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Construction of Human Monocyte Derived Macrophages Armed with Oncolytic Viruses
溶瘤病毒活化的人单核细胞源性巨噬细胞的构建   

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Abstract

Macrophages are involved in many key physiological processes and complex responses such as inflammatory, immunological, infectious and neoplastic diseases. The appearance and activation of macrophages are thought to be rapid events in the development of many pathological lesions, including malignant tumours, atherosclerotic plaques, and arthritic joints. This has prompted recent attempts to use macrophages as novel cellular vehicles for gene therapy, in which macrophages are genetically modified ex vivo and then reintroduced into the body with the hope that a proportion will then home to the diseased site. Here, we describe a protocol for preparing monocyte-derived macrophages (MDM) and arming these with oncolytic viruses (OV) as a novel way for delivering anti-cancer therapies. In this approach, proliferation of macrophages co-transduced with a hypoxia-regulated E1A/B construct and an E1A-dependent oncolytic adenovirus, is restricted to prostate tumour cells using prostate-specific promoter elements from the TARP, PSA, and PMSA genes (Muthana et al., 2013; Muthana et al., 2011). When such co-transduced cells reach an area of extreme hypoxia (like that found in tumours), the E1A/B proteins are expressed, thereby activating replication of the adenovirus. The virus is subsequently released by the host macrophage and infects neighboring tumour cells. The virus then infects neighboring cells but only proliferates and is cytotoxic in prostate tumour cells. OV kill cancer cells by a number of mechanisms, including direct lysis, apoptosis, autophagy and shutdown of protein synthesis, as well as the induction of anti-tumoural immunity. Using macrophages to deliver OV ensures that they are protected from the many hazards they face in circulation including neutralizing antibodies, complement activation and non-specific uptake by other tissues such as the liver and spleen.

Materials and Reagents

  1. Blood (Sheffield Blood Transfusion Service)
  2. 50 ml falcon tubes (BD Biosciences, Falcon®, catalog number: 734-0451 )
  3. Hank's Balanced Salt Solution (HBSS) (without Calcium, Ca2+ and Magnesium, Mg2+) (Lonza BioWhittaker Ltd., catalog number: 10-543Q )
  4. Ficoll-Hypaque (GE Healthcare, catalog number: 17-1440-02 )
  5. IMDM medium (Lonza BioWhittaker Ltd., catalog number: 12-722 )
  6. Human AB serum (Lonza BioWhittaker Ltd., Wokingham, UK)
  7. L-Glutamine (Lonza BioWhittaker Ltd., catalog number: 17-605 )
  8. RPMI medium (Lonza BioWhittaker Ltd., catalog number: 12-115 )
  9. Fetal bovine serum (FBS) (Biosera, catalog number: FB-1000 )
  10. Phosphate buffered saline (PBS) (Lonza BioWhittaker Ltd., catalog number: 17-516F )
  11. Trypsin/EDTA (Promocell Ltd., catalog number: C-41000 )
  12. Adenoviruses (Adenoviruses used in our experiment include AdCMV-GFP, and oncolytic Ad[1/PPTE1A]) (kind gift from professor Magnus Essand, Uppsala, Sweden)
  13. Hypoxia driven plasmid (HRE-E1A/B)
  14. Macrophage Amaxa Nucleofector® kit (Lonza BioWhittaker Ltd.)

Equipment

  1. T75 tissue culture flask (BD Biosciences, Falcon®)
  2. 12-well plate (BD Biosciences, Falcon®, catalog number: 734-2324 )
  3. Centrifuge (MSE Mistral 2000R)
  4. Haemocytometer (Camlab Limited, catalog number: 1127884 )
  5. Nucleofector® machine (Lonza BioWhittaker Ltd.)

