
material has a molecular weight of at least about 30 kd.
58. The conditioned culture medium of claim 54, wherein said
entrapped cancer cells are epithelial cells.
59. The conditioned culture medium of claim 54, wherein said
entrapped cancer cells are breast cancer cells, renal cancer cells,
prostate cancer cells or choriocarcinoma cells.
60. The conditioned culture medium of claim 54, wherein said
entrapped cancer cells are human cancer cells.
61. The conditioned culture medium of claim 54, wherein said
entrapped cancer cells are mouse cancer cells.
62. The conditioned culture medium of claim 54, wherein said
structure contains from about 10,000 to about 500,000 cancer
cells.
63. The conditioned culture medium of claim 54, wherein said
structure contains from about 30,000 to about 250,000 cancer
cells.
64. A frozen conditioned culture medium which has a cancer cell
proliferation-inhibiting effect produced by a process comprising
the steps of entrapping cancer cells in a biocompatible,
selectively-permeable structure comprising agarose, culturing the
entrapped cancer cells in culture medium to restrict proliferation
of said entrapped cancer cells, wherein growth of said cancer cells
is restricted by entrapment in said structure so that they produce
a material that suppresses the proliferation of cancer cells which
permeates through said structure into said culture medium to
produce conditioned culture medium, recovering the conditioned
culture medium and freezing the recovered conditioned culture
medium.
65. The frozen conditioned culture medium of claim 64, wherein said
biocompatible, selectively-permeable structure is a bead.
66. The frozen conditioned culture medium of claim 65, wherein said
bead is a solid, agarose coated, agarose containing bead.
67. The frozen conditioned culture medium of claim 64, wherein said
material has a molecular weight of at least about 30 kd.
68. The frozen conditioned culture medium of claim 64, wherein said
entrapped cancer cells are epithelial cells.
69. The frozen conditioned culture medium of claim 64, wherein said
entrapped cancer cells are breast cancer cells, renal cancer cells,
prostate cancer cells or choriocarcinoma cells.
70. The frozen conditioned culture medium of claim 64, wherein said
entrapped cancer cells are human cancer cells.
71. The frozen conditioned culture medium of claim 64, wherein said
entrapped cancer cells are mouse cancer cells.
72. The frozen conditioned culture medium of claim 64, wherein said
structure contains from about 10,000 to about 500,000 cancer
cells.
73. The frozen conditioned culture medium of claim 64, wherein said
structure contains from about 30,000 to about 250,000 cancer
cells.
74. The conditioned culture medium of claim 55, wherein said bead
is a solid, agarose and collagen, agarose coated bead.
75. The frozen conditioned culture medium of claim 65, wherein said
bead is a solid, agarose and collagen, agarose coated bead.
Description
RELATED APPLICATION
[0001] This application is a continuation in part of allowed patent
application Ser. No. 08/745,063, filled on Nov. 7, 1996, which is a
continuation-in-part of co-pending application Ser. No. 08/625,595,
filed Apr. 3, 1996 now abandoned. Both are incorporated by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to the restriction of the
proliferation of cancer cells to produce material which suppresses
proliferation of unrestricted cancer cells. The structures which
are one feature of the invention can be used "as is," or to produce
material such as concentrates with a minimum approximate molecular
weight, which also have an anti-proliferative effect on cancer.
BACKGROUND AND PRIOR ART
[0003] The encapsulation of various biological materials in
biologically compatible materials, which is well documented in the
literature, is a technique that has been used for some time, albeit
with limited success. Exemplary of the art are U.S. Pat. No.
5,227,298 (Weber, et al.); U.S. Pat. No. 5,053,332 (Cook, et al.);
U.S. Pat. No. 4,997,443 (Walthall, et al.); U.S. Pat. No. 4,971,833
(Larsson, et al.); U.S. Pat. No. 4,902,295 (Walthall, et al.); U.S.
Pat. No. 4,798,786 (Tice, et al.); U.S. Pat. No. 4,673,566 (Goosen,
et al.); U.S. Pat. No. 4,647,536 (Mosbach, et al.); U.S. Pat. No.
4,409,331 (Lim); U.S. Pat. No. 4,392,909 (Lim); U.S. Pat. No.
4,352,883 (Lim); and U.S. Pat. No. 4,663,286 (Tsang, et al.). Also
of note is U.S. Pat. No. 5,643,569 to Jain, et al., incorporated by
reference herein. Jain, et al. discuss, in some detail, the
encapsulation of islets in various biocompatible materials. Islets
produce insulin, and the use of the materials disclosed by Jain, et
al. in the treatment of diabetes is taught therein.
[0004] The Jain, et al. patent discusses, in some detail, the prior
approaches taken by the art in transplantation therapy. These are
summarized herein as well.
[0005] Five major approaches to protecting the transplanted tissue
from the host's immune response are known. All involve attempts to
isolate the transplanted tissue from the host's immune system. The
immunoisolation techniques used to date include: extravascular
diffusion chambers, intravascular diffusion chambers, intravascular
ultrafiltration chambers, microencapsulation, and
macroencapsulation. There are many problems associated with methods
of the prior art, including a host fibrotic response to the implant
material, instability of the implant material, limited nutrient
diffusion across semi-permeable membranes, secretagogue and product
permeability, and diffusion lag-time across semi-permeable membrane
barriers.
[0006] For example, a microencapsulation procedure for enclosing
viable cells, tissues, and other labile membranes within a
semipermeable membrane was developed by Lim in 1978. (Lim, Research
report to Damon Corporation (1978)). Lim used microcapsules of
alginate and poly L-lysine to encapsulate the islets of Langerhans.
In 1980, the first successful in vivo application of this novel
technique in diabetes research was reported (Lim, et al., Science
210: 908 (1980)). The implantation of these microencapsulated
islets of Langerhans resulted in sustaining a euglycemic state in
diabetic animals. Other investigators, however, repeating these
experiments, found the alginate to cause a tissue reaction and were
unable to reproduce Lim, et al.'s results (Lamberti, et al. Applied
Biochemistry and Biotechnology 10: 101 (1984); Dupuy, et al., J.
Biomed. Material and Res. 22: 1061 (1988); Weber, et al.,
Transplantation 49: 396 (1990); and Doon-shiong, et al.,
Transplantation Proceedings 22: 754 (1990)). The water solubility
of these polymers is now considered to be responsible for the
limited stability and biocompatibility of these microcapsules in
vivo (Dupuy, et al., supra, Weber et al., supra, Doon-shiong, et
al., supra, and Smidsrod, Faraday Discussion of Chemical Society
57: 263 (1974)).
[0007] Iwata et al., (Iwata, et al. Jour. Biomedical Material and
Res. 26: 967 (1992)) utilized agarose for microencapsulation of
allogeneic pancreatic islets and discovered that it could be used
as a medium for the preparation of microbeads. In their study,
1500-2000 islets were microencapsulated individually in 5% agarose
and implanted into streptozotocin-induced diabetic mice. The graft
survived for a long period of time, and the recipients maintained
normoglycemia indefinitely.
[0008] Their method, however, suffers from a number of drawbacks.
It is cumbersome and inaccurate. For example, many beads remain
partially coated and several hundred beads of empty agarose form.
Additional time is thus required to separate encapsulated islets
from empty beads. Moreover, most of the implanted microbeads gather
in the pelvic cavity, and a large number of islets in completely
coated individual beads are required to achieve normoglycemia.
Furthermore, the transplanted beads are difficult to retrieve, tend
to be fragile, and will easily release islets upon slight
damage.
[0009] A macroencapsulation procedure has also been tested.
