Designing ageing conditions in tumour microenvironment—A new possible modality for cancer treatment

Mechanisms of Ageing and Development 130 (2009) 76–85

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Designing ageing conditions in tumour microenvironment—A new possible modality for cancer treatment Judith Leibovici *, Orit Itzhaki, Tatiana Kaptzan, Ehud Skutelsky, Judith Sinai, Moshe Michowitz, Raida Asfur, Annette Siegal, Monica Huszar, Ginnette Schiby Department of Pathology, Sackler Faculty of Medicine, Tel-Aviv University, 69978 Tel-Aviv, Israel

A R T I C L E I N F O

A B S T R A C T

Article history:

While tumour incidence is known to augment with age, paradoxically tumour growth and metastasis were often found to proceed at a slower rate at late ages. This age-related biological behaviour of tumours actually imposes a differential therapeutic approach to the old cancer patient. Several mechanisms of the age-related reduced tumour progression have been demonstrated: decreased tumour cell proliferation, increased apoptotic cell death, decreased angiogenesis and anti-tumoural immune response changes. We postulated that it might be possible to design age-adjusted treatment modalities based on the mechanisms responsible for the reduced tumour progression rate in the aged. Based on these mechanisms, we compared the effect of different treatments (apoptosis-inducing agents, Hydrocortisone and Adriamycin, anti-angiogenic agent, TNP-470, and immunomodulators-Levamisole and BCG) on two experimental tumours (B16 melanoma and AKR lymphoma) growing in young and old mice. Most treatments showed, in both tumours, a higher inhibitory effect on tumours growing in old mice than on those developing in young ones, to our knowledge, a feature not described before for anti-tumoural agents. We suggest that designing ageing conditions in tumours of young patients might possibly alleviate neoplastic aggressiveness in these patients as well. ß 2008 Elsevier Ireland Ltd. All rights reserved.

Available online 25 March 2008 Keywords: Cancer Ageing Age-adjusted therapy

1. Introduction The proportion of aged individuals in the population is constantly rising. This fraction of the population is the most afflicted by cancer. The absolute number of cancer patients in the USA is expected to double by year 2030 (Mandelblatt, 2006). In Europe, over 45% of all cancers occur in patients over 70 years of age (Lonardi et al., 2007). It is expected that 70% of all neoplasms will occur in persons 65 years and over by the year 2020, leading to increased cancer-related morbidity among the elderly (Balducci and Extermann, 2000). Therefore research on tumour development and its treatment in the elderly should become of greater importance. However, only few studies have been devoted to the effect of ageing on tumour biology and even fewer on age-adjusted cancer therapy. In fact, most experimental studies on cancer are, inappropriately, performed on young animals, which may be irrelevant for the aged organism. While treatment of cancer has improved in persons under 50, mortality has not been reduced for older cancer patients (Balducci

* Corresponding author. Tel.: +972 3 6409630; fax: +972 3 6409141. E-mail address: [email protected] (J. Leibovici). 0047-6374/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2008.03.004

and Lyman, 1997). This is thought to be due to the fact that physicians display a reluctance to undertake aggressive therapy in elderly cancer patients (Siu, 2007). Aged cancer patients have actually often been offered suboptimal treatment (Balducci, 2007), due to the numerous pathologies of aged individuals and to their higher susceptibility to the aggressive anti-tumoural treatments in use. The ageing host is definitely very different from the young one. What is less known, or anyway, not at all considered, is that tumour behaviour in the aged is also different. While cancer incidence is known to augment with age, paradoxically, tumour growth and metastasis were often found to proceed at a slower rate in aged organisms, in both humans (Ershler, 1986) and experimental models (Ershler et al., 1984). Bronchogenic cancer (Ershler et al., 1983) and cancers of breast (Fisher et al., 1997) and colon (Calabrese et al., 1973) were reported to grow and metastasize at a lower rate in old patients. With regard to experimental tumours, a reduced aggressiveness in aged as compared to young animals was found as well (Peto et al., 1975; Ershler et al., 1984). We have also reported this phenomenon in the B16 melanoma (Donin et al., 1997) and in the AKR lymphoma (Itzhaki et al., 2000). Not all tumours display an age-related reduced tumour progression. This phenomenon is very frequent in human

