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Determining Leukocyte Origins Using Parabiosis in the PyMT Breast Tumor Model
采用联体模型在PyMT乳腺肿瘤模型中测定白血球起源   

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Abstract

Tumors develop in a complex microenvironment alongside numerous cell types that impact their survival. Immune cells make up a large proportion of these accessory cells and many are known to promote tumor progression. Macrophages, in particular, are associated with poor patient prognosis and are therefore potential candidates for therapeutic targeting in cancer. However, to develop successful strategies to target macrophages, it is important to clarify whether these cells are derived from blood-borne precursors or a tissue-resident population. Parabiosis, or the surgical connection of two mice resulting in a shared blood circulation, allows the distinction between these two cellular sources. Here, we describe the use of parabiosis to define cell ontogeny in a mouse model of breast cancer.

Keywords: Tumor-associated macrophages(肿瘤相关巨噬细胞), Monocytes(单核细胞), Breast cancer(乳腺癌), Oncogene-driven tumor model(癌基因驱动的肿瘤模型)

Materials and Reagents

  1. Mice: Female age- and weight-matched congenically-marked MMTV-PyMT mice [see Franklin et al. (2014) for specific strain information]
  2. Injectable anesthesia (ketamine/xylazine cocktail: 150 mg/kg/15 mg/kg)
  3. Antiseptic solution (Betadine)
  4. 70% ethanol
  5. Ophthalmic ointment (Paralube)
  6. Analgesic (Buprenorphine, 0.1 mg/kg)
  7. Non-wetting sterile water (HydroGel)
  8. Sterile saline solution (0.9% NaCl)
  9. 1x PBS
  10. 10x PBS
  11. Bovine serum albumin (Sigma-Aldrich, catalog number: A2153 )
  12. Sodium azide (Sigma-Aldrich, catalog number: S2002 )
  13. Collagenase Type III (Worthington Biochemicals, catalog number: LS004182 )
  14. Percoll (Sigma-Aldrich, catalog number: P1644 )
  15. HBSS + calcium chloride + magnesium chloride (Life Technologies, Gibco®, catalog number: 14025 )
  16. Deoxyribonuclease I from bovine pancreas (Sigma-Aldrich, catalog number: DN25 )
  17. CD45.1 APC-eFluor® 780 (eBioscience, catalog number: 47-0453-82 )
  18. CD45.2 PerCP-Cyanine5.5 (eBioscience, catalog number: 45-0454-82 )
  19. MHC Class II (I-A/I-E) FITC (eBioscience, catalog number: 11-5321-82 )
  20. CD11b Pacific Blue™ (Life Technologies, InvitrogenTM, catalog number: RM2828 )
  21. Ly-6C APC (BD Pharmingen, catalog number: 560595 )
  22. Ly-6G PE (BD Pharmingen, catalog number: 551461 )
  23. Fc block (2.4G2), (BD Pharmingen, catalog number: 553142 )
  24. RPMI 1640 (Life Technologies, Gibco®, catalog number: 11875-093 )
  25. Digestion buffer (see Recipes)
  26. 100% Percoll (see Recipes)
  27. 44% Percoll (see Recipes)
  28. 66% Percoll (see Recipes)
  29. FACS buffer (see Recipes)

Equipment

  1. Non-absorbable black nylon suture (4-0, 13mm, 3/8 circle) (Ethicon, catalog number: 1854G )
  2. Wound clip applier, wound clip remover, wound clips (Autoclip, 9 mm) (Braintree Scientific, catalog number: ACS KIT )
  3. Cotton-tipped applicators (3 inch, wood shaft) (Fisherbrand, catalog number: 23-400-105 )
  4. Sterile gauze (Fisherbrand, catalog number: 22-028-558 )
  5. Needle holder with locking mechanism (Fine Science Tools)
  6. Scissors (Fine Science Tools)
  7. Curved forceps (2 pairs) (Fine Science Tools)
  8. Razor blades
  9. Slides (Corning Incorporated, catalog number: 2948-75X25 )
  10. Heating lamp
  11. Heating pads
  12. Electrical shaver
  13. Surgical drapes/sterile pads (Fisherbrand)
  14. 6 well non-tissue culture treated plates (Falcon®)
  15. 70 micron cell strainers (Falcon®, catalog number: 352350 )
  16. 50 ml conical tubes (Falcon®)
  17. 15 ml conical tubes (Falcon®)
  18. 3 ml syringe (BD Bioscience)
  19. U-bottom 96 well plates (Falcon®)
  20. Desktop centrifuge (Sorvall)
  21. 37 °C water bath
  22. Flow cytometer (BD LSR II or similar)

