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Mar 2020
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Tyramide Signal-Amplified Immunofluorescence of MYCN and MYC in Human Tissue Specimens and Cell Line Cultures
酪胺信号放大的人体组织标本和细胞系培养物中的MYCN和MYC免疫荧光   

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Abstract

MYC family members, MYC, MYCN, and MYCL, are oncogenic transcription factors that regulate the expression of genes involved in normal development, cell growth, proliferation, metabolism, and survival. While MYC is amplified and/or overexpressed across a variety of tissue types, MYCN is often overexpressed in tumors of the nervous system (neuroblastoma and medulloblastoma) or with neuroendocrine features (neuroendocrine prostate cancer). Given recent reports that MYCN expression is also deregulated in a variety of non-neuronal tissue types, we investigated whether MYCN was also deregulated in triple-negative breast cancer (TNBC). In contrast to previous individual immuno-fluorescence (IF) stains against higher expressing MYC family isoform protein, we developed an IF stain to simultaneously detect both MYCN- and MYC-expressing cells within the same tumor cell population. Our methodology allows for the detection of low level MYCN and MYC expression and can be multiplexed with additional protein probes. Herein, using tyramide signal amplification (TSA), we present two protocols for the IF detection of MYCN and MYC on formalin-fixed paraffin embedded (FFPE) tumor sections and in cell lines fixed in situ after growth as adherent cultures on chambered microscope slides.

Keywords: Cancer biology (肿瘤生物学), Tyramide signal amplification (酪胺信号放大), Immunofluorescence (免疫荧光), Chamber slides (腔体切片), MYCN (MYCN), MYC (MYC)

Background

Previous studies have demonstrated MYCN and MYC preferentially regulate the same set of core genes involved in metabolism and cell growth, and while the MYCN allele can functionally replace MYC in murine development (Malynn et al., 2000), MYCN and MYC have separate temporal regulation over organogenesis in early vertebrate development (Hurlin, 2013). MYCN expression is essential for initial establishment of stem and progenitor populations; over the course of organ system development, MYCN expression switches to low MYC expression to support stem and progenitor cell maintenance, and during cell lineage commitment and expansion, elevated MYC levels drive highly proliferative cells until they reach terminal differentiation (Hurlin, 2013).

A similar relationship has been observed in the development of TNBC metastases where MYCN expression is elevated in newly seeded metastatic lesions that expand and differentiate into highly proliferative MYC-expressing tumors (Lawson et al., 2015). Given the near ubiquitous nature of MYC deregulation across tumor tissue types, MYC is the most studied MYC family member. However, due to a correlation between deregulated MYCN expression and a poor prognosis (Beltran, 2014), MYCN has been increasingly studied in both neuronal and non-neuronal cancers, including acute myeloid leukemia (Kawagoe et al., 2007), small-cell lung cancer (Funa et al., 1987), ovarian cancer (Baratta et al., 2015), and now TNBC (Schafer et al., 2020). Therefore, we began to explore the relationship between MYCN and MYC by developing an IF stain that could simultaneously detect both isoforms within the same tumor tissue section. Established protocols that involve the IF detection of MYCN or MYC have been separate stains that primarily recognizes each isoform at highly amplified/overexpressed levels. Given that TNBC and other non-neuronal tissue types exhibit aberrant low-level MYCN expression, we developed a method to detect lower expression of MYCN and MYC using TSA. Further, we have two protocol versions of the method: application of the dual MYC family isoform (MYCN and MYC) TSA-IF to FFPE tumor sections, and a modified version of the stain for evaluation MYC family isoforms expression in cell line cultures fixed in situ after growth as adherent cultures on chambered microscope slides. We anticipate both MYCN and MYC are coexpressed in a variety of cancer tissue types and the advent of these IF methodologies will allow investigators to further characterize MYCN- versus MYC-expressing cells in tumor development and disease etiology.

Materials and Reagents

  1. Microscopy slides (VWR, catalog number: 48311-703 )
  2. Coverslips (Fisherbrand, catalog number: 12-545K )
  3. HistoPrep pen (Fischer Scientific, catalog number: 14-905-30 )
  4. PAP pen (Electron Microscopy Sciences, catalog number: 71310 )
  5. 10% neutral buffered formalin (Thermo scientific, catalog number: 5701 ), store at room temperature
  6. Xylene (Fisher Scientific, catalog number: X3P-1GAL ), store at room temperature
  7. Ethanol (Pharmco, catalog number: 111000200 ), store at room temperature
  8. Citra plus antigen retrieval buffer (BioGenex, catalog number: HK080-9K ), store at 4 °C
  9. Phosphate-buffered saline (Gelifesciences, HyClone, catalog number: SH30013.03 ), store at 4 °C
  10. Tween-20 (Acros Organics, catalog number: 23336-0010 ), store at room temperature
  11. Triton X-100 (Millipore, OmniPur, catalog number: 9410 ), store at room temperature
  12. 30% hydrogen peroxide (H2O2) (Fisher Chemical, catalog number: H325-100 ), store at 4 °C
  13. Image-iT FX Signal Enhancer (Invitrogen, catalog number: I36933 ), store at 4 °C
  14. Goat serum (Gemini, catalog number: 100-109 ), store at -20 °C
  15. DMSO (Sigma, catalog number: D2650-100ml ), store at room temperature
  16. FITC TSA reagent (PerkinElmer, catalog number: NEL741B001KT ), store at 4 °C
  17. Cy3 TSA reagent (PerkinElmer, catalog number: NEL744B001KT ), store at 4 °C
  18. Optional: Cy5 TSA reagent (PerkinElmer, catalog number: NEL745B001KT ), store at 4 °C
  19. DAPI (Sigma-Aldrich, catalog number: D9542 ), store at -20 °C, or Hoechst (Invitrogen, catalog number: H3570 ), store at 4 °C
  20. SlowFade Gold Antifade Mountant (ThermoFisher Scientific, catalog number: S36937 ), store at room temperature
  21. Nail polish (any kind or brand will suffice)
  22. Antibodies
    1. Anti-MYCN antibody (Cell Signaling, catalog number: 51705S ), store at -20 °C
    2. Anti-MYC antibody (Abcam, catalog number: ab32072 ), store at -20 °C
    3. Anti-rabbit HRP secondary (Thermo Fisher Scientific, catalog number: 31462 ), store at 4 °C
  23. Permeabilization buffer (see Recipes)
  24. Blocking buffer (see Recipes)
  25. Antibody diluent (see Recipes)
  26. Wash buffer (see Recipes)
  27. FITC signal amplification buffer (see Recipes)
  28. Cy3 signal amplification buffer (see Recipes)