Macrocapsules of various different materials, such as
poly-2-hydroxyethyl-methacrylate, polyvinylchloride-c-acrylic acid,
and cellulose acetate were made for the immunoisolation of islets
of Langerhans. (See Altman, et al., Diabetes 35: 625 (1986);
Altman, et al., Transplantation: American Society of Artificial
Internal Organs 30:
[0010] 382 (1984); Ronel, et al., Jour. Biomedical Material
Research 17: 855 (1983); Klomp, et al., Jour. Biomedical Material
Research 17: 865-871 (1983)). In all these studies, only a
transitory normalization of glycemia was achieved.
[0011] Archer, et al., Journal of Surgical Research 28: 77 (1980),
used acrylic copolymer hollow fibers to temporarily prevent
rejection of islet xenografts. They reported long-term survival of
dispersed neonatal murine pancreatic grafts in hollow fibers which
were transplanted into diabetic hamsters. Recently Lacy, et al.,
Science 254: 1782-1784 (1991) confirmed their results, but found
the euglycemic state to be a transient phase. They found that when
the islets are injected into the fiber, they aggregate within the
hollow tube with resultant necrosis in the central portion of the
islet masses. The central necrosis precluded prolongation of the
graft. To solve this problem, they used alginate to disperse the
islets in the fiber. However, this experiment has not been repeated
extensively. Therefore, the membrane's function as an islet
transplantation medium in humans is questionable.
[0012] The Jain, et al. patent discussed reports that encapsulating
secretory cells in a permeable, hydrophilic gel material results in
a functional, non-immunogenic material, that can be transplanted
into animals, can be stored for long lengths of time, and is
therapeutically useful in vivo. The macroencapsulation of the
secretory cells provided a more effective and manageable technique
for secretory cell transplantation.
[0013] The patent does not discuss at any length the incorporation
of cancer cells. A survey of the literature on encapsulation of
cells reveals that, following encapsulation, cells almost always
produce less of materials than they produce when not encapsulated.
See Lloyd-George, et al., Biomat. Art. Cells & Immob. Biotech.
21(3): 323-333 (1993); Schinstine, et al., Cell Transplant 4(1):
93-102 (1995); Chicheportiche, et al., Diabetologica 31:54-57
(1988); Jaeger, et al., Progress In Brain Research 82:41-46 (1990);
Zekorn, et al., Diabetologica 29:99-106 (1992); Zhou, et al., Am.
J. Physiol. 274: C1356-1362 (1998); Darquy, et al., Diabetologica
28:776-780 (1985); Tse, et al., Biotech. & Bioeng. 51:271-280
(1996); Jaeger, et al., J. Neurol. 21:469-480 (1992); Hortelano, et
al., Blood 87(12): 5095-5103 (1996); Gardiner, et al., Transp.
Proc. 29:2019-2020 (1997). None of these references deal with the
incorporation of cancer cells into a structure which entraps them
and restricts their growth, but nonetheless permit diffusion of
materials into and out of the structure.
[0014] One theory relating to the growth of cancerous masses likens
such masses, e.g., tumors, to normal organs. Healthy organs, e.g.
the liver, grow to a particular size, and then grow no larger;
however, if a portion of the liver is removed, it will regenerate
to a certain extent. This phenomenon is also observed with tumors.
To summarize, it has been noted that, if a portion of a tumor is
removed, the cells in the remaining portion of the tumor will begin
to proliferate very rapidly until the resulting tumor reaches a
particular size, after which proliferation slows down, or ceases.
This suggests that there is some internal regulation of cancer
cells.
[0015] The invention, which will be seen in the following
disclosure, shows that when cancer cells are restricted by being
entrapped, their proliferation is halted, and they produce
unexpectedly high amounts of material which, when applied to
non-restricted cancer cells, inhibits the proliferation of these
non-restricted cancer cells. The ability to retard proliferation of
cancer cells has been a goal of oncology since its inception.
Hence, the therapeutic usefulness of this invention will be clear
and will be elaborated upon herein. The material produced does not
appear to be limited by the type of cancer cell used, nor by the
animal species from which the cancer cells originate. Further, the
effect does not appear to be species specific, as restricted cells
from a first species produce material which inhibits proliferation
of unrestricted cells from a second species. Also, the effect does
not appear to be specific to the type of cancer, as restricted
cells from a first cancer type produce material which inhibits
proliferation of unrestricted cells from another cancer type.
[0016] Nor does the effect appear to require an immune response.
The antiproliferative effect is seen in in vitro systems, where no
immune cells are used. Hence the antiproliferative effect cannot be
attributed to classical immunological responses.
[0017] Thus, a preferred embodiment of the invention relates to a
composition of matter having a biocompatible,
proliferation-restrictive, selectively-permeable structure. The
structure restricts cancer cells which then produce more of a
material which suppresses cancer cell proliferation compared to an
equal number of the same cancer cells when unrestricted.
[0018] Another preferred embodiment of the present invention
relates to a process for preparing a biocompatible,
proliferation-restrictive, selectively-permeable structure, by
forming a structure by contacting cancer cells with biocompatable,
proliferation-restrictive matter to form the structure, and
culturing the structures for a sufficient period of time to
restrict the cancer cells such that they produce a material which
suppresses cancer cell proliferation compared to an equal number of
unrestricted cancer cells of the same cancer type.
[0019] Yet another preferred embodiment relates to a method of
increasing the production of material that suppresses cancer cell
growth by a cancer cell, comprising restricting cancer cells in a
structure-forming material to form a biocompatable,
selectively-permeable, proliferation-restrictive structure and
culturing the cancer cells until they are restricted and produce
the material.
[0020] It has also been found that a powerful antiproliferative
effect can be achieved by subjecting conditioned medium obtained by
culturing the structures of the invention in culture medium to
filtration. The resulting concentrates have extremely strong
anti-proliferative effects.
[0021] The material, the conditioned medium, and/or the
concentrates derived therefrom may also be useful for inducing the
production of the anti-proliferative material by other
non-restricted cancer cells.
[0022] These, and other features of the invention, will be seen
from the disclosure which follows.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
EXAMPLE 1
[0023] This example, and those which follow, employ RENCA cells.
These are spontaneous renal adenocarcinoma cells of BALB/C mice,
which are widely available, having been maintained in both in vitro
cultures and in vivo. See Franco, et al., Cytokine Included Tumor
Immunogenecity, 181-193 (1994).
[0024] Samples of frozen RENCA cells were thawed at 37.degree. C.,
and then placed in tissue culture flasks containing Dulbecco's
Modified Medium (D-MEM), which had been supplemented with 10%
bovine serum, penicillin (100 u/ml) and streptomycin (50 ug/ml), to
give what will be referred to as "complete medium" hereafter.
[0025] Cells were grown to confluence, and then trypsinized,
followed by washing with Hank's Balanced Salt Solution, and then
with the complete medium referred to supra.
[0026] In order to determine if the RENCA cells produced tumors
efficiently, two BALB/C mice were injected, intraperitoneally, with
10.sup.6 of these cells. The mice were observed, over a 3-4 week
period. Clinically, they appeared healthy for the first two weeks,
and exhibited normal activity. Thereafter, the clinical
manifestations of cancer became evident. One mouse died after 23
days, and the second, after 25 days. Following death, the mice were
examined, and numerous tumors of various size were observed. Some
of the tumors exhibited hemorrhaging as well.
[0027] A sample of one tumor, taken from one of the mice, was fixed
in 10% formalin for later histological examination.
EXAMPLE 2
[0028] Following the showing that the RENCA cells did grow in vivo,
studies were carried out to determine if these cells grew when
restricted in the structure of the invention.
[0029] RENCA cells were grown to confluency, as described supra,
trypsinized, and washed, also as described above. Samples of
between 60,000 and 90,000 cells were then prepared. The cells were
then centrifuged, at 750 RPMs, and fluid was removed. The cells
were then suspended in solutions of 1% atelocollagen, in phosphate
buffered saline solution, at a pH of 6.5.
