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A Murine Orthotopic Allograft to Model Prostate Cancer Growth and Metastasis
使用鼠原位移植建立前列腺癌生长和转移模型   

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Abstract

Prostate cancer is one of the most common cancers in men in the United States. Comprehensive understanding of the biology contributing to prostate cancer will have important clinical implications. Animal models have greatly impacted our knowledge of disease and will continue to be a valuable resource for future studies. Herein, we describe a detailed protocol for the orthotopic engraftment of a murine prostate cancer cell line (Myc-CaP) into the anterior prostate of an immune competent mouse.

Keywords: Orthotopic allograft(原位同种异体移植物), Myc-CaP(Myc-CaP), Prostate(前列腺), Cancer(癌症), Metastasis(转移), in vivo(体内), Mouse model(小鼠模型)

Background

Prostate cancer is a leading cause of cancer death in men due to a subset of cancers that metastasize. The genetic and molecular factors that drive local tumor development and progression to metastatic disease, however, remain incompletely understood. Both genetically engineered mouse (GEM) models and xenograft models of prostate cancer have contributed to our understanding of the genetics of prostate cancer (Ittmann et al., 2013; Park et al., 2010). Genetic manipulation, either by prostate specific transgenic overexpression such as in Hi-Myc mice (Ellwood-Yen et al., 2003) or by prostate specific deletion such as in Pten-/- mice (Wang et al., 2003), is advantageous because it models tumor development and progression in the organ microenvironment in an immune competent mouse. Development of metastatic prostate cancer is variable among these GEM models, with a low frequency in some such as the Pten-/- model (Wang et al., 2003), and a higher frequency in other models such as TRAMP (transgenic adenocarcinoma mouse prostate) (Greenberg et al., 1995) and Hi-Myc/Pten-/- (Hubbard et al., 2016). Despite their great utility for prostate cancer research, it is difficult, time-consuming, and costly to further genetically manipulate GEM models. To overcome some of these limitations, researchers have relied on both subcutaneous and orthotopic xenografts of human cell lines. Cell lines can be genetically manipulated in vitro in a variety of ways. While subcutaneous xenografts are advantageous due to their ease of injection and monitoring, orthotopic xenografts better recapitulate the local tumor microenvironment which may affect sensitivity to drugs (Wilmanns et al., 1992; Kuo et al., 1993), methylation patterns (Fleming et al., 2010), growth rate (Fleming et al., 2010), and ultimately predictions for clinical response (Killion et al., 1998; Hoffman, 1999). In addition, some human prostate cancer cell line models metastasize from xenografts implanted orthotopically. A limitation of all xenograft models is that they require immunocompromised mice making it difficult to model tumor progression in an intact immune system. The Myc-CaP cell line (Watson et al., 2005) allows for engraftment either subcutaneously or orthotopically in immune competent syngeneic (FVB/N) mice (Watson et al., 2005; Hurley et al., 2015). Myc-CaP was derived from a prostate carcinoma from a Hi-Myc mouse (Watson et al., 2005). When engrafted orthotopically, Myc-CaP cells metastasize to abdominal lymph nodes, liver, and lung (Hurley et al., 2015). Additionally, Myc-CaP is amenable to in vitro manipulation of gene expression (Hurley et al., 2015). Thus, Myc-CaP can be used as an easily manipulable model for both tumor growth in the prostate and metastatic growth in mice with an intact immune system. Herein, we describe the methods for orthotopic engraftment of Myc-CaP cells into the mouse anterior prostate.

Materials and Reagents

  1. Institutional ACUC approval of orthotopic allograft protocol
  2. Mask, cap, clean lab coat, arm covers, and sterile gloves
  3. Tissue culture flasks
  4. 70% alcohol wipes (DUKAl, catalog number: 852 )
  5. Cotton swab
  6. 30 G ½” needle (BD, catalog number: 305106 ) – orthotopic engraftment
  7. Reflex 9 mm stainless steel wound clips (Roboz Surgical Instrument, catalog number: RS-9262 )
  8. 21 G 1” needle & 3 ml syringe (BD, catalog number: 309575 ) – lung inflation
  9. POLYSORBTM size 3-0 USP (2 metric), 30 inches (75 cm) UNDYED on V-20 needle absorbable surgical sutures (COVIDIEN, catalog number: GL322 )
  10. Myc-CaP cells (ATCC, catalog number: CRL-3255 )
  11. FVB/N male mice (THE JACKSON LABORATORY), 10 weeks and older
    Note: Post-pubescent mice have androgen signaling and a larger prostate that facilitates ease of engraftment. We also recommend using mice that are approximately the same age and weight. Male GEM models can also be used as long as they are fully back-crossed to FVB/N.
  12. Matrigel (Corning, catalog number: 354234 )
  13. 1x phosphate buffered saline (PBS), pH 7.4 (Thermo Fisher Scientific, GibcoTM, catalog number: 10010-023 )
  14. 1x trypsin-EDTA (Mediatech, catalog number: 25-053-CI )
  15. Fluriso isoflurane (VetOne, catalog number: 501017 )
  16. 10% povidone-iodine ‘Betadine’ prep solution, 16 oz. (Henry Schein, catalog number: 6906950 )
  17. 50 mg/ml injectable Carprofen (Zoetis, catalog number: 07-844-7425 )
  18. Ethanol, reagent alcohol 200 proof, PHARMCO-AAPERTM (Thermo Fisher Scientific, Fisher Scientific, catalog number: 16-100-824 )
  19. ImmPRESS HRP Anti-Rabbit Polymer Detection Kit (Vector Labs, catalog number: MP-7401 )
  20. ImmPACT DAB Peroxidase Substrate (Vector Labs, catalog number: SK-4105 )
  21. Myc-CaP growth medium (see Recipes)
    1. Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11965-092 )
    2. Fetal bovine serum (Gemini Bio-Products, catalog number: 100-106 )
    3. Penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
    4. Bovine pituitary extract (BPE) (Thermo Fisher Scientific, GibcoTM, catalog number: 13028-014 )
    5. Recombinant human EGF (R&D Systems, catalog number: 236-EG )
    6. Bovine insulin powder (Gemini Bio-Products, catalog number: 700-112 )

