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Cancer Gene Therapy (2005) 12, 54–60 All rights reserved 0929-1903/05 $30.00

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Growth hormone releasing hormone plasmid supplementation, a potential treatment for cancer cachexia, does not increase tumor growth in nude mice Amir S Khan,1 Louis C Smith,1 Ingrid W Anscombe,1 Kathleen K Cummings,1 Melissa A Pope,1 and Ruxandra Draghia-Akli1,2 1

ADViSYS, Inc., The Woodlands, Texas 77381, USA; and 2Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

Growth hormone releasing hormone (GHRH) is known to have multiple anabolic effects and immune-stimulatory effects. Previous studies suggest that treatment with anabolic hormones also has the potential to mitigate the deleterious effects of cancer cachexia in animals. We studied the effects of plasmid-mediated GHRH supplementation on tumor growth and the role of antitumor immune cells with two different human tumor cell lines, NCI-H358 human bronchioalveolar carcinoma and MDA-MB-468 human breast adenocarcinoma, subcutaneously implanted in nude mice. GHRH supplementation by delivery of human GHRH from a musclespecific GHRH expression plasmid did not increase tumor progression in tumor-bearing nude mice. Male animals implanted with the NCI-H358 tumor cell line and treated with the GHRH-expressing plasmid exhibited a 40% decrease in the size of the tumors (Po.02), a 48% increase in white blood cells (Po.025) and a 300% increase in monocyte count (Po.0001), as well as an increase in the frequency of activated CD3 þ and CD4 þ cells in the tumors, compared to tumors of control animals. No adverse effects were observed in animals that received the GHRH-plasmid treatment. The present study shows that physiological stimulation of the GHRH–GH–IGF-I axis in mice with cancer does not promote tumor growth and may provide a viable treatment for cancer cachexia in humans. Cancer Gene Therapy (2005) 12, 54–60. doi:10.1038/sj.cgt.7700767 Published online 17 September 2004 Keywords: plasmid; GHRH; cachexia; electroporation; muscle

hree of the hormones largely responsible for postnatal T growth in animals and humans include growth hormone releasing hormone (GHRH), which stimulates growth hormone (GH) production and secretion from the anterior pituitary,1 and insulin-like growth factor-I (IGFI) that is responsible for many of the indirect effects of GH.2 The effects of these hormones on development, growth, metabolism and regeneration have been widely documented.3,4 Recent studies in different animal models and humans have also shown that GHRH has immunestimulatory effects, both through stimulation of the GH axis and direct actions as an immune-modulator.5 Conflicting data exist regarding the role of the GHRH– GH–IGF-I axis in tumorigenesis and cancer-associated pathology. Some studies have suggested that carcinogenesis is dependent upon critical plasma levels of GH and IGF-I.6 GH- or IGF-I-deficient animals are resistant to chemically induced carcinogenesis.7 Circulating IGF-I Received April 9, 2004.

Address correspondence and reprint requests to: Dr Ruxandra Draghia-Akli, MD, PhD, Vice President of Research, ADViSYS, Inc., 2700 Research Forest Drive, Suite 180, The Woodlands, TX 77381, USA. E-mail: [email protected] or [email protected]

levels play an important role in tumor development and metastasis8 and IGF-II mRNA levels are increased in some tumor lines.9 By contrast, other studies have failed to demonstrate an effect of GH on cancer development10 or have concluded that GH may actually improve the efficacy of cancer chemotherapy.11 Preclinical studies in rodents have suggested that anabolic hormones, such as GH and IGF-I, may reverse the catabolic state associated with cachexia, one of the major complications of cancer and cancer therapies,12 as well as inhibit metastases in tumor-bearing animals.13,14 In this study, the use of species-specific GHRH was not necessary. Numerous studies have shown that GHRH of different mammalian origin or GHRH analogs exert similar effects in species such as dogs, pigs, cattle and rodents.15,16 In a previous study in immunocompetent mice with implanted LL-2 adenocarcinoma cell line, we showed that stimulation of the GH axis by intramuscular delivery of a GHRH plasmid increased serum IGF-I concentrations by 13% (an indicator of GHRH activity), decreased growth of the tumor by 20% in males and 11% in females, attenuated tumor metastases by up to 57%, and prevented muscle atrophy. These results suggest a role for plasmid-mediated GHRH therapy in reversing the catabolic processes associated with cancer cachexia.17

