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Xenograft Mouse Model of Human Uveal Melanoma
人葡萄膜黑色素瘤异种移植小鼠模型   

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Abstract

Uveal melanoma (UM) is a malignant intraocular tumor in adults. Metastasis develops in almost half of the patients and over 90% of the metastases are in the liver. With the advances in molecular targeting therapy for melanoma, a proper metastasis animal model is of increasing importance for testing the accuracy and effectiveness of systemic therapies. Here, we describe a xenograft model for mimicking human UM liver metastasis by injecting human UM cells into the vitreous cavity in nude mice. The athymic nude mice are immunocompromised and suitable for xenograft tumor growth and metastasis, and intravitreal injection of cells is a quicker and easier operation under a binocular scope, thereby it is simple and effective to test human UM growth and metastasis.

Keywords: Human uveal melanoma(人葡萄膜黑色素瘤), Xenograft mouse model(异种移植小鼠模型), Intravitreal injection(玻璃体内注射), Ocular tumor growth(眼肿瘤生长), Liver metastasis(肝转移)

Background

UM is the most common primary intraocular tumor in adults, with an incidence rate varying from 5 to 10 cases per million in the world (Singh et al., 2011). Almost half of the patients develop metastasis within 15 years from initial diagnosis even after treatment or/and removal of primary tumor (Kujala et al., 2003; Weis et al., 2016). In over 4% of the patients, micrometastasis already exists at the time of diagnosis (Finger et al., 2005). The number might be underestimated because of limitations in detection of early UM. Current medical treatments such as enucleation, plaque brachytherapy, proton beam irradiation have been successful in removing or repressing early focal ocular UMs (Dogrusoz et al., 2017). But in general, UM is resistant to the standard chemotherapies and to date no effective systemic treatment is available for metastatic lesions (Goh and Layton, 2016; Carvajal et al., 2017). Although various advances have been made in UM treatment over decades, one-year survival rate after metastasis remains unchanged at 10-15% (Woodman, 2012).

UM arises from uveal melanocytes. Enriched with blood supply and lack of ocular lymph ducts, UM cells mainly spread to distant organs hematogenously. As a result, 95% of UM metastases have a predilection for the liver (Woodman, 2012). The mechanism underlying metastatic transformation of UM is still not clear. Since the liver metastasis is the leading cause of UM-related death (Collaborative Ocular Melanoma Study, 2011), current studies have been focused on the mechanism and molecular targeted prevention of tumor metastasis. Based on the metastatic proclivity, UMs are divided into two categories: class 1 and 2. Class 2 UMs are more inclined to metastasis with primitive stem cell-like gene expression pattern (Harbour and Chao, 2014). The activation of RB/P53, PI3K/AKT and MAPK signaling pathways leads to tumor overgrowth and anti-apoptosis (Coupland et al., 2013; Reichstein, 2017). 85% of primary and metastatic UMs are presented with gain-function mutations in either of two G-protein genes, GNAQ and GNA11 (Shoushtari and Carvajal, 2014). Loss of chromosome 3 or loss-function mutation in BAP1 gene indicates a poor prognosis and metastatic UM (Damato et al., 2011; van Essen et al., 2014). Transcription factors such as ID2, ZEB1 and TWIST1 are involved in UM growth and invasiveness (Chen et al., 2017), expression of PD-1 in UM cells avoids immune destruction by suppressing T cell, facilitating tumor dissemination, and resistance to chemotherapies (Komatsubara and Carvajal, 2017).

Appropriate animal models for UM are critical in understanding molecular mechanisms and evaluating therapeutic effectiveness. Mice are most commonly utilized for tumor models. No spontaneous UM was found in wild-type mice (Stei et al., 2016). Mutation in GNAQ gene could generate choroidal melanoma in mice, but the tumor exclusively metastasizes to the lung (Huang et al., 2015). To date, no genetic animal model mimicking the aforementioned biological and molecular features of human UM has been generated (Stei et al., 2016). By contrast, mouse intraocular xenograft tumor is a widely accepted UM animal model with an effective formation of primary ocular tumors and potential to metastasize to the liver. This article details a protocol to xenograft human UM cells in the vitreous cavity of nude mice, which develops primary tumors in the eye and metastases in the liver in a relatively short period of time.

