A magnetic microrheometry method for measuring cell-size scale viscoelasticity

Abstract

There is provided a magnetic microrheometry method for measuring viscoelasticity of extracellular matrices at a cell-size scale. The method comprises obtaining collagen type 1 matrix encapsulating magnetic probes having a radius of R, applying X-ray irradiation on the collagen type 1 matrices. After the X-ray irradiation, magnetic forces ( are exerted on the magnetic probes within the collagen type 1 matrices using magnetic microrheology and detecting displacements of the magnetic probes. Cell-size scale viscoelasticity of collagen type 1 matrices is measured based at least on the application of X-rays, the exerted controlled magnetic force ( and the detected displacements of the magnetic probes.

Description

TITLE Amagnetic microrheometry method for measuring cell-size scaleviscoelasticity TECHNICAL FIELD

[0001] The present invention relates to a magnetic microrheometry method formeasuring viscoelasticity of extracellular matrices at a cell-size scale, and uses of themethod. BACKGROUND

[0002] Alterations of extracellular matrix mechanics play a key role in breast-cancerprogression, as cancer cells sense mechanical cues of the matrix along their migration route

[0017] –

[0019] . Collagen type 1 (col-1) is typically the most abundant structural constituent ofthe extracellular matrix in the tissue area called stroma that surrounds a breast tumor. Inadvancing breast cancer toward metastasis, the tumor’s cancer cells migrate outward through this collagen-rich stromal matrix, mechanically interacting with the matrix

[0020] . Infact, the col-1 matrix mediates invasion of breast cancer cells mechanically (via stiffness inparticular

[0021] ,

[0022] ), and physiochemically

[0023] –

[0025] .SUMMARY

[0003] According to some aspects, there is provided the subject-matter of theindependent claims. Some embodiments are defined in the dependent claims.

[0004] According to a first aspect of the present invention, there is provided amagnetic microrheometry method for measuring viscoelasticity of extracellular matrices ata cell-size scale, comprising obtaining collagen type 1 matrix encapsulating magneticprobes having a radius of R, applying X-ray irradiation on the collagen type 1 matrices,after the X-ray irradiation, exerting magnetic forces ( on the magnetic probes within thecollagen type 1 matrices using magnetic microrheology, detecting displacements of themagnetic probes, and measuring cell-size scale viscoelasticity of collagen type 1 matricesbased at least on the application of X-rays, the exerted controlled magnetic force ( andthe detected displacements of the magnetic probes.

[0005] According to a second aspect of the present invention, there is provided useof the method according to the first aspect to analyze cell-scale viscoelasticity of thefurther components in a breast-cancer tissue.

[0006] According to a third aspect of the present invention, there is provided use ofthe method according the first aspect combined with live-cell experiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] In the following embodiments are discussed in more detail with reference tothe attached drawings, of which:

[0008] FIGURES 1A, 1B, 1C and 1D illustrate a technique to quantify the impact ofX-ray radiation on cell-scale viscoelasticity.

[0009] FIGURES 2A, 2B, 2C and 2D illustrate a higher dose of X-ray irradiationaffects the cell-size-scale viscoelasticity of col-1 matrices, specifically, by softening thematrices, and increasing their loss tangent.

[0010] FIGURES 3A, 3B, 3C and 3D illustrate a lower, clinically relevant dose ofX-ray radiation has an insignificant effect on viscoelasticity of col-1 matrices.

[0011] Figures are presented as illustrative examples and embodiments may not belimited solely to the illustrated parts, but modifications may be made under the scope as defined in the claims. Figures that may not fully present the claimed invention, aim toprovide better understanding on the context and relating technical field.DESCRIPTION OF EMBODIMENTS

[0012] X-rays are widely used in mammography and radiotherapy of breast cancer.The research has focused on the effects of X-rays on cells in breast tissues, instead of thetissues’ non-living material, extracellular matrix. It is unclear what is the influence of X-ray irradiation on the matrix’s mechanical cues, known to regulate malignant cancer-cellbehaviors. Here, we developed a technique based on magnetic microrheology that canquantify the influence of X-ray irradiation on matrix viscoelasticity––or (solid-like) elasticand (liquid-like) viscous characteristics––at cell-size scales. To model breast-tissueextracellular matrix, we used the primary component of the tissue matrix, collagen type 1,as it is for control, and as irradiated by X-rays (tube voltage 50 kV). We used a magneticmicrorheometer to measure collagen matrices using 10-μm-diameter magnetic probes. Ineach matrix, the probes were nanomanipulated using controlled magnetic forces by themicrorheometer, while the probes displacements were detected, to measure theviscoelasticity. The collagen-matrix data involves with a typical spatial variation inviscoelasticity. We find that higher irradiation doses (320 Gy) locally reduce stiffness(soften) collagen matrices, and increase their loss tangent, indicating an elevated liquid-like nature. For lower, clinically relevant irradiation doses (54 Gy), we find insignificantmatrix-viscoelasticity changes. We provide this irradiation-related technique for detection,and modification, of matrix viscoelastic cues at cell-size scales. The technique enablesenhanced characterization of irradiated tissue constituents in a variety of breast-cancerradiotherapy types.

[0013] X-rays are commonly used in breast-cancer-relevant clinical applications,including mammography for screening, and radiotherapy for treatment. In mammography, technological advances have enabled an enhanced imaging resolution while minimizing theabsorbed X-ray radiation dose below 3 gray (Gy) in breast tissue [1]–[2]. Radiotherapy isused to kill cancer cells within the breast tissue by administering a significantly higherdoses of radiation, typically, up to 63 Gy of the radiation type (i.e. X-rays, gamma rays orelectrons / protons) [3]–[7]. X-rays are the most common type of radiotherapy radiation,often operating with accelerating voltage ranging from 40 to 300 kV [8], while also higheraccelerating voltages are recently used [9]. The radiotherapy has not only the therapeuticimpact

[0010] –

[0011] , but also carcinogenic

[0012] and immunosuppressive

[0013] side effects, aswell as a physical impact to tissues due to tissue–X-ray interactions

[0014] . In fact, thisphysical impact of X-rays has been studied by few researchers focusing on radiotherapy(e.g. in respect to tissue structures and proteins

[0015] ,

[0016] ). Qayyum et al. [9] reported onhow X-ray irradiation alters the macroscale stiffness of breast tissue’s non-living part,extracellular matrix that encapsulated breast tumor cells. To date, there is little quantitativeinformation on the changes in matrix mechanics upon X-ray irradiation, thus, the influenceof X-rays on the breast-tissue-relevant matrix remains inadequately understood.

