Nonlinear elasticity of biological basement membranerevealed by rapid inflation and deflationHui Lia,b,1,2, Yue Zhengc,1, Yu Long Hanb, Shengqiang Caic,2, and Ming Guob,2

aSchool of Systems Science, Beijing Normal University, Beijing 100875, China; bDepartment of Mechanical Engineering, Massachusetts Institute ofTechnology, Cambridge, MA 02139; and cDepartment of Mechanical and Aerospace Engineering, University of California San Diego, La Jolla, CA 92093

Edited by Yonggang Huang, Northwestern University, Glencoe, IL, and approved February 2, 2021 (received for review October 27, 2020)

Basement membrane (BM) is a thin layer of extracellular matrixthat surrounds most animal tissues, serving as a physical barrierwhile allowing nutrient exchange. Although they have importantroles in tissue structural integrity, physical properties of BMsremain largely uncharacterized, which limits our understandingof their mechanical functions. Here, we perform pressure-controlledinflation and deflation to directly measure the nonlinear mechanics ofBMs in situ. We show that the BMs behave as a permeable, hypere-lastic material whose mechanical properties and permeability can bemeasured in amodel-independent manner. Furthermore, we find thatBMs exhibit a remarkable nonlinear stiffening behavior, in contrast tothe reconstituted Matrigel. This nonlinear stiffening behavior helpsthe BMs to avoid the snap-through instability (or structural softening)widely observed during the inflation of most elastomeric balloonsand thus maintain sufficient confining stress to the enclosed tissuesduring their growth.

basement membrane | nonlinear mechanics | strain stiffening |extracellular matrix | permeability

Basement membrane (BM) is a thin layer of fibrous matrixseparating cells from the connecting tissues, which functions

as a physical barrier and widely exists across multicellular or-ganisms (1). The BM is typically composed of laminins, collagenIV, nidogens, and proteoglycans; laminin and collagen IV are themajor components that constitute networks forming the struc-ture of the BM, and nidogen and proteoglycans are associatedwith the laminin and collagen IV networks. As a physical barrier,the structural and mechanical properties of BM are important inthe organization and morphogenesis of tissues and organs as wellas in the maintenance of adult functions (2); abnormal BM hasbeen associated with a variety of diseases such as cancer (3). Forexample, in metastasis, cancer cells must invade through BMs toescape from the primary tumor—a process that causes 90% ofcancer-related death (4). Indeed, breaks in BMs can be observedin malignant tumors (5). Thus, mechanical properties of the BMare considered to play important roles in regulating cancer cellinvasion (6, 7). Furthermore, as a physical barrier differentiatingdifferent parts of tissues, BMs are required to be permeable tosmall molecules to allow exchange of water and nutrients; thepermeability of BM is thus one of the essential kinetic parame-ters regulating biomolecule exchange and activities of internalcells (8, 9). Given the importance of BMs as a semipermeablebarrier maintaining tissue structural integrity, however, theirpermeability and mechanical properties remain largely unknown,mainly due to the lack of direct measurement methods, espe-cially in situ. This limits our understanding of the physical role ofBMs in various physiological and pathological processes such astumor development and angiogenesis.Determining the mechanical properties of intact BMs in situ is

challenging because of their irregular shape, small thickness, andtight connection to the cells inside. Due to these limitations, con-ventional mechanical tests such as tensile, compression, and bend-ing tests are difficult to be applied to characterize the mechanicalbehavior of the BM in situ. Instead, previous measurements hadbeen carried out on fragmented BMs isolated from various tissues