Additional materials and reagents required for cell line TSA-IF:
  1. Eight-well chamber microscope slides (Corning, catalog number: 354108 )
  2. Methanol (Millipore, catalog number: MX0485-7 ), store at room temperature
  3. Antibodies
    1. Anti-Histone H3 antibody (Abcam, catalog number: ab1791 ), store at -20 °C
    2. Cy5-conjugated anti-rabbit secondary antibody (Invitrogen, catalog number: A10523 ), store at 4 °C

Equipment

  1. Slide Warmer (e.g., Lab-Line Instruments, model: CPC-600N1 )
  2. Electric pressure cooker (e.g., Cuisinart, model: CPC-600N1 )
  3. Microscope slide moisture chamber (e.g., Ted Pella, catalog number: 21053 )
  4. Slide staining station (e.g., Tissue-Tek, catalog number: 4451 )

Procedure

Tissue sections

  1. Sample fixation and preparation
    1. When harvesting tissue, quickly place specimen (with a maximum thickness of 3 mm) directly into 10% neutral buffered formalin and incubate tissue for 24 h at room temperature.
      Note: Invert the container and/or mix contents every 30 min for the first few h to ensure even exposure and proper fixation.
    2. Transfer specimens to 70% ethanol and take tissue to appropriate facility for FFPE sample preparation.
      Note: If unable to process samples immediately, place specimens at 4 °C.
    3. Use standard microtone sectioning procedures to section tissue and dry specimens on positively charged microscope slides overnight.
      Note: This protocol has been optimized for staining 4 micron thick FFPE tissue sections. If tissues are not to be stained immediately, dip slides in paraffin and store at 4 °C.

  2. Antigen retrieval
    1. Label slides as desired with HistoPrep pen.
    2. Melt paraffin wax by placing slides on slide warmer at 60 °C for 60 min.
    3. Quickly transfer slides to vertical slide holder already submerged in xylene.
    4. Incubate slides in xylene four times for 10 min each.
    5. Rehydrate tissue with graded alcohol incubations:
      1. 100% ethanol: two separate incubations for 1 min each
      2. 90% ethanol: two separate incubations for 1 min each
      3. 75% ethanol: two separate incubations for 1 min each
      4. 50% ethanol: two separate incubations for 1 min each
      5. dH2O: two separate incubations for 5 min each
    6. Transfer slides into 1x citra plus antigen retrieval buffer.
    7. Add 175 ml of dH2O to the base of pressure cooker and set program to high pressure (or 125 °C) for 4 min.
    8. Allow slides to remain in pressure cooker for another 30 min for depressurization and cooling.
    9. Remove slides from pressure cooker and allow slides to cool further at room temperature (RT) for 45 min.
    10. Wash slides with 1x PBS twice for 5 min each.

  3. Blocking steps (do not allow your tissue to dry out):
    1. Inactivate endogenous peroxidases by incubating slides in 3% H2O2 (1:10 dilution of 30% H2O2 in 1x PBS) at RT for 10 min.
    2. Wash slides with 1x PBS twice for 5 min each.
    3. Permeabilize tissue with permeabilization buffer at RT for 20 min with gentle rocking.
    4. Wash slides with 1x PBS twice for 5 min each.
    5. Quickly vacuum off PBS around tissue, add PAP circle, place slide in microscope slide moisture chamber, and add PBS to tissue inside PAP circle until all slides are in place.
      Note: Add 0.5-1 inch of water to bottom of microscope slide moisture chamber and keep chamber covered during incubations.
    6. Aspirate PBS and incubate slides in Image-it Enhancer at RT for 30 min.
    7. Transfer slides to slide holder already submerged in blocking buffer and incubate at RT for 1 h.
    8. Wash slides with 1x PBS twice for 5 min each.
    9. Return slides to microscope slide moisture chamber and add PBS to each slide until all slides have been transferred.

  4. MYC staining (TSA-IF round 1)
    1. Prepare the appropriate amount of antibody diluent with anti-MYC antibody at a 1:500 dilution.
    2. Incubate slides with the diluted anti-MYC antibody at 4 °C overnight.
    3. Transfer slides to slide holder already submerged in wash buffer and incubate 5 min.
    4. Wash slides further with 1x PBS three times for 5 min each.
    5. Return slides to microscope slide moisture chamber and add PBS to each slide until all slides have been transferred.
    6. Apply the anti-rabbit HRP secondary antibody at 1:1,000 in antibody diluent for 1 h.
    7. Transfer slides to slide holder already submerged in wash buffer and incubate 5 min.
    8. Wash slides further with 1x PBS three times for 5 min each.
    9. Return slides to microscope slide moisture chamber and add PBS to each slide until all slides have been transferred.
    10. Prepare the FITC SAB.
    11. One slide at a time, aspirate PBS and apply the FITC SAB to tissue.
      Note: This protocol has been optimized to apply the FITC SAB for 6 min. Incubation times may need to be optimized for a given experiment to ensure a robust signal and minimal background. The latter can be achieved by using positive and negative controls that differ in the amount of MYC transcript. When applying the stain across multiple slides, be extremely precise with timing. Start timer when FITC SAB is applied to first slide. Continue to replace PBS with FITC SAB and stop reaction at the exact designating time for each slide.
    12. To stop reaction, transfer slide to wash buffer. Incubate 5 min in wash buffer after all slides have been transferred.
    13. Wash slides with 1x PBS twice for 5 min each.
    14. Wash slides with dH2O once for 5 min.
    15. To prepare for the second antibody probe, perform the antigen retrieval Steps B6-B10.
      Note: At this point, FITC has been selectively conjugated to MYC-proximal tyrosine residues. Use of the pressure cooker at this step serves as the stripping method to remove the previously applied primary and secondary antibodies. Stated further in the Notes section, be sure to include appropriate controls to ensure the antibodies have been properly stripped.
    16. Reblock tissue by performing Steps C1-C2 and C5-C9.