Procedure

  1. Parabiosis
    This surgical procedure will result in the formation of microvasculature at the connection site and thus a shared blood circulation between two animals during the development of oncogene-induced mammary tumors.
    1. Sterilize all surgical tools by autoclaving or soaking in 70% ethanol for at least 30 min.
    2. Lay out all sterile tools and necessary materials on clean surgical drapes/sterile pads.
    3. Warm a clean cage with heating lamp for use during recovery.
    4. Anesthetize mice using ketamine/xylazine cocktail (IP). The animals should be fully sedated (as measured by no response to a toe pinch) before proceeding. Throughout anesthesia, apply ophthalmic ointment to eyes as needed.
    5. After mice are anesthetized, shave the right flank of one partner and the left flank of the other partner. All mice should be kept on heating pads during surgery.
    6. Start procedure on first partner. Lay the mouse on its side on sterile gauze and sterilize the shaved area to prepare for incision, alternating antiseptic solution and 70% ethanol three times, using a fresh cotton-tipped applicator for each application. Make a small incision in the skin over the thigh, about 0.5 cm dorsally from the knee joint. Insert the tip of the scissors into the incision and open and close the nose of the scissors to pull the skin away from the inner fascia covering the leg muscles. This will allow you to expose the muscles and patellar tendon of the hindlimb for suturing in later steps.
    7. Cut in a straight line from this incision up to the shoulder just past the elbow joint. Repeat opening and closing of the scissors the entire length of the incision, being careful to leave the body wall intact.
    8. Holding the ankle joint of the hindlimb between your thumb and forefinger, carefully maneuver the knee out from the skin so that the patella is exposed and turned upwards, facing you. Insert the needle of the non-absorbable suture between the tibia and fibula from the inside of the leg to the outside, about 1/3 of the way below the knee. Avoid piercing major blood vessels. Pull the suture through until 2-3 inches of the free end remain (this free end will be used to tie to the other partner, so make sure there is enough length for you to comfortably perform two throws of an instrument tie square knot). Tie off the suture with the needle holder, using an instrument tie square knot (two throws of the knot is sufficient). There will be two ends of the suture radiating from the knot, the 2-3 inch free end and the end connected to the needle. Cut the needle end as close to the knot as possible, leaving the free end available to tie in subsequent steps. Try to secure the knots on the outside of the thigh as this will make connecting the two partners easier. Detailed instructions on how to perform surgical ties can be found at http://www.bumc.bu.edu/surgery/training/technical-training/instrument-tie/.
    9. Repeat this procedure on the forelimb, using the remaining length of the suture (needle end) from step A8. Insert the suture needle between the radius and ulna, right below the elbow, from inside to outside.
    10. Keeping the 1st partner on the heating pad, laying on its side with the incision facing upwards, make a matching incision on the 2nd partner. Repeat the same procedure as the 1st partner. You will now have 4 loose suture ends with all of the knots facing upwards.
    11. To connect the two mice, lay them both on their backs, with their abdomens facing upwards, and their incisions facing each other. Insert the suture needle under the patellar tendon of the 1st partner from the inside of the knee to the outside and then under the patellar tendon of the 2nd partner from the outside to the inside (one continuous motion). Tie off suture (two throws) and cut both loose ends as close to the knot as possible to minimize irritation from suture ends. Be careful not to accidentally tie the suture ends from step A8 into the knot.
    12. To connect the hindlimb sutures, flip mice over so they are laying on their stomachs. Use the needle holder to perform 2 throws of an instrument tie square knot on the suture ends from step A8 and cut loose ends. Keeping the mice on their stomachs, use the needle holder to tie together the forelimb sutures from step A9 (2 throws of an instrument tie square knot) and cut loose ends.
    13. After these sutures are tied together, the mice will be in their final connected position so take care to line up forelimbs and hindlimbs in a way that will make ambulation as easy as possible. The two partners should now be tightly and stably held together with their body walls in contact with each other.
    14. Close the skin incisions using 9 mm wound clips. This procedure is best performed by two people, one person to hold the skin together, and one to apply the wound clips. Using two curved end forceps, hold the skin flaps from the dorsal sides of both mice up and away from their body walls. At this step it is very important to roll the outer edges of the skin flaps away each other as to avoid clipping any remaining hair into the wound. This also prevents the skin from curling into the wound. Apply the first wound clip where the inside of the skin flaps touch each other, approximately 0.25 cm from the edge of the incision, starting from the tail end and working towards the head, placing each wound clip right next to the previous clip. By pulling the skin up while clipping, you prevent clipping into the body wall or connecting the mice too tightly together. Once clipping on the dorsal side of the wound is complete, flip the mice onto their backs and apply clips to the ventral side of the wound, holding the skin together during clipping as previously described. Extra clips may need to be applied near the forelimbs and hindlimbs to fully close the wound.
    15. After clipping is complete, lay the mice on their stomachs, on sterile gauze, in the warmed cage. The heating lamp must be used until the mice are fully awake and ambulating.
    16. Inject each partner with 500 μl sterile saline (sub-Q) immediately following surgery and 2x daily for 4-7 days post-surgery.
    17. Inject each partner with analgesic (IP) immediately following surgery and 1x daily for 1-2 days post-surgery.
    18. Provide non-wetting sterile water in the bottom of the cage for 1 week following surgery and provide moistened food for the duration of the experiment.
    19. If maintaining the mice for more than 1 month, remove the wound clips at 3-4 weeks using a wound clip remover.