[0014] Recently, the col-1 matrix has been studied using clinical doses ofradiotherapy based on gamma rays that has been found to alter the stiffness and chemicalstructures of the matrix, resulting to changing malignancy-relevant cancer cell behaviors

[0024] . However, these col-1 matrices are mechanically and structurally heterogeneous

[0023] ,

[0026] ,

[0027] , and each location of a col-1 matrix has unique, complex mechanical properties,described by viscoelasticity, the (solid-like) elastic and (liquid-like) viscous characteristics.Particularly, the spatially varying elasticity / stiffness changes regulate and direct cancer-cellmigration in a variety of matrices

[0028] –

[0030] . So far, the changes of these cell-size-scaleviscoelastic properties in the col-1 matrix, due to application of X-rays, remain unknown.

[0015] The current literature includes knowledge on the influence of irradiation oncol-1 matrices, based on macroscale stiffness / elasticity-related properties, but lacksinformation on the cell-size-scale viscoelastic cues. Miller at al.

[0031] report on themacroscale stiffness reduction of col-1 matrices upon irradiation (i.e. by high-energygamma rays at ≃0.7 MV). Previous techniques capable of quantifying the cell-size-scale,microscale viscoelastic cues of col-1 matrices include optical tweezers

[0027] ,

[0032] andatomic force microscopy

[0033] , however, they measure mainly at the surface proximity ofthe matrices. Recently, magnetic microrheometry has been shown to measure the cell-size- scale viscoelastic cues inside matrices at the stiffness levels as found in breast tissue

[0034] and breast tumor tissue

[0035] .

[0016] FIGURES 1A, 1B, 1C and 1D illustrate a technique to quantify the impact ofX-ray radiation on cell-scale viscoelasticity. In FIGURE 1A schematic of themicrorheometer, with electromagnets, electromagnet cores, a microscope objective. A col-1 sample including magnetic and reference probes is shown. FIGURE 1B Illustrationshows the different types of samples, namely: (i) unirradiated control, (ii) higher dose ofirradiation, and (iii) lower dose of irradiation. We account only for the absorbed radiation,whereas a part of the total radiation is scattered. In FIGURE 1C microrheometer’sworkspace with a sample holder is shown. The width of the workspace (WS) is 3.5 mm. InFIGURE 1D sinusoidal forces are exerted on the probes while the probes displacesinusoidally, with a phase shift of

[0017] Here, our objective is to provide a technique that can quantify the effects ofX-ray irradiation on col-1 matrix viscoelasticity at the cell-size scale. We report aboutdeveloping the technique and quantification of how an X-ray irradiation dose, produced ata tube voltage of 50 kV, affects the cell-size-scale viscoelasticity (FIGURE 1A). Thistechnique uses prepared col-1 matrices for irradiation, together with unirradiated matrixreplicas, both encapsulating 10-μm-diameter magnetic probes for measuring viscoelasticcues (FIGURE 1B). We have focused on investigating two different irradiation doses,applied on the col-1 matrices. To induce viscoelasticity alterations, we selected a higherdose of irradiation (320 Gy) that is roughly a five-fold value of a typical upper limit ofclinically relevant irradiation (63 Gy [3]–[7]). A lower dose (54 Gy) was chosen to beclose to this upper limit of clinically relevant irradiation. To compare the cell-size-scaleviscoelasticity between the irradiated and unirradiated matrices, we used magneticmicrorheometry to exert controlled magnetic forces on the magnetic probes within thematrices, while the probes displacements within the matrices were detected, to measure theviscoelasticity. This introduced technique enables enhanced cell-size-scale quantificationof irradiated tissue constituents in breast-cancer radiotherapy for varied irradiation dosesand energies. MATERIALS AND METHODS COLLAGEN TYPE 1 (COL-1) MATRIX PREPARATION

[0018] The breast tissue’s key component, col-1, was modeled using a standardproduct of col-1 from rat tail (Corning cat. #354249). To produce the col-1 matrices, we diluted the product into a concentration of 2.2 mg / mL, since typical collagen concentrations of breast tissue are reported to be between 1 mg / mL (normal conditions) and 4 mg / mL (pre-malignant and malignant conditions)

[0036] –

[0038] . Further, we controlled the pH and the temperature that also affect polymerization and the final stiffness of the col-1 sample. During the preparation, we introduced magnetic and reference probes within col-1 samples for microrheometry measurements (FIGURE 1A). The magnetic probes (Sigmacat. #49664) had a calibrated mean diameter of 10.24 μm, with a standard deviation (SD)of 0.14 μm. Non-magnetic probes (Polysciences cat. #15714), serving as the referenceprobes to subtract environmental motion in the measurements, had a nominal diameter of6.0 μm. Both probe types were diluted into 0.3 wt% solutions.

[0019] The morphology of the col-1 matrices and the magnetic probes within thematrices were analyzed by scanning electron microscopy (SEM) (Zeiss Sigma VP). Thegelled matrix specimens were dehydrated by gradual water–ethanol exchange, dried bycritical-point process and snap-cracked in liquid nitrogen. Finally, the samples weremounted on stubs and coated with 5-nm-layer of Au / Pd (using Leica EM ACE600 highvacuum sputter) and then imaged on the SEM.

