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Adoptive Transfer of Myeloid-Derived Suppressor Cells and T Cells in a Prostate Cancer Model
前列腺癌模型中骨髓来源的抑制性细胞和T淋巴细胞的过继转移   

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Abstract

The adoptive transfer of immune cells for cancer, chronic infection, and autoimmunity is an emerging field that has shown promise in recent trials. The transgenic adenocarcinoma mouse prostate (TRAMP) is a classical mouse model of prostate cancer (PCa) and TRAMP cell lines were derived from a TRAMP mouse tumor. TRAMP-C2 is tumorigenic when (subcutaneously) s.c. grafted into syngeneic C57BL/6 host mice (Foster et al., 1997). This protocol will describe the adoptive transfer of purified CD11b+Gr1+ double positive (DP) myeloid-derived suppressor cells (MDSC) and CD3+ T cells in the TRAMP-C2 prostate cancer mouse model in order to establish the intrinsic functionality of these immune cells and to determine their role in tumorigenesis in vivo (Yan et al., 2014).

Materials and Reagents

  1. RPMI 1640 (Life Technologies, Gibco®, catalog number: 22400-089 )
  2. Trypan blue 0.4% solution (Lonza, catalog number: 17-942E )
  3. Myeloid-Derived Suppressor Cell Isolation Kit (mouse) (Miltenyi Biotec, catalog number: 130-094-538 )
  4. Pan T Cell Isolation Kit II, mouse (Miltenyi Biotec, catalog number: 130-095-130 )
  5. Antibodies for flow cytometry: Ly-6G-FITC (Gr1, RB6-8C5), CD11b-PE (M1/70), and CD3-FITC (17A2) (Biolegend, catalog numbers: 108405 , 01207 , and 100203 respectively)
  6. MACS® BSA Stock Solution (Miltenyi Biotec, catalog number: 130-091-376 )
  7. AutoMACS® Rinsing Solution (Miltenyi Biotec, catalog number: 130-091-222 )
  8. Phosphate buffer saline (PBS) (see Recipes)
  9. Sterile red blood cell lysis buffer (RBC lysis buffer) (see Recipes)
  10. MACS buffer (see Recipes)

Equipment

  1. LS column (Miltenyi Biotec, catalog number: 130-042-401 )
  2. MidiMACS Separator (Miltenyi Biotec, catalog number: 130-042-302 )
  3. MACS MultiStand (Miltenyi Biotec, catalog number: 130-042-303 )
  4. Wide field microscope (Nikon Diaphot Phase Contrast Inverted Laboratory Microscope, catalog number: 805426 )
  5. Sterile forceps and scissors
  6. Flow cytometer
  7. 1 ml syringes (29 G) (BD Biosciences, catalog number: 329410 )
  8. Sterile Cell strainers 70 μm (BD Biosciences, catalog number: 352350 )
  9. 15 ml conical tubes (BD Biosciences, catalog number: 352095 )
  10. Tabletop centifuge  
  11. Cell culture centrifuge
  12. Sterile culture hood
  13. Hemocytometer
  14. 60 mm cell culture dish

Procedure

  1. Isolation of splenocytes
    1. Prepare a single cell suspension from mouse (TRAMP-C2 tumor bearing, about 4 months old) spleens in the sterile culture hood. Disrupt the spleen with the plunger of a 1 ml syringe against a 70-μm cell strainer in a 60 mm petri dish filled with 2 ml of RPMI1640.
    2. Centrifuge single cell suspensions in 15 ml conical tubes at 300 x g for 10 min at RT.
    3.  Re-suspend the splenocytes with 5 ml of RBC lysis buffer and incubate 5 min at RT. Dilute with 10 ml PBS and centrifuge for 10 min at 300 x g. Re-suspend cell pellet in 5 ml MACS buffer (4 °C) and count viable cell numbers using a 0.4% Trypan blue solution. Each spleen yields about 200 x 106 splenocytes.
    4. One spleen can provide enough Gr1+CD11b+ DP cells for transplantation of 3 experimental mice; the CD3+ cells isolated from one spleen is also enough for transplantation of 3 experimental mice. Splenocytes from 2-3 individual spleens can be pooled before immune cell purification.

