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Relative Stiffness Measurements of Tumour Tissues by Shear Rheology
通过剪切流变学测量肿瘤组织的相对刚度   

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Abstract

The microenvironment of solid tumours is a critical contributor to the progression of tumours and offers a promising target for therapeutic intervention (Cox and Erler, 2011; Barker et al., 2012; Cox et al., 2016; Cox and Erler, 2016). The properties of the tumour microenvironment vary significantly from that of the original tissue in both biochemistry and biomechanics. At present, the complex interplay between the biomechanical properties of the microenvironment and tumour cell phenotype is under intense investigation. The ability to measure the biomechanical properties of tumour samples from cancer models will increase our understanding of their importance in solid tumour biology. Here we report a simple method to measure the viscoelastic properties of tumour specimens using a controlled strain rotational rheometer.

Keywords: Shear rheology(剪切流变学), Tissue stiffness(组织刚度), Matrix remodeling(基质重塑), Collagen cross-linking(胶原蛋白交联), Lysyl oxidase(赖氨酰氧化酶), Breast cancer(乳腺癌)

Background

The growth of solid tumours is accompanied by pathological remodelling of the native tissue (Cox and Erler, 2011; Bonnans et al., 2014). During progression, the local tissue environment experiences physical as well as biological changes, resulting in increased tissue stiffness (elastic modulus) (Humphrey et al., 2014). Alterations in the extracellular matrix lead to the generation of new tissue properties, which activate mechano-signalling pathways within tumour cells (DuFort et al., 2011). This outside-in signalling leads to altered behaviour, cell morphology, differentiation, proliferation, migration and stemness. In preclinical animal models of cancer, these changes have been shown to drive malignant progression and metastatic spread (Erler et al., 2006; Levental et al., 2009; Bonnans et al., 2014). Thus, as a result, the targeting of matrix remodelling and in particular stiffening has received substantial attention in recent years, and several clinical trials have been initiated (Barker et al., 2012; Baker et al., 2013; Cox et al., 2013; Miller et al., 2015; Madsen et al., 2015; Cox and Erler, 2016; Kai et al., 2016).

The mechanical properties of the tumour microenvironment can readily be examined using approaches such as atomic force microscopy (AFM) and nanoindentation (Akhtar et al., 2009). These approaches provide nanometre resolution and concurrent measurement of the applied force with picoNewton resolution (Kasas and Dietler, 2008). However, AFM is not applicable to understand the elastic properties of larger 3D samples. The mechanical properties of bulk 3D tumour samples can be more accurately examined using shear rheology (Picout and Ross-Murphy, 2003). Rheology is the study of how a material deforms when forces are applied to them. Thus applying shear stress to a 3D matrix can determine the elastic modulus (stiffness) as well as viscous properties of a bulk 3D tumour tissue. In this protocol, we describe a method to measure changes on tumour stiffness by shear rheology.

Materials and Reagents

  1. 1,000 μl sterile pipet tip
  2. 1.5 ml sterile microcentrifuge tubes
  3. Cell strainer, 70 µm (VWR, catalog number: 734-0003 )
  4. Hypodermic needles, 27 G (Fine-JectR) (VWR, catalog number: 613-2012 )
  5. 8 mm disposable biopsy punch (KAI, catalog number: BP-80F )
  6. Precision syringes, 1 ml (VWR, catalog number: 613-3908 )
  7. Standard scalpel #11 (Fine Science Tools, catalog number: 10011-00 )
  8. The 4T1 wild-type cell line was obtained from F. Miller at the University of Michigan
  9. The SW480, early-stage colon adenocarcinoma (Duke stage B) cell line was obtained from the American Type Culture Collection (ATCC) (LGC Standards) (ATCC, catalog number: CCL-228 )
  10. The SW480 + LOX cell line was derived from the parent line and has been previously described (Baker et al., 2011; Baker et al., 2013)
  11. For the human colorectal cancer model, 8-week-old female immunodeficient MF1 nude mice were used (Envigo [formerly known as Harlan Laboratories Inc.])
  12. For the murine mammary carcinoma model, 8-week-old female BALB/c mice were used (Taconic Biosciences)
  13. DMEM (Thermo Fisher Scientific, GibcoTM, catalog number: 31966047 )
  14. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
  15. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  16. Sterile PBS, pH 7.2 (Thermo Fisher Scientific, GibcoTM, catalog number: 20012068 )
  17. Trypsin-EDTA (0.25%), phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  18. Ethanol
  19. Growth medium (see Recipes)