[0020] To mechanically measure the col-1 matrices, custom-made sample holderswere prepared with outer dimensions (width length heigth) of 3.4 mm 24.5 mm 3.25mm, and inner dimensions of 2.2 mm 18.9 mm 3.1 mm. The frames of the sampleholders were cut with a laser cutter out of polymethyl methacrylate (PMMA), a standard material in laser cutting, with inertness to collagen matrix. Microscopy glass slides with a 0.15 mm thickness were glued on the bottom of the sample holders for imaging. The col-1samples, placed in the sample holders, originated from separate col-1 batches (i.e. collagenin an Eppendorf tube, taken out directly from a fridge-stored commercial collagen bottle).

[0021] For each col-1 batch, we prepared a volume of 510 μL of col-1 dilution at aconcentration of 2.20 mg / mL, by adding the following ingredients in the correspondingorder: 259 μL of DI water, 64 μL of magnetic-probe solution, 64 μL of non-magneticprobe solution, and 102 μL of the col-1 product. Then, a volume of 20 μL sodiumhydroxide (NaOH 10.5 g / L) was added to elevate the pH of the batch above 7 to initiate thecol-1 polymerization, followed by a vortex mixing for 15 s. The batch was aliquoted into three samples, each containing 160 μL of the dilution, and the three samples were placedon a heat plate at 33–34°C for 20 minutes. In comparison to col-1 polymerization in bodytemperature (37°C), validation experiments show negligible differences in rheology between the heat plate temperatures of 34°C and 37°C. After the heating, the first sample was irradiated (referred to as ‘irradiated sample’), the second sample served as a control (referred to as ‘unirradiated sample’), and the third sample was spared to serve as anadditional control in case the unirradiated sample preparation would fail. These threesamples from each batch had a volume of 160 μL.X-RAY IRRADIATION

[0022] We irradiated the col-1 matrix samples (FIGURE 1B) using a computedtomography (CT) instrument (Fraunhofer EZRT CTportable #012; X-ray tube #19620).The CT instrument was used to provide X-rays at a 50 kV tube voltage for 30 s. Toirradiate the col-1 matrix samples with two different doses, we adjusted the X-ray tubecurrent. For the higher (320 Gy) and lower (54 Gy) irradiation doses, we used tube currentsof 400 µA and 65 µA, respectively (FIGURE 1B). The dose calculations use theattenuation coefficients of the medium (col-1 matrix and the sample-holder wall) that weremeasured using the CT device. We accounted for the X-ray radiation attenuating in themedia via photoelectric absorption, incoherent scattering and coherent scattering. Toextract the absorbed energy, we excluded the coherent scattering from the attenuatedenergy, since coherent scattering is unable to transmit energy to matter. For the purpose,the proportion of the coherent scattering was estimated based on the attenuation statisticsof water

[0039] . After the irradiation, microrheometry measurements of unirradiated and irradiated col-1 matrix samples were carried out.CELL-SCALE VISCOELASTICITY MEASUREMENTS AND DATA ANALYSIS

[0023] The cell-size-scale viscoelasticity measurements of the col-1 matrix sampleswere performed using a microscope-integrated microrheometer developed by Pokki et al.

[0034] (FIGURE 1A). Briefly, the microrheometer (micromanipulator type 2

[0034] ) uses twoelectromagnets mounted on a microscope (Olympus CK2) having a dedicated camera (Basler acA2040) imaging at 20 fps, controlled by a custom-made C++ program for precise timing of each image’s capture. The electromagnets exert forces on magneticprobes that are controlled using a data acquisition card (National Instruments PCIe6341),and the probe displacements are recorded using the microscope camera. The workspace forthe samples (FIGURE 1B) between the microrheometer’s electromagnets is shown inFIGURE 1C. The measurements of unirradiated col-1 matrix samples by themicrorheometer have been validated against a parallel-plate rheometer in

[0034] , and thevalidation showed matching results between the mean values by the microrheometer andthe rheometer.

[0024] To measure cell-size-scale viscoelasticity, we used the microrheometer’selectromagnets to generate sinusoidal magnetic forces onto the magnetic probes withineach col-1 matrix sample at room temperature of 22 3 . These forces were used todisplace the probes with less than 2.1 μm in amplitude, as in our previous cell-size-scalemeasurements that align with standard parallel-plate rheometry experiments at a strain of1% at relevant concentrations

[0034] . Such small displacements and strains are known to beat the linear viscoelasticity regime

[0040] . Further, we used an oscillatory frequency of 0.05Hz, relevant to breast-cancer-cell dynamics

[0041] (FIGURE 1D). The matrices were placedin sample holders between the electromagnets (FIGURE 1C). For these samples, the forces(with an amplitude of relate to a <2.1 µm displacement of the magnetic probes (with aradius of R). Specifically, the probe displacement has an amplitude of , and a phase shiftin relation to the forces (FIGURE 1D). We extracted the values of , , and from thesinusoidal displacement / force data, as in

[0034] , using nonlinear least squares fitting. NB: Toobtain signals with sinusoidal displacements, the experiments are sensitive to the probepositioning between the two electromagnets. For data collection, Tables 1-2 show thegeneral data structure: the number of probes / samples (1-2 probes in each sample) in theviscoelasticity measurements, and the direct measurement parameter, probe displacementamplitude ( ). For every magnetic probe at a defined location within each col-1 matrixsample, we performed 1-3 repetitions using the probe to calculate the local viscoelasticityvalue(s): the absolute shear modulus ( ), and the loss tangent ( ). Specifically, theabsolute shear modulus in each repetition is:

[0026] Next, we computed mean values of the repetitions to obtain cell-scaleviscoelasticity values for each probe at its location (i.e. andForevery col-1 sample, the viscoelasticity values are based on a viscoelasticity measurementin one location, or the mean value of the viscoelasticity measurements in two locations (i.e.andfor unirradiated samples). Typically, one location could bemeasured in each sample, because the used col-1 volumes of 160 μL experienced drainage while we had carried out the 1-3 repetitions. Thus, the measurement at the second locationwas often unfeasible. When it was possible, the second location was randomly chosen bymoving the sample with roughly a field of view (i.e. 0.5-1 mm).

