INTRODUCTION

Section:

ChooseTop of pageABSTRACTINTRODUCTION <<RESULTSDISCUSSIONMETHODSSUPPLEMENTARY MATERIALMore than 1.9 × 106 new cancer cases and 600 thousand cancer deaths are projected to occur in the United States in 2022.11. R. L. Siegel, K. D. Miller, H. E. Fuchs, and A. Jemal, “ Cancer statistics, 2022,” CA: Cancer J. Clin. 72, 7–33 (2022). https://doi.org/10.3322/caac.21708 The major cause of cancer morbidity and mortality is metastasis, a process of several intricate steps including tumor cells (TCs) detaching from the primary tumor, intravasating into and extravasating out of the circulatory system before invading a secondary tissue. Tumor cells achieve intravasation and extravasation by exploiting the abnormal leakage of pathological blood vessels.2,32. H. Hashizume et al., “ Openings between defective endothelial cells explain tumor vessel leakiness,” Am. J. Pathol. 156, 1363–1380 (2000). https://doi.org/10.1016/S0002-9440(10)65006-73. D. M. McDonald and P. Baluk, “ Significance of blood vessel leakiness in cancer,” Cancer Res. 62, 5381–5385 (2002). Although the exact mechanism remains unclear, it has been observed that endothelial functions are altered during the early and late stages of cancer progression.44. J. Suraj et al., “ Early and late endothelial response in breast cancer metastasis in mice: Simultaneous quantification of endothelial biomarkers using a mass spectrometry-based method,” Dis. Model. Mech. 12, dmm036269 (2019). https://doi.org/10.1242/dmm.036269The endothelial cells (ECs) lining the lumen surface of blood vessels control the passage of molecules and cells in and out of the bloodstream, and they, hence, regulate the environment in biological tissues. Normally, the EC layer serves as a barrier with adherens junctions and tight junctions between cells that regulate the permeability of blood vessels. However, the barrier functions can be disrupted remarkably in pathological processes.5,65. E. Dejana, “ Endothelial cell-cell junctions: Happy together,” Nat. Rev. Mol. Cell Biol. 5, 261–270 (2004). https://doi.org/10.1038/nrm13576. E. Vandenbroucke, D. Mehta, R. Minshall, and A. B. Malik, “ Regulation of endothelial junctional permeability,” Ann. N. Y. Acad. Sci. 1123, 134–145 (2008). https://doi.org/10.1196/annals.1420.016 In metastasis, breakdown of the endothelial barrier is critical for the transmigration of TCs during intravasation and extravasation. It is attributable to both the paracrine7–127. E. T. Roussos, J. S. Condeelis, and A. Patsialou, “ Chemotaxis in cancer,” Nat. Rev. Cancer 11, 573–587 (2011). https://doi.org/10.1038/nrc30788. J. A. Joyce and J. W. Pollard, “ Microenvironmental regulation of metastasis,” Nat. Rev. Cancer 9, 239–252 (2009). https://doi.org/10.1038/nrc26189. D. Quail and J. Joyce, “ Microenvironmental regulation of tumor progression and metastasis,” Nat. Med. 19, 1423–1437 (2013). https://doi.org/10.1038/nm.339410. N. Ferrara, “ VEGF and the quest for tumour angiogenesis factors,” Nat. Rev. Cancer 2, 795–803 (2002). https://doi.org/10.1038/nrc90911. J. Fan et al., “ Integrin β4 signaling promotes mammary tumor cell adhesion to brain microvascular endothelium by inducing ErbB2-mediated secretion of VEGF,” Ann. Biomed. Eng. 39, 2223–2241 (2011). https://doi.org/10.1007/s10439-011-0321-612. J. Folkman, “ What is the