Materials and Reagents

  1. 100-mm culture plate (Corning, catalog number: 430293 )
  2. 15-ml tube (Corning, catalog number: 430052 )
  3. 30 G blunt needle (BD, catalog number: 305106 )
  4. Cotton swabs (VWR, catalog number: 89031-270 )
  5. Glass coverslips (Fisher Scientific, catalog number: 12-550-15 )
  6. Diapers (VWR, catalog number: 82020-845 )
  7. Surgeon masks (VWR, catalog number: 10843-149)
    Manufacturer: KCWW, Kimberly-Clark, catalog number: 47500 .
  8. Gloves (VWR, catalog number: 82026-426 )
  9. Head cover (3M, catalog number: S-133S-5 )
  10. 1-ml syringe (BD, catalog number: 309628 )
  11. 27 G needle (BD, catalog number: 305109 )
  12. 200-μl tips (USA Scientific, catalog number: 1111-1700 )
  13. Athymic nude mice (THE JACKSON LABORATORY, catalog number: 002019 )
  14. Human UM cell line OCM1 (provided by Dr. Klara Valyi-Nagy in the University of Illinois at Chicago)
  15. 0.25% trypsin (Mediatech, catalog number: 20-053-Cl )
  16. Phosphate-buffered saline (PBS) (Mediatech, catalog number: 21-040-CV )
  17. Mydriatic eye drops are the mixture of Phenylephrine Hydrochloride Ophthalmic Solution (Paragon, NDC 42702-102-15) and Tropicamide Ophthalmic Solution (Bausch & Lomb, NDC 24208-585-64) at the ratio of 1:1
  18. Hypromellose ophthalmic demulcent solution (Akorn, NDC 17478-064-12)
  19. GONAK Lubricant (2.5% Hypromellose ophthalmic demulcent solution) (Akorn, NDC 17478-064-12)
  20. Povidone-Iodine solution (Dynarex, NDC 67777-100-03)
  21. Ketamine hydrochloride (Hospira, NDC 0409-2053-10)
  22. Xylazine sterile solution (Akorn, AnaSed®, NDC 59399-110-20)
  23. Neomycin and polymyxin B sulfates and dexamethasone ophthalmic ointment (Fera, NDC 48102-003-35)
  24. Meloxicam (Henry Schein, NDC 11695-6925-2)
  25. 10% neutral buffered formalin (Sigma-Aldrich, catalog number: F5554-4L )
  26. Ethanol (Sigma-Aldrich, catalog number: 459836-1L )
  27. Dulbecco’s modified Eagle’s medium (DMEM) (Mediatech, catalog number: 10-013-CV )
  28. Fetal bovine serum (FBS) (GE Healthcare, HycloneTM, catalog number: SH30070.03HI )
  29. Penicillin-streptomycin solution (Mediatech, catalog number: 30-002-Cl )
  30. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D5879-500ML )
  31. Cell culture medium (see Recipes)
  32. Cell cryopreservation medium (see Recipes)

Equipment

  1. Cell culture incubator (Thermo Fisher Scientific, Thermo ScientificTM, model: Series 8000 Water-Jacketed , catalog number: 3423)
  2. Bench top centrifuge (Fisher Scientific, model: accuSpin 24CTM )
  3. -80 °C freezer (Thermo Fisher Scientific, Thermo ScientificTM, model: TSUTM Series , catalog number: TSU400A-EA)
  4. 5-μl syringe (Hamilton, catalog number: 87900 )
  5. 50-ml beaker (VWR, catalog number: 10754-946 )
  6. Forceps (VWR, catalog number: 82027-404 )
  7. Scissors (VWR, catalog number: 89259-982 )
  8. 200-µl pipet (Eppendorf, catalog number: 3123000055 )
  9. Liquid nitrogen tank (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: CY50945 )
  10. Surgical microscope (Olympus, model: SZ61/51 )
  11. Warm pad (Pristech Products, catalog number: 20414 )
  12. Autoclave (SOMA Technology, model: Steris Amsco Century V116 )
  13. -20 °C freezer (Kelvinator Commercial, model: KCBM180FQY )
  14. Sterile hood (Labconco, model: Class II, Type B2 (Total Exhaust) )

Procedure

Note: Athymic nude mice were purchased from the Jackson’s Laboratory, breeding, husbandry, and surgical procedures were in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Central South University or/and by University of Louisville Institutional Animal Care and Use Committee (IACUC).

  1. Preparation of UM cells
    1. Dr. Klara Valyi-Nagy in the University of Illinois at Chicago generously provided the human UM cell line OCM1.
    2. The OCM1 cells are cultured in the medium (Dulbecco’s modified Eagle’s medium [DMEM] with 10% heat-inactivated fetal bovine serum [FBS], 100 U/ml penicillin and 0.1 mg/ml streptomycin, see Recipes) in a 37 °C and 5% CO2 incubator.
    3. Cells are passaged at 1:3 ratio when confluent by trypsinization with enough 0.25% trypsin solution to cover cell culture plate surface after removal of the culture medium and rinsing of cells twice with phosphate-buffered saline (PBS).
    4. Trypsin is inactivated by adding the culture medium. Add twice the amount of the medium than trypsin solution used above.
    5. The trypsinized cells are transferred to a 15-ml tube, centrifuged at 500 x g at 4 °C for 5 min, and resuspended in PBS to make a final concentration of 5 x 104 cells/μl.
    6. If not used immediately, cells can be resuspended in a cryopreservation medium (the above culture medium with 10% dimethyl sulfoxide, see Recipes) at the concentration of 1 x 106 cells/ml, and then stored in a -80 °C freezer for short-term storage (< one month) or in a liquid nitrogen tank for long-term storage (> one month).

