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Experimental Therapeutics, Preclinical Pharmacology |
General Pharmacology, DuPont Pharmaceuticals Co., Wilmington, Delaware 19880-0400 [E. J. W., P. M. C., J. J. D., A. M. S., J. S. K.]; New England Baptist Bone and Joint Institute and Department of Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02115 [E. M. G.]; Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts 02115 [J. L.]; and Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213 [M. K. K. W.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The human tumor xenograft model has been used as a preclinical model for the discovery of anticancer drugs (1) . Evaluation of tumor growth has relied on the overall size and weight changes of the tumors. Because tumor growth is regulated through the balance of cell proliferation and cell death, measurement of these cellular processes is critical in assessing the kinetics of tumor growth. One, therefore, needs methods to quantitate cellular proliferation and cell death (both apoptosis and necrosis) within the tumors. The ability to create large montages from many high-resolution images (tiling) makes the investigation of entire tumor tissue sections possible and characterization of areas of distinct tumor morphology feasible. One purpose of the present study was to develop a method using "tiling" applied to entire tumor sections to identify such regional morphology. In addition, we sought to identify changes in regional morphology and kinetics of tumor growth after treatment with selected chemotherapeutic agents. The tiling of tumor sections requires more time than conventional analyses but allows visualization and quantitation of distinct areas of tumor morphology.
We have focused on two human cell lines grown in a traditional xenograft model: colon adenocarcinoma cells (HCT-116) and non-small cell carcinoma cells (NCI-H460). Three therapeutic agents were dosed in the model: cyclophosphamide (Cytoxan), gemcitabine (Gemzar), and mitomycin C. Using standard immunohistochemical methods, we have assessed cell proliferation and apoptosis to evaluate parameters of cell kinetics. Use of entire tumor sections, coupled with information gained from this analysis, permit a more complete understanding of the balance of cell proliferation and cell death that regulate tumor growth.
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MATERIALS AND METHODS |
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Cell Lines and Cell Culture.
HCT-116, a human colon adenocarcinoma cell line, and NCI-H460, a human
non-small cell lung carcinoma cell line, were purchased from
American Type Culture Collection (Manassas, VA). HCT-116 cells
were maintained in culture in McCoy’s medium supplemented with 10%
heat-inactivated fetal bovine serum, 2% L-glutamine, and
2% penicillin/streptomycin. NCI-H460 cells were maintained in culture
in DMEM supplemented with 10% FCS, 2% L-glutamine, an 2%
penicillin/streptomycin. Both cell lines were maintained at 37°C with
5% CO2. Subconfluent cultures were trypsinized
using 0.25% trypsin-EDTA, pelleted using centrifugation, counted with
a hemocytometer, resuspended in complete media, and diluted to
appropriate concentrations.
Animal Care.
All animal studies were conducted in a facility accredited by the
American Association for the Accreditation of Laboratory Animal Care.
Female athymic Swiss nu/nu mice at 6–8 weeks of age were used for the
xenograft studies (Taconic Farms, Germantown, NY). They were housed
five to a cage in sterile, polycarbonate filter-capped microisolator
cages. All animals were kept in temperature-controlled rooms,
maintained in a barrier facility on 12-h light/dark cycles, and
provided with food and water ad libitum.
Human Cell Implantation and Xenograft Studies.
Either HCT-116 cells or NCI-H460 cells were implanted s.c. into the
inguinal area at 1 x 107 cells (0.1 ml) per
mouse. At the end of 7–10 days postinjection, tumor volumes were
determined by measuring the tumors in two dimensions, and the volume
was calculated using the formula for a prolate ellipsoid: length
(mm) x width2
(mm)/2 = mm3.
Because the tumors were not removed from the animals, tumor volume was
converted to weight by assuming unit density (i.e., 1.0
cm3 = 1.0 g; Ref. 2
). Animals
were placed in groups so the tumor weights were not statistically
different among the groups prior to treatment. Selected
chemotherapeutic drugs (cyclophosphamide, Cytoxan; gemcitabine, Gemzar;
and mitomycin C) were administered daily i.p. over 14–15 days. At the
end of treatment, tumors were removed and weights were determined and
expressed as mean ± SE. TGI was determined as the ratio of the
percent tumor weight from treated animals divided by tumor weights from
control animals x 100. TGI >60% was considered significant.