Equipment

  1. Benchtop Centrifuge capable of holding 15 ml tubes
  2. Pipette, P1000 (Gilson)
  3. Cell incubator at 37 °C, 5% CO2 (HERAcell)
  4. Standard glass haematocytometer
  5. Calipers
  6. Stainless Steel Spatula, One End Flat, One End Bent, 6 in. in length (United Scientific Supplies, model: SSFB06 )
  7. Standard pattern surgical scissors blunt/blunt (Fine Science Tools, catalog number: 14000-18 )
  8. 8 mm sand-blasted smart-swap upper geometry, Figure 1A (arrowhead) (TA Instruments)
  9. 8 mm sand-blasted stepped lower geometry, Figure 1A (arrow) (TA Instruments)
  10. Discovery Series Hybrid rheometer (TA Instruments, model: dhr-2 )

Procedure

This protocol describes the biomechanical interrogation of tissue samples, which can be obtained from a variety of sources. Here, we utilise the 4T1/BALB/c syngeneic orthotopic model of murine mammary carcinoma (Miller and Heppner, 1979; Cox et al., 2013; Cox et al., 2015), and a subcutaneous SW480/Nude model of human colorectal cancer which has been engineered to overexpress the matrix cross-linking enzyme lysyl oxidase (LOX) (Baker et al., 2011; Baker et al., 2013). High LOX expression in primary tumours has been shown to cross-link extracellular matrix components, in particular collagens, leading to increases in tensile strength of the tissues (Levental et al., 2009; Baker et al., 2011; Baker et al., 2013). Tumour tissue can be collected from any source, including Genetically Engineered Mouse Models (GEMMS), spontaneous models of cancer, as well as orthotopic and subcutaneous models of cancer (see Note 1). Whilst we demonstrate the approach with both a human colorectal cancer model and murine breast cancer model, any solid tumour tissue, including patient material, could be used providing tissue samples meet the following criteria:
A. The tissue must be easily accessible for intact surgical resection.
B. The tissue can be processed fresh immediately (see Notes 2-4).
C. A minimum biopsy size of 8 mm in diameter and ≥ 1 mm thick can be obtained.
D. Samples can be measured immediately (see Notes 2-4).

  1. Measuring relative stiffness of tumour tissues
    Rheological characterization was performed on all tumour samples using a TA Instruments DHR-2 controlled strain rotational rheometer fitted with an 8 mm sand-blasted parallel plate upper geometry, and an 8 mm sand-blasted lower stepped geometry (Figure 1A). Table 1 below outlines the testing parameters that we have determined to be optimal for measuring tumour tissue using a Discovery Series Hybrid rheometer (TA Instruments, model: DHR-2) under the conditions described (see Notes 5-7).

    Table 1. Rheometer settings
    Parameter
    Value
    Temperature (°C)
    21
    Temperature soak time (sec)
    0
    Oscillation frequency (rad/sec)
    1.0
    Oscillation strain (%)
    0.2-2.0
    Data points per decade
    15
    Controlled strain type
    Continuous Oscillation [direct strain]
    Axial force (N)
    0.03-0.05
    Conditioning time (sec)
    2.0
    Sampling time (sec) 
    3.0

  2. Tumour implantation 
    1. 4T1 and SW480 cell lines were maintained in DMEM/10% FBS/Pen-Strep at 37 °C and 5% CO2.
    2. Aspirate the growth medium from the cells and wash the cells once briefly with PBS.
    3. Aspirate the PBS, and add trypsin-EDTA (0.25%) enough to just cover the cells.
    4. Once the cells have detached, centrifuge at 300 x g for 5 min and resuspend in normal growth medium and count the cells.
    5. Wash the cells in PBS, then remove cell clusters by passing through a cell strainer (70 µm).
    6. Clean and sterilize the inoculation area of the mice with ethanol.
    7. Mix the cells and draw the cells into a syringe without a needle. Place a 27 G needle on the syringe and make sure to remove any air and bubbles.
    8. Inject 100 µl tumour cells into the anatomical location of interest (see Note 8).
    9. Following implantation, regular monitoring of the animals as per institutional guidelines is required, and tumour size and body weight is measured three times a week (see Note 9).

  3. Experimental setup
    1. Startup and calibrate the rheometer according to manufacturer’s guidelines.
    2. Attach the stepped lower geometry to the heated peltier plate (Figure 1A) (arrow).
    3. Attach the 8 mm diameter upper geometry (Figure 1A) (arrowhead).
    4. Set the peltier temperature to the required temperature (Table 1).
    5. Surgically excise the tumour tissue to be measured (see Notes 2, 3, 4 and 10). Figures 1B-1D show a primary orthotopic fatpad-implanted 4T1 mammary carcinoma tumour at 3 weeks post implantation being excised. When measured externally using calipers the tumour measures approximately 520 mm3, which is sufficient to allow an 8 mm diameter tissue sample to be collected (see conditions above under Procedures).
    6. A ≥ 1 mm thick section is cut from the region of interest for measurement (Figures 1E and 1F). The remaining tissue not used for rheology can be used for additional analysis including RNA/DNA/protein extraction, or fixed/frozen and processed for immunohistochemistry/immunofluorescence (see Note 11).
    7. Using an 8 mm disposable biopsy punch, a circular biopsy is cut and extraneous material is removed (Figure 1G).