  2. Tumor isolation and processing
    1. After mammary tumors become palpable, euthanize the mice and remove the tumors from the unconnected side of each partner. Do not isolate mammary glands/tumors from the connected side. Special care must be taken to avoid disrupting the lymph nodes in order to prevent contamination of the tumor prep with lymph node leukocytes. This is especially important for the mammary gland that is found next to the inguinal lymph node. It is often best to avoid removing this tumor entirely. In addition, some mammary glands will not have palpable tumors, yet the pair of mice will have to be euthanized due to the size of one or more tumors. In this case, all tumors and mammary glands may be pooled (only pool tumors within one individual mouse), or analyzed separately.
    2. Remove tumors and place in 1x PBS in a 6 well plate on ice.
    3. Process tumors by cutting into small pieces with a razor blade on top of a glass slide and incubate tumors in 10-15 ml digestion buffer (see Recipes), for 1¼ h in 37 °C water bath. Vortex tubes every 15-20 min.
    4. After digestion, pour tumors and digestion buffer over a 70 micron cell strainer into a 6 well plate. Mash tumors through cell strainer with the back of a plunger from a 3 ml syringe. Wash filter with 1x PBS and move cell strainer to the next well. Continue mashing, washing, and moving the cell strainer to the next well of the plate until the majority of tumor tissue has been dissociated.
    5. Transfer digested and filtered tumor tissue to a 50 ml conical tube using a serological pipette, wash all wells with 1x PBS to collect any remaining cells, and spin down at 650 x g for 6 min in desktop centrifuge (total volume will be 40-50 ml).
    6. Pour off supernatant and resuspend cell pellet in 10 ml 44% Percoll and transfer to a 15 ml conical tube.
    7. Slowly underlayer with 3 ml 66% Percoll in the bottom of the tube using a 5 ml serological pipette, pulling out the pipette as the Percoll is deposited, taking care not to disturb the interface between the 44% and 66% Percoll.
    8. Spin at 1,500 x g, 30 min, 4 °C, NO BRAKE/lowest brake setting.
    9. Remove debris at the top of the tube, along with 3-5 ml 44% Percoll. Collect cells at interface “buffy coat” using a p1000 micropipette and transfer into a clean 15 ml conical tube. Be generous with the volume collected at this step (4-5 ml total), while avoiding the red blood cell pellet at the bottom of the tube. Spin down at 650 x g for 5 min.
    10. Wash with 10 ml 1x PBS, spin down at 650 x g for 5 min.