  2. Instrument sterilization
    1. Prepare a 30 G blunt needle and a 5-µl syringe, place them into a 50-ml beaker with 30 ml of distilled water and boil for 30 min.
    2. Prepare forceps and scissors, clean the instruments with running water for 5 min, disinfect all the instruments in an autoclave with a standard program (121 °C for 20 min).
    3. Prepare cotton swabs, 30 G needles, mydriatic eye drops, Povidone-Iodine and erythromycin eye ointment, glass coverslips.
    4. Place a sterilized diaper underneath a surgical microscope or any binocular scope, establish and maintain a sterile field to minimize possible contamination.
    5. The operator should wear head cover, mask and sterilized gloves before the operation.

  3. Preparation of anesthesia and positioning of the animal
    1. Prepare 6-week-old athymic nude mice, by weighing all mice individually to ensure accurate medication dosage for each of them.
    2. Give an intraperitoneal (IP) anesthesia shot with a 27 G needle and a 1-ml syringe containing a mixture of 100 mg of ketamine and 10 mg of xylazine per kg of the body weight.
    3. Monitor the effectiveness of the anesthesia until the animal completely falls to sleep prior to following procedure.
    4. Give a drop of mydriatic eye drop to dilate the pupil.
    5. Trim eye lashes with a pair of scissors (with some ophthalmic ointment on the blades).
    6. Give 2 drops of Povidone-Iodine to disinfect the eyes.
    7. Give a drop of eye lubricant drop to moisture the eyes.
    8. Place the animal on the diaper and let it lay on one side to facilitate the operation.

  4. Intravitreal injection of tumor cell (see Video 1)
    1. Put a sterilized glass coverslip on the eyeball and focalize the surgical microscope to see the fundus clearly.
    2. Use a 30 G needle to poke a hole about 1 mm behind the limbus.
    3. Before injection, use a 200-μl tip to pipet up and down to ensure a single-cell suspension.
    4. Use Hamilton microliter syringe to inject 2 μl of the above prepared cells into the vitreous, and place the needle posteriorly to avoid touching the lens to avoid cataract formation.
    5. Press a cotton swab on the incision for 1 min and cover the eye with erythromycin ointment to stop possible bleeding after the injection.
    6. Inject 2 µl of PBS in the same way into the other eye as a shamed control.

      Video 1. Resuspending pellet in buffer I or buffer II: Gently rotate bottles in an ice-bucket to resuspend the bacterial pellet in buffer

  5. Animal care after the operation
    1. Keep the grafted mice on a warm pad, check their respiration and activity hourly until they revive.
    2. Keep the mice under specific-pathogen-free condition, maintain the warmth, ventilation and clean the cage as usual.
    3. Check the wound and add ointment daily (while under anesthesia) for the next 5 days to prevent infection.
    4. Give 0.1 ml of Meloxicam at 5 mg/ml subcutaneously per 0.25 kg of body weight daily for at least 2 days to reduce wound pain.

  6. Tumor growth assessment
    1. Prepare intraperitoneal anesthesia with 1-ml syringe and 27 G needle containing a mixture of 100 mg of ketamine and 10 mg of xylazine per kg of the body weight.
    2. Use a drop of mydriatic eye drop to dilate the pupil.
    3. Put the animal to lay on one side.
    4. Give a drop of eye lubricant drop and place a glass coverslip on the eyeball.
    5. Place the animal under the surgical microscope or check the fundus with an indirect ophthalmoscope. The tumor size is estimated in relation to the entire vitreous cavity.

  7. Potential metastasis assessment
    1. Euthanize the mice 4 weeks later by CO2 inhalation followed by a cervical dislocation.
    2. Isolate the eye, liver, lung, and kidney for paraffin-embedded sections to check formation of primary tumor and metastasis by histology haemotoxylin and eosin (H&E) staining.

  8. Tumor dissection and histology
    1. Put all collected tissues in 10% neutral buffered formalin and then in 70% ethanol until further processing for embedding in paraffin.
    2. Section the tissues at 10-μm for histopathological and immunostaining analyses.

Data analysis

  1. Representative results
    The above-described procedure on 16 nude mice resulted in both tumor formation in the eye (87.5%) and the liver (50%). The average time for the tumor to be discovered intraocularly under the ophthalmoscope or outside of the eye was 12.4 days and 23.8 days, respectively (Figures 1A-1C). The mice were euthanized within a week after tumor grew outside of the eyes. Extraocular tumors usually displayed with bleeding and atrophy of the eyes (Figures 1C and 2A), though the retina structure was still maintained relatively intact (Figure 2A). Micrometastasis was detected in the liver tissues around the blood vessels (Figure 2B) (Chen et al., 2017).


