Note: Descriptions are shown in the official language in which they were submitted.
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INCREASING TUMOR OXYGEN CONTENT BY
ADMINISTRATION OF STRESSED CELLS
FIELD OF THE INVENTION
This invention pertains to the field of medicine and in particular to the
treatment
of tumors.
BACKGROUND OF THE INVENTION
Tumors are a rapidly expanding mass originating from one transformed cell.
This rapid expansion does not allow for proper vascularization to occur. At
the size of
2-3 mm3 the tumor would hypothetically stop growing because of its need for a
blood
supply to provide oxygen and nutrients (1). Unfortunately, the tumor can
coerce the
host endothelial cells to enter the growing mass and provide life support.
This is
accomplished, in part, by release of chemoattractant compounds from the
growing
tumor or from immune system cells which have entered the growing tumor. These
compounds activate neighboring endothelial cells to migrate to the tumor, to
cut through
host tissue so that they can arrive at the tumor, and to start proliferating
and forming
new blood vessels for the tumor. This process is called angiogenesis (3).
Although the host-derived endothelium allows the tumor to grow, its vascular
structure is much different from that of normal tissue. The tumor has no
intratumor
lymphatics, this does not permit fluid draining from tumour tissue and as a
result high
interstitial fluid pressure develops (2). The high interstitial pressure
inhibits drugs from
penetrating the whole tumor tissue, as well as forcing death of some tumor
cells. This
alteration of interstitial pressure combined with the rapid rate of tumor cell
proliferation
ends up forming a situation where the growing tumor is vascularized, but only
to the
limited extend that it needs for it's own survival. Tumor blood vessels do not
contain
smooth muscle lining, are resistant to control by the nervous system, and grow
in a
disorganized manner compared to vasculature in non-tumor tissue (3). An
example of
the difference between tumor and non-tumor vasculature is that the former
relies on
tumor secretion of vascular endothelial growth factor (VEGF) for its survival,
whereas
the later is insensitive to withdraw) of VEGF (4).
Tumors contain areas of hypoxia (4a). The cause of this is multifactorial and
includes poor tumor perfusion by the blood (5), clotting of tumor blood
vessels due to
activated clotting factors on tumor endothelium (6), and the rapid rate of
tumor growth.
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Hypoxia, and poor perfusion have been shown to negatively correlate with
prognosis (7,
g). Cancer cells under hypoxic environments secrete matrix.metalloproteases,
which
allow them to metastasize (9). In addition, hypoxia programs cancer cells and
macrophages to secrete VEGF, a protein that stimulates angiogenesis as well as
immune
suppression (10). Hypoxia activates hypoxia inducible factor (HIF-1) a nuclear
transcription factor which is important in promotion of angiogenesis (11).
Besides local hypoxia, late stage cancer patients have lower systemic
hemoglobin levels compared to healthy controls, this is due in part to lower
renal
production of erythropoietin (1la). Lower hemoglobin implies less oxygen
transport
and therefore reduced tumor oxygenation. Studies aimed at increasing
hemoglobin
levels by administration of erythropoietin have shown increased efficacy of
radiotherapy
and chemotherapy (llb).
An important consideration of tumor hypoxia is the role it plays in protection
of
tumors from innate anti-tumor defense systems. For example tumor necrosis
factor
alpha (TNF-a) is a cytokine secreted by activated macrophages, which as the
name
implies has antitumor activity. Interestingly, the cytotoxicity of TNF-oc to
tumor cells is
reduced under hypoxic conditions (12). Hypoxia also stimulates production of
soluble
TNF-oc receptors which hypothetically may block systemic activities of this
cytokine
(13). Lymphokine activated killer (LAK) cells are effective in killing certain
types of
tumor cells in vitv~o and in vivo. The ability to generate these cells is
depressed under
conditions of hypoxia (14). Proliferation of lymphocytes in response to
interleukin-2 is
also inhibited during hypoxia (15).