  3. FACS analysis of immune infiltrates
    1. Resuspend the immune cell pellet in an adequate volume of 1x PBS to transfer into 96 well plate for FACS staining, approximately 100 μl/well, and place on ice.
    2. Prepare antibody cocktail in FACS buffer, 50 μl/well. The antibodies listed below are for the identification of myeloid cells, including macrophages, monocytes, and neutrophils.
      CD45.1 APC-eFluor780, 1:400
      CD45.2 PerCP-Cyanine5.5, 1:300
      MHC Class II (I-A/I-E) FITC, 1:1,200
      CD11b Pacific Blue, 1:800
      Ly-6C APC, 1:500
      Ly-6G PE, 1:500
      Fc block (2.4G2), 1:400
    3. Spin down plate in desktop centrifuge at 500 x g for 3 min and discard supernatant by flicking the plate. Resuspend each cell pellet in 50 μl antibody cocktail. Incubate plate, covered, on ice for 30 min.
    4. Wash 2x in 150 μl of 1x PBS and resuspend in 1x PBS for analysis using a flow cytometer.

Representative data



Figure 1. Blood-borne precursors contribute to macrophage populations in developing PyMT tumors. Representative data expected from a parabiosis experiment. Two weight-matched, 8-week-old, congenically-marked female PyMT mice were surgically connected and maintained for approximately 3 months. The mammary tissue/tumors were removed from the unconnected side of each partner and pooled (tissue/tumors were pooled within each mouse, but not between mice). Tumors were processed as described above. The gated populations are defined as follows: CD45+MHCII+CD11bhi, mammary tissue macrophage (MTMs), CD45+MHCII+CD11blo, tumor-associated macrophages (TAMs), CD45+MHCII-CD11bhiLy6C+Ly6G+, neutrophils, and CD45+MHCII-CD11bhiLy6C+Ly6G-, monocytes.

Notes

  1. This protocol was developed using the MMTV-PyMT tumor model, however it can likely be utilized to investigate other models. Regardless of the model used, all mice must be congenically-marked in order to distinguish cells originating from the individual partners. Additionally, the use of male mice should be avoided if possible due to the likelihood of fighting after surgical connection.
  2. If the MMTV-PyMT model is used, the animals must be connected as early as possible (6-8 weeks) so that the surgery is performed before the tumors have become palpable. This is important from both a practical standpoint, as the surgery is performed near the mammary glands, and an experimental standpoint, as it would be difficult to interpret data from mice connected during later stages of tumor development.
  3. The relative percentage of CD45.1/CD45.2 chimerism in the mammary tissue/tumors depends on both tumor stage of each mouse upon surgery (6-8 weeks is optimal) and upon sacrifice. Invariably, one partner will have more developed tumors than the other and this will be reflected in the degree of chimerism, number of infiltrating cells obtained, and mammary tissue macrophage (MTM):tumor-associated macrophage (TAM) ratio. For example, in the representative data shown above, the CD45.1+ partner had more advanced tumors and therefore had a lower degree of chimerism, contained more infiltrating cells, and had a lower MTM:TAM ratio (TAMs, rather than MTMs, are the dominant macrophage population in advanced PyMT tumors [see Franklin et al. (2014) for additional information].
  4. The CD45.1-CD45.2- population observed after gating on single, live cells (see Representative data) represents tumor cells and is more prominent in advanced or necrotic tumors.

Recipes

  1. Digestion buffer
    1x HBSS
    Collagenase III (~280 U/ml)
    DNase I (4 μg/ml)
  2. 100% Percoll
    45 ml Percoll
    5 ml 10x PBS
  3. 44% Percoll
    22 ml “100%” Percoll
    28 ml 1x PBS
  4. 66% Percoll
    33 ml “100%” Percoll
    17 ml RPMI (to provide color, makes interface more clear)
  5. FACS buffer
    1x PBS
    1% BSA
    0.02% Sodium azide

Acknowledgments

We thank Kang Liu for her teaching of the parabiosis technique. This work was supported
by the Cancer Research Institute Tumor Immunology Predoctoral Fellowship Training Grant (R.A.F), Cancer Research Institute Clinic and Laboratory Integration Program Grant (M.O.L.), and the American Cancer Society Research Scholar Award (M.O.L.).

References

  1. Franklin, R. A., Liao, W., Sarkar, A., Kim, M. V., Bivona, M. R., Liu, K., Pamer, E. G. and Li, M. O. (2014). The cellular and molecular origin of tumor-associated macrophages. Science 344(6186): 921-925.

