- Email: [email protected]
Creation and characterization of a xenograft model for human cervical cancer
Gynecologic Oncology 118 (2010) 76–80
Contents lists available at ScienceDirect
Gynecologic Oncology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y g y n o
Creation and characterization of a xenograft model for human cervical cancer Corinna Hoffmann a, Christopher Bachran b, Jonas Stanke a, Sefer Elezkurtaj c, Andreas M. Kaufmann a, Hendrick Fuchs b, Christoph Loddenkemper c, Achim Schneider a, Günter Cichon a,⁎ a b c
Charité – Campus Benjamin Franklin, Dept. of Obstetrics and Gynecology, Hindenburgdamm 30, 12200 Berlin, Germany Charité – Campus Benjamin Franklin, Dept. of Biochemistry, Hindenburgdamm 30, 12200 Berlin, Germany Charité – Campus Benjamin Franklin, Dept. of Pathology, Hindenburgdamm 30, 12200 Berlin, Germany
a r t i c l e
i n f o
Article history: Received 4 August 2009 Available online 2 May 2010 Keywords: Cervical cancer Xenograft model Scid beige mice
a b s t r a c t Objective. Most of primary human cancer tissues show effective engraftment and proliferation after transplantation onto Scid mice. However xenotransplantation of vital specimens of cervical carcinoma has not been successful in the past, also the generation of cell lines from primary cervical cancer has hardly ever been possible. The lack of appropriate xenograft models impedes the search for improved specific therapeutic agents. Methods. We explored the efficiency of different techniques for tumor transplantation and describe the first protocol to enable reliable and efficient engraftment of human cervical cancer in Scid beige mice. To demonstrate the value of this tumor model, we explored the therapeutic potency of a novel immunotoxin (SA2E). SA2E is a chimeric protein constructed by fusing the human epidermal growth factor and the plant protein toxin saporin. Results. About 70% of transplanted tumors exhibited potent proliferation, and multiple retransplantation was possible in 40%. Local treatment with the immunotoxin SA2E had a dose dependent therapeutic effect and achieved a tumor volume reduction of up to 60%. Conclusions. Reliable engraftment and high reproducibility make this novel xenograft model an attractive test system to identify new therapeutic agents for cervical cancer. © 2010 Published by Elsevier Inc.
Introduction Cervical cancer is one of the most common cancers and a major cause of morbidity and mortality among women worldwide [1]. In 70% of the cases, it is associated with HPV 16 and 18 infections [2]. Most HPV-related cancers originate in the uterine cervix, although HPV is probably also a causal factor in some head and neck cancers as well as in a number of other anogenital cancers [3–5]. Despite surgical interventions and application of combined radio- chemotherapy 30% of the affected women die from cervical cancer. A rapidly growing number of novel oncotherapeutic agents like immunotoxins, therapeutic antibodies, cell-based immunotherapies and chemotherapies are reaching the market, but exploring their value for treating single tumor entities like cervical cancer requires reliable and clinically relevant in vivo test systems [6–9]. Xenograft models are already available for most of the common human malignancies like breast cancer [10], leukemia [11–13], colorectal cancer [14,15], head and neck cancer [16], prostate cancer [17,18] and ovarian cancer [19,20]. However, a xenograft model for cervical cancer could not be created until now. The small number of established tumorigenic cervical
⁎ Corresponding author. E-mail address: [email protected] (G. Cichon). 0090-8258/$ – see front matter © 2010 Published by Elsevier Inc. doi:10.1016/j.ygyno.2010.03.019
cancer lines does not meet the criteria. Some of them, like Hela or CaSki, were established decades ago. Thus an extended passage number and altered expression patterns reduce their value for preclinical testing. This study provides a protocol designed to ensure reliable and efficient transplantation and proliferation of human cervical cancer tissue in Scid beige mice. We show that tumor cells retain their expression patterns after retransplantation, and we demonstrate the therapeutic effect of a newly developed immunotoxin in this xenograft model. Material and methods Transplantation of primary human cervical cancer tissue Eight- to twelve-week-old female Scid beige mice (C.B-17/lcrHsdPrkcdscid Lystbg) were purchased from Taconic (Sweden). Mice were kept under specific-pathogen-free (SPF) conditions. Following surgery, freshly isolated cervical cancer tissue was transferred into transport medium (RPMI) and stored at room temperature. Within 2–5 h after tumor isolation, the tissue was minced with small scissors in PBS under sterile conditions. The tumor cell suspension was then transplanted by syringe (18-gauge needle) in a volume of 200 μl. It was subcutaneously inoculated into the left dorsal region of mice. The xenotransplanted