    Figure 1. Time-course of tumor growth in nude mice. A. Tumor development inside and outside the eye; B. 12 days after intravitreal injection of tumor cells; C. 25 days after intravitreal injection of tumor cells. PID, post injection day.


    Figure 2. Hematoxylin and eosin (H&E) staining of the tumors in the eye and in the liver. A. A xenograft tumor in the eye; B. Tumor micrometastasis in the liver. T, Tumor. R, Retina. LC, Liver cell. MetT, Metastasized tumor. BV, Blood vessel.

  2. Discussion
    1. This protocol describes a xenograft tumor model in nude mice to enable study of UM liver metastasis. Nude mice are commonly used in tumor studies for they are immune deficient. Xenograft tumors are generated fast in nude mice comparing to other mice such as C57BL/6, in which we did not observe any intraocular tumor formation by intravitreal injection with OCM1 cells (data not shown). In contrast to genetic and chemical-induced UM models, in which metastatic tumors are rarely generated or the metastases occur solely in other organs such as in the lung, but not in the liver as expected to be, the described xenograft UM model is simple and effective in generating liver metastasis in a relative short period of time. There are other methods using cutaneous melanoma cells for xenograft tumor study; however, the metastasis lesion was detected usually in the lung (Kilian et al., 2016). Human UM cell xenograft tumor model manifests more similarities with human UM development in terms of biological and pathological features. The critical step of this protocol is intravitreal injection. Mice are cost-efficient and easy for care and experimental operations. However, their lenses are extremely large, occupying almost 90% of the vitreous while the vitreous cavity of mice is relatively small, compared to other popular animal models. As a result, the number of implanted UM cells is limited, and the accidental touch of the lens would produce cataract that hampers the observation on follow-up tumor growth. Pointing the needle posteriorly towards the retina can avoid touching the lens. UM xenograft models include anterior chamber or subcutaneous injection of UM tumor cells (de Lange et al., 2012; Kilian et al., 2016), but the vitreous injection of tumor cells may have more effects on the ocular posterior segment, which would mimic the context where most human UMs develop. Although the best option for choroidal melanoma is the sub-RPE injection, the technique is more technically challenging to comprehend with a risk of misinjection into subretinal space or perforating the sclera, and therefore would significantly reduce the success rates.
    2. Liver micrometastasis was detected in this model when the tumor grew outside of the eye. Nevertheless, no similar micrometastasis was found in the lung, kidney and spleen, which might be due to the short-term observation. Actually, some nude mice died when the tumor grew out of the eye, the reason of death might be ascribed to the bleeding or infection from necrosis of extraocular tumor. The described UM liver metastasis mouse model can be applied for evaluating systemic therapies for UM, which would provide new prospect for preventing metastasis of UM.

Notes

  1. Directly putting live cells, through cryopreservation medium in a vial, into liquid nitrogen results in cell death in large number. Thus, gradually decreasing temperature by leaving the cells first in a -20 °C freezer for one day, and then in a -80 °C freezer for an additional day before storing them in liquid nitrogen significantly preserves cell viability in their long-term cryopreservation.
  2. Intravitreal injections must be administered under aseptic conditions to avoid ocular infection. Aseptic technique includes the use of topical disinfectant, sterile coverslip, gloves, and surgical draping. All cell culture-related solutions and plastic wares must be sterile, and manipulations must be conducted in a sterile hood.
  3. Use of xylazine-ketamine mixture for anesthesia may cause acute and reversible cataract that impacts follow-up intravitreal injection of tumor cells. To avoid such a problem, operator may anesthetize mice by IP injection of an alternative drug like avertin at an appropriate dose (i.e., use 350 μl for a 20-g mouse).
  4. Mouse lens is relatively large while mouse vitreous cavity is extremely small. Accidentally touching the lens by the intravitreal injection needle would cause cataract that hampers follow-up observation of ocular tumor formation. Care therefore must be taken to avoid touching the lens by pointing the needle posteriorly towards the retina, and gently push cells out of the syringe into the vitreous.

Recipes

  1. Cell culture medium
    Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS) with 100 U/ml penicillin and 0.1 mg/ml streptomycin
  2. Cell cryopreservation medium
    Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS) with 10% dimethyl sulfoxide (DMSO)

Acknowledgments

We are grateful to Drs. Klara Valyi-Nagy and Tibor Valyi-Nagy in University of Illinois at Chicago for providing us with OCM1 human UM cell line. This work was supported by the National Natural Science Foundation of China (No. 3087282 and No. 81072221) and the Natural Science Foundation of Hunan Province (14JJ2005) and by the Basic Research Grant of University of Louisville School of Medicine (E0819 to YL), and Research to Prevent Blindness (to DOVS at Louisville), and partially published in Scientific Reports (Chen et al., 2017). The authors have nothing to disclose.

