(CANCER RESEARCH 50. 3339-3344. June 1. 1WO]
Classification of Antineoplastic Treatments by Their Differential Toxicity toward
Putative Oxygenated and Hypoxie Tumor Subpopulations in Vivo in the
FSalIC Murine Fibrosarcoma1
Beverly A. Teicher,2 Sylvia A. Holden, Antoine Al-Achi, and Terence S. Herman
Dana-Farber Cancer Institute ¡B.A. T., S. A. H., A. A-A., T. S. H.I and Joint Center for Radiation Therapy ¡B.A. T., T. S. H.], Boston, Massachusetts 02115
ABSTRACT
In order to investigate the effect of environmentally determined con
ditions on the cytotoxicity of anticancer treatments, Hoechst 33342 dye
selected tumor subpopulations were separated after in vivo treatment and
plated for single cell colony survival. The 10% brightest cells were
assayed as putative normally oxygenated cells and the 20% dimmest as
putative hypoxic cells. At single therapeutic doses, cyclophosphamide
treatment resulted in the largest differential killing between bright and
dim cells (6.3-fold bright > dim); l,3-bis(2-chloroethyl)-l-nitrosourea
was 3.2-fold more cytotoxic toward bright cells and carboplatin was 2.4fold more toxic toward bright cells. Both radiation (10 Gy) and melphalan
were 2.2-fold more toxic to bright cells, while m-diamimnedichloroplatinum(II) was 1.8-fold, thiotepa was 1.2-fold and procarbazine was 1.3fold more toxic to bright cells. Actinomycin D was 3.4-fold more toxic to
bright cells. Adriamycin was 2.2-fold, vincristine was 2.1-fold, and etoposide was 1.6-fold more toxic to bright cells. Bleomycin and 5-fluorouracil were also tested and were 1.5- and 2.3-fold more toxic to bright cells,
respectively. Only four treatments were more toxic to dim cells: mitomycin C (3.5-fold), misonidazole (1.5-fold), etanidazole (3.5-fold), and
43°C,30 min local hyperthermia (2.6-fold). In an attempt to shift the
pattern of dim cell sparing, Fluosol-DA plus carbogen (95% O2/5% CO2)
breathing was added to treatment with radiation (10 Gy), melphalan, cndiamminedichloroplatinum(II), and etoposide. Although each of these
treatments became significantly more toxic with the addition of FluosolDA/carbogen, only with melphalan did the combination overcome the
sparing of dim cells. These results indicate that cells located distally
from the tumor vasculature are significantly less affected by most anticancer drugs and suggest that successful therapeutic strategies against
solid tumors will involve greater use of the few treatments which are
more toxic toward this tumor subpopulation.
hyperthermia have been shown to be affected by changes in
both intra- and extracellular pH (25-28).
Position in the cell cycle is also critical to the cytotoxic action
of some chemotherapeutic agents and is an important variable
in the actions of many others (29, 30). Although a difficult
question to approach experimentally (31), it is likely that the
great proportion of cells which are distal from the vasculature
and in a hypoxic and acidotic environment are noncycling. Cells
which are noncycling would be expected to be less sensitive to
many agents but may be more sensitive to nitrosoureas and
bleomycin (32-35).
The ability of drugs to penetrate through cell layers to reach
cells farther from the vasculature in concentrations adequate to
be therapeutically effective is a variable dependent to a large
degree on the lipophilicity and metabolic stability of the drug
molecule. Furthermore, the intracellular concentrations of the
cytotoxic agents which can be achieved may differ for oxygen
ated and hypoxic cells. Although molecular oxygen diffuses
only a short distance through tumor tissue because of its rapid
metabolic utilization, some dyes and some drugs can diffuse
into the tumor mass over much greater distances (8, 9, 36).
In this study, the cytotoxic effects of antineoplastic agents
from several different classes and treatment modalities on the
survival of tumor subpopulations near to and distal from the
tumor vasculature from FSalIC tumors treated in vivo were
examined. The effect of Fluosol-DA with air or carbogen
breathing on several of these treatments was also examined.
MATERIALS AND METHODS
INTRODUCTION
Drugs. CDDP' and carboplatin were gifts from Drs. Donald Picker
It is difficult to cure most solid tumors by treatment with
nonsurgical therapeutic modalities. Intrinsic resistance of the
tumor cells to treatment agents provides a partial explanation
for treatment failure (1-3); heterogeneity in several physiolog
ical properties of solid tumors resulting from inadequate and
nonuniform vascularization is also a contributing factor (4-9).