简介

肿瘤在复杂的微环境中发展,伴随许多影响其存活的细胞类型。 免疫细胞构成这些附属细胞的大部分,并且已知许多细胞促进肿瘤进展。 巨噬细胞,特别是与不良的患者预后相关,因此是癌症中治疗靶向的潜在候选者。 然而,要开发成功的策略,以靶向巨噬细胞,重要的是要澄清这些细胞是否源自血源性前体或组织驻留人群。 Parabiosis或两个小鼠的外科连接导致共享的血液循环,允许这两个细胞来源之间的区别。 在这里,我们描述在乳腺癌的小鼠模型中定义细胞个体发育的拟杆菌的使用。

关键字:肿瘤相关巨噬细胞, 单核细胞, 乳腺癌, 癌基因驱动的肿瘤模型

材料和试剂

  1. 小鼠:女性年龄和体重匹配的共生标记的MMTV-PyMT小鼠[具体应变信息参见Franklin等人(2014)]
  2. 注射麻醉(氯胺酮/赛拉嗪混合物:150mg/kg/15mg/kg)
  3. 防腐液(Betadine)
  4. 70%乙醇
  5. 眼用软膏(Paralube)
  6. 镇痛药(丁丙诺啡,0.1mg/kg)
  7. 无润湿无菌水(HydroGel)
  8. 无菌盐水溶液(0.9%NaCl)
  9. 1x PBS
  10. 10x PBS
  11. 牛血清白蛋白(Sigma-Aldrich,目录号:A2153)
  12. 叠氮化钠(Sigma-Aldrich,目录号:S2002)
  13. 胶原酶III型(Worthington Biochemicals,目录号:LS004182)
  14. Percoll(Sigma-Aldrich,目录号:P1644)
  15. HBSS +氯化钙+氯化镁(Life Technologies,目录号:14025)
  16. 来自牛胰腺的脱氧核糖核酸酶I(Sigma-Aldrich,目录号:DN25)
  17. CD45.1 APC-eFluor 780(eBioscience,目录号:47-0453-82)
  18. CD45.2 PerCP-Cyanine5.5(eBioscience,目录号:45-0454-82)
  19. MHC II类(I-A/I-E)FITC(eBioscience,目录号:11-5321-82)
  20. CD11b Pacific Blue TM(Life Technologies,InvitrogenTM,目录号:RM2828)
  21. Ly-6C APC(BD Pharmingen,目录号:560595)
  22. Ly-6G PE(BD Pharmingen,目录号:551461)
  23. Fc嵌段(2.4G2),(BD Pharmingen,目录号:553142)
  24. RPMI 1640(Life Technologies,目录号:11875-093)
  25. 消化缓冲液(参见配方)
  26. 100%Percoll(见配方)
  27. 44%Percoll(见配方)
  28. 66%Percoll(见配方)
  29. FACS缓冲区(参见配方)

设备

  1. 不可吸收的黑色尼龙缝合线(4-0,13mm,3/8圈)(Ethicon,目录号:1854G)
  2. 伤口夹钳(伤口钳)(伤口钳(Autoclip,9mm)(Braintree Scientific,目录号:ACS KIT)
  3. 棉针头(3英寸,木杆)(Fisherbrand,目录号:23-400-105)
  4. 无菌纱布(Fisherbrand,目录号:22-028-558)
  5. 带锁定机构的针夹(Fine Science Tools)
  6. 剪刀(精细工具)
  7. 弯曲镊子(2对)(Fine Science Tools)
  8. 剃刀片
  9. 幻灯片(Corning Incorporated,目录号:2948-75X25)
  10. 加热灯
  11. 加热垫
  12. 电动剃须刀
  13. 手术巾/无菌垫(Fisherbrand)
  14. 6孔非组织培养处理的平板(Falcon )
  15. 70微米细胞过滤器(Falcon ,目录号:352350)
  16. 50ml锥形管(Falcon )
  17. 15ml锥形管(Falcon )
  18. 3ml注射器(BD Bioscience)
  19. U-底96孔板(Falcon )
  20. 台式离心机(Sorvall)
  21. 37°C水浴
  22. 流式细胞仪(BD LSR II或类似)