GHRH does not increase tumor growth in nude mice AS Khan et al

Similar results were obtained in dogs with spontaneous malignancies.18 Long-term evaluation of the cancerafflicted dogs that received plasmid-mediated GHRH supplementation showed a significant improvement in hematological parameters and quality of life.19 Clinical use of GHRH-expressing plasmids for cancer cachexia, however, requires that ectopic GHRH expression does not upregulate tumor growth or increase tumor progression. In lieu of a constitutively active system of GHRH delivery, regulated expression of GHRH may be required under some circumstances. A mifepristone (MFP)-inducible plasmid vector system was used in this case.20 An earlier version of this MFP-inducible GHRH system has been shown to increase serum IGF-I, lean body mass, body weight, and bone mineral density in SCID mice.21 In the present study on nude mice with implanted NCIH358 human bronchioalveolar carcinoma22 or MDAMB-468 breast adenocarcinoma cells,23 we tested the hypothesis that physiologic stimulation of the GHRHGH-IGF-I axis through a plasmid-based GHRH expression system does not stimulate tumor growth in immunedeficient animals, and thus may prove beneficial as a treatment for cancer cachexia.

Materials and methods

Cell culture NCI-H358 cells (ATCC CRL-5807) and MDA-MB-468 cells (ATCC HTB-132) were obtained from ATCC (Manassas, VA) and stored in liquid nitrogen. Cells were rapidly thawed and plated in DMEM media (GIBCO, Grand Island, NY) with 10% fetal bovine serum (GIBCO, Grand Island, NY) and 1% penicillin/streptomycin. Cells were grown to 80% confluence, removed by adding 0.1% trypsin-EDTA (Gibco), centrifuged and resuspended in 1 PBS. Cells were counted before implantation.

DNA constructs The plasmid pSPc5-12 contained a 360 bp SacI/BamHI fragment of the SPc5-12 synthetic promoter.24 To generate pSP-hGHRH(1-40), the human GHRH cDNA was modified by site-directed mutagenesis of human (144)OH GHRH cDNA, and cloned into the BamHI/Hind III sites of pSP-GHRH, followed by the 30 untranslated region and poly(A) signal of hGH gene.25 The GHRHinducible (IS) is a two-plasmid system. Plasmid pGS1633 codes for GeneSwitch regulatory protein, version 4.0 and is controlled by a muscle-specific promoter (Valentis, Burlingame, CA). Plasmid pGHRH(1-40) is an inducible human GHRH plasmid. The GeneSwitch and inducible GHRH plasmids were mixed to generate a 1:10 mol/mol solution.21 Control plasmid, pSP-b-gal, contained the Escherichia. coli b-galactosidase gene under control of the same muscle-specific promoter. Plasmids were grown in E. coli DH5a, (GIBCO, Grand Island, NY). Endotoxinfree plasmid (Qiagen Inc., Chatsworth, CA) preparations

were diluted to 0.8 mg/mL in sterile water and stored at 801C prior to use. Mifepristone (Sigma, St. Louis, MO) was diluted in sesame oil and 360 mg/kg was administered by gavage three times a week starting at Day 7.

Animals Hsd:Athymic Nude-nu mice were obtained from Harlan (Indianapolis, IN) or Charles River (Raleigh, NC) and allowed to acclimate for 2 weeks. Rodents were housed five per cage on Tek-Fresh Autoclavable Bedding (Harlan) and given ad libitum access to food (Autoclavable Rodent Lab Diet 5010, Harlan) and water (reverseosmosis, UV-treated, sterile-filtered from municipal water supply). No known contaminants that would interfere with the outcome of the study were present in feed or water. Animals were housed at 22731C, with a relative humidity of range between 30 and 80% on a 12- hour light/dark cycle.