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malignancies. But indeed, in experimental tumours, opposite results were reported even in the same tumour, such as B16 melanoma (Hirayama et al., 1985; Baumgart et al., 1988; Anisimov, 2006). This may be due to various experimental conditions, such as size of inoculum and site of injection. Our impression (according to unpublished data) is that the lower the inoculum, the lower is the malignant behaviour of tumours in old mice. The fact that the inoculum size can determine the biological behaviour of tumours in old animals can be explained in the following way: the immune response in old mice can be more efficient than in the young ones at low tumour cell inocula but not at high inocula. This may be the case for human tumours, where (in contrast to studies with most experimental tumours), neoplastic growth begins from a single cell and tumours then develop during long years. In mice, we usually use inocula of 103 to 106 cells, not single cells. Therefore, tumour development following low inocula may be closer to tumour evolution in humans. We suggest that the age-dependent differential biological behaviour of tumours imposes a differential therapeutic approach to young and old cancer patients. The mechanisms underlying the reduced tumour aggressiveness in aged compared to young organisms have not yet been established. Decreased proliferative capacity in the old has been suggested (Cameron, 1972). The rate of DNA synthesis in tumours from old animals in organ culture was found to be lower than in tumours from young animals (Holbrook and Ikeyama, 2002) and decreases in growth factor and hormone availability with age have also been documented (Roth, 1979; Chahal and Drake, 2007). We have demonstrated an increased apoptotic cell death in tumours from old as compared to those from young mice bearing the B16 melanoma and the AKR lymphoma (Itzhaki et al., 2004). Reduced angiogenesis with age has also been suggested to constitute a possible mechanism of the decreased tumour progression rate in old as compared to young organisms (Kreisle et al., 1990; Pili et al., 1994). Modifications in anti-tumoural immune reactions with age have been demonstrated by the group of Ershler (Ershler, 1986; Kaesberg and Ershler, 1989) and by our group (Donin et al., 1995; Kaptzan et al., 2004). Klement et al. (2007) found that atherosclerosis in old Apo E/ mice can also reduce neovascularization in tumours. We have recently found incidentally another possible mechanism, passage from tetraploidy in B16 melanoma of young mice to diploidy in tumours of old animals (Itzhaki et al., 2008). In fact, most of these features (among many other features) characterize normal ageing at the molecular, cellular, tissue and organismal level. Actually, opposite features characterize tumour progression, namely increased tumour cell proliferation, decreased tumour cell apoptosis, increased angiogenesis and changes in immune responses. While the problems imposed by age on cancer therapy have been (and should be) considered, the differential behaviour of tumours in function of age has not been taken into account. We considered that the diminished aggressiveness of tumours often observed in the aged, and the elucidation of the mechanisms underlying this interesting phenomenon, might suggest new therapeutic modalities, more appropriate for the old organism. We postulated that it may be possible to design age-adjusted treatment modalities based on the mechanism(s) responsible for the reduced tumour progression rate in the aged. Based on these mechanisms of the reduced tumour malignancy in the aged, demonstrated by other groups and by ourselves, we tested the therapeutic effect of apoptosis-inducing drugs, an angiogenesis-inhibitor and immunomodulators on the development of tumours in young and old mice.

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2. Materials and methods 2.1. Mice and tumours Studies on animal models were performed according to the guidelines of the TelAviv University. C57BL/6J and AKR/J mice were purchased from the Tel-Aviv University Breeding Center. Two age groups were used: young mice: 6–8 weeks for both C57BL/6J and AKR/J mice; old mice: 16–24 months for C57BL/6J mice and 7–9 months for AKR/J mice. We chose this age as ‘‘old’’ for the AKR mice, because the lifespan of this strain of mice is short, and in order to avoid the use of mice with spontaneous tumours which begin to appear at around 6 months (Wainfan et al., 1990). Mice were examined (by palpation of inguinal lymph nodes) for presence of spontaneous tumours and those which developed such tumours were not included in the experiments. Tumour cell suspensions of both AKR lymphoma and B16 melanoma were prepared as previously described (Leibovici et al., 1980). Tumour cells suspended in 0.2 ml RPMI-1640 medium (Sigma–Aldrich, Rehovot, Israel) were inoculated subcutaneously (s.c.) in the back of mice. For AKR lymphoma, the inoculum of 1  104 cells was chosen in order to obtain the maximum differential biological behaviour of the tumour between young and old mice. In the case of B16 melanoma the inoculum of 2.2  105 cells was chosen so that there were (usually) no survivors in the non-treated groups but there were survivors in the treated mice groups. Tumour growth was evaluated by recording the incidence and by measuring 2–3 times a week the diameter of the tumours formed at the s.c. site of inoculation. Mice mortality was recorded daily. Long-term survival was followed for 100 days at least. 2.2. Histological examination 2.2.1. H&E staining The resected primary tumours were fixed in formalin, embedded in paraffin and 4 mm-thick sections were stained by ordinary H&E. 2.2.2. Giemsa staining Tumours were excised and tumour cell suspensions were prepared, spread on slides, fixed with methanol and stained with May–Grunwald–Giemsa. 2.3. ApopTag staining Apoptosis was identified by labelling the DNA 30 -OH nick-ends using a variant of TUNEL staining. ApopTag staining was carried out according to the manufacturer’s instructions using the materials provided in the kit (ApopTag, Intergen Company, New York). After deparaffinization in xylene and rehydration in a series of decreasing concentrations of alcohol, the 5 mm thick tissue sections were incubated at 37 8C for 1 h in the presence of terminal deoxynucleotidyl transferase (TdT) and digoxigenin-labelled nucleotides. After washing, anti-digoxigenin-peroxidase was added to the slides which were incubated in a humidified chamber for 30 min at room temperature. After washing in PBS, the slides were stained with diaminobenzidine (Sigma–Aldrich, Rehovot, Israel) and then counterstained with 1% methylgreen (Vector, Burlingame, CA). The percentage of apoptotic bodies was determined at high power fields. A total of 300 cells were counted. 2.4. Factor VIII immunostaining Slides with sections (4 mm thick) of the paraffin-embedded tumours were prepared, deparaffinized by three washes in xylene, and rehydrated through a graded ethanol series (100%, 90% and 70%). Endogenous peroxide was blocked with 3% H2O2 for 20 min. Factor VIII was detected by a rabbit polyclonal antibody, prediluted read-to-use solution (ImmuStain, CA, USA) after predigestion with trypsin (Zymed Laboratories Inc., CA, USA) and by a standard streptavidin–biotin horseradish peroxidase complex detection method (Histostain-Plus Bulk Kit) with 3,3-diaminobenzidine (DAB) as a chromogen (Zymed Laboratories Inc., CA, USA). Faint nuclear counterstaining was achieved with Meyer’s hematoxylin solution and coverslipped with permanent mounting media. 2.5. Assessment of microvessel density The comparison of microvessel density in the B16 melanoma and AKR lymphoma grown in young or old mice was assessed in sections of the subcutaneous primary tumours following formalin fixation, paraffin embedding and routine staining by H&E as well as by Factor VIII immunostaining. 2.6. Assessment of host response: macrophage content in tumours Host response to tumours was assessed in situ following H&E staining in sections of B16 melanoma and AKR lymphoma local primary tumours and their environment. For macrophage percentage evaluation, local s.c. tumours were excised and tumour cell suspensions were prepared, spread on slides, fixed with methanol and stained with Giemsa. Two hundred cells were counted and the percentage of macrophages was calculated.