[0027] To minimize the influence of col-1 batch-to-batch differences, we havepaired an irradiated and an unirradiated sample from each batch, to extract relative valuesof viscoelasticity, or the relative modulus ( ),relative loss tangent

[0030] NB: We require that every probe-based measurement within an unirradiatedsample has a minimum of 2 repetitions, because the relative viscoelasticity values areexpected to be sensitive to the unirradiated sample values that are used in the equations’ denominator to calculate relative viscoelasticity (Eqs.3.2-3.3).

[0031] Table 1. General data structure for the higher X-ray irradiation dose. Thenumber of probes / samples for absolute shear modulus (|G|) and loss tangent (tan(j)), aswell as the directly measured probe displacement amplitude ( ). The probes refer to the 10-mm-diameter spherical probes that are used to extract cell-scale viscoelasticity at eachlocation within every sample. The data consists of 1-2 probes within each sample.Number of probes Number of probes Probe displacement (and samples) in (and samples) inamplitude ( ) asMatrix |G| measurements tan(^) mean±SD values [^m] measurements Unirradiated14 (10) 12 (10) 0.1509 ^ 0.0889control Irradiated 10 (8) 8 (7) 0.4530 ^ 0.5035

[0032] Table 2. General data structure for the lower, clinically relevant X-rayirradiation dose. The number of probes / samples for absolute shear modulus (|G|) and losstangent (tan(j)), as well as the directly measured probe displacement amplitude ( ). Theprobes refer to the 10-mm-diameter spherical probes that are used to extract cell-scalevisco-elasticity at each location within every sample (i.e. 1-2 probes in each sample).Number of probes Number of probes Probe displacement (and samples) in (and samples) inamplitude ( ) asMatrix |G| measurements tan(^) mean±SD values [^m] measurements Unirradiated8 (8) 6 (6) 0.6247 ^ 0.4210control Irradiated 9 (8) 7 (6) 0.4725 ^ 0.2858MACROSCALE VISCOELASTICITY BY PARALLEL-PLATE RHEOMETRY

[0033] We carried out supplementary parallel-plate rheometry (Physica MCR 302rheometer, Anton Paar) measurements that were compared with the cell-scaleviscoelasticity results by microrheometry. Additionally, we performed rheometrymeasurements as in

[0035] that enable us to compare col-1 viscoelasticity with the probes andwithout the probes (at 20°C and 37°C).

[0034] For a macroscale comparison with cell-scale microrheometry data, we haveperformed time sweeps at a strain amplitude of 1%, a frequency of 0.05 Hz, and a gapheight of 1 mm. We made an oil enclosure around each sample to prevent waterevaporation. NB: these comparative measurements were performed within 90 min from the preparation of irradiated / unirradiated samples, which could have caused some evaporation / drainage, although the samples were covered.

[0035] The macroscale viscoelasticity, absolute shear modulus and the loss tangent,were measured every 1 min for a duration of 5 min at 22°C. While our microrheometrysystem has previously been validated using the rheometry plate of 25 mm in diameter

[0034] ,we needed to use a smaller plate of 8 mm to measure the irradiated / unirradiated samples,due to the limited volume of each col-1 sample fitting to the irradiation system. Here, asvalidation of our measurements using this smaller plate, the smaller plate (8.0 mm)provides 2.20-fold absolute shear modulus values on average, compared to the 25-mm-diameter plate (i.e. 4 measurements; the conversion factor’s mean±SD= 2.20±0.027). Thus,here, we do not only report the raw viscoelasticity values but also the values that have beenconverted to correspond measurements using the 25-mm-diameter plate. NB: Such plateprovides viscoelasticity measurements that match with microrheometry results

[0034] .STATISTICAL METHODS

[0036] We studied the absolute and relative values of viscoelasticity using thefollowing statistical analysis protocols, and we defined significance at the 95% confidencelevel. We apply a logarithmic transformation to the data because the transformed data iscloser to normal distribution than the data as they are. In contrast, the data as they areexhibit skewed distribution behaviors. The Welch T test for non-equal variances was usedfor the transformed data (referred to as ‘T test’). We used a one-sided T test to evaluate thestatistical differences between the probes in the irradiated samples, and the probes in theunirradiated samples. For the one-sided T test, we have set the test direction in theexperiments using the higher dose and used the same test direction also for the lower,clinically relevant dose. Specifically, we tested whether the irradiation softens col-1matrices (as for gamma-rays in

[0031] ), and elevates col-1 loss tangent, increasing liquid-likenature, due to potential breaking of col-1 fibril bond

[0031] .

[0037] For the absolute and relative values of viscoelasticity, there were separatestatistical approaches to further analyze the data as previously in Pokki et al.

[0034] : one thatconsiders each probe-based measurements as an independent measurement within itsirradiated or unirradiated group (absolute values of viscoelasticity), and another thatconsiders the data to be paired between each irradiated and unirradiated sample that have been drawn from the same col-1 batch (relative values of viscoelasticity). Therefore,unpaired (in the first approach) and paired (in the second approach) versions of the T testwere applied for analyzing the absolute and relative values of viscoelasticity, respectively. For the relative values of viscoelasticity, the specific pairing is between the irradiatedsample (each cell-scale viscoelasticity value within the sample in microrheometry, or thesample value in parallel-plate rheometry) and the corresponding unirradiated sample (themean value of the locations within the sample in microrheometry, or the sample value inparallel-plate rheometry), from the same batch as the irradiated sample. The viscoelasticity value––absolute shear moduli and loss tangent––of the unirradiated sample corresponds toa level of 100%. These statistical approaches have been employed both for the cell-scaledata (microrheometry) and the macroscale data (parallel-plate rheometry).RESULTS HIGHER X-RAY IRRADIATION DOSE SOFTENS COLLAGEN TYPE 1 (COL-1) AT CELL SCALES