      Figure 1. Rheology set-up. A. 8 mm sand-blasted lower stepped geometry (arrow), peltier plate and 8 mm sand-blasted upper geometry (arrowhead); B. Primary orthotopic 4T1 tumour (circled) at 3-week post-implantation into the mammary fatpad of a BALB/c mouse; C and D. Post-mortem surgical excision of the whole tumour (arrowhead) from the fatpad (E and F). A ≥ 1 mm section is cut using a scalpel from the region of interest in the tumour (see Notes 2-4). G. Using an 8 mm disposable biopsy punch, a circular biopsy is cut and extraneous material is removed. H. The tissue sample is then placed on the lower geometry (arrow) and the upper geometry (arrowhead) lowered so that it comes into contact with the sample. The gap between the upper geometry (arrowhead) and lower geometry (arrow) is slowly reduced until a stable axial force (see Table 1) is achieved and the measurement commences. I. Representative example of measurements taken from 4T1 mammary carcinoma tumours at 3 weeks post implantation compared to normal mammary fat pad tissue bearing no tumour. The storage modulus is measured over one decade of strain from 0.2 to 2% (left). The right-hand graph represents the storage modulus at 1% strain showing that tumour tissue is significantly stiffer than healthy control tissue. µ represents the mean storage modulus (G’) ± standard deviation. J. Representative example of how over-expression of lysyl oxidase (LOX), a potent extracellular matrix cross-linker, leads to increases in the stiffness of tumour tissue in a human colorectal cancer model. The storage modulus (G’) of tumour tissue is measured 4 weeks after implantation subcutaneously (left). The right-hand graph represents the storage modulus at 1% strain, and illustrates that over-expression of LOX in tumour cells leads to stiffening of their environment. µ represents the mean storage modulus (G’) ± standard deviation.

    8. Zero the axial force on the rheometer.
    9. Carefully transfer the tumour tissue to the lower geometry (arrow) using a flat-ended spatula.
    10. Move the 8 mm upper geometry (arrowhead) down until it just contacts the top surface of the tissue (Figure 1H).
    11. Decrease the gap by 50 µm increments to increase the axial force applied to the tissue and continue until a stable axial force is reached as detailed in Table 1 (see Note 7).
    12. Set an oscillation strain sweep as per Table 1.
    13. Set an oscillation frequency as per Table 1.
    14. Begin measurement.

  4. Analysis of the relative stiffness
    1. Ensure a linear viscoelastic response [storage modulus (G’)] within the strain range evaluated (Figures 1I and 1J) (see Notes 6 and 12).
    2. Extract the storage modulus (G’) at a matched strain between samples (example here is 1% strain) when comparing multiple tissue measurements (Figures 1I and 1J).
    3. If required, the elastic moduli (E) can be determined from the storage modulus (G’) using:
      E = 2 x G’ (1 + υ)
      where, υ = Poisson’s ratio of 0.5 for most tissues.

Data analysis

To ensure reliable data, be sure to perform at least five biological repeats within each experimental group with the appropriate controls. Extract the storage modulus (G’) at 1% strain for each repeat when comparing multiple tissue measurements (Figures 1I and 1J, right panels). Ensure a linear viscoelastic (storage modulus [G’]) response within the strain range evaluated (Figures 1I and 1J, left panels) (see Notes 5 and 6). If this is not the case, disregard the measurement. If this is a recurrent issue, frequencies and strains will need to be optimised for the specific tissue under evaluation (see Note 5).