C. Hoffmann et al. / Gynecologic Oncology 118 (2010) 76–80
tumors were passaged after reaching a volume of 1 cm³. The volume was measured by a caliper. For xenograft retransplantation, mice were killed by isofluorane inhalation, and the tissue was isolated and applied again as described above. These studies were approved by the local Ethics Committee and the Animal Welfare Committee (Landesamt für Tierschutz – State Bureau of Animal Protection). Monitoring tumor engraftment Survival, general performance, and tumor growth were monitored twice a week. Engraftment was considered successful when tumor tissue was macroscopically detectable. Tumors were collected after reaching a volume of 1 cm. They were snap-frozen in liquid nitrogen or preserved in 5% formamide for histological analysis. Liver, spleen, kidney, heart and lung tissues were also preserved in 5% formamide for further analysis. HPV typing of tumor tissue HPV typing of primary tumors as well as xenotransplanted and passaged tumor tissue was determined by bead-based multiplex genotyping [21]. DNA was extracted from cryostat sections by the QIamp DNA-extraction kit (Qiagen, Germany) as described in the customer instruction manual. Sample DNA was subjected to GP5+/6+PCR as previously described [21]. Briefly, amplification of HPV L1 fragment was performed in 50 μl PCR mixture containing 50 mM KCl, 0.8 g/liter Nonidet P40, 10 mM Tris–HCl, pH 8.8 (MBI Fermentas GmbH, St. Leon Roth, Germany), 200 μM of each deoxynucleoside triphosphate, 3.5 mM MgCl2, 1 U of DNA AmpliTaq polymerase (Roche Applied Biosystems, Mannheim, Germany), and 25 pmol each of the GP5+ (5´-TTT GTT ACT GTG GTA GAT ACT AC-3´) and 5´-biotinylated GP6+ (5´-GAA AAA TAA ACT GTA AAT CAT ATT C-3´) primers (MWGBiotech AG, Ebersberg, Germany). A 4-min denaturation step at 94 °C was followed by 40 cycles of amplification with a PCR thermocycler (Gene Amp PCR System 2400; Perkin-Elmer, Wellesley, MA). Each cycle included a denaturation step at 94 °C for 20 s, an annealing step at 38 °C for 30 s, and an elongation step at 71 °C for 80 s. The final elongation step was prolonged for a further 4 min. Following PCR amplification, HPV types were determined in a bead based hybridisation assay. A volume of 10 μl of each reaction mixture was transferred to 96-well plates (96-well PCR thermo polystyrene plates; Costar, Wiesbaden, Germany) containing 33 μl of tetramethylammonium chloride (TMAC) hybridization solution (0.15 M TMAC, 75 mM Tris– HCl, 6 mM EDTA, 1.5 g/liter Sarkosyl, pH 8.0), a mixture of 2,000 probe-coupled beads of HPV16, HPV18 and HPV33 set and 7.0 μl TE buffer. Hybridization was performed at 41 °C for 30 min in a thermomixer (Eppendorf, Hamburg, Germany). Beads were washed and resuspended for 20 min in 75 μl of streptavidin-R-phycoerythrin (Strep-PE; Molecular Probes, Eugene, OR) diluted 1:1,600 in 2.0 M TMAC, 75 mM Tris–HCl, 6 m MEDTA, 1.5 g/liter Sarkosyl, 1.0 g/liter casein, pH 8.0. After a final washing step and block beads were analyzed for internal bead color and Rphycoerythrin reporter fluorescence on a Luminex 100 analyzer. The median reporter fluorescence intensity (MFI) of at least 100 beads was computed for each bead set in the sample [21]. Immunohistochemical analysis Formalin-embedded or cryostat sections from primary and xenotransplanted tumor tissue were mounted on glass slides, fixed in acetone and air-dried. The following antibodies were used for tissue staining: EGF Pharm Dx (Code K1494, DakoCytomation), p16INK4a (CINtec Histology Kit), HLA-ABC (DakoCytomation), HLA-DP, DQ, DR (Clone CR3/43, DakoCytomation), and anti-human Ki-67 (DakoCytomation). anti-HPV16 E7 (was kindly provided by Pidder Jansen-Dürr; Institute for Biomedical Ageing Research; Austrian Academy of
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Sciences; Rennweg 10, 6020 Innsbruck, Austria). Staining was performed as described in the customer instruction manual. Treatment with SAE2 immunotoxin Twenty female Scid beige mice were retransplanted with comparable amounts of minced tumor tissue. Two weeks later when tumors had grown to a volume of 125 mm³, 4 groups of animals each consisting 5 mice were set up (n = 5). Animals in the control group received 6 subcutaneous injections of saporin alone (15 μg in 20 μl PBS) in adjacent to the tumor every third day. The other three groups were treated at the same intervals with 6 injections of 5, 10 or 15 μg of SAE2. Statistical analysis Mean values of at least 5 tumors per group were used to calculate standard deviations. Results Development of a transplantation protocol In initial trials, freshly operated solid tumor pieces were transplanted as solid 3-5 mm cubes. This technique required anesthesia, shaving, skin incision and subsequent suturing. The outcome was unsatisfactory, since only a low percentage of tumor biopsies showed engraftment and