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2.7. Anti-tumoural treatments 2.7.1. Treatment of young and old tumour-bearing mice by apoptosis-inducing agents Treatment was performed with single doses of 500 mg of HC and 25 mg per mouse of ADR. Single doses were administered 24 h following tumour inoculation. Each experimental group consisted of 5 mice and each experiment was repeated with Hydrocortisone, 5 times for B16 melanoma and 4 times with AKR lymphoma and with Adriamycin 7 times for B16 melanoma and 5 times for AKR lymphoma. 2.7.2. Treatment of young and old tumour-bearing mice with the anti-angiogenic agent TNP-470 Treatment with TNP-470 was performed by injecting 400 mg per mouse intratumourally 3 times a week, beginning 24 h after tumour inoculation and continued for the duration of the experiment. Each experimental group consisted in 5 mice and each experiment was repeated 4 and 3 times with the B16 melanoma and AKR lymphoma, respectively. The authors wish to thank Takeda Pharmaceutical Company, Ltd. (Osaka, Japan) for the generous gift of TNP-470. 2.7.3. Treatment of young and old tumour-bearing mice by immunomodulating agents Treatment with Levamisole was performed with 25 mg per mouse intratumourally 3 times a week, beginning 24 h after tumour inoculation and continued for the duration of the experiment. Treatment with BCG was performed with single doses of 25 mg per mouse 24 h following tumour inoculation. Each experimental group consisted of 5 mice and each experiment was repeated for Levamisole, 5 and 3 times for B16 melanoma and AKR lymphoma, respectively. 2.8. Statistical evaluation Statistical evaluation was performed for most data by Student’s t-test. The x2 test was used to test the significance of the differences between the treated and nontreated groups of mice in long term survivors.

3. Results 3.1. Comparison of biological behaviour of B16 melanoma and AKR lymphoma in young and old mice Fig. 1 presents a comparison of the biological behaviour of the tumours in young and old mice. Both the B16 melanoma and the AKR lymphoma grow at a slower rate in old as compared to young mice. The differences are statistically significant at all points, in the two tumours. Similar differences were observed according to other criteria of tumour growth, such as tumour incidence and mice mortality as well as by histopathological criteria. 3.2. Mechanisms responsible for the reduced malignancy of tumours in old as compared to young mice Evidence for three mechanisms of the reduced tumour growth in aged mice is presented in Fig. 2. Increased apoptotic cell death in old mice-derived tumours is shown in Fig. 2a, reduced angiogenesis in tumours from aged mice is seen in Fig. 2b and c shows increased macrophage infiltration around tumours of old as compared to those derived from young animals. The differences are statistically significant to highly significant. It seems therefore that three mechanisms (in addition to the decreased tumour cell proliferation which is not shown) can be responsible for the reduced malignancy of our two experimental tumours when growing in old mice as compared to those developing in young animals: increased tumour cell apoptosis, reduced angiogenesis and increased macrophage infiltration. 3.3. Comparison of the effect of treatments based on the mechanisms of the age-related reduced tumour aggressiveness on young and old tumour-bearing mice Table 1 and Fig. 3 present a comparison of the therapeutic effect of several treatment modalities acting on the basis of the mechanisms proven to be responsible for the reduced malignancy of tumours in aged as compared to young mice. We used as