Notes

  1. All animal experiments were carried out under authorisation and guidance from the Danish Inspectorate for Animal Experimentation.
  2. Avoid storing tissues and especially prepared biopsies for measurement in buffers such as PBS for long periods of time (in excess of 1 h) prior to measurement as this can result in the tissue swelling and breaking up affecting mechanical properties.
  3. Do not allow tissues or prepared biopsies to dry out as this causes shrinkage and will also affect mechanical properties.
  4. Store whole tissues on ice prior to preparation. Avoid storing for more than 1 h prior to measurements.
  5. Optimisation of measurement parameters may be required for extremely soft or extremely stiff samples. It may be necessary to determine the optimal frequency and strain parameters to ensure measurements are collected within the linear viscoelastic response of the tissue.
  6. The point at which a Storage modulus (G’) deviates by more than 10% from a constant (plateau) value indicates departure from linear viscoelastic behaviour.
  7. When applying an axial force to the samples prior to starting the measurements, ensure this is consistent across measurements. A value of 0.03-0.05 N for tumour tissues described above is sufficient.
  8. The number of implanted tumour cells depends on the set-up of the experiment. We implant 0.5 x 106 4T1 cells resuspended in 100 µl PBS per mouse in the 4th mammary fat pad of 8-week old BALB/c mice. We implant 1 x 106 SW480 cells resuspended in 100 µl PBS per mouse subcutaneously into the flank of 8-week old immunodeficient MF1 nude mice. In both models general anesthesia is not required in mice for implantation as approved by ethics (see Note 1). It is important to prepare a sufficient amount of cells resuspended in PBS to include the dead volume of the needle (typically 100 µl). We recommend to prepare approximately twice as much as needed. It is also important that the cells are actively growing (exponential growth) before harvesting the cells for implantation.
  9. Make sure to monitor the health state (body weight and respiration) of animals following tumour implantation on a regular basis. The external/superficial tumour size can be monitored using Calipers and should not exceed institutional guidelines.
  10. The time taken for the tumours to reach a suitable size will depend on the cell line in question and should be optimised by the researcher prior to the commencement of rheology experiments. Typically the 4T1-Balb/c syngeneic mouse model of mammary carcinoma will reach 8-10 mm in diameter within 21-24 days post implantation. The SW480/Nude subcutaneous model of colorectal cancer takes approximately 25-40 days from implantation. When collecting samples, be sure to collect via surgical resection under sterile conditions. Tissues should be minimum 8 mm in diameter and minimum 1 mm in thickness (see Figure 1). Typically a tissue thickness of 2-4 mm is ideal.
  11. Following rheological characterisation, tissues can be fixed with formalin or PFA and used in routine histopathological staining (such as haematoxylin and eosin staining for cells or Picrosirius Red staining for collagens). The use of these samples for immunohistochemistry, or protein/DNA/RNA extraction is cautioned against, since rheological characterisation is carried out fresh and may lead to changes in gene and protein expression during measurement.
  12. Tissues are typically only minimally frequency dependent within the range of 0.1 to 10 rad/sec (data not shown) and an oscillation frequency of 1 rad/sec is preferred when using the DHR2. At this frequency tumour samples show a linear viscoelastic response within the strain range evaluated (see Figures 1I and 1J).

Recipes

  1. Growth medium
    DMEM
    Fetal bovine serum (10%)
    Penicillin-streptomycin (100 U/ml)

Acknowledgments

This protocol has been adapted from previous published papers (Baker et al., 2013; Cox et al., 2013; Madsen et al., 2015). TRC is supported by an NHMRC New Investigator grant, Australia. CDM is supported by the Ragnar Söderberg Foundation, BioCARE, Cancerfonden, and Åke Wiberg foundation, all Sweden. We thank Lena Wullkopf, Biotech Research Innovation Centre, University of Copenhagen for assistance and also thank Professor Janine Erler at the Biotech Research & Innovation Centre, University of Copenhagen for providing access to the rheometer.