evidence that tumors are angiogenesis dependent?,” JNCI J. Natl. Cancer Inst. 82, 4–7 (1990). https://doi.org/10.1093/jnci/82.1.4 and physical receptor-ligand communications between the tumor and ECs,13–1613. G. Sökeland and U. Schumacher, “ The functional role of integrins during intra- and extravasation within the metastatic cascade,” Mol. Cancer 18, 12 (2019). https://doi.org/10.1186/s12943-018-0937-314. D. Wirtz, K. Konstantopoulos, and P. C. Searson, “ The physics of cancer: The role of physical interactions and mechanical forces in metastasis,” Nat. Rev. Cancer 11, 512–522 (2011). https://doi.org/10.1038/nrc308015. R. H. Kramer and G. L. Nicolson, “ Interactions of tumor cells with vascular endothelial cell monolayers: A model for metastatic invasion,” Proc. Natl. Acad. Sci. 76, 5704–5708 (1979). https://doi.org/10.1073/pnas.76.11.570416. F. W. Orr, H. H. Wang, R. M. Lafrenie, S. Scherbarth, and D. M. Nance, “ Interactions between cancer cells and the endothelium in metastasis,” J. Pathol. 190, 310–329 (2000). https://doi.org/10.1002/(SICI)1096-9896(200002)190:3<310::AID-PATH525>3.0.CO;2-P which consequently triggers a cascade of intracellular signals in ECs, not only affecting their growth but also altering their cytoskeletal proteins and promoting asymmetric cell morphology and migration.7,177. E. T. Roussos, J. S. Condeelis, and A. Patsialou, “ Chemotaxis in cancer,” Nat. Rev. Cancer 11, 573–587 (2011). https://doi.org/10.1038/nrc307817. L. Lamalice, F. Le Boeuf, and J. Huot, “ Endothelial cell migration during angiogenesis,” Circ. Res. 100, 782–794 (2007). https://doi.org/10.1161/01.RES.0000259593.07661.1eThe handedness of the cell, termed cell chirality, has been recently discovered as an intrinsic property of the cell.1818. L. Q. Wan et al., “ Micropatterned mammalian cells exhibit phenotype-specific left-right asymmetry,” Proc. Natl. Acad. Sci. U. S. A. 108, 12295–12300 (2011). https://doi.org/10.1073/pnas.1103834108 In other words, the cell is left–right asymmetrically structured and functions in a similar way of a human body, but on a much smaller scale. Cell chirality has been found phenotype-specific and seen as left–right biased cell alignment,18,1918. L. Q. Wan et al., “ Micropatterned mammalian cells exhibit phenotype-specific left-right asymmetry,” Proc. Natl. Acad. Sci. U. S. A. 108, 12295–12300 (2011). https://doi.org/10.1073/pnas.110383410819. T.-H. Chen et al., “ Left-right symmetry breaking in tissue morphogenesis via cytoskeletal mechanics,” Circ. Res. 110, 551–559 (2012). https://doi.org/10.1161/CIRCRESAHA.111.255927 rotation,20–2320. Y. H. Tee et al., “ Cellular chirality arising from the self-organization of the actin cytoskeleton,” Nat. Cell Biol. 17, 445–457 (2015). https://doi.org/10.1038/ncb313721. W. Liu et al., “ Nanowire magnetoscope reveals a cellular torque with left-right bias,” ACS Nano 10, 7409–7417 (2016). https://doi.org/10.1021/acsnano.6b0114222. A. Davison et al., “ Formin is associated with left-right asymmetry in the pond snail and the frog,” Curr. Biol. 26, 654–660 (2016). https://doi.org/10.1016/j.cub.2015.12.07123. A. S. Chin et al., “ Epithelial cell chirality revealed by three-dimensional spontaneous rotation,” Proc. Natl. Acad. Sci. U. S. A. 115, 12188–12193 (2018). https://doi.org/10.1073/pnas.1805932115 migration,24–2624. J. Xu