  5. MYCN staining (TSA-IF round 2)
    1. Prepare appropriate amount of antibody diluent with anti-MYCN antibody at a 1:500 dilution.
    2. Incubate slides with the diluted anti-MYCN antibody at 4 °C overnight.
    3. Transfer slides to slide holder already submerged in wash buffer and incubate 5 min.
    4. Wash slides further with 1x PBS three times for 5 min each.
    5. Return slides to microscope slide moisture chamber and add PBS to each slide until all slides have been transferred.
    6. Apply the anti-rabbit HRP secondary antibody at 1:1,000 in antibody diluent for 1 h.
    7. Transfer slides to slide holder already submerged in wash buffer and incubate 5 min.
    8. Wash slides further with 1x PBS three times for 5 min each.
    9. Return slides to microscope slide moisture chamber and add PBS to each slide until all slides have been transferred.
    10. Prepare the Cy3 SAB.
    11. One slide at a time, aspirate PBS and apply the Cy3 SAB to tissue.
      Note: This protocol has been optimized to apply the CY3 SAB for 8 min. Incubation times may need to be optimized for a given experiment to ensure a robust signal and minimal background. The latter can be achieved by using positive and negative controls that differ in the amount of MYCN transcript. When applying the stain across multiple slides, be extremely precise with timing. Start timer when CY3 SAB is applied to first slide. Continue to replace PBS with CY3 SAB and stop reaction at the exact designating time for each slide.
    12. To stop reaction, transfer slide to wash buffer. Incubate 5 min in wash buffer after all slides have been transferred.
    13. Wash slides with 1x PBS twice for 5 min each.
    14. Apply either DAPI at 1:50,000 or Hoechst at 1:2,000 at RT for 15 min.
      Note: Increase length of incubation if a shorter exposure time at the microscope is needed.
    15. Wash slides with 1x PBS three times for 5 min each.
    16. Seal tissue by applying 2-4 drops of SlowFade Gold Antifade Mountant, laying on cover slip, gently applying pressure to roll out bubbles and wicking off excess mounting medium.
    17. Lay flat and allow slides to dry overnight at RT shielded from light.
      Note: For longer term use, seal edges of coverslip with nail polish and store at 4 °C.

Chamber slides
  1. Slide preparation
    1. Optimize seeding density for chamber slides so cells are near confluent at time of fixation.
      Note: This protocol has been optimized for 8-well chamber slides and fixation 4-5 days after cell seeding.
    2. Perform a quick wash with 1x PBS.
    3. Incubate slides in 100% methanol for 10 min.
    4. Wash slides with 1x PBS twice for 5 min each.
      Note: Depending on experimental design, chambers can be removed off of slides at this point or anytime here after. Remove chambers according to manufacturer’s recommendations in the presence of PBS and do not let cells dry out.

  2. Blocking and antibody incubations
    1. Perform blocking steps and antibody incubations as you would for FFPE tissue described in the previous section, Procedures C, D and Steps E1-E13.
    2. Wash slides with dH2O once for 5 min.
    3. To prepare for the third antibody probe, perform the antigen retrieval and reblocking steps as you would for FFPE tissue described in the previous section, Steps B6-B10, C1-C2, and C5-C9.
    4. Apply the histone H3 antibody at 1:100 and incubate at 4 °C overnight.
      Note: Application of the histone H3 antibody acts as the nuclear counterstain. After the antigen retrieval step(s), nuclear dyes such as DAPI and Hoechst will no longer completely localize to the nucleus.
    5. Transfer slides to slide holder already submerged in wash buffer and incubate 5 min.
    6. Wash slides further with 1x PBS three times for 5 min each.
    7. Return slides to microscope slide moisture chamber and add PBS to each slide until all slides have been transferred.
    8. Apply the Cy5-conjugated anti-rabbit secondary antibody at 1:200 and incubate at RT for 1 h.
    9. Transfer slides to slide holder already submerged in wash buffer and incubate 5 min.
    10. Wash slides further with 1x PBS three times for 5 min each.
    11. Seal tissue by applying 4-6 drops of SlowFade Gold Antifade Mountant, laying on cover slip, gently applying pressure to roll out bubbles and wicking off excess mounting medium.
    12. Lay flat and allow slides to dry overnight at RT shielded from light.
    13. Seal edges of coverslip with nail polish (apply at least three coats to ensure cells are fully sealed and the mountant does not leak) and store at 4 °C.

Data analysis

  1. FFPE tissue sections: manually take pictures or perform whole slide fluorescence imaging without overexposing each fluorophore. Composite FITC [excitation (494 nM), emission (517 nM)], Cy3 [excitation (550 nM), emission (570 nM)] and DAPI [excitation (358 nM), emission (461 nM)] images as single channel and overlaid images (Figure 1).
  2. Chamber slides: while cells primarily grow as a monolayer, we recommend whole slide Z-stack fluorescence imaging.


Any cell/tissue sections that dry out during the staining procedure will cause increased fluorescence intensity and background. Use MYCN and MYC positive and negative controls to dictate fluorophore exposure levels. Avoid capturing images near the edge of tissue.


Figure 1. TSA-IF of MYCN and MYC in human tissue specimens and cell line cultures. A-B. Representative TSA-IF stains of MYCN and MYC in an TNBC FFPE clinical specimen (A) and TNBC cell line grown in 8-well chamber slides for five days (B). Colors represent cell nuclei (blue), MYCN (magenta), and MYC (green). Scale bars = 20 μm.

Notes

  1. Do not allow the tissue to dry out.
  2. Protect fluorophores from light exposure by covering the microscope slide moisture chamber during incubations and after tissue sections or chamber slides have been cover slipped.
  3. All washes and blocking steps are conducted at RT.
  4. Always include verified positive and negative controls for MYCN and MYC. Design the experiment so each control tissue is subjected to the entire protocol as well as individual primary stains. While antibody cross reactivity has not been observed between MYCN and MYC using this protocol, we suggest the investigator includes controls to confirm the absence of cross reactivity.
  5. Incubations times with FITC and Cy3 SAB may need to be optimized for a given experiment.
  6. Single or dual MYC family isoform TSA-IF can be multiplexed with other antibodies by performing additional rounds of antigen retrieval to strip previously conjugated antibodies. After which, a third TSA-IF reaction can be performed with Cy5 TSA reagent (PerkinElmer, catalog number: NEL745B001KT ), or non-specialized IF protocols can be applied with secondary antibodies of any species.

