INTRODUCTION

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ChooseTop of pageABSTRACTINTRODUCTION <<RESULTSDISCUSSIONCONCLUSIONSMETHODSSUPPLEMENATRY MATERIALCancer is the leading cause of death worldwide, accounting for nearly 10 × 106 deaths in 2020.11. H. Sung, J. Ferlay, R. L. Siegel, M. Laversanne, I. Soerjomataram, A. Jemal, and F. Bray, “ Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries,” Ca-Cancer J. Clin. 71, 209–249 (2021). https://doi.org/10.3322/caac.21660 Progression to the metastatic stage is one of the primary causes for this.2,32. X. Guan, “ Cancer metastases: Challenges and opportunities,” Acta Pharm. Sin. B 5, 402–418 (2015). https://doi.org/10.1016/j.apsb.2015.07.0053. D. Crosby, S. Bhatia, K. M. Brindle, L. M. Coussens, C. Dive, M. Emberton, S. Esener, R. C. Fitzgerald, S. S. Gambhir, P. Kuhn et al., “ Early detection of cancer,” Science 375, 80 (2022). https://doi.org/10.1126/science.aay9040 Cancer cells differ from normal cells in their genetic, molecular, and morphological features, as well as their biomechanical properties. At the single-cell level, the mechanical deformability of cancer cells (ability to change shape) was found to correlate with malignancy potential and cell function.4–114. J. Guck, S. Schinkinger, B. Lincoln, F. Wottawah, S. Ebert, M. Romeyke, S. Ulvick, and C. Bilby, “ Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence,” Biophys. J. 88, 3689–3698 (2005). https://doi.org/10.1529/biophysj.104.0454765. W. Xu, R. Mezencev, B. Kim, L. Wang, J. McDonald, and T. Sulchek, “ Cell stiffness is a biomarker of the metastatic potential of ovarian cancer cells,” PLoS One 7(10), e46609 (2012). https://doi.org/10.1371/journal.pone.00466096. S. Suresh, J. Spatz, J. P. Mills, A. Micoulet, M. Dao, C. T. Lim, M. Beil, and T. Seufferlein, “ Connections between single-cell biomechanics and human disease states: Gastrointestinal cancer and malaria,” Acta Biomater. 1, 15–30 (2005). https://doi.org/10.1016/j.actbio.2004.09.0017. M. Beil, A. Micoulet, G. Von Wichert, S. Paschke, P. Walther, M. B. Omary, P. P. Van Veldhoven, U. Gern, E. Wolff-Hieber, J. Eggermann et al., “ Sphingosylphosphorylcholine regulates keratin network architecture and visco-elastic properties of human cancer cells,” Nat. Cell Biol. 5, 803–811 (2003). https://doi.org/10.1038/ncb10378. M. Prabhune, G. Belge, A. Dotzauer, J. Bullerdiek, and M. Radmacher, “ Comparison of mechanical properties of normal and malignant thyroid cells,” Micron 43, 1267–1272 (2012). https://doi.org/10.1016/j.micron.2012.03.0239. J. S. Park, C. J. Burckhardt, R. Lazcano, L. M. Solis, T. Isogai, L. Li, C. S. Chen, B. Gao, J. D. Minna, R. Bachoo et al., “ Mechanical regulation of glycolysis via cytoskeleton architecture,” Nature 578, 621–626 (2020). https://doi.org/10.1038/s41586-020-1998-110. V. Papalazarou, T. Zhang, N. R. Paul, A. Juin, M. Cantini, O. D. K. Maddocks, M. Salmeron-Sanchez, and L. M. Machesky, “ The creatine–phosphagen system is mechanoresponsive in pancreatic adenocarcinoma and fuels invasion and metastasis,” Nat. Metab. 2, 62–80 (2020). https://doi.org/10.1038/s42255-019-0159-z11. A. N. Gasparski, J. T. Wilson, A. Banerjee, and K. A. Beningo, “ The role of PAK1 in the maturation of invadopodia during transient mechanical stimulation,” Front. Cell Dev. Biol. 7, 1–10 (2019). https://doi.org/10.3389/fcell.2019.00269. It is well established, for example, that mechanical forces in the tumor niche affect various biological functions of cancer cells, such as their proliferation and level of differentiation.12,1312. S. M. Pupa, S. Ménard, S. Forti, and E. Tagliabue, “ New insights into role extracellular matrix during tumor onset progression,” J. Cell. Physiol. 