程序

  1. Parabiosis
    这种外科手术将导致在连接部位处形成微血管,从而在癌基因诱导的乳腺肿瘤的发生过程中在两只动物之间共享血液循环。
    1. 通过高压灭菌或浸泡在70%乙醇中至少30分钟灭菌所有手术工具。
    2. 将所有无菌工具和必要材料布置在干净的手术帘/无菌垫上
    3. 用加热灯加热清洁的笼子,以便在恢复期间使用。
    4. 使用氯胺酮/赛拉嗪混合物(IP)麻醉小鼠。 动物们 应该完全镇静(通过对趾压缩没有反应来测量) 然后继续。 在整个麻醉,应用眼膏软膏 眼睛。
    5. 小鼠麻醉后,刮胡子 一个配偶体的侧翼和另一个配偶体的左侧翼。 所有小鼠 应在手术期间保持在加热垫上
    6. 启动过程开   第一个合作伙伴。 将鼠标放在其侧面的无菌纱布和消毒   剃须区域以准备切口,交替消毒 溶液和70%乙醇3次,使用新鲜棉头 涂抹器。 在皮肤上做一个小切口 大腿,距离膝关节约0.5厘米。 插入尖端 将剪刀插入切口并打开和关闭鼻子 剪刀将皮肤拉离覆盖腿部的内筋膜 肌肉。 这将允许你暴露肌肉和髌腱 的后肢在后续步骤中缝合
    7. 切直 线从这个切口到肩部刚好通过肘关节。 重复打开和关闭剪刀的整个长度 切口,小心地保持身体壁完整
    8. 保持 您的拇指和食指之间的后肢的踝关节, 小心翼翼地从皮肤上操纵膝盖,使髌骨成为 暴露和向上转,面向你。插入针 不可吸收的缝合在胫骨和腓骨之间从里面 腿向外,约在膝下方1/3处。避免 刺穿主要血管。拉动缝合线直到2-3英寸 的自由端保留(该自由端将用于连接到另一端 合作伙伴,所以确保有足够的长度,你舒适地 执行两次仪器领带平结)。绑扎缝线  用针架,用仪器领结方形结(两针  的结是足够的)。将有两端的缝合线 辐射从结,2-3英寸自由端和端部连接 针。尽可能靠近结,切开针头,离开  自由端可用于结合后续步骤。尝试保护 在大腿外侧的结,这将使连接两个 合作伙伴更容易。关于如何进行手术结合的详细说明 可以在 http://www.bumc.bu。教育/外科/培训/技术培训/仪器领带/
    9. 在前肢上重复此过程,使用剩余长度   来自步骤A8的缝合线(针端)。 插入缝合针 半径和尺骨,在肘部的正下方,从内到外
    10. 保持第一伴侣在加热垫上,躺在它的一边 切口朝上,在第二个匹配的切口 伙伴。 重复与第1个伙伴相同的步骤。 你现在有   4宽松的缝合线,所有的结都朝上。
    11. 至 连接两个老鼠,把他们两个在他们的背上,与他们的腹部 面向上,并且它们的切口彼此面对。 插入缝线   针下髌骨腱的第一伙伴从里面 膝盖到外面,然后在第二的髌骨腱下 伙伴从外部到内部(一个连续运动)。 绑定 缝合(两次),并切割两个松散的末端,靠近结 可能使缝合线末端的刺激最小化。 小心不要 意外地将步骤A8的缝合线结扎到结中
    12. 至 连接后肢缝线,翻转老鼠,使他们躺在他们   胃。 使用针托执行2次仪器 在来自步骤A8的缝合线端部上打结方形结,并切割松散端。 保持小鼠在他们的胃,使用持针器绑 一起来自步骤A9的前肢缝合(2次的仪器 领结方形结)和切开松散端。
    13. 这些缝线后 绑在一起,小鼠将处于其最终连接位置 注意排列前肢和后肢的方式,将使 步行尽可能容易。 两个伙伴现在应该紧紧 并且与它们的体壁稳定地保持在一起,与其接触 其他。
    14. 使用9 mm伤口夹闭合皮肤切口。这个 程序最好由两个人进行,一个人握住皮肤 一起施加伤口夹子。使用两个弯曲端 镊子,握住皮肤襟翼从两侧的小鼠向上和 远离他们的身体墙壁。在这一步,滚动非常重要 皮肤的外边缘彼此分开以避免夹紧 任何剩余的毛发进入伤口。这也防止皮肤 卷曲入伤口。应用第一个伤口夹在里面 皮肤襟翼彼此接触,距离边缘大约0.25cm 切口,从尾端​​开始朝向头部工作, 将每个伤口夹子紧挨着前一个夹子。通过拉 皮肤,同时剪裁,你防止剪辑到身体的墙壁或 连接鼠标太紧密在一起。一次在背部的剪报 侧的伤口是完整的,翻转小鼠到他们的背部和应用 夹子到伤口的腹侧,在皮肤保持在一起  如前所述。可能需要应用额外的剪辑 靠近前肢和后肢,以完全闭合伤口
    15. 后   剪辑完成,将小鼠放在他们的胃,在无菌纱布上,   在温暖的笼子里。 加热灯必须使用,直到老鼠 完全清醒和行走
    16. 注射每个合作伙伴与500微升 无菌盐水(sub-Q),手术后立即每天2次 手术后4-7天。
    17. 手术后立即注射每个合作伙伴止痛剂(IP),术后1-2天每天1次。
    18. 在笼子底部提供无润湿的无菌水1 周手术后,提供持续的润湿食物 实验
    19. 如果维持小鼠超过1个月,使用伤口夹子去除剂在3-4周去除伤口夹。