Recipes

  1. Permeabilization buffer (0.1% Tween-20)
    1 ml Tween-20
    999 ml 1x PBS
  2. Blocking buffer (0.3% Triton X-100, 5% goal serum, 1x PBS)
    3 ml Triton X-100
    50 ml goat serum
    947 ml 1x PBS
  3. Antibody diluent (5% goat serum, 1x PBS)
    25 ml goat serum
    475 ml 1x PBS
  4. Wash buffer (0.1% Triton X-100, 0.25% goat serum, 1x PBS)
    1 ml Triton X-100
    2.5 ml goat serum
    996.5 ml 1x PBS
  5. FITC signal amplification buffer (SAB)
    1. Reconstitute hydrolyzed FITC with 150 μl DMSO
    2. Immediate before each application of SAB, add 1 μl of 30% H2O2 to 200 μl of 1x Plus Amplification Diluent (H2O2 Additive)
    3. To create the working SAB, calculate the volume needed for a given experiment and add FITC at 1:50 and H2O2 Additive at 1:100. For example, for 1 ml of SAB, add 20 μl of FITC and 10 μl of H2O2 Additive to 970 μl of 1x Plus Amplification Diluent
    Note: Prepare the FITC SAB immediately before use.
  6. Cy3 signal amplification buffer (SAB)
    1. Reconstitute hydrolyzed Cy3 with 150 μl DMSO
    2. Immediate before each application of SAB, add 1 μl of 30% H2O2 to 200 μl of 1x Plus Amplification Diluent (H2O2 Additive)
    3. To create the working SAB, calculate the volume needed for a given experiment and add Cy3 at 1:50 and H2O2 Additive at 1:100. For example, for 1 ml of SAB, add 20 μl of Cy3 and 10 μl of H2O2 Additive to 970 μl of 1x Plus Amplification Diluent
    Note: Prepare the Cy3 SAB immediately before use.

Acknowledgments

We thank the patients who contributed tissue used in this study. This research was supported by: a grant from Incyte Corporation as part of the Incyte-Vanderbilt Alliance; an NCI grant CA068485; and a Susan G. Komen grant SAC110030. We thank the Translational Pathology Shared Resources Core, supported by the Vanderbilt-Ingram Cancer Center (P30 CA068485), and the Pathology and Tissue Informatics Core of the Specialized Program of Research Excellence (SPORE) in Breast Cancer (P50 CA098131) for their histopathological assistance and expertise.

Competing interests

Funding for this research was in part provided by a research grant through Incyte Corporation. In reference to our corresponding manuscript, M.C.S. is a current employee of Incyte Corporation. P.C.C.L. and P.S. are former employees of Incyte Corporation; current affiliations are Kymera Therapeutics and Prelude Therapeutics, respectively.

Ethics

Tissue specimens used for development of the FFPE TSA-IF staining procedure were originally collected by clinical providers at the Vanderbilt-Ingram Cancer Center (VICC) and processed at the Vanderbilt University Medical Center (VUMC) Translational Pathology Shared Resources Core. Specimens were de-identified and data prohibited from release by established regulations and policies by the VUMC Institutional Review Board. VICC is an NCI-designated Comprehensive Cancer Center supported by P50 CA098131 that uses best practices for management of core services in accordance with NCI policy.

References

  1. Baratta, M. G., Schinzel, A. C., Zwang, Y., Bandopadhayay, P., Bowman-Colin, C., Kutt, J., Curtis, J., Piao, H., Wong, L. C., Kung, A. L., Beroukhim, R., Bradner, J. E., Drapkin, R., Hahn, W. C., Liu, J. F. and Livingston, D. M. (2015). An in-tumor genetic screen reveals that the BET bromodomain protein, BRD4, is a potential therapeutic target in ovarian carcinoma. Proc Natl Acad Sci U S A 112(1): 232-237.
  2. Beltran, H. (2014). The N-myc oncogene: maximizing its targets, regulation, and therapeutic potential. Mol Cancer Res 12(6): 815-822.
  3. Funa, K., Steinholtz, L., Nou, E. and Bergh, J. (1987). Increased expression of N-myc in human small cell lung cancer biopsies predicts lack of response to chemotherapy and poor prognosis. Am J Clin Pathol 88(2): 216-220.
  4. Hurlin, P. J. (2013). Control of vertebrate development by MYC. Cold Spring Harb Perspect Med 3(9): a014332.
  5. Kawagoe, H., Kandilci, A., Kranenburg, T. A. and Grosveld, G. C. (2007). Overexpression of N-Myc rapidly causes acute myeloid leukemia in mice. Cancer Res 67(22): 10677-10685.
  6. Lawson, D. A., Bhakta, N. R., Kessenbrock, K., Prummel, K. D., Yu, Y., Takai, K., Zhou, A., Eyob, H., Balakrishnan, S., Wang, C. Y., Yaswen, P., Goga, A. and Werb, Z. (2015). Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells. Nature 526(7571): 131-135.
  7. Malynn, B. A., de Alboran, I. M., O'Hagan, R. C., Bronson, R., Davidson, L., DePinho, R. A. and Alt, F. W. (2000). N-myc can functionally replace c-myc in murine development, cellular growth, and differentiation. Genes Dev 14(11): 1390-1399.
  8. Schafer, J. S., Lehmann, B. D., Gonzalez-Ericsson, P. I., Marshall, C. B., Beeler, J. S., Redman, L. N., Jin, H., Sanchez, V., Stubbs, M. C., Scherle, P., Johnson, K. N., Sheng, Q., Roland, J. T., Bauer, J. A., Shyr, Y., Chakravarthy, B., Mobley, B. C., Hiebert, S. W., Balko, J. M., Sanders, M. E., Liu, P. C. C., Pietenpol, J. A. (2020). Targeting MYCN-expressing triple-negative breast cancer with BET and MEK inhibitors. Sci Transl Med 12(534).