It has been well established that oxygen is rapidly metabolized
by cells and, therefore, in tissues has a limited diffusion distance
from vasculature (8-14). Regions of hypoxia have been dem
onstrated in many solid tumor model systems (10-13, 15, 16)
and in human solid tumors (5, 17) by several different methods.
In cell culture and in some cases in vivo, significant differences
in the effectiveness of many antineoplastic agents and treatment
modalities have been demonstrated to be dependent on cellular
oxygénation(7, 18-23). Solid tumors may also have regions of
more acidic or more basic pH than are found in normal tissues
(10, 14, 24). The actions of some chemotherapeutic agents and
Received 8/7/89; revised 2/16/90.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Cancer Institute Grant POI CAI9589
and a grant from Bristol-Myers. Inc.. Wallingford, CT.
2To whom requests for reprints should be addressed.
and Michael Abrams, Johnson Matthey, Inc. (West Chester, PA).
Bleomycin (Blenoxane) was a gift from Bristol Laboratories (Syracuse,
NY). MISO and ETA were obtained as gifts from the Drug Synthesis
and Chemistry Branch, Developmental Therapeutics Program, Na
tional Cancer Institute (Bethesda, MD). L-PAM and Mito C were
purchased from Sigma Chemical Co. (St. Louis, MO). Fluosol-DA,
20% (manufactured by Green Cross Corp.) was provided by Alpha
Therapeutics Corp. (Los Angeles, CA). The stem emulsion was stored
frozen and the complete emulsion was prepared immediately prior to
use. Carbogen is 95% oxygen and 5% carbon dioxide. All other drugs
were obtained from the Dana-Farber Cancer Institute pharmacy.
Tumor. The FSall fibrosarcoma (37) adapted for growth in culture
(FSalIC) (38, 39) was carried in male C3H/He mice (The Jackson
Laboratory, Bar Harbor, ME). For the experiments, 2 x 10* tumor
cells prepared from a brei of several stock tumors were implanted i.m.
into the legs of male C3H/He mice 8 to 10 weeks of age.
Tumor Subpopulation Studies: Tumor Growth and Hoechst 33342
Labeling. When the tumor volumes were approximately 100 mm3
(about 1 week after tumor cell implantation), animals were treated with
a single dose of each drug, with a single dose of hyperthermia (43°C,
'The abbreviations used are: CDDP. m-diamminedichloroplatinum(II);
B
cells, bright cell subpopulation: D cells, dim cell subpopulation; BCNU, carmustine. l,3-bis(2-chloroethyl)-l-nitrosourea;
L-PAM, melphalan. L-phenylalanine
mustard; Procarb. procarbazine; 5-FUra, 5-fluorouracil; VP-16, etoposide. VP16-213: Mito C, mitomycin C; ETA. etanidazole, SR-2508; MISO, misonidazole;
S.F.. surviving fraction.
3339
TREATMENT TUMOR SUBPOPULATION SELECTIVITY
30 min) by immersion of the tumor-bearing limb in a specially designed
Plexiglas water bath at 44°Cwhich allows the centers of tumors to
reach 43 ±0.2T as measured by a digital readout thermistor (Sensortech, Inc., Clifton, NJ) placed into the center of the tumor in selected
control animals or with radiation ( 10 Gy) delivered locally to the tumorbearing limb ("7C 7-rays at a dose rate of 88 rads/min; Gamma Cell
40; Atomic Energy of Canada, Ltd.). Several groups also received
Fluosol-DA (0.3 ml, 12 ml/kg) with air or carbogen breathing (1 or 6
h) after drug administration or prior to (1 h) and during radiation.
