It is generally accepted that histologically similar tumors behave less aggressively in the aged. This longstanding impression arose from clinical observations in humans1, 2, 3 and was further supported by animal models, in which young and aged mice received identical inocula of tumor cells and were subsequently monitored for tumor growth and aggressiveness.4, 5, 6, 7, 8, 9 Tumors examined, including the B16/F10 melanoma, the Lewis Lung Carcinoma, the EHS sarcoma, the SP1 fibrosarcoma and the AKR lymphoma; all demonstrated significantly delayed and slower growth in the aged relative to young mice.4, 5, 6, 7, 8, 9 Proposed mechanisms have focused on age-related reductions in cell proliferation, deficits in immune mediated responses that promote tumor growth (such as a lack of inflammatory cells and their secreted chemokines), increased tumor cell apoptosis and decreased angiogenesis (thereby providing less nutrients to the tumor bed).3, 4, 5, 6, 7, 10, 11, 12, 13, 14 Although the tumors that were used in animal studies do not reflect the group of tumors that occur most commonly in the elderly (such as colon, breast and prostate),15, 16 the results mimic that of clinical observations in humans.1, 2, 3, 15, 16 It has been argued that the less permissive environment of aged tissues is an adaptive response to the greater risk of cancer conferred by age-related and environmentally induced increased risk of genetic mutations.17 On the other hand, others have suggested that the microenvironment of aged animals can be conducive to tumor growth.18, 19
Age-related influences on tumor biology have implications for both the growth and treatment of histologically similar cancers in the young and aged. At least 2 observational studies have noted reduced numbers of blood vessels, independent of tumor cell characteristics, in the breast cancers of aged women compared to young women.20, 21 Although the connections between patient age, tumor vessel density, growth rate and clinical outcomes are still being established, the advent of angiogenesis inhibitors as adjuvant treatments for cancer underscores the need to determine if aging confers broad deficits in tumor vascularization.22, 23, 24
In our study we examined the effect of aging on tumor onset, growth and angiogenesis in TRAMP-C2 prostate tumors. Although the prognosis from prostate cancer is dependent more on tumor grade than patient age,25 the incidence and prevalence of prostate cancer is highly associated with aging15, 16 and it is surprising that prostate cancer has not been previously examined in an aged animal model. We unexpectedly found robust growth and angiogenesis in the TRAMP-C2 tumors in the aged mice. Subsequent experiments using the B16/F10 melanoma cell line, a well-studied tumor that is highly representative of other tumors that have been shown to grow slowly in aged mice, showed a significant lack of growth in the aged mice relative to the young mice. Our data demonstrate that tumor characteristics can be the primary determinant of the influence of aging on tumor angiogenesis and growth.
Material and methods
Both B16/F10 melanoma and TRAMP-C2 prostate cells form tumors when injected subcutaneously into syngeneic C57Bl/6 hosts. B16/F10 cells were obtained from the ATCC (ATCC® Number: CRL-6475) and grown as previously described.4 TRAMP-C2 cells were a generous gift from Dr. Norman Greenberg (Fred Hutchinson Cancer Research Center, Seattle, WA) and were derived from the prostate tumors of TRAMP mice. The latter were generated when a transgene carrying the −426/+28 fragment of the rat probasin gene fused to the SV40 T antigen resulted in an independent transgenic autochthonous model for prostate cancer in the C57Bl/6 inbred strain. The prostate tumors of the TRAMP mice are the source of the prostate cancer epithelial cell line, TRAMP-C2, which retain the histologic and biologic features of prostate cancer.26, 27, 28 For tumor implantation, cell lines were grown to 80% confluency in 100 mm culture dishes, trypsinized, washed with phosphate-buffered saline (PBS) and resuspended in PBS at a concentration of 1 × 105 (B16/F10) or 1 × 106 cells (TRAMP-C2)/100 μl.