apoptosis-inducing agents Hydrocortisone and Adriamycin, as anti-angiogenic agent TNP-470 and as immunostimulators, Levamisole and BCG. Table 1 shows the effect of the different treatments on the growth of B16 melanoma and AKR lymphoma, according to average tumour size and Fig. 3 presents the effect according to long term surviving mice. As seen in Table 1, all treatments except ADR in B16 melanoma resulted in a more pronounced growth inhibition of tumours in old than in young mice. For instance, treatment with Levamisole inhibited the AKR lymphoma in young mice by 13% while in old mice the inhibition was of 55%. The tumour-inhibitory effect of all these therapeutic modalities, except one treatment in one tumour (Adriamycin in B16 melanoma) was more pronounced against tumours of old mice than towards those of young animals. The highest percentage of long term surviving mice was observed in all but one case (mentioned above) in the group of old-treated mice. Moreover, in AKR lymphoma, with most treatments (four out of five treatments), this group of oldtreated mice was the only one in which long term survivors were observed (except ADR treatment, which affected also tumours of young mice although less than those of old animals). In B16 melanoma, treatment with Levamisole and BCG also resulted in long-term survivors in old-treated mice only. It should be noted that delay in mice mortality was seen, in most cases, in young mice as well, but this delay was less pronounced than in old mice. The inhibitory effect on the mortality of tumour-bearing mice was statistically significant in the case of adriamycin in AKR lymphoma ( p < 0.0125 in young mice and p < 0.0005 in old animals). In B16 melanoma, the effect was significant in young mice only ( p < 0.00025). Treatment with TNP-470 resulted in statistically significant inhibition of mice mortality in both young and old mice in the case of B16 melanoma (for both p < 0.0025) and in the case of AKR lymphoma, only in old mice ( p < 0.025). The inhibitory effect of Levamisole and BCG was significant only in oldtreated mice, in both tumours ( p < 0.05–p < 0.0125). The inhibitory effect of Hydrocortisone on mice mortality was not significant statistically. However, according to the data of two other criteria, incidence and size of tumours, the differences were significant particularly for the AKR lymphoma, with a higher significance in old mice. Of note, with almost all treatments, the statistical significance of tumour inhibition, according to all criteria was more marked in old mice. The differences in mice mortality between the young-treated and the old-treated mice in days of survival (according to mean survival time) ranged from 0 in one case (AKR lymphoma treated with Hydrocortisone) to 16 days in the B16 melanoma treated with TNP470. However, the differences in long-term surviving mice between old-treated and young-treated mice, which is more important, were much more pronounced and observed in all but one treatment. 4. Discussion In view of the demographic changes which have taken place during the last century in relation to the rapid rise in the proportion of the elderly population, the search for adequate treatment of aged cancer patients is of great importance. It is, however, very problematic mainly due to the low resistance of the aged towards the aggressive therapies currently used against cancer. Numerous problems are encountered in the treatment of old cancer patients, such as numerous pathologies, higher susceptibility to surgical procedures and higher sensitivity to the toxicity of the aggressive chemotherapeutic treatments now in use. In addition, chemo- and radio-therapy display a lower effectiveness on tumours of the aged which have a lower proliferative capacity, an important feature not often mentioned.

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Fig. 1. Comparison of biological behaviour of B16 melanoma and AKR lymphoma in young and old mice: (a) kinetics of tumour development-comparison of average diameter of tumours. The error bars indicate S.D. *, p < 0.01; **, p < 0.025; ***, p < 0.0005. (b) Comparison of tumour growth of B16 melanoma and AKR lymphoma in young and old mice—representative animals. Mice were photographed on day 16 after tumour inoculation in the case of B16 melanoma and on day 13 in the case of AKR lymphoma. Two age groups of mice were used: young mice: 6–8 weeks for both C57BL/6J and AKR/J mice; old mice: 16–24 months for C57BL/6J mice and 7–9 months for AKR/J mice. C57BL/6J mice were inoculated s.c. with 2.2  105 cells of B16 melanoma and AKR/J mice were injected by the same route with 1  104 cells of AKR lymphoma. Groups of 5 mice were inoculated s.c. with tumour cells on day 0. The data represent the average diameter of tumours (mm). Student’s t test was used for calculating statistical significance.