References

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  2. Chen, Y., Lu, X., Montoya-Durango, D. E., Liu, Y. H., Dean, K. C., Darling, D. S., Kaplan, H. J., Dean, D. C., Gao, L. and Liu, Y. (2017). ZEB1 regulates multiple oncogenic components involved in uveal melanoma progression. Sci Rep 7(1): 45.
  3. Collaborative Ocular Melanoma Study, G. (2001). Assessment of metastatic disease status at death in 435 patients with large choroidal melanoma in the Collaborative Ocular Melanoma Study (COMS): COMS report no. 15. Arch Ophthalmol 119(5): 670-676.
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简介

葡萄膜黑色素瘤(UM)是成人中的恶性眼内肿瘤。 几乎一半的患者发生转移,90%以上的转移灶位于肝脏。 随着分子靶向治疗黑色素瘤的进展,正确的转移动物模型对于检测全身治疗的准确性和有效性越来越重要。 在这里,我们描述了一个模仿人类UM肝转移的异种移植模型,通过将人类UM细胞注入裸鼠的玻璃体腔中。 无胸腺裸鼠具有免疫功能低下,适合异种移植瘤的生长和转移,在双眼镜下,玻璃体内注射细胞更快,更容易手术,因此简单有效地检测人类的UM生长和转移。

【背景】UM是成人中最常见的原发性眼内肿瘤,发病率在世界范围内为5至10个/百万(Singh et al。,2011)。几乎一半的患者在初始诊断即使在治疗或/和移除原发性肿瘤后15年内也发生转移(Kujala等人,2003; Weis等人, 2016)。在超过4%的患者中,诊断时已经存在微转移(Finger等人,2005)。这个数字可能会被低估,因为早期的UM检测的限制。目前的药物治疗,例如眼球摘除术,斑块近距离放疗,质子束照射已经成功地去除或抑制早期的局灶性眼内UMs(Dogrusoz等人,2017)。但是一般来说,UM对标准的化学疗法具有抗性,迄今为止还没有有效的全身治疗可用于转移性病变(Goh和Layton,2016; Carvajal等人,2017)。虽然UM治疗已有数十年的进展,但转移后一年生存率仍保持在10-15%(Woodman,2012)。

UM来自葡萄膜黑素细胞。 UM细胞富含血供,缺乏眼淋巴管,主要传播到远处器官。因此,95%的UM转移瘤倾向于肝脏(Woodman,2012)。 UM的转移转化机制尚不清楚。由于肝转移是导致UM相关性死亡的主要原因(Collaborative Ocular Melanoma Study,2011),目前的研究集中在肿瘤转移的机制和分子靶向预防方面。根据转移倾向,将UM分为两类:1类和2类。2类UM更倾向于具有原始干细胞样基因表达模式的转移(Harbour和Chao,2014)。 RB / P53,PI3K / AKT和MAPK信号传导途径的激活导致肿瘤过度生长和抗细胞凋亡(Coupland等人,2013; Reichstein,2017)。 85%的原发性和转移性UM在两种G蛋白基因GNAQ和GNA11中都有增益功能突变(Shoushtari和Carvajal,2014)。 BAP1基因中染色体3丢失或丢失功能突变表明预后不良和转移性UM(Damato等人,2011; van Essen等人,2014)。转录因子如ID2,ZEB1和TWIST1参与了UM的生长和侵袭(Chen等人,2017),UM细胞中PD-1的表达通过抑制T细胞避免免疫破坏,促进肿瘤传播和对化疗的抵抗(Komatsubara和Carvajal,2017)。

UM的合适动物模型对于理解分子机制和评估治疗有效性至关重要。小鼠最常用于肿瘤模型。在野生型小鼠中没有发现自发的UM(Stei等人,2016)。 GNAQ基因的突变可能在小鼠中产生脉络膜黑色素瘤,但是肿瘤完全转移到肺部(Huang等人,2015)。迄今为止,尚未产生模仿上述人类UM的生物学和分子特征的基因动物模型(Stei等人,2016)。相比之下,小鼠眼内异种移植肿瘤是广泛接受的UM动物模型,有效形成原发性眼部肿瘤并可能转移至肝脏。本文详细介绍了在裸鼠的玻璃体腔中异种移植人UM细胞的方案,其在相对短的时间内在眼中发生原发性肿瘤并在肝中发生转移。