192, 259–267 (2002). https://doi.org/10.1002/jcp.1014213. J. M. Northcott, I. S. Dean, J. K. Mouw, and V. M. Weaver, “ Feeling stress: The mechanics of cancer progression and aggression,” Front. Cell Dev. Biol. 6, 1–12 (2018). https://doi.org/10.3389/fcell.2018.00017Phagocytosis is a natural mechanism performed by immune cells that act as professional phagocytes such as macrophages and neutrophils and is largely employed as a defense mechanism against microbes.14,1514. J Günther and H. M. Seyfert, “ The first line of defence: Insights into mechanisms and relevance of phagocytosis in epithelial cells,” Semin. Immunopathol. 40, 555–565 (2018). https://doi.org/10.1007/s00281-018-0701-115. V. Jaumouillé and C. M. Waterman, “ Physical constraints and forces involved in phagocytosis,” Front. Immunol. 11, 1–20 (2020). https://doi.org/10.3389/fimmu.2020.01097 In these cells, phagocytosis is mediated by receptor–ligand interactions as well as the mechanical parameters of the phagocytic target.1616. K. A. Beningo and Y. Wang, J. Cell Sci. 115, 849–856 (2002). https://doi.org/10.1242/jcs.115.4.849 In contrast, for nonprofessional phagocytes such as fibroblasts, epithelial cells, and cancer cells, no specific receptors involved in the engulfment of microparticles have been identified.1717. J. C. Seeberg, M. Loibl, F. Moser, M. Schwegler, M. Büttner-Herold, C. Daniel, F. B. Engel, A. Hartmann, U. Schlötzer-Schrehardt, M. Goppelt-Struebe et al., “ Non-professional phagocytosis: A general feature of normal tissue cells,” Sci. Rep. 9(1), 8 (2019). https://doi.org/10.1038/s41598-019-48370-3 Therefore, for these cases, the term phagocytosis is used to designate any cellular internalization of particles that are larger than the endocytosis particle-size threshold and is independent of biological mediators. Small nanoparticles (18–2018. S. Zhang, H. Gao, and G. Bao, “ Physical principles of nanoparticle cellular endocytosis,” ACS Nano 9, 8655–8671 (2015). https://doi.org/10.1021/acsnano.5b0318419. D. Vorselen, R. L. D. Labitigan, and J. A. Theriot, “ A mechanical perspective on phagocytic cup formation,” Curr. Opin. Cell Biol. 66, 112–122 (2020). https://doi.org/10.1016/j.ceb.2020.05.01120. D. Manzanares and V. Ceña, “ Endocytosis: The nanoparticle and submicron nanocompounds gateway into the cell,” Pharmaceutics 12, 371 (2020). https://doi.org/10.3390/pharmaceutics12040371 Mechanically, endocytosis is predominantly governed by membrane fluidity and local distortions rather than massive deformations of the cell body.2121. Y. Brill-Karniely, “ Mechanical measurements of cells using AFM: 3D or 2D physics?,” Front. Bioeng. Biotechnol. 8, 1–5 (2020). https://doi.org/10.3389/fbioe.2020.605153 However, in the case of phagocytosis, which is the dominant uptake pathway for particles larger than 500 nm, the biomechanical mechanisms primarily involve massive and global cell distortions and cytoskeletal remodeling. This means that in these cases, the engulfment process is affected more by cell stiffness than by membrane fluidity. Our recent work demonstrated a Triangular Correlation (TrC) at the single-cell level between cancer cells' deformability, the extent of phagocytosis, and malignancy.2222. Y. Brill-Karniely, D. Dror, T. Duanis-Assaf, Y. Goldstein, O. Schwob, T. Millo, N. Orehov, T. Stern, M. Jaber, N. Loyfer et al., “ Triangular correlation (TrC) between cancer aggressiveness, cell uptake capability, and cell deformability,” Sci. Adv. 6, eaax2861 (2020). https://doi.org/10.1126/sciadv.aax2861 This key finding implies that cells that are more deformable have a greater capacity to engulf large particles, but it also suggests that the temporal mechanical state of cells may affect this capacity as well.