Several therapeutic interventions such as radiation and certain types of
chemotherapy are oxygen dependent. In 1953 the impact of tumor oxygenation on
efficacy of radiation therapy was described by Gray et al (16). Since then a
great
number of studies confirming that tumor sensitivity to radiotherapy positively
correlates
with tumor oxygenation (reviewed in 17). Several other interventions such as
etoposide,
doxorubicin, camptothecin and vincristine therapy are oxygen dependent (17a).
Methods of increasing tumor oxygenation have been described. One such
method involves exposing a patient to higher oxygen tension by use of a
hyperbaric
chamber. This approach has shown marginal increases in oxygenation of some
tumors
although clinical efficacy is a matter of debate. A randomized trial of
squamous cell
sarcoma patients treated with radiotherapy in the presence of air or
hyperbaric oxygen
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demonstrated a significantly greater number of patients achieving clinical
response in
the hyperbaric oxygen group (18). Another randomized study assessing the
ability of
hyperbaric oxygen to increase efficacy of radiotherapy in cervical carcinoma
patients
demonstrated no beneficial effects (19). In addition to questionable clinical
efficacy
hyperbaric oxygen is a costly and sometimes dangerous procedure, which is not
commonly used.
Inhalation of carbogen, a mixture of 95% oxygen and 5% carbon dioxide has
also been shown to increase tumor oxygenation both in animal models (20,21)
and in
the clinical situation (22-24).. An explanation far this effect is that carbon
dioxide has
vasodilatory functions in this setting which allows for better tumor perfusion
of the high
concentration of inhaled oxygen (25). Therapeutic benefits of carbogen therapy
are
mixed although some radiosensitizing effects have been observed alone (26-28),
or in
combination with nicotinamiide in Phase I/II trials (29).
Due to the lack of established evidence of efficacy of the above methods used,
it
becomes apparent that novel methods of increasing tumor oxygen content are
needed.
This background information is provided for the purpose of making known
information believed by the applicant to be of possible relevance to the
present
invention. No admission is necessarily intended, nor should be construed, that
any of
the preceding information constitutes prior art against the present invention.
SUMMARY OF THE INVENTION
The invention discloses a method of using agents which stress mammalian cells
in such a manner so that administration of these stressed cells into a tumor
bearing host
will allow for increased oxygen content in the tumor. Another embodiment of
the
invention is stressing cells of a mammal in vivo through the administration of
ozone
gas. By increasing tumor oxygenation, therapeutic index of oxygen-dependent
treatment modalities will be increased.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 illustrates administration of ozone stressed cells reduces tumor
hypoxia
as measured by EppendorfT"" probe.
Figure 2 illustrates administration of ozone stressed cells enhances the
ability of
radiotherapy to decrease clonogenicity of tumors.
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Figure 3 illustrates administration of ozone stressed cells enhances ability
of
radiotherapy to inhibit tumor growth.
Figure 4 illustrates administration of ozone stressed cells enhances ability
of
melphalan, an alkylating agent, to inhibit tumor growth.
Figure 5 illustrates that administration of UVA + 8-methoxypsoralen stressed
cells increases tumor sensitivity to radiotherapy.
Figure 6 illustrates that administration of UVA + 8-methoxypsoralen stressed
cells increases tumor sensitivity to melphalan, an alkylating agent.
Figure 7 illustrates stressed mononuclear cells inhibits proliferation of
endothelial cells
Figure 8 illustrates intravenous administration of ozone enhances tumor
inhibitory effects of melphalan, an alkylating agent.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides a method for overcoming hypoxia in tumors through
administration of a cell mixture or whole blood, which has been subjected ex
vivo to
biological stress and/or oxidative agents. These cells, chemical intermediates
produced
by the cells, or oxygen carried by these cells, induce biological cascades,
which result in
reduction of tumor hypoxia. The present invention provides various agents that
can be
used to stimulate this biological cascade. Increasing oxygen content of tumors
renders
them susceptible to various interventions including chemotherapy, radiotherapy
and
immune therapy.