关键字:人葡萄膜黑色素瘤, 异种移植小鼠模型, 玻璃体内注射, 眼肿瘤生长, 肝转移

材料和试剂

  1. 100毫米培养皿(康宁,目录号:430293)
  2. 15毫升管(康宁,目录号:430052)
  3. 30 G钝针(BD,目录号:305106)
  4. 棉签(VWR,目录号:89031-270)
  5. 玻璃盖玻片(Fisher Scientific,目录号:12-550-15)
  6. 尿布(VWR,目录号:82020-845)
  7. 外科口罩(VWR,目录号:10843-149)
    制造商:KCWW,Kimberly-Clark,目录号:47500。
  8. 手套(VWR,目录号:82026-426)
  9. 头罩(3M,产品目录号:S-133S-5)
  10. 1毫升注射器(BD,目录号:309628)
  11. 27 G针(BD,目录号:305109)
  12. 200-μl吸头(USA Scientific,目录号:1111-1700)
  13. 无胸腺裸鼠(THE JACKSON LABORATORY,目录号:002019)
  14. 人类UM细胞系OCM1(由芝加哥伊利诺伊大学的Klara Valyi-Nagy博士提供)
  15. 0.25%胰蛋白酶(Mediatech,目录号:20-053-Cl)
  16. 磷酸盐缓冲盐水(PBS)(Mediatech,目录号:21-040-CV)
  17. 散瞳眼药水是盐酸去氧肾上腺素眼用溶液(Paragon,NDC 42702-102-15)和托卡罗胺眼用溶液(Bausch&amp; Lomb,NDC 24208-585-64)的混合物,比例为1:1。
  18. 羟丙甲纤维素眼用缓和溶液(Akorn, NDC 17478-064-12
  19. GONAK润滑剂(2.5%羟丙甲纤维素眼用缓冲溶液)(Akorn, NDC 17478-064 -12
  20. 聚维酮碘溶液(Dynarex,NDC 67777-100-03)
  21. 盐酸氯胺酮(Hospira,NDC 0409-2053-10)
  22. 甲苯噻嗪无菌溶液(Akorn,AnaSed , NDC 59399- 110-20
  23. 新霉素和多粘菌素B硫酸盐和地塞米松眼膏(Fera,NDC 48102-003-35)
  24. 美洛昔康(Henry Schein,NDC 11695-6925-2)
  25. 10%中性缓冲福尔马林(Sigma-Aldrich,目录号:F5554-4L)
  26. 乙醇(Sigma-Aldrich,目录号:459836-1L)
  27. 达尔伯克改良伊格尔培养基(DMEM)(Mediatech,目录号:10-013-CV)
  28. 胎牛血清(FBS)(GE Healthcare,Hyclone TM,目录号:SH30070.03HI)。
  29. 青霉素 - 链霉素溶液(Mediatech,目录号:30-002-Cl)
  30. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D5879-500ML)
  31. 细胞培养基(见食谱)
  32. 细胞冷冻保存液(见食谱)

设备

  1. 细胞培养孵育器(Thermo Fisher Scientific,Thermo Scientific TM,型号:Series 8000 Water-Jacketed,目录号:3423)
  2. 台式离心机(Fisher Scientific,型号:accuSpin 24C TM)
  3. -80℃冷冻机(Thermo Fisher Scientific,Thermo Scientific TM,型号:TSU TM TM Series,目录号:TSU400A-EA)
  4. 5-μl注射器(汉密尔顿,目录号:87900)
  5. 50毫升烧杯(VWR,目录号:10754-946)
  6. 镊子(VWR,目录号:82027-404)
  7. 剪刀(VWR,目录号:89259-982)
  8. 200μl移液器(Eppendorf,目录号:3123000055)
  9. 液氮罐(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:CY50945)
  10. 手术显微镜(奥林巴斯,型号:SZ61 / 51)
  11. 暖垫(Pristech Products,目录编号:20414)
  12. 高压灭菌器(SOMA Technology,型号:Steris Amsco Century V116)
  13. -20°C冰柜(Kelvinator商业,型号:KCBM180FQY)
  14. 无菌罩(Labconco,型号:II级,B2型(全部排气))

程序

注:无性生殖的裸鼠购自杰克逊实验室,育种,饲养和手术程序符合视觉和眼科学研究协会(ARVO)在眼科和视力研究中使用动物的声明,由中南大学或路易斯维尔大学动物管理与使用委员会(IACUC)批准。

  1. UM细胞的制备
    1. 芝加哥伊利诺斯大学的Klara Valyi-Nagy博士慷慨提供了人类UM细胞系OCM1。
    2. 将该OCM1细胞在含有10%热灭活的胎牛血清[FBS],100U / ml青霉素和0.1mg / ml链霉素的Dulbecco改良伊格尔培养基[DMEM]的培养基(参见食谱)中于37℃培养, 5%CO 2培养箱。
    3. 细胞以1:3的比例进行传代,通过用足够的0.25%胰蛋白酶溶液进行胰蛋白酶消化以除去培养基后覆盖细胞培养皿表面,并用磷酸盐缓冲盐水(PBS)冲洗细胞两次。
    4. 加入培养基使胰蛋白酶失活。
      加入比上面使用的胰蛋白酶溶液多两倍的培养基
    5. 将胰蛋白酶消化的细胞转移至15ml管中,在500℃下在500rpm下离心5分钟,并重悬于PBS中以使终浓度为5×10 4 /细胞/μl。
    6. 如果不立即使用,细胞可以以1×10 6个细胞/ ml的浓度重新悬浮在冷冻保存培养基(含有10%二甲基亚砜的上述培养基,参见食谱)中,然后保存在用于短期储存(<1个月)的-80℃冷冻机或用于长期储存(> 1个月)的液氮罐中。