et al., “ Polarity reveals intrinsic cell chirality,” Proc. Natl. Acad. Sci. U. S. A. 104, 9296–9300 (2007). https://doi.org/10.1073/pnas.070315310425. K. E. Worley, D. Shieh, and L. Q. Wan, “ Inhibition of cell-cell adhesion impairs directional epithelial migration on micropatterned surfaces,” Integr. Biol. 7, 580–590 (2015). https://doi.org/10.1039/c5ib00073d26. K. G. Sullivan, L. N. Vandenberg, and M. Levin, “ Cellular migration may exhibit intrinsic left-right asymmetries: A meta-analysis,” bioRxiv (2018). and organelle positioning.27–2927. K. Taniguchi et al., “ Chirality in planar cell shape contributes to left-right asymmetric epithelial morphogenesis,” Science 333, 339–341 (2011). https://doi.org/10.1126/science.120094028. J. Fan, H. Zhang, T. Rahman, D. N. Stanton, and L. Q. Wan, “ Cell organelle-based analysis of cell chirality,” Commun. Integr. Biol. 12, 78–81 (2019). https://doi.org/10.1080/19420889.2019.160527729. P. Ray et al., “ Intrinsic cellular chirality regulates left-right symmetry breaking during cardiac looping,” Proc. Natl. Acad. Sci. U. S. A. 115, E11568–E11577 (2018). https://doi.org/10.1073/pnas.1808052115 Cell chirality can notably influence the planar cellular organization in monolayers and account for the organ-specific asymmetries such as the heart,2929. P. Ray et al., “ Intrinsic cellular chirality regulates left-right symmetry breaking during cardiac looping,” Proc. Natl. Acad. Sci. U. S. A. 115, E11568–E11577 (2018). https://doi.org/10.1073/pnas.1808052115 gut,2727. K. Taniguchi et al., “ Chirality in planar cell shape contributes to left-right asymmetric epithelial morphogenesis,” Science 333, 339–341 (2011). https://doi.org/10.1126/science.1200940 and genitalia.3030. K. Sato et al., “ Left-right asymmetric cell intercalation drives directional collective cell movement in epithelial morphogenesis,” Nat. Commun. 6, 10074 (2015). https://doi.org/10.1038/ncomms10074 In blood vessels, ECs have been found possessing a strong clockwise (CW, or rightward) chirality,18,3118. L. Q. Wan et al., “ Micropatterned mammalian cells exhibit phenotype-specific left-right asymmetry,” Proc. Natl. Acad. Sci. U. S. A. 108, 12295–12300 (2011). https://doi.org/10.1073/pnas.110383410831. J. Fan et al., “ Cell chirality regulates intercellular junctions and endothelial permeability,” Sci. Adv. 4, eaat2111 (2018). https://doi.org/10.1126/sciadv.aat2111 which may lead to a helically aligned tubular sheet. Enervating the rightward chirality of ECs through protein kinase C alpha (PKCα) activation impairs the endothelial barrier function by changing the cell shape, alignment, and cell junctional morphology.3131. J. Fan et al., “ Cell chirality regulates intercellular junctions and endothelial permeability,” Sci. Adv. 4, eaat2111 (2018). https://doi.org/10.1126/sciadv.aat2111 Activation of PKCα in ECs has been found in various vascular conditions, including tumor adhesion.3232. P. Zhang, C. Goodrich, C. Fu, and C. Dong, “ Melanoma upregulates ICAM-1 expression on endothelial cells through engagement of tumor CD44 with endothelial E-selectin and activation of a PKCα–p38-SP-1 pathway,” FASEB J. 28, 4591–4609 (2014). https://doi.org/10.1096/fj.11-202747 However, it is unclear of the alteration of the CW chirality of ECs when interacting with TCs during the metastasis.