Procedure


I.   Monocyte Isolation

  1. Venous blood collected by Sheffield Teaching Hospitals (STH) blood bank was collected from healthy donors and consolidated into one bag of waste buffy coat.
  2. 25 ml of this blood was poured into 50 ml Falcon tubes and then filled up to the 40 ml line with 15 ml of HBSS (without Calcium, Ca2+ and Magnesium, Mg2+).
  3. The tubes were then spun at 1,200 x g for 20 min (4 °C and Brake Rate of 0).
  4. After spinning, the cloudy white blood cell layer (below the plasma) was removed by pipette and collecting the transferred into two new 50 ml Falcon tubes where it was made up to 30 ml with HBSS (w/o Ca2+/Mg2+).
  5. The 30 ml of white cells were then carefully layered on top of 20 ml of Ficoll-Hypaque (in 50 ml Falcon tubes).
  6. These tubes were then placed in the centrifuge and once again spun at 1,200 x g (4 °C and Brake Rate of 0) for 20 min.
  7. Once again, the cloudy white cell layer was removed and made up to 40 ml with HBSS (w/o Ca2+/Mg2+) in a new 50 ml Falcon tube.
  8. This tube was then spun at 400 x g for 5 min (4 °C and Brake Rate of 3) to wash and pellet the cells.
  9. This process was repeated three times.
  10. The cells were then counted using a haemocytometer and diluted with IMDM (supplemented with 2% Human AB serum and 4 mM L-Glutamine) so that there were 5-7 x 106 cells /ml.
  11. To plate the cells down 1 ml of this cell suspension was added to each T75 (BD falcon-these flasks are superior when it comes to infecting with virus) flask and topped up to 10 ml with supplemented IMDM. The cells were then incubated for two hours (37 °C, 5% CO2). After two hours the flasks were washed with HBSS to remove the non-adherent cells and then refilled with 10 ml of supplemented IMDM.


II.  Subculture of monocytes and differentiation into monocyte derived macrophages (MDM)

Monocytes isolated as above and cultured in 10 ml of IMDM, supplemented with L-glutamine (4 mM) and Human AB serum (2%), for the first 24 h. After this point the medium was replaced with 5 ml of RPMI, supplemented with L-glutamine (4 mM) and 10% FBS. Medium was replaced every three days depending on which other procedures were performed and monocyte derived MDM. Macrophages are visible after 24 hours these have a different morphology to monocytes, which are non-adherent and spherical. Macrophages on the other hand are larger and have irregular features. The can possess dendrites and appear spindle shaped.


III. Co-transduction of MDM

MDM were co-transduced with a therapeutic oncolytic adenovirus and a hypoxia-regulated plasmid carrying the replication component of the virus E1A. A description of the virus and plasmid can be found in (Muthana et al., 2013; Muthana et al., 2011).

Note: The adenoviruses used in this study include AdCMV-GFP, and oncolytic Ad[1/PPTE1A].

  1. MDM infection with human adenovirus
    1. The plated monocytes were washed the following day with HBSS and re-suspended in 5 ml of RPMI (supplemented with 10% FBS and L-Glutamine). This prevents the virus binding to the Human AB serum in the IMDM and neutralising the viral particles.
    2. The viral particles were added at a multiplicity of infection (MOI) 100 and placed in the incubator over-night.
    3. After an overnight incubation the cells were washed in PBS and fresh supplemented RPMI medium was added. Virus infection was assessed by flow cytometry and fluorescent microscopy for expression of green fluorescent protein (GFP) reporter gene.
  2. Transfection of MDM using Amaxa Nucleofection
    1. 24 h later infected macrophages were harvested using trysin/EDTA and then transfected with a hypoxia driven plasmid (HRE-E1A/B) using the macrophage Amaxa Nucleofector® kit. This was carried out according to the manufacturer’s protocol. In brief, RPMI (1.5 ml) supplemented with 10% FCS and L-glutamine was added to the wells of a 12-well plate and pre-incubated at 37 °C.
    2. Three-day-old MDM were trypsinised, centrifuged at 400 x g for 5 min (at room temperature) and resuspended at 8 x 105 cells/ ml. The supernatant was completely discarded and 100 μl of Amaxa Nucleofector® solution added as well as 3 μg plasmid (HRE-E1A/B). The cell suspension was then inserted into the Nucleofector® machine and the Y-10 program selected. Once the machine displayed “OK”, the sample was removed, 500 μl of pre-warmed RPMI supplemented with 10% FCS and L-glutamine was added and the entire cell suspension was transferred to the appropriately labelled well. The 12-well plate was then incubated for 24 h. Plasmid transfection was optimised using the pmaxGFP plasmid supplied with the kit according to the manufacturer’s protocol. In brief, GFP expression was assessed 24-48 h after transfection by fluorescent microscopy and flow cytometry.
    3. Co-transduced macrophages can be delivered to tumour cells in vitro (e.g. 3D tumour spheroids) or intravenously to tumour bearing mice. Tumour cell death as a result of the OV killing can be determined.