References

  1. Akhtar, R., Schwarzer, N., Sherratt, M. J., Watson, R. E., Graham, H. K., Trafford, A. W., Mummery, P. M. and Derby, B. (2009). Nanoindentation of histological specimens: Mapping the elastic properties of soft tissues. J Mater Res 24(3): 638-646.
  2. Baker, A. M., Bird, D., Lang, G., Cox, T. R. and Erler, J. T. (2013). Lysyl oxidase enzymatic function increases stiffness to drive colorectal cancer progression through FAK. Oncogene 32(14): 1863-1868.
  3. Baker, A. M., Cox, T. R., Bird, D., Lang, G., Murray, G. I., Sun, X. F., Southall, S. M., Wilson, J. R. and Erler, J. T. (2011). The role of lysyl oxidase in SRC-dependent proliferation and metastasis of colorectal cancer. J Natl Cancer Inst 103(5): 407-424.
  4. Barker, H. E., Cox, T. R. and Erler, J. T. (2012). The rationale for targeting the LOX family in cancer. Nat Rev Cancer 12(8): 540-552.
  5. Bonnans, C., Chou, J. and Werb, Z. (2014). Remodelling the extracellular matrix in development and disease. Nat Rev Mol Cell Biol 15(12): 786-801.
  6. Cox, T. R., Bird, D., Baker, A. M., Barker, H. E., Ho, M. W., Lang, G. and Erler, J. T. (2013). LOX-mediated collagen crosslinking is responsible for fibrosis-enhanced metastasis. Cancer Res 73(6): 1721-1732.
  7. Cox, T. R. and Erler, J. T. (2011). Remodeling and homeostasis of the extracellular matrix: implications for fibrotic diseases and cancer. Dis Model Mech 4(2): 165-178.
  8. Cox, T. R. and Erler, J. T. (2016). Fibrosis and cancer: Partners in crime or opposing forces? Trends Cancer 2: 279-282.
  9. Cox, T. R., Gartland, A. and Erler, J. T. (2016). Lysyl oxidase, a targetable secreted molecule involved in cancer metastasis. Cancer Res 76(2): 188-192.
  10. Cox, T. R., Rumney, R. M., Schoof, E. M., Perryman, L., Hoye, A. M., Agrawal, A., Bird, D., Latif, N. A., Forrest, H., Evans, H. R., Huggins, I. D., Lang, G., Linding, R., Gartland, A. and Erler, J. T. (2015). The hypoxic cancer secretome induces pre-metastatic bone lesions through lysyl oxidase. Nature 522(7554): 106-110.
  11. DuFort, C. C., Paszek, M. J. and Weaver, V. M. (2011). Balancing forces: architectural control of mechanotransduction. Nat Rev Mol Cell Biol 12(5): 308-319.
  12. Erler, J. T., Bennewith, K. L., Nicolau, M., Dornhöfer, N., Kong, C., Le, Q. T., Chi, J. T., Jeffrey, S. S. and Giaccia, A. J. (2006). Lysyl oxidase is essential for hypoxia-induced metastasis. Nature 440(7088):1222-1226.
  13. Humphrey, J. D., Dufresne, E. R. and Schwartz, M. A. (2014). Mechanotransduction and extracellular matrix homeostasis. Nat Rev Mol Cell Biol 15(12): 802-812.
  14. Kai, F., Laklai, H. and Weaver, V. M. (2016). Force matters: biomechanical regulation of cell invasion and migration in disease. Trends Cell Biol 26(7): 486-497.
  15. Kasas, S. and Dietler, G. (2008). Probing nanomechanical properties from biomolecules to living cells. Pflugers Arch 456(1): 13-27.
  16. Levental, K. R., Yu, H., Kass, L., Lakins, J. N., Egeblad, M., Erler, J. T., Fong, S. F., Csiszar, K., Giaccia, A., Weninger, W., Yamauchi, M., Gasser, D. L. and Weaver, V. M. (2009). Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell 139(5): 891-906.
  17. Madsen, C. D., Pedersen, J. T., Venning, F. A., Singh, L. B., Moeendarbary, E., Charras, G., Cox, T. R., Sahai, E. and Erler, J. T. (2015). Hypoxia and loss of PHD2 inactivate stromal fibroblasts to decrease tumour stiffness and metastasis. EMBO Rep 16(10): 1394-1408.
  18. Miller, B. W., Morton, J. P., Pinese, M., Saturno, G., Jamieson, N. B., McGhee, E., Timpson, P., Leach, J., McGarry, L., Shanks, E., Bailey, P., Chang, D., Oien, K., Karim, S., Au, A., Steele, C., Carter, C. R., McKay, C., Anderson, K., Evans, T. R., Marais, R., Springer, C., Biankin, A., Erler, J. T. and Sansom, O. J. (2015). Targeting the LOX/hypoxia axis reverses many of the features that make pancreatic cancer deadly: inhibition of LOX abrogates metastasis and enhances drug efficacy. EMBO Mol Med 7(8): 1063-1076.
  19. Miller, F. R. and Heppner, G. H. (1979). Immunologic heterogeneity of tumor cell subpopulations from a single mouse mammary tumor. J Natl Cancer Inst 63(6): 1457-1463.
  20. Picout, D. R. and Ross-Murphy, S. B. (2003). Rheology of biopolymer solutions and gels. ScientificWorldJournal 3: 105-121.

简介

实体肿瘤的微环境是肿瘤进展的关键因素,为治疗干预提供了有希望的靶点(Cox和Erler,2011; Barker等人,2012; Cox等人,2016; Cox和Erler,2016)。肿瘤微环境的特性与生物化学和生物力学中原始组织的性质差异显着。目前,微环境生物力学特性与肿瘤细胞表型之间的复杂相互作用正在进行深入的研究。从癌症模型中测量肿瘤样本的生物力学性质的能力将增强我们对于在实体肿瘤生物学中的重要性的理解。在这里,我们报告一种使用受控应变旋转流变仪测量肿瘤标本的粘弹性的简单方法。

背景 实体瘤的生长伴随着天然组织的病理重塑(Cox和Erler,2011; Bonnans等人,2014)。在进行期间,局部组织环境经历物理和生物变化,导致组织刚度(弹性模量)增加(Humphrey等人,2014)。细胞外基质的改变导致新的组织特性的产生,其激活肿瘤细胞内的机械信号通路(DuFort等人,2011)。这种外部信号传导导致行为改变,细胞形态,分化,增殖,迁移和干性。在癌症的临床前动物模型中,这些变化已被证明可以驱动恶性进展和转移性扩散(Erler等人,2006; Levental等人,2009; Bonnans 等,,2014)。因此,结果,近年来,基质重塑,特别是硬化的靶向受到了极大的关注,并且已经开始了若干临床试验(Barker等人,2012; Baker等人2013年; Cox等人,2013年; Miller等人,2015年; Madsen等人,2015年,2015年; ; Cox和Erler,2016; Kai等人,2016)。
 使用诸如原子力显微镜(AFM)和纳米压痕(Aktar等人,2009)的方法可以容易地检查肿瘤微环境的机械性质。这些方法提供了纳米分辨率,并用picoNewton分辨率同时测量了所施加的力(Kasas和Dietler,2008)。然而,AFM不适用于了解较大3D样品的弹性特性。使用剪切流变学可以更准确地检查体积3D肿瘤样品的机械性能(Picout和Ross-Murphy,2003)。流变学是研究当施加力时材料如何变形。因此,将剪切应力施加到3D矩阵可以确定体积3D肿瘤组织的弹性模量(刚度)以及粘性。在本方案中,我们描述了一种通过剪切流变学测量肿瘤僵硬度变化的方法。