Equipment

  1. Incubator
  2. Centrifuge
  3. Cell culture laminar flow hood, vacuum suction, centrifuge, cell counter
  4. 2 L induction chamber
  5. Scissors (Roboz Surgical Instrument, catalog number: RS-5914 )
  6. Fine forceps (Roboz Surgical Instrument, catalog number: RS-5132 )
  7. Mouse housing and handling facility
  8. Isoflurane gas anesthetic system (VetEquip, catalog number: 922100 ) including a vaporizer, induction chamber, tubing, nose cones, and oxygen tank
  9. Glass bead sterilizer (GERMINATOR 500) (Roboz Surgical Instrument, catalog number: DS-401 )
  10. Rechargeable shaver
  11. Space gel heating pads (Braintree Scientific, catalog number: SPGL )
  12. 25 µl, model 702 RN syringe (Hamilton, catalog number: 7636-01 )
  13. Syringe guide 25 µl-500 µl/7000 SYR (Hamilton, catalog number: 14906 )

Procedure

Note: All procedures conducted in mice must first have institutional ACUC approval.

  1. Culture and preparation of Myc-CaP cells for injection
    1. Propagate Myc-CaP cells in tissue culture flasks with Myc-CaP growth media and passage as per ATCC guidelines. Engraft 1 x 106 Myc-CaP cells per mouse. Due to ‘dead space’ within the needle, overestimate the number of allografts needed by 20% or a minimum of two extra allografts.
      Note: Injecting more than 1 x 106 Myc-CaP cells per mouse is not recommended as it will accelerate primary tumor growth and shorten the time between engraftment and euthanasia due to morbidity. This may decrease the likelihood of observing metastatic disease.
    2. Prior to harvesting Myc-CaP cells for injection, thaw Matrigel on ice.
    3. Remove media from flasks containing 80-90% confluent monolayers of Myc-CaP cells, wash cells with 1x PBS, and detach cells by incubating in 0.05% trypsin-EDTA for 3-6 min in 37 °C incubator. Neutralize trypsin by adding growth media.
    4. Transfer cells to a 50 ml conical tube and centrifuge at 300 x g for 5 min. Remove trypsin/media and then resuspend the cell pellet in fresh growth media.
    5. Count cells to isolate 1 x 106 cells per allograft. Centrifuge isolated cell suspension at 300 x g for 5 min.
    6. Perform the following step on ice. Remove media and resuspend cell pellet in Matrigel to a volume of 10 μl per 1 x 106 cells. Keep cell:Matrigel mixture on ice until engraftment.
      Note: Thoroughly mix cell:Matrigel prior to engraftment.