Elderly patients tolerate chemotherapy poorly compared to young patients because of the progressive reduction in organ function and comorbidities so often related to age. For this reason, elderly patients have been excluded from or underrepresented in

clinical trials and often receive inadequate treatment (Rossi et al., 2005). Very often, elderly cancer patients have not been offered lifesaving interventions due to the assumption (the just assumption)

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Fig. 2. Mechanisms responsible for the reduced malignancy of tumours in old as compared to young mice: (a) comparison of apoptotic cell content in sections of metastatic tumours of AKR lymphoma derived from young and old mice (ApopTag staining). Left side: representative picture. Magnification: 400. Right side: quantitative evaluation of apoptotic cell content of AKR lymphoma growing in young and old mice. Ten areas from each specimen were chosen randomly and the number of cells stained positive with ApopTag (brown) was counted. (b) Comparison of angiogenesis in B16 melanoma of young and old mice Left side: immunostaining with factor VIII. Magnification: 400. Right side: microvessel density (MVD). Tumours were excised on day 18 after inoculation. Microvessel density was assessed quantitatively by counting microvessels in a light microscope at 200 magnification in 10 fields. Microvessel density is represented by the number of blood vessels per 200 field. (c) Comparison of macrophage (Mf) content in metastatic tumours (spleen) of AKR lymphoma derived from young and aged mice. Left side: H&E staining. Magnification: 400. Note that in the old mice a large part of the tumour section is occupied by macrophages, part of them phagocytosing lymphoma cells. Right side: quantitative comparison of macrophage content in AKR lymphoma derived from young and old mice, according to cell morphology. For assessing macrophage content by cell morphology, tumour cells, freshly extracted from young and old mice, were spread on slides, fixed by methanol and stained with Giemsa. The data represent average values of macrophage number as observed in 10 high power fields.

Table 1 Effect of different treatments on B16 melanoma and AKR lymphoma growth in young and old mice: size of tumours Young mice

Old mice

Non-treated

Treated

Inhibition (%)

p

Non-treated

Treated

Inhibition (%)

p

B16 Melanoma HC ADR TNP-470 LEV BCG

40.7  7.2 48.8  8.1 15.4  11.4 28.6  8.8 40.0  14.6

43.5  4.6 11.1  10.6 14.8  5.5 32.6  10.2 31.8  12.7

0 77 4 0 19

N.S <0.0005 N.S N.S N.S

36.2  1.9 45.9  0.0 4.0  3.6 27.2  3.4 32.8  4.0

21.7  8.3 22.2  14.0 1.14  2.3 12.9  12.2 13.3  10.9

40 52 72 53 59

<0.005 <0.0025 <0.025 <0.0125 <0.01

AKR lymphoma HC ADR TNP-470 LEV BCG

26.1  2.4 18.5  3.7 17.9  4.1 26.2  2.2 18.3  5.1

26.1  2.7 7.0  3.7 5.6  1.9 22.9  3.5 25.5  2.5

0 62 69 13 0

N.S <0.0025 <0.0005 <0.05 <0.001

16.1  2.4 11.5  2.9 11.0  3.8 16.9  2.8 16.6  .2.5

9.8  4.3 1.5  3.1 0.8  1.8 7.6  2.3 2.5  1.9

39 87 93 55 85

<0.005 <0.0005 <0.0005 <0.0025 <0.0005

The data represent the average diameter of tumours (in mm)  S.D. Each experimental group consisted in 5 mice. The data presented are those observed on day 14 after tumour cell inoculation for AKR lymphoma in all experiments and for B16 melanoma, on day 22 for the experiment with Levamisole (LEV), on day 24 for the experiments with Hydrocortisone (HC) and TNP-470 and on day 28 for the experiments with Adriamycin (ADR) and BCG. The kinetics of growth differed from one experiment to another.

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Fig. 3. Comparison of the efficiency of treatments based on the mechanisms of the age-related reduced tumour aggressiveness on young and old tumour-bearing mice. The age-dependent effect of treatment with apoptosis-inducing drugs, Hydrocortisone (HC) and Adriamycin (ADR), an angiogenesis inhibitor (TNP-470) and immunomodulators, Levamisole (LEV) and BCG, was tested on B16 melanoma and AKR lymphoma. The data of the effect on long-term survival is presented. Control-non-treated tumour-bearing mice; HC—the mice were treated with 500 mg/mouse of Hydrocortisone at a single dose, injected intratumourally on day 1 after tumour cell inoculation; ADR—the mice were treated with 25 mg/mouse of adriamycin at a single dose, injected intratumourally on day 1 after tumour cell inoculation. TNP-470—the mice were treated with 400 mg/ mouse of TNP-470, injected intratumourally 3 times a week for the duration of the experiment. LEV-the mice were treated with Levamisole intratumourally with doses of 25 mg 3 times a week for the duration of the experiment; BCG-the mice were treated with 25 mg BCG intratumourally on day 1 after tumour cell inoculation. The data represent the percentage of surviving mice per total number of mice. These are cumulative results of several experiments, each containing usually 5 mice per group. In the case of Hydrocortisone, 5 experiments were done with B16 melanoma and 4 with AKR lymphoma, the effect of Adriamycin was tested in 7 experiments in B16 melanoma and in 5 in AKR lymphoma, TNP-470 was examined in 4 and 3 experiments in B16 melanoma and AKR lymphoma, respectively, Levamisole effect was tested in 5 and 3 experiments in B16 melanoma and AKR lymphoma, respectively, and the effect of BCG was examined in 4 and 2 experiments in B16 melanoma and AKR lymphoma, respectively. The total number of mice was for instance for Hydrocortisone treatment, 25 animals for each experimental group in the case of B16 melanoma.