关键字:剪切流变学, 组织刚度, 基质重塑, 胶原蛋白交联, 赖氨酰氧化酶, 乳腺癌

材料和试剂

  1. 1,000μl无菌移液管尖端
  2. 1.5 ml无菌微量离心管
  3. 细胞过滤器,70μm(VWR,目录号:734-0003)
  4. 皮下注射针,27 G(Fine-Ject R )(VWR,目录号:613-2012)
  5. 8毫米一次性活检穿孔器(KAI,目录号:BP-80F)
  6. 精密注射器,1 ml(VWR,目录号:613-3908)
  7. 标准手术刀#11(精细科学工具,目录号:10011-00)
  8. 4T1野生型细胞系从密歇根大学的F. Miller获得
  9. SW480,早期结肠腺癌(Duke B期)细胞系获自美国典型培养物保藏中心(ATCC)(LGC Standards)(ATCC,目录号:CCL-228)
  10. SW480 + LOX细胞系来源于母系,之前已经描述过(Baker等人,2011; Baker等人,2013)
  11. 对于人类结肠直肠癌模型,使用8周龄的女性免疫缺陷型MF1裸鼠(Envigo [以前称为Harlan Laboratories Inc.])
  12. 对于鼠乳腺癌模型,使用8周龄的雌性BALB/c小鼠(Taconic Biosciences)
  13. DMEM(Thermo Fisher Scientific,Gibco TM ,目录号:31966047)
  14. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10270106)
  15. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  16. 无菌PBS,pH 7.2(Thermo Fisher Scientific,Gibco TM,目录号:20012068)
  17. 胰蛋白酶-EDTA(0.25%),苯酚红(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  18. 乙醇
  19. 生长培养基(见食谱)

设备

  1. 台式离心机能够容纳15毫升管子
  2. 移液器,P1000(Gilson)
  3. 细胞培养箱37℃,5%CO 2(HERAcell)
  4. 标准玻璃血细胞计数器
  5. 卡尺
  6. 不锈钢螺旋桨,一端扁平,一端弯头,6英寸(联合科学用品,型号:SSFB06)
  7. 标准图案手术剪刀钝/钝(Fine Science Tools,目录号:14000-18)
  8. 8毫米喷砂智能插拔上几何,图1A(箭头)(TA Instruments)
  9. 8毫米喷砂阶梯下几何形状,图1A(箭头)(TA仪器)
  10. 发现系列混合流变仪(TA仪器,型号:dhr-2)

程序

该协议描述了可以从各种来源获得的组织样本的生物力学询问。在这里,我们利用鼠乳腺癌的4T1/BALB/c同基因原位模型(Miller和Heppner,1979; Cox等人,2013; Cox等人, 2015),以及已经被设计为过表达基质交联酶赖氨酰氧化酶(LOX)的人结肠直肠癌的皮下SW480 /裸体模型(Baker等人,2011; Baker& et al。,2013)。已经显示原发性肿瘤中的高LOX表达可以交联细胞外基质成分,特别是胶原,导致组织的拉伸强度增加(Levental等人,2009; Baker等人[ al。,2011; Baker等人,2013)。可以从任何来源收集肿瘤组织,包括基因工程小鼠模型(GEMMS),癌症的自发模型,以及原位和皮下的癌症模型(见注1)。虽然我们用人类结肠直肠癌模型和鼠乳腺癌模型证明了这种方法,但是可以使用包括患者材料在内的任何实体肿瘤组织,提供符合以下标准的组织样本:
A.对于完整的手术切除,组织必须易于接近。
B.组织可立即进行新鲜处理(见注2-4)。
C.可以获得直径8 mm,厚度≥1 mm的最小活检尺寸。
D.可以立即测量样品(参见注释2-4)。

  1. 测量肿瘤组织的相对硬度
    使用配备有8mm喷砂平行板上几何形状的TA Instruments DHR-2控制应变旋转流变仪和8mm喷砂下阶梯几何形状(图1A),对所有肿瘤样品进行流变学表征。下表1概述了使用Discovery Series混合流变仪(TA)测定肿瘤组织的最佳测试参数(TA仪器,型号:DHR-2)在所述条件下(见注释5-7)
    表1.流变仪设置
    参数