  2. Engraftment of Myc-CaP cells into anterior prostate of > 10 weeks male FVB/N mice
    1. Perform engraftment in a laminar flow hood using aseptic techniques, as per your institutional Animal Care and Use Committee (ACUC) guidelines. Wear a mask, cap, clean lab coat, arm covers, and sterile gloves throughout the procedure. Sterilize instruments using a glass bead sterilizer, and disinfect with 70% ethanol between animals.
    2. Induce general anesthesia using a 2 L induction chamber by inhalation of 2% isoflurane in oxygen. Following complete anesthesia induction, maintain anesthesia by nose cone at approximately 1.5% isoflurane, adjusted as needed to maintain a deep plane of anesthesia (Figure 1). The level of anesthesia is sufficient for surgery when pinching of the skin between the digits does not stimulate pedal withdrawal.
    3. Provide preemptive analgesia according to your institutional guidelines.
      Note: As per our institutionally approved animal protocol, we inject mice subcutaneously with 5 mg/kg Carprofen diluted in sterile saline to a total volume of 0.5 ml.
    4. Place the anesthetized mouse in dorsal recumbency, shave hair over the surgical site, and swab the surgical site with Betadine followed by 70% alcohol wipes (Figure 1).
    5. Using sterile scissors and fine forceps, make a horizontal incision 1 cm in length at 2 mm above the preputial gland (Figure 1, and Bertrand et al., 2016). Once the abdominal wall is exposed, make a similar incision to open the abdomen (Figure 1).
    6. Use fine forceps to manipulate the bladder gently out of the abdomen and caudally toward the prepuce. This exposes the seminal vesicles and attached anterior prostate lobes (Figure 1, and Oliveira et al., 2016). Gently coax one seminal vesicle and anterior prostate out of the abdomen with fine forceps, cotton swabs, or gentle pressure on the abdomen (Figure 1). Take special care to prevent damage to the seminal vesicles, as leakage of seminal fluid into the abdomen will incite an intense inflammatory reaction.
    7. After exposing and exteriorizing the seminal vesicle, insert a 25 µl Hamilton syringe preloaded with the Myc-CaP cell:Matrigel mixture and fitted with a 30 G ½” needle longitudinally into the anterior prostate (parallel to the length of the seminal vesicle). Slowly inject 10 μl of the mixture (equal to 1 x 106 Myc-CaP cells) into the prostate (Figure 1). Take care to avoid spillage of the injected contents when removing the needle from the anterior prostate as spillage may result in the seeding of multiple tumors in the abdomen. To avoid spillage, keep the needle in place for 1-2 sec after injection (giving the Matrigel time to solidify within the lobe) and apply gentle pressure to the injection site with a cotton swab after removing the needle.
      Note: Injecting more than 10 μl into the anterior lobe increases the likelihood of spillage. Other lobes of the mouse prostate can be used for injection, however, the anterior lobe offers ease of location and a reduced likelihood of urethra constriction upon growth. Lobe choice for engraftment site should remain consistent throughout the experiment.
    8. Gently return the seminal vesicle and bladder to the abdomen and then close the abdominal wall with 3-0 continuous, absorbable sutures. Close skin with 9 mm steel wound clips (Figure 1).
      Note: For blinded studies, mark animals with toe tattoo or ear punch as per institutional guidelines. Give each animal a unique number for identification.


      Figure 1. Engraftment of Myc-CaP cells into the mouse anterior prostate. A. Following induction of general anesthesia (step B2), the surgical site is shaved and cleaned with Betadine followed by alcohol wipes (step B4). A 1 cm incision is made above the preputial gland (step B5), and the anterior prostate is exposed (step B6). Myc-CaP cells are injected into the anterior prostate (step B7), and then the incision is closed with sutures and surgical clips (step B8); B. Anatomic approximation of preputial glands (P.G., dotted lines) and incision site (solid horizontal black line). Scrotum (S) and prepuce (P) are labeled for reference; C. Organ anatomy of mouse at the incision site. Bladder (B, shown emptied of urine) and seminal vesicles (S.V., left-sided exposed only) are easily identified at the incision site for their midline location and white horn-shaped structure, respectively. The anterior prostate (A.P.) lobes run parallel to the seminal vesicles attached along their lesser curvature. D. Enlarged view of Myc-CaP cell injection into the anterior prostate.

  3. Post-operative care
    1. After surgical site closure, place animal in a clean cage on a disinfected heating pad to maintain body temperature during recovery.
    2. Mice should be active and alert within 2 h of surgery. Monitor mice intermittently until they are alert.
    3. Monitor mice daily for signs of distress or infection such as failure to groom, weight loss, reluctance to move, labored breathing.
    4. If animals show signs of pain or distress, administer 5 mg/kg Carprofen subcutaneously every 12 h up to 72 h postoperatively.

  4. Necropsy, processing and staining
    1. Length of engraftment depends upon experimental design and endpoints.
      Note: In our experience, engraftment of 1 x 106 Myc-CaP cells into the anterior prostate yields allografts approximately 60 mm3 in size and micro-metastatic disease at 21 days post engraftment (Hurley et al., 2015). Micro-metastatic disease is often difficult to appreciate grossly.
      Following engraftment, euthanize mice according to your institutional guidelines for necropsy. Dissect the primary tumor away from the seminal vesicle and weigh it. If the engrafted tumor cannot be dissected from the adjacent seminal vesicle, remove the tumor along with the seminal vesicle and weigh en bloc. Inspect mice for gross evidence of metastatic disease and, if present, quantify the number and location of visible macro-metastatic lesions. Photograph both the engrafted lesion and any macro-metastatic lesions.
    2. Remove the abdominal organs for formalin-fixation and paraffin embedding. Section organs for hematoxylin and eosin (H&E) staining (Slaoui and Fiette, 2011). Additionally, remove the lungs and inflate with formalin for optimal pathological examination prior to formalin fixation. To inflate the lungs, a 21 G needle attached to a 3 ml syringe filled with fixative is introduced into the trachea at its open end, forceps are used to clamp gently around the needle, and fixative is introduced until excess refluxes up the trachea (Fiette and Slaoui, 2011). A certified pathologist should analyze H&E stained slides for micro-metastatic disease.
    3. The presence of Myc-CaP cells in metastases can be confirmed via immunohistochemical (IHC) staining of formalin-fixed and paraffin embedded (FFPE) tissue for androgen receptor (AR) and Myc positivity (Figure 2). IHC staining protocol was adapted from a previously published study (Simons et al., 2015).