Using a breast cancer model, we focus on studying the changes in EC chirality caused by paracrine signaling and physical contact of TCs. Any changes or disruptions of intrinsic CW chirality of EC into non-chiral (NC) or counterclockwise (CCW) chirality may compromise the overall endothelial integrity which potentially increases the risk of metastasis. We hypothesized that either physical contact or paracrine signaling between TCs and ECs disrupts the chirality of the endothelial barrier. We hope to provide insight into how TC–EC interaction modulated endothelial morphogenesis may affect trans-endothelial migration during extravasation and aid the identification of therapeutic strategies to stop metastasis.

RESULTS

Section:

ChooseTop of pageABSTRACTINTRODUCTIONRESULTS <<DISCUSSIONMETHODSSUPPLEMENTARY MATERIAL

The intrinsic clockwise chirality of endothelial cells is disrupted in TC–EC co-culture

Oncogenic activation of GTPase HRas (HRAS) and human epidermal growth factor receptor 2 (HER2) has been frequently found in breast cancers, leading to tumor initiation, progression, and metastasis [Fig. S1(a)].33–3533. M. Galiè, “ RAS as supporting actor in breast cancer,” Front. Oncol. 9, 1199 (2019). https://doi.org/10.3389/fonc.2019.0119934. E. Y. H. P. Lee and W. J. Muller, “ Oncogenes and tumor suppressor genes,” Cold Spring Harbor Perspect. Biol. 2, a003236 (2010). https://doi.org/10.1101/cshperspect.a00323635. M. M. Moasser, “ The oncogene HER2: Its signaling and transforming functions and its role in human cancer pathogenesis,” Oncogene 26, 6469–6487 (2007). https://doi.org/10.1038/sj.onc.1210477 In this study, the MCF10A human breast epithelial cell line and its malignant mutants, MCF10A-HRAS (or MCF10AT1, HRAS overexpression) and MCF10A-HER2 (HER2 overexpression) [Fig. S1(b)], were co-cultured with ECs in a transwell system to simulate the non-contact or contact TC–EC interactions during the metastasis [Fig. 1(a)]. In addition, each type of TCs was used at a high, mid, or low concentration to simulate different degrees of tumorous conditions. The ECs in co-culture were seeded on a ring-shaped micropattern, and their chirality, including the numbers and percentages of CW, CCW, NC rings [Figs. 1(b)–1(e) and S2], the chiral factor [Fig. 1(d)], and the mean circumferential angle of cell alignment on the micropattern [Fig. 1(f)], were calculated using a custom-written MATLAB (MathWorks) program as described previously.1818. L. Q. Wan et al., “ Micropatterned mammalian cells exhibit phenotype-specific left-right asymmetry,” Proc. Natl. Acad. Sci. U. S. A. 108, 12295–12300 (2011). https://doi.org/10.1073/pnas.1103834108

FIG. 1. (a) Models of co-cultured ECs and TCs used to study the chirality change of ECs caused by TC paracrine signaling and physical contact during extravasation. (b) Top row: typical images of clockwise (CW), non-chiral (NC) micropatterns formed by ECs under the control condition, or counterclockwise (CCW) micropattern under the physical contact of HRAS TCs; bottom row: the color bars, generated by a pre-developed MATLAB program, show the aligning direction of each cell on the micropattern. (Scale bar: 100 μm). (c) The definition of circumferential angle of each bar [blue bar, cell alignment in (b)] deviated from the circumferential direction (red line) and its relationship to chirality. (d) Numbers of the CW, CCW, and NC rings analyzed in each group and the corresponding chiral factors of ECs treated by paracrine signaling or physical contact of TCs. Chiral factor: defined by comparing ring numbers from the formula: (number of CW rings − number of CCW rings)/number of total rings, with CF = +1 standing for complete CW and CF = −1 for complete CCW. (e) Percentage of CW, NC, and CCW rings of ECs from table (d). (f) Mean circumferential angle of ECs on the ring-shaped micropatterns [illustrated in (c)]. The “low,” “mid,” and “high” in figures. (d)–(f) represent the TC densities (20, 100, and 200k/ml) introduced to ECs in the different co-culture groups. Data in (f) are represented as mean ± SD. Under the control condition, 633 rings of ECs were analyzed; under the non-contact condition of MCF10A cells, 50 (low), 44 (mid), and 84 (high) rings were analyzed; under the contact condition of MCF10A cells, 62 (low), 67 (mid), and 76 (high) rings were analyzed; under the non-contact condition of HRAS cells, 115 (low), 114 (mid), and 134 (high) rings were analyzed; under contact condition of HRAS cells, 64 (low), 69 (mid), and 65 (high) rings were analyzed; under the non-contact condition of HER2 cells, 24 (low), 52 (mid), and 35 (high) rings were analyzed; under contact condition of HER2 cells, 70 (low), 74 (mid), and 78 (high) rings were analyzed. “*” in (f) indicates significant differences between the experimental group and control; “*” or “#” indicates P < 0.05; “**” or “##” indicates P <0.01; and “***” or “###” indicates P <0.001 by one-way analyses of variance (ANOVAs) with the Tukey method between groups.