  2. CD11b+Gr1+ DP cells purification from splenocytes using Miltenyi Myeloid-Derived Suppressor Cell Isolation Kit (mouse, a kit for positive isolation of cells)
    1. Centrifuge cell suspension at 300 x g for 10 min at 4 °C in the 15 ml conical tubes. Aspirate supernatant completely.
    2. Re-suspend cell pellet in 350 µl of MACS buffer per 108 total cells.
    3. Add 50 µl of FcR Blocking Reagent per 108 total cells.
    4. Mix well and incubate for 10 min in the refrigerator (2-8 °C).
    5. Add 100 µl of Anti-Ly-6G-Biotin (MDSC-Kit).
    6. Mix well and incubate for 10 min in the refrigerator (2-8 °C).
    7.  Wash cells by adding 10 ml of MACS buffer per 108 cells and centrifuge at 300 x g for 10 min at 4 °C. Aspirate supernatant completely.
    8. Re-suspend up to 108 cells in 800 µl of MACS buffer.
    9. Add 200 µl of Anti-Biotin MicroBeads.
    10. Mix well and incubate for 15 min in the refrigerator (2-8 °C).
    11. Wash cells by adding 10 ml of MACS buffer per 108 cells and centrifuge at 300 x g for 10 min at 4 °C. Aspirate supernatant completely.
    12. Re-suspend up to 108 cells in 500 µl of MACS buffer.
    13. Place the LS column in the magnetic field of a MidiMACS separator.
    14. Equilibrate the column by rinsing with 3 ml of MACS buffer.
    15. Apply the cell suspension onto the column; collect flow-through containing unlabeled cells.
    16.  Wash the column with 3 x 3 ml of MACS buffer and collect unlabeled cells that pass through and combine with the effluent from step B15; keep unlabeled cells on ice until further processing.
    17. Remove the column from the separator and place it in a 15 ml conical tube.
    18.  Pipette 5 ml of MACS buffer onto the column; immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column and collect CD11b+Gr1+ DP cells.
    19. Count viable cell numbers using a 0.4% Trypan blue solution. Set aside 2 x 105 cells for evaluating purification efficiency as described below.

  3. Miltenyi T cells purification from splenocytes using Pan T Cell Isolation Kit II (mouse, a kit for negative isolation of cells)
    1. Count and centrifuge unlabeled cell suspension from steps B15-16; re-suspend cell pellet in 400 µl MACS buffer per 108 total cells.
    2. Add 100 μl of Biotin-Antibody Cocktail per 108 total cells.
    3. Mix well and incubate for 5 min in the refrigerator (2-8 °C).
    4. Add 300 μl of MACS buffer per 108 total cells.
    5. Add 20 μl of Anti-Biotin MicroBeads per 108 total cells.
    6. Mix well and incubate for 10 min in the refrigerator (2-8 °C).
    7. Place a LS Column in the magnetic field of a MidiMACS Separator.
    8. Prepare the column by rinsing with 3 ml of MACS buffer.
    9. Apply cell suspension onto the column and collect flow-through containing unlabeled cells, representing the enriched T cells.
    10.  Wash the column with 3 ml of MACS buffer and collect unlabeled cells that pass through, representing the enriched T cells; combine with the effluent from step C9.
    11. Count viable cell numbers using 0.4% Trypan Blue solution. Set aside 2 x 105 cells for evaluating purification efficiency as described below.

  4. Control of purification efficiency by flow cytometry
    1. Stain 2 x 105 total cells (step B19) with 20 μl of a suspension contained pre-titrated amounts of anti-mouse Ly-6G and anti-mouse CD11b. The antibodies are 1:100 diluted in PBS with 1% BSA.
    2. Stain 2 x 105 total cells (step C11) with 20 μl of a suspension contained pre-titrated amounts of anti-mouse CD3.
    3. Incubate 30 min at 4 °C and wash in 150 μl of washing buffer (PBS with 1% BSA).
    4. Centrifuge cell suspension at 4 °C and 500 x g for 5 min. Discard supernatant and keep the cell pellet.
    5. Re-suspend in 200 μl of washing buffer (PBS with 1% BSA) and analyze in a flow cytometer.


    Figure 1. The purity of CD11b+Gr1+ DP cells (Gr1 and CD11b antibodies) and CD3+ T cells (CD4 and CD3 antibodies) can be assessed by FACS analyses

  5. Mouse prostate cancer model and tail vein injection (≥ 5 mice/group)
    1. Mice with C57/Bl6 background are subcutaneously (s.c.) injected with TRAMP-C2 cells (3 x 106 cells in 0.2 ml PBS per mouse) on the same day of the adoptive transfer of immune cells (Yan et al., 2014). On day 7 and day 14 post-injections, an additional two doses of purified CD11b+Gr1+ DP cells (5 x 106 cells per mouse) or purified CD3+ T cells (5 x 106 cells per mouse) need to be adoptively transferred via intravenous injection. Mice will be sacrificed when they appeared moribund (45 days).
    2. Warm up the mice under a lamp for 5 min to achieve vasodilation for tail vein injection. Inject purified CD11b+Gr1+ DP cells (5 x 106 cells/200 μl PBS per mouse) or purified CD3+ T cells (5 x 106 cells/200 μl PBS per mouse) in the lateral tail vein with a 1 ml-syringe with a 29 G needle.
    3. Tumor development will be closely monitored, and tumor size will be measured every 7 days.