      Figure 2. Myc-CaP orthotopic allograft and staining by H&E and IHC. A. Myc-CaP orthotopic allograft (arrow) in the mouse anterior prostate. Seminal vesicle (S.V.) and anterior prostate (A.P.). H&E and IHC staining for Myc and androgen receptor (AR) of a Myc-CaP orthotopic allograft tumor; B. H&E and IHC staining of a Myc-CaP orthotopic allograft tumor (T) and adjacent seminal vesicle containing secretions (S, bright pink staining); C. H&E and IHC staining of a lung metastasis from a Myc-CaP orthotopic allograft. Positive Myc and AR staining, shown in brown, was visualized with an ImmPRESS Polymer Detection Kit and ImmPACT DAB (Vector Laboratories). IHC slides were counterstained with hematoxylin. Images at 10x magnification (20x inset) and black bars are 100 µm.

Data analysis

We recommend considering the ARRIVE Guidelines (Kilkenny et al., 2010) for in vivo animal study design, analyses, and reporting. As outlined in the ARRIVE Guidelines, take comprehensive records of study design, experimental procedures, experimental animals, animal housing and husbandry, sample size, allocation of animals to experimental groups, experimental outcomes, and statistical methods (Kilkenny et al., 2010). To determine sample size (number of animals per experimental and control groups), first calculate the required effect size for both continuous and categorical measurements. For example, if during a pilot study we determine that the control group’s primary tumor weighs on average (0.122 g ± 0.072 g) and the experimental group’s primary tumor weighs (0.038 g ± 0.0133 g), then the effect size, d = 1.622467. Using a two-sided test with α error of probability = 0.05, power = 0.8, and an allocation ratio of control/experimental = 1, the sample size for both groups should be 8 animals to demonstrate a statistically significant difference between the groups (Festing and Altman, 2002). To account for a 20% error rate, we would use 10 animals per group.
Note: A pilot study should be performed first in order to determine the appropriate sample size for a given experiment. We strongly recommend consulting with a biostatistician during the project design phase of any animal study.
The appropriate statistical methods for data analysis will depend on the experimental design and experimental outcomes assessed. To improve experimental robustness, we recommend two independent experimental replications and blinded data analysis. If any animals are excluded from the final analyses, provide sound rationale for exclusion such as cell leakage into the body cavity as evidenced by multiple metastatic lesions on the body cavity wall.

Recipes

  1. Myc-CaP growth medium
    500 ml DMEM
    50 ml fetal bovine serum
    5 ml penicillin-streptomycin
    25 µg/ml bovine pituitary extract
    5 µg/ml bovine insulin
    6 ng/ml recombinant human epidermal growth factor
    Note: Myc-CaP cells can be also cultured in DMEM with bovine serum to a final concentration of 10%

Acknowledgments

This protocol was adapted from a previously published study (Hurley et al., 2015). This work was supported by The Prostate Cancer Foundation Hagen Challenge Award; The Patrick C. Walsh Prostate Cancer Fund, and The Hinman Urologic Endowed Fund Educational Scholarship.