(e.g., via atomic force microscopy [AFM] indentation) and foundthat the BM stiffness ranges from ∼kPa to ∼MPa (10–17). In ad-dition, a constitutive relationship is required to extract the materialparameters such as elastic modulus and permeability from theseexperimental measurements. However, like most biological tissues,a reliable constitutive model for the BM is not yet available, causingadditional difficulties in obtaining its mechanical parameters frommost traditional experiments.In this work, we demonstrate an in situ method to simulta-

neously measure both the elastic properties and permeability ofintact BM in breast cancer spheroid by recording the deflationprocess of an inflated BM filled with phosphate buffered saline(PBS) by microinjection without requiring complex samplepreparation and post-data processing. During the deflation ofthe BM, its elastic retraction generates a pressure difference todrive the liquid flow through the membrane; the liquid flux canbe calculated from the reduction of the intact BM diameter.With the BM thickness measured by transmission electron mi-croscopy (TEM), we can determine the shear modulus, perme-ability, and diffusivity of the intact BM. Moreover, we find fromour measurements that the elasticity of BM is highly nonlinearwith a strong strain-stiffening effect. Furthermore, we discuss thepossible impact of the strain-stiffening effects of BM on itsfunctions.

Significance

Basement membranes (BMs) are thin layers of extracellularmatrix ubiquitously found in animals surrounding various tis-sues. As a physical barrier, their mechanical properties are im-portant in maintaining structural integrity of tissues, and theirpermeabilities are essential for molecule exchange and internalcell activities. However, due to the lack of direct measurementmethods, the physical properties of BMs remain largely un-clear, limiting our understanding of BMs in various physiolog-ical and pathological processes such as tumor development.Here, we apply pressure-controlled inflation/deflation tomeasure the stress–strain behaviors of intact BM in situ and todetermine the mechanical properties in a model-independentmanner. We discover a strong strain-stiffening effect of intactBM, which is essential for preventing its snap-through instability.

Author contributions: H.L., S.C., and M.G. designed research; H.L. and Y.Z. performedresearch; H.L., Y.Z., and Y.L.H. contributed new reagents/analytic tools; H.L. and Y.Z. an-alyzed data; and H.L., Y.Z., S.C., and M.G. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).1H.L. and Y.Z. contributed equally to this work.2To whom correspondence may be addressed. Email: huili@bnu.edu.cn, shqcai@ucsd.edu,or guom@mit.edu.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2022422118/-/DCSupplemental.

Published March 8, 2021.

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ResultsTo investigate the mechanics of BM, we fabricate three-dimensional breast cancer spheroids (18, 19). Briefly, individu-al human breast cancer MDA-MB-231 cells are seeded onto agelled Matrigel bed (10 mg/mL) and are cultured to allow pro-liferation for 5 d to form clusters. BM is secreted and generatedby cells during the growth of these multicellular cancer spher-oids. After that, the intact cancer spheroids with BM are isolatedfrom the Matrigel and seeded on a glass-bottom Petri dish forsubsequent microinjection. The BM remains intact on thesecancer spheroids (Fig. 1 A and C).To examine the structure of the BM of these cancer spheroids,

we first perform immunofluorescent imaging of the BM inextracted spheroids by labeling laminin-5, one of the majorcomponents of BM (Fig. 1C). Indeed, we see a continuous layerof laminin-5 surrounding the cancer spheroid, suggesting that theBM is nicely formed on these spheroids. To further investigatethe structure and thickness of these BMs, we perform TEMimaging on in situ BM sections, which could preserve the originalcondition of the BM. To do so, cancer cell spheroids that partlyembed in Matrigel are fixed on day 5, then sectioned and stainedaccording to established protocols (20). The cross-sections of thespheroid above the Matrigel surface are collected and used forTEM imaging. It is apparent that a gray layer of BM surroundsthe cell clusters inside (Fig. 1A). As we zoom in, clear fibrousstructures of the BM can be observed (Fig. 1B). From theseimages, we find that the thickness of BM in our cancer spheroidsystem is 2.5 ± 1.2 μm (Fig. 1D). Interestingly, the thickness ofBMs has obvious variations, even on the same spheroid. This islikely to be a result of cell-to-cell variation as the BM is locallygenerated by adjacent cells.