  2. 肿瘤隔离和治疗
    1. 乳房肿瘤变得可触摸后,安乐死小鼠和删除 肿瘤从每个伙伴的未连接的一侧。 不要隔离 乳腺/肿瘤。 必须特别小心 以避免破坏淋巴结以防止 用淋巴结白细胞污染肿瘤制备物。 这是 特别是对于发现旁边的乳腺非常重要 腹股沟淋巴结。 通常最好避免去除这种肿瘤 完全。 此外,一些乳腺不会触摸 肿瘤,然而由于大小,该对小鼠将必须实施安乐死 的一种或多种肿瘤。 在这种情况下,所有肿瘤和乳腺可以 (仅在一只个体小鼠中的池肿瘤),或分析
    2. 取出肿瘤,并置于1×PBS中的6孔板在冰上
    3. 通过用剃刀刀片切割成小块来处理肿瘤 并在10-15ml消化缓冲液中孵育肿瘤 (参见Recipes),在37℃水浴中1小时。 涡流管每15-20 min。
    4. 消化后,将肿瘤和消化缓冲液倒在70℃ 微米细胞滤器过滤到6孔板中。 通过细胞的Mash肿瘤 过滤器与来自3ml注射器的柱塞的背部。 洗涤过滤器 用1×PBS并将细胞过滤器移动到下一个孔。 继续糖化, 洗涤,并将细胞过滤器移动到板的下一个孔 直到大多数肿瘤组织已经解离
    5. 转让   消化并过滤的肿瘤组织至50ml锥形管 血清移液管,用1x PBS洗涤所有孔以收集任何剩余的   细胞,并在台式离心机中以650×g离心6分钟(总共 体积将为40-50ml)
    6. 倒出上清液并将细胞沉淀重悬于10ml 44%Percoll中,并转移至15ml锥形管中。
    7. 在管底部缓慢下层,用3ml 66%Percoll 使用5ml血清移液管,拔出吸管作为Percoll   沉积,注意不要扰乱44% 和66%Percoll。
    8. 1500分钟旋转,30分钟,4℃,无制动/最低制动设置。
    9. 除去管顶部的碎片,以及3-5 ml 44%Percoll。   收集细胞在界面"血沉棕黄层"使用p1000微量移液管和 转移到干净的15ml锥形管中。 大方的体积 在此步骤收集(总共4-5ml),同时避免红细胞   在管的底部沉淀。 在650分钟x 下旋转5分钟。
    10. 用10ml 1x PBS洗涤,以650×g离心5分钟。

  3. 免疫浸润的FACS分析
    1. 将免疫细胞沉淀重悬于足够体积的1×PBS中 转移到96孔板用于FACS染色,约100 μl/孔,并置于冰上
    2. 在FACS中制备抗体混合物 缓冲液,50μl/孔。 下面列出的抗体适用于 鉴定骨髓细胞,包括巨噬细胞,单核细胞和 中性粒细胞 CD45.1 APC-eFluor780,1:400
      CD45.2 PerCP-Cyanine5.5,1:300
      MHC II类(I-A/I-E)FITC,1:1,200
      CD11b Pacific Blue,1:800
      Ly-6C APC,1:500
      Ly-6G PE,1:500
      Fc嵌段(2.4G2),1:400
    3. 在桌上型离心机中以500×g离心3分钟 通过轻拂板丢弃上清液。重悬每个细胞沉淀  50μl抗体鸡尾酒。孵育板,覆盖,在冰上30分钟。
    4. 在150μl的1×PBS中洗涤2x,并重悬于1×PBS中用于使用流式细胞仪进行分析。