Patient cells drawn from peripheral blood, or whole blood itself, is subjected
to
the stressful conditions. Alternatively, distinct subtypes of blood cells may
be purified
and subjected to stressful conditions. These conditions include but are not
limited to
alterations in temperature, alterations in osmotic balance, irradiation of the
cells, serum
depravation, treatment with gamma or ultraviolet radiation, treatment with
ozone gas, or
treatment with other agents which induce oxidative stress such as, but not
limited to
hydrogen peroxide. The stressed cells, intermediates released from the cells,
or the
mixture of the oxidative agents with cell-free portion of the blood are
reintroduced to
the patient either intravenously, subcutaneously, intramuscularly, or by other
means of
introducing agents into patients. The invention teaches that this treatment of
purified
cells or whole blood to stressors, will induce biological cascades which
result in
reduction of tumor hypoxia. Alternatively, injection of ozone gas at
appropriate
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concentration in medical grade oxygen can result in stressing of cells in
vivo, also
causing biological cascades which result in the inhibition of tumor hypoxia.
Such
reduction of hypoxia is useful as an adjuvant to various cancer treatments
known in the
art or can be used alone to inhibit tumor growth by increasing oxygenation of
tumors.
A method is provided for increasing oxygen content in tumor cells by cells
which have been subjected to stress either outside of the body and
subsequently
reintroduced into the body, or by cells which hare been stressed inside the
body by
administration of a stressing agent. As used herein, the term "stress" refers
to
conditions which alter the normal functions of cells. More specifically,
several such
conditions are described, without limiting the invention to specifics, these
conditions
include: exposure to oxidative agents such as ozone, treatment of cells with
unphysiological conditions such as alterations in temperature, exposure of
cells to
radiation, or utilization of a chemosensitizer together with radiation such as
the
combination of 8-methoxypsoralen with ultraviolet A irradiation. Effects of
stress on
cells include alterations of normal functions, and in some cases apoptosis.
The amount
of stress administered to a cell to achieve the desired effect of a cell
population capable
of increasing oxygen content of tumors once the cells are introduced into the
tumor
bearing animal, is decided by the tumor type, type of cell stressed, and
individual
characteristics of the patient. The practitioner of the invention disclosed
will possess
access to techniques that allow for individual determination of the most
optimum
method of practicing the invention. For example, quantification of tumor
oxygenation
can be performed using EppendorfT"" probes in order to modify the types and
amounts of
the indicated components of this invention to achieve maximal oxygenation of
the
tumor(s).
In one embodiment, whole blood is taken from the patient in a desired volume,
preferentially but not limited to a volume of 10 ml and ozone gas is bubbled
through it
in a sterile container. Cells can be stressed by bubbling 5-20 pg of ozone per
ml of
oxygen at a flow rate of 220 ml/minute for 2 minutes in a sterile glass flask.
Ozone gas
may also be mixed with the blood using methods known in the art, including
those
described by Wainwright (30), Muller (31), or by Williams (32) The
concentration of
ozone gas being bubbled through the blood, as well as, the amount of time that
it is
bubbled through, is determined on the appropriate amount of stressed cells
needed to
achieve a decrease in tumor hypoxia. Stressing of blood cells can be measured
by
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surrogate markers such as secretion of interleukin 10 using ELISA, release of
heat shock
proteins, or amount of apoptotic lymphocytes using AnnexinV-FITC staining. The
treated blood is then administered in the patient intramuscularly or
subcutaneously.
Such treatment can be performed on a schedule depending on the needs of the
patient.
A typical treatment protocol is administration of the stressed blood cells
three times a
week for several months. This embodiment can be practiced by one skilled in
the art
with various modifications while not departing from the spirit of the
invention, that is,
the administration of the stressed cells in order to decrease tumor hypoxia.
Another embodiment of the invention involves the extracting 50 to 100 ml of
the
patient's blood, ozonating it ex vivo, using an appropriate concentration of
ozone and an
appropriate time span to induce formation of stressed cells, and
readministering it to the
same patient. Following ozonation, the stressed blood cells may be
readministered in
the form of a normal drip-infusion. Blood cells can be stressed by bubbling 5-
20 ~,g of
ozone per ml of oxygen at a flow rate of 220 ml/minute for 2 minutes in a
sterile glass
flask. The rate of reinfusion is preferable, but not limited to 90 drips per
minute.