To begin to characterize the interplay between the processes of tumor growth and tumor cell death, a time course study was performed, dosing 120 mg/kg Q3Dx3 gemcitabine and evaluating tumors after drug treatment. The BrdUrd-LI and apoptotic index at 1, 2, 3, and 4 days after completion of the third drug dose were determined, as described below.
Immunohistochemistry: BrdUrd Incorporation.
BrdUrd (0.2 ml/mouse) was injected i.p. 2 h before mice were
sacrificed. This allowed time for BrdUrd incorporation into
proliferating cells. To ensure BrdUrd was consistently incorporated,
additional tissues from the small intestine were collected from all
animals injected with BrdUrd. Tumor and small intestine sections were
excised and trimmed of fat, and serial sections no thicker than 0.5 cm
were processed for paraffin embedding by fixing in 10% buffered
formalin for 4 h and dehydrated with graded alcohols: 80%, 95%
twice, and 100% three times for 60 min per bath. The samples were
cleared with three baths of xylene and then infused with paraffin via
two additional baths. Using a microtome (Olympus America Inc.,
Melville, NY), tissues were sectioned at 4 µm and placed onto
silane-coated slides and air-dried. To ensure consistency of the
tissue sections and to maximize differences among treatments, the
largest cross-sectional diameter of each tumor was selected. The slides
were stored at room temperature.
The Zymed BrdUrd staining kit was used for visualization of incorporated BrdUrd. BrdUrd labeling identifies cells in the S phase of the cell cycle. The slides were deparaffinized, hydrated in PBS, and processed through 0.3% hydrogen peroxide to quench endogenous peroxides. Tissues were then treated with 0.125% trypsin and denatured. Sections were then blocked using the kit blocking reagent, incubated with biotinylated mouse anti-BrdUrd for 60 min at room temperature, followed by streptaviden-peroxidase incubation for 10 min, and 2–5 min with the diaminobenzidine chromagen solution. The sections were counterstained with hematoxylin and coverslipped with a permanent mounting medium. The three negative controls used were: an isotype control, a non-BrdUrd injected tumor, and a no primary antibody section; BrdUrd-injected mouse intestine was used as the positive control. Five areas of the same size (0.05 mm) and at least four tumors per group were analyzed.
PCNA Staining.
The Zymed PCNA staining kit was used as a marker for cell
proliferation, because PCNA levels are elevated in actively
proliferating cells during the S, G2, and M
phases of the cell cycle. The tissue preparation was performed as
outlined above for BrdUrd, and the staining and visualization were done
according to the manufacturer’s specifications.
Apoptosis.
Invitrogen Apoptag plus kit was used to visualize and quantify the
number of cells undergoing apoptosis. Tissues were fixed in 10%
neutral buffered formalin, paraffin embedded, and sectioned at 4 µm,
and the kit protocol was followed. This kit uses the method of terminal
deoxynucleotidyl transferase-mediated dUTP-nick end
labeling for in situ labeling of fragmented DNA. The
slides were counter-stained with methyl green. The negative control
tissue section was the primary antibody solution with the dUTP
substrate but without dUTP, and the positive control tissue was tissue
from the mouse mammary gland. Five areas of the same tumor and at least
four tumors per group were analyzed.
Image Analysis and Ratio of Tumor Areas.
Image analysis was performed with the M2 MCID Image Analysis System
(Imaging Research, Toronto, Canada), coupled with the Olympus AX70
microscope.
BrdUrd-LI.