The mechanical state of cell results from the combined effects of native structural effectors such as the cytoskeleton, membrane, water content, and adaptive effectors that are resulting from external mechanical inputs. Given the relationship between cell mechanics and phagocytosis, we hypothesize that the cell's capacity to engulf colloidal particles (sub-micron and micrometer particles) may be affected by cells' adherence to surfaces. Despite the significant biological implications of this relationship, particularly in relation to cancer cell phagocytosis of synthetic or natural particles, this aspect of cell uptake behavior has not been thoroughly researched. While the extent and nature of cell adhesion to surfaces may be affected by many surface properties, such as texture, composition, and rigidity, our current study focuses specifically on the effect of surface rigidity.

The rigidity of the surface to which cells adhere affects both the physical properties of cells, such as spreading area, stretching, and Young's modulus,19–2219. D. Vorselen, R. L. D. Labitigan, and J. A. Theriot, “ A mechanical perspective on phagocytic cup formation,” Curr. Opin. Cell Biol. 66, 112–122 (2020). https://doi.org/10.1016/j.ceb.2020.05.01120. D. Manzanares and V. Ceña, “ Endocytosis: The nanoparticle and submicron nanocompounds gateway into the cell,” Pharmaceutics 12, 371 (2020). https://doi.org/10.3390/pharmaceutics1204037121. Y. Brill-Karniely, “ Mechanical measurements of cells using AFM: 3D or 2D physics?,” Front. Bioeng. Biotechnol. 8, 1–5 (2020). https://doi.org/10.3389/fbioe.2020.60515322. Y. Brill-Karniely, D. Dror, T. Duanis-Assaf, Y. Goldstein, O. Schwob, T. Millo, N. Orehov, T. Stern, M. Jaber, N. Loyfer et al., “ Triangular correlation (TrC) between cancer aggressiveness, cell uptake capability, and cell deformability,” Sci. Adv. 6, eaax2861 (2020). https://doi.org/10.1126/sciadv.aax2861 as well as their biological activities, including proliferation, motility, morphology, differentiation, and adhesion.2525. D. M. Richards and R. G. Endres, “ The mechanism of phagocytosis: Two stages of engulfment,” Biophys. J. 107, 1542–1553 (2014). https://doi.org/10.1016/j.bpj.2014.07.070 Therefore, mechanical-mediated phagocytosis may occur in a variety of clinical scenarios. Examples include cancer metastases, in which cancer cells are attached to secondary organs, each with distinct rigidity, or when cells are located on the outer lining of a tissue that becomes stiffer over time due to fibrosis or desmoplasia.Here, we investigated the adhesion of cancer cells to surfaces of distinct rigidities in pancreatic ductal adenocarcinoma (PDAC) and breast adenocarcinoma cell lines, both of which are known for their adaptability to complex mechanical desmoplastic niches in vivo.23,2423. M. J. Paszek, N. Zahir, K. R. Johnson, J. N. Lakins, G. I. Rozenberg, A. Gefen, C. A. Reinhart-King, S. S. Margulies, M. Dembo, D. Boettiger et al., “ Tensional homeostasis and the malignant phenotype,” Cancer Cell 8, 241–254 (2005). https://doi.org/10.1016/j.ccr.2005.08.01024. C. J. Whatcott, C. H. Diep, P. Jiang, A. Watanabe, J. Lobello, C. Sima, G. Hostetter, H. M. Shepard, and D. D. Hoff, Von Pancreatic Cancer 21, 3561–3568 (2016). https://doi.org/10.1158/1078-0432.CCR-14-1051 These tumors are distinguished by abnormal tissue stiffening, which affects the cellular phenotypes of cancer and stromal cells in tumors as well as cancer progression in general.Detailed uptake experiments were performed on gel surfaces with distinct rigidities mimicking natural tissues. Fabricated polyacrylamide hydrogels and commercial silicone gels provided the necessary physiologically relevant range of Young's modulus values, thereby rendering a well-controlled mechanically mediated platform. The uptake experiments were performed with inert fluorescently labeled sub-micron and micrometer particles (0.8 and 2.4 μm) that were incubated with the cells for different durations and taken up via a phagocytic-like mechanism, as previously shown.2222. Y. Brill-Karniely, D. Dror, T. Duanis-Assaf, Y. Goldstein, O. Schwob, T. Millo, N. Orehov, T. Stern, M. Jaber, N. Loyfer et al., “ Triangular correlation (TrC) between cancer aggressiveness, cell uptake capability, and cell deformability,” Sci. Adv. 6, eaax2861 (2020). https://doi.org/10.1126/sciadv.aax2861

Using imaging flow cytometry, which allows cell imaging and particle localization analysis, we quantified the efficiency of particle uptake within the cells. Our data revealed that the cells' uptake pattern showed a wave-like (meandering) dependence on the rigidity of the substrate on which they were cultured. This meandering behavior indicates the presence of opposing physical and biological factors that control the cells' uptake capacity, as shown experimentally and theoretically.