Acknowledgments

This protocol is adapted from Muthana et al. (2011) and Muthana et al. (2013).

References

  1. Muthana, M., Rodrigues, S., Chen, Y. Y., Welford, A., Hughes, R., Tazzyman, S., Essand, M., Morrow, F. and Lewis, C. E. (2013). Macrophage delivery of an oncolytic virus abolishes tumor regrowth and metastasis after chemotherapy or irradiation. Cancer Res 73(2): 490-495.
  2. Muthana, M., Giannoudis, A., Scott, S. D., Fang, H. Y., Coffelt, S. B., Morrow, F. J., Murdoch, C., Burton, J., Cross, N., Burke, B., Mistry, R., Hamdy, F., Brown, N. J., Georgopoulos, L., Hoskin, P., Essand, M., Lewis, C. E. and Maitland, N. J. (2011). Use of macrophages to target therapeutic adenovirus to human prostate tumors. Cancer Res 71(5): 1805-1815.

简介

巨噬细胞参与许多关键的生理过程和复杂的反应,例如炎症,免疫,感染和肿瘤疾病。巨噬细胞的出现和活化被认为是许多病理损伤的发展中的快速事件,包括恶性肿瘤,动脉粥样硬化斑块和关节炎性关节。这已经促使最近尝试使用巨噬细胞作为用于基因治疗的新型细胞载体,其中巨噬细胞在体外进行遗传修饰,然后重新引入体内,希望一部分随后归巢到患病部位。在这里,我们描述了制备单核细胞衍生的巨噬细胞(MDM)和武装与溶瘤病毒(OV)作为一种新的方式提供抗癌治疗的协议。在这种方法中,使用来自TARP的前列腺特异性启动子元件将与缺氧调节的E1A/B构建体和E1A依赖性溶瘤腺病毒共转导的巨噬细胞的增殖限制于前列腺肿瘤细胞, , ,2011; Muthana等人。当这种共转导的细胞到达极端缺氧区域(如在肿瘤中发现的)时,E1A/B蛋白被表达,从而激活腺病毒的复制。该病毒随后被宿主巨噬细胞释放并感染相邻的肿瘤细胞。然后病毒感染邻近细胞,但仅在前列腺肿瘤细胞中增殖并且是细胞毒性的。 OV通过许多机制杀死癌细胞,包括直接裂解,凋亡,自噬和蛋白质合成的关闭,以及诱导抗肿瘤免疫。使用巨噬细胞递送OV确保它们被保护免于在循环中面临的许多危险,包括中和抗体,补体激活和其他组织例如肝和脾的非特异性摄取。