To study the mechanics of BM, we perform an inflation anddeflation experiment of the BM surrounding the spheroid in situ.To do so, we use a microneedle (2 μm in diameter) to penetratethe surrounding BM and place the needle tip inside the spheroid.We then apply a constant pressure to inject PBS solution into thespheroid via the microneedle. Both the injection pressure and itsduration are precisely controlled by a microinjector that connectsto the microneedle (Fig. 2A). Interestingly, upon a constantpressure, we observe that the BM detaches from the inside cellaggregations and inflates into a spherical shape like a latex bal-loon; it rapidly inflates to a steady state (Fig. 2B), at which thecontinuous PBS influx is balanced by the outflow through thesemipermeable membrane. When the fluid influx is turned off,the inflated BM gradually deflates back to its initial size as aresult of the elasticity of the membrane, which drives the outflowof liquid through the semipermeable BM. We find this inflationand deflation process is highly reversible, suggesting that the BMis deformed elastically during this process. Typically, to fullyseparate the BM from the cell cluster, we perform several roundsof inflation/deflation prior to final experiments. The sphericalshape of an inflated BM suggests that the BM is fully isolatedfrom cells (Fig. 2B); thus, its shape is dominated by the balanceof membrane tension and cross-membrane pressure difference.To quantitatively investigate the inflation and deflation pro-

cess, we perform continuous imaging at 14 Hz via bright-fieldmicroscopy to record the shape and size of a BM during thisprocess (Fig. 2C and Movie S1). This allows us to measure theradius of the BM and its change over time, as shown by thekymograph in Fig. 2C and quantification in Fig. 2D. The quan-titative inflation and deflation curves indeed remain the sameover different loading cycles, demonstrating good resilience ofBM. Although the inflation process is rapid, especially underlarge injection pressure, the deflation process is relatively slow,as shown by the rapid increase and gradual decrease of BM ra-dius (Fig. 2D). Furthermore, we repeat this experiment usingdifferent injection pressures; as the injection pressure is in-creased, the radius of the inflated BM at steady state is increasedaccordingly (Fig. 2D and SI Appendix, Fig. S1).To understand the steady-state inflation as well as the defla-

tion process of a BM, we consider a spherical membrane withinner radius R0 and membrane thickness H prior to deformationwhile no pressure is applied. With the inner fluid pressure p, theinner radius increases to r, and wall thickness reduces to h. Forsimplicity, in the following analysis, we assume the fluid pressureis homogenous and neglect the inertial effect of the membraneduring the deflation because the process is relatively slow.For an inflated spherical membrane, any point in the mem-

brane is in an equal-biaxial stress (and stretch) state. We denotethe equal-biaxial hoop stretch as λ and the stress as σ. Simplegeometrical analysis shows that the biaxial hoop stretch equalsthe radial expansion, namely λ = r=R0. With different levels ofthe inflation pressure p, the corresponding maximal stretches inthe steady state λmax = rmax=R0 of the membrane before the de-flation begins are plotted in Fig. 3A. During the deflation pro-cesses, the decreases of the radial expansion with time for fivedifferent inflation pressures are shown in Fig. 3B. The water fluxJ is related to the radius of the BM as J = −dr=dt with the as-sumption of the incompressibility of water, thus the flux can beplotted as a function of stretch for different experiments, asJ = J(λ) shown in Fig. 3C. Notably, the relationships between Jand λ obtained under different injection pressures on the sameBM overlap.Next, we will show that the constitutive behavior of the

membrane can be directly deduced from the results shown inFig. 3 A and C. We assume the constitutive behavior of themembrane subject to equal-biaxial stress (σ) is given by σ = σ(λ).The force balance requires σ = pr=2h. With the commonlyadopted assumption for soft tissues that the membrane material

Fig. 1. TEM and fluorescence images of BMs in cancer spheroids. (A) In situTEM images of a cell spheroid partly embedded in Matrigel matrix. The topcross-sections of the spheroid above the Matrigel surface are collected andimaged. The gray layer surrounding the aggregated cells (marked as C) is theBM. (Scale bar, 10 μm.) (B) A magnified image of the BM shows the fiber-likestructure. (Scale bar, 500 nm.) (C) Immunofluorescence images of laminin-5in the BM (green) from an extracted cell spheroid. The cell nuclei are labeledby Hoechst 33342 (blue). (Scale bar, 10 μm.) (D) The thickness of BMs mea-sured from TEM images, with an average value 2.5 ± 1.2 μm. n = 85 mea-surements from 11 spheroids.