There are two phases in ligand-mediated phagocytosis as performed by professional phagocytes. The first is a contact phase that begins slowly, possibly due to the passive cell–particle interaction, while the second is an active stage, during which particle engulfment rapidly develops.2525. D. M. Richards and R. G. Endres, “ The mechanism of phagocytosis: Two stages of engulfment,” Biophys. J. 107, 1542–1553 (2014). https://doi.org/10.1016/j.bpj.2014.07.070 Therefore, one of our goals in the present work was to determine whether the two phases that occur in phagocytosis, i.e., the passive and active phases, are also present during the uptake of bare particles by cancer cells. To elucidate this, we measured the expression of phosphorylated paxillin in cells grown on surfaces of various rigidities. Since phosphorylated paxillin is expected to be negatively correlated with passive absorbance and positively correlated with active engulfment, its expression levels can be used to determine which of these two processes is primarily responsible for determining the extent of uptake. Interestingly, we found that the expression of phosphorylated paxillin was inversely correlated with microparticle uptake, indicating that the first stage, at which passive adhesion occurs, is the main limiting step.Taken together, our findings indicate that the tissues' mechanical traits can significantly influence the phagocytic behavior of adherent cancer cells. This suggests that cancer cell localization in the body, for example, in primary and metastatic organs, may significantly affect cell function, as indicated by the mechanically mediated uptake of particles. Further studies could explore the effect of various external mechanical cues on the cellular mechanical uptake of natural microparticles or synthetic microcarriers such as microbubbles, which are used for imaging or theranostics. Our findings may be further developed as a tool for the selective delivery of microparticles to specific organs in the body, such as tumor tissues. Moreover, based on the stiffness-dependent cellular interactions with drug carriers seen in physical models that account for 3D, additional physical parameters may be identified and used to improve targeted drug delivery systems and their selective uptake, substantially reducing the off-target drug exposure and increasing the success rate of cancer treatments. Figure 1 illustrates the general hypothesis and experimental outline to identify the mechanical effectors that are involved in phagocytosis in cells attached to various surfaces.

RESULTS

Section:

ChooseTop of pageABSTRACTINTRODUCTIONRESULTS <<DISCUSSIONCONCLUSIONSMETHODSSUPPLEMENATRY MATERIAL

Measurement of polyacrylamide gel stiffness using a MicroTester

Hydrogels serve as a valuable and accessible culture model for extracellular matrices. Figure 2 shows the mechanical characterization of fabricated polyacrylamide (PAA) hydrogels via compressive experiments. The elastic modulus of the hydrogel was calculated from the applied stress and the resultant strain of the material within the linear elastic region of deformation.2929. S. R. Caliari and J. A. Burdick, “ A practical guide to hydrogels for cell culture,” Nat. Methods 13, 405–414 (2016). https://doi.org/10.1038/nmeth.3839 The gels were custom-fabricated using 3D-printed molds with dimensions compatible with the MicroTester scale analyzer. Polyacrylamide was cast into the molds and placed on cover glasses. After polymerization, complementary molds were used to remove the polyacrylamide gel cubes. Cubic samples of 1 mm3 were compressed with a tungsten compression platen attached to the edge of a microbeam (see Methods). The mean Young's moduli of polyacrylamide cubes were 1.59 ± 0.37, 9.19 ± 1.77, and 31.34 ± 0.50 kPa. Our elasticity results are generally consistent with previously reported studies, while minor discrepancies may be attributable to the selected measurement techniques.30,3130. A. K. Denisin and B. L. Pruitt, “ Tuning the range of polyacrylamide gel stiffness for mechanobiology applications,” ACS Appl. Mater. Interfaces 8, 21893–21902 (2016). https://doi.org/10.1021/acsami.5b0934431. A. Elosegui-Artola, E. Bazellières, M. D. Allen, I. Andreu, R. Oria, R. Sunyer, J. J. Gomm, J. F. Marshall, J. L. Jones, X. Trepat et al., “ Rigidity sensing and adaptation through regulation of integrin types,” Nat. Mater. 13, 631–637 (2014). https://doi.org/10.1038/nmat3960 Despite the fact that the percentages of acrylamide and bisarylamide used in our studies were almost identical to the concentrations used in other studies, it is important to note that Young's modulus studies in the literature were conducted with cantilever-based micro-indentation mechanical testing, while our measurements were conducted using macroscale compression testing on bulk gel samples. However, there is generally good consistency between micromechanical measurements and bulk measurements, as shown in the study by Kain et al.,3232. L. Kain, O. G. Andriotis, P. Gruber, M. Frank, M. Markovic, D. Grech, V. Nedelkovski, M. Stolz, A. Ovsianikov, and P. J. Thurner, “ Calibration of colloidal probes with atomic force microscopy for micromechanical assessment,” J. Mech. Behav. Biomed. Mater. 85, 225–236 (2018). https://doi.org/10.1016/j.jmbbm.2018.05.026 where the microscale mechanics of agarose samples measured using Atomic Force Microscopy showed good correlation with bulk compression tests.