To calculate the LI, viable cells (BrdUrd-positive cells plus unstained
cells) were counted from five same-sized areas (0.05
mm2
), containing no edges, artifacts, or necrotic
portions, and from at least four separate tumors per group. Using 20
individual cells, the average nuclear size was calculated to determine
the number of cells if clumps of cells were counted. Automated counting
was done using hue, intensity, and saturation as criteria for
separating labeled from unlabeled cells. The ratio of BrdUrd-labeled
cells to the total number of cells counted (both labeled and unlabeled
cells in histologically viable areas of tumor determined by hematoxylin
staining) was determined, and this ratio was multiplied by 100. This is
defined as the BrdUrd-LI. For both the proliferating cells and the
apoptotic cells the data are expressed as percentages rather than total
number of positive cells per field. We reasoned that sampling errors
were due to the heterogeneity of the tumors and, therefore, the
selected fields would be minimized.
Percentage of Apoptotic Cells.
The percentage of labeled cells was determined using the same
methodology as for the BrdUrd-LI: percentage of labeled cells =
positive labeled cells/(positive labeled cells + unstained cells) x 100.
Characterization of Tumor Areas.
Tiled images of tumor sections were created, which resulted in
resolution of the entire tumor section at x10 (16–100 individual
frames captured and stitched together). The different regions of the
tiled tumor were traced manually. Because the tumors were variable in
size, each tumor section was standardized to 100
mm2
and the percentage of necrotic and viable
areas per section were calculated based on 100
mm2
. The tiled tumor section was divided into two
distinct areas: (a) the area containing BrdUrd-stained cells
plus the area containing histologically viable but unstained cells; and
(b) the necrotic area determined by morphological changes
via hematoxylin staining. To confirm that many of the unstained cells
within the tumor were still within the cell cycle, additional studies
were performed to determine whether the cells not labeled with BrdUrd
would label with PCNA.
Using standardized tumor areas, two percentages were derived: viable tumor (areas containing both unlabeled but histologically viable cells plus BrdUrd-labeled cells) and necrotic tumor determined by H&E. The percentage of viable tumor area was calculated as the ratio of viable BrdUrd-labeled cells plus unlabeled cells to the total tumor area, including necrotic portions of the tumor, x 100. The percentage of necrotic tumor area was calculated as the ratio of the necrotic area to the total tumor area x 100.
Statistical Methods.
Statistical differences were determined using the Student’s
t test and the nonparametric Mann-Whitney test. A
P < 0.05 was considered significant.
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RESULTS |
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and 2
show tiled images of HCT-116 and NCI-H460 untreated tumors with serial
sections of H&E staining or hematoxylin staining and BrdUrd labeling.
These sections clearly show the heterogeneity within the HCT-116 tumor
(Fig. 1
, A and B). The HCT-116 tumor grew in
organized sheets composed of areas of poorly differentiated cells with
a high nuclear:cytoplasmic ratio and prominent nucleoli. There was some
inflammatory response. Central areas of the tumor contained frankly
necrotic cells. Viable tumor areas were predominately located around
the tumor periphery. BrdUrd-stained cells were concentrated in the
central portions of the viable areas.
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, A and B)
had viable tumor cells present in the peripheral areas of the specimen.
Tumor growth was in fronds, and occasional mitotic figures were
present. Tumor cells contained large nuclei with prominent nucleoli.
Deep to the viable tumor was an admixture of areas containing clearly
necrotic cells and areas containing cells that, by routine histology,
appeared viable but smaller. BrdUrd labeling was seen again,
predominantly in central areas of clearly viable tumor fronds. The
BrdUrd-labeled HCT-116 and NIH-H460 tumors under x100 oil demonstrate
the distribution of nuclei, nucleoli, and mitotic figures within these
representative tumor sections (Fig. 3
, A and B). The BrdUrd-labeled intestine clearly
demonstrates positively stained cells in intestinal crypts (Fig. 4
B).
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shows serial sections of an untreated HCT-116 tumor with PCNA-labeled
cells (A) and with BrdUrd-labeled cells (B).
Serial sections show that many tumor cells not labeled with BrdUrd did
label with PCNA and, therefore, were within the cell cycle, confirming
cells not labeled with BrdUrd were viable. We used this criterion in
addition to cellular morphology with hematoxylin staining to delineate
the viable portions of tumor sections.