Injection, electroporation, and experimental procedure Animal groups were: Group 1 (constitutive pSP-GHRH), Group 2 (control pSP-bgal), Group 3 (GHRH-inducible system (GHRH-IS), no MFP), Group 4 (GHRH-IS, MFP), Group 5 (control pSP-bgal, MFP), and Group 6 (no plasmid). All animals received a pre-experiment physical examination by a registered veterinary technician prior to selection for testing. At Day 7, all animals were weighed, bled, and randomly assigned to one of six groups (n ¼ 20/group, 10 of each sex). On Day 1, animals were weighed, bled, and injected subcutaneously in the flank with tumor cells in 30 mL PBS. Nude mice received either 2 107 NCI-H358 cells or 1 107 MDAMB-468 cells. Mice were anesthetized on Day 0 with 0.5– 0.7mL/kg of a combination anesthetic: ketamine (42.8 mg/mL), xylazine (8.2 mg/mL) and acepromazine (0.7 mg/mL). A measure of 25 mL of the thawed plasmid stock was injected into the lateral gastrocnemius muscle using 3/10 cm3 syringe with 26-gauge needle. At 2 minutes after injection, the injected muscle was electroporated (3 pulses, 150 V/cm, 50 milliseconds) with a BTX ECM 830 electroporator and two-needle electrodes (BTX, San Diego, CA), as described.26 Animals were weighed and bled once a week, while tumor volume was evaluated twice a week using Promax NSK Electronic Digital Calipers (Fred Fowler Co., Newton, MA) and Gage Wedge for Sylvac Measuring Tools software (TAL Technologies, Philadelphia, PA). Length, width, and depth of the tumors were separately measured and then used to calculate tumor volume.

Necropsy and histopathology Animals were weighed and then euthanized by CO2 inhalation on Days 42–43 (NCI-H358 mice) and Days 33– 34 (MDA-MB-468 mice). Organs (lungs, heart, liver, spleen, kidneys, and the injected gastrocnemius) were excised, weighed and checked for gross pathologies. Gastrocnemius and any of the excised organs with macroscopic abnormalities were fixed in 10% buffered

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formalin overnight, washed in PBS, and transferred to 70% ethanol for storage. Tumor was also excised, weighed, fixed in 10% buffered formalin overnight, and stored in 70% ethanol. For MDA-MB-468 males, a complete histopathological examination was performed on internal organs (brain, heart, lung, liver, spleen, and kidneys), injected muscle, and tumor (IDEXX Laboratories, Inc., West Sacramento, CA). The tissues were paraffin-embedded, sectioned at 4–5 mm, stained with hematoxylin/eosin and examined microscopically by IDEXX Laboratories. An independent licensed veterinary pathologist read slides of each organ, including tumors of each animal and data were recorded. Tissue from NCIH358 mice was paraffin embedded, sectioned at 4–5 mm, stained with hematoxylin/eosin and examined microscopically for micrometastases.

transferase), creatinine kinase, albumin, total protein, bilirubin, cholesterol, glucose, calcium, phosphorous, bicarbonate, chloride, potassium, and sodium.

IGF-I radioimmunoassay Serum was aliquoted for serum IGF-I measurement using a mouse-specific IGF-I kit (Diagnostic Systems Laboratories, Inc., Webster, TX). The intra-assay variability was 6.6% for NCI-H358 males and 5.0% for NCI-H358 females.

Statistical analysis A Microsoft Excel statistics analysis package was used. The mean values were compared with paired t-test or ANOVA with subsequent Student’s t-test as post hoc test. Po.05 was taken as the level of statistical significance.