Substrate stiffness affects AsPC-1 cell metabolic activity

Matrix stiffness regulates cell behavior in various ways. Cell metabolism reflects the cells' viability on different surfaces and may modify the cell's capacity to engulf particles. To evaluate the relationship between matrix stiffness and the cells' metabolic activity, AsPC-1 cells were grown on polyacrylamide-fabricated matrices of different rigidities for 12 or 96 h, followed by a 3–(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay [Fig. 3(a)]. In addition, the metabolic activity of cells grown on commercial substrates for 12 h was measured [Fig. 3(b)].We found a maximum decrease in 30% in metabolic activity, which was observed with the soft substrates [Fig. 3(b)]. Thus, most cells maintained high functionality on fabricated polyacrylamide [Fig. 3(a)] as well as on the commercial [Fig. 3(b)] substrates tested.

To further assess the link between surface rigidity and cell death, we applied Annexin V–APC and PI double staining to AsPC-1 cells grown for 12 h on silicone surfaces of various stiffnesses. Figure S2 shows that more than 90% of the cells remained viable after 12 h of growth on different surfaces.

Higher cell spreading on stiff surfaces

The exposed cell surface available for particle absorption is a critical steric factor that greatly influences the extent of particle uptake. Fluorescent images [Figs. 4(a) and S3] show that cells cultured on soft substrates displayed a round morphology, whereas those on intermediate and stiff substrates appeared to be more spread out. These findings are in agreement with previous studies.33–3533. Q. Wei, C. Huang, Y. Zhang, T. Zhao, P. Zhao, P. Butler, and S. Zhang, “ Mechanotargeting: Mechanics-dependent cellular uptake of nanoparticles,” Adv. Mater. 30, e1707464 (2018). https://doi.org/10.1002/adma.20170746434. C. Huang, P. J. Butler, S. Tong, H. S. Muddana, G. Bao, and S. Zhang, “ Substrate stiffness regulates cellular uptake of nanoparticles,” Nano Lett. 13, 1611–1615 (2013). https://doi.org/10.1021/nl400033h35. Y. Wang, T. Gong, Z. R. Zhang, and Y. Fu, “ Matrix stiffness differentially regulates cellular uptake behavior of nanoparticles in two breast cancer cell lines,” ACS Appl. Mater. Interfaces 9, 25915–25928 (2017). https://doi.org/10.1021/acsami.7b08751Moreover, the surface area of cells grown on intermediate substrates was smaller than those grown on stiff substrates. The exposed cell surface of cells plated on different substrates for 12 h was evaluated using Adobe Photoshop software [Figs. 4(b) and 4(c)]. Generally, we found a clear rigidity-dependent pattern of an increase in the exposed cell area with increasing surface rigidity, with cell area saturation for rigidities larger than 16 kPa. However, the exposed cell area of AsPC-1 cells grown on a 0.5 kPa surface deviated from this trend. This deviation, which, to the best of our knowledge, has not been previously observed, may result from the non-conformational behavior of the cells on very soft substrates, and it is probably not an indicative case. The cell area was measured both including and excluding the nuclei since particle uptake likely occurs predominantly outside of the nuclear region. In both cases, we observed very similar dependencies of the cells' surface area on surface rigidity, indicating that the issue of whether particles are or are not adsorbed on top of the nuclei does not qualitatively affect the dependence of uptake on the surface rigidity.The average pixel-per-cell ratio is higher for silicone surfaces than for polyacrylamide surfaces of similar stiffness [Figs. 4(b) and 4(c)], implying that cells on silicone surfaces tend to have larger surface areas. This finding suggests that the material composition of surfaces influences cell spreading and attachment. To further investigate this, the adherence of AsPC-1 cells was evaluated after short incubation times (2 and 6 h after seeding), which showed that the cells adhere better to the silicone surfaces compared to the polyacrylamide ones (Fig. S6).