[0038] FIGURES 2A, 2B, 2C and 2D illustrates a higher dose of X-ray irradiationaffects the cell-size-scale viscoelasticity of col-1 matrices, specifically, by softening thematrices, and increasing their loss tangent. The reference level in relation to unirradiatedmatrix is noted with a red dashed line. FIGURE 2A shows stiffness of the col-1 matrices asirradiated (higher dose, 320 Gy), and as they are (unirradiated, 0 Gy). Absolute shearmodulus approximately halves on average for the higher irradiation dose (** Pr < 0.01,unpaired t-test, probes in irradiated matrices n=10, probes in unirradiated matrices n=14). Squares represent viscoelasticity values, measured by separate magnetic probes, used for statistical analysis. The mean values are shown in wide bar plots, and their error barsindicate standard deviations (SD). FIGURE 2B shows relative value of shear modulus, orrelative modulus, is the ratio between probe-based stiffness in the irradiated col-1 matrices,and unirradiated matrices’ mean value, from the same batch. After irradiating the matriceswith the higher dose, the relative modulus shows a softening of 64 29 (mean SD) (** Pr< 0.01, paired t-test, probes in irradiated matrices n=6, probes in unirradiated matricesn=8). FIGURE 2C shows loss tangent of the col-1 matrices as irradiated with the higherdose, and as unirradiated as they are. The higher dose increases the matrices loss tangenton average (* Pr < 0.05, unpaired t-test, probes in irradiated matrices n=8, probes inunirradiated matrices n=12). FIGURE 2D shows relative loss tangent is the ratio betweenprobe-based loss tangent values in the irradiated col-1 matrices, and unirradiated matrices’mean value, from the same batch. Irradiating with the higher dose increases the relativeloss tangent, specifically, by an increase of 144 102% (mean SD) (* Pr < 0.05, paired t-test, probes in irradiated matrices n=5, probes in unirradiated matrices n=8).

[0039] We have quantified for the first time how X-ray irradiation of col-1 matriceschanges the matrices’ cell-size-scale viscoelasticity. For the quantification, we haveinitially used an X-ray dose of 320 Gy (referred to as ‘higher dose’) (FIGURES 2A-D).This higher dose increases probe displacements in the col-1 matrices, specifically, wedetect an increased mean displacements amplitude as a response to controlled forces(FIGURE 1D and Table 1). Thus, the higher dose of irradiation reduces the absolute shearmodulus (softens) in the col-1 matrices (Eq. 3.1). This softening by the irradiation,characterized by approximately a half value of the absolute shear modulus, is shown inFIGURE 2A. To obtain relative modulus, these probe-based absolute shear modulus valuesin each irradiated sample have been normalized by the mean of the same batch’sunirradiated sample’s locations moduli. The relative moduli have a mean SD value of36 29% compared to the mean of unirradiated matrices (FIGURE 2B). For validation, ourmacroscale measurements show similar softening as these cell-scale data (Table 3) andconfirmed by statistical analysis. Further, our macroscale measurements with probes andwithout probes match, and the SEM analysis shows probe integration within col-1matrices. These confirm our findings on cell-scale col-1 softening upon the higherirradiation dose. The softening may occur via varied mechanisms includingphysicochemical changes in col-1 fibrils within the matrix

[0031] , suggesting that there could be alterations of further viscoelastic properties, such as the loss tangent.Table 3. Comparison of cell-scale microrheometry and macroscale, parallel-platerheometry for the higher dose of the X-ray irradiation. These data consist of mean±SDresults. The notation ‘n’ denotes for the number of probes to measure different locations inmicrorheometry, and ‘N’ is the number of samples in parallel-plate rheometry. Based onthe relative modulus and loss tangent definitions (Eqs. 3.2-3.3), the value of 100% is the level of the unirradiated control samples. The characterindicates the absolute shear modulus that has been converted to correspond values as obtained by the macrorheometry plate of 25 mm in diameter.Parameter Cell-scaleMacroscale microrheometry parallel-plate rheometry Irradiated Unirradiated Irradiated Unirradiatedcontrol control Absolute shear 9.40±8.34 19.03±10.79 23.94±4.47 86.68±26.83 modulus [Pa] N / A N / A (10.91±2.04)* (39.49±12.22) * n=10 n=14 N=4 N=3Relative modulus [%] 36±29100 28±3 100 n=6 N / A N=3 N / ALoss tangent [ ] 0.213±0.0890.161±0.143 0.288±0.010 0.127±0.015 n=8 n=12 N=4 N=3 Relative loss tangent244 115100232 98100 [%] n=5 N / A N=3 N / ACOL-1 SOFTENING BY HIGHER DOSE IS ACCOMPANIED BY ELEVATED LOSSTANGENT

[0040] We report for the first time that the higher dose of X-ray radiation increasesthe cell-scale loss tangent of col-1 matrices (FIGURE 2C) (i.e. the loss tangent increasecorresponds to elevated liquid-like nature of the elastically dominated matrix). Tominimize col-1 batch-to-batch variation, we analyzed next the relative loss tangent values(Eq. 3.3). The higher dose of X-ray radiation increases the cell-scale relative loss tangent(FIGURE 2D). The relative loss tangent values have a mean SD value of 244 102%compared to the mean of unirradiated matrices. Thus, the higher dose not only softens col-1 matrices but also increases the matrices loss tangent. For validation, our macroscalemeasurements show a similar increase in the loss tangent as these cell-scale data.Motivated by the results, we decreased the irradiation dose and investigated whetherviscoelasticity alterations take place. COL-1 SOFTENING AND INCREASE IN LOSS TANGENT DISAPPEARS WITH LOWER DOSE

[0041] FIGURES 3A, 3B, 3C and 3D illustrate a lower, clinically relevant dose ofX-ray radiation has an insignificant effect on viscoelasticity of col-1 matrices. Thereference level in relation to unirradiated matrix is noted with a red dashed line. FIGURE3A shows stiffness of the col-1 matrices as irradiated (clinically relevant dose, 54 Gy), andas they are (unirradiated, 0 Gy). Squares represent viscoelasticity values, measured by separate magnetic probes, which are used for statistical analysis. The mean values areshown in wide bar plots, and their error bars indicate SD. There were statisticallyinsignificant differences between irradiated and unirradiated matrices (n.s. Pr > 0.05,unpaired t-test, probes in irradiated matrices n=9, probes in unirradiated matrices n=8). InFIGURE 3B relative value of shear modulus, or relative modulus, is shown. The irradiationof the matrices causes insignificant differences in relative modulus (n.s. Pr > 0.05, paired t-test, probes in irradiated matrices n=8, probes in unirradiated matrices n=7). FIGURE 3Cshows loss tangent of the col-1 matrices remains unchanged (n.s. Pr > 0.05, unpaired t-test,probes in irradiated matrices n=7, probes in unirradiated matrices n=6). In FIGURE 3Drelative loss tangent is preserved (n.s. Pr > 0.05, paired t-test, probes in irradiated matrices n=6, probes in unirradiated matrices n=5).