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shows xenograft results from mitomycin C-, cyclophosphamide (Cytoxan)-,
and gemcitabine (Gemzar)-treated mice with colonic adenocarcinoma
(HCT-116) or non-small cell lung (NCI-H460) tumors. Mitomycin C (1
mg/kg/day for 14 days) consistently reduced tumor growth compared with
controls in animals with both HCT-116 and NCI-H460 tumors.
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Because we determined the LI only at one time point, we have compared
control and treated groups within the same experiment (Table 1, A and B)
. In the HCT-116 tumors cyclophosphamide
at 50 mg/kg/day reduced the LI by 30%, from 28% in the control group
to 20% (P < 0.05, Table 1
). Gemcitabine dosed at 120
mg/kg Q3Dx5 lowered LI to 21%, a 26% decrease. Treatment with
mitomycin C dosed daily did not significantly change the LI. In
contrast, in the NCI-H460 tumor group mitomycin C treatment reduced the
LI from 
22% in controls to 10%, a 55% decrease in proliferation
(P < 0.05), whereas gemcitabine and cyclophosphamide
treatments did not significantly affect this index. Therefore, the LI
does not always correlate with the change in tumor weight.
With both cyclophosphamide and gemcitabine, the percentage of apoptotic
cells (apoptotic index) significantly increased in the HCT-116 tumors,
suggesting this process significantly contributes to the reduction in
tumor growth. (Table 1B)
. The apoptotic indices were not
significantly different in the mitomycin C-treated animals with either
tumor, although the mitomycin C-treated NCI-H460 tumors were
significantly smaller than untreated controls (Table 1A)
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demonstrating the apoptotic index does not always correlate with the
change in tumor weight.
Morphology of the drug-treated tumors differs from that of the
controls. To ensure consistency of the tissue sections and to maximize
differences among treatments, the largest cross-sectional diameter
of each tumor was selected. The tiled sections of HCT-116 tumors
treated with gemcitabine have smaller necrotic areas than untreated
controls (Fig. 6
, A and B). The NCI-H460 tumors treated with
mitomycin C had very few, if any, distinct areas of necrosis (Fig. 7
, A and B). The cells stained with BrdUrd were
uniformly distributed throughout viable areas of the tumor sections,
demonstrating that cells throughout the treated tumors continued to
proliferate. Because cyclophosphamide was not active against either
tumor, the tiled image was similar to the control tumors, with a
central necrotic area on H&E and with BrdUrd-stained sections (data not
shown).
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, whereas the area of necrosis decreased, coupled with significant
reduction in tumor weights (Table 1)
. Tumors not significantly smaller
than controls did not exhibit the same quantitative morphological
changes in the viable and necrotic areas (mitomycin C-treated HCT-116
tumors; cyclophosphamide- and gemcitabine-treated NCI-H460 tumors).
Such analysis demonstrates different patterns of tumor responses to
chemotherapeutic agents.
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. A comparison of Fig. 8
and Table 1
clearly demonstrates that different
time points give different cellular responses. Gemcitabine (120 mg/kg
Q3Dx5) increased the apoptotic index to 4.3 ± 0.2% from 1.9 ± 0.4%. The BrdUrd-LI initially decreased within 24 h and then
rebounded to 23.5 ± 4.5%, moving toward the control level of
31%.
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DISCUSSION |
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To the best of our knowledge, this is the first report of complete
tumor sections that have been both visualized and analyzed with such
high resolution, allowing appreciation of the complexity of the tumor.