Hoechst 33342 dye (2 mg/kg; Aldrich Chemical Co., Milwaukee, WI)
dissolved in phosphate-buffered saline was administered by tail vein
injection (0.25 ml) into tumor-bearing mice 24 h after treatment. Tumor
cell suspensions were prepared by excising the tumor 20 min after i.v.
administration of the dye (31, 40-45) under sterile conditions, and
single cell suspensions of tumor cells were prepared for the colonyforming assay (46). To remove contaminating erythrocytes, 0.17 M
NH4C1 was added to the tumor cell suspension for 3 min at room
temperature just after filtering through gauze. The cells were then
washed once with a minimum essential medium, supplemented with
10% fetal bovine serum, filtered through a syringe fitted with a 40-um
nylon mesh filter to remove cell clumps, counted, and centrifuged at
200 x g. Cells were then resuspended at a concentration of 2 x 106
cells/ml in complete medium. No significant difference in the number
of cells recovered in experimental versus untreated tumors was found.
The number of cells per tumor was independent of the interval between
treatment and excision. Since the numbers of recovered cells per
treatment group typically varied by 15%, however, only cell losses of
greater than 20% would have been detectable.
Flow Cytometry and Sorting. The fluorescence of the cells from
tumors was analyzed and sorted using the Coulter Epics V instrument
(Hialeah, FL). Hoechst 33342 dye intensity was measured using an
argon ion laser with excitation at 350-360 nm (40 mW power) and
emission monitored with a 457-nm long pass and a 530-nm short pass
filter. The fluorescence distributions were divided into ten fractions
based on Hoechst 33342 intensity. Two sort fractions of cells were
collected, one which contained the brightest 10% of cells and the other
containing the dimmest 20% of cells. For any tumor cell population
fluorescence intensity spans about 3 logs on a scale of 1 to 1000. The
10% brightest cells are collected over a fluorescence range from about
75 to 550 and the 20% dimmest cells are collected over a fluorescence
range from about 2 to 4. No significant difference in the fluorescent
patterns of the sorted cellular populations at 24 h after treatment was
observed as compared with cells from untreated control tumors. The
cells were washed once with complete medium. After 1 week colonies
were stained with crystal violet, and colonies of >50 cells were counted
by eye. The plating efficiency for the unsorted population was 15.5 ±
2.7%. For the 10% brightest cells, the plating efficiency was 9.2 ±
1.6%, and for the 20% dimmest cells the plating efficiency was 5.5 ±
1.4%. The survival results are expressed as the surviving fraction ±SE
of the treated bright and dim fractions compared to the bright and dim
untreated controls, respectively.
Table 1 Survival of subpopulations based on Hoechst 3.1342fluorescence
intensity ofFSallC cells from FSallC tumors treated with a single dose of an
antitumor agent or treatment modality
Subpopulation surviving
fraction"
TreatmentDose(mg/kg)BrightDimFolddifferential*
Alkylating agents
CyclophosphamideBCNUCarboplatinX-raysMelphalanCDDPThiotepaProcarbazineISO5050(lOGy)1010
0.046.33.22.42.22.21.81.71.2AntibioticsBleomycinActinomycin
0.081.000.74
±
DAdriamycinVincristineVP0.061.000.62
±
16101252200.440.290.330.470.38-t-±-4-+±0.050.040.050.050.030.66
±0.051.53.42.22.11.6
Antimetabolite
5-Fluorouracil
40
0.97 ±0.03
2.3
Hypoxic cell selective treatments
Mitomycin C
5
0.37 ±0.05 0.11 ±0.02
Etanidazole
1000
0.82 ±0.04 0.24 ±0.04
Misonidazole
1000
0.84 ±0.06 0.56 ±0.04
(43°C,
3
0
min)
0.36 ±0.04
0.14 ±0.02
Hyperthermia
" Data are means of three independent experiments ±SEM.
* Ratio of D-cell survival to B-cell survival.
0.29
0.29
0.66
0.39
0.43 ±0.06
cells. It has been well established that the toxicity of radiation
is diminished under hypoxic conditions (6, 16, 39). Radiation
(10 Gy) was 2.2-fold more cytotoxic toward the B cells than
toward the D cells. L-PAM (10 mg/kg), a relatively lipophilic
nitrogen mustard, was also 2.2-fold more cytotoxic toward the
B cells than toward the D cells. CDDP (10 mg/kg) which is a
relatively readily water soluble small molecule, was about 1.8fold more cytotoxic toward the B cells than toward the D cells.