active proliferation. Moreover, transplants from the same tumor differed substantially with regard to growth behavior, environmental influences, and blood vessel development, which made serial testing of therapeutic agents very difficult. Retransplantation of engrafted tumors regularly failed. In further attempts single cell suspensions of primary tumor tissues comprising additional supplements like matrigel or growth factors were applied subcutaneously. But again no engraftment was detectable (0/6). For these reasons we altered the protocol. In the next trial, tumor pieces were minced with scissors and aspirated into a syringe prior to transplantation. The tumor cell suspension was subcutaneously inoculated into the dorsal region of mice. This procedure was gentle and well tolerated by the animals. Moreover, it did not require anesthesia or skin surgery and thus had lower infection rates. Most importantly though, this approach led to successful engraftment and proliferation of more than 70% of transplanted tumors. Palpable or visible tumors appeared 6–8 weeks after transplantation, on the average. The tumors showed reproducible growth behavior, were encapsulated, and exhibited good vascularization. Xenotransplantation achieved highly efficient engraftment in 70 % (Table 1, Fig. 1). No differences in engraftment were observed
Table 1 General outcome. Carcinoma type
Transplant at
HPV-type
Engraftment
Passage
Adenocarcinoma
1 2 3 4 5 6 7 8 9 10
16 16 16 18 16 16 16 18 33 16
+ + + + + − − + − +
+ − − − + − − + − +
Squamocolumnar carcinoma
Neuroendocrine carcinoma
Successful engraftment of primary tumor transplants occurred in about 70% of attempts. Forty percent of tumors could be retransplanted. The time span for tumor engraftment and outgrowth of primary xenotransplants varied from 40 days to 5 months. Outgrowth was more rapid after retransplantation. On the average, xenografts reached a size of 1 cm3 40–60 days after retransplantation.
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xenotransplants occurred in a time span that varied from 40 days to 5 months. Only high-risk HPV types 16 and 18 were identified by HPV typing in xenotransplanted tumors. Tumor biopsy specimens were classified as adenocarcinomas, squamocolumnar carcinomas or neuroendocrine carcinomas. The established tumors were retransplanted to ensure preservation and proliferation of tissue. Retransplantation was feasible in 40% of transplanted tumors. Outgrowth of passaged xenotransplanted tumors with a volume of up to 1 cm³ occurred within 40 to 60 days. Early and late passage tumors developed within the same time span. The new animal model presented here enables efficient and reproducible engraftment and is easy to prepare. Tumor tissue retains its characteristics after xenotransplantation and passage Fig. 1. EGF receptor expression on human xenografts (5 days after transplantation) is restricted to human cervical cancer cells. Engrafted tumor tissue displays high mitotic activity (black arrows).
between squamous cell carcinomas and adenocarcinomas. Analysis of engrafted tumor tissue five days after transplantation revealed high mitotic activity (Fig. 1). Moreover, immunohistological staining showed HLA class I expression in strongly differentiated tumors (Fig. 2). The transplanted tissues displayed high infiltration rates of antigen-presenting cells five days after transplantation (Fig. 2). Tumor development was accompanied by tissue encapsulation and extensive vascularization (Fig. 1). Engraftment and outgrowth of primary
A prerequisite for creating an efficient replicative xenomodel is to ensure that the transplanted cervical cancer tissue retains its properties after multiple passages. Analysis of therapeutics requires an adequate amount of tumor tissue with the same characteristics. Retransplantation and multiple passages of xenotransplants have not been previously described. We were able to achieve replication of the outgrown cancer tissues. After efficient engraftment of primary tumors, xenotransplanted grown tissues were retransplanted. The xenotumors were treated as primary tumors. Outgrowth of retransplanted tumors occurred in 4 of 10 carcinomas (Table 1). Tumor markers such as EGF receptor and p16 were preserved after early and late tumor passages (Fig. 3). Human cancer tissue showed permanent and constant proliferation after multiple passages (Figs. 1 and 3). Moreover, the HPV type and HLA class I expression remained constant during passages (data not shown). The findings demonstrate that xenotransplanted tumor tissue retains its tumor characteristics after multiple passages in mice. This enables analysis of therapeutic agents in a broad range of experimental settings. Therapeutic treatment of transplanted tumor tissue
Fig. 2. Immunohistochemical staining for MHC class I and MHC class II complexes in cervical cancer xenografts 5 days after transplantation.