that such treatments would be for them too toxic (Balducci, 2007). Nevertheless, according to Balducci, most elderly cancer patients can benefit from cancer treatment to an extent comparable to that of younger patients, and only a minority should be excluded from treatment. Since older populations are very heterogeneous, he

proposes that management of cancer in the elderly should be guided by individual estimates of life expectancy and functional reserve rather than by chronological age. Kurtz and Dufour (2002) suggested that chemotherapy should not be denied to elderly patients with metastatic breast cancer,

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provided a prior geriatric assessment is performed to evaluate the risk-benefit ratio. Cinamon et al. (2006) found that senior patients with head and neck squamous cell carcinoma can benefit from curative aggressive treatment. They concluded that exclusion should be based, as for younger subjects, on an individual basis. Ramesh et al. (2005) suggest that surgeons are called upon to provide optimal cancer management for the heterogeneous group that elderly people constitute. They state that delivery of suboptimal surgical cancer treatment due to ageist attitudes or personal beliefs is no more acceptable. Sawhney et al. (2005) propose that the increase in the aged cancer patient population necessitates that oncology professionals become familiar with ageassociated changes in organ physiology and with the impact of these changes on cancer treatment and toxicity. Increasing interest in designing chemotherapy suitable for the elderly and the poor performance status patients has lead to clinical trials in advanced NSCLC, for instance (Gridelli and Hainsworth, 2002). It was suggested that to avoid treatment complications, therapeutic decisions should be based on an estimation of the patient’s life expectancy and risks and benefits should be weighed up accordingly. In addition to Comprehensive Geriatric Assessment (CGA), comorbidity, polypharmacy, cognition and social support should also be considered (Repetto and Balducci, 2002). The problems of the old cancer patient treatment are all at the disadvantage of the patient. One positive feature in aged cancer patients is the reduced aggressiveness of their tumours. We suggest that this feature should also be considered when designing age-adjusted anti-tumoural therapy, eventually to the advantage of the aged patient. A therapeutic approach could be to target the tumour microenvironment, enforcing properties of ageing tissues which reduce tumour aggressiveness. Understanding the mechanisms of the differential biological behaviour of tumours in relation to host age is of importance in itself but it may also be important for the eventual exploitation of these mechanisms for a rational design of cancer therapy appropriate for the aged organism. The age-related differential biological behaviour of tumours actually implies the necessity of a differential therapy for cancer patients of different ages. Very few experiments of this type have been performed (Yuhas and Ullrich, 1976; Takeichi et al., 1990; Watanabe, 1996; Katz and Boylan, 1987; Gravekamp, 2007). If we take into account, not only the poor condition of the elderly patient, but also the differential malignant behaviour of many tumours in the aged, the old patients may possibly profit from drugs addressed towards mechanisms underlying the reduced tumour progression rate in the aged organism. We reasoned that, based on the mechanism(s) of the reduced aggressiveness of tumours in the old, a rational design of agerelated therapy can be envisaged. We have shown that three treatment modalities, based on mechanisms underlying the reduced tumour progression in aged organisms (drugs inducing apoptotic cell death, an anti-angiogenic agent and immunomodulators) display an age-related differential effect on two experimental tumours. We have demonstrated that these three treatment modalities which induce elements of an ‘‘ageing microenvironment’’ exert an inhibitory effect on tumours of young and old mice. Notably, almost all these treatment modalities proved to be more efficient against tumours in old than against those growing in young mice, to our knowledge, not described before for anti-tumoural agents.The single exception was treatment by Adriamycin on B16 melanoma. In this case ADR had a higher inhibitory effect on young than on old tumour-bearing mice. It is now well established that many chemotherapeutic drugs, including Adriamycin, exert their effect, at least partially, by