[0042] The application of lower, clinically relevant dose of X-ray radiation had aninsignificant influence on cell-scale viscoelasticity within col-1 matrices (FIGURES 3A-D). Specifically, after the use of X-ray dose of 54 Gy (lower dose), the col-1 matricesmaintained their original stiffness (i.e. absolute shear modulus and relative modulus;FIGURE 3A-B), as well as their liquid-like characteristics (loss tangent and relative losstangent; FIGURES 3C-D). For validation, our macroscale measurements also showinsignificant differences in the absolute / relative values of viscoelasticity. DISCUSSION AND CONCLUSIONS

[0043] In this article, we develop for the first time a technique to quantifyviscoelasticity of irradiated matrices at the cell-size scale. We report about X-rayirradiation conditions that relate to col-1 matrix’s cell-scale softening (loweredabsolute / relative values of absolute shear modulus) and increases of its liquid-like nature(raised absolute / relative values of loss tangent). Considerably elevated SD values are present in the data that is consistent with the known batch-to-batch differences and spatialheterogeneity of col-1 matrices

[0023] ,

[0026] ,

[0027] ,

[0034] ,

[0042] . We find these changes in thematrices that have been irradiated by higher doses of X-rays (320 Gy) (FIGURES 2A-D).This dose is roughly a five-fold value of a clinically relevant dose (63 Gy) that –– foranother radiation type, gamma rays–– can also comparably soften col-1 matrices at the macroscale

[0031] . In contrast, we findthat reducing the X-ray irradiation dose to a lower, clinically relevant value (54 Gy) showsinsignificant effects on col-1 matrices viscoelasticity (FIGURES 3A-D). Theseviscoelasticity results are consistent at the cell scale (FIGURES 2A-D, 3A-D) andmacroscale (Tables 3–6), although –– for all measured viscoelasticity parameters –– thecoefficient of variation (SD normalized by the mean) is mostly larger in the measurementsat the cell scale than at the macroscale (Tables 4 and 6). This is likely due to the internalheterogeneity within col-1 samples [3]–[5]. Importantly, we find the relative values ofviscoelasticity to quantify the cell-scale alterations due to col-1 irradiation: for the higherdose, relative modulus and relative loss tangent data have mean SD values of 36 29%and 244 102%, compared to the mean of unirradiated matrices, respectively. The data israther log-normally distributed than normally distributed and have expectedly considerableSDs (i.e. likely due to internal heterogeneity of each col-1 matrix

[0023] ,

[0026] ,

[0027] ,

[0034] ,

[0042] ).

[0044] Table 4. Coefficient of variation between cell-scale microrheometry andmacroscale rheometry for the higher dose of the X-ray irradiation. The notation ‘n’ denotesfor the number of probes to measure different locations in microrheometry, and ‘N’ is thenumber of samples in parallel-plate rheometry. All the values are percentages.Coefficient of variation Cell-scale Macroscale [%] microrheometry parallel-plate rheometry Irradiated Unirradiated Irradiated Unirradiatedcontrol control Absolute shear modulus 88.7 56.7 18.7 31.0 n=10 n=14 N=4 N=3 Relative modulus 80.6 N / A 10.7 N / A n=6 N / A N=3 N / A Loss tangent 72.4 88.8 3.5 11.8 n=8 n=12 N=4 N=3 Relative loss tangent 47.1 N / A 42.2 N / A n=5 N / A N=3 N / A

[0045] Table 5. Comparison of cell-scale microrheometry and macroscale, parallel-plate rheometry for the lower, clinically relevant dose of the X-ray irradiation. These dataconsists of mean±SD results. The notation ‘n’ denotes for the number of probes to measuredifferent locations in microrheometry, and ‘N’ is the number of samples in parallel-plate rheometry. Based on the relative modulus and loss tangent definitions (Eqs. 3.2-3.3), the value of 100% is the level of the unirradiated control samples. The characterindicatesthe absolute shear modulus that has been converted to correspond values as obtained by themacrorheometry plate of 25 mm in diameter. Cell-scale Macroscale Parameter microrheometry parallel-plate rheometry Irradiated Unirradiated Irradiated Unirradiatedcontrol control Absolute shear 7.77±7.87 5.65±4.24 52.25±14.86 63.51±36.35 modulus [Pa] N / A N / A (23.80±6.77)* (28.93±16.56) * n=9 n=8 N=6 N=5Relative modulus [%] 176±118100 93±65 100 N=5 n=8 N / A N / ALoss tangent [ ] 0.264 ± 0.1320.498 ± 0.266 0.175 ± 0.053 0.177 ± 0.079 n=7 n=6 N=6 N=5 Relative loss tangent 71±58 100103 47100 [%] n=6 N / A N=5 N / A

[0046] Table 6. Coefficient of variation between cell-scale microrheometry andmacroscale rheometry for the lower, clinically relevant dose of the X-ray irradiation. The notation ‘n’ denotes for the number of probes to measure different locations in microrheometry, and ‘N’ is the number of samples in parallel-plate rheometry. All the values are percentages. Coefficient of variation Cell-scale Macroscale [%] microrheometry parallel-plate rheometry Irradiated Unirradiated Irradiated Unirradiatedcontrol control Absolute shear 101 75.0 28.4 57.2 modulus n=9 n=8 N=6 N=5 Relative modulus 67.0 N / A 70.0 N / A N / A N=5 n=8 N / A Loss tangent 50.0 53.4 30.3 44.6 n=7 n=6 N=6 N=5Relative loss tangent 81.7N / A 45.6 N / A n=6 N / A N=5 N / A

[0047] The reported alterations in col-1 viscoelasticity upon irradiation could relateto a variety of structural changes

[0043] . For the col-1 fibers, the viscoelasticity alterationsmay relate to breaking / formation of crosslinks, macromolecular folding / unfolding, andmacro-molecular entanglement

[0044] . Interestingly, we find col-1 softening upon the X-rayirradiation dose of 320 Gy that compares to the reduced relative stiffness upon irradiationby another irradiation type and dose, reported by Miller et al.