[0030] A 1% solution of low viscosity agarose was prepared in
minimal essential medium (MEM), maintained at 60.degree. C., and
then 100 ul of this was added to the suspension of RENCA cells and
atelocollagen, described supra. The materials were then
transferred, immediately, as a single large droplet, into sterile,
room-temperature mineral oil. The mixture formed a single, smooth,
semi-solid bead. This procedure was repeated to produce a number of
beads.
[0031] After one minute, the beads were transferred to complete
medium, as described supra, at 37.degree. C. The beads were then
washed three times in Minimal Essential Medium (MEM) containing the
antibiotics listed supra. The beads were then incubated overnight
at 37.degree. C., in a humidified atmosphere of air and 5%
CO.sub.2. Following the incubation the beads, now solid, were
transferred to a sterile spoon which contained 1 ml of 5% agarose
in MEM. Beads were rolled in the solution 2-3 times to uniformly
coat them with agarose. The beads were transferred to mineral oil
before the agarose solidified, to yield a smooth outer surface.
After 60 seconds, the beads were washed, five times, with complete
medium at 37.degree. C. to remove the oil. Overnight incubation
(37.degree. C., humidified atmosphere of air, 5% CO.sub.2)
followed.
[0032] These RENCA containing beads were used in the experiments
which follow.
EXAMPLE 3
[0033] Prior to carrying out in vivo investigations, it was
necessary to determine if the RENCA cells would grow in beads
prepared in the manner described supra.
[0034] To do this, beads prepared as discussed in example 2 were
incubated in the medium described in example 2, for a period of
three weeks, under the described conditions. Three of the beads
were then cut into small pieces, and cultured in standard culture
flasks, affording direct contact with both the flask and culture
medium.
[0035] Observation of these cultures indicated that the cells grew
and formed standard RENCA colonies. This indicated that the cells
had remained viable in the beads.
EXAMPLE 4
[0036] In vivo experiments were then carried out. In these
experiments, the beads were incubated for seven days, at 37.degree.
C. Subject mice then received bead transplants. To do this, each of
four mice received a midline incision, carried through
intraperitoneally. Three beads, each of which contained 60,000
RENCA cells were transplanted. Incisions were then closed
(two-layer closure), using an absorbable suture. The four mice
(BALB/C) were normal, male mice, weighing between 24-26 grams, and
appeared to be healthy. Two sets of controls were set up. In the
first set, two mice received three beads containing no RENCA cells,
and in the second, two mice were not treated with anything.
[0037] Three weeks after the implantation, all of the mice received
intraperitoneal injections of 1O6 RENCA cells. Eighteen days later,
one control mouse died. All remaining mice were then sacrificed,
and evaluated for the presence or absence of tumor.
[0038] Control mice showed numerous tumors, while the mice which
received the implants of bead-encapsulated cells showed only
isolated small nodules throughout the cavity.
[0039] These encouraging results suggested the design of the
experiments set forth in the following example.
EXAMPLE 5
[0040] In these experiments, established cancers were simulated by
injecting RENCA cells under one kidney capsule of each of six
BALB/C mice. Fifteen days later, mice were divided into two groups.
The three mice in the first group each received three beads, as
described in example 4, supra. The second group (the control group)
received beads which did not contain RENCA cells.
[0041] For the initial 4-5 days, mice which had received RENCA cell
containing implants looked lethargic, and their fur had become
spiky. Thereafter, they returned to normal. The control group
remained energetic, with no change in condition of fur.
[0042] Ten days after implantation (25 days after injection of
RENCA cells), however, the control mice became sluggish and
exhibited distended abdomens. One of the three control mice died at
fourteen days following bead transplantation. Sacrifice of the mice
followed.
[0043] The body cavities of the control mice showed profuse
hemorrhaging, with numerous tumors all over the alimentary canal,
liver, stomach and lungs. All organs of the abdominal cavity had
become indistinguishable due to rampant tumor growth. The mice
which had received beads with encapsulated RENCA cells, however
showed no hemorrhaging, and only a few nodules on the alimentary
canal. In addition, comparison of test and control groups showed
that in the test group, nodules had not progressed beyond their
initial growth under the kidney capsule and before macrobead
implantation.
EXAMPLE 6
[0044] In vitro, freely inoculated RENCA cell growth is inhibited
when such cells are incubated along with macrobead encapsulated
RENCA cells. A further set of experiments was carried out to
determine if this effect was observable with other cells.
[0045] An adenocarcinoma cell line, i.e., MMT (mouse mammary
tumor), was obtained from the American Type Culture Collection.
Encapsulated MMT cells were prepared, as described, supra with MMT
cells, to produce beads containing 120,000 or 240,000 cells per
bead. Following preparation of the beads, they were used to
determine if they would inhibit proliferation of RENCA cells in
vitro. Specifically, two six-well petri plates were prepared, via
inoculation with 1.times.10.sup.4 RENCA cells per well, in 4 ml of
medium. In each plate, three wells served as control, and three as
test. One of the three control wells in each plate received one
empty bead. Each of the other wells received either two or three
empty beads. The second set of wells was treated similarly, with
wells receiving one, two or three beads containing 120,000 or
240,000 MMT cells. Wells were incubated at 37.degree. C. for one
week, after which RENCA cells were trypsinized, washed, and
counted, using a hemocytometer. Results are shown in Table 1:
TABLE-US-00001 TABLE 1 DISH #1 DISH #2 # of cells retrieved # of
cells retrieved after one week after one week Control Control
(Empty 120,000 (Empty 240,000 Well# macrobead) MMT cells Macrobead)
MMT cells 1 2.4 .times. 10.sup.5 1.4 .times. 10.sup.5 2.8 .times.
10.sup.5 1 .times. 10.sup.5 2 2.0 .times. 10.sup.5 1.2 .times.
10.sup.5 3.6 .times. 10.sup.5 7 .times. 10.sup.4 3 4.4 .times.
10.sup.5 1.25 .times. 10.sup.5 2.5 .times. 10.sup.5 9 .times.
10.sup.4
EXAMPLE 7
[0046] Following the results in example 6, the same experiments was
carried out using 1.times.10.sup.4 MMT cells as the inoculant
(i.e., the free cells) rather than RENCA cells. The experiment was
carried out precisely as example 6. Results are set forth in Table
2 below. TABLE-US-00002 TABLE 2 DISH #1 DISH #2 Control 120,000
Control 240,000 (Empty MMT cells in (Empty MMT cells in Well#
macrobead) macrobeads Macrobead) macrobeads 1 3.1 .times. 10.sup.6
1.6 .times. 10.sup.6 2.8 .times. 10.sup.6 1.3 .times. 10.sup.6 2
3.3 .times. 10.sup.6 1.0 .times. 10.sup.6 2.6 .times. 10.sup.6 1.1
.times. 10.sup.6 3 3.0 .times. 10.sup.6 6.0 .times. 10.sup.5 2.8
.times. 10.sup.6 5.0 .times. 10.sup.5
[0047] These results encouraged an in vivo experiment. This is
presented in example 8.
EXAMPLE 8
[0048] The mouse mammary tumor cell line (MMT) described supra was
used. Using the protocols set forth, supra, implants were prepared
which contained 120,000 cells per bead, and 240,000 cells per
bead.
[0049] The experimental model used was the mouse model, supra.
Twenty-two mice were divided into groups of 4 (control), 9 and 9.
The first group, i.e., the controls, were further divided into
three groups: two received implants of one empty bead, one received
two empty beads, and one received three empty beads.
[0050] Within experimental Group A (9 animals), the beads contained
120,000 cells, while in experimental Group B, the beads contained
240,000 cells. Within Groups A and B, there were three
subdivisions, each of which contained three mice. The subgroups
received one, two, or three beads containing MMT cells.