All of the drugs used in these studies have been or are being used in
the clinic against colon and non-small lung tumors. Cyclophosphamide
inhibits DNA cross-linking and synthesis (3)
; mitomycin C
inhibits DNA cross-linking and slows the rate of cellular proliferation
(4)
, whereas gemcitabine is an antimetabolite that
inhibits cellular proliferation in S phase (5)
. These
drugs had different effects on the inhibition of tumor growth as well
as cellular proliferation (the BrdUrd-LI) and the percentage of
apoptotic cells (apoptotic index). Although cyclophosphamide was not
effective in inhibiting growth against either solid tumor (Table 1)
, it
did inhibit the proliferative index in the HCT-116 tumor cells and
increased the apoptotic index in the same cells. Cyclophophamide is
primarily used clinically against leukemias and lymphomas
(6)
. Gemcitabine, an effective drug against a broad base
of human tumors in xenografts (7)
, was efficacious against
colon but not non-small cell lung tumor growth dosed at 120 mg/kg
Q3Dx5. Although cell proliferation was decreased in both tumors with
gemcitabine treatment, these reductions were not significant. The
percentage of apoptotic cells increased significantly in the HCT-116
tumors, suggesting that cell death was the primary contributor to the
reduction in tumor size with this dosing regimen. This drug is used
against aggressive tumors such as adenocarcinomas of the pancreas in
single and combination therapies (8)
. Mitomcycin C reduced
both colon and lung tumor growth, but significantly reduced cellular
proliferation only in the NCI-H460 lung tumors.
These data demonstrate that assessing TGI by simply measuring changes
in tumor weight of untreated animals compared with controls does not
accurately assess the kinetics of tumor growth and response to
treatment. Apoptosis is an important end point because more anticancer
drugs have been reported to induce this process in tumor cells
(9)
. We quantified apoptosis, coupled with cellular
morphological changes (10)
, to detect the percentage of
apoptotic cells in a mixed cell population at the end of 14–15 days of
daily dosing. We counted the total number of cells in each field
(2000) and expressed the terminal deoxynucleotidyl
transferase-mediated dUTP-nick end labeling-positive cells as a
percentage of the total. We reasoned that counting total cells within
each selected field was more consistent than counting only the positive
cells per field, because the tumors were heterogeneous. The percentage
of apoptotic cells in the tumors from untreated animals was higher
but in the range reported for other tumors (11)
. The
percentage of apoptotic cells within the tumor cell population of the
HCT-116 tumors treated with cyclophosphamide or gemcitabine was
significantly increased compared with controls, which was not seen with
mitomycin C-treated tumors, suggesting that this mechanism may be
important for the response of these tumor cells to those drugs. It is
possible that the apoptotic cell fragments from the mitomycin C-treated
cells may be more efficiently phagocytosed by neighboring cells
(12)
, so that changes in the apoptotic cell numbers
were not observed. Because the percentage of apoptotic cells was
determined at only one time point, such a possibility cannot be ruled
out.
The BrdUrd-LI has been used to investigate the proliferative characteristics of tumor cell populations to evaluate tumor growth (13) . The percentage of proliferating cells in the HCT-116 and NCI-H460 tumors ranged from 22–35%, which is higher than reported in human melanoma (11) . However, the majority of cells (65–78%) were not labeled with BrdUrd; a pulse exposure was used, so only those cells rapidly synthesizing DNA used the BrdUrd. A significant reduction in cellular proliferation does not consistently lead to a significant decrease in tumor weight as shown by the cyclophosphamide-treated HCT-116 tumors. On the other hand, a significant inhibition of tumor growth was demonstrated in the gemcitabine-treated HCT-116 tumors, and this was not accompanied by a statistically significant decrease in cellular proliferation. These data, again, suggest that analyzing only a single parameter does not accurately reflect overall kinetics of tumor growth.
Through the use of tiled images, a more complete picture of tumor
morphology can be gained. The tiled images demonstrate differences in
the patterns of proliferation and cell death within the tumors, from
the extremes of the HCT-116-gemcitabine-treated and NCI-H460 mitomycin
C-treated tumors that were essentially uniformly labeled with BrdUrd
(Figs. 6
and 7)
to the large control tumors with necrotic centers
(Figs. 1
and 2)
. Standardizing these tissues to 100
mm2
reflects these morphological observations
(Table 2)
. HCT-116 tumor growth was inhibited by >90% after treatment
with gemcitabine (Table 1)
, coupled with a 26% reduction in the
percentage of proliferating cells and a significant increase in the
percentage of apoptotic cells, which contribute to the reduction in
tumor growth (14)
. The percentage of viable tumor after
gemcitabine treatment was >85%, indicating that most of the remaining
tumor was viable after drug treatment. Similar findings were present in
the NCI-H460 mitomycin C-treated tumors.