Young (4 month) and aged (20 month) male mice of the C57Bl/6 strain were obtained from the NIA Rodent Colony at Harlan Sprague Dawley (Chicago, IL). Animals were housed in the SPF facility at the Harborview Research and Training building at the University of Washington and the University of Washington Animal Welfare Committee approved all animal procedures. Senescence associated β galactosidase analysis was performed on primary skin fibroblasts obtained from the mice and demonstrated that the older mice had the phenotypic changes of aging.29
After the animals had acclimated for 5 days, 1 × 106 TRAMP-C2 cells (n = 19 young and n = 21 aged mice) or 1 × 105 B16/F10 cells (n = 10 young and 10 aged mice) were injected subcutaneously into the left flank. Tumors were monitored by caliper measurements in 2 dimensions every 2–3 days after the tumor was palpable. Volume calculation (mm3) was based on the formula 0.52 × a2 × b, where a = short axis and b = long axis.30 Mass (mg) was obtained at the time of sacrifice. Mice were followed for a maximum of 30 days (B16/F10) and 35 days (TRAMP-C2). B16/F10 tumors were harvested, on average, 5 days earlier than the TRAMP-C2 tumors due to the size of the tumors in some of the young mice.
All tumors were removed (except for n = 3 B16/F10 tumors in aged mice that grew only as a sheet of pigmented cells in the subcutaneous space), embedded in paraffin or OCT and sectioned. Sections from each tumor were stained with Masson's trichrome. For immunohistochemistry, paraffin sections were dewaxed in xylene and hydrated in a graded series (100–70%) of ethanol solutions. Frozen sections were air-dried and fixed in acetone. Both types of sections were pretreated with 3% hydrogen peroxide, blocked overnight in PBS with 2% normal goat serum and incubated with 2–5 μg/ml of specified primary antibodies for 2 hr at room temperature. The antibodies used were rat anti-mouse endothelium (MECA32, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), rabbit anti-laminin (Sigma, St. Louis, MO), rabbit anti-VEGF (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-VEGFR2,31 goat anti-CD3 (Santa Cruz), rat anti-CD8 (eBioscience, San Diego, CA) and rat anti-monocyte/macrophage (Serotec, Raleigh, NC). Slides were incubated in secondary antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch Labs, West Grove, PA or Amersham Biosciences, Piscataway, NJ) or where amplification was needed, 1 μg/ml biotinylated secondary antibody (Vector, Burlingame, CA) followed by treatment with avidin/biotin complex (Vector). For immunostain with the monoclonal antibody against alpha smooth muscle actin (Sigma), the Mouse on Mouse kit (Vector) was followed according to the manufacturer's recommendation. Uniformly timed application of the peroxidase substrate 3,3-diaminobenzidine (Vector) was used for all studies. Immunostained sections were counterstained with hematoxylin, dehydrated with a graded series of ethanol, cleaned with xylene, mounted and visualized by light microscopy. In all experiments, secondary antibody alone served as a negative control.
Quantitative image analysis
For image analysis of vessel area, images were captured using a Nikon Eclipse 50i and Q-Imaging Digital Camera with Q-capture software (Q-Imaging, Burnaby, BC, Canada). For each sample, at least 4 arbitrary and equivalent divisions were created and representative images for each division were captured using the 10× objective lens. Images were subsequently analyzed using Adobe Photoshop (Adobe, San Jose, CA) with Fovea Pro plug-in (Reindeer Graphics, Asheville, NC). Briefly, equivalent thresholds were applied to all images to generate the region of interest, which included only areas of positive staining. The measured image area was obtained and subsequently used to infer relative areas of staining as of function of the mean of these image area values for a given sample.
Vessel size analysis
Digital images of MECA32-stained samples were collected as described earlier. For each sample, at least 4 arbitrary and equivalent divisions were created and representative images for each division were captured using the 10× objective lens. Images were examined in Adobe Photoshop (Adobe) and discrete noncontiguous areas of staining were counted with each notated as a singular “vessel.” Digital scalars were then calibrated to a stage micrometer and vessels were further categorized by size in their greatest dimension according to the following definitions: Small (less than 35 μm), medium (greater than 35 μm, but less than 70 μm) and large (greater than 70 μm).