apoptosis (Hannun, 1997). Due to their ‘‘lympholytic effect’’, adrenal corticosteroids have been used to treat lymphomas since the late 1940s (Distelhorst and Dubyak, 1998). Lymphoid and leukemic cells are generally prone to apoptosis and, moreover, uniquely sensitive to the lytic action of glucocorticoid hormones (Smets et al., 1999). The different action exerted by ADR on B16 melanoma can be explained as following: glucocorticoids can kill their sensitive target cells only by inducing programmed cell death whereas the contribution of apoptosis to the total level of tumour cell kill by chemotherapeutic drugs such as Adriamycin and radiation is a matter of debate (Smets, 1994). Mechanisms other than apoptosisinduction (such as evidently an anti-proliferative effect) may be responsible for the higher effect of ADR on B16 melanoma of young mice, an effect which may be less efficient against tumours of old mice. In this sense, Adriamycin (and cytotoxic agents in general) is not a purely apoptosis-inducing agent. In addition, the B16 melanoma appears to be less prone to apoptosis than the AKR lymphoma. In fact, it is not expected that all apoptosis-inducing treatments will be more effective in old mice against each tumour. In view of the rapidly increasing aged population and the paucity of efficient antitumoural treatments in the aged, it may be of importance if such mechanisms of treatment will be effective in some tumours of the elderly. Except our published results in two tumour models, B16 melanoma and AKR lymphoma, with 5 different treatments (Itzhaki et al., 2004; Kaptzan et al., 2004, 2006, and summarized here), the few studies which compared the effectiveness of anticancer treatment in relation to age, were, to our knowledge, mainly with immunotherapy, except one recent study which applied an anti-angiogenic treatment to young and old tumour-bearing mice (Klement et al., 2007) and none with apoptosis inducers. These few studies generally found a lower response to treatment in old than in young animals (Yuhas and Ullrich, 1976; Katz and Boylan, 1987; Takeichi et al., 1990; Watanabe, 1996; Gravekamp, 2007). Joudi et al. (2006) reported that patient age had a negative effect on the efficiency of intravesical immunotherapy – BCG – against superficial bladder carcinoma. Interestingly however, the same group found a low efficacy in patients younger than 50 years. Only very recently, Klement et al. (October 2007) have reported a higher effect of an anti-angiogenic treatment (metronomic chemotherapy with cyclophosphamide) on Lewis lung carcinoma and B16 melanoma of old mice than on tumours of young animals. The often found lower aggressiveness of tumours in old as compared to young patients might impose a new concept in the treatment of elderly cancer patients. While ageing is considered to preclude current (mainly anti-proliferative) antitumoural treatments, we supposed that therapeutic modalities based on other mechanisms (those shown to be responsible for the reduced tumour progression rate in the old) might be as efficient as or even more efficient in aged than in young organisms. In view of the problems encountered in the treatment of elderly cancer patients, finding treatment modalities which might be as efficient or even more efficient against tumours of aged patients may be of importance. Moreover, designing ageing microenvironmental conditions in tumours of young patients might possibly alleviate neoplastic aggressiveness in these patients as well. The young mice were less susceptible (but in most cases not unsusceptible) than the old ones to the drugs inducing an ‘‘ageing microenvironment’’. Except Hydrocortisone and Levamisole, both in B16 melanoma only, young mice were affected (although at a lower extent than young ones) by the other treatments. AKR lymphoma was inhibited in young mice with all treatments, although, as we anticipated, at a lesser extent than in old mice. This lower sensitivity possibly occurs because normally young organ-

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isms (without relation to tumours) have a lower tendency to apoptosis (Muskhelishvili et al., 1995; Zang and Herman, 2002; Itzhaki et al., 2003), a higher propensity for angiogenesis (Rivard et al., 1999; Sadoun and Reed, 2003) and a different immune response, showing decreased T cell-mediated function (Pawelec and Solana, 1997) but conserved or even augmented innate immunity (Franceschi et al., 2000). Therefore probably, induction of apoptosis, reduction of angiogenesis and possibly stimulation of the innate immune system (in our case, mainly macrophages), are more difficult but still possible to achieve in young animals. It is possible that higher doses are needed for young than for old animals. Immune modulation should certainly be carefully tested in view of the complexity of the immune system in general and in young as compared to old patients in particular. While our results show a proof of concept, it is evident that many more studies have to be done in order to find optimal conditions of treatment in both old and young organisms. The immune system of aged humans as well as that of animals is known to undergo alterations, usually described as a decline in function (Shigemoto et al., 1975; Makinodan and Kay, 1980; Callard et al., 1980; Schwab et al., 1989). Immune impairment accompanying aging is mainly due to thymus involution (Pawelec and Solana, 1997). Only few studies have compared the efficiency of cancer immunotherapy as a function of age and contradictory results have been reported. Of these few studies, most have shown a lower capacity of immunotherapy to inhibit tumour growth in old as compared to young animals (Urban and Schreiber, 1984; Simova et al., 1989; Miller et al., 1991; Dunn and North, 1991a,b; Dussault and Miller, 1995; Watanabe, 1996). However, some other studies found either similar effects (Ho et al., 1990) or, like us (Kaptzan et al., 2004 and data presented here), a higher inhibition of tumours in old than in those of young mice (Strausser and Rosenstein, 1979; Goldstein et al., 1986). We suggest that the apparently contradictory results of cancer immunotherapy in old as compared to young mice can possibly be explained by the fact that not all host defense systems decline with increasing age. In addition, certain depletions (of T suppressor cells, of regulatory T cells) may have a positive effect on the immune defense against tumours by avoiding tumour enhancement. While very numerous studies have demonstrated decreased T cell functions during aging, the function of nonspecific immune cells (polymorphonuclear leukocytes (PMN), macrophages (Mf) and NK cells) was found to be more preserved in later life (Provinciali et al., 1998; Takiguchi et al., 2002). No differences were found in LAK cell number and activity between old and young healthy humans (Provinciali et al., 1995; Kawakami and Bloom, 1987). NK cells actually increased in number during human aging, although their activity decreased in the elderly (Facchini et al., 1987; Mariani et al., 1994). Recently various specific functions of the cells comprised in the innate immune system were shown to be reduced by age (Solana et al., 2006). However, these modifications seem to be only moderate (Provinciali and Smorlesi, 2005). Macrophages have been shown to be important in the defense against B16 melanoma (Garcia-Hernandez et al., 2002) and in the malignant behaviour of AKR lymphoma (Kaptzan et al., 2000). The increased number of macrophages that we found in old as compared to young mice might possibly be involved in the increased efficiency of immunotherapy that we demonstrated in aged tumour-bearing mice. The stronger therapeutic effect of Levamisole in aged mice might be due to an increased macrophage-mediated anti-tumoural effect. The more or less accepted dogma pretends that the immunosenescence-accompanying aging should reduce the efficiency of immunotherapy in the old. This was indeed reported in various