  2. 仪器灭菌
    1. 准备一个30 G钝针和一个5微升的注射器,将它们放入一个50毫升烧杯与30毫升蒸馏水煮沸30分钟。
    2. 准备钳子和剪刀,用流水清洗仪器5分钟,用标准程序(121℃20分钟)对高压灭菌器中的所有仪器进行消毒。
    3. 准备棉签,30 G针,散瞳滴眼液,聚维酮碘和红霉素眼膏,玻璃盖玻片。
    4. 将无菌尿布置于手术显微镜或任何双目镜下,建立并保持无菌区域,以尽量减少可能的污染。

    5. 在操作前,操作人员应戴头罩,面罩和消毒手套
  3. 麻醉的准备和动物的定位
    1. 准备6周龄的无胸腺裸鼠,分别称量所有小鼠,以确保每个小鼠的准确药物剂量。
    2. 用27G针头和1毫升注射器腹腔(IP)麻醉注射,注射器中含有每公斤体重100毫克氯胺酮和10毫克甲苯噻嗪的混合物。
    3. 监测麻醉的有效性,直到动物完全堕入睡眠状态,然后再进行手术。

    4. 给一滴散瞳药滴眼液来扩张瞳孔。
    5. 用一把剪刀修剪眼睫毛(刀片上有一些眼药膏)。
    6. 给2滴聚维酮碘消毒眼睛。
    7. 给一滴眼睛润滑剂滴眼睛湿润。
    8. 将动物放在尿布上,让它躺在一边,以方便手术。

  4. 玻璃体内注射肿瘤细胞(见视频1)
    1. 将消毒的玻璃盖玻片放在眼球上,使手术显微镜聚焦清晰。

    2. 使用30G针在角膜缘后约1毫米处戳一个孔
    3. 在注射之前,使用200-μl吸头上下移动以确保单细胞悬液。
    4. 使用汉密尔顿微量注射器将2μl上述制备的细胞注射到玻璃体中,并将针头放在后面以避免接触镜片以避免白内障形成。
    5. 在切口处按棉签1分钟,用红霉素软膏覆盖眼睛,以防止注射后出血。

    6. 注射2μl的PBS以相同的方式进入另一只眼睛作为羞耻的控制。


  5. 手术后的动物护理
    1. 保持移植的老鼠在温暖的垫上,检查他们的呼吸和活动每小时,直到他们恢复。
    2. 保持小鼠处于无特定病原体状态,照常保持温暖,通风和清洁笼子。
    3. 检查伤口,并在接下来的5天内每天(在麻醉状态下)加入软膏以防止感染。
    4. 给予每毫升0.25毫克皮下注射5毫克/毫升美洛昔康0.1毫克至少2天,以减轻伤口疼痛。

  6. 肿瘤生长评估
    1. 准备1毫升注射器和27克针头,每公斤体重含有100毫克氯胺酮和10毫克赛拉嗪的混合物腹腔麻醉。

    2. 使用一滴散瞳药滴眼液来扩张瞳孔。
    3. 把动物放在一边。
    4. 给一滴眼药水,并在眼球上放一个玻璃盖玻片。
    5. 将动物置于手术显微镜下或用间接检眼镜检查眼底。估计肿瘤大小与整个玻璃体腔相关。

  7. 潜在的转移评估

    1. 4周后通过CO 2吸入使颈椎脱臼安乐死小鼠。
    2. 用石蜡包埋切片分离眼,肝,肺和肾,通过组织学的血吸虫和曙红(H&amp; E)染色来检查原发肿瘤和转移的形成。

  8. 肿瘤解剖和组织学
    1. 将所有收集的组织放入10%中性缓冲的福尔马林中,然后加入70%乙醇中,直至进一步处理以包埋在石蜡中。
    2. 切片10微米的组织进行组织病理学和免疫染色分析。

数据分析

  1. 代表结果
    对16只裸鼠的上述程序导致眼睛(87.5%)和肝脏(50%)两者形成肿瘤。检眼镜下或眼外发现肿瘤的平均时间分别为12.4天和23.8天(图1A-1C)。肿瘤在眼睛外生长一周后使小鼠安乐死。尽管视网膜结构仍保持相对完整(图2A),但眼外肿瘤通常伴有出血和眼萎缩(图1C和2A)。在血管周围的肝组织中检测到微转移(图2B)(Chen等人,2017)。

    图1.裸鼠肿瘤生长的时间过程A.眼睛内外的肿瘤发展; B.玻璃体内注射肿瘤细胞12天后; C.玻璃体内注射肿瘤细胞25天后。 PID,注射后一天。


    图2.眼睛和肝脏中肿瘤的苏木精和伊红(H&amp; E)染色。 :一种。眼中的异种移植肿瘤; B.肝脏中的肿瘤微转移。 T,肿瘤。 R,视网膜。 LC,肝细胞。 MetT,肿瘤转移。 BV,血管。