References

  1. Bertrand, H. G., Thomas, A. A., Ellen, Y. C., Dorward, R. S. and Flecknell, P. A. (2016). A surgical approach in the treatment of preputial gland abscesses in mice. BMC Vet Res 12: 16.
  2. Ellwood-Yen, K., Graeber, T. G., Wongvipat, J., Iruela-Arispe, M. L., Zhang, J., Matusik, R., Thomas, G. V. and Sawyers, C. L. (2003). Myc-driven murine prostate cancer shares molecular features with human prostate tumors. Cancer Cell 4(3): 223-238.
  3. Festing, M. F. and Altman, D. G. (2002). Guidelines for the design and statistical analysis of experiments using laboratory animals. ILAR J 43(4): 244-258.
  4. Fiette, L. and Slaoui, M. (2011). Necropsy and sampling procedures in rodents. Methods Mol Biol 691: 39-67.
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  6. Greenberg, N. M., DeMayo, F., Finegold, M. J., Medina, D., Tilley, W. D., Aspinall, J. O., Cunha, G. R., Donjacour, A. A., Matusik, R. J. and Rosen, J. M. (1995). Prostate cancer in a transgenic mouse. Proc Natl Acad Sci U S A 92(8): 3439-3443.
  7. Hoffman, R. M. (1999). Orthotopic metastatic mouse models for anticancer drug discovery and evaluation: a bridge to the clinic. Invest New Drugs 17(4): 343-359.
  8. Hubbard, G. K., Mutton, L. N., Khalili, M., McMullin, R. P., Hicks, J. L., Bianchi-Frias, D., Horn, L. A., Kulac, I., Moubarek, M. S., Nelson, P. S., Yegnasubramanian, S., De Marzo, A. M. and Bieberich, C. J. (2016). Combined MYC activation and Pten loss are sufficient to create genomic instability and lethal metastatic prostate cancer. Cancer Res 76(2): 283-292.
  9. Hurley, P. J., Hughes, R. M., Simons, B. W., Huang, J., Miller, R. M., Shinder, B., Haffner, M. C., Esopi, D., Kimura, Y., Jabbari, J., Ross, A. E., Erho, N., Vergara, I. A., Faraj, S. F., Davicioni, E., Netto, G. J., Yegnasubramanian, S., An, S. S. and Schaeffer, E. M. (2015). Androgen-regulated SPARCL1 in the tumor microenvironment inhibits metastatic progression. Cancer Res 75(20): 4322-4334.
  10. Ittmann, M., Huang, J., Radaelli, E., Martin, P., Signoretti, S., Sullivan, R., Simons, B. W., Ward, J. M., Robinson, B. D., Chu, G. C., Loda, M., Thomas, G., Borowsky, A. and Cardiff, R. D. (2013). Animal models of human prostate cancer: the consensus report of the New York meeting of the Mouse Models of Human Cancers Consortium Prostate Pathology Committee. Cancer Res 73(9): 2718-2736.
  11. Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M. and Altman, D. G. (2010). Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol 8(6): e1000412.
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  16. Simons, B. W., Durham, N. M., Bruno, T. C., Grosso, J. F., Schaeffer, A. J., Ross, A. E., Hurley, P. J., Berman, D. M., Drake, C. G., Thumbikat, P. and Schaeffer, E. M. (2015). A human prostatic bacterial isolate alters the prostatic microenvironment and accelerates prostate cancer progression. J Pathol 235(3): 478-489.
  17. Slaoui, M. and Fiette, L. (2011). Histopathology procedures: from tissue sampling to histopathological evaluation. Methods Mol Biol 691: 69-82.
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简介

前列腺癌是美国男性最常见的癌症之一。对前列腺癌生物学的全面了解将具有重要的临床意义。动物模型大大影响了我们对疾病的了解,并将继续成为未来研究的宝贵资源。在这里,我们描述了将一种鼠前列腺癌细胞系(Myc-CaP)原位植入免疫能力小鼠前列腺的详细方案。

背景 由于转移的癌症的一小部分,前列腺癌是男性癌症死亡的主要原因。然而,驱动局部肿瘤发展和进展为转移性疾病的遗传和分子因素仍未完全了解。基因工程小鼠(GEM)模型和前列腺癌的异种移植模型都有助于我们对前列腺癌遗传学的了解(Ittmann等人,2013; Park等人,2010)。通过前列腺特异性转基因过表达如Hi-Myc小鼠(Ellwood-Yen等人,2003)或通过前列腺特异性缺失例如在Pten中的遗传操作, - 小鼠(Wang等人,2003)是有利的,因为它模拟了免疫能力小鼠的器官微环境中的肿瘤发展和进展。转移性前列腺癌的发展在这些GEM模型中是可变的,其中一些低频率, 其他模型如TRAMP(转基因腺癌​​小鼠前列腺)(reenberg等,1995)和Hi-Myc / Pten < / - (Hubbard等人,2016)。尽管他们对前列腺癌研究有很大的实用价值,但进一步的遗传操纵GEM模型是困难,耗时和昂贵的。为了克服这些局限性,研究人员依赖于人类细胞系的皮下和原位异种移植物。细胞系可以以多种方式在体外进行遗传操作。虽然皮下异种移植物由于其易于注射和监测而是有利的,但原位异种移植物更好地概括了可能影响药物敏感性的局部肿瘤微环境(Wilmann等人,1992; Kuo等人,1993),甲基化模式(Fleming等人,2010),增长率(Fleming等,2010),最终预测临床反应(Killion等人,1998; Hoffman,1999)。此外,一些人类前列腺癌细胞系模型从原位植入异种移植物转移。所有异种移植模型的限制是它们需要免疫受损的小鼠,使得难以在完整的免疫系统中模拟肿瘤进展。 Myc-CaP细胞系(Watson等人,2005)允许在免疫能力同基因(FVB / N)小鼠(Watson等人)中皮下或原位植入。 ,2005; Hurley等人,2015)。 Myc-CaP衍生自Hi-Myc小鼠的前列腺癌(Watson等人,2005)。当植入原位时,Myc-CaP细胞转移到腹部淋巴结,肝和肺(Hurley等,2015)。另外,Myc-CaP可以在体外实施基因表达的操纵(Hurley等人,2015)。因此,Myc-CaP可以用作前列腺肿瘤生长和具有完整免疫系统的小鼠转移生长的易于操作的模型。在这里,我们描述了将Myc-CaP细胞原位植入小鼠前列腺的方法。