[0051] For the first few days, the mice in Groups A and B were
lethargic, with spiky hair. This persisted for about five days,
after which normal behavior was observed. Twenty-one days following
implantation, all animals received injections of 40,000 RENCA
cells.
[0052] After another twenty days, the control mice exhibited
distended abdomens, and extremely spiky hair. One control mouse
died twenty-five days following injection, while the remaining
control mice appeared terminal. All mice were sacrificed, and tumor
development was observed. These observations are recorded in Table
3 infra: TABLE-US-00003 TABLE 3 NUMBER OF MACROBEADS CON-
EXPERIMENTAL EXPERIMENTAL IN MICE TROL GROUP A GROUP B 1 ++++ - - 1
++++ - - 1 + ++ 2 ++++ - - 2 - - 2 ++ ++ 3 ++++ - - 3 - - 3 -
+++
[0053] These results show that, of eighteen mice treated, thirteen
showed no disease. Of the mice in Group A, one mouse exhibited a
few small nodules (+), and another mouse showed a few tumors
(++).
[0054] Within Group B, one mouse which had received one bead, and
one mouse which received two beads showed a few tumors, entangled
with intestine. One of the mice which received three beads had
developed a large solid tumor and was apparently very sick (+++).
All control mice had numerous tumors (++++). The results showed
that the encapsulated mouse mammary tumor cells inhibited tumor
formation.
EXAMPLE 9
[0055] As suggested, supra, the practice of the invention results
in the production of material which inhibits and/or prevents tumor
cell proliferation. This was explored further in the experiment
which follows.
[0056] Additional beads were made, as described supra in example 2,
except that atelocollagen was not included. Hence, these beads are
agarose/agarose beads. RENCA cells, as described, supra, were
incorporated into these beads, again as described supra.
[0057] Two sets of three six-well plates were then used as control
and experimental groups. In the control group, wells were filled
with 4 ml of RPMI complete medium (10% fetal calf serum and 11 ml/l
of penicillin). Each control group well was then inoculated with
10,000 RENCA cells.
[0058] In the experimental group, the RPMI complete medium was
conditioned, by adding material secured by incubating ten RENCA
containing beads (120,000 cells per bead), in a 35.times.100 mm
petri plate containing 50 ml of the RPMI complete medium. Following
five days of incubation, medium was collected from these plates,
and 4 ml of it was placed in each test well. These wells were then
inoculated with 10,000 RENCA cells in each.
[0059] All plates (both control and experimental) were incubated at
37.degree. C. for five days. Following the incubation period, cells
were trypsinized, washed, pooled, and counted using a
hemocytometer. The results are shown in Table 4: TABLE-US-00004
TABLE 4 RENCA CELLS RENCA CELLS TEST WITH WITH CONDITIONED WELL #
CONTROL MEDIUM MEDIUM 1 7 .times. 10.sup.5 3 .times. 10.sup.5 2 8
.times. 10.sup.5 2.5 .times. 10.sup.5 3 7 .times. 10.sup.5 3.4
.times. 10.sup.5
[0060] These results show that the cells, when restricted in, e.g.,
the beads of the examples, produced some material which resulted in
suppression of tumor cell proliferation.
EXAMPLE 10
[0061] The experiment set forth supra showed that RENCA cell
growth, in conditioned medium, was about half the growth of the
cells in control medium. The experiments set forth herein examined
whether the suppression of proliferation would continue after the
conditioned medium was frozen.
[0062] RENCA conditioned medium was prepared by incubating ten
RENCA containing beads for five days. Incubation was in
35.times.100 mm petri plates, with 50 ml RMPI complete medium, at
37.degree. C. Following the incubation, the medium was collected
and stored at -20.degree. C. Conditioned medium was prepared by
incubating MMT (mouse mammary tumor) cell containing beads. The
beads contained 240,000 cell per bead; otherwise all conditions
were the same.
[0063] Frozen media were thawed at 37.degree. C., and then used in
the following tests. Three six-well plates were used for each
treatment, i.e., (i) RMPI control medium, (2) RENCA frozen
conditioned medium, and (3) MMT frozen conditioned medium. A total
of 4 ml of medium was dispensed into each well. All wells were then
inoculated with 10,000 RENCA cells, and incubated at 37.degree. C.,
for five days. Following incubation, two plates of samples were
taken from each well, trypsinized, washed, pooled, and counted in a
hemocytometer. At eight days, the remaining three plates of each
well were tested in the same way.
[0064] Results follow: TABLE-US-00005 TABLE 5 FROZEN FROZEN CONTROL
CONDITIONED CONDITIONED MEDIUM MEDIUM OF RENCA MEDIUM OF MMT DISH 5
DAYS OLD 1 6 .times. 10.sup.5 5 .times. 10.sup.5 8 .times. 10.sup.4
2 6.8 .times. 10.sup.5 4.2 .times. 10.sup.5 8.5 .times. 10.sup.4 8
DAYS OLD 3 2.8 .times. 10.sup.6 2 .times. 10.sup.6 8 .times.
10.sup.4
[0065] When these results are compared to those in example 6,
supra, it will be seen that, while the frozen/thawed RENCA
conditioned medium did not suppress proliferation to the same
extent that frozen/thawed MMT conditioned medium did (compare
examples 6 and 7), it did, nonetheless, suppress proliferation.
EXAMPLE 11
[0066] The experiments set forth supra showed that frozen
conditioned medium from RENCA- or MMT-containing macrobeads
inhibits the proliferation of RENCA cells in vitro. The experiments
set forth herein examined whether RENCA- or MMT-macrobead
conditioned medium, prepared as 30 kd or 50 kd concentrates by
filtration, would inhibit the proliferation of RENCA cells in
vitro. The effects of macrobead conditioned media were compared to
the effects of media conditioned in the presence of unrestricted
RENCA and MMT cells growing in monolayer cultures, to determine
whether unrestricted tumor cells grown to confluence also make
proliferation regulating material.
[0067] For these experiments, 10 macrobeads, each containing
120,000 RENCA or MMT cells (i.e., 1.2.times.10.sup.6 cells total)
were used to condition the medium (complete RPMI) over a period of
5 days. In parallel, 1.2.times.10.sup.6 RENCA or MMT cells, i.e.,
the same number of cells, were plated in a culture dish and allowed
to proliferate as a monolayer over a period of 4 days in complete
RPMI medium. Medium was then changed, and this medium was collected
twenty-four hours later. The reason for the different length of
time of exposure of the beads and unrestricted cells was the
difference in cell numbers in the monolayers vs. the beads (3- to
5-fold more cells in the monolayers) at the end of the 5-day
period. In other words, unrestricted cells grew so much more
rapidly than encapsulated cells, that there were 3-5 times more
cells. 30 kd and 50 kd filters were used to prepare concentrates of
the conditioned media that would, presumably, contain the active
material, and would also eliminate toxic metabolic and/or waste
materials as confounding factors in the experiments. These
contaminants, which are well known, are too small to be retained on
a 30 kd filter. Filtrates were also tested, but any interpretation
of the results with this material is complicated by the presence of
the cellular waste products. A serum-free medium (AIM V) was also
used in some experiments to be certain that any effects of serum
per se were controlled.
[0068] Essentially, conditioned medium was collected, either three
to five days after the macrobeads had been added to it, or
twenty-four hours after new medium had been added to the
unrestricted cells. The medium was then placed in a test tube
filter with an appropriate filter (either a 30 kd or 50 kd filter),
and centrifuged for 90 minutes. Material which remained on the
filter is referred to as the "concentrate," while that which spins
through the filter and collects at the bottom of the tube is the
filtrate.
[0069] The results, summarized in the Table 6 which follow, show
that when the conditioned medium resulting from the restricted
RENCA cells in the macrobeads was used, this inhibited RENCA cell
proliferation by about 52% in two separate experiments. The 50 kd
concentrate inhibited proliferation by about 99%, in both cases,
while the 30 kd concentrate inhibited proliferation by about 97%.