代表数据



图1.血源性前体在发展PyMT肿瘤中有助于巨噬细胞群体。代表性的数据预期来自于仿生实验。将两个重量匹配的8周龄,同系标记的雌性PyMT小鼠手术连接并维持约3个月。从每个配偶体的未连接侧移除乳腺组织/肿瘤并合并(将组织/肿瘤合并在每只小鼠内,但不在小鼠之间)。 如上所述处理肿瘤。门控群体定义如下:CD45 +,MHCII +,CD11b,sup,乳腺组织巨噬细胞(MTM),CD45 +肿瘤相关巨噬细胞(TAM),CD45 +和MHCII抗体,CD11b肿瘤相关巨噬细胞(TAM),CD45 - 肿瘤相关巨噬细胞> hi Ly6C + ,中性粒细胞和CD45 + MHCII -CD11b 单核细胞。单核细胞。单核细胞。

笔记

  1. 这个协议是使用MMTV-PyMT肿瘤模型开发的,但是它可能被用于研究其他模型。不管使用的模型,所有小鼠必须进行先天标记以区分源自个体伴侣的细胞。此外,如果可能,应避免使用雄性小鼠,因为外科手术后可能发生战斗
  2. 如果使用MMTV-PyMT模型,动物必须尽早(6-8周)连接,以便在肿瘤变得可触知之前进行手术。从实践的角度来看,这是重要的,因为在乳腺附近进行手术和实验观点,因为很难解释在肿瘤发展的后期阶段连接的小鼠的数据。
  3. CD45.1/CD45.2嵌合体在乳腺组织/肿瘤中的相对百分比取决于每只小鼠在手术(6-8周是最佳的)和处死后的肿瘤阶段。不变地,一个伙伴将具有比另一个更成熟的肿瘤,并且这将反映在嵌合度,获得的浸润细胞的数量和乳腺组织巨噬细胞(MTM):肿瘤相关巨噬细胞(TAM)比率上。例如,在上面所示的代表性数据中,CD45.1 + 伴侣具有更晚期的肿瘤,因此具有较低程度的嵌合现象,包含更多的浸润细胞,并且具有较低的MTM:TAM比率TAM,而不是MTM,是主导 巨噬细胞群体[参见Franklin等人(2014)了解更多信息]。
  4. 在对单个活细胞门控后观察到的CD45.1 - CD45.2 - 群体(参见代表性数据)表示肿瘤细胞,并且在晚期或坏死肿瘤中更加突出。

食谱

  1. 消化缓冲区
    1x HBSS
    胶原酶III(〜280U/ml) DNase I(4μg/ml)
  2. 100%Percoll
    45 ml Percoll
    5ml 10×PBS
  3. 44%Percoll
    22ml"100%"Percoll
    28 ml 1x PBS
  4. 66%Percoll
    33ml"100%"Percoll
    17ml RPMI(提供颜色,使界面更清晰)
  5. FACS缓冲区
    1x PBS
    1%BSA
    0.02%叠氮化钠

致谢

我们感谢刘刘的她的教学的抛物线技术。 这项工作是由癌症研究所肿瘤免疫学前基金奖学金培训补助金(R.A.F),癌症研究所诊所和实验室集成计划授予(M.O.L.)和美国癌症协会研究学者奖(M.O.L.)支持。

参考文献

  1. Franklin,R.A.,Liao,W.,Sarkar,A.,Kim,M.V.,Bivona,M.R.,Liu,K.,Pamer,E.G.and Li,M.O。 肿瘤相关巨噬细胞的细胞和分子起源 em> 344(6186):921-925。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Franklin, R. A. and Li, M. O. (2015). Determining Leukocyte Origins Using Parabiosis in the PyMT Breast Tumor Model. Bio-protocol 5(16): e1567. DOI: 10.21769/BioProtoc.1567.
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