材料和试剂

  1. 血(谢菲尔德输血服务)
  2. 50ml falcon管(BD Biosciences,Falcon ,目录号:734-0451)
  3. Hank's平衡盐溶液(HBSS)(不含钙,Ca 2+和Mg 2+,Mg 2+)(Lonza BioWhittaker Ltd.,目录号:10-543Q)
  4. Ficoll-Hypaque(GE Healthcare,目录号:17-1440-02)
  5. IMDM培养基(Lonza BioWhittaker Ltd.,目录号:12-722)
  6. 人AB血清(Lonza BioWhittaker Ltd.,Wokingham,UK)
  7. L-谷氨酰胺(Lonza BioWhittaker Ltd.,目录号:17-605)
  8. RPMI培养基(Lonza BioWhittaker Ltd.,目录号:12-115)
  9. 胎牛血清(FBS)(Biosera,目录号:FB-1000)
  10. 磷酸盐缓冲盐水(PBS)(Lonza BioWhittaker Ltd.,目录号:17-516F)
  11. 胰蛋白酶/EDTA(Promocell有限公司,目录号:C-41000)
  12. 腺病毒(我们实验中使用的腺病毒包括AdCMV-GFP和溶瘤Ad [1/PPTE1A])(来自瑞典乌普萨拉的Magnus Essand教授的赠品)
  13. 缺氧驱动质粒(HRE-E1A/B)
  14. 巨噬细胞Amaxa Nucleofector 试剂盒(Lonza BioWhittaker Ltd.)

设备

  1. T75组织培养瓶(BD Biosciences,Falcon )
  2. 12孔板(BD Biosciences,Falcon ,目录号:734-2324)。
  3. 离心机(MSE Mistral 2000R)
  4. 血球计(Camlab有限公司,目录号:1127884)
  5. Nucleofector 机器(Lonza BioWhittaker Ltd.)

程序


I.   单核细胞分离

  1. 谢菲尔德教学医院(STH)采集的静脉血 银行从健康捐助者收集并合并成一袋 废白毛皮。
  2. 将25ml该血液倒入50ml中 Falcon管中,然后用15ml HBSS填充至40ml管线 (不含钙,Ca 2+和镁,Mg 2+和)。
  3. 然后将管在1,200×g下旋转20分钟(4℃,制动速率为0)。
  4. 后   纺丝,多云白细胞层(在血浆下) 通过移液管取出并收集转移到两个新的50ml中 Falcon管中,其中用HBSS(w/o Ca 2+ + Mg 2+/sup>)补充至30ml。
  5. 然后将30ml白细胞小心地铺在20ml Ficoll-Hypaque(在50ml Falcon管中)的顶部。
  6. 然后将这些管置于离心机中,并再次以1,200×g(4℃,制动速率为0)旋转20分钟。
  7. 再次,除去混浊的白色细胞层,并在新的50ml Falcon中用HBSS(w/o Ca 2+ +/Mg 2+ 2+)补充至40ml 管。
  8. 然后将该管在400×g下旋转5分钟(4℃,制动速率为3),以洗涤细胞并使其沉淀。
  9. 该过程重复三次。
  10. 的   然后使用血细胞计数器计数细胞,并用IMDM稀释 (补充有2%人AB血清和4mM L-谷氨酰胺),使得在那里   为5-7×10 6个细胞/ml。
  11. 平板细胞下1毫升 将该细胞悬浮液加入到每个T75(BD falcon-这些烧瓶 优越的感染病毒)烧瓶和加满 10毫升与补充的IMDM。 然后将细胞孵育2小时 小时(37℃,5%CO 2)。 2小时后,用HBSS洗涤烧瓶 以除去非粘附细胞,然后用10ml的补充物补充 补充IMDM。


II。  单核细胞亚培养和分化成单核细胞衍生的巨噬细胞(MDM)

如上所述分离的单核细胞,并在补充有L-谷氨酰胺(4mM)和人AB血清(2%)的10ml IMDM中培养第一个24小时。此后,用5ml补充有L-谷氨酰胺(4mM)和10%FBS的RPMI替换培养基。每三天更换培养基,取决于进行的其他操作和单核细胞衍生的MDM。巨噬细胞在24小时后可见,它们具有与单核细胞不同的形态,其是非粘附的和球形的。另一方面,巨噬细胞较大且具有不规则的特征。可以拥有树突和出现纺锤形。