Normally, human umbilical vein endothelial cells (hUVECs) are strongly CW dominant with a chiral factor above 0.90 [Figs. 1(d) and 1(e)], consistent with previous reports.3131. J. Fan et al., “ Cell chirality regulates intercellular junctions and endothelial permeability,” Sci. Adv. 4, eaat2111 (2018). https://doi.org/10.1126/sciadv.aat2111 Non-contacting co-culture with wildtype MCF10A and HRAS cells did not change the strong CW chirality of ECs, while increasing concentration of HER2 cells induces a 27% decrease in CW chirality of EC shown as the chiral factor decreasing from 0.95 to 0.69 [Figs. 1(d) and 1(e)]. For TC–EC contact groups, the ECs formed notable NC and CCW rings when co-cultured with TCs. In addition, their chiral factor is decreased to 0.83 when co-cultured with MCF10A, and down to a 0.60 level with HRAS or HER2 cells [Figs. 1(d) and 1(e)]. The results from the mean circumferential angle give details about the chiral alignment of cells on the micropattern [Fig. 1(f)]. In both TC–EC non-contacting groups and contacting groups, it shows an overall similar decreasing trend along with the increase in TC density. [Note that the circumferential angle for CW cell alignment is negative in Fig. 1(f).] Under non-contacting co-culture, it requires a high TC concentration to induce a significant decrease in the chiral alignment of ECs; however, under TC–EC contacted co-culture, low density of HRAS or HER2 overexpression cells was adequate to make differences [Fig. 1(f)], but not the wildtype. Particularly for HRAS cells, the physical contact caused a significantly stronger interruption of the CW chirality of ECs than paracrine signaling.Lung is one of the most common target organs for metastasis of malignant breast cancer.3636. W. Chen, A. D. Hoffmann, H. Liu, and X. Liu, “ Organotropism: New insights into molecular mechanisms of breast cancer metastasis,” npj Precis. Oncol. 2, 4 (2018). https://doi.org/10.1038/s41698-018-0047-0 Therefore, we also examined the effects of breast cancer cells on the chirality of the human lung microvascular endothelial cells (hLMVECs). Under the normal condition, the hLMVECs exhibit a strong CW chirality with a chiral factor of 0.88 [Fig. S3(a)], comparable to 0.95 of the hUVECs. Co-cultured with different TCs, the hLMVECs exhibit quite similar patterns of CW chirality weakening [Figs. S3(a) and S3(b)]. Particularly under the physical contact of the HRAS and HER2 cells, the chiral factors of hLMVECs dropped to 0.66 and 0.50 [Fig. S3(a)], along with significant decreases in the mean circumferential angles [Fig. S3(c)], again consistent with the observations from hUVECs. These together suggest the TCs could cause the weakness of the CW chirality of ECs by interacting with them under a physical contact condition. This effect could be significantly enhanced by the oncogenic expression of HRAS and HER2, which may contribute to their malignancy in metastasis.

Tumor secretion plays a limited role in endothelial cell chirality

In the TC–EC contact condition, the cell interactions are not only from physical bindings but also from the paracrine signaling of TCs simultaneously. In addition, the TC–EC communications are bi-directional even without the physical contact of the two cell types.99. D. Quail and J. Joyce, “ Microenvironmental regulation of tumor progression and metastasis,” Nat. Med. 19, 1423–1437 (2013). https://doi.org/10.1038/nm.3394 It means TC secretions are modifying ECs, while EC secretions are simultaneously stimulating TCs in a cycle. Therefore, we further investigated the EC chirality changes in the TC conditioned medium (CM) to understand whether the weakness of EC chirality results from inherent tumor secretions which do not require the EC induction [Fig. 2(a)]. When cultured in TC conditioned medium, the ECs formed an increased percentage of NC rings, and their chiral cell alignment was significantly decreased compared with control [Figs. 2(b)–2(d)]. However, the decreases of either overall EC chiral factors or the chiral cell alignment caused by the inherent tumor secretions are not comparable with the physical contact groups in Figs. 1(d)–1(f). In addition, neither HRAS nor HER2 conditioned medium induces significantly weaker CW chiral alignment of ECs compared with the wildtype [Fig. 2(d)]. Considering the highest density of TCs was used to generate this conditioned medium, therefore, the inherent secretions of TCs play a limited role in EC chirality change under the TC–EC physical contact condition.

FIG. 2. (a) Schematic representation of testing the chirality of ECs treated by TC conditioned medium. (b) Numbers of the CW, CCW, and NC rings and the corresponding chiral factors of ECs treated by TC conditioned medium. “CM” is an abbreviation for “conditioned medium” in group names. (c) Percentage of CW, NC, and CCW rings of ECs from table (b), showing the increased non-chiral chirality of ECs treated with HRAS or HER2 TC conditioned medium. (d) Mean circumferential angle of ECs on the ring-shaped micropatterns, which, however, does not show significant differences. Data are represented as mean ± SD. Under normal medium, 633 rings of ECs were analyzed; under MCF10A