    Figure 2. DP, but not T cells from tumor challenged WT mice were sufficient to permit tumor growth in ogr1-/- mice injected with TRAMP0C2 cells. (Yan et al., 2014)

Recipes

  1. RBC lysis buffer
    0.15 M NH4Cl
    1 mM NaHCO3
    0.1 mM EDTA dissolved in sterile double distilled water
    Adjust pH to 7.2-7.4 with 1 M HCl
    Filter sterilize
  2.  Phosphate buffer saline (PBS)
    136 mM NaCl
    8.2 mM Na2HPO4
    1.5 mM KH2PO4
    2.7 mM KCl (pH 7.4)
  3. MACS buffer
    Prepare a solution containing phosphate-buffered saline (PBS) (pH 7.2), 0.5% bovine serum albumin (BSA), and 2 mM EDTA by diluting MACS® BSA Stock Solution1:20 with autoMACS® Rinsing Solution.
    Keep buffer cold (2-8 °C).
    Degas buffer before use, as air bubbles may block the column.

Acknowledgments

This work was supported in part by the National Institutes of Health (RO1 155145 to YX); and the Mary Fendrich-Hulman Charitable Trust Fund to YX.

References

  1. Foster, B. A., Gingrich, J. R., Kwon, E. D., Madias, C. and Greenberg, N. M. (1997). Characterization of prostatic epithelial cell lines derived from transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Cancer Res 57(16): 3325-3330.
  2. Yan, L., Singh, L. S., Zhang, L. and Xu, Y. (2014). Role of OGR1 in myeloid-derived cells in prostate cancer. Oncogene 33(2): 157-164.

简介

免疫细胞用于癌症,慢性感染和自身免疫的过继转移是一个新兴领域,其在最近的试验中显示出希望。 转基因腺癌小鼠前列腺(TRAMP)是前列腺癌(PCa)的经典小鼠模型,TRAMP细胞系衍生自TRAMP小鼠肿瘤。 当皮下注射时,TRAMP-C2是致瘤的。 移植到同基因C57BL/6宿主小鼠中(Foster等人,1997)。 该方案将描述纯化的CD11b +(Ad)双重阳性(DP)骨髓衍生的抑制细胞(MDSC)和CD3 + sup/+的过继转移。 T细胞在TRAMP-C2前列腺癌小鼠模型中的表达,以便建立这些免疫细胞的内在功能并且确定它们在体内的肿瘤发生中的作用(Yan等人)。 ,2014)。

材料和试剂

  1. RPMI 1640(Life Technologies,Gibco ,目录号:22400-089)
  2. 台盼蓝0.4%溶液(Lonza,目录号:17-942E)
  3. 骨髓来源的抑制细胞分离试剂盒(小鼠)(Miltenyi Biotec,目录号:130-094-538)
  4. Pan T Cell Isolation Kit II,小鼠(Miltenyi Biotec,目录号:130-095-130)
  5. 流式细胞术的抗体:Ly-6G-FITC(Gr1,RB6-8C5),CD11b-PE(M1/70)和CD3-FITC(17A2)(Biolegend,目录号分别为108405,01207和100203) />
  6. MACS BSA Stock Solution(Miltenyi Biotec,目录号:130-091-376)
  7. AutoMACS 冲洗溶液(Miltenyi Biotec,目录号:130-091-222)
  8. 磷酸盐缓冲盐水(PBS)(见配方)
  9. 无菌红细胞裂解缓冲液(RBC裂解缓冲液)(参见配方)
  10. MACS缓冲区(参见配方)