TABLE-US-00006 TABLE 6 Inhibition of RENCA Cell Growth in RENCA
Macrobead Conditioned Medium and Reconstituted Concentrates RENCA
Macrobead Unconditioned Conditioned 30K Concentrate Concentrate
Plate RPMI Medium Medium of this Medium 50K of this Medium Number #
of Cells # of Cells Inhibition # of Cells Inhibition # of Cells
Inhibition 1 1.6 .times. 10.sup.6 7.8 .times. 10.sup.5 51.3% 4.2
.times. 10.sup.4 97% 2.0 .times. 10.sup.4 99% 2 1.65 .times.
10.sup.6 8.0 .times. 10.sup.5 51.5% 5.0 .times. 10.sup.4 97% 2.0
.times. 10.sup.4 99%
[0070] TABLE-US-00007 TABLE 7 Inhibition of RENCA Cell Growth in
RENCA Cell Culture Conditioned Medium and Reconstituted
Concentrates RENCA Cell Culture Unconditioned Conditioned 30K
Concentrate 50K Concentrate Plate Medium Medium of this Medium of
this Medium Number # of Cells # of Cells Inhibition # of Cells
Inhibition # of Cells Inhibition 1 1.6 .times. 10.sup.6 1.3 .times.
10.sup.6 18.8% 1.1 .times. 10.sup.6 31.3% 9.0 .times. 10.sup.5
43.8% 2 1.6 .times. 10.sup.6 1.2 .times. 10.sup.6 25.0% 1.0 .times.
10.sup.6 37.5% 9.5 .times. 10.sup.5 40.6%
[0071] TABLE-US-00008 TABLE 8 Inhibition of RENCA Cell Growth in
RENCA Macrobead Conditioned Medium and Concentrate (AIM V Medium)
AIM V CONDITIONED 30K 50K PLATE CONTROL MEDIUM CONCENTRATE
CONCENTRATE NUMBER MEDIUM # cells % inhibition # cells % inhibition
# cells % inhibition 1 1.3 .times. 10.sup.6 6.0 .times. 10.sup.5
54% .about.5.0 .times. 10.sup.4 96% .about.4.0 .times. 10.sup.4 97%
2 1.3 .times. 10.sup.6 5.5 .times. 10.sup.5 58% .about.5.0 .times.
10.sup.4 96% .about.4.0 .times. 10.sup.4 97%
[0072] An important point of the experiment is that MMT cells and
RENCA cells, when entrapped and restricted in the macrobeads both
suppress RENCA cell proliferation, indicating that the
proliferation-restrictive effect is not specific to tumor type.
These experiments confirm those of Example 8 in which
MMT-containing macrobeads suppressed the proliferation of RENCA
cells in vivo. In addition, they extend the findings to indicate
that the material released from the macrobeads into the medium
contains molecules that are at least 30 kd in molecular weight
which are responsible, in part, for the proliferation-restrictive
effect. Finally, these experiments show that the
macrobead-restricted RENCA and MMT cells produce far more of the
proliferation-suppressing material than the same cells grown to
confluency in monolayer cultures.
EXAMPLE 12
[0073] The experiments set forth above show that both MMT- and
RENCA-macrobead conditioned media contain material released from
the proliferation-restricted cells in the macrobead that can
inhibit the proliferation of RENCA cells in vivo and in vitro.
Importantly, the experiments show that the proliferation-inhibitory
effect is not specific to tumor type. The experiments set forth
herein examine whether the effect is also independent of the
species in which the tumor originally arose. Here, the tumor cell
proliferation-inhibitory effects of a human breast cancer-derived
cell line on RENCA cells (using macrobeads and
macrobead-conditioned media) and also MMT cells (using
macrobead-conditioned media only) in vitro were examined.
[0074] The methodologies for these in vitro studies were similar to
those described in the examples above. 100,000 MCF-7 cells, (human
breast cancer cells) were encapsulated in macrobeads, and the
resulting MCF-7 macrobeads were incubated with RENCA cells (10,000
per well) for 5 days to evaluate the proliferation-inhibitory
effects of the macrobeads. In addition, MCF-7 macrobead-conditioned
medium was prepared over a 5-day incubation period and tested on
both RENCA and MMT cells. Cell proliferation was measured over a
5-day period.
[0075] The results are set forth below: TABLE-US-00009 TABLE 9
RESULTS OF MCF-7 MACROBEADS ON RENCA TARGET CELLS CONTROL MCF-7
Well # (Empty Macrobeads) MACROBEADS 1 8.4 .times. 10.sup.5 4.4
.times. 10.sup.5 2 8.0 .times. 10.sup.5 4.4 .times. 10.sup.5 3 7.4
.times. 10.sup.5 3.8 .times. 10.sup.5
[0076] TABLE-US-00010 TABLE 10 RESULTS OF MCF-7 CONDITIONED MEDIUM
ON RENCA TARGET CELLS RPMI Conditioned RPMI Control Medium Plate
Medium MCF-7 1 9.0 .times. 10.sup.5 5.0 .times. 10.sup.5 2 8.8
.times. 10.sup.5 4.8 .times. 10.sup.5
[0077] TABLE-US-00011 TABLE 11 RESULTS OF MCF-7 CONDITIONED MEDIUM
ON MMT TARGET CELLS RPMI Control RPMI Conditioned Plate Medium
Medium: MCF-7 1 5.0 .times. 10.sup.5 1.5 .times. 10.sup.5 2 6.0
.times. 10.sup.5 1.8 .times. 10.sup.5
[0078] The results show that MCF-7, a human breast adenocarcinoma
cell line, when proliferation-restricted in macrobeads, produces a
material that inhibits the proliferation of mouse renal
adenocarcinoma cells and mouse breast cancer tumor cells to a
significant degree (30-70%) as demonstrated by both the macrobeads
themselves and conditioned media derived therefrom. This indicates
that the proliferation-inhibitory effect of growth-restricted
cancer cells is independent of both tumor type and species of tumor
origin, i.e., mouse and human.
EXAMPLE 13
[0079] The experiments set forth above demonstrate that a
human-derived breast adenocarcinoma cell line (MCF-7), when
growth-restricted in macrobeads, produces proliferation inhibition
of mouse renal and mouse breast adenocarcinoma cells in vitro. The
experiments set forth herein examine whether a parallel effect of
MCF-7-containing macrobeads on RENCA cell tumor growth in vivo
exists.
[0080] Eighteen Balb/c mice were injected with 20,000 RENCA cells
intraperitoneally. After three days the mice were divided into two
groups. Group 1 had six mice and Group 2 had the remaining twelve
mice. Group 1 mice, the controls, were transplanted with three
empty macrobeads each. Group 2 received three MCF-7-containing
macrobeads (100,000 cells per bead). After twenty-five days, 2 mice
from Group 1 and three mice from Group 2 were sacrificed. The same
number were sacrificed on day twenty-six and the remaining mice
were sacrificed on day twenty-seven.
[0081] On necroscopy, the peritoneal cavities of the control mice
were observed to be completely packed with tumor, and the normal
organs were difficult to identify. We classified this as ++++
(100%) tumor intensity. In the treated mice, tumor intensity was
rated at +(10-20%).
[0082] These results show that macrobeads containing human breast
adenocarcinoma cells are capable of inhibiting renal cell
adenocarcinoma tumor growth in mice, confirming again that the
cancer-cell proliferation/tumor growth-inhibitory effect is neither
type-specific nor species-specific.
EXAMPLE 14
[0083] The experiments set forth above demonstrate that the cell
proliferation/tumor growth inhibitory effect of macrobead
growth-restricted tumors is neither tumor-type nor species
specific. The experiments set forth herein examine whether
(macrobead) proliferation-restricted mouse breast adenocarcinoma
cells can inhibit the growth of both spontaneous mammary tumors and
tumors resulting from the injection of MMT cells.