关键字:原位同种异体移植物, Myc-CaP, 前列腺, 癌症, 转移, 体内, 小鼠模型

材料和试剂

  1. 机构ACUC批准原位同种异体移植协议
  2. 面罩,帽子,干净的实验室外套,手臂盖和无菌手套
  3. 组织培养瓶
  4. 70%酒精擦拭纸(DUKA1,目录号:852)
  5. 棉签
  6. 30 G½"针(BD,目录号:305106) - 原位植入
  7. 反射9毫米不锈钢伤口夹(Roboz手术器械,目录号:RS-9262)
  8. 21 G 1"针& 3 ml注射器(BD,目录号:309575) - 肺膨胀
  9. POLYSORB TM 尺寸3-0 USP(2公制),30英寸(75厘米)在V-20针吸收性外科缝合线上接触(COVIDIEN,目录号:GL322)
  10. Myc-CaP细胞(ATCC,目录号:CRL-3255)
  11. FVB/N雄性小鼠(THE JACKSON LABORATORY),10周龄以上
    注意:后短柔毛小鼠具有雄激素信号传导和较大的前列腺,便于移植。我们还建议使用与年龄和体重大致相同的小鼠。男性GEM模型也可以使用,只要它们完全反向FVB/N。
  12. Matrigel(康宁,目录号:354234)
  13. 1×磷酸缓冲盐水(PBS),pH 7.4(Thermo Fisher Scientific,Gibco TM,目录号:10010-023)
  14. 1x胰蛋白酶-EDTA(Mediatech,目录号:25-053-CI)
  15. Fluriso异氟烷(VetOne,目录号:501017)
  16. 10%聚维酮碘"Betadine"制剂溶液,16盎司(Henry Schein,目录号:6906950)
  17. 50mg/ml可注射Carprofen(Zoetis,目录号:07-844-7425)
  18. 乙醇,试剂酒精200,PHARMCO-AAPER TM (Thermo Fisher Scientific,Fisher Scientific,目录号:16-100-824)
  19. ImmPRESS HRP抗兔聚合物检测试剂盒(Vector Labs,目录号:MP-7401)
  20. ImmPACT DAB过氧化物酶底物(载体实验室,目录号:SK-4105)
  21. Myc-CaP生长培养基(见食谱)
    1. Dulbecco改良的Eagle's培养基(DMEM)(Thermo Fisher Scientific,Gibco TM,目录号:11965-092)
    2. 胎牛血清(Gemini Bio-Products,目录号:100-106)
    3. 青霉素 - 链霉素(10,000U/ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140-122)
    4. 牛垂体提取物(BPE)(Thermo Fisher Scientific,Gibco TM,目录号:13028-014)
    5. 重组人EGF(R& D Systems,目录号:236-EG)
    6. 牛胰岛素粉(Gemini Bio-Products,目录号:700-112)

设备

  1. 孵化器
  2. 离心机
  3. 细胞培养层流罩,真空抽吸,离心机,细胞计数器
  4. 2 L感应室
  5. 剪刀(Roboz手术器械,目录号:RS-5914)
  6. 精镊(Roboz手术器械,目录号:RS-5132)
  7. 鼠标房屋和搬运设施
  8. 包括蒸发器,感应室,管道,鼻锥和氧气罐的异氟烷气体麻醉系统(VetEquip,目录号:922100)
  9. 玻璃珠灭菌器(GERMINATOR 500)(Roboz Surgical Instrument,目录号:DS-401)
  10. 充电剃须刀
  11. 空间凝胶加热垫(Braintree Scientific,目录号:SPGL)
  12. 25μl,702 RN型注射器(Hamilton,目录号:7636-01)
  13. 注射器导管25μl-500μl/7000 SYR(Hamilton,目录号:14906)

程序

注意:所有在小鼠中进行的手术必须首先获得ACUC机构的批准

  1. 用于注射的Myc-CaP细胞的培养和制备
    1. 在具有Myc-CaP生长培养基的组织培养瓶中传播Myc-CaP细胞并按照ATCC指南进行传代。每只小鼠移植1×10 6个Myc-CaP细胞。由于针内的"死空间",高估了20%或至少两个额外同种异体移植物所需的同种异体移植物数量。
      注意:不推荐每只小鼠注射超过1×10 6个/m 2的Myc-CaP细胞,因为它会加速原发性肿瘤的生长,并缩短由于发病而导致的移植和安乐死之间的时间。这可能会降低观察转移性疾病的可能性。
    2. 在收获Myc-CaP细胞注射之前,在冰上解冻Matrigel。
    3. 从含有80-90%Myc-CaP细胞汇合单层的培养瓶中取出培养基,用1×PBS洗涤细胞,并在37℃培养箱中,在0.05%胰蛋白酶-EDTA中孵育3-6分钟来分离细胞。通过添加生长培养基中和胰蛋白酶。
    4. 将细胞转移到50ml锥形管中,并以300×g离心5分钟。取出胰蛋白酶/培养基,然后将细胞沉淀重新悬浮于新鲜生长培养基中
    5. 计数细胞以每个同种异体移植物分离1×10 6个细胞。将分离的细胞悬浮液以300×g离心5分钟。
    6. 在冰上执行以下步骤。取出培养基并将基质胶中的细胞沉淀重悬于每1×10 6细胞10μl的体积中。保持细胞:Matrigel混合物在冰上直到植入。
      注意:在植入之前彻底混合细胞:Matrigel。