简介

[摘要] MYC家族成员MYC、MYCN和MYCL是一类致癌转录因子,它们调节与正常发育、细胞生长、增殖、代谢和生存有关的基因的表达。虽然MYC在多种组织类型中扩增和/或过度表达,但MYCN通常在神经系统肿瘤(神经母细胞瘤和髓母细胞瘤)或具有神经内分泌特征(神经内分泌前列腺癌)中过度表达。鉴于最近的报道,MYCN在多种非神经元组织中的表达也被解除了调控,我们研究了MYCN在三阴性乳腺癌(TNBC)中是否也被解除了调控。与以往针对高表达MYC家族亚型蛋白的个体免疫荧光(IF)染色不同,我们开发了一种IF染色法来同时检测同一肿瘤细胞群中MYCN-和MYC表达细胞。我们的方法允许检测低水平的MYCN和MYC表达,并且可以与额外的蛋白质探针复合。在此,我们利用酪酰胺信号放大(TSA),提出了两种检测MYCN和MYC的方案,用于在福尔马林固定石蜡包埋(FFPE)肿瘤切片和生长后原位固定的细胞系中检测MYCN和MYC。

[背景] 先前的研究表明,MYCN和MYC优先调节参与新陈代谢和细胞生长的同一组核心基因,虽然MYCN等位基因在小鼠发育过程中可以在功能上取代MYC(Malynn等人,2000),但MYCN和MYC在脊椎动物早期发育中对器官发生有单独的时间调节作用(Hurlin,2013年)。MYCN的表达对于干细胞和祖细胞群体的初步建立是必不可少的;在器官系统发育过程中,MYCN表达转为低MYC表达,以支持干细胞和祖细胞的维持,以及在细胞系分化和扩展过程中,升高的MYC水平驱动高增殖细胞直到它们达到终末分化(Hurlin,2013)。

在TNBC转移的发展过程中也观察到了类似的关系,其中MYCN表达在新的转移病灶中升高,这些转移灶扩展并分化为高增殖性MYC表达的肿瘤(Lawson,2015)。鉴于MYC在不同肿瘤组织类型中几乎无处不在,MYC是研究最多的MYC家族成员。然而,由于MYCN表达解除调控与预后不良之间的相关性(Beltran,2014),MYCN在神经和非神经细胞癌中的研究越来越多,包括急性髓系白血病(Kawago,2007)、小细胞肺癌(Funa,1987)、卵巢癌(Baratta,2015)和现在的TNBC(Schafer,2020)。因此,我们开始研究MYCN和MYC之间的关系,通过开发一种可以同时检测同一肿瘤组织切片中两种异构体的IF染色。已建立的涉及MYCN或MYC检测的方案是单独的染色剂,主要识别高度放大/过度表达水平的每种异构体。鉴于TNBC和其他非神经元组织表现出异常的低水平MYCN表达,我们开发了一种用TSA检测MYCN和MYC低表达的方法。此外,我们有两个方案版本的方法:双MYC家族亚型(MYCN和MYC)TSA-IF在FFPE肿瘤切片上的应用,以及一种用于评估MYC家族亚型在原位固定的细胞系培养物中表达的改良型染色剂。我们预计MYCN和MYC在多种肿瘤组织中共表达,而这些IF方法的出现将使研究者进一步确定MYCN-与MYC表达细胞在肿瘤发展和疾病病因中的特性。等等。

关键字:肿瘤生物学, 酪胺信号放大, 免疫荧光, 腔体切片, MYCN, MYC

材料和试剂


 


1.     显微镜载玻片(VWR,目录号:48311-703)


2.     盖玻片(Fisherbrand,目录号:12-545K)


3.     HistoPrep笔(Fischer Scientific,目录号:14-905-30)


4.     PAP-pen(电子显微镜科学,目录号:71310)


5.     10%中性缓冲福尔马林(Thermo scientific,目录号:5701),室温下储存


6.     二甲苯(Fisher Scientific,目录号:X3P-1GAL),室温下储存


7.     乙醇(Pharmco,目录号:111000200),室温储存


8.     Citra plus抗原回收缓冲液(BioGenex,产品目录号:HK080-9K),储存于4°C


9.     磷酸盐缓冲盐水(Gelifesciences,HyClone,产品目录号:SH30013.03),储存于4°C


10.  吐温-20(Acros有机物,目录号:23336-0010),室温储存


11.  Triton X-100(Millipore,OmniPur,目录号:9410),室温储存


12.  30%过氧化氢(H2O2)(费希尔化学公司,产品目录号:H325-100),储存于4°C


13.  Image-iT FX信号增强器(Invitrogen,产品目录号:I36933),储存于4°C


14.  山羊血清(双子座,目录号:100-109),储存于-20°C


15.  二甲基亚砜(Sigma,目录号:D2650-100ml),室温下储存


16.  FITC TSA试剂(PerkinElmer,目录号:NEL741B001KT),储存于4°C


17.  Cy3 TSA试剂(PerkinElmer,目录号:NEL744B001KT),储存于4°C


18.  可选:Cy5 TSA试剂(PerkinElmer,目录号:NEL745B001KT),保存在4°C


19.  DAPI(Sigma-Aldrich,目录号:D9542),储存于-20°C,或Hoechst(Invitrogen,目录号:H3570),储存于4°C


20.  SlowFade Gold防褪色贴片(ThermoFisher Scientific,目录号:S36937),室温下储存


21.  指甲油(任何种类或品牌都可以)


22.  抗体


a、 抗MYCN抗体(细胞信号,目录号:51705S),储存于-20°C


b、 抗MYC抗体(Abcam,目录号:ab32072),储存于-20°C


c、 二级抗兔HRP(赛默飞世尔科技公司,目录号:31462),储存于4°c


23.  渗透缓冲液(见配方)


24.  阻塞缓冲区(见配方)


25.  抗体稀释剂(见配方)


26.  洗涤缓冲液(见配方)


27.  FITC信号放大缓冲器(见配方)


28.  Cy3信号放大缓冲器(见配方)


 


细胞系TSA-IF所需的其他材料和试剂:


1.     8个井室显微镜载玻片(康宁,目录号:354108)


2.     甲醇(Millipore,目录号:MX0485-7),室温储存


3.     抗体


a、 抗组蛋白H3抗体(Abcam,目录号:ab1791),储存于-20°C


b、 Cy5结合抗兔二级抗体(Invitrogen,目录号:A10523),保存于4°C


 


设备


 