Surface stiffness affects the uptake capacity of cells

To investigate the relationship between surface stiffness and cellular uptake capacity, we measured the uptake of fluorescent polystyrene particles by AsPC-1 [Figs. 5 and S5(a), S5(b), S5 (d), S5(f)] and MDA-MB 231 [Figs. S5(c) and S5(e)] cells cultured on fabricated and commercial matrices [Figs. 5(c) and S5] of different rigidities.We tested several incubation periods of AsPC-1 cells on surfaces of various stiffnesses and found that 12 h is an optimal time interval for cells to attach and spread on the gel surface while minimizing their penetration into the gel (Fig. S4). After the cells had been cultured for 12 or 96 h on surfaces of different rigidities, 0.8 or 2.4 μm fluorescent polystyrene beads were added for a predetermined incubation time of 12 h. A non-monotonic dependence between the cell uptake capacity and the surface rigidity of commercial silicone substrates is observed in Fig. 5(c). The percentage of cells grown on surfaces with low rigidities, that internalized any number of beads (one or more), was lower compared with cells grown on surfaces of high rigidity. Whereas the percentage of cells cultured on surfaces with medium rigidity was the highest. When comparing the uptake capacity of 0.8 μm beads with 2.4 μm beads, both kinds of cells internalized larger beads less efficiently. Although the trend of the non-monotonic dependence of the cell uptake capacity on surface rigidity was preserved across both bead sizes, the difference in the uptake capacity of cells cultured on different surface rigidities became more apparent with larger beads. Similarly, to the commercial silicone substrates, we found that the fabricated polyacrylamide surfaces with increased surface rigidities did not induce a monotonic increase in the cell uptake capacity [Fig. S5(a)]; rather, there were alternating results.Different cells have varied uptake kinetics for the same particles.3636. T. Dos Santos, J. Varela, I. Lynch, A. Salvati, and K. A. Dawson, “ Quantitative assessment of the comparative nanoparticle-uptake efficiency of a range of cell lines,” Small 7, 3341–3349 (2011). https://doi.org/10.1002/smll.201101076 When AsPC-1 cells were cultured with beads for 6 h, a maximum of 1.5% of the total cell population ingested one or more particles, as opposed to MDA-MB 231 cells, in which over 50% of the cell population ingested beads [Figs. S5(c) and S5(d)]. Incubation of MDA-MB 231 cells with beads for 12 h resulted in bead internalization by ∼95% of the cell population on all the different surface rigidities [Fig. S5(e)].

In most of the cases studied here, meandering uptake patterns were observed. The “wavelength” and “phase” of the wave-like dependence varied with cell type and particle size. Different cell lines responded differently to the mechanical and biochemical cues mediated by substrate rigidity. These parameters affect the cells' specific interactions with beads of different sizes. Among all the cases studied, the only one in which a monotonic pattern was found was in the MDA-MB 231 cells when incubated with 2.4 μm beads [Fig. S5(c)]. This observation does not contradict a large wavelength situation, where non-monotonicity would be observed in a broader range of surface rigidities.

Opposing trends controlling the dependence of the uptake capacity of cells on the surface rigidity