CD3 and CD4 immunohistochemistry Tumor sections were selected from NCI-H358 male animals from Groups 1 and 2. Sections were deparaffinized and subsequently washed in PBS. Slides were stained using goat ABC staining system (Santa Cruz Biotechnology, Santa Cruz, CA) following the manufacturer’s instructions with slight modifications. Briefly, the sections were first incubated in 0.03% hydrogen peroxide in methanol solution to block endogenous peroxidases, then incubated in the blocking solution (1.5% donkey serum (Santa Cruz Biotechnology, Santa Cruz, CA) in PBS), and finally, incubated overnight at 41C in the primary antibody, CD3-e0 (M-20) or CD-4 (C-18) (Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1:2000 (CD3-e0 ) and 1:1500 (CD-4) in blocking solution. After PBS washes, the secondary antibody was applied for 30 minutes at room temperature and slides were incubated in ABC solution for 30 minutes, as per kit instructions. Slides were washed in PBS between each step of the procedure. Peroxidase activity was revealed using diaminobenzidine (DAB) as substrate (Vector Laboratories, Burlingame, CA) for 4 minutes. The stained sections were visualized on an Olympuss BX51 microscope (Leeds Instruments, Irving, TX) with a 20 objective, and digital images of the sections were captured using an Optronics MagnaFire digital color camera with the MagnaFire 2.0 software (Optronics, Goleta, CA). The observer was blinded to the treatment groups and counted a random set of 9–18 fields per group. The within-animal average was corrected for tumor volume, and then used to calculate an average of the CD3 þ and CD4 þ cell counts/ tumor volume per group.

CBC and biochemistries At necropsy, whole blood was collected in Microtainer Brand tubes with EDTA (Becton Dickinson, Franklin Lakes, NJ) for CBC analysis and in Microtainer Serum Separator tubes (Becton Dickinson, Franklin Lakes, NJ) for serum biochemistries. All tests were performed by IDEXX Contract Research Services (West Sacramento, CA). Parameters tested in the biochemical analysis were: ALT (alanine aminotransferase), AST (aspartate amino-

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Results

Body weight No significant differences in body weight were observed among groups during the treatment period.

Tumor volume As an initial observation, tumor growth rate was higher for the NCI-H358 males (Fig 1a) than for the females (Fig 1b). In NCI-H358 males, animals treated with the constitutive GHRH plasmid exhibited a 40% decline in tumor volume by Day 40 (Po.02), compared to bgalactosidase (pSP-b-gal) plasmid controls. In NCI-H358 animals, the females treated with the constitutive GHRH plasmid exhibited a 33% decrease in tumor volume by Day 40 (P ¼ .14), compared to pSP-b-gal plasmid controls and 37% decline (P ¼ .15) in final tumor weight at necropsy. Male and female mice that received the GHRHIS activated at 7 days after tumor implantation, had a 23% reduction in tumor growth (Po.05 for males, and P ¼ .15 for females, due to high variance within the groups). Tumor volumes in MDA-MB-468 males were not statistically significantly different (data not shown) among groups. Female animals implanted with MDAMB-468 were removed from analysis due to abnormalities in growth rate of MDA-MB-468 cells in culture and, unlike other experiments, had inconsistent tumor development.

CBC and serum biochemistry In the NCI-H358 male mice treated with the constitutively active GHRH plasmid, white blood count (WBC) was increased 48.8% (Po.025) (Fig 2a) and percent monocytes were increased 300% (Po.00005) (Fig 2b) compared to pSP-b-gal plasmid controls. In MDA-MB-468 males treated with the constitutively active GHRH plasmid, lymphocyte count was increased by 20% (Po.045) relative to pSP-b-gal plasmid controls (Fig 2c). In the NCI-H358 female mice, with overall smaller tumors and tumor growth rates, no biologically or


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Figure 1 (a) Tumor growth as measured by tumor volume in MALE mice implanted with NCI-H358 human bronchioalveolar carcinoma cells. Tumors were measurable from Day 5 of the experiments, and tumor volume measurements were taken until Day 40. (b) Tumor growth as measured by tumor volume in Groups 1 and 2 of the FEMALE mice implanted with NCI-H358 human bronchioalveolar carcinoma cells. Tumors were measurable from Day 4 of the experiments, and tumor volume measurements were taken until Day 40. *Po.02.

statistically significant changes were found in the CBC or biochemistry values.