[0084] C3H mice have a very high incidence of the development of
mammary tumors over their life span. Seven mice at risk for the
development of such tumors showed tumors at sixteen months of age.
At this time, five of the seven mice were implanted with four MMT
macrobeads containing 100,000 cells each. The remaining two control
mice received four empty macrobeads each. The two control mice
developed large tumors and died within three months after the bead
implants. The treated mice were sacrificed eleven months after the
MMT macrobead implants. The retrieved macrobeads, organs and tumors
were examined grossly and histologically. Hemotoxylin & Eosin
staining of the MMT macrobeads showed viable cells. The
pre-existing tumors had not increased in size, and there was no
evidence of any new tumor development.
[0085] Experiments in which MMT tumor cells were injected
subcutaneously in the thoracic region were also performed. Fourteen
C3H mice were divided into two groups. The five control group mice
were implanted with three empty macrobeads each. The nine treated
mice received three MMT-containing macrobeads (240,000 cells each).
Three weeks after implantation all fourteen mice were injected
subcutaneously in the mammary area with 20,000 MMT cells each.
[0086] Within twenty-five to thirty days, the five control group
mice became ill with evident tumor formation, and all were dead by
thirty-five days post-injection. The nine treated mice, observed
weekly, continued without any evidence of tumor formation or ill
health during this period. Ten to twelve months after tumor
injection, four of the nine treated mice developed lumps and lost
their fur in patches. The remaining five mice were implanted again
with three MMT macrobeads thirteen months after the initial tumor
injection. One mouse died three days after this surgery, but on
necropsy was completely free of tumor. The four surviving mice were
sacrificed eight months after the second macrobead implant.
Necropsy showed minimal or no tumor proliferation.
[0087] An additional observation from these experiments was that
the beads retrieved from the first implantation contained viable
tumor cells based both on histology and their ability to resume
aggressive tumor growth patterns in tissue culture after removal
from the bead.
[0088] The results of these experiments show that the cell
proliferation/tumor growth-inhibiting effects of
macrobead-restricted cancer cells, in this case mouse mammary
adenocarcinoma cells, can influence the development and growth of
both spontaneously arising tumors and experimentally induced tumors
arising from the injection of tumor cells into the mammary
area.
EXAMPLE 15
[0089] The experiments set forth above demonstrate a tumor cell
proliferation/tumor growth-inhibitory effect of macrobead
proliferation-restricted cancer cells that is characterized by its
effectiveness across tumor types and across species, as well as in
both spontaneous and artificially-induced tumors. The experiments
described herein extend these findings to examine the effects of
macrobead-entrapped, proliferation-restricted human prostate
adenocarcinoma-derived cells (ARCap10), mouse (Balb/c) renal
adenocarcinoma cells (RENCA cells), and mouse (C3H) mammary
adenocarcinoma cells (MMT) on the proliferation of ARCaP10 tumor
cells and ARCaP10 tumor growth in nude (Nu/Nu) mice.
[0090] In the first series of experiments, fifteen Nu/Nu mice were
injected with 2.5.times.10.sup.6 ARCaP10 cells subcutaneously in
the flank. On the twentieth day after injection, at which time the
average maximal tumor diameter was 0.5 cm, the mice were divided
into two groups. Nine were implanted with four ARCaP10 macrobeads
(1.0.times.10.sup.5cells per macrobead) each, and six control mice
received four empty macrobeads each.
[0091] Ten weeks after implantation, five of the control mice had
very large vascularized tumors (average 2.5 cm in diameter) and one
mouse showed a slightly smaller tumor (less than 0.5 cm). In the
treated group, five mice showed complete regression of the initial
tumors, and all remained tumor free until sacrifice at eight
months. Two mice showed no tumor growth, i.e., their tumors had the
same maximal diameter as they had had at the time of implantation
of the macrobeads, and two mice showed tumors that had enlarged
since implantation of the macrobeads.
[0092] The results (tumor volume and size (l.times.w.times.h)) of
an experiment in which RENCA-containing macrobeads
(1.2.times.10.sup.5) were implanted eighteen days after
subcutaneous flank injection of 3.0.times.10.sup.6 ARCaP10 tumor
cells per animal in 4 Nu/Nu mice are set forth below:
TABLE-US-00012 TABLE 12 SIZE OF TUMORS OBSERVED IN TREATED MICE (in
mm) 10 Days 14 Days Treated 3 Days Before Day of 3 Days After 6
Days After After After Mouse Transplant Transplant Transplant
Transplant Transplant Transplant Number (Mar. 3, 1998) (Mar. 6,
1998) (Mar. 9, 1998) (Mar. 12, 1998) (Mar. 16, 1998) (Mar. 20,
1998) 1 3.5 .times. 3 .times. flat 6.2 .times. 5.4 .times. flat 4
.times. 4 .times. flat disappearing 0 0 2 3 .times. 3 .times. 1.5
5.1 .times. 2.2 .times. 2 4 .times. 2 .times. 0.5 3 .times. 3
.times. 0.4 2 .times. 2 .times. 0.3 2 .times. 2 .times. 0.3 3 3
.times. 2.5 .times. 1 3.1 .times. 3.3 .times. 1 3 .times. 2 .times.
0.5 3 .times. 2 .times. 0.2 3 .times. 2 .times. 0.2 3 .times. 2
.times. 0.2 4 2.5 .times. 2.5 .times. flat 3.2 .times. 3.4 .times.
0.5 speck under skin 0 0 0
[0093] TABLE-US-00013 TABLE 13 VOLUME OF TUMORS OBSERVED IN TREATED
MICE 3 Days Before Day of 3 Days After 6 Days After 10 Days After
14 Days After Treated Mouse Transplant Transplant Transplant
Transplant Transplant Transplant Number (Mar. 3, 1998) (Mar. 6,
1998) (Mar. 9, 1998) (Mar. 12, 1998) (Mar. 16, 1998) (Mar. 20,
1998) 1 2.76 8.81 1.68 0 0 0 2 7.10 11.81 2.10 1.89 0.63 0.63 3
3.95 5.38 1.58 0.63 0.63 0.63 4 1.64 2.86 0 0 0 0
[0094] In another experiment 10 Nu/Nu mice were injected with
2.5.times.10.sup.6APCaP10 cells, with six of the mice showing tumor
development sixty-four days after injection. Three of these mice
were given four MMT macrobeads (2.4.times.10.sup.5 cells each) and
three received empty macrobeads. The results are set forth below:
TABLE-US-00014 TABLE 14 SIZE OF TUMORS OBSERVED IN TREATED MICE (in
mm) 5 Days Before Day of 18 Days After 22 Days After 27 Days After
30 Days After Treated Mouse Transplant Transplant Transplant
Transplant Transplant Transplant Number (Feb. 5, 1998) (Feb. 10,
1998) (Feb. 28, 1998) (Mar. 4, 1998) (Mar. 9, 1998) (Mar. 12, 1998)
1 2 .times. 2 .times. 1 3 .times. 3 .times. 1.5 1 .times. 1 .times.
0.5 0 0 0 2 3 .times. 2 .times. 1 3 .times. 2.5 .times. 1 2 .times.
2 .times. flat <1 mm <0.8 mm <0.8 mm 3 4 .times. 4 .times.
1.5 6 .times. 6 .times. 1.5 6 .times. 2 .times. flat 4 .times. 1
.times. flat 3 .times. 1 .times. flat 3 .times. 1 .times. flat
[0095] TABLE-US-00015 TABLE 15 SIZE OF TUMORS OBSERVED IN CONTROL
MICE (in mm) Control 5 Days Before 18 Days After 22 Days After 27
Days After 30 Days After Mouse Transplant Day of Transplant
Transplant Transplant Transplant Transplant Number (Feb. 5, 1998)
(Feb. 10, 1998) (Feb. 28, 1998) (Mar. 4, 1998) (Mar. 9, 1998) (Mar.