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is incompressible, the thickness of the membrane after inflation canbe related to the stretch as h = Hλ−2. Consequently, the relation-ship between biaxial membrane stress and internal fluid pressurebecomes σ(λ) = R0

2Hλ3p(λ). Without requiring any material model or

assumption, using measured function p(λ) as shown in Fig. 3A, theconstitutive function σ(λ) can be fully determined with up to oneconstant R0

2H. Both R0 and H can be easily obtained from bright-fieldor TEM imaging. With measured R0 = 32.5μm andH = 2.5μm, weobtain biaxial stress–stretch relationship σ(λ) of BM frominflation–deflation tests, as shown by black circles in Fig. 3D.We next compare the above experimentally determined con-

stitutive relationship with existing hyperelastic models. Based onthe shape of the function σ = σ(λ) shown in Fig. 3D, we chooseFung’s hyperelastic model as an example. It can be shown thatwith strain energy function proposed by Fung (21):W = G

2b (eb(λ21+λ22+λ23−3) − 1), the relationship between the equal-

biaxial stress and stretch is given by the following equation:

σ = G(λ2 − 1λ4)eb(2λ2+ 1

λ4−3)

, [1]

where G and b are two material constants. G can be regarded asshear modulus of the material and b is a dimensionless param-eter related to the strain stiffening of the material. By fitting theprediction from Eq. 1 to the experimental data in Fig. 3D, weobtain these material constants for this particular BM, as G =82.30 kPa and b = 2.10. The measured G is consistent with pre-vious measurements on isolated BMs, for example, the elasticmodulus of corneal BM (80 kPa, which is roughly three times ofthe shear modulus for an incompressible solid) (11) and BMfrom Drosophila eggs (70 kPa) (12). Moreover, we compare

the predictions of different hyperelastic models with our exper-imental measurements, in addition to Fung’s model. We findthat Fung’s model gives a better description to the experimentalstress–strain relationship of BMs as compared to several othermodels such as neo-Hookean, Mooney–Rivlin model, or theGent model (SI Appendix, Fig. S2).To understand the kinetic properties of the BM, we adopt

Darcy’s law to determine the water flux J passing through themembrane as J = k

μph, where k is diffusivity of the membrane

while μ is dynamic viscosity of the fluid. The equations aboveenable us to further obtain that σ = μR0

2k λJ. So with knowing thenumber μ

2k, we can also obtain the constitutive function σ = σ(λ)from J = J(λ) shown in Fig. 3C. By choosing μ

2k to be 0.72 kPa · s/μm2,

the constitutive relation determined from Fig. 3C agrees extremelywell with the constitutive relation shown in Fig. 3D. Finally, with thedynamic viscosity of the aqueous solution as μ = 1mpa · s, we canestimate the permeability of the BM k = 6.95 × 10−19 m2. This isconsistent with previous measurement on isolated lens BMs frombovine eye (22). Furthermore, if we adopt the diffusivity asD = kG=μ, we can obtain the poroelastic diffusion coefficient D =5.72 × 10−11 m2/s.By comparing in situ inflation and deflation curves in experiments

to analytical solution, we can obtain several key materials parame-ters of the BM, as summarized in SI Appendix, Table S1. We indeedobserve a significant variation of the measured values. These vari-ations reflect structural and mechanical differences among theseBMs as they are locally produced by each cancer cell spheroid andthus may vary both BM thickness and membrane structures (11, 23).The inflation–deflation process reveals the nonlinear elastic

behavior of the BM. From the measured biaxial stress–straindiagram of BMs shown in Fig. 3 A and D, we observe significantnonlinearity and strain-stiffening effects. Based on Fung’s model,

Fig. 2. Experimental setup. (A) A schematic of the experimental setup. (B) Spherical BM before (Left) and under the injection at 20 kPa (Middle). (Scale bar,10 μm.) The inside cell spheroid is predetached from the surrounding BM by several injections. (C) Kymograph of two inflation and deflation processes of theBM under an injection pressure 20 kPa. (Scale bars, vertically 10 μm and horizontally 5 s.) (D) The measured radius of BM during inflations and deflations underincreased injection pressures.