Although the meandering uptake pattern may seem like wave dependence, there is no reason to assume that infinite periodicity is present in cell uptake behavior as a function of substrate stiffness. Instead, the non-monotonic dependence most likely results from a superposition of discrete opposing effects. To better understand the mechanical role in the meandering uptake pattern, we introduced a simple physical scenario that accounts for two conflicting trends, as illustrated in Fig. 6(a). The basic assumption, whose validity is examined below, and is by previous studies,22,2522. Y. Brill-Karniely, D. Dror, T. Duanis-Assaf, Y. Goldstein, O. Schwob, T. Millo, N. Orehov, T. Stern, M. Jaber, N. Loyfer et al., “ Triangular correlation (TrC) between cancer aggressiveness, cell uptake capability, and cell deformability,” Sci. Adv. 6, eaax2861 (2020). https://doi.org/10.1126/sciadv.aax286125. D. M. Richards and R. G. Endres, “ The mechanism of phagocytosis: Two stages of engulfment,” Biophys. J. 107, 1542–1553 (2014). https://doi.org/10.1016/j.bpj.2014.07.070 is that phagocytosis begins as a slow thermodynamic process and that stable particle absorbance is a limiting step toward full particle penetration. Based on this assumption, understanding the physical interplay which governs static particle adhesion to the cell membrane provides valuable insights into the cell uptake dependence on surface rigidity. On the one hand, the cells are more spread out on stiffer substrates; thus, each cell has more surface area available to adsorb particles. However, cells are less deformable on more rigid substrates, and are therefore less likely to perform the shape deformation needed for contact with the micrometer-scale particles.21,2221. Y. Brill-Karniely, “ Mechanical measurements of cells using AFM: 3D or 2D physics?,” Front. Bioeng. Biotechnol. 8, 1–5 (2020). https://doi.org/10.3389/fbioe.2020.60515322. Y. Brill-Karniely, D. Dror, T. Duanis-Assaf, Y. Goldstein, O. Schwob, T. Millo, N. Orehov, T. Stern, M. Jaber, N. Loyfer et al., “ Triangular correlation (TrC) between cancer aggressiveness, cell uptake capability, and cell deformability,” Sci. Adv. 6, eaax2861 (2020). https://doi.org/10.1126/sciadv.aax2861 For a simple intuitive understanding of the latter argument, it is assumed here that the uptake probability density function is linearly proportional to the initial cell-particle contact area, ɑ(ξ), where ξ is the substrate rigidity. The more elastic the cells are, the larger the particle wrapping by the cell membrane is during the initial absorbance. For micrometer-scale particles, this step is governed by the three-dimensional deformation of the cell bulk. It, therefore, depends on the cell's Young's modulus, E(ξ), which increases with the substrate rigidity.37,3837. S. Y. Tee, J. Fu, C. S. Chen, and P. A. Janmey, “ Cell shape and substrate rigidity both regulate cell stiffness,” Biophys. J. 100, L25–L27 (2011). https://doi.org/10.1016/j.bpj.2010.12.374438. K. Pogoda, R. Bucki, F. J. Byfield, K. Cruz, T. Lee, C. Marcinkiewicz, and P. A. Janmey, “ Soft substrates containing hyaluronan mimic the effects of increased stiffness on morphology, motility, and proliferation of glioma cells,” Biomacromolecules 18, 3040–3051 (2017). https://doi.org/10.1021/acs.biomac.7b00324 Thus, the more rigid the substrate, the smaller the ɑ(ξ); this tends to reduce the probability of proceeding into complete active particle internalization. This is in contrast to the increase in the cell spreading area, which increases the number of particles that are in contact with the cell and thus contributes to elevated uptake on more rigid substrates. Based on these arguments, the dependence of the uptake probability density on the substrate stiffness can be written as Puptake(ξ)∝A(ξ)·a(ξ).As we found in the fluorescent image analysis (Fig. 4), and in accordance with previous findings,37,3837. S. Y. Tee, J. Fu, C. S. Chen, and P. A. Janmey, “ Cell shape and substrate rigidity both regulate cell stiffness,” Biophys. J. 100, L25–L27 (2011). https://doi.org/10.1016/j.bpj.2010.12.374438. K. Pogoda, R. Bucki, F. J. Byfield, K. Cruz, T. Lee, C. Marcinkiewicz, and P. A. Janmey, “ Soft substrates containing hyaluronan mimic the effects of increased stiffness on morphology, motility, and proliferation of glioma cells,” Biomacromolecules 18, 3040–3051 (2017). https://doi.org/10.1021/acs.biomac.7b00324 the spreading area Aξ of cells increases with ξ until saturated (excluding the divergent case of 0.5 kPa on commercial substrates). We use the approximate expression Aξ∝tanh(0.2ξ) to describe the trend of the results in Fig. 4(c). We further assume that Eξ∝tanh(0.05ξ), based on previous experimental findings.3838. K. Pogoda, R. Bucki, F. J. Byfield, K. Cruz, T. Lee, C. Marcinkiewicz, and P. A. Janmey, “ Soft substrates containing hyaluronan mimic the effects of increased stiffness on morphology, motility, and proliferation of glioma cells,” Biomacromolecules 18, 3040–3051 (2017). https://doi.org/10.1021/acs.biomac.7b00324 The cell-particle contact area during static absorbance was obtained using a thermodynamic model that we previously published,2222. Y. Brill-Karniely, D. Dror, T. Duanis-Assaf, Y. Goldstein, O. Schwob, T. Millo, N. Orehov, T. Stern, M. Jaber, N. Loyfer et al., “ Triangular correlation (TrC) between cancer aggressiveness, cell uptake capability, and cell deformability,” Sci. Adv. 6, eaax2861 (2020). https://doi.org/10.1126/sciadv.aax2861 where the dependence on ξ is assumed to be governed by the increase in the elastic cell moduli, which controls the contact angle θξ=0.75πEξR3(Δγ−4κR2). Here, typical values were taken for the work of adhesion per unit area Δγ=0.002 κPa  μm and the membrane bending modulus κ=1.9·10−5 κPa μm3.22,3922. Y. Brill-Karniely, D. Dror, T. Duanis-Assaf, Y. Goldstein, O. Schwob, T. Millo, N. Orehov, T. Stern, M. Jaber, N. Loyfer et al., “ Triangular correlation (TrC) between cancer aggressiveness, cell uptake capability, and cell deformability,” Sci. Adv. 6, eaax2861 (2020). https://doi.org/10.1126/sciadv.aax286139. C. Händel, B. U. S. Schmidt, J. Schiller, U. Dietrich, T. Möhn, T. R. Kießling, S. Pawlizak, A. W. Fritsch, L. C. Horn, S. Briest et al., “ Cell membrane softening in human breast and cervical cancer cells,” New J. Phys. 17, 083008 (2015). https://doi.org/10.1088/1367-2630/17/8/083008Using this general scheme, Puptake (ξ) is plotted in Fig. 6(b) for beads of 0.8 or 2.4 μm, as used in our experiments. The opposing effects detailed above resulted in an initial increase in uptake, followed by a decline as a function of the substrate rigidity. The meandering patterns are found in Fig. 5(b) indicate additional contributions that may not be physical. The dashed lines in the plots of Fig. 6(b) indicate that the increase in uptake with large rigidities can result from the increased functionality of the cells (as found in Fig. 4), which is manifested in elevated uptake activity on the more rigid substrates.