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tumour systems as mentioned above. We suppose, however, that this must not be the case for all tumour systems and with all treatment modalities and conditions. Indeed several studies showed either no difference (Ho et al., 1990) or indeed a higher efficiency of immunotherapy in old as compared to young mice, at least in certain conditions (Goldstein et al., 1986; Kaptzan et al., 2004). More and more data confirm that the immune characteristics of the elderly are not the expression of a deficit but rather an adjustment to a modified homeostasis. Natural immunity is often conserved or even upregulated with age (Franceschi et al., 2000). It has been suggested that the reshaping of the immune response during aging could actually play a major role in the decreased susceptibility to cancer of very old people (Bonafe et al., 2002). It has been proposed that, paradoxically, immunosenescence might create an environment unfavorable for tumour growth in centenarians (Migliore and Coppede, 2002). The fact that immunotherapy (or, for that matter, any therapy) may be more effective against tumours growing in old than in young organisms might seem counterintuitive. However, since we used treatments based on the lower malignancy of tumours in the old, which implies (as we have shown) that they are more prone to apoptosis, less prone to angiogenesis and more sensitive to macrophages, a rational (though counterintuitive) design, we anticipated, may result in a higher therapeutic effect on the more indolent tumours of old mice than on those more aggressive of young animals. Since most cancer patients are old, it is important to try to find suitable treatment modalities particularly for this population. It is with this idea in mind that we started this series of studies. These studies lead us however to the possibility of envisaging treatment of young cancer patients with those agents which induce an ageing microenvironment which favours, in certain tumours, a reduced aggressive behaviour. Even if such treatments will be less efficient in young than in old patients, they may still contribute to cancer treatment efficacy since they act according to a new different reasoning than that of the usual treatments. The title of one of the articles of Hayflick was: ‘‘Ageing is not a disease’’ (Hayflick, 1998). According to our results, ageing may even be a remedy! References Anisimov, V.N., 2006. Effect of host age on tumor growth rate in rodents. Front Biosci. 11, 412–422. Balducci, L., 2007. Aging, frailty, and chemotherapy. Cancer Control 14, 7–12. Balducci, L., Extermann, M., 2000. Cancer and aging. An evolving panorama. Hematol. Oncol. Clin. North Am. 14, 1–16. Balducci, L., Lyman, G.H., 1997. Cancer in the elderly. Epidemiologic and clinical implications. Clin. Geriatr. Med. 13, 1–14. Baumgart, J., Zhukovskaya, N.V., Anisimov, V.N., 1988. Carcinogenesis and aging.VIII. Effect of host age on tumour growth, metastatic potential, and chemotherapeutic sensitivity to 1.4 benzoquinone-guaninehydrazonethiosemicarbazone (ambazone) and 5-fluorouracil in mice and rats. Exp. Pathol. 33, 239–248. Bonafe, M., Barbi, C., Storci, G., Salvioli, S., Capri, M., Olivieri, F., Valensin, S., Monti, D., Gonos, E.S., De Benedictis, G., Franceschi, C., 2002. What studies on human longevity tell us about the risk for cancer in the oldest old: data and hypotheses on the genetics and immunology of centenarians. Exp. Gerontol. 37, 1263–1271. Calabrese, C.T., Adam, Y.G., Volk, H., 1973. Geriatric colon cancer. Am. J. Surg. 125, 181–184. Callard, R.E., Fazekas, d.S.G., Basten, A., McKenzie, I.F., 1980. Immune function in aged mice. V. Role of suppressor cells. J. Immunol. 124, 52–58. Cameron, I.L., 1972. Cell proliferation and renewal in aging mice. J. Gerontol. 27, 162–172. Chahal, H.S., Drake, W.M., 2007. The endocrine system and aging. J. Pathol. 211, 173–180. Cinamon, U., Hier, M.P., Black, M.J., 2006. Age as a prognostic factor for head and neck squamous cell carcinoma: should older patients be treated differently? J. Otolaryngology 35, 8–12. Distelhorst, C.W., Dubyak, G., 1998. Role of calcium in glucocorticosteroidinduced apoptosis of thymocytes and lymphoma cells: resurrection of old theories by new findings. Blood 91, 731–734.

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