Thiotepa (/V,/V',/V"-triethylenethiophosphoramide)
(10 mg/
kg), which may undergo extratumoral metabolism to the active
alkylating species (22, 53-55), was about 1.7-fold more cyto
toxic toward the B cells than toward the D cells. Finally,
procarbazine (20 mg/kg) which is a lipophilic hydrazine that
undergoes a complex metabolism/chemical degradation to al
kylating species (56, 57), was about 1.3-fold more cytotoxic
toward the B cells than toward the D cells where it had only a
marginal effect.
The antitumor activity of actinomycin D is believed to depend
primarily on its ability to intercalate into the DNA helix (58,
59). Actinomycin D (l mg/kg) was at least 3.4-fold more
RESULTS
cytotoxic toward the B cells than toward the D cells where no
toxicity was observed (Table 1). Adriamycin (25 mg/kg) was
Each drug or treatment modality was examined at a single
dose. The dose chosen in each case was in the single dose about 2.2-fold more cytotoxic toward the B cells than toward
the D cells. The Vinca alkaloid vincristine (2 mg/kg) was at
therapeutic range for that particular agent or modality. The
least 2.1-fold more cytotoxic toward the B cells than toward
largest differential tumor cell kill between the bright (presum
ably oxygenated) and dim (presumably hypoxic) tumor cell the D cells where no tumor cell killing was observed. The
epipodophyllotoxin
derivative (60) VP-16 (20 mg/kg) was
subpopulations was observed after treatment with cyclophosabout
1.6-fold
more
cytotoxic toward the B cells than toward
phamide (150 mg/kg (Table 1). Cyclophosphamide (150 mg/
kg), which is a prodrug for the short-lived alkylating species
the D cells. The antitumor peptide bleomycin (10 mg/kg) was
about 1.5-fold more cytotoxic toward the B cells than toward
phosphoramide mustard (47-49), was 6.3-fold more toxic to
the D cells. The antimetabolite 5-FUra (40 mg/kg) was about
ward the B cells than toward the D cells. The nitrosourea
BCNU (50 mg/kg), which is also relatively short-lived in the 2.3-fold more cytotoxic toward the B cells than toward the D
circulation (50-52), was about 3.2-fold more toxic toward the cells where almost no cytotoxicity was seen.
Only four treatments were found to be more cytotoxic toward
B cells than toward the D cells. Carboplatin (50 mg/kg), a
platinum complex which is more lipophilic than CDDP, was the dim (hypoxic) tumor cell subpopulation than toward the
about 2.4-fold more toxic toward the B cells than toward the D bright (oxygenated) tumor cell subpopulation (Table 1). Mito
3340
TREATMENT TUMOR SUBPOPULATION SELECTIVITY
C (5 mg/kg) which has been well established as a hypoxic cell
selective cytotoxic agent through predominantly in vitro studies
(4-7, 23, 40), was about 3.5-fold more cytotoxic toward the D
cells than toward the B cells. Both of the 2-nitroimidazole
radiosensitizers, MISO (1 g/kg) and ETA (1 g/kg), were more
cytotoxic toward the D cells. MISO (l g/kg) was about 1.5-fold
more cytotoxic toward the D cells than toward the B cells;
while ETA (1 g/kg) was about 3.5-fold more cytotoxic toward
the D cells than toward the B cells. Hyperthermia (43°C,30
min) was about 2.6-fold more cytotoxic toward the D cells than
toward the B cells.
The effect of Fluosol-DA (12 ml/kg)/carbogen breathing on
the cytotoxicity of radiation (10 Gy) toward the B and D cells
of the FSalIC fibrosarcoma is shown in Table 2. Fluosol-DA
and air breathing had no significant effect on the cytotoxicity
of radiation in either tumor cell subpopulation. With carbogen
breathing there was about a 2-fold increase in the killing of
both tumor cell subpopulations. Fluosol-DA/carbogen breath
ing in combination with radiation resulted in about a 5.0-fold
increase in the killing of the B cells compared to radiation
alone. Fluosol-DA/carbogen
breathing in combination with
radiation produced about a 3.1-fold increase in the killing of
the D cells compared to radiation alone. Therefore, the addition
of Fluosol-DA/carbogen breathing to 10 Gy was about 3.6-fold
more cytotoxic toward the B cells than toward the D cells.