12, 1998) 1 4 .times. 4 .times. 1.5 5 .times. 5 .times. 2 6.5
.times. 6 .times. 3 6.5 .times. 6 .times. 3 6.5 .times. 6 .times. 3
7 .times. 7 .times. 3 2 3 .times. 2 .times. 1 4 .times. 6 .times. 3
4.5 .times. 7 .times. 3 5 .times. 8 .times. 3 11 .times. 12 .times.
5 13.3 .times. 13.3 .times. 6.5 2.sup.nd tumor: 6 .times. 6 .times.
1 3 5 .times. 4 .times. 1 5 .times. 4 .times. 2 5 .times. 4.6
.times. 2.5 5 .times. 5 .times. 2.5 6 .times. 6 .times. 2.5 7
.times. 7 .times. 2.5 (multilobe) 2.sup.nd tumor: 2.sup.nd tumor: 2
.times. 2 .times. 1 3 .times. 3 .times. 0.5
[0096] The results of these experiments further confirm the
cross-species, cross-tumor nature of the tumor growth-inhibiting
effect of proliferation restriction on tumors of various types. In
addition, these experiments demonstrate the ability of the
proliferation-restricted cancer cells not only to suppress tumor
growth and to prevent tumor formation, but also to cause actual
regression of in vivo tumors.
EXAMPLE 16
[0097] The experiments set forth above showed that
proliferation-restricted cancer cells from several types of tumors
and species can inhibit the proliferation of the same and different
cancer cell types in vitro and prevent the formation of both
spontaneous and induced tumors, prevent the growth of tumors, and
cause tumors to regress in vivo in an effect that is independent of
species and cancer type. The experiment set forth herein describes
the extension of the findings to another species (rabbit) and a
rabbit tumor known to have been induced virally (VX2).
[0098] In this experiment, a New Zealand White Rabbit (2.5 lbs.)
was injected intramuscularly in one thigh (two sites) with 0.5 ml
of a VX2 tumor slurry (characterized as being able to pass through
a #26 gauge needle) at each site. At 3.5 weeks, a 5 cm.times.2.5 cm
(l.times.w) tumor had appeared on the dorsal thigh and two 3
cm-diameter tumors were present on the ventral thigh. At this
point, 211 macrobeads (108 RENCA cell beads, 63 MMT cell beads, and
40 MCF-7 human breast cancer cell-containing beads) were implanted
intraperitoneally. Within two days, the tumor on the dorsal thigh
had shrunk by approximately 50%; however, the two ventral tumors
did not change. The animal was sacrificed ten days after macrobead
implantation. On necropsy, there was a clear difference between the
dorsal and ventral tumors in that the former was much smaller than
it had been at the time of macrobead implantation, whereas the two
ventral tumors were both hemorrhagic and necrotic.
[0099] This experiment extends the findings of the effectiveness of
proliferation restriction of various types of cancer cells in
relation to the prevention, arrest, and even regression of tumor
growth to another species, the rabbit, adds a tumor of known viral
origin to the list of cancer types, and further supports the
cross-tumor and cross-species nature of the growth inhibiting
effect, since a combination of mouse renal, mouse breast and human
breast cancer cell-containing macrobeads were used. In addition,
the experiment adds a larger animal model to the in vivo testing of
the effectiveness of proliferation-restriction of cancer cells for
the treatment of cancer.
EXAMPLE 17
[0100] The experiments set forth above show that
proliferation-restriction of various types of tumor cells results
in their ability to inhibit the growth of cells of the same or
different type in vitro and to prevent the formation of, suppress
the growth of, or cause regression of various types of tumors in
vivo and that the effects seen are independent of tumor type and
species. The experiments set forth herein evaluated the long-term
viability of the proliferation-restricted RENCA cancer cells in
agarose-agarose macrobeads maintained in culture over periods of 1
month, 6 months, 2 years, and 3 years using histological, culture,
and in vivo techniques. MMT-containing macrobeads were maintained
in culture for up to six months. In addition, RENCA- and
MMT-containing macrobeads retrieved from Balb/c and C3H mice
respectively after periods of 2 to 8 months after implantation were
examined for viable tumor cells by both histological and culture
techniques.
[0101] For these experiments the agarose-agarose macrobeads were
prepared with either 1.2.times.10.sup.5 RENCA cells or
2.4.times.10.sup.5 MMT cells. They were examined histologically
(hermatoxylin & eosin staining) and by culture techniques for
cell viability and tumor characteristics at the intervals described
supra. For the RENCA macrobeads, cell numbers increased
approximately 3- to 5-fold over the first month with a subsequent
additional doubling in six months. After one year, there was a
continued increase in cellular mass, but the rate of cell
proliferation had decreased. After two years, amorphous material
had begun to appear in the center of the bead, and the cell
mass/numbers did not appear to be increasing, although mitotic
figures are still evident. After three years, there appeared to be
somewhat more amorphous material in the center of the bead, but the
cell mass/number was stable. MMT macrobeads have been followed for
only six months, but the early pattern of cell proliferation and
bead appearance is similar to that of RENCA.
[0102] For evaluation of the viability and biological behavior of
the RENCA and MMT cells at the intervals described above, ten beads
were crushed and plated in two or more 25 cm.sup.2 tissue culture
flasks in complete RPMI medium. The flasks were then observed for
cell growth. At one and six month intervals, the number of viable
cells retrievable from the beads increases. At one year, the number
of RENCA cells growing from the crushed bead appears to be similar
to that at six months. At two and three years, the proportion of
viable cells appears to be somewhat less, dropping to approximately
20% of the maximum number they reached in the bead (i.e., in their
restricted state) after three years in culture.
[0103] For the evaluation of the retrieved RENCA and MMT macrobeads
after in vivo implantation (periods of 1-4 years for RENCA
macrobeads and up to 8 months for MMT macrobeads), histological
techniques have been utilized to date. The patterns of cell
proliferation and mass are very similar to those of the beads
maintained in culture for the corresponding periods of time, i.e.,
the cells increase in number at least up to 4 months for RENCA and
8 months for MMT.
[0104] For the other cancer cell lines-with which we have been
working, such as MCF-7 and ARCaP10, the viability patterns in
macrobeads are similar to those observed for RENCA and MMT.
[0105] These experiments show that cancer cells can be maintained
in vitro for periods of up to 3 years and in vivo for periods of at
least 8 months in a proliferation-restricting environment and that
they maintain their viability for these periods with clear
demonstration of increasing cell numbers up to at least one year.
This is important not only for the ability to create and store
cancer treatment materials, but also for the ability of the
proliferation-restricted cells to put out tumor growth suppressing
material in warm-blooded animals over the continuous, prolonged
periods likely to be necessary for the successful treatment of
experimental or naturally-occurring cancer.
EXAMPLE 18
[0106] The experiments set forth above show that cancer cells of
various types can be maintained under proliferation-restricted
conditions for long periods of time (up to 3 years) with retention
of their ability to proliferate, form tumors, and release
cell-proliferation-inhibiting and tumor-growth preventing,
suppressing, and even regressive materials. The experiments set
forth herein evaluate the possible toxicity of long-term (one-year)
implants of cancer cell-containing, agarose-agarose macrobeads in
Balb/c mice.
[0107] Seven Balb/c mice were implanted with 3 RENCA macrobeads
each (1.2.times.10.sup.5 cells per bead). Immediately after surgery
the mice appeared ill (spiky fur and lethargy) for a few days, but
became healthy again after this. All mice survived in apparent good
health for a period of at least one year, with one mouse dying of
old age and another of unrelated causes. All mice were sacrificed.