[0031] (i.e. 35% for a col-1concentration of 2.5mg / mL; irradiation by gamma rays at a dose of 63 Gy). NB: Thehigher-energy gamma rays can pass through the matrix more easily than the X-rays,however, when the gamma rays absorb, they are energetically expected to cause morechanges than X-rays, although the changes may be distinct. As in this previous literatureabout comparable stiffness changes

[0031] , here, a potential mechanism behind the observedsoftening and increased liquid-like nature is a reduced number of bonds, relevant tocollagen crosslinking and backbone (C–N, C–C, and C–O). On the other hand, ourirradiation doses could also involve the formation of crosslinks between amino acidresidues (e.g. new C–N and C–C bonds)

[0045] ,

[0046] , which is expected to stiffen the matrix.We cannot rule out that such competitive, matrix-stiffening bonds could partially beformed, while the col-1 matrix is softening overall.

[0048] The development of cell-scale mechanical quantification of col-1 matrices iscrucial to breast cancer research focusing on tumor tissue and stroma. We have developeda method that provides irradiation-relevant, cell-scale mechanical information on stroma- related col-1 matrices. These cell-scale, spatial data is useful in breast cancer research,because breast cancer cells not only sense the matrix mechanical cues but also respond tothem with altered behaviors (e.g. increased / decreased migration

[0047] ,

[0048] ,

[0049] , directedmigration in durotaxis

[0028] –

[0030] , and changes in adhesion

[0029] ). In this work, we find that,for the higher doses of X-ray irradiation, col-1 matrix softening is accompanied byelevated loss tangent, indicating increased liquid-like nature, which is relevant to breast-cancer tissue properties. Previously, Sinkus et al.

[0050] and Balleyguier

[0051] have shownthat breast cancer tissue’s liquid-like nature, and elevated loss tangent, correlates with themalignancy of patients. Further, as the focus of this work, we report on the mechanicaleffects by irradiation on the breast-cancer tissue’s most abundant component: col-1. Thus,this work has a limitation in quantification of the actual tissue’s response, because theother tissue components than col-1 may have distinct responses to irradiation, possessingcharacteristic biomechanical- and chemical properties

[0020] . In the future research, thismethod has the potential to analyze cell-scale viscoelasticity of the further components inthe breast-cancer tissue upon irradiation and utilizing our recent quantification technique ofunbiased mechanical heterogeneity (i.e. to account for the measurement uncertainty

[0035] ).Further, this method is plausibly combinable with live-cell experiments (i.e. we havemeasured the viscoelasticity during multiple hours of live-cell time lapses in a fluorescencemicroscope

[0052] ).

[0049] We have established a new technique and an experimental protocol thatenables future studies on breast-cancer tissue components, irradiated by selected irradiationenergy / dose. This technique could be used to detect, and modify, matrix viscoelastic cuesat cell-size scales, adjacent to live-cell microscopy

[0052] , for in vitro modeling ofradiotherapy in 3D cell culture. Such tools to account for the contribution of matrix inbreast-cancer tissues paves the way for better understanding of elimination and recurrenceof breast cancer after radiotherapy, as well as developing more precise and effectiveradiotherapy. CITATION LIST[1] R. L. Kruger and B. A. Schueler, “A survey of clinical factors and patient dose inmammography,” Med Phys, vol.28, no.7, pp.1449–1454, 2001.[2] T. M. Svahn, N. Houssami, I. Sechopoulos, and S. Mattsson, “Review of radiation doseestimates in digital breast tomosynthesis relative to those in two-view full-field digital mammography,” The Breast, vol.24, no.2, pp.93–99, 2015.[3] E. Evans and J. Staffurth, “Principles of cancer treatment by radiotherapy,” Surgery(Oxford), vol.36, no.3, pp.111–116, 2018.[4] S. Galper et al., “Second nonbreast malignancies after conservative surgery and radiationtherapy for early-stage breast cancer,” Int J Radiat Oncol Biol Phys, vol.52, no.2, pp.406– 414, 2002.[5] S. M. Pierce et al., “Long-term radiation complications following conservative surgery (CS)and radiation therapy (RT) in patients with early stage breast cancer,” Int J Radiat Oncol Biol Phys, vol.23, no.5, pp.915–923, 1992.[6] X. S. Qi, J. White, and X. A. Li, “Is $α$ / $β$ for breast cancer really low?,” Radiotherapy andOncology, vol.100, no.2, pp.282–288, 2011.[7] H. B. Kal and H. Struikmans, “Radiotherapy during pregnancy: fact and fiction,” LancetOncol, vol.6, no.5, pp.328–333, 2005.[8] A. L. Palmer, M. Pearson, P. Whittard, K. E. McHugh, and D. J. Eaton, “Current status ofkilovoltage (kV) radiotherapy in the UK: installed equipment, clinical workload, physics quality control and radiation dosimetry,” Br J Radiol, vol.89, no.1068, p.20160641, 2016.[9] M. A. Qayyum, J. T. Kwak, and M. F. Insana, “Stromal-epithelial responses to fractionatedradiotherapy in a breast cancer microenvironment,” Cancer Cell Int, vol.15, no.1, pp.1– 10, 2015.