To evaluate the xenotransplantation model in a therapeutic setting, we analyzed the effect of a novel immunoprotein that targets the EGF receptor on the tumor cell surface. The therapeutic agent is SE, a chimeric toxin (CT) consisting of the epidermal growth factor and the plant protein toxin saporin from Saponaria officinalis [22–24]. This construct (SA2E) additionally contains a peptidic adapter that improves cytosolic uptake, mediates retention of the toxin in the cytosol, and detoxifies the drug after cell death. Cervical cancer xenografts were highly appropriate tumor models for analyzing immunoprotein properties. Results obtained by immunohistochemical staining of xenotumors have shown that EGF receptor as a target for SA2E is strongly and constantly expressed in xenotransplanted tissues (Figs. 1 and 3). In the experimental setting, a xenotransplanted tumor was allowed to expand in 20 mice. After the tumor volume had increased to 125 cm³, three groups of four mice each received different concentrations of SA2E. The last group served as a control group. SA2E was administered every third day for 6 times, starting on day 13 after tumor transplantation. The tumor volume of xenotransplanted mice was monitored for 45 days. Results of the treatment at day 30 are demonstrated in Fig. 4. Control mice showed rapid tumor outgrowth within 30 days (Fig. 4). Treatment with 5 and 10 μg dose of the immunoprotein decelerated tumor growth by about 20-30%, while treatment with 15 μg of SA2E reduced tumor growth on average by 5060% compared to the control group. The decelerating effect of SA2E on tumor was restricted to treatment periode and after discontinuation of SA2E applications tumors showed rapid outgrow (Fig. 4). We
C. Hoffmann et al. / Gynecologic Oncology 118 (2010) 76–80
Fig. 3. Expression of marker proteins on primary tumor tissue, primary transplant and re-transplants. (p.t.: post transplantation). 79
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C. Hoffmann et al. / Gynecologic Oncology 118 (2010) 76–80
Acknowledgment This study was supported by the Wilhelm Sander Stiftung (Munich, Germany). We thank Ursula Schulz for excellent technical assistance. References
Fig. 4. Effect of immunotoxin SA2E on tumor growth 30 days after tumor transplantation. A dose-dependent deceleration of tumor growth was seen in animals receiving 5 and 10 μg of SA2E, while in the highest dose group (15 μg) partial tumor regression (60% by volume) was noticed.
demonstrated the efficacy of this cervical cancer xenotransplantation model, which enables testing of agents for their effect on human primary cervical cancers. High engraftment efficiency, exact reproducibility, massive tumor expansion, and easy preparation make this newly created tumor model attractive for analysis of immunotherapeutics. Discussion In the presented xenograft model rates for successful engraftment were about 70%. Reasons for the failure of engraftment are not evident, but since they were not correlated to certain tumor subtypes, we assume that the vitality of the primary tissue was most likely responsible for this phenomenon. The vitality of primary tumor tissue is closely linked to duration of surgical procedures, individual differences in tumor localization, accessibility, tumor volume and rates of necrosis and varies between each trial. We have created a cervical cancer xenotransplantation model that allows analysis of human primary tumor growth, including infiltration of surrounding tissue and interaction of tumor cells with their stromal environment. It offers high engraftment efficiency, preservation of relevant tumor features, reproducible tumor growth, and massive tumor expansion. It thus enables testing and optimization of several tumor treatment modalities like cytostatic therapy, gene therapy or immunotherapy in a broad range of experimental settings. This transplantation method also has the advantage of minimal invasiveness and easy preparation, unlike xenotransplantation models described for other tumor entities [10,16,17,25]. Moreover, this model can be used to tailor therapies to individual patients. This means that patient-derived tumor tissue can be tested in the mouse xenotransplant model for the most effective treatment strategy. This in vivo model should enable monitoring of tumor cell differentiation and escape variants during chemotherapy or radiotherapy, which will facilitate the development of new treatment concepts and strategies. The utilization of multiply passaged tumor tissue will presumably simplify the establishment of new stable cancer cell lines for in vitro culturing. In further studies the model should be improved by confinement to a specific mouse strain. Patient-derived immune cells will thus be able to create a new human immune environment in the mice, which will enable the analysis of immunotherapies such as vaccines. This newly created tumor model is an attractive system for analyzing treatment strategies. Conflict of interest statement The authors declare that there are no financial conflicts of interest in regards to this work.