设备

  1. LS柱(Miltenyi Biotec,目录号:130-042-401)
  2. MidiMACS分离器(Miltenyi Biotec,目录号:130-042-302)
  3. MACS MultiStand(Miltenyi Biotec,目录号:130-042-303)
  4. 宽视场显微镜(Nikon Diaphot Phase Contrast Inverted Laboratory Microscope,目录号:805426)
  5. 无菌镊子和剪刀
  6. 流式细胞仪
  7. 1ml注射器(29G)(BD Biosciences,目录号:329410)
  8. 无菌细胞过滤器70μm(BD Biosciences,目录号:352350)
  9. 15ml锥形管(BD Biosciences,目录号:352095)
  10. 桌面centifuge
  11. 细胞培养离心机
  12. 无菌培养罩
  13. 血细胞计数器
  14. 60 mm细胞培养皿

程序

  1. 脾细胞的分离
    1. 准备单个细胞悬浮液从小鼠(支持TRAMP-C2肿瘤, 约4个月大)脾脏在无菌培养罩中。 中断 脾用1ml注射器的柱塞对着70-μm细胞过滤器 在装有2ml RPMI1640的60mm培养皿中。
    2. 将单细胞悬浮液在15ml锥形管中在300×g离心10分钟,在室温下离心。
    3.  用5ml RBC裂解缓冲液重新悬浮脾细胞,孵育5   min。 用10ml PBS稀释并在300×g离心10分钟。 重悬细胞沉淀在5ml MACS缓冲液(4℃),并计数活细胞 数字使用0.4%台盼蓝溶液。 每个脾产生约200×   10 6脾细胞。
    4. 一个脾可以提供足够的Gr1 + CD11b + DP 用于移植3只实验小鼠的细胞; CD3 + 细胞 从一个脾脏分离也足够移植3 实验小鼠。 可以汇集来自2-3个单独脾脏的脾细胞   免疫细胞纯化前。

  2. 使用Miltenyi髓样衍生的抑制细胞分离试剂盒(小鼠,用于细胞的阳性分离的试剂盒)从脾细胞中纯化CD11b + CD11b + +/- DP1 +细胞。
    1. 在4℃下在15ml锥形管中以300×g离心细胞悬浮液10分钟。 完全吸出上清液
    2. 重悬细胞沉淀在350微升MACS缓冲区每10 <支柱> 8 总细胞
    3. 每10 8个总细胞加入50μlFcR封闭试剂。
    4. 充分混合并在冰箱(2-8℃)中孵育10分钟
    5. 加入100μl的抗Ly-6G-生物素(MDSC-Kit)
    6. 充分混合并在冰箱(2-8℃)中孵育10分钟
    7.  通过每10 8个细胞加入10 ml MACS缓冲液洗涤细胞并离心 在300×g下在4℃温育10分钟。 完全吸出上清液
    8. 在800μlMACS缓冲液中重悬最多10个 8 细胞
    9. 加入200μl抗生物素MicroBeads。
    10. 充分混合并在冰箱(2-8℃)中孵育15分钟
    11. 通过每10 8个细胞加入10ml的MACS缓冲液洗涤细胞并离心 在300×g下在4℃温育10分钟。 完全吸出上清液
    12. 在500μlMACS缓冲液中重悬最多10个 8 细胞
    13. 将LS柱置于MidiMACS分离器的磁场中。
    14. 通过用3ml MACS缓冲液冲洗平衡柱
    15. 将细胞悬浮液应用到柱上; 收集含有未标记细胞的流过物
    16.  用3×3ml MACS缓冲液洗涤柱子,收集未标记的 细胞,其通过并与来自步骤B15的流出物合并; 保持未标记的细胞在冰上,直到进一步处理
    17. 从分离器中取出色谱柱,并将其放入15 ml锥形管中
    18.  移取5 ml MACS缓冲液至柱上; 立即冲出来 通过将柱塞牢固地推入柱中而被磁性标记的细胞   并收集CD11b + Gr1 + DP细胞
    19. 使用计数活细胞数   0.4%台盼蓝溶液。 放置2×10 5个细胞用于评价 净化效率如下所述。

  3. 使用Pan T细胞分离试剂盒II(小鼠,用于细胞阴性分离的试剂盒)从脾细胞中纯化Miltenyi T细胞
    1. 计数和离心来自步骤B15-16的未标记的细胞悬浮液; 重悬细胞沉淀在400μlMACS缓冲液每10 8总细胞
    2. 每10个 8个细胞加入100μl生物素 - 抗体混合物。
    3. 充分混合,并在冰箱(2-8℃)中孵育5分钟
    4. 每10个 8个总细胞加入300μlMACS缓冲液。
    5. 每10 8个细胞添加20微升抗生物素微珠。
    6. 充分混合并在冰箱(2-8℃)中孵育10分钟
    7. 将LS柱放在MidiMACS分离器的磁场中。
    8. 通过用3ml MACS缓冲液冲洗来制备柱
    9. 将细胞悬液应用到柱上,收集含有未标记细胞的流通液,代表富集的T细胞
    10.  用3 ml MACS缓冲液洗涤柱子,收集未标记的细胞 通过,代表富集的T细胞; 结合 步骤C9的流出物
    11. 计数活细胞数使用0.4% 台盼蓝溶液。 放置2×10 5个细胞用于评价 净化效率如下所述。