Necropsy Tumor weights at necropsy were 45% smaller (P ¼ .017) in Group 1 relative to Group 2 in NCI-H358 male animals (Fig 3a), and were 37% smaller in the NCI-H358 females, although this did not attain statistical significance due to individual variation of tumor size (P ¼ .15) (Fig 3b). Tumor weights were not significantly different between Groups 1 and 2 in the MDA-MBA-468 animals. No biologically significant changes were found in the mean necropsy organ weights (mean organ weight/mean body weight) of male or female mice implanted with either cell line.

Histopathology A complete histopathological examination was performed on internal organs (brain, heart, lung, liver, spleen, and kidneys), injected muscle and tumor of MDA-MBA-468 animals killed at Days 41–42. Analysis showed similar

Figure 2 CBC values in tumor-bearing mice either treated with constitutively active GHRH plasmid (pSP-GHRH) or nontreated controls: (a) White blood cell counts (WBC) in NCI-H358 males, *Po.025 (b) monocyte percentage in NCI-H358 males, *Po.00005 and (c) lymphocyte percentage in MDA-MB-468 male mice, *Po.045.

tumors in all examined animals, with expansile and locally invasive nodular tumors composed of cords of neoplastic epithelial cells. There was no difference in tumor vascularization between groups. Only one micrometastasis was found, in the liver of a control animal. Extramedullary hematopoiesis was noted in the spleen of all animals. Two animals in the pSP-bgal plasmid group also exhibited lymphoid hyperplasia in the spleen. Focal areas of generally mild muscle atrophy were present

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Figure 4 Intratumoral CD3 þ and CD4 þ cell counts corrected for tumor volume in NCI-H358 male mice treated with constitutive GHRH plasmid, as compared to nontreated controls.

Figure 3 Tumor weights at necropsy (Day 42 in males, Day 43 in females) in (a) NCI-H358 male, *P ¼ .017 and in (b) NCI-H358 female mice.

in many animals across all groups. Macrophage infiltration was also found in most animals and did not appear to be treatment related. A survey of liver and lung sections from NCI-H358 mice did not reveal any apparent differences in the numbers of micrometastases.

Immunohistochemistry Tumors collected at necropsy (Days 41–42) were examined for tumor-infiltrating T cells. CD3 þ cell counts corrected for tumor volume were increased 66.7% (P ¼ .18) and CD4 þ cell counts corrected for tumor volume were increased 87.2% (P ¼ .15) in Group 1 animals versus Group 2 animals (Fig 4).

Serum IGF-I values Serum IGF-I levels, while increased in the GHRH-treated groups, were not significantly different among treatment groups for the duration of the experiments.

Discussion

NCI-H358 human bronchioalveolar carcinoma and MDA-MB-468 human breast adenocarcinoma cell lines

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have previously been used to investigate tumor progression in mice.27,28 This report demonstrates that plasmidmediated, physiologic GHRH supplementation did not increase tumor progression in tumor-bearing nude mice. Furthermore, male animals implanted with the NCI-H358 tumor cell line and treated with a GHRH-expressing plasmid showed a decrease in tumor volume, increased white blood cells and monocyte count, as well as increased intratumoral activated CD3 þ and CD4 þ cells, as compared to control animals. Finally, this work confirms earlier work in immunocompetent animals17 and suggests that the mechanism responsible for tumor growth reduction involves immune stimulation. These results provide support for the use of GHRH-expressing plasmids in the treatment of cancer cachexia. The present data confirm the results of a previous study that used LL-2 adenocarcinoma cell line in immunocompetent mice.17 In both studies, pSP-GHRH-treated male mice exhibited the highest decrease in tumor volume at the end of the study compared to controls. In the present study, tumor weights at necropsy were smaller in pSPGHRH-treated animals as compared to animals that received GHRH-IS inducible system that was activated at 7 days after tumor implantation. Thus, during tumorigenesis, it is preferable to increase circulating GHRH levels early rather than late for therapeutic success. Body weight was not changed in any of the experiments, confirming that at lower doses the direct effects of GHRH on tissues may be more important than the effects mediated through the GH axis. It is known that IGF-I is highly dependent on metabolic state, and substantially decreased in subjects with cancer cachexia.29 Importantly, the nude mice in the present experiment maintained circulating IGF-I concentrations within physiologic limits. As seen in GH-deficient patients administered physiologic doses of GH,30 the maintenance of normal IGF-I levels results in improved clinical outcome, while the adverse effects of IGF-I overstimulation are avoided. Sexual dimorphism in the neuroendocrine regulation of the GH axis in nude mice is largely unknown. In humans, a gender dissociation within the GH/IGF-I axis is evident in protracted critical illness, with men showing greater