  2. 讨论
    1. 该协议描述了在裸鼠中的异种移植肿瘤模型以使得能够研究UM肝转移。裸鼠通常用于肿瘤研究,因为它们是免疫缺陷的。与其他小鼠如C57BL / 6相比,异种移植肿瘤在裸鼠中快速生成,其中我们没有观察到通过OCM1细胞玻璃体内注射的任何眼内肿瘤形成(数据未显示)。与遗传和化学诱导的UM模型(其中转移性肿瘤很少产生或者转移仅在其它器官如肺中而不在肝中发生)相反,所描述的异种移植UM模型是简单的并且有效地在相对较短的时间内产生肝转移。还有其他方法使用皮肤黑素瘤细胞进行异种移植肿瘤研究;然而,通常在肺中检测到转移病灶(Kilian等人,2016)。人类UM细胞异种移植肿瘤模型在生物学和病理学特征方面表现出与人类UM发展更为相似。该协议的关键步骤是玻璃体内注射。小鼠具有成本效益,易于护理和实验操作。然而,与其他流行的动物模型相比,它们的镜片非常大,几乎占据了玻璃体的90%,而小鼠的玻璃体腔相对较小。因此,植入的UM细胞的数量是有限的,并且镜片的偶然接触会产生白内障,这阻碍了对随访肿瘤生长的观察。将针头向后指向视网膜可避免接触镜头。 UM异种移植模型包括前房或UM皮下注射UM肿瘤细胞(de Lange等人,2012; Kilian等人,2016),但玻璃体内注射肿瘤细胞可能对眼后段有更多的影响,这将模仿大多数人类UMs发展的背景。虽然脉络膜黑色素瘤的最佳选择是次级RPE注射,但是技术上更难以理解误入视网膜下腔或穿孔巩膜的风险,因此会显着降低成功率。
    2. 当肿瘤生长在眼外时,在该模型中检测到肝脏微转移。然而,在肺,肾和脾中未发现类似的微转移,这可能是由于短期的观察。实际上,有些裸鼠在肿瘤生长的时候死亡,死亡的原因可能是由于眼外肿瘤坏死引起的出血或感染。所描述的UM肝转移小鼠模型可用于评价UM的全身治疗,这将为预防UM的转移提供新的前景。

笔记

  1. 直接将活细胞通过小瓶中的冷冻保存介质置于液氮中导致大量细胞死亡。因此,通过将细胞首先放置在-20℃的冷冻器中一天,然后在-80℃的冷冻器中保存一天,然后将它们储存在液氮中,从而显着地保持了细胞在其长期冷冻保存中的活力。
  2. 必须在无菌条件下进行玻璃体内注射以避免眼部感染。无菌技术包括使用局部消毒剂,无菌盖玻片,手套和外科手术。所有细胞培养相关的解决方案和塑料制品必须是无菌的,操作必须在无菌罩中进行。
  3. 使用赛拉嗪 - 氯胺酮混合物进行麻醉可能引起急性和可逆性白内障,影响随后的玻璃体内注射肿瘤细胞。为了避免这样的问题,操作者可以通过以适当的剂量IP注射替代药物如阿佛丁来麻醉小鼠(即,使用350μl用于20-g小鼠)。
  4. 鼠标透镜比较大,而小鼠玻璃体腔则非常小。不小心通过玻璃体内注射针头接触晶状体会导致白内障,妨碍随后的眼部肿瘤形成的观察。因此必须注意避免将针头向后指向视网膜,并将细胞从注射器中轻轻推入玻璃体内,以免触及镜片。

食谱

  1. 细胞培养基
    Dulbecco改良Eagle培养基(DMEM),含10%热失活胎牛血清(FBS),含100U / ml青霉素和0.1mg / ml链霉素
  2. 细胞低温保存培养基
    达尔伯克改良伊格尔培养基(DMEM)与10%热灭活胎牛血清(FBS)与10%二甲基亚砜(二甲基亚砜)

致谢

我们感谢Drs。芝加哥伊利诺伊大学的Klara Valyi-Nagy和Tibor Valyi-Nagy为我们提供了OCM1人类UM细胞系。本研究由国家自然科学基金(No.3087282和81072221)和湖南省自然科学基金(14JJ2005)和路易斯维尔医学院基础研究基金(E0819〜YL)资助, ,以及防止盲目性的研究(在路易斯维尔的DOVS),并部分刊登在“科学报告”(Chen等人,2017年)中。作者没有什么可以披露的。

参考

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chen, Y., Liu, X., Gao, L. and Liu, Y. (2017). Xenograft Mouse Model of Human Uveal Melanoma. Bio-protocol 7(21): e2594. DOI: 10.21769/BioProtoc.2594.
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