The other drug-treated tumors did not exhibit any significant
changes in percent area of tumor viability or necrosis, as shown in
Table 2
. Gemcitabine was clearly inactive against the NCI-H460 tumors
in all parameters measured. Mitomycin C reduced the NCI-H460 tumor
necrotic area. Clearly, the 50-mg/kg/day dose of cyclophosphamide had
activity against the HCT-116 tumors, with a significant reduction in
the proliferative index and a significant increase in the apoptotic
index. However, when the tissue areas were normalized, the areas of
tumor necrosis and viability in cyclophosphamide-treated tumors were
not different from controls, suggesting that the overall tissue
morphology remained constant. Although there is no direct evidence that
these specific measurements, including standardizing the tumor tissues
for viable and necrotic areas, lead to a more predictable assessment of
the effects of chemotherapeutic agents on tumors, additional
information does permit a more comprehensive assessment of the
relationship between tumor morphology and treatment outcome.
The observation that the NCI-H460-mitomycin C-treated and HCT-116 gemcitabine-treated tumors were the smallest in size, with the least cellular proliferation, increased number of apoptotic cells, and virtually no necrotic areas within the tumor, is interesting. The relevance of these observations is unclear, but one could speculate that the treated tumors were not hypoxic or acidotic and had adequate blood supply; they remain viable tumors. What happens in the tumors with continued treatment with these chemotherapeutic agents remains to be determined.
Evaluation of tumors at several time points after drug treatment, shown in the gemcitabine time course study, clearly generates additional information about the effect of the drug on the tumor. More of the tumor cells in the treated animals undergo apoptosis, while fewer cells are proliferating. By day 4 after the termination of gemcitabine treatment, the proliferative index is not significantly different from this index after 15 days of drug treatment, but the apoptotic index is significantly elevated.
In summary, we have described a new approach for investigating tumor responsiveness to chemotherapeutic agents. Through the use of tiling, we have demonstrated the heterogeneity within the tumors. Because of this heterogeneity, we have used the largest cross-sectional diameter of each tumor, reasoning that this section should be morphologically the most variable. We have identified two distinctly different areas within each tumor, an area containing proliferating and viable cells, and a necrotic area. As a result of treatment with the chemotherapeutic agents, the percentage of these areas within the tumor sections changed when the tumors were standardized to 100 mm2 . No areas of necrosis were found in the HCT-116 gemcitabine-treated and mitomycin C-treated NCI-H460 tumors. Tumors remained viable. Certainly, different drugs, dosing regimens, time points, and tumors will lead to different results, as shown by the two different dosing regimens with gemcitabine. Comparisons among groups at a single time point give valuable information. We used standard therapeutic agents, dosing regimens, and selected time points as tools to demonstrate the broad applicability of the methodology.
This methodology also may be applied in the clinical analysis of human tumors. For example, an analysis of tumors treated with chemotherapy before and after surgical removal, as in neoadjuvant or induction protocols, would provide information about the efficacy of treatment. This approach could also help answer questions related to the dose and fractionation of radiation treatments by providing insight into relative intratumor growth and death postradiation. Lastly, emerging concepts about the action of anticancer agents such as antiangiogenesis drugs suggest their effect may be to induce a state of tumor dormancy or stability through the balance of intratumor cell death and proliferation. The approaches outlined could be applied to these new agents and lead to a more rational design of treatment.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at General Pharmacology, DuPont Pharmacueticals Co., Route
141 and Henry Clay Road, P.O. Box 80400, Wilmington, DE 19880-0400. 
2 The abbreviations used are: BrdUrd,
bromodeoxyuridine; PCNA, proliferating cell nuclear antigen; TGI, tumor
growth inhibition; LI, labeling index.
Received 3/ 6/00; revised 5/25/00; accepted 5/26/00.
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