loss of pulsatility and regularity within the GH secretory pattern than women (despite indistinguishable total GH output) and concomitantly lower IGF-I levels;31 as a clinical consequence, females appear to be protected against, at least in part, adverse outcome from prolonged critical illness. In the present study, we found that certain tumors develop at a slower rate in female compared to male mice, remarkably consistent with our previously published study.17 Additional studies are required to elucidate the mechanism of sexual dimorphism of the GHRH axis on tumor progression. Enhancement of immune function is also one of the possible mechanisms of the decline in tumor growth observed in the present study. A substantial body of research exists to support the production of GHRH, GH and IGF-I by cells of the immune system.32 In the present study, tumor-bearing male mice treated with GHRH plasmid demonstrated significant increases in WBC, monocytes, and lymphocytes, confirming a similar observation in dogs with late-stage malignancy.18 Furthermore, the numbers of intratumoral CD3 þ and CD4 þ cells were higher in male NCI-H358 tumor-bearing animals treated with GHRH plasmid compared to controls, which could reflect an immune response against the tumor itself.33 The data support the presence of an antitumor immune function in athymic nude mice. Since nude mice lack a normal thymus, they are characterized by low numbers of mature T cells.34 These animals have measurable numbers of Thy-1 þ and CD8 þ and small numbers of CD4 þ T cells can be found. In addition, a second T-cell receptor (TCR), gamma delta-TCR, is expressed in the spleens of nude mice. Lake et al35 demonstrate the quantitative measurement of TCR expression by T cells that mature in athymic nude mice, and they suggest that the extrathymic environment, although inefficient, is nevertheless permissive for the maturation of alpha/beta and gamma/delta TCR-expressing T cells. Kennedy et al36 provide further evidence of extrathymic T-cell maturation and that nude mice accrue increasing numbers of lymphocytes bearing Thy-1, CD3, CD4, and CD8 with age. Radzikowski et al37 have reported that splenocytes from young immunodeficient mice retain their cytotoxic immune response to a challenge. This suggests a possible mechanism for the antitumor response demonstrated by the mice injected with GHRH-expressing plasmid in this study. Schally and colleagues have introduced evidence that GHRH antagonists may inhibit tumor growth,38 through specific splice-variant GHRH receptors on tumor cells.39 Synthetic GHRH antagonists do not inhibit the bioactivity of endogenous GHRH, but act primarily through the inhibition of autocrine production of growth factors in tumor cells.40,41 Thus, an endocrine stimulation of the GHRH axis that results in augmentation of immune function will likely benefit affected patients, while not stimulating malignant cell growth. GHRH expression does not increase tumor growth in immunodeficient, tumor-bearing mice. The benefits of plasmid-mediated GHRH treatment may be related to increased immune activity against the tumor in addition

to anabolic effects on the tumor-bearing animal. An improved metabolic status of a patient in advanced stages of cancer may increase the likelihood of the patient surviving the radiotherapy and/or chemotherapy cancer treatment. The findings in the present study suggest that physiological stimulation of the GHRH-GH-IGF-I axis in patients with cancer will likely enhance the quality of life and may increase survival time.

Acknowledgments

We particularly thank Dr Malcolm Brenner and the Center for Cell and Gene Therapy for continuous support and useful discussions. We also thank Dr Jeff Nordstrom and Ms Catherine Tone for the editorial correction of this manuscript. We acknowledge support for this study from ADViSYS, Inc. (The Woodlands, TX).

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