[1] Marshall K. Cervical dysplasia: early intervention. Altern Med Rev May 2003;8(2): 156–70. [2] Bosch FX, de Sanjose S. The epidemiology of human papillomavirus infection and cervical cancer. Dis Markers 2007;23(4):213–27. [3] Paavonen J. Human papillomavirus infection and the development of cervical cancer and related genital neoplasias. Int J Infect Dis Nov 2007;11(Suppl 2):S3–9. [4] Psyrri A, DiMaio D. Human papillomavirus in cervical and head-and-neck cancer. Nat Clin Pract Oncol 2008 Jan;5(1):24–31. [5] Ragin CC, Taioli E. Survival of squamous cell carcinoma of the head and neck in relation to human papillomavirus infection: review and meta-analysis. Int J Cancer Oct 15 2007;121(8):1813–20. [6] Bellone S, Pecorelli S, Cannon MJ, Santin AD. Advances in dendritic-cell-based therapeutic vaccines for cervical cancer. Expert Rev Anticancer Ther Oct 2007;7(10): 1473–86. [7] Hung CF, Ma B, Monie A, Tsen SW, Wu TC. Therapeutic human papillomavirus vaccines: current clinical trials and future directions. Expert Opin Biol Ther Apr 2008;8(4):421–39. [8] Kummel S, Thomas A, Jeschke S, Hauschild M, Sehouli J, Lichtenegger W, et al. Postoperative therapy modalities for cervical carcinoma. Anticancer Res Mar-Apr 2006;26(2C):1707–13. [9] Stanley M. Prevention strategies against the human papillomavirus: the effectiveness of vaccination. Gynecol Oncol Nov 2007;107(2 Suppl 1):S19–23. [10] Beckhove P, Schutz F, Diel IJ, Solomayer EF, Bastert G, Foerster J, et al. Efficient engraftment of human primary breast cancer transplants in nonconditioned NOD/ Scid mice. Int J Cancer Jul 1 2003;105(4):444–53. [11] Durig J, Ebeling P, Grabellus F, Sorg UR, Mollmann M, Schutt P, et al. A novel nonobese diabetic/severe combined immunodeficient xenograft model for chronic lymphocytic leukemia reflects important clinical characteristics of the disease. Cancer Res Sep 15 2007;67(18):8653–61. 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Combination of thalidomide and cisplatin in an head and neck squamous cell carcinomas model results in an enhanced antiangiogenic activity in vitro and in vivo. Int J Cancer Oct 15 2007;121(8):1697–704. [17] Patrawala L, Calhoun-Davis T, Schneider-Broussard R, Tang DG. Hierarchical organization of prostate cancer cells in xenograft tumors: the CD44+alpha2beta1+ cell population is enriched in tumor-initiating cells. Cancer Res Jul 15 2007;67(14): 6796–805. [18] Zhang AL, Russell PJ, Knittel T, Milross C. Paclitaxel enhanced radiation sensitization for the suppression of human prostate cancer tumor growth via a p53 independent pathway. Prostate Nov 1 2007;67(15):1630–40. [19] Chiriva-Internati M, Grizzi F, Weidanz JA, Ferrari R, Yuefei Y, Velez B, et al. A NOD/SCID tumor model for human ovarian cancer that allows tracking of tumor progression through the biomarker Sp17. J Immunol Methods Apr 10 2007;321(1–2):86–93. [20] Lee CH, Xue H, Sutcliffe M, Gout PW, Huntsman DG, Miller DM, et al. 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Contents lists available at ScienceDirect
Gynecologic Oncology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y g y n o
Creation and characterization of a xenograft model for human cervical cancer Corinna Hoffmann a, Christopher Bachran b, Jonas Stanke a, Sefer Elezkurtaj c, Andreas M. Kaufmann a, Hendrick Fuchs b, Christoph Loddenkemper c, Achim Schneider a, Günter Cichon a,⁎ a b c
Charité – Campus Benjamin Franklin, Dept. of Obstetrics and Gynecology, Hindenburgdamm 30, 12200 Berlin, Germany Charité – Campus Benjamin Franklin, Dept. of Biochemistry, Hindenburgdamm 30, 12200 Berlin, Germany Charité – Campus Benjamin Franklin, Dept. of Pathology, Hindenburgdamm 30, 12200 Berlin, Germany
a r t i c l e
i n f o
Article history: Received 4 August 2009 Available online 2 May 2010 Keywords: Cervical cancer Xenograft model Scid beige mice
a b s t r a c t Objective. Most of primary human cancer tissues show effective engraftment and proliferation after transplantation onto Scid mice. However xenotransplantation of vital specimens of cervical carcinoma has not been successful in the past, also the generation of cell lines from primary cervical cancer has hardly ever been possible. The lack of appropriate xenograft models impedes the search for improved specific therapeutic agents. Methods. We explored the efficiency of different techniques for tumor transplantation and describe the first protocol to enable reliable and efficient engraftment of human cervical cancer in Scid beige mice. To demonstrate the value of this tumor model, we explored the therapeutic potency of a novel immunotoxin (SA2E). SA2E is a chimeric protein constructed by fusing the human epidermal growth factor and the plant protein toxin saporin. Results. About 70% of transplanted tumors exhibited potent proliferation, and multiple retransplantation was possible in 40%. Local treatment with the immunotoxin SA2E had a dose dependent therapeutic effect and achieved a tumor volume reduction of up to 60%. Conclusions. Reliable engraftment and high reproducibility make this novel xenograft model an attractive test system to identify new therapeutic agents for cervical cancer. © 2010 Published by Elsevier Inc.
