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Cancer Patent 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.
Cancer Patent Claims
We claim:
1. A composition of matter comprising a solid, agarose coated,
agarose and collagen bead, wherein said bead contains cancer cells
isolated from an animal which, when restricted by being entrapped
in said bead, produce more of a material that suppresses cancer
cell proliferation, wherein said material diffuses through said
solid, agarose coated, agarose and collagen bead.
2. The composition of matter of claim 1, wherein said cancer cells
are renal cancer cells.
3. The composition of matter of claim 1, wherein said bead contains
from about 10,000 to about 200,000 cells.
4. The composition of matter of claim 3, wherein said bead contains
from about 30,000 to about 100,000 cells.
5. Method for suppressing cancer cell proliferation in a subject,
comprising implanting a sufficient amount of the composition of
matter of claim 1 in said subject to suppress the proliferation
of cancer cells in subject.
6. A process for making a solid bead which comprises agarose and
collagen, and is coated with agarose, wherein said solid bead contains
cancer cells which, when restricted by being entrapped in said bead
produce material that suppresses cancer cell proliferation and diffuses
through said bead, comprising: (a) adding agarose and collagen to
a solution which contains a sample of cancer cells isolated from
an animal which are capable of producing material that suppresses
cancer cell proliferation which diffuses through said bead when
said cancer cells are restricted by being entrapped by the bead,
(b) forming a semi-solid bead comprising said agarose and collagen
and said cancer cells, (c) polymerizing the collagen in said semi-solid
bead to form a solid, agarose and collagen bead containing and thereby
restricting said cancer cells, and (d) coating said solid, agarose
and collagen containing bead containing the restricted cancer cells
with agarose, wherein said restricted cancer cells produce more
of said material than when said cancer cells are not entrapped in
said coated bead.
7. The process of claim 6, wherein said solution contains from
about 10,000 to about 200,000 cells.
8. The process of claim 7, wherein said solution contains from
about 30,000 cells to about 100,000 cells.
Cancer Patent Description
FIELD OF THE INVENTION
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
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.
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.
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.
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)).
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.
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.
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: 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.
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.
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.
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.
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.
SUMMARY OF THE INVENTION
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.
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.
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.
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.
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.
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.
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.
These, and other features of the invention, will be seen from the
disclosure which follows.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
EXAMPLE 1
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 Induced Tumor Immunogenecity,
181-193 (1994).
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.
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.
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.
A sample of one tumor, taken from one of the mice, was fixed in
10% formalin for later histological examination.
EXAMPLE 2
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.
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.
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.
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.
These RENCA containing beads were used in the experiments which
follow.
EXAMPLE 3
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.
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.
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
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.
Three weeks after the implantation, all of the mice received intraperitoneal
injections of 10.sup.6 RENCA cells. Eighteen days later, one control
mouse died. All remaining mice were then sacrificed, and evaluated
for the presence or absence of tumor.
Control mice showed numerous tumors, while the mice which received
the implants of bead-encapsulated cells showed only isolated small
nodules throughout the cavity.
These encouraging results suggested the design of the experiments
set forth in the following example.
EXAMPLE 5
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.
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.
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.
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
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.
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 (Empty 120,000
Control (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
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
These results encouraged an in vivo experiment. This is presented
in example 8.
EXAMPLE 8
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.
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.
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.
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.
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 EXPERI- MACROBEADS MENTAL EXPERIMENTAL
IN MICE CONTROL GROUP A GROUP B 1 ++++ - - 1 ++++ - - 1 + ++ 2 ++++
- - 2 - - 2 ++ ++ 3 ++++ - - 3 - - 3 - +++
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 (++).
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
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.
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.
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.
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.
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 TEST RENCA CELLS WITH CONDITIONED
WELL # WITH 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
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
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.
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.
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.
Results follow:
TABLE-US-00005 TABLE 5 FROZEN FROZEN CONTROL CONDITIONED CONDITIONED
DISH MEDIUM MEDIUM OF RENCA MEDIUM OF MMT 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
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
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.
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.
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.
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 Unconditioned
RENCA Macrobead 30K Concentrate of this 50K Concentrate of this
Plate RPMI Medium Conditioned Medium Medium 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%
TABLE-US-00007 TABLE 7 Inhibition of RENCA Cell Growth in RENCA
Cell Culture Conditioned Medium and Reconstituted Concentrates Unconditioned
RENCA Cell Culture 30K Concentrate of 50K Concentrate of Plate Medium
Conditioned Medium this Medium 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%
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% ~5.0
.times. 10.sup.4 96% ~4.0 .times. 10.sup.4 97% 2 1.3 .times. 10.sup.6
5.5 .times. 10.sup.5 58% ~5.0 .times. 10.sup.4 96% ~4.0 .times.
10.sup.4 97%
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
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.
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.
The results are set forth below:
TABLE-US-00009 TABLE 9 RESULTS OF MCF-7 MACROBEADS ON RENCA TARGET
CELLS CONTROL Well # (Empty Macrobeads) MCF-7 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
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
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
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
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.
Eighteen Balb/c mice were injected with 20,000 RENCA cells intraperitoneally.
After three days the mice were divided into twogroups. 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.
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%).
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
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.
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. Hernotoxylin & 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.
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.
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.
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.
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
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.
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.5
cells per macrobead) each, and six control mice received four empty
macrobeads each.
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.
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
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 Trans- plant 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
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 Trans- plant 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
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
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
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).
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.
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
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.
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.
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.
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.
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Other facets of the invention will be clear to the skilled artisan,
and need not be set out here.
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. |