In contrast, Fluosol-DA (12 ml/kg) with carbogen breathing
had a marked effect on the cytotoxicity of L-PAM (10 mg/kg)
in the D cells (Table 2). Fluosol-DA with air breathing, carbo
gen breathing for 1 h, or carbogen breathing for 6 h in combi
nation with L-PAM (10 mg/kg) resulted in small, progressive
increases in the kill of the B cells of about 1.3-, 1.4-, and 1.5fold, respectively. On the other hand, the addition of FluosolDA to L-PAM (10 mg/kg) resulted in about 1.6-, 9.5-, and
11.7-fold increases in the killing of the D cells when used with
air breathing, carbogen for 1 h, and carbogen for 6 h, respec
tively. Carbogen breathing in the absence of Fluosol-DA had
no significant effect on tumor cell killing by L-PAM (data not
shown). L-PAM with Fluosol-DA and carbogen breathing (6 h)
was about 3.6-fold more cytotoxic toward the D cells than
toward the B cells.
The effect of Fluosol-DA (12 ml/kg) and carbogen breathing
on the cytotoxicity of CDDP (10 mg/kg) in the bright and dim
tumor cell subpopulations is also shown in Table 2. In the B
cells, Fluosol-DA and air breathing produced only about a 1.3fold increase in tumor cell killing. When 1 h of carbogen
breathing followed administration of Fluosol-DA and CDDP,
about a 1.7-fold increase in the killing of the B cells was
observed compared to CDDP alone. When carbogen breathing
was maintained for 6 h following Fluosol-DA and CDDP
treatment, there was about a 2.5-fold increase in the killing of
the B cells compared with CDDP alone. In the D cells there
was no change in tumor cell killing with Fluosol-DA and air
breathing in addition to CDDP treatment. There was about a
1.2-fold increase in the killing of the D cells with 1 h of carbogen
breathing following Fluosol-DA and CDDP treatment and
about a 3.3-fold increase in the killing of the D cells with 6 h
of carbogen breathing following Fluosol-DA and CDDP treat
ment compared to CDDP alone. Therefore, CDDP with Fluo
sol-DA and carbogen breathing for 6 h was about 1.4-fold more
cytotoxic toward the B cells than toward the D cells.
Fluosol-DA (12 ml/kg) and carbogen breathing had a marked
effect on the cytotoxicity of VP-16 (20 mg/kg) in both the
bright and dim tumor cell subpopulations (Table 2). FluosolDA and air breathing increased VP-16 killing of the B cells by
about 3.5-fold and increased the killing of the D cells by about
2.0-fold compared with VP-16 alone. Carbogen breathing for 1
h increased the killing of the B cells by VP-16 plus Fluosol-DA
by about 5.7-fold compared with VP-16 alone. Six h of carbogen
breathing further increased the killing of the B cells so that
there was about 21.1-fold greater killing of the bright cell
subpopulation by Fluosol-DA/VP-16/carbogen
(6 h) than by
VP-16 alone. The addition of carbogen breathing for 1 h
following Fluosol-DA and VP-16 resulted in about a 3.3-fold
increase in the killing of the D cells compared to VP-16 alone.
Increasing the carbogen breathing time to 6 h following FluosolDA and VP-16 further increased the killing of the D cells, so
Table 2 Sun'ival of subpopulations based on Hoechst 33342 fluorescence intensity ofFSallC cells from FSallC tumors treated with a single dose of radiation (10 Gy)
or an anticancer drug with or without Fluosol-DA and carbogen or air breathing
differentialTreatment
fraction"TreatmentlOGy'
Subpopulations surviving
alone
(combination)Dim/bright2.3
10 Gy/Fluosol-DAc
lOGy/Carbogen''
Gy/Fluosol-DA/carbogenMelphalan
10
±0.03
0.10 ±0.03
0.07 ±0.01
0.022 ±
0.0040.025
(10 mg/kg)
Melphalan/Fluosol-DA
Melphalan/Fluosol-DA/carbogen
h)CDDP
Melphalan/Fluosol-DA/carbogen
±0.005
0.019 ±0.005
0.018 ±0.004
0.017
0.0050.15
±
(10 mg/kg)
CDDP/Fluosol-DA
CDDP/Fluosol-DA/carbogen(l
CDDP/Fluosol-DA/carbogen
h)VP-
(1 h)
(6
h)
(6
±0.04
0.22 ±0.04
0.14 + 0.04
0.010.055
0.08 ±
±0.010
0.035 ±0.007
0.0058 ±0.0012
0.0047
120.27±0.00
±0.05
0.28 ±0.07
0.22 ±0.04
0.083
0.0090.62
±
±0.02
0.12 ±0.02
0.088 ±0.009
0.059
0.0050.38
±
2.2
2.0
3.62.2
1.8
0.32
0.281.8
2.3
2.5
1.41.62.8
1.65.01.3
1.8
3.11.69.5
1.4
11.71.0
1.51.31.72.53.55.7
1.2
3.32.0
16 (20 mg/kg)
±0.03
±0.05
VP-16/Fluosol-DA
0.11 ±0.02
0.31 ±0.04
VP-16/Fluosol-DA/carbogen(l
h)
0.19 ±0.04
2.8
0.067 ±0.008
3.311.9
VP-16/Fluosol-DA/carbogen
(6 h)Bright0.11
0.01 8 ±0.003Dim0.25 0.052 ±0.006Fold
2.9Bright1.1
21.1Dim1.1
" Data are means of three independent experiments ±SEM.