Introduction Cervical cancer is one of the most common cancers and a major cause of morbidity and mortality among women worldwide [1]. In 70% of the cases, it is associated with HPV 16 and 18 infections [2]. Most HPV-related cancers originate in the uterine cervix, although HPV is probably also a causal factor in some head and neck cancers as well as in a number of other anogenital cancers [3–5]. Despite surgical interventions and application of combined radio- chemotherapy 30% of the affected women die from cervical cancer. A rapidly growing number of novel oncotherapeutic agents like immunotoxins, therapeutic antibodies, cell-based immunotherapies and chemotherapies are reaching the market, but exploring their value for treating single tumor entities like cervical cancer requires reliable and clinically relevant in vivo test systems [6–9]. Xenograft models are already available for most of the common human malignancies like breast cancer [10], leukemia [11–13], colorectal cancer [14,15], head and neck cancer [16], prostate cancer [17,18] and ovarian cancer [19,20]. However, a xenograft model for cervical cancer could not be created until now. The small number of established tumorigenic cervical
⁎ Corresponding author. E-mail address: [email protected] (G. Cichon). 0090-8258/$ – see front matter © 2010 Published by Elsevier Inc. doi:10.1016/j.ygyno.2010.03.019
cancer lines does not meet the criteria. Some of them, like Hela or CaSki, were established decades ago. Thus an extended passage number and altered expression patterns reduce their value for preclinical testing. This study provides a protocol designed to ensure reliable and efficient transplantation and proliferation of human cervical cancer tissue in Scid beige mice. We show that tumor cells retain their expression patterns after retransplantation, and we demonstrate the therapeutic effect of a newly developed immunotoxin in this xenograft model. Material and methods Transplantation of primary human cervical cancer tissue Eight- to twelve-week-old female Scid beige mice (C.B-17/lcrHsdPrkcdscid Lystbg) were purchased from Taconic (Sweden). Mice were kept under specific-pathogen-free (SPF) conditions. Following surgery, freshly isolated cervical cancer tissue was transferred into transport medium (RPMI) and stored at room temperature. Within 2–5 h after tumor isolation, the tissue was minced with small scissors in PBS under sterile conditions. The tumor cell suspension was then transplanted by syringe (18-gauge needle) in a volume of 200 μl. It was subcutaneously inoculated into the left dorsal region of mice. The xenotransplanted
C. Hoffmann et al. / Gynecologic Oncology 118 (2010) 76–80
tumors were passaged after reaching a volume of 1 cm³. The volume was measured by a caliper. For xenograft retransplantation, mice were killed by isofluorane inhalation, and the tissue was isolated and applied again as described above. These studies were approved by the local Ethics Committee and the Animal Welfare Committee (Landesamt für Tierschutz – State Bureau of Animal Protection). Monitoring tumor engraftment Survival, general performance, and tumor growth were monitored twice a week. Engraftment was considered successful when tumor tissue was macroscopically detectable. Tumors were collected after reaching a volume of 1 cm. They were snap-frozen in liquid nitrogen or preserved in 5% formamide for histological analysis. Liver, spleen, kidney, heart and lung tissues were also preserved in 5% formamide for further analysis. HPV typing of tumor tissue HPV typing of primary tumors as well as xenotransplanted and passaged tumor tissue was determined by bead-based multiplex genotyping [21]. DNA was extracted from cryostat sections by the QIamp DNA-extraction kit (Qiagen, Germany) as described in the customer instruction manual. Sample DNA was subjected to GP5+/6+PCR as previously described [21]. Briefly, amplification of HPV L1 fragment was performed in 50 μl PCR mixture containing 50 mM KCl, 0.8 g/liter Nonidet P40, 10 mM Tris–HCl, pH 8.8 (MBI Fermentas GmbH, St. Leon Roth, Germany), 200 μM of each deoxynucleoside triphosphate, 3.5 mM MgCl2, 1 U of DNA AmpliTaq polymerase (Roche Applied Biosystems, Mannheim, Germany), and 25 pmol each of the GP5+ (5´-TTT GTT ACT GTG GTA GAT ACT AC-3´) and 5´-biotinylated GP6+ (5´-GAA AAA TAA ACT GTA AAT CAT ATT C-3´) primers (MWGBiotech AG, Ebersberg, Germany). A 4-min denaturation step at 94 °C was followed by 40 cycles of amplification with a PCR thermocycler (Gene Amp PCR System 2400; Perkin-Elmer, Wellesley, MA). Each cycle included a denaturation step at 94 °C for 20 s, an annealing step at 38 °C for 30 s, and an elongation step at 71 °C for 80 s. The final elongation step was prolonged for a further 4 min. Following PCR amplification, HPV types were determined in a bead based hybridisation assay. A volume of 10 μl of each reaction mixture was transferred to 96-well plates (96-well PCR thermo polystyrene plates; Costar, Wiesbaden, Germany) containing 33 μl of tetramethylammonium chloride (TMAC) hybridization solution (0.15 M TMAC, 75 mM Tris– HCl, 6 mM EDTA, 1.5 g/liter Sarkosyl, pH 8.0), a mixture of 2,000 probe-coupled beads of HPV16, HPV18 and HPV33 set and 7.0 μl TE buffer. Hybridization