1.     滑盖加热器(例如,实验室线路仪器,型号:CPC-600N1)


2.     电压力锅(如Cuisinart,型号:CPC-600N1)


3.     显微镜载玻片湿度室(,Ted Pella,目录号:21053)例如


4.     玻片染色站(例如,Tissue-Tek,目录号:4451)


 


程序


 


组织切片


A、 样品固定和制备


1.     收获组织时,迅速将样本(最大厚度为3 mm)直接放入10%中性缓冲福尔马林中,并在室温下培养24小时。


注意:在最初的几个小时内,每隔30分钟翻转容器和/或混合内容物,以确保均匀暴露和正确固定。


2.     将标本转移到70%的乙醇中,并将组织带到适当的设施中进行FFPE样品制备。


注:如果无法立即处理样本,则将样本放在4 °C。


3.     使用标准的microtone切片程序在带正电的显微镜载玻片上进行组织切片和干燥。


注:本方案已优化用于4微米厚的FFPE组织切片染色。如果组织不需要立即染色,则将切片浸入石蜡中并保存在4 摄氏度.


 


B、 抗原回收


1.     根据需要用HistoPrep笔标记载玻片。


2.     将载玻片放在载玻片加热器上,在60°C下加热60分钟,融化石蜡。


3.     快速将载玻片转移到已浸没在二甲苯中的垂直载玻片架上。


4.     在二甲苯中培养载玻片四次,每次10分钟。


5.     用分级酒精培养法对组织进行再水化:


a、 100%乙醇:两次单独培养,每次1分钟


b、 90%乙醇:两次单独培养,每次1分钟


c、 75%乙醇:两次单独培养,每次1分钟


d、 50%乙醇:两次单独培养,每次1分钟


e、 dH2O:两次单独培养,每次5分钟


6.     将载玻片转移到1x citra plus抗原回收缓冲液中。


7.     向压力锅底部加入175ml的dH2O,并将程序设置为高压(或125°C)4分钟。


8.     让载玻片在压力锅中再保持30分钟,以便减压和冷却。


9.     从压力锅中取出载玻片,让载玻片在室温下进一步冷却45分钟。


10.  用1x PBS清洗载玻片两次,每次5分钟。


 


C、 阻塞步骤(不要让组织变干):


1.     将载玻片在3%过氧化氢(1:10稀释30%过氧化氢于1x PBS中)中培养10分钟,使内源性过氧化物酶失活。


2.     用1x PBS清洗载玻片两次,每次5分钟。


3.     在室温下用渗透缓冲液使组织渗透20分钟,轻轻摇动。


4.     用1x PBS清洗载玻片两次,每次5分钟。


5.     迅速抽干组织周围的PBS,加入PAP圆,将载玻片放入显微镜载玻片湿润室,并将PBS加入PAP圆内的组织中,直到所有载玻片就位。


注:在底部加入0.5-1英寸的水显微镜下滑动湿室,并在孵育期间保持密封。


6.     抽吸PBS,在图像增强剂中培养载玻片30分钟。


7.     将载玻片转移到已浸没在阻挡缓冲液中的载玻片支架上,并在室温下培养1小时。


8.     用1x PBS清洗载玻片两次,每次5分钟。


9.     将载玻片放回显微镜载玻片湿度室,并向每个载玻片中添加PBS,直到所有载玻片转移完毕。


 


D、 MYC染色(TSA-IF第1轮)


1.     用抗MYC抗体配制适量的抗体稀释液,稀释倍数为1:500。


2.     用稀释的抗MYC抗体在4℃下孵育过夜。


3.     将载玻片转移到已浸没在冲洗缓冲液中的载玻片支架上,并培养5分钟。


4.     用1x PBS进一步清洗载玻片三次,每次5分钟。


5.     将载玻片放回显微镜载玻片湿度室,并向每个载玻片中添加PBS,直到所有载玻片转移完毕。


6.     将抗兔HRP二级抗体按1:1000的比例加入抗体稀释液中1h。


7.     将载玻片转移到已浸没在冲洗缓冲液中的载玻片支架上,并培养5分钟。


8.     用1x PBS进一步清洗载玻片三次,每次5分钟。


9.     将载玻片放回显微镜载玻片湿度室,并向每个载玻片中添加PBS,直到所有载玻片转移完毕。


10.  准备FITC SAB。


11.  一次一片,抽吸PBS并在组织上涂抹FITC SAB。


注:本方案已优化为应用FITC-SAB 6分钟。可能需要针对给定实验优化培养时间,以确保信号稳定且背景最少。后者可以通过使用不同MYC转录量的阳性和阴性对照来实现。在多张幻灯片上涂抹色斑时,要非常精确地把握时间。当FITC SAB应用于第一张幻灯片时启动计时器。继续用FITC-SAB代替PBS,并在每个玻片的确切指定时间停止反应。


12.  为了停止反应,将载玻片转移到冲洗缓冲液中。转移所有载玻片后,在洗涤缓冲液中培养5分钟。


13.  用1x PBS清洗载玻片两次,每次5分钟。


14.  用dH2O清洗载玻片一次5分钟。


15.  为准备第二抗体探针,执行抗原检索步骤B6-B10。


注:在这一点上,FITC被选择性地与MYC近端酪氨酸残基结合。在这一步中使用压力锅作为剥离方法来去除先前应用的一级和二级抗体。在注释部分有进一步说明,一定要包括适当的控制措施,以确保抗体被适当地剥离。


16.  通过执行步骤C1-C2和C5-C9重新锁定组织。


 


E、 MYCN染色(TSA-IF第2轮)