    温度(°C)
    21
    温度浸泡时间(秒)
    0
    振荡频率(rad/sec)
    1.0
    振荡应变(%)
    0.2-2.0
    每十年数据点
    15
    受控应变型
    连续振荡[直接应变]
    轴向力(N)
    0.03-0.05
    调节时间(秒)
    2.0
    抽样时间(秒) 
    3.0

  2. 肿瘤植入
    1. 将4T1和SW480细胞系在37℃和5%CO 2维持在DMEM/10%FBS/Pen-Strep中。
    2. 从细胞吸出生长培养基,并用PBS短暂洗涤细胞。
    3. 吸出PBS,并加入足以覆盖细胞的胰蛋白酶-EDTA(0.25%)。
    4. 一旦细胞分离,以300×g离心5分钟,并重悬于正常生长培养基中并计数细胞。
    5. 在PBS中洗涤细胞,然后通过细胞过滤器(70μm)去除细胞簇。
    6. 用乙醇清洁和灭菌小鼠的接种面积。
    7. 混合细胞并将细胞抽入注射器而不用针头。将27 G针放在注射器上,确保清除任何空气和气泡。
    8. 将100μl肿瘤细胞注入感兴趣的解剖位置(见注8)
    9. 在植入后,需要按照制度指南定期监测动物,并且每周测量三次肿瘤大小和体重(见注9)。

  3. 实验设置
    1. 根据制造商的指导启动和校准流变仪。
    2. 将步进的下几何形状连接到加热的珀耳帖板(图1A)(箭头)。
    3. 连接8 mm直径的上几何(图1A)(箭头)。
    4. 将珀尔帖温度设置为所需温度(表1)
    5. 手术切除要测量的肿瘤组织(见注2,3,4和10)。图1B-1D显示植入后3周切除原发性原位脂肪垫植入的4T1乳腺癌肿瘤。当使用卡尺测量时,肿瘤测量大约520 mm 3,这足以允许收集8 mm直径的组织样本(参见上述程序下的条件)。
    6. 从感兴趣区域切割≥1mm厚的部分用于测量(图1E和1F)。不用于流变学的剩余组织可用于进一步分析,包括RNA/DNA /蛋白质提取,或固定/冷冻,并进行免疫组织化学/免疫荧光(见附注11)。
    7. 使用8毫米一次性活检穿孔器切割圆形活检,并移除外来材料(图1G)。


      图1.流变学设置 A. 8 mm喷砂下阶梯几何(箭头),peltier板和8 mm喷砂上部几何(箭头); B.在BALB/c小鼠的乳腺脂肪垫植入后3周的原发性原位4T1肿瘤(圆圈); C和D.从脂肪垫(E和F)进行的死后手术切除整个肿瘤(箭头)。使用来自肿瘤中感兴趣区域的手术刀切割≥1mm的切片(参见注释2-4)。 G.使用8毫米一次性活检穿孔器,切割圆形活检,除去外来材料。然后将组织样品放置在下几何形状(箭头)上,并且上部几何形状(箭头)降低,使得其与样品接触。上部几何(箭头)和下几何形状(箭头)之间的间隙缓慢减小,直到达到稳定的轴向力(见表1)并开始测量。 I.与没有肿瘤的正常乳腺脂肪垫组织相比,在植入后3周从4T1乳腺癌肿瘤获得的测量的代表性实例。在十年的应变下测量储能模量为0.2至2%(左)。右图表示1%应变下的储能模量,表明肿瘤组织比健康对照组织显着更硬。 μ表示平均储能模量(G')±标准偏差。 J.代表性的例子是如何过度表达赖氨酰氧化酶(LOX),一种有效的细胞外基质交联剂,导致人类结肠直肠癌模型中肿瘤组织刚度的增加。皮下植入后4周测量肿瘤组织的储存模量(G')(左)。右图表示1%应变下的储能模量,说明LOX在肿瘤细胞中的过度表达导致其环境僵化。 μ表示平均储存模量(G')±标准差。

    8. 将流变仪上的轴向力置零。
    9. 使用平头刮刀小心地将肿瘤组织转移到较低的几何形状(箭头)。
    10. 向上移动8 mm的上几何(箭头),直到它接触到组织的顶部表面(图1H)
    11. 将间隙减小50μm增加以增加施加到组织的轴向力,并持续到达到稳定的轴向力,如表1所示(见注7)。
    12. 根据表1设置振荡应变扫描。
    13. 根据表1设置振荡频率。
    14. 开始测量。

  4. 相对刚度分析
    1. 确保在评估的应变范围内的线性粘弹性响应[储能模量(G')](图1I和1J)(见注6和12)。
    2. 当比较多个组织测量时(图1I和1J),在样品之间的匹配应变(示例为1%应变)下提取储能模量(G')。
    3. 如果需要,弹性模量(E)可以使用:
      从储能模量(G')确定 E = 2×G'(1 +υ)
      其中,υ=大多数组织的泊松比为0.5。