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we can obtain the stress–strain relationship of the BM underuniaxial tension as σ = G(λ2 − λ−1)eb(λ2+2λ−1−3). Using the mea-sured material parameters, we further plot both uniaxial andequal-biaxial tangent modulus of the BM (defined as K = dσ=d«with σ representing the true stress and « = lnλ representing thetrue strain for either uniaxial or equal-biaxial deformation) as afunction of strain in Fig. 4A. With a slight increase of strain, thetangent modulus K of BM increases by several times for bothuniaxial and biaxial deformation. This is in direct contrast to thewide linear elastic regime observed on reconstituted Matrigel(24, 25), indicating distinct structural characteristics betweennatural BMs and Matrigel reconstituted from BM extracts. In-deed, clear fibrous network structures can be observed in theintact BMs, as shown by the TEM images in Fig. 1C. These fi-brous networks of biopolymers are known to cause a strong,nonlinear stiffening effect (26), due to various mechanisms in-cluding entropic elasticity of individual filaments, geometric

effects due to fiber bending and buckling, and collective networkeffects governed by critical phenomena (27–33).Strain stiffening of soft biological tissues has been widely ob-

served and known to play important yet different roles in variousbiological processes (24, 34, 35). Herein, we discuss the possibleeffect of the strain stiffening of BMs on their functions. It hasbeen well known that a hyperelastic balloon under inflation oftensuffers from snap-through instability when the internal pressureor the radial expansion is beyond a critical value (36–38). Fig. 4Bplots the relationship between the inflating pressure and the radialexpansion of a balloon made up of a neo-Hookean membrane (suchas a latex membrane). The relationship between the internal pres-sure and the radial expansion of the balloon is nonmonotonic, andthere is a peak pressure with a corresponding critical radial ex-pansion: λ=1.38. As a result, when the radial expansion ratio rea-ches the critical value, the balloon can dramatically increase its sizewithout requiring a further increase of the pressure, as shown by thehorizontal line in Fig. 4B. For a latex balloon, such drastic expansionduring the snap-through process often leads to rupture. More in-terestingly, if the volume of the balloon is controlled to increasegradually, the internal pressure drops with the further increase ofthe volume after the balloon expansion exceeds the critical value. Ifthe mechanical behavior of the BM is similar to that of a latexballoon, after the radial expansion of the BM exceeds 1.38, furthergrowth of tissue inside (such as a tumor) reduces mechanical con-straint from the BM, which may lead to irregular tissue morphologyor rapid tumor expansion (39).It is worth noting that the nonmonotonic relationship between

the inflating pressure and radial expansion of a hyperelasticballoon (and the resulting snap-through instability) is not aconsequence of special mechanical properties of the material.Instead, it is due to the significant reduction of the balloonthickness with its radial expansion. So, the descending part of thepressure versus the radial expansion curve (for a neo-Hookeanelastomeric balloon) in Fig. 4B can be referred to as geometricalsoftening. One way to avert such snap-through instability is to in-troduce significant strain stiffening in the material at relatively smalldeformation (with biaxial stretch smaller than the critical stretch:1.38) to compensate the geometrical softening effect. As shown inFig. 4A, the BM indeed exhibits a strong strain-stiffening effectstarting at a small deformation. As a result, the relationship be-tween the inflating pressure and the radial expansion of the BM ismonotonic, and snap-through instability does not occur, as shown inFigs. 3A and 4B. These results indicate the importance of thenonlinear elasticity of BMs in maintaining tissue mechanical in-tegrity during growth and development. In the following, we willfurther elaborate the necessary strain stiffening of the material forpreventing snap-through instability of the balloon.