Analysis of sera testosterone levels
Sera testosterone levels from aged mice (n = 7) and young mice (n = 9) were measured with a radioimmunoassay kit (DSL-4100, Diagnostic Systems Laboratories, Webster, Texas). The assay has an intra-assay coefficient of variation of 6.2% and an inter-assay coefficient of variation of 21%. The lower limit of detection in mouse plasma is 0.05 ng/ml. Young mice (n = 2) with sera levels of testosterone greater than 2.0 ng/ml were not included in the comparison. All data from aged mice were included.
ELISA for VEGF
Tumor lysates (n = 21 aged and n = 18 young mice) and plasma samples (n = 6 aged and n = 7 young mice) containing equivalent amounts of total protein were analyzed for VEGF content by ELISA per the manufacturer's recommendation (R&D Systems, Minneapolis, MN). The assay's lower limit of detection for mouse VEGF is <3.0 pg/ml in both tissue lysates and plasma.
Total RNA was extracted from TRAMP-C2 (n = 12) and B16/F10 (n = 9) tumors removed from aged mice using RNeasy kits (Qiagen, Valencia, CA). RNA was subsequently analyzed with spectrophotometry and nonreducing agarose gels to verify integrity. cDNA was then generated using the SuperScript First-Strand Synthesis System (Invitrogen, Carlsbad, CA). Custom primer sets (Invitrogen) were created using Primer 3 software with nucleotide sequences generated from GenBank. Multiple primer sets were generated for each gene of interest. Primer sets were then screened at universal settings (40 cycles of: 15 sec at 95°C, 60 sec at 62°C) using the ABI 7700 RT-PCR instrument with SYBR Green Master Mix chemistries for detection (Applera, Norwalk, CT). Primer sets were chosen according to the analysis of melting curves and agarose electrophoresis. Sequences for primer sets are available upon request. Amplification efficiencies were then calculated for these primer sets using a dilution series of pooled RNA.
To analyze pro- and active MMP2 (matrix metalloproteinase) and MMP9, lysates representing equivalent amounts of total protein from TRAMP-C2 (n = 11) or B16/F10 (n = 5) tumors removed from aged mice were solubilized in nonreducing Laemmli buffer, and subjected to SDS-PAGE zymography. Stained gels were then digitally imaged and optical densities were generated for each band using LabWorks software (UVP, Upland, CA).
Comparisons between the young and aged mice were performed with unpaired 2-tailed t-tests. All data are represented as mean ± standard error. The study was adequately powered to detect a 40% difference between the young and aged mice with respect to tumor size and the angiogenic response. The % difference chosen reflects the size of the difference previously reported in studies of tumor growth, wound healing and angiogenesis in young and aged mice.34, 35
Growth of TRAMP-C2 prostate tumors is robust in the aged mice
We wished to examine the growth of TRAMP-C2 tumor cells, an epithelial prostate tumor line that has not been previously examined, in aged and young mice. Unexpectedly and in contrast to prior studies with other tumors, TRAMP-C2 tumors in the aged mice (n = 21) and the young mice (n = 19), were palpable at similar timepoints (∼20 days), grew as rapidly (Fig. 1a), and had similar volume at sacrifice (1,200 ± 209 mm3 and 822 ± 157 mm3, respectively) (Fig. 1b).
In contrast to the TRAMP-C2, B16/F10 tumors demonstrate decreased growth in aged mice relative to young mice
To confirm that our model system gave similar responses in tumors that have been previously examined, we implanted B16/F10 melanoma cells in young (n = 10) and aged (n = 10) mice. As expected, tumors were delayed in onset (3 aged and 7 young mice had palpable tumors at 17 days and 7 aged and all young mice had palpable tumors by 23 days) and grew less rapidly in the aged mice relative to the young mice (Fig. 2a). Three of the aged mice did not have palpable tumors and had only a layer of melanoma cells in their subcutaneous space at the time of sacrifice. Figure 2b shows the significantly smaller volume of the B16/F10 tumors in aged mice, relative to young mice, at the time of sacrifice (117 ± 51 mm3 and 1,738 ± 444 mm3, respectively).