Ratio of the surviving fraction for each treatment alone (10 Gy, melphalan. CDDP. or VP-16) with each treatment in combination with Fluosol-DA with or
without carbogen breathing in the bright and dim tumor subpopulations.
' Radiation was delivered locally to the tumor-bearing limb and drugs were injected i.p.
'' Fluosol-DA (0.3 ml, 12 ml/kg) was administered i.v. in the tail 1 h prior to radiation treatment and immediately prior to drug administration.
' Carbogen (95% oxygen/5% carbon dioxide) breathing was maintained for 1 h prior to and during X-ray delivery and for 1 or 6 h after drug administration.
3341
TREATMENT TUMOR SUBPOPULATION SELECTIVITY
that there was about 12-fold greater killing of the D cells than
with VP-16 alone. The combination of Fluosol-DA/VP-16/
carbogen (6 h) was about 2.9-fold more cytotoxic toward the B
cells than toward the D cells.
DISCUSSION
The effect of the level of oxygénationon the cytotoxicity of
several antineoplastic agents and treatment modalities has been
determined previously in cell culture (4-7, 18-23). Of the
treatments examined in the current study, thiotepa, ionizing
radiation, procarbazine, bleomycin, actinomycin D, vincristine
and VP-16 were preferentially cytotoxic toward normally oxy
genated cells in vitro (7, 18, 22). L-PAM, BCNU, CDDP, and
5-FUra showed no selectivity based on cellular oxygénation(7,
19, 20); and Mito C, MISO, and ETA have been shown to be
selectively cytotoxic toward hypoxic cells in culture (4-7, 23).
Only a few treatments have been found to be more cytotoxic
toward stationary phase cells in culture; these include bleomy
cin, BCNU, and hyperthermia (25, 29, 30). Although in vivo
the direct cellular effects of these treatments as measured in
vitro pertain to cytotoxicity, other properties of the drugs in
cluding metabolic stability, lipophilicity, and pH effects are
relevant.
All of the drugs tested which had no cytotoxic selectivity in
vitro based on cellular oxygénationwere more cytotoxic toward
the B cells than to the D cells in vivo. This differential may well
have been primarily due to lesser penetration of active drug to
the D cells, but an additional factor could also have been
diminished activity under acidic pH conditions in tumor regions
distal from the vasculature (24-28). All of those treatments
that showed selective in vitro cytotoxicity toward normally
oxygenated cells were also more toxic toward the B cells in
vivo. The magnitude of the difference between the killing of B
versus D cells was not greater for these drugs than for those
without preferential cytotoxicity toward normally oxygenated
cells in vitro. This finding may indicate that the collective
factors noted above are at least as important as the level of
cellular oxygénationin determining in vivo effectiveness. In
contrast, all of the drugs which were selectively cytotoxic toward
hypoxic cells in culture were more cytotoxic toward the D cells
in vivo. These correlations tend to validate both the Hoechst
dye methodology and the notion that oxygénationeffects, as
studied in culture, are predictive of in vivo efficacy and indicate
that significant concentrations of some drugs can reach poorly
perfused tumor subpopulations.