was performed at 41 °C for 30 min in a thermomixer (Eppendorf, Hamburg, Germany). Beads were washed and resuspended for 20 min in 75 μl of streptavidin-R-phycoerythrin (Strep-PE; Molecular Probes, Eugene, OR) diluted 1:1,600 in 2.0 M TMAC, 75 mM Tris–HCl, 6 m MEDTA, 1.5 g/liter Sarkosyl, 1.0 g/liter casein, pH 8.0. After a final washing step and block beads were analyzed for internal bead color and Rphycoerythrin reporter fluorescence on a Luminex 100 analyzer. The median reporter fluorescence intensity (MFI) of at least 100 beads was computed for each bead set in the sample [21]. Immunohistochemical analysis Formalin-embedded or cryostat sections from primary and xenotransplanted tumor tissue were mounted on glass slides, fixed in acetone and air-dried. The following antibodies were used for tissue staining: EGF Pharm Dx (Code K1494, DakoCytomation), p16INK4a (CINtec Histology Kit), HLA-ABC (DakoCytomation), HLA-DP, DQ, DR (Clone CR3/43, DakoCytomation), and anti-human Ki-67 (DakoCytomation). anti-HPV16 E7 (was kindly provided by Pidder Jansen-Dürr; Institute for Biomedical Ageing Research; Austrian Academy of
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Sciences; Rennweg 10, 6020 Innsbruck, Austria). Staining was performed as described in the customer instruction manual. Treatment with SAE2 immunotoxin Twenty female Scid beige mice were retransplanted with comparable amounts of minced tumor tissue. Two weeks later when tumors had grown to a volume of 125 mm³, 4 groups of animals each consisting 5 mice were set up (n = 5). Animals in the control group received 6 subcutaneous injections of saporin alone (15 μg in 20 μl PBS) in adjacent to the tumor every third day. The other three groups were treated at the same intervals with 6 injections of 5, 10 or 15 μg of SAE2. Statistical analysis Mean values of at least 5 tumors per group were used to calculate standard deviations. Results Development of a transplantation protocol In initial trials, freshly operated solid tumor pieces were transplanted as solid 3-5 mm cubes. This technique required anesthesia, shaving, skin incision and subsequent suturing. The outcome was unsatisfactory, since only a low percentage of tumor biopsies showed engraftment and active proliferation. Moreover, transplants from the same tumor differed substantially with regard to growth behavior, environmental influences, and blood vessel development, which made serial testing of therapeutic agents very difficult. Retransplantation of engrafted tumors regularly failed. In further attempts single cell suspensions of primary tumor tissues comprising additional supplements like matrigel or growth factors were applied subcutaneously. But again no engraftment was detectable (0/6). For these reasons we altered the protocol. In the next trial, tumor pieces were minced with scissors and aspirated into a syringe prior to transplantation. The tumor cell suspension was subcutaneously inoculated into the dorsal region of mice. This procedure was gentle and well tolerated by the animals. Moreover, it did not require anesthesia or skin surgery and thus had lower infection rates. Most importantly though, this approach led to successful engraftment and proliferation of more than 70% of transplanted tumors. Palpable or visible tumors appeared 6–8 weeks after transplantation, on the average. The tumors showed reproducible growth behavior, were encapsulated, and exhibited good vascularization. Xenotransplantation achieved highly efficient engraftment in 70 % (Table 1, Fig. 1). No differences in engraftment were observed
Table 1 General outcome. Carcinoma type
Transplant at
HPV-type
Engraftment
Passage
Adenocarcinoma
1 2 3 4 5 6 7 8 9 10
16 16 16 18 16 16 16 18 33 16
+ + + + + − − + − +
+ − − − + − − + − +
Squamocolumnar carcinoma
Neuroendocrine carcinoma
Successful engraftment of primary tumor transplants occurred in about 70% of attempts. Forty percent of tumors could be retransplanted. The time span for tumor engraftment and outgrowth of primary xenotransplants varied from 40 days to 5 months. Outgrowth was more rapid after retransplantation. On the average, xenografts reached a size of 1 cm3 40–60 days after retransplantation.
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xenotransplants occurred in a time span that varied from 40 days to 5 months. Only high-risk HPV types 16 and 18 were identified by HPV typing in xenotransplanted tumors. Tumor biopsy specimens were classified as adenocarcinomas, squamocolumnar carcinomas or neuroendocrine carcinomas. The established tumors were retransplanted to ensure preservation and proliferation of tissue. Retransplantation was feasible in 40% of transplanted tumors. Outgrowth of passaged xenotransplanted tumors with a volume of up to 1 cm³ occurred within 40 to 60 days. Early and late passage tumors developed within the same time span. The new animal model presented here enables efficient and reproducible engraftment and is easy to prepare. Tumor tissue retains its characteristics after xenotransplantation and passage Fig. 1. EGF receptor expression on human xenografts (5 days after transplantation) is restricted to human cervical cancer cells. Engrafted tumor tissue displays high mitotic activity (black arrows).