On necropsy, no abnormalities, such as fibrosis, peritonitis, or
tumor growth were observed. All organs observed appeared normal,
although some adherence of the beads to the serosal surfaces of the
intestines were observed, especially where there were intestinal
loops. No interference with the normal function or structure of the
intestines has been observed.
[0108] These results show that cancer cell-containing
agarose-agarose macrobeads are well tolerated in experimental
animals over a one-year period. These findings show that the
proliferation-restricting cancer-cell beads can be utilized in vivo
for the prevention, suppression and regression of the growth of in
vivo tumors of various types.
[0109] The foregoing examples describe the invention, which
includes, inter alia, compositions of matter which can be used to
produce material which suppresses proliferation of cancer. These
compositions comprise cancer cells entrapped in a
selectively-permeable material to form a structure which restricts
the proliferation of the entrapped cells. As a result of their
being restricted, the cells produce unexpectedly high amounts of
material which suppresses proliferation of cancer cells. The
restricted cells produce more of the material than comparable,
non-restricted cancer cells.
[0110] The matter used to make the structures of the invention
include any biocompatible matter which restricts the growth of
cancer cells, thereby inducing them to produce greater amounts of
cancer cell proliferation/tumor growth-suppressing material. The
structure has a suitable pore size such that the above material can
diffuse to the external environment, and prevent products or cells
from the immune system of the host from entering the structure and
causing the rejection of or otherwise impair their ability to
survive and continue to produce the desired material. The matter
used to form the structure will also be capable of maintaining
viable (proliferation-restricted, but surviving) cells both in
vitro and in vivo, preferably for periods of up to several years by
providing for the entrance of proper nutrients, the elimination of
cellular waste products, and a compatible physico-chemical
intra-structural environment. The matter used to prepare the
structure is preferably well tolerated when implanted in vivo, most
preferably for the entire duration of implantation in the host.
[0111] A non-limiting list of materials and combinations of
materials that might be utilized includes alginate-poly-(L-lysine);
alginate-poly-(L-lysine)-alginate;
alginate-poly-(L-lysine)-polyethyleneimine; chitosan-alginate;
polyhydroxylethyl-methacrylate-methyl methacrylate;
carbonylmethylcellulose; K-carrageenan; chitosan;
agarose-polyethersulphone-hexadi-methirine-bromide (Polybrene);
ethyl-cellulose; silica gels; and combinations thereof.
[0112] The structures which comprise the compositions of matter may
take many shapes, such as a bead, a sphere, a cylinder, a capsule,
a sheet or any other shape which is suitable for implantation in a
subject, and/or culture in an in vitro milieu. The size of the
structure can vary, depending upon its eventual use, as will be
clear to the skilled artisan.
[0113] The structures of the invention are selectively permeable,
such that nutrients may enter the structure, and so that the
proliferation-inhibiting material as well as cellular waste may
leave the structure. For in vivo use, it is preferred that the
structures prevent the entry of products or cells of the immune
system of a host which would cause the rejection of the cancer
cells, or otherwise impair their ability of the cancer cells
producing the proliferation-suppressive material.
[0114] Another aspect of the invention includes compositions which
are useful in suppressing cancer cell proliferation. These
compositions are prepared by culturing restricted cells as
described supra in an appropriate culture medium, followed by
recovery of the resultant conditioned medium. Concentrates can then
be formed from the conditioned medium, e.g., by separating
fractions having molecular weight of greater than 30 kd or greater
than 50 kd, which have high anti-proliferative effect on cancer
cells.
[0115] As the examples show, the invention is not limited to any
particular type of cancer; any neoplastic cell may be used in
accordance with the invention. Exemplary types of cancer cells
which can be used are renal cancer cells, mammary cancer cells,
prostate cancer cells, choriocarcinoma cells and so forth. The
cancer cells may be of epithelial, mesothelial, endothelial or germ
cell origin, and include cancer cells that generally do not form
solid tumors such as leukemia cells.
[0116] As will be clear from this disclosure, a further aspect of
the invention is therapeutic methods for treating individuals
suffering from cancer. When used in a therapeutic context, as will
be elaborated upon infra, the type of cancer cell restricted in the
structure need not be the same type of cancer from which the
subject is suffering, although it can be. One such method involves
inserting at least one of the structures of the invention into the
subject, in an amount sufficient to cause suppression of
cancer-cell proliferation in the subject. Preferably, the subject
is a human being, although it is applicable to other animals, such
as domestic animals, farm animals, or any type of animal which
suffers from cancer.
[0117] The composition of the present invention can be used as
primary therapy in the treatment of cancer, and as an adjunct
treatment in combination with other cancer therapies. For example,
patients may be treated with compositions and methods described
herein, in conjunction with radiation therapy, chemotherapy,
treatment with other biologically active materials such as
cytokines, anti-sense molecules, steroid hormones, gene therapy,
and the like. Additionally, the compositions and methods of the
invention can be used in conjunction with surgical procedures to
treat cancer, e.g., by implanting the macrobeads after resection of
a tumor to prevent regrowth and metastases. Cancers which present
in an inoperable state may be rendered operable by treatment with
the anti-proliferative compositions of the invention.
[0118] The compositions of the invention can also be used
prophylactically in individuals at risk for developing cancer,
e.g., presence of individual risk factors, family history of cancer
generally, family history of cancer of a specific type (e.g. breast
cancer), and exposure to occupational or other carcinogens or
cancer promoting agents. For prophylaxis against cancer, a
prophylactically effective amount of the structures of the
invention are administered to the individual upon identification of
one or more risk factors.
[0119] As indicated by the examples, supra, the antiproliferative
effect is not limited by the type of cancer cell used, nor by the
species from which the cancer cell originated. Hence, one can
administer structures which contain cancer cells of a first type to
a subject with a second, different type of cancer. Further, cancer
cells of a species different from the species being treated can be
used in the administered structures. For example, mouse cancer
cells may be restricted in the structures of the invention, and
then be administered to a human. Of course, the structures may
contain cancer cells from the same species as is being treated.
Still further, the cancer cells may be taken from the individual to
be treated, entrapped and restricted, and then administered to the
same individual.
[0120] Yet another aspect of the invention is the use of
concentrates, as described herein, as a therapeutic agent. These
concentrates may be prepared as described herein, and then be
administered to a subject with cancer. All of the embodiments
described supra may be used in preparing the concentrates. For
example, following in vitro culture of structures containing mouse
cancer cells, concentrates can be prepared and then administered to
humans. Similarly, the structures can contain human cells, and even
cells from the same individual. Also, as discussed supra, the type
of cancer cell used to prepare the concentrate may be, but need not
be, the same type of cancer as the subject suffers from. Hence,
murine mammary cancer cells may be used, e.g., to prepare a
concentrate to be used to treat a human with melanoma, or an
individual with prostate cancer may have some of his prostate
cancer cells removed, entrapped in a structure of the invention,
cultured in an appropriate medium, and then have resulting
conditioned medium filtered to produce a concentrate. It should be
borne in mind that the conditioned media resulting from in vitro
cultures of the structures of the invention is also a part of the
invention.
[0121] Processes for making the structures of the invention, as
well as the concentrates of the invention, are also a part of the
invention. In the case of the concentrates, one simply cultures the
structures of the invention for a time sufficient to produce a
sufficient amount of antiproliferative material and then separates
the desired portions from the resultant conditioned medium, e.g.,
by filtration with a filter having an appropriate cut off point,
such as 30 kilodaltons or 50 kilodaltons.
[0122] Other facets of the invention will be clear to the skilled
artisan, and need not be set out here.
[0123] The terms and expression which-have been employed are used
as terms of description and not of limitation, and there is no
intention in the use of such terms and expression of excluding any
equivalents of the features shown and described or portions
thereof, it being recognized that various modifications are
possible within the scope of the invention.
* * * * *