  4. 通过流式细胞术来控制纯化效率
    1. 用20μl悬浮液染色2×10 5个总细胞(步骤B19) 含有预滴定量的抗小鼠Ly-6G和抗小鼠CD11b。   将抗体在含有1%BSA的PBS中1:100稀释
    2. 染色2×10 5个总细胞(步骤C11),其中20μl的悬浮液含有预滴定量的抗小鼠CD3。
    3. 在4℃孵育30分钟,并在150μl洗涤缓冲液(含1%BSA的PBS)中洗涤
    4. 在4℃和500×g离心细胞悬浮液5分钟。 弃去上清液并保留细胞沉淀。
    5. 重悬在200μl洗涤缓冲液(含1%BSA的PBS)中,并在流式细胞仪中分析。


    图1. CD11b +细胞(Gr1和CD11b抗体)和CD3 sup + T细胞(CD4和CD4)的纯度, CD3抗体)可通过FACS分析
    评估
  5. 小鼠前列腺癌模型和尾静脉注射(≥5只小鼠/组)
    1. 具有C57/B16背景的小鼠皮下(s.c.)注射 TRAMP-C2细胞(每只小鼠在0.2ml PBS中的3×10 6个细胞) 的免疫细胞的过继转移(Yan等人,2014)。在第7天 和第14天注射后,另外两个剂量的纯化的 (每只小鼠5×10 6个细胞)或纯化的CD3 +淋巴细胞的培养物上清液中培养CD11b + (每只小鼠5×10 6个细胞)需要通过过继转移 静脉注射。 小鼠出现时将被牺牲 濒死(45天)。
    2. 在灯下预热小鼠5分钟 实现尾静脉注射的血管舒张。 注射纯化的CD11b +细胞(每只小鼠5×10 6个细胞/200μlPBS)或纯化的CD3 +细胞,/sup> T细胞 (5×10 6个细胞/200μlPBS /小鼠)的尾静脉中 ml注射器用29 G针
    3. 将密切监测肿瘤发展,并且每7天测量肿瘤大小。


    图2.来自肿瘤攻击的WT小鼠的DP,但不是T细胞足以允许用TRAMP0C2细胞注射的ogr1 -/-/-/- 小鼠中的肿瘤生长。 strong>(Yan 等人,2014)

食谱

  1. RBC裂解缓冲液
    0.15 M NH 4 Cl
    1mM NaHCO 3/v/v 0.1mM EDTA溶于无菌双蒸水中 用1M HCl将pH调节至7.2-7.4 过滤灭菌
  2.  磷酸盐缓冲盐水(PBS)
    136 mM NaCl 8.2mM Na 2 HPO 4
    1.5mM KH 2 PO 4 4/v/v 2.7mM KCl(pH7.4)
  3. MACS缓冲区
    通过用autoMACS稀释MACS原液1:20制备含有磷酸盐缓冲盐水(PBS)(pH 7.2),0.5%牛血清白蛋白(BSA)和2mM EDTA的溶液。 ®冲洗溶液。
    保持缓冲液冷(2-8°C)。
    使用前脱气缓冲液,因为气泡可能堵塞色谱柱

致谢

这项工作部分得到国家卫生研究院(RO1155145至YX)的支持; 和YX的Mary Fendrich-Hulman慈善信托基金。

参考文献

  1. Foster,B.A.,Gingrich,J.R.,Kwon,E.D.,Madias,C.and Greenberg,N.M。(1997)。 源自小鼠前列腺(TRAMP)转基因腺癌的前列腺上皮细胞系的表征。 Cancer Res 57(16):3325-3330。
  2. Yan,L.,Singh,L. S.,Zhang,L.and Xu,Y.(2014)。 OGR1在前列腺癌的髓系细胞中的作用 癌基因 33(2):157-164。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Yan, L. and Xu, Y. (2015). Adoptive Transfer of Myeloid-Derived Suppressor Cells and T Cells in a Prostate Cancer Model. Bio-protocol 5(16): e1557. DOI: 10.21769/BioProtoc.1557.
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