Yet another embodiment of the invention disclosed involves ozonation of blood
through a extra-corporeal loop apparatus where a tube is placed in the artery
of the
patient, the blood flows through an ozone generator, the blood is mixed with
the ozone
ex vivo, and returned to the patient via a tube placed in a vein. Such an
apparatus has
been previously described in the art hich has been described in the art (30).
In the above embodiments care must be taken not to administer ozone at a high
enough concentration or for a long enough duration to cause hemolysis. Ozone
is
usually administered to the blood as a mixture with pure oxygen but other
methods of
administration may be devised by one skilled in the art without departing from
the spirit
of the disclosed invention. A concentration of ozone used by our group, which
was
found optimal was 5-70 p,g/ml, although 30-300 p,g/ml may also be used.
An embodiment of the invention involves administration of apoptotic cells to
the
patient. Purification of lymphocytes may easily be performed by centrifugation
of
whole blood on a density gradient. Cells from the patient may then be purified
of
erythrocytes and treated by various means such that apoptosis is induced. Such
means
are commonly known in the art, examples of which include administration of
protein
synthesis inhibitors or nucleoside analogues, physical changes in osmotic
pressure,
ligation of death receptors, or treatment with radiation. Using the system
described for
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treatment of psoriasis, cells may be sensitized with a chemical and then
exposed to UV
irradiation as described by McLaughlin et al (33). These cells can then be
purified for
apoptotic bodies, or used as an unpurified inoculum. Administration of these
cells may
take place via subcutaneous, intramuscular, or intravenous route depending on
the
condition of the patient and the effect desired.
The work of Bolton (34) described a novel means of inducing systemic
antiinflammatory biological responses by administration of cells stressed with
heat
and/or UV radiation, while being exposed to ozone. An embodiment of the
invention
described herein is to use the method of Bolton for reduction of tumor
hypoxia.
Another embodiment involves stressing of distinct cell populations found in
circulation. Purified cell populations from whole blood can have potent
effects at
decreasing tumor hypoxia. An embodiment of this invention involves
purification of a
cell population, stressing of the cell, and readministration in order to
reduce tumor
hypoxia. These cells may also be grown, expanded, and/or differentiated ex
vivo before
administration of the stressor. A preferred embodiement involves purification
of
circulating dendritic cells from the patient according to methods such as
Magnetic
Activated Cell Sorting (MACS) or flow sorting, stressing the dendritic cells
with ozone,
or any of the stressors mentioned above, and readministration of the cells to
the patient.
In the cases where substantial numbers of dendritic cells can not be purified,
patient
monocytes can be induced to differentiate into dendritic cells by treatment
with
granulocyte-macrophage colony stimulating factor in conjunction with
interleukin-4 as
previously described (35). Another purified population of cells may be T
cells. Further
purification based on phenotypic markers can be performed according to the
amount and
type of response desired in order to reduce tumor hypoxia.
Another embodiment involves stressing of cells through subjecting them to
conditions which are unphysiological. The stress signaling pathways seem to
converge
to common intracellular mechanisms and nuclear transcription factors. For this
reason
cells which are subjected to serum starvation turn on the same, or very
similar apoptotic
pathways as cells which are "stressed" by radiation or hyperthermia. The
cancer anti-
hypoxic utility of administration of cells stressed by unphysiological
conditions is an
embodiment of the invention described herein.
Another embodiment involves administration of an ozone/oxygen mixture
directly into circulation of a mammal through intravenous injection. This
procedure
increases tumor oxygenation by inducing formation of stressed cell in vivo.
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EXAMPLES
EXAMPLE I
Reduction of Hypoxia In the KHT Fibrosarcoma Model by Ozone Stressed Blood
ml of blood was pooled from 10 C3H mice collected by cardiac puncture in
sodium citrate tubes. Cells were stressed by bubbling 5 or 20 p,g of ozone per
ml of
oxygen at a flow rate of 220 ml/minute for 2 minutes in a sterile glass flask.