The angiogenic response and distribution of vessel sizes were similar in prostate tumors from the aged mice and young mice
It has previously been reported that a decrease in the amount of angiogenesis is responsible, in part, for the deficit in B16/F10 tumor growth in aged mice.4, 6, 9 Frozen sections of TRAMP-C2 tumors from aged (Fig. 3a) and young (Fig. 3b) mice were examined by immunostain for the presence of mouse endothelial cell antigen 32 (MECA32), which reacts only with mouse endothelial cells.36 Quantitative analysis of digital images demonstrated that the percent area of the tumor that was comprised of blood vessels was similar in tumors grown in aged mice and young mice (Fig. 3c).
We have previously reported that angiogenesis in aging is characterized by both delays in vessel appearance and deficits in vessel density.37 However, it is not known if aged tissues might have a greater proportion of large dysmorphic vessels.7 The presence of fewer vessels of larger size would result in similar areas of vascularization in the tumors of young and aged mice irrespective of decreased vessel number in aged tissues. To address this possibility, vessels in the tumors from young and aged mice were analyzed for size distribution by analysis of digital images. The relative distribution of small, medium and large vessels was similar in both age groups (Fig. 3d).
Features of vessel maturity are similar in the prostate tumors in the aged mice and young mice
The deposition of basement membrane and investment with mural cells are indicative of vessel maturation.38 Sections were analyzed by immunostain for laminin, to represent basement membrane, and actin, to assess the appearance of mural cells (for example, pericytes and smooth muscle cells). Not surprisingly, the vasculature of the tumors in both the aged and young mice had robust immunostaining for laminin (Figs. 4a and 4b). Concurrent analysis for the presence of actin positive cells demonstrated that they were present only in the larger vessels near the tumor capsule and in equivalent number in both the aged and the young mice (Figs. 4c and 4d).
The presence of immune cells is not different between the young and the aged
Others have proposed that decreased tumor onset and slowed tumor growth in aged mice reflects a deficit in the immune response.5, 11, 14 In this context the lack of cytokines and chemokines that induce angiogenesis and tumor proliferation in the aged would result in a less permissive milieu. Sections were analyzed for the presence of monocytes/macrophages, CD3+, and CD8+ cells and were found to be similar in the young and aged mice (data not shown).
Measures of testosterone and VEGF are similar in the aged and young mice
Androgen levels are positively correlated with the growth of prostate tumor cells.39 Although the TRAMP-C2 cells express the androgen receptor, the growth of these cells in vitro is not known to be responsive to androgen levels. However, since the effect of testosterone on the growth of TRAMP-C2 in vivo is unknown, levels of testosterone in sera collected at sacrifice were measured in young and aged mice with and without TRAMP-C2 tumors. As expected, the aged mice had significantly lower levels of serum testosterone than the young mice (0.137 ± 011 ng/ml and 0.23 ± 032 ng/ml, respectively), but levels were decreased in both groups relative to controls with no tumors (Fig. 5). Accordingly, robust androgen levels did not account for the response in the aged mice.
The density of vessels in prostate cancers is correlated with poor prognosis and is directly associated with greater degrees of VEGF expression.40 Moreover, levels of VEGF are induced by the presence of sex steroids.24, 39, 41 VEGF as measured by ELISA did not differ significantly in either the sera or TRAMP-C2 tumors of young and aged mice (211.1 ± 50.3 pg/mg total protein and 210.4 ± 32.5 pg/mg total protein in the tumors from aged and young mice, respectively). Immunostaining for VEGFR2 in the TRAMP-C2 tumors of aged and young mice showed similar degrees of expression (data not shown).