  2. 将Myc-CaP细胞移植到前列腺中> 10周雄性FVB/N小鼠
    1. 根据您的机构动物护理和使用委员会(ACUC)指南,使用无菌技术在层流罩中进行移植。在整个程序中佩戴面具,帽子,干净的实验室外套,手臂盖和无菌手套。使用玻璃珠灭菌器灭菌仪器,并在动物之间用70%乙醇消毒。
    2. 通过在氧气中吸入2%异氟烷,使用2L诱导室诱导全身麻醉。完全麻醉诱导后,通过鼻锥保持约1.5%异氟烷麻醉,根据需要进行调整,以保持深层麻醉平面(图1)。麻醉水平足以在手指之间捏住皮肤之间的数字不刺激踏板撤退。
    3. 根据您的制度准则提供抢先镇痛。
      注意:根据我们制定上认可的动物方案,我们将5毫克/千克卡洛芬的皮下注射到无菌盐水中,总体积为0.5毫升。
    4. 将麻醉的老鼠置于背侧卧位,将头发刮到手术部位,用Betadine擦拭手术部位,然后用70%酒精擦拭拭子(图1)。
    5. 使用无菌剪刀和细镊子,在横截面上方2厘米长的水平切口(图1和Bertrand等人,2016)。一旦腹壁暴露,做一个类似的切口打开腹部(图1)。
    6. 使用细镊子将膀胱轻轻地从腹部操作,并朝向包皮。这暴露了精囊和附着的前前叶(图1,和Oliveira等人,2016)。用细镊子,棉签轻轻地将一个精液囊泡和前列腺从肛门排出腹部,或轻轻地压在腹部(图1)。特别注意防止精液囊泡的损伤,因为精液渗入腹部会引起强烈的炎症反应。
    7. 在将精囊露出和外观后,插入一个预先装有Myc-CaP细胞的25μlHamilton注射器:Matrigel混合物,并将30G½"针纵向配合到前列腺前(平行于精囊的长度)。慢性注射10μl混合物(等于1×10 6个Myc-CaP细胞)到前列腺中(图1)。当从前列腺去除针头时,注意避免注射的内容物溢出,因为溢出可能导致腹部多个肿瘤的播种。为了避免溢出,注射后保持针头1-2秒(使Matrigel时间在叶片内固化),并在取出针后用棉签轻轻按压注射部位。
      注意:在前叶中注入10μl以上会增加溢出的可能性。小鼠前列腺的其他裂片可以用于注射,然而,前叶提供了位置的便利性,并且在生长时尿道收缩的可能性降低。在实验过程中,移植网站的选择应保持一致。
    8. 轻轻将精囊和膀胱返回腹部,然后用3-0连续可吸收的缝合线封闭腹壁。用9毫米钢伤口夹紧紧皮肤(图1)。
      注意:对于盲目的研究,按照制度指导标记具有脚趾纹身或耳朵的动物。给每只动物一个唯一的编号进行识别。


      图1.将Myc-CaP细胞移植到小鼠前列腺中A.在诱导全身麻醉后(步骤B2),用Betadine剃去手术部位,然后用酒精擦拭(步骤B4 )。在注射腺上方形成1cm切口(步骤B5),暴露前列腺前(步骤B6)。将Myc-CaP细胞注射到前列腺前(步骤B7),然后用缝线和手术夹闭合切口(步骤B8); B.假性腺体的解剖近似(P.G.,虚线)和切口部位(实线黑线)。阴囊(S)和包皮(P)标记为参考; C.切口部位的小鼠器官解剖。膀胱(B,显示为尿液)和精囊(S.V.,仅左侧暴露)分别容易地在切口部位识别其中线位置和白色喇叭形结构。前列腺前叶(A.P.)叶平行于沿其较小曲率附着的精囊运动。 D.增加Myc-CaP细胞注射入前列腺的观点
  3. 术后护理
    1. 手术部位闭合后,将动物放在消毒的加热垫上的干净的笼子中,以恢复恢复期间的体温
    2. 小鼠应在手术后2小时内积极进行警惕。间歇地监视鼠标,直到警惕。
    3. 每天监测小鼠遇难或感染的迹象,如新郎失败,体重减轻,不情愿移动,呼吸困难。
    4. 如果动物出现疼痛或痛苦迹象,术后每72小时皮下注射5毫克/千克卡洛芬。