As a
10 control, blood was placed in the glass flask for 2 minutes and medical
grade oxygen was
bubbled through it at the same flow rate.
For evaluating the effect of stressed cells on tumor oxygenation, 9-12 week
old
male C3H mice were divided into groups of 10, all of which were inoculated
with 2 x
105 KHT sarcoma cells into the left gastrocnemius muscle. Treatments were
performed
for 5 consecutive days starting two days after tumor cell inoculation.
Group I was given no treatment. Group II was treated with a 50p1 intramuscular
injection of untreated blood. Group III was treated with a 50p,1 intramuscular
injection
of blood treated with ozone at 5p.g/ml (described above). Group IV was treated
with a
50p,1 intramuscular injection of blood treated with ozone at 20pg/ml
(described above).
On day 7 after tumor inoculation, an Eppendorf computerized fine-needle
polarographic electrode probe (Eppendorf, Hamburg, Germany) was used for
intratumor
measurement of hypoxia. In order to assess hypoxic areas, the electrode was
inserted 1
mm into the tumor. The probe was moved through the tumor in increments of
0.8mm
followed by withdrawing the probe 0.3 mm before taking the measurement. 1.4
second
intervals were allowed between re-measurements. An average of 5 parallel
insertions
were performed in each tumor with an average of 50 measurements taken per
tumor.
Oxygenation is reported as mean p02 (mmHg).
As illustrated in Figure 1, administration of ozonated blood from syngeneic
mice
for 5 consecutive days resulted in increased tumor oxygenation compared to
mice
treated with blood that was not ozonated. Increased oxygen content in tumors
was
higher in animals which received blood that was treated with 20p,g/ml compared
to
those treated with 5 pg/ml.
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EXAMPLE II
Reduction of Tumor Clonogenicity in the ICHT Fibrosarcoma Model by Ozone
Stressed Blood
9-12 week old male C3H mice were divided into groups of 10, all of which were
inoculated with 2 x 105 KHT sarcoma cells into the left gastrocnemius muscle.
Treatments were performed for 5 consecutive days starting two days after tumor
cell
inoculation.
Group I was given no treatment. Group II was treated with a 50p,1
intramuscular
injection of untreated blood. Group III was treated with a 50p,1 intramuscular
injection
of blood treated with ozone at 5pg/ml (described above). Group IV was treated
with a
50p1 intramuscular injection of blood treated with ozone at ZOp,g/ml
(described above).
One day 7 post-tumor inoculation, tumors from mice in all groups were
irradiated with a ~3~Cs source at 3 Gy/min for varying time points to
administer doses of
0-25 Gy. Tumors were extracted from mice and made into single cell suspensions
by
mechanical disassociation, and treatment with trypsin and DNase I. Single
cells were
washed with phosphate buffered saline and plated at 5 X 105 cells/ml in a 24
well plate.
As feeder cells 104 lethally irradiated KHT cells/well were plated. The
culture media
was 0.2% agar in a-MEM media with 10% fetal calf serum. Cells were incubated
for
14 days at 37 Celsius, with 5% CO~ in a fully humidified atmosphere. Colonies
with
greater than 50 cells were enumerated.
As illustrated in Figure 2, the higher concentration of ozone which was
applied
to the blood cells, the more sensitive the tumor cells became to radiotherapy.
In
contrast, administration of unstressed mouse blood did not affect
radiosensitivity.
EXAMPLE III
Enhanced Inhibition of Tumor Growth By Ozone Stressed Cells
C3H mice were inoculated with KHT tumors as described above. At about day
7-8 when tumors reached 8 mm in circumference mice were divided into 4 groups
of 10
mice per group. Group I was the untreated control, Group II was administered
15 cGy,
Group III was administered lScGy together with an intramuscular injection of
50 ~l of
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untreated syngeneic blood. Group IV was injected with 50 pl of syngeneic blood
treated with 20 p,l ozone/ml of oxygen as described above. Injections of
untreated, and
treated blood were administered one hour before radiotherapy, and for 4
subsequent
days. Tumor size was evaluated daily and the time until tumors reached 16 mm
was
recorded.
As illustrated in Figure 3, treatment with ozone-stressed blood cells resulted
in a
potent prolongation of time it took for tumor growth to occur.
EXAMPLE IV
Administration of Ozone Stressed Cells Increases Sensitivity of Tumors To
AIkyIating Agents
8 week old mate C3H mice were inoculated with 2 x 106 FSaIIC marine
fibrosarcoma cells in the hind limb. When tumors reached a volume of 50mm3,
animal
were divided into 4 groups of 10 mice. Group I was untreated, Group II was
administered a dose of 10 mg/kg melphalan by intraperitoneal injection. Group
III was
administered melphalan together with 50 p,l of untreated syngeneic blood
injected .
intramuscularly in the non-tumor bearing hind limb. Group IV was administered
melphalan with 50 pl of syngeneic blood treated with 20 pl ozone/ml of oxygen.
Following the initial melphalan injection, animals in Groups III and IV
continued
receiving intramuscular injections of 50 p.l of untreated syngeneic blood, or
50 pl of
syngeneic blood treated with 20 p.l ozone/ml of oxygen, respectively for 4
days, one
injection per day. All animals were observed until tumors attained a volume of
500mm3. The delay in tumor time to achieve 500m3 volume is illustrated in
Figure 4.
E~1MPLE Y
Administration of Cells Stressed By UVA Together With 8-methoxypsoralen
Increases Sensitivity of Tumor Cells To Radiotherapy
C3H spleens were extracted from male mice and made into a single cell
suspension by mechanical dissociation. Erythrocytes were lysed using hypotonic
solution, and mononuclear cells were washed in phosphate buffered saline.
These
mononuclear cells were subsequently incubated, at 37 Celsius for 20 minutes
with 200
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nglml of 8-methoxypsoralen in complete RPMI media at a concentration of 106
cells per
ml. Cells were subsequently exposed to a 2 Jlcm2 dose of UVA (peak of
emission: 365
nm). Exposed cells were allowed to incubate for 18 hours, at 37 Celsius,
followed by
washing in phosphate buffered saline and subcutaneous injection into the non-
tumor
bearing hind limb of the C3H mice.
C3H mice were inoculated with KHT tumors as described above. At about day
7-8 when tumors reached 8 mm in circumference mice were divided into 4 groups
of 10
mice per group. Group I was the untreated control, Group II was administered
15 cGy,
Group III was administered lScGy together with an intramuscular injection of
1X106
syngeneic untreated mononuclear cells. Group IV was injected with 1X106
syngeneic
mononuclear cells that were stressed using UVA and 8-methoxypsoralen as
described
above. Injections of untreated, and treated mononuclear cells were
administered one
hour before radiotherapy, and for 4 subsequent days, once per day. Tumor size
was
evaluated daily and the time until tumors reached 16 mm was recorded.
As illustrated in Figure 5, treatment with stressed lymphocytes resulted in a
potent prolongation of time it took for tumor growth to occur.
EXAMPLE YI
Administration of Cells Stressed By UVA Together With 8-methoxypsoralen
Increases Sensitivity of Tumor Cells To Alkylating Agents
8 week old male C3H mice were inoculated with 2 x 106 FSaIIC murine
fibrosarcoma cells in the hind limb. When tumors reached a volume of 50mm3,
animal
were divided into 4 groups of 10 mice. Group I was untreated, Group II was
administered a dose of 10 mg/kg melphalan by intraperitoneal injection. Group
III was
administered melphalan together with an intramuscular injection of 1X106
syngeneic
untreated mononuclear cells. Group IV was administered melphalan with 1X106
syngeneic mononuclear cells that were stressed using UVA and 8-methoxypsoralen
as
described above. Following the initial melphalan injection, animals in Groups
III and
IV continued receiving intramuscular injections of 1X106 syngeneic untreated
mononuclear cells, or 1X106 syngeneic mononuclear cells that were stressed
using UVA
and 8-methoxypsoralen, respectively for 4 days, one injection per day. All
animals were
observed until tumors attained a volume of 500mm3. The delay in tumor time to
achieve 500m3 volume is illustrated in Figure 6.
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EXAMPLE YII
Stressed Mononuclear Cells Inhibits Proliferation of Endothelial Cells
Murine mononuclear cells were stressed with 5 p,g/ml or 20 p.g/ml of ozone, or
stressed by treatment with 8-methoxypsoralen and UVA, as described in the
previous
examples. 5X10ø stressed, or untreated mononuclear cells were added to
confluent
human umbilical vein endothelial cells (HUVEC) in 96 well plates. Cells were
cultured
in EBM-2T"" media. Co-culture of stressed cells with HUVEC was performed for
24
hours. Proliferation of HUVEC was assessed by pulsing the 96 well plate with 1
p.Ci
tritiated thymidine per well for 18 hours. Cells were then harvested on a
WallacTM
harvester, and radioactivity was measured using scintillation.counting.
As seen in Figure 7, mononuclear cell had minimal activity on proliferation of
HUVEC cells. In contrast cells stressed by ozone and UVA+8-methoxypsoralen had
an
antiproliferative effect.
EXAMPLE VIII
In Vivo Stressing of Cells Increases Sensitivity of Tumors To Alkylating
Agents
In order to stress cells in vivo, ozone at 5 p.g per ml of oxygen, or 20p,g
per ml of
oxygen was injected directly into the tail vein of tumor bearing mice. Such
injections
are performed by filling a 1 cc syringe with the indicated concentration of
ozone in
medical grade oxygen. The gas is slowly injected into the tail vein so not to
induce
embolism. Due to the high solubility of oxygen and ozone in blood, embolism
formation is not usually a problem.
8 week old male C3H mice were inoculated with 2 x 106 FSaIIC murine
fibrosarcoma cells in the hind limb. When tumors reached a volume of 50mm3,
animal
were divided into 4 groups of 10 mice. Group I was untreated, Group II was
administered a dose of 10 mg/kg melphalan by intraperitoneal injection. Group
III was
administered melphalan together with 50 p.l of 5pg of ozone per ml of oxygen
by tail
vein injection. Group IV was administered melphalan with 50 p,l of 20p,g of
ozone per
ml of oxygen by tail vein injection. Following the initial melphalan
injection, animals
in Groups III and IV continued receiving intravenous injections of 50 pl of
5p,g of ozone
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per ml of oxygen, or 50 pl of 20p.g of ozone per ml of oxygen, respectively
for 4 days,
one injection per day. All animals were observed until tumors attained a
volume of
500mm3. The delay in tumor time to achieve 500m3 volume is illustrated in
Figure 8.
Treatment with intravenous ozone was well tolerated and both concentrations
increased
S the tumor inhibitory effects of melphalan.
The invention discloses several biotherapeutic utilities of DU-145
supernatant.
It is to be understood that the above examples are presented only for clarity
and that the
invention may take other embodiments as practiced by one skilled in the art.
Through
routine experimentation, one skilled in the arts will recognize many
equivalents to the
specific materials and procedures described herein. These equivalents are
intended to
be encompassed with the scope of the claims of the disclosed invention.
The invention being thus described, it will be obvious that the same may be
varied in many ways. Such variations ar not to be regarded as a departure from
the
spirit and scope of the invention, and all such modifications as would be
obvious to one
skilled in the art are intended to be included within the scope of the
following claims.
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Int J Radiat Biol. 1991 Jul-Aug;60(1-2):85-100
3. Tumour angiogenesis / edited by Roy Bicknell, Claire, E. Lewis, and
Napoleone
Ferrara. Oxford, [England] ; New York : Oxford University Press, 1997.
4. Benjamin LE, Golijanin D, Itin A, Pode D, Keshet E. Selective ablation of
immature
blood vessels in established human tumors follows vascular endothelial growth
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