TRAMP-C2 tumors have extracellular matrix and high levels of MMP2 and MMP9 expression and activity
The activity of MMPs is necessary for the creation of a permissive environment that allows for cell migration, invasion, proliferation and angiogenesis.42, 43, 44 Of primary interest in solid tumors are the gelatinases, MMP2 and MMP9.42, 43, 45 The expression of MMPs is determined, in part, by the presence of a matrix that requires their activity. Whereas the matrix of the TRAMP-C2 tumors (Fig. 6a) had a significant amount of collagenous material as reflected in Masson's trichrome stain, the B16F10 tumors consisted primarily of sheets of melanoma cells with minimal evidence of collagen (Fig. 6b). Tissue from both the TRAMP-C2 and the B16/F10 tumors of aged mice were analyzed for MMP2 and MMP9 gene expression and enzymatic activity. Analysis of tumor tissue with RT-PCR demonstrated significantly greater levels of transcripts for MMP2 and MMP 9 in the TRAMP-C2 tumors in comparison to B16/F10 tumors (Fig. 6c). Not surprisingly, MMP2 and MMP9 enzyme activity was also significantly greater in the TRAMP-C2 tumors, relative to B16/F10 tumors, reflecting higher availability for enzymatic digestion of the matrix in the stroma of the TRAMP-C2 tumors (Fig. 6d).
In our study, we found that the TRAMP-C2 prostate tumor, a cancer cell line not previously examined in healthy rodent models of aging, grew as well in aged mice as in the young mice. Moreover, the tumors had a similar time of onset and amount of angiogenesis in aged mice relative to young mice. These results are in marked contrast to previously reported models of tumor growth (such as those using the B16/F10 melanoma line, the LLC line, the EHS sarcoma line and the AKR lymphoma line) that made comparisons among animals that differed only with respect to age and found significantly decreased tumor growth in the aged mice.4, 5, 6, 7, 8, 10, 11, 13, 14
The TRAMP-C2 tumors in our study began as identical inoculants implanted into the subcutaneous space. The latter was chosen as the implantation site to minimize the stress to the animal and allow for ease of monitoring tumor growth. In addition, the skin and the subcutaneous space has been one of the best characterized tissues with respect to the effect of aging on the angiogenic response.34, 35, 37, 46 It is generally accepted that angiogenesis is both delayed and impaired in the skin and subcutaneous space of aged mice by ∼30–40% during wound healing.35 Accordingly, when tumor onset and growth in the aged mice occurred in a similar fashion to that of the young mice, we focused on vascularization within the tumors. Analysis of immunostained sections showed that the angiogenic response did not differ between the young and aged mice. These data are the first to show equivalent tumor angiogenesis in the tissues of aged mice relative to young mice. It is noted that vessel area alone cannot be used as a measure of the angiogenic response in aging. The possibility of a greater presence of large, dysmorphic vessels in aged tissues can alter calculations based on area. However, the distribution of vessel sizes within the tumors did not differ between the young and aged mice in our study. Moreover, the presence of actin-positive cells, often used to indicate vessel maturation and development, was only noted near the capsule of the tumors in both age groups.38 This area represents the point of contact between the tumor and the vessels of the subcutaneous space and would be expected to have more mature vascular structures independent of the age of the mice.
The ability of the aged mice to develop large, highly vascularized tumors has implications from both the therapeutic and gerontologic perspective. The therapeutic importance is prompted by the advent of inhibitors of angiogenesis as adjunctive agents to surgery and chemotherapy for the treatment of colon, lung and possibly other cancers including prostate tumors.22, 23, 24, 47 Whether these agents will be more or less efficacious in tumors in the very elderly depends on whether tumor angiogenesis is impaired in the aged, and the subsequent impact that might have on the response to angiogenesis inhibitors. If tumors in the aged have fewer vessels, then a relative decrease in vessel number after treatment with these agents would result in a similar effect in young and aged subjects. In contrast, if the effect is an absolute reduction in the number of vessels, then the aged tissues with their fewer vessels might benefit to a greater degree. Our finding that vessel density can be as high in prostate tumors in the aged mice demonstrates that the clinical importance of this issue may only be relevant in certain cancers. Independent of the age of the host, the relative contribution of mature and precursor endothelial cells to blood vessel formation in a given tumor remains to be established and may also influence the efficacy of an angiogenesis inhibitor.48
From the gerontologic perspective, one wonders how a mouse near the end of its life expectancy (23–24 months for the C57/Bl6) could generate such an exuberant vascular response in the TRAMP-C2 tumors. Examples of aged tissues undergoing robust formation of vessels are rare in the literature. One exception are diseases of the eye in which decreases in the angiogenesis inhibitor pigment epithelium derived factor (EPC-1/PEDF), at the same time hypoxia stimulates the production of proangiogenic molecules, results in a proangiogenic milieu even in the very aged.49, 50 In our study, levels of VEGF were highly variable and did not differ significantly in the TRAMP-C2 tumors or in the sera of the aged mice in comparison to that of the young mice. Analysis of other possible mechanisms showed that the aged mice with tumors had markedly decreased levels of testosterone relative to its young counterpart. Moreover, as expected, both groups of mice with tumors had significantly less testosterone than age-matched controls without tumors, thereby further discounting the role of androgens in our results. Other variables that have previously been shown to be deficient in tumors in aged mice relative to young mice, such as the influx of inflammatory cells, were not altered in the TRAMP-C2 tumors of the aged mice relative to those of the young mice.