数据分析

为了确保可靠的数据,请务必在每个实验组中进行至少5次生物学重复,并进行相应的控制。当比较多个组织测量时(图1I和1J,右图),为每个重复提取1%应变下的储能模量(G')。确保在评估的应变范围内的线性粘弹性(储能模量[G'])响应(图1I和1J,左图)(见注5和6)。如果不是这种情况,不考虑测量。如果这是一个复发性问题,频率和应变将需要针对特定组织进行优化(见注5)。

笔记

  1. 所有动物实验均在丹麦动物实验检验局的授权和指导下进行
  2. 避免在测量前长时间(超过1小时),在缓冲液(如PBS)中储存组织和特别准备的活组织检查用于测量缓冲液,因为这可能导致组织肿胀和破坏影响机械性能。
  3. 不要让组织或准备好的活组织检查干燥,因为这会导致收缩,也会影响机械性能
  4. 在准备之前将整个组织储存在冰上。避免在测量前储存超过1小时。
  5. 对于极软或非常硬的样品,可能需要优化测量参数。可能需要确定最佳频率和应变参数,以确保在组织的线性粘弹性响应内收集测量结果。
  6. 储能模量(G')从恒定(平稳)值偏离超过10%的点表示偏离线性粘弹性行为。
  7. 在开始测量之前向样品施加轴向力时,请确保测量结果一致。上述肿瘤组织的0.03-0.05N值是足够的
  8. 植入肿瘤细胞的数量取决于实验的设置。在8周龄的BALB/c小鼠的第4个乳腺脂肪垫中,将0.5×10 6个细胞重新悬浮于每只小鼠100μlPBS中。我们将1×10 6细胞重新悬浮在每只小鼠100μlPBS中,皮下注射到8周龄免疫缺陷型MF1裸鼠的侧腹。在两种模式下,通过道德准入的小鼠进行全身麻醉并不是必需的(见注1)。重要的是准备足够量的细胞重悬于PBS中以包括针的死体积(通常为100μl)。我们建议根据需要准备大约两倍。在收获细胞进行植入之前,细胞也积极生长(指数生长)也很重要
  9. 确保定期监测肿瘤植入后动物的健康状况(体重和呼吸)。可以使用卡尺来监测外部/表面肿瘤大小,不应超过制度指导。
  10. 肿瘤达到合适大小所需的时间将取决于所讨论的细胞系,并应在研究人员开始流变学实验之前进行优化。通常,植入后21-24天,乳腺癌的4T1-Balb/c同基因小鼠模型的直径将达到8-10毫米。结直肠癌的SW480 /裸体皮下模型大约需要25-40天的植入。收集样品时,请务必在无菌条件下进行手术切除。组织的直径应为最小8 mm,厚度最小为1 mm(见图1)。通常,2-4毫米的组织厚度是理想的
  11. 在流变学表征之后,组织可以用福尔马林或PFA固定,并用于常规组织病理学染色(如苏木精和细胞的伊红染色或胶原的Picrosirius红染色)。注意使用这些样品进行免疫组织化学,或蛋白质/DNA/RNA提取,因为流变学表征是新鲜的,并且可能导致测量过程中基因和蛋白质表达的变化。
  12. 组织通常在0.1至10弧度/秒(数据未显示)的范围内最低频率依赖,并且当使用DHR2时,组织优选为1弧度/秒的振荡频率。在这个频率下,肿瘤样品显示出在所评估的应变范围内的线性粘弹性响应(见图1I和1J)。

食谱

  1. 生长培养基
    DMEM
    胎牛血清(10%)
    青霉素 - 链霉素(100U/ml)

致谢

该协议已经从先前发表的论文(Baker等人,2013; Cox等人,2013; Madsen等人, 2015年)。 TRC由澳大利亚NHMRC新研究者补助金支持。 CDM由瑞典所有的RagnarSöderberg基金会,BioCARE,Cancerfonden和ÅkeWiberg基金会提供支持。我们感谢哥本哈根大学生物技术研究创新中心Lena Wullkopf的协助,并感谢Janine Erler教授在生物技术研究与生物技术研究所哥德堡大学创新中心提供流变仪。

参考

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  3. Barker,HE,Cox,TR和Erler,JT(2012)。  在癌症中靶向LOX家族的理由。 Nat Rev Cancer 12(8):540-552。
  4. Bonnans,C.,Chou,J. and Werb,Z.(2014)。  重塑细胞外基质在发育和疾病中的应用。 Nat Rev Mol Cell Biol 15(12):786-801。
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  6. Cox,TR和Erler,JT(2011)。  重塑和细胞外基质的体内平衡:对纤维化疾病和癌症的影响。 Dis Model Mech 4(2):165-178。
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引用:Madsen, C. D. and Cox, T. R. (2017). Relative Stiffness Measurements of Tumour Tissues by Shear Rheology. Bio-protocol 7(9): e2265. DOI: 10.21769/BioProtoc.2265.
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