Fig. 3. Mechanical and kinetic properties of BM determined from experi-mental results. (A) The stretch of the membrane at steady state (maximumstretch) for different injection pressure. (B) Deflation curves in term of radialexpansion λ = r=R0 at varying injection pressure. (C) Fluid flux of BM as afunction of the stretch of the membrane. During the deflation, fluid fluxJ = dr=dt. Insert, schematics of BM: inner fluid pressure, p; biaxial stress onspherical membrane, σ; fluid flux, J. (D) Constitutive behavior of the BMobtained from (A) by using R0 = 32.5μm and H = 2.5μm. The dashed curve isthe fitting result from Fung’s model. Using Darcy’s law, we can convert thefluid flux curve in C to the constitutive relation of the BM with the fittedpermeability of the membrane k = 6.95 × 10−19 m2.

Fig. 4. Nonlinear elastic behavior of BMs. (A) Tangent modulus of BMs (defined in the text) as a function of true strain for equal-biaxial tension and uniaxial tension.(B) The relationship between the inflating pressure as a function of radial expansion of a balloon made up of a neo-Hookean material compared to that of BMs. (C) Ifthe strain-stiffening curve of the material such as BM does not intersect the dashed line (K = 3σ), snap-through instability can be avoided. If the strain-stiffening curveof the material, such as neo-Hookean material or linear elastic material, intersect with the dashed line, snap-through instability of the balloon can occur. It is notedthat in C, both of the tangent modulus and stress are normalized by the tangent modulus of the material in free-standing state.

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To avoid the snap-through or structural softening of a balloon,the relationship between the internal pressure and its radial ex-pansion needs to be monotonic, namely dp=dλ> 0. With the ma-terial incompressibility and force balance, we can show that thecondition above is equivalent to K > 3σ, where K is biaxial tangentmodulus defined above and σ is the true equal-biaxial stress. InFig. 4C, K = 3σ is shown by the dashed line. We can also plot bi-axial tangent modulus as a function of the true biaxial stress fordifferent hyperelastic material models in Fig. 4C. For a hyperelasticmaterial whose strain stiffening is significant enough like the BM, itsstrain-stiffening curve does not intersect the dashed line (K = 3σ),as shown in Fig. 4C; therefore, snap-through instability or structuralsoftening can be prevented. In contrast, if the strain-stiffening curveintersects the dashed line (e.g., for neo-Hookean material markedor materials with no strain-stiffening effect), snap-through instabilityor structural softening occurs. It is noted that using the fitted pa-rameters of the BM as shown above, we predict the mechanicalbehavior of the BM with the deformation much larger than the oneshown in the experiment, which may be beyond the rupturing pointof the BM.

DiscussionIn this work, we introduce a facile method based on inflation–deflation experiments to in situ measure the mechanics of BM inbreast cancer spheroid. We find that BMs can be well capturedas a permeable hyperelastic material. As the inflation pressure isreleased, the inflated BM fully returns to its initial state; thisoutstanding elastic property may provide mechanical protectionto its internal structures. Moreover, the inflation–deflationmethod we introduce here explores the global mechanics ofBMs, in contrast to other local measurements on fragmentedBM such as AFM. Two critical mechanical properties, elasticmodulus and permeability, can be directly determined from thedeflation process independent of specific material models. Webelieve this is an important advantage of the current inflation–deflation method compared with other methods such as inden-tation test. For an indentation test, a specific constitutive modelfor the BM has to be assumed in the first place to enable theconversion of the experimental measurements (e.g., indentationforce versus indentation depth) to the stress–strain relationshipof a BM. Justifying an appropriate selection of the constitutivemodel can be very challenging given the highly nonlinear elasticbehavior and the poroelastic nature of BMs. Consequently, suchjustification is often missing or inadequate in the previousstudies. Thus, the inflation–deflation method developed in thecurrent work may be used to measure mechanics of intact BMs indifferent systems and during both healthy and disease states.Furthermore, our in situ measurement reveals marked non-

linear elasticity of the BMs whose onset is below 10% of strain.This behavior is in direct contrast to the large linear elastic re-gime observed in reconstituted Matrigel (24, 25) yet is consistentwith a wide variety of biological tissues and biopolymer networks(26, 40, 41). This nonlinear stiffening behavior of the BM mayhave an important role in maintaining tissue mechanical integrityduring growth and deformation (e.g., to avoid structural insta-bility), which is a classical behavior leading to drastic expansionor even rupture when inflating most elastomeric balloons.