between squamous cell carcinomas and adenocarcinomas. Analysis of engrafted tumor tissue five days after transplantation revealed high mitotic activity (Fig. 1). Moreover, immunohistological staining showed HLA class I expression in strongly differentiated tumors (Fig. 2). The transplanted tissues displayed high infiltration rates of antigen-presenting cells five days after transplantation (Fig. 2). Tumor development was accompanied by tissue encapsulation and extensive vascularization (Fig. 1). Engraftment and outgrowth of primary
A prerequisite for creating an efficient replicative xenomodel is to ensure that the transplanted cervical cancer tissue retains its properties after multiple passages. Analysis of therapeutics requires an adequate amount of tumor tissue with the same characteristics. Retransplantation and multiple passages of xenotransplants have not been previously described. We were able to achieve replication of the outgrown cancer tissues. After efficient engraftment of primary tumors, xenotransplanted grown tissues were retransplanted. The xenotumors were treated as primary tumors. Outgrowth of retransplanted tumors occurred in 4 of 10 carcinomas (Table 1). Tumor markers such as EGF receptor and p16 were preserved after early and late tumor passages (Fig. 3). Human cancer tissue showed permanent and constant proliferation after multiple passages (Figs. 1 and 3). Moreover, the HPV type and HLA class I expression remained constant during passages (data not shown). The findings demonstrate that xenotransplanted tumor tissue retains its tumor characteristics after multiple passages in mice. This enables analysis of therapeutic agents in a broad range of experimental settings. Therapeutic treatment of transplanted tumor tissue
Fig. 2. Immunohistochemical staining for MHC class I and MHC class II complexes in cervical cancer xenografts 5 days after transplantation.
To evaluate the xenotransplantation model in a therapeutic setting, we analyzed the effect of a novel immunoprotein that targets the EGF receptor on the tumor cell surface. The therapeutic agent is SE, a chimeric toxin (CT) consisting of the epidermal growth factor and the plant protein toxin saporin from Saponaria officinalis [22–24]. This construct (SA2E) additionally contains a peptidic adapter that improves cytosolic uptake, mediates retention of the toxin in the cytosol, and detoxifies the drug after cell death. Cervical cancer xenografts were highly appropriate tumor models for analyzing immunoprotein properties. Results obtained by immunohistochemical staining of xenotumors have shown that EGF receptor as a target for SA2E is strongly and constantly expressed in xenotransplanted tissues (Figs. 1 and 3). In the experimental setting, a xenotransplanted tumor was allowed to expand in 20 mice. After the tumor volume had increased to 125 cm³, three groups of four mice each received different concentrations of SA2E. The last group served as a control group. SA2E was administered every third day for 6 times, starting on day 13 after tumor transplantation. The tumor volume of xenotransplanted mice was monitored for 45 days. Results of the treatment at day 30 are demonstrated in Fig. 4. Control mice showed rapid tumor outgrowth within 30 days (Fig. 4). Treatment with 5 and 10 μg dose of the immunoprotein decelerated tumor growth by about 20-30%, while treatment with 15 μg of SA2E reduced tumor growth on average by 5060% compared to the control group. The decelerating effect of SA2E on tumor was restricted to treatment periode and after discontinuation of SA2E applications tumors showed rapid outgrow (Fig. 4). We
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Fig. 3. Expression of marker proteins on primary tumor tissue, primary transplant and re-transplants. (p.t.: post transplantation). 79
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Acknowledgment This study was supported by the Wilhelm Sander Stiftung (Munich, Germany). We thank Ursula Schulz for excellent technical assistance. References
Fig. 4. Effect of immunotoxin SA2E on tumor growth 30 days after tumor transplantation. A dose-dependent deceleration of tumor growth was seen in animals receiving 5 and 10 μg of SA2E, while in the highest dose group (15 μg) partial tumor regression (60% by volume) was noticed.
demonstrated the efficacy of this cervical cancer xenotransplantation model, which enables testing of agents for their effect on human primary cervical cancers. High engraftment efficiency, exact reproducibility, massive tumor expansion, and easy preparation make this newly created tumor model attractive for analysis of immunotherapeutics. Discussion In the presented xenograft model rates for successful engraftment were about 70%. Reasons for the failure of engraftment are not evident, but since they were not correlated to certain tumor subtypes, we assume that the vitality of the primary tissue was most likely responsible for this phenomenon. The vitality of primary tumor tissue is closely linked to duration of surgical procedures, individual differences in tumor localization, accessibility, tumor volume and rates of necrosis and varies between each trial. We have created a cervical cancer xenotransplantation model that allows analysis of human primary tumor growth, including infiltration of surrounding tissue and interaction of tumor cells with their stromal environment. It offers high engraftment efficiency, preservation of relevant tumor features, reproducible tumor growth, and massive tumor expansion. It thus enables testing and optimization of several tumor treatment modalities like cytostatic therapy, gene therapy or immunotherapy in a broad range of experimental settings. This transplantation method also has the advantage of minimal invasiveness and easy preparation, unlike xenotransplantation models described for other tumor entities [10,16,17,25]. Moreover, this model can be used to tailor therapies to individual patients. This means that patient-derived tumor tissue can be tested in the mouse xenotransplant model for the most effective treatment strategy. This in vivo model should enable monitoring of tumor cell differentiation and escape variants during chemotherapy or radiotherapy, which will facilitate the development of new treatment concepts and strategies. The utilization of multiply passaged tumor tissue will presumably simplify the establishment of new stable cancer cell lines for in vitro culturing. In further studies the model should be improved by confinement to a specific mouse strain. Patient-derived immune cells will thus be able to create a new human immune environment in the mice, which will enable the analysis of immunotherapies such as vaccines. This newly created tumor model is an attractive system for analyzing treatment strategies. Conflict of interest statement The authors declare that there are no financial conflicts of interest in regards to this work.
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