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Claims

CLAIMS:

1. A magnetic microrheometry method for measuring viscoelasticity of extracellularmatrices at a cell-size scale, comprising< / sub>- obtaining collagen type 1 matrix encapsulating magnetic probes having a radiusof R,- applying X-ray irradiation on the collagen type 1 matrices,- after the X-ray irradiation, exerting magnetic forces ( on the magnetic probeswithin the collagen type 1 matrices using magnetic microrheology,- detecting displacements of the magnetic probes,- measuring cell-size scale viscoelasticity of collagen type 1 matrices based atleast on the application of X-rays, the exerted controlled magnetic force ( andthe detected displacements of the magnetic probes.

2. A method according to the claim 1, wherein the X-ray irradiation dose is producedat an X-ray tube voltage of 50 kV.

3. A method according to the claim 1, comprising introducing the irradiated magneticprobes and reference probes for magnetic microrheometry measurements.

4. A method according to any of the preceding claims, comprising analysingmorphology of the collagen type 1 matrices and the magnetic probes encapsulatedtherein.

5. A method according to any of the preceding claims, comprising X-ray irradiatingthe collagen type 1 matrices, or samples of such, using a computed tomography,CT, instrument, or another irradiation source.

6. A method according to any of the preceding claims, comprising adjusting the X-raycurrent to irradiate the collagen type 1 matrices, or samples of such, with differentdoses, optionally with two different doses.

7. A method according to the claim 6, comprising, for a higher irradiation dose of 320Gy and for a lower irradiation dose of 54 Gy, and X-ray tube currents of 400 µAand 65 µA, respectively.

8. A method according to any of the preceding claims, wherein the magneticmicrorheometer comprises electromagnets configured to exert forces on themagnetic probes that are controlled using a data acquisition card, and a microscopiccamera configured to record the magnetic probe displacements.

9. A method according to any of the preceding claims, wherein measuring cell-sizescale viscoelasticity of the collagen type 1 matrices provides quantitativeinformation on changes in the matrix mechanics upon X-ray irradiation.

10. A method according to any of the preceding claims, wherein the magnetic probeshave a radius (R).

11. A method according to any of the preceding claims, wherein the magnetic forces( are controlled magnetic forces.

12. A method according to any of the preceding claims, wherein the magnetic forces( relate to a displacement of less than 2.1 µm of the magnetic probes.

13. A method according to any of the preceding claims, wherein the displacements of themagnetic probes have an amplitude ( ), and a phase shift ( ) in relation to the magneticforces ( .

14. A method according to any of the preceding claims, comprising calculating thelocal viscoelasticity value(s) of at least one of the following: an absolute shearmodulus ( ), and a loss tangent ( ).

15. A method according to any of the preceding claims, wherein the collagen type 1matrices encapsulate 10-μm-diameter magnetic probes.

16. A method according to any of the preceding claims, wherein in each matrix, themagnetic probes are nanomanipulated using controlled magnetic forces ( by themagnetic microrheometer.

17. A method according to any of the preceding claims, comprising usingelectromagnets of the magnetic microrheometer to generate sinusoidal magneticforces onto the magnetic probes within each collagen type 1 matrix sample at roomtemperature of 22 3 .

18. A method according to claim 18, comprising using oscillatory frequency of 0.05Hz.

19. A method according to any of the preceding claims 17-18, comprising extractingthe values for the controlled force ( ), an amplitude ( ), and a phase shift ( ) fromthe sinusoidal displacement per force data, and using nonlinear least squares fittingthe forces ( .

20. A method according to any of the preceding claims, wherein the viscoelasticityvalues are based on analysing a viscoelasticity measurement in one location, or themean value of the viscoelasticity measurements in two locations.

21. A method according to any of the preceding claims, comprising calculating a localviscoelasticity value(s) of at least one of the following: the absolute shear modulus( ), and the loss tangent ( ).

22. A method according to any of the preceding claims, comprising computing meanvalues of repetitive detections to obtain cell-scale viscoelasticity values for eachmagnetic probe at its location (23. A method according to any of the preceding claims, wherein a higher dose of X-rayirradiation increases displacements of the magnetic probes in the collagen type 1matrices.

24. A method according to any of the preceding claims, wherein a higher dose of X-rayirradiation causes increased mean displacements of the magnetic probes as aresponse to the magnetic forces ( .

25. A method according to any of the preceding claims, wherein higher dose of X-rayirradiation reduces the absolute shear modulus in the collagen type 1 matrices.

26. A method according to any of the preceding claims, wherein the higher dose of X-ray radiation increases the cell-scale loss tangent of the collagen type 1 matrices.

27. A method according any of the preceding claims, wherein the higher X-rayirradiation doses locally reduce stiffness of the collagen type 1 matrices, andincrease loss tangent of the collagen type 1 matrices.

28. A method according any of the preceding claims, wherein responsive to the higherdoses of X-ray irradiation, collagen type 1 matrices softening is accompanied byelevated loss tangent.

29. A method according any of the preceding claims, wherein relative values ofviscoelasticity to quantify the cell-scale alterations due to collagen type 1irradiation for the higher dose, relative modulus and relative loss tangent data havemean SD values of 36 29% and 244 102%, compared to the mean ofunirradiated matrices, respectively.

30. A method according to any of the preceding claims 23-29, wherein a higher dose ofX-ray irradiation is 320 Gy.

31. A method according to any of the preceding claims, comprising a method fordetection and modification of matrices viscoelastic cues at the cell-size scales.

32. A method according any of the preceding claims, wherein the X-ray irradiationconditions relate to the collagen type 1 matrices’ cell-scale softening via loweredabsolute / relative values of absolute shear modulus; and increase of its liquid-likenature via raised absolute / relative values of a loss tangent.

33. A method according to any of the previous claims, wherein the method iscombinable with live-cell experiments.

34. Use of the method according to any of the previous claims, to analyze cell-scaleviscoelasticity of the further components in a breast-cancer tissue.

35. Use of the method according to any of the previous claims, as combined with live-cell experiments.

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