Materials and MethodsCancer Spheroid Culture and Isolation. The human breast cancer MDA-MB-231cells (ATCC) are cultured in Dulbecco’s Modified Eagle Medium (Gibco) with10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Gibco) at37 °C with 5% CO2. The individual cells at the log phase are seeded onto thegelled Matrigel bed (10 mg/mL, Corning, 354234) and are cultured to allowproliferation for 5 d to form clusters. Culture medium supplemented with0.2 mg/mL Matrigel is replaced every 2 d. BM is secreted and generated bycells during the growth of these multicellular cancer spheroids.

To isolate these cancer spheroids, samples are placed in ice-cold PBS tobreak down the Matrigel, and then centrifuged at 100 g to remove thesupernatant Matrigel fragments. The intact cancer spheroids out of Matrigelare collected by resuspending them in PBS and are then seeded on a glass-bottom Petri-dish for immunofluorescence staining or microinjectionexperiments.

Immunofluorescence Staining. The isolated cancer spheroids are firstly fixedwith 4% paraformaldehyde at room temperature for 30 min. Then, thespheroids are permeabilized with 0.2% Triton X-100 in PBS for 15 min. Afterthat, the samples are blocked with 5% bovine serum albumin (BSA) in PBSfor 1 h at room temperature and subsequently incubated with the primaryantibody for laminin-5 (1:100 in PBS with 0.1% BSA, Santa Cruz Biotech-nology, sc-13578) overnight at 4 °C. The samples are then incubated with thesecond antibody Alexa-488 goat anti-mouse (1:300, Thermo Fisher, A-11001)for 1 h at room temperature in darkness. The nuclei are stained with Hoechst33342 (Sigma, 14533) before imaged under a confocal microscopy(Leica SP8).

Microinjection. The FemtoJet microjector (5248, Eppendorf) is mounted on aconfocal microcopy (Lecia TCL SP8) equipped with a 25× water-immersedobjective (0.9 numerical aperture) and a cage incubator (37 °C, Okolab). Aglass-made microneedle (2 μm in diameter, Eppendorf) is loaded with PBSbuffer. Then it is manipulated to penetrate the surrounding BM, placing theneedle tip inside the spheroid. To perform an inflation–deflation cycle, theinjection pressure and its duration are predefined in the microinjector con-necting to the microneedle. After manually starting the injection protocol,the BM would quickly inflate to a steady state, and then gradually deflateback to its initial size. Under each pressure, two or more inflation–deflationcycles are performed for one sample. Bright-field imaging at 14 Hz is appliedto record the shape and size of a BM. From the recorded movies, the radiusof BMs is determined by using a customized algorithm in MATALB.

Electron Microscopy. The sample preparation follows a previously establishedprotocol (20). Briefly, the cancer spheroids cultured on a Matrigel bed arefixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate for 1 h. Then,samples are postfixed by 1% osmium tetroxide in 0.1 M sodium cacodylateat 4 °C for 30 min and dehydrated in graded ethanol. After that, the samplesare embedded in epoxy resin, followed by sectioning. Only cross-sections ofthe spheroid above the Matrigel surface are collected and stained with 1.4%uranyl acetate and then with lead citrate. Sections are imaged with a HitachiH-7650. The BM thicknesses are measured from the TEM images byusing ImageJ.

Data Availability. All study data are included in the article and/or supportinginformation.

ACKNOWLEDGMENTS. We thank Ying Li for the assistance in TEM samplepreparations. Y.L.H. and M.G. would like to acknowledge the support fromMathWorks. M.G. is supported by the Sloan Research Fellowship. H.L.acknowledges support from National Natural Science Foundation of China(Grant Numbers 11874415 and 12074043).

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