1.     用抗MYCN抗体配制适量的抗体稀释液,稀释倍数为1:500。


2.     用稀释的抗MYCN抗体在4℃孵育载玻片过夜。


3.     将载玻片转移到已浸没在冲洗缓冲液中的载玻片支架上,并培养5分钟。


4.     每片用PBS冲洗5分钟。


5.     将载玻片放回显微镜载玻片湿度室,并向每个载玻片中添加PBS,直到所有载玻片转移完毕。


6.     将抗兔HRP二级抗体按1:1000的比例加入抗体稀释液中1h。


7.     将载玻片转移到已浸没在冲洗缓冲液中的载玻片支架上,并培养5分钟。


8.     用1x PBS进一步清洗载玻片三次,每次5分钟。


9.     将载玻片放回显微镜载玻片湿度室,并向每个载玻片中添加PBS,直到所有载玻片转移完毕。


10.  准备Cy3 SAB。


11.  一次一片,抽吸PBS并在组织上涂抹Cy3 SAB。


注:本方案已优化为应用CY3-SAB 8分钟。可能需要针对给定实验优化培养时间,以确保信号稳定且背景最少。后者可以通过使用不同MYCN转录量的阳性和阴性对照来实现。在多张幻灯片上涂抹色斑时,要非常精确地把握时间。当CY3 SAB应用于第一张幻灯片时启动计时器。继续用CY3-SAB代替PBS,并在每个玻片的确切指定时间停止反应。


12.  为了停止反应,将载玻片转移到冲洗缓冲液中。转移所有载玻片后,在洗涤缓冲液中培养5分钟。


13.  用1x PBS清洗载玻片两次,每次5分钟。


14.  在1:50000条件下使用DAPI或1:2000在RT条件下使用Hoechst 15分钟。


注:如果需要缩短显微镜下的曝光时间,则增加培养时间。


15.  用1x PBS清洗载玻片三次,每次5分钟。


16.  通过涂抹2-4滴Slowfed Gold防腐蚀贴片,放置在盖玻片上,轻轻施加压力使气泡滚出,并吸干多余的安装介质,从而密封纸巾。


17.  平放,让载玻片在避光的室温下干燥一夜。


注意:若要长期使用,请用指甲油密封封盖条边缘,并存放在4号°C。


 


腔室滑道


A、 幻灯片准备


1.     优化室内载玻片的播种密度,使细胞在固定时接近汇合。


注:本方案已优化为8孔室载玻片和细胞接种后4-5天的固定。


2.     用1x PBS快速冲洗。


3.     在100%甲醇中培养载玻片10分钟。


4.     用1x PBS清洗载玻片两次,每次5分钟。


注:根据实验设计,可以在此时或之后的任何时候从载玻片上取下腔室。在PBS存在的情况下,根据制造商的建议移除腔室,不要让电池干燥。


 


B、 阻断和抗体孵育


1.     如前一节程序C、D和步骤E1-E13所述,对FFPE组织进行阻断步骤和抗体培养。


2.     用dH2O清洗一次玻片。


3.     为准备第三个抗体探针,执行与上一节步骤B6-B10、C1-C2和C5-C9中描述的FFPE组织相同的抗原检索和重新锁定步骤。


4.     在1:100的条件下应用组蛋白H3抗体,并在4℃下培养过夜。


注:应用组蛋白H3抗体作为核复染。抗原回收步骤后,核染料如DAPI和Hoechst将不再完全定位于细胞核。


5.     将载玻片转移到已浸没在冲洗缓冲液中的载玻片支架上,并培养5分钟。


6.     用1x PBS进一步清洗载玻片三次,每次5分钟。


7.     将载玻片放回显微镜载玻片湿度室,并向每个载玻片中添加PBS,直到所有载玻片转移完毕。


8.     取Cy5结合的抗兔二级抗体1:200,RT孵育1h。


9.     将载玻片转移到已浸没在冲洗缓冲液中的载玻片支架上,并培养5分钟。


10.  用1x PBS进一步清洗载玻片三次,每次5分钟。


11.  通过涂抹4-6滴Slowfed Gold防腐蚀贴片,放置在盖玻片上,轻轻施加压力使气泡滚出,并吸干多余的安装介质,从而密封纸巾。


12.  平放,让载玻片在避光的室温下干燥一夜。


13.  用指甲油封住盖玻片的边缘(至少涂三层以确保电池完全密封且贴片不会泄漏),并在4°C下储存。


 


数据分析


 


1.     FFPE组织切片:手动拍照或进行全玻片荧光成像,每个荧光团不过度曝光。复合FITC[激发(494 nM),发射(517 nM)],Cy3[激发(550 nM),发射(570 nM)]和DAPI[激发(358 nM),发射(461 nM)]图像作为单通道和叠加图像(图1)。


2.     室内载玻片:虽然细胞主要以单层形式生长,但我们建议使用全载玻片Z-stack荧光成像。


 


任何细胞/组织切片在染色过程中变干将导致荧光强度和背景增强。使用MYCN和MYC阳性和阴性对照来指示荧光粉的暴露水平。避免在组织边缘附近拍摄图像。


 






图1。人组织标本和细胞系培养中MYCN和MYC的TSA-IF。A-B.TNBC-FFPE临床标本中MYCN和MYC的代表性TSA-IF染色(A)和在8孔室玻片中生长的TNBC细胞系(B)。颜色代表细胞核(蓝色)、MYCN(洋红色)和MYC(绿色)。比例尺=20米。μ


 


笔记


 


1.     不要让纸巾变干。


2.     在培养期间和组织切片或培养箱载玻片盖好后,通过覆盖显微镜载玻片水分室来保护荧光团免受光照。


3.     所有冲洗和堵塞步骤均在室温下进行。


4.     始终包括MYCN和MYC的经验证的阳性和阴性对照品。设计实验,使每一个对照组织都符合整个实验方案以及单独的原始染色。虽然使用该方案在MYCN和MYC之间没有观察到抗体交叉反应性,我们建议研究者包括控制,以确认没有交叉反应性。


5.     可能需要对Cy3进行优化和培养。


6.     单或双MYC家族亚型TSA-IF可通过执行额外的抗原回收以去除先前的结合抗体而与其他抗体复合。之后,第三次TSA-IF反应可使用Cy5 TSA试剂(PerkinElmer,目录号:NEL745B001KT)进行,也可对任何物种的次级抗体应用非专用IF协议。


 


食谱


 


1.     渗透缓冲液(0.1%吐温-20)


1毫升吐温-20


999毫升1x PBS


2.     阻断缓冲液(0.3%Triton X-100,5%目标血清,1x PBS)


3毫升Triton X-100


50ml山羊血清


947毫升1x PBS


3.     抗体稀释液(5%山羊血清,1x PBS)


25毫升山羊血清


475毫升1x PBS


4.     洗涤缓冲液(0.1%Triton X-100,0.25%山羊血清,1x PBS)