An inverse correlation exists between the levels of phosphorylated paxillin and the extent of microparticle uptake in AsPC-1 cells

To gain mechanistic insights and further investigate the reliability of the assumption that stable particle adhesion to the cell membrane is required prior to complete cell penetration, we examined the expression of phosphorylated paxillin in AsPC-1 cells that were plated on substrates of varying rigidities [Fig. 7(a)]. Integrin binding to ECM promotes paxillin phosphorylation, activating numerous signaling cascades that have been shown to promote cell migration and adhesion dynamics.40–4240. A. M. López-Colomé, I. Lee-Rivera, R. Benavides-Hidalgo, and E. López, “ Paxillin: A crossroad in pathological cell migration,” J. Hematol. Oncol. 10, 50 (2017). https://doi.org/10.1186/s13045-017-0418-y41. T. Iwasaki, A. Nakata, M. Mukai, K. Shinkai, H. Yano, H. Sabe, E. Schaefer, M. Tatsuta, T. Tsujimura, N. Terada et al., “ Involvement of phosphorylation of Tyr-31 and Tyr-118 of paxillin in MM1 cancer cell migration,” Int. J. Cancer 97, 330–335 (2002). https://doi.org/10.1002/ijc.160942. V. Petit, B. Boyer, D. Lentz, C. E. Turner, J. P. Thiery, and A. M. Valles, “ Phosphorylation of tyrosine residues 31 and 118 on paxillin regulates cell migration through an association with CRK in NBT-II cells,” J. Cell Biol. 148, 957–969 (2000). https://doi.org/10.1083/jcb.148.5.957 Inhibited levels of phosphorylated paxillin results in decreased cell motility and is correlated with weaker integrin-mediated matrix adhesion, resulting in an increased incidence of stress fiber breaks.42,4342. V. Petit, B. Boyer, D. Lentz, C. E. Turner, J. P. Thiery, and A. M. Valles, “ Phosphorylation of tyrosine residues 31 and 118 on paxillin regulates cell migration through an association with CRK in NBT-II cells,” J. Cell Biol. 148, 957–969 (2000). https://doi.org/10.1083/jcb.148.5.95743. N. O. Deakin and C. E. Turner, “ Paxillin comes of age Nicholas,” J. Cell Sci. 121, 2435–2444 (2008). https://doi.org/10.1242/jcs.018044 The effect of phosphorylated paxillin on cell-matrix adhesion is expected to contribute mainly to the static phase of cell uptake, since reducing cell adhesiveness releases cell tension, manifesting in the formation of membrane ruffles.44,4544. M. Skalski, Q. Yi, M. J. Kean, D. W. Myers, K. C. Williams, A. Burtnik, and M. G. Coppolino, “ Lamellipodium extension and membrane ruffling require different SNARE-mediated trafficking pathways,” BMC Cell Biol. 11, 62 (2010). https://doi.org/10.1186/1471-2121-11-6245. S. B. Azimifar, R. T. Böttcher, S. Zanivan, C. Grashoff, M. Krüger, K. R. Legate, M. Mann, and R. Fässler, “ Induction of membrane circular dorsal ruffles requires co-signalling of integrin-ILK-complex and EGF receptor,” J. Cell Sci. 125, 435–448 (2012). https://doi.org/10.1242/jcs.091652 This thermal effect can reduce the need for the elastic alterations in cell morphology that are necessary for particle adhesion, as illustrated in Fig. 7(b). Notably, phosphorylated paxillin was also cor