In subsequent experiments we sought to find tumor cell characteristics that are unique to the TRAMP-C2 prostate line relative to the oft-cited B16/F10 melanoma line. Comparison with the latter is highly relevant, as B16/F10 tumors grew poorly in aged mice relative to young mice, in all the published studies.4, 5, 6, 7, 10, 11, 13, 14 Moreover, the behavior of B16/F10 tumors was representative of other tumor lines examined in young and aged mice.6, 7, 8, 10, 13, 14 We first noted that the TRAMP-C2 tumors had a highly developed extracellular matrix that stained for the presence of collagen and other matrix proteins. In contrast, the B16/F10 tumors had minimal evidence of an extracellular matrix and were comprised primarily of sheets of tumor cells. In conjunction with the presence of a well formed matrix, the TRAMP-C2 tumors had significantly greater amounts of gelatinase (MMP2 and MMP9) expression and activity. This is expected as extracellular matrix proteins regulate, in part, the production of the enzymes responsible for their turnover and degradation.51 Once tumor cells express MMPs, they can induce MMP secretion from their associated stromal cells thereby further amplifying their potency.52 It is generally accepted that the ability of malignancies, especially solid cancers, to express gelatinases is positively correlated with their invasive potential and subsequent poor clinical outcomes.45 Prostate tumors express increasing amounts of MMP2 and MMP9 as they progress to higher-grade tumors and greater degrees of metastatic potential, including the strongly prognostic Gleason score.53, 54 The influence of MMPs on tumor propagation results from both direct and indirect mechanisms.44 Direct effects, via degradation of the matrix, result in a more permissive environment for tumor cell migration and invasion. MMP activity also allows endothelial cells to migrate and proliferate more efficiently. The subsequent facilitation of the angiogenic response results in a greater blood supply to the tumor. Indirect effects of MMP activity include: activation of other pro-MMPs, cleavage of regulatory precursor molecules at the cell surface and induction of nascent chemokines and growth factors that require enzymatic activation for optimal effect. In the current study, the high expression of MMP2 and MMP9 in TRAMP-C2 tumors provides a potential explanation for the observed differences between the growth of B16/F10 and TRAMP-C2 tumors in aged mice. Moreover, MMP activity has been shown to be a key component of VEGF-induced angiogenesis in tumors55 and might be responsible, in part, for the robust angiogenic response observed in TRAMP-C2 tumors from young and old animals.
In conclusion, we have found that TRAMP-C2 prostate tumors grow efficiently with a robust angiogenic response in aged mice. This finding is highly significant and unique relative to other tumor models in young and aged mice. The results may reflect the fact that TRAMP-C2 cells produce high amounts of MMP2/MMP9 activity. The latter could result in specific stimulation of tumor growth and angiogenesis by the interaction of TRAMP-C2 cells with the extracellular matrix, thereby creating a more permissive environment independent of the age of the host.
The authors wish to thank Dr. E. Helene Sage and Dr. Jennifer Wu for thoughtful discussions. The authors also thank Ms. Lily Higgins and Ms. Ayaka Iwata for technical assistance with the experiments.