  4. 尸检,加工染色
    1. 植入长度取决于实验设计和终点。
      注意:根据我们的经验,将1×10 6个Myc-CaP细胞移植到前列腺前列腺中产生约60mm 3的大小和微转移性疾病的同种异体移植物在移植后21天(Hurley等,2015)。微转移性疾病往往很难得到很大的欣赏。
      植入后,根据您的尸体解剖制度准则安乐死小鼠。解剖原发性肿瘤远离精囊,称重。如果移植的肿瘤不能从相邻的精囊清除,请与精囊一起去除肿瘤并整体称重。检查小鼠是否有转移性疾病的证据,如果有的话,可以量化可见的大转移性病变的数量和位置。照相移植的病变和任何宏观转移性病变。
    2. 取出腹部器官用于福尔马林固定和石蜡包埋。苏木精和伊红(H& E)染色的部分器官(Slaoui和Fiette,2011)。此外,在福尔马林固定前,取出肺部并用福尔马林充气以进行最佳病理检查。为了使肺膨胀,将连接到装有固定剂的3ml注射器的21G针头在其开放端引入气管中,使用镊子轻轻地夹在针头周围,并引入固定剂直至气管多余回流(Fiette和Slaoui,2011)。经认证的病理学家应分析H& E染色的幻灯片用于微转移性疾病。
    3. Myc-CaP细胞在转移灶中的存在可以通过用于雄激素受体(AR)和Myc阳性的福尔马林固定和石蜡包埋(FFPE)组织的免疫组织化学(IHC)染色来证实(图2)。 IHC染色方案从先前发表的研究(Simons等人,2015)中改编。


      图2.Myc-CaP原位同种异体移植物和H& E和IHC染色。
      A.Myc-CaP小鼠前列腺前列腺同种异体移植物(箭头)。精囊(S.V.)和前列腺(A.P.)。 Myc和CaP原位异体移植肿瘤的Myc和雄激素受体(AR)的H& E和IHC染色; B.Myc-CaP原位同种异体移植肿瘤(T)和相邻精囊分泌物(S,亮粉色染色)的H& E和IHC染色; C.H& E和IHC染色的来自Myc-CaP原位同种异体移植物的肺转移。用ImmPRESS聚合物检测试剂盒和ImmPACT DAB(Vector Laboratories)显现阳性Myc和AR染色,显示为棕色。 IHC载玻片用苏木精复染。 10倍放大倍数(20x插图)和黑色条纹的图像为100μm。

数据分析

我们建议您考虑动物研究设计,分析和报告中的"体内动物研究指南(Kilkenny等,2010)。如"ARRIVE指南"中所概述的,综合记录研究设计,实验程序,实验动物,动物住房和畜牧业,样本量,动物分配到实验组,实验结果和统计方法(Kilkenny等人, em>。,2010)。为了确定样本大小(每个实验组和对照组的动物数量),首先计算连续和分类测量所需的效应大小。例如,如果在试点研究中,我们确定对照组的原发性肿瘤平均重(0.122 g±0.072 g),实验组原发肿瘤重(0.038 g±0.0133 g),则效应大小 = 1.622467。使用α误差概率为0.05,功率= 0.8,对照/实验的分配比为1的双侧测试,两组的样本量应为8只动物,以显示组间差异有统计学意义(Festing和Altman,2002)。要占20%的错误率,我们每组使用10只动物。
注意:首先应进行初步研究,以确定给定实验的适当样本量。我们强烈建议在任何动物研究的项目设计阶段咨询生物统计学家。
数据分析的适当统计方法将取决于评估的实验设计和实验结果。为了提高实验的鲁棒性,我们推荐两个独立的实验重复和盲目的数据分析。如果任何动物被排除在最终分析之外,提供排除的良好理由,例如细胞渗漏到体腔中,如体腔壁上的多发性转移病变所证明的。

食谱

  1. Myc-CaP生长培养基
    500毫升DMEM
    50ml胎牛血清
    5 ml青霉素 - 链霉素
    25μg/ml牛垂体提取物 5μg/ml牛胰岛素 6ng/ml重组人表皮生长因子
    注意:Myc-CaP细胞也可以在含有牛血清的DMEM中培养至终浓度为10%。

致谢

该协议由先前发表的研究(Hurley等人,2015年)改编而成。这项工作得到前列腺癌基金会哈根挑战奖的支持;帕特里克·沃尔什前列腺癌基金,以及欣曼泌尿基金教育奖学金。

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引用:Hughes, R. M., Simons, B. W. and Hurley, P. J. (2017). A Murine Orthotopic Allograft to Model Prostate Cancer Growth and Metastasis. Bio-protocol 7(4): e2137. DOI: 10.21769/BioProtoc.2137.
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