1毫升Triton X-100


2.5毫升山羊血清


996.5毫升1x PBS


5.     FITC信号放大缓冲器(SAB)


a、 用150μl二甲基亚砜重组水解FITC


b、 每次施用SAB前,立即向200μl 1x Plus放大稀释剂(H2O2添加剂)中加入1μl 30%H2O2


c、 要创建工作SAB,计算给定实验所需的体积,并添加1:50的FITC和1:100的H2O2添加剂。例如,对于1 ml SAB,在970μl 1x Plus放大稀释剂中添加20μl FITC和10μl H2O2添加剂


注意:使用前应立即准备FITC SAB。


6.     Cy3信号放大缓冲器(SAB)


a、 用150μl二甲基亚砜重组水解Cy3


b、 每次施用SAB前,立即向200μl 1x Plus放大稀释剂(H2O2添加剂)中加入1μl 30%H2O2


c、 为了创建工作SAB,计算给定实验所需的体积,并添加1:50的Cy3和1:100的H2O2添加剂。例如,对于1 ml SAB,向970μl 1x Plus扩增稀释剂中添加20μl Cy3和10μl H2O2添加剂


注意:使用前应立即准备Cy3 SAB。


 


致谢


 


我们感谢为本研究提供组织的患者。这项研究得到了以下方面的支持:Incyte公司作为Incyte-Vanderbilt联盟的一部分;NCI的一份CA068485;以及一份susang.Komen-grant SAC110030。我们感谢Vanderbilt Ingram癌症中心(P30 CA068485)支持的翻译病理学共享资源核心,以及乳腺癌卓越研究专业计划(SPORE)的病理学和组织信息学核心(P50 CA098131)提供的组织病理学帮助和专业知识。




 


相互竞争的利益


 


这项研究的部分资金是由Incyte公司的一笔研究补助金提供的。本公司现有员工的手稿与本公司现有员工的手稿相对应。P、 C.C.L.和P.S.是Incyte公司的前雇员;目前的附属公司分别是Kymera Therapeutics和Prelude Therapeutics。


 


伦理学


 


用于发展FFPE TSA-IF染色程序的组织标本最初由范德比尔特英格拉姆癌症中心(VICC)的临床提供者收集,并在范德比尔特大学医学中心(VUMC)翻译病理学共享资源核心进行处理。VUMC机构审查委员会根据既定法规和政策,对样本进行了去鉴定,禁止发布数据。VICC是NCI指定的综合癌症中心,由P50 CA098131支持,根据NCI政策,采用最佳实践管理核心服务。


 


工具书类


 


1.     Baratta,M.G.,Schinzel,A.C.,Zwang,Y.,Bandopadhayay,P.,Bowman Colin,C.,Kutt,J.,Curtis,J.,Piao,H.,Wong,L.C.,Kung,A.L.,Beroukhim,R.,Bradner,J.E.,Drapkin,R.,Hahn,W.C.,Liu,J.F.和Livingston,D.M.(2015年)。肿瘤内基因筛查显示,BET溴化物主蛋白BRD4是卵巢癌的潜在治疗靶点。美国自然科学院学报112(1):232-237。


2.     Beltran,H.(2014年)。N-myc癌基因:最大化其靶点、调节和治疗潜力。分子癌症研究12(6):815-822。


3.     富纳,K.,斯坦霍尔茨,L.,诺伊,E.和伯格,J.(1987年)。N-myc在人小细胞肺癌活检组织中的表达增加预示着化疗缺乏反应和预后不良。美国临床病理杂志88(2):216-220。


4.     Hurlin,P.J.(2013年)。用MYC控制脊椎动物的发育。冷泉哈勃透视医学3(9):a014332。


5.     Kawagoe,H.,Kandilci,A.,Kranenburg,T.A.和Grosveld,G.C.(2007年)。N-Myc过表达可迅速导致小鼠急性髓系白血病。癌症研究67(22):10677-10685。


6.     Lawson,D.A.,Bhakta,N.R.,Kessenbrock,K.,Prummel,K.D.,Yu,Y.,Takai,K.,Zhou,A.,Eyob,H.,Balakrishnan,S.,Wang,C.Y.,Yaswen,P.,Goga,A.和Werb,Z.(2015年)。单细胞分析揭示了人类转移性乳腺癌细胞的干细胞程序。自然526(7571):131-135。


7.     Malynn,B.A.,de Alboran,I.M.,O'Hagan,R.C.,Bronson,R.,Davidson,L.,DePinho,R.A.和Alt,F.W.(2000年)。在小鼠的发育、细胞生长和分化过程中,N-myc可替代c-myc。基因进化14(11):1390-1399。


8.     沙弗、J、S、Lehmann、B.D、冈萨雷斯—爱立信、P.I、马歇尔、C.B、Beeler、J.S、Redman、L.N、金、H、桑切斯、V、Stubbs、M.C、Scherle、P、强生、K.N、盛生、Q、Roland、J.T、Bauer、J.A、A、Shyr、Y、查克拉瓦瓦、B、Mobley、B、B、B、B、C、C、Hiebert、S、W、Balko、Balko、J.M、Sanders、M、E、刘、P.C、P.C、Pietenpol、J.A(2020年2020年2020年),2020年(2020年)年(2020年)P.)中。用BET和MEK抑制剂靶向表达MYCN的三阴性乳腺癌。Sci翻译医学12(534)。
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Copyright: © 2020 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Schafer, J. M. and Pietenpol, J. A. (2020). Tyramide Signal-Amplified Immunofluorescence of MYCN and MYC in Human Tissue Specimens and Cell Line Cultures. Bio-protocol 10(13): e3677. DOI: 10.21769/BioProtoc.3677.
  2. Schafer, J. S., Lehmann, B. D., Gonzalez-Ericsson, P. I., Marshall, C. B., Beeler, J. S., Redman, L. N., Jin, H., Sanchez, V., Stubbs, M. C., Scherle, P., Johnson, K. N., Sheng, Q., Roland, J. T., Bauer, J. A., Shyr, Y., Chakravarthy, B., Mobley, B. C., Hiebert, S. W., Balko, J. M., Sanders, M. E., Liu, P. C. C., Pietenpol, J. A. (2020). Targeting MYCN-expressing triple-negative breast cancer with BET and MEK inhibitors. Sci Transl Med 12(534).
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