III。 MDM的共转导

MDM与治疗性溶瘤腺病毒和携带病毒E1A的复制组分的低氧调节的质粒共转导。病毒和质粒的描述可以在(Muthana等人,2013; Muthana等人,2011)中找到。

注意:用于本研究的腺病毒包括AdCMV-GFP和溶瘤Ad [1/PPTE1A]。

  1. 人类腺病毒的MDM感染
    1. 第二天用HBSS洗涤接种的单核细胞 重悬于5ml RPMI(补充有10%FBS, L-谷氨酰胺)。 这防止病毒与人AB血清结合 IMDM和中和病毒颗粒
    2. 以多重感染(MOI)100加入病毒颗粒,并置于孵育箱中过夜
    3. 后   过夜温育,将细胞在PBS中洗涤并且新鲜 补充的RPMI培养基。 通过流量评估病毒感染   细胞计数和荧光显微镜用于表达绿色 荧光蛋白(GFP)报告基因。
  2. 使用Amaxa核转染法转染MDM
    1. 24小时后,使用trysin/EDTA收获感染的巨噬细胞,然后使用巨噬细胞Amaxa Nucleofector 试剂盒用缺氧驱动的质粒(HRE-E1A/B)转染。这根据制造商的方案进行。简言之,将补充有10%FCS和L-谷氨酰胺的RPMI(1.5ml)加入12孔板的孔中并在37℃下预孵育。
    2. 将三日龄MDM胰蛋白酶化,在400×g离心5分钟(在室温下),并以8×10 5个细胞/ml重悬。完全弃去上清液,加入100μlAmaxa Nucleofector溶液以及3μg质粒(HRE-E1A/B)。然后将细胞悬浮液插入Nucleofector 机器中,并选择Y-10程序。一旦机器显示"OK",取出样品,加入500μl预热的补充有10%FCS和L-谷氨酰胺的RPMI,将整个细胞悬浮液转移到适当标记的孔中。然后将12孔板孵育24小时。使用根据制造商的方案提供的试剂盒提供的pmaxGFP质粒优化质粒转染。简言之,在荧光显微镜和流式细胞术转染后24-48小时评估GFP表达。
    3. 共转导的巨噬细胞可以在体外(例如3D肿瘤球体)递送至肿瘤细胞或静脉内递送至荷瘤小鼠。可以确定作为OV杀伤的结果的肿瘤细胞死亡。

致谢

该协议改编自Muthana等人(2011)和Muthana等人(2013)。

参考文献

  1. Muthana,M.,Rodrigues,S.,Chen,Y. Y.,Welford,A.,Hughes,R.,Tazzyman,S.,Essand,M.,Morrow,F.and Lewis,C.E。 巨噬细胞溶瘤病毒的递送消除了化疗或照射后的肿瘤再生和转移。 em> Cancer Res 73(2):490-495。
  2. Muthana,M.,Giannoudis,A.,Scott,SD,Fang,HY,Coffelt,SB,Morrow,FJ,Murdoch,C.,Burton,J.,Cross,N.,Burke,B.,Mistry, ,Hamdy,F.,Brown,NJ,Georgopoulos,L.,Hoskin,P.,Essand,M.,Lewis, C. E.和Maitland,N.J。(2011)。 使用巨噬细胞将治疗性腺病毒靶向人前列腺肿瘤 癌症研究 71(5):1805-1815。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Muthana, M., Richardson, J., Rodrigues, S. and Lewis, C. (2013). Construction of Human Monocyte Derived Macrophages Armed with Oncolytic Viruses. Bio-protocol 3(13): e809. DOI: 10.21769/BioProtoc.809.
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