Fluosol-DA/carbogen breathing provides a means of increas
ing oxygen levels in tumor regions (12, 13, 39, 61-66). In a
whole tumor cell survival assay the addition of Fluosol-DA and
carbogen breathing for l h prior to and during radiation therapy
(10 Gy, single dose) reduced tumor cell survival from about
S.F. 0.08 to about S.F. 0.03 and increased tumor growth delay
from 1.6 ±0.5 days to 5.8 ±1.5 days (39, 67, 68). In the tumor
subpopulation survivals, there was a greater increase in the
killing of the B cells than in the D cells indicating that even the
addition of supplemental oxygen delivery by Fluosol-DA/car
bogen cannot fully radiosensitize the most poorly perfused
tumor cells (12, 13). Instead, Fluosol-DA/carbogen was prob
ably more effective in reoxygenating intermittently hypoxic
cells which may well be more plentiful in the Hoechst dye bright
subpopulation.
In the whole FSallC tumor cell survival assay, Fluosol-DA/
carbogen (1 or 6 h) increased tumor cell killing by L-PAM (10
mg/kg) from about S.F. 0.03 to about S.F. 0.003 and increased
3342
tumor growth delay from 2.9 ±0.3 days to 9.5 ±1.4 days (19,
20). It is interesting that most of this change appears to be
reflected in an increase in the killing of the D cells where there
was about 1 log increase in tumor cell killing while the change
in the B cells was very modest.
VP-16 may operate through a topoisomerase II-mediated
pathway activating an oxygen-requiring cascade leading to cell
death and/or may be toxic through a microsomal activation to
reactive free radical species (18, 60). In a multiple dose VP-16
protocol, Fluosol-DA/carbogen markedly increased the tumor
growth delay in the FSallC tumor (18). Fluosol-DA/carbogen
(6 h) increased the tumor cell killing by VP-16 in both the B
and D cells by 1 log or more indicating that oxygen availability
may be a greater limiting factor in the antitumor action of this
drug than is the ability of the drug to penetrate deeply into the
tumor. Additionally, in view of the relative lesser effect of
Fluosol-DA/carbogen with radiation in the D cells, it may be
that VP-16 requires less oxygen to optimize its cytotoxicity in
vivo than does radiation.
The most modest changes in tumor cell killing by the addition
of Fluosol-DA/carbogen were obtained with CDDP. In a tumor
growth delay study in the FSallC fibrosarcoma, Fluosol-DA/
carbogen (6 h) increased the tumor growth delay produced by
a single dose of CDDP (10 mg/kg) by about 1.6-fold (69). This
increase reflected a 2-3-fold increase in the killing of both
tumor cell subpopulations. CDDP does not require oxygen for
optimum cytotoxicity in vitro at 37°C(7, 45). In addition, it is
a small molecule and probably penetrates well into the region
of the D cells so that the increase in tumor oxygénationand/
or tumor blood flow caused by Fluosol-DA/carbogen would not
be expected to markedly improve CDDP cytotoxicity.
A useful clinical approach may be to use in combination
treatments which are selectively cytotoxic to both the oxygen
ated (bright) tumor subpopulation and to the hypoxic (dim)
tumor subpopulation (7, 25, 27, 40, 44, 67, 70). The combina
tion of Mito C and radiation led to an increase in disease-free
survival in a recently reported head and neck clinical trial (71).
In laboratory studies, combinations of Fluosol-DA/carbogen/
radiation with Mito C, porfiromycin, MISO, and ETA have
produced at least additive effects in tumor growth delay and
tumor cell survival studies (67, 68). Similarly, combinations of
CDDP with ETA, MISO, hyperthermia, and Mito C have led
to marked increases in tumor growth delay as well as at least
additive increases in tumor cell killing (69, 72). The addition
of hyperthermia to treatment with radiation has established
benefit clinically (25) and is further supported by the finding
that hyperthermia is more cytotoxic toward the dim tumor cell
subpopulation and appears to prevent repair of radiation dam
age (27). Similar effects have been seen with combinations of
hyperthermia and BCNU or Mito C (40).
In conclusion, treatment regimens directed toward solid tu
mors can be designed based upon a consideration of the phys
iological status of tumor subpopulations to be attacked. Such a
therapeutic approach would require a combination of agents
and/or modalities directed toward cycling and noncycling pop
ulations of oxygenated and hypoxic cells at normal and acidic
pH. Only through a thorough knowledge of the in vivo effects
of potential treatments on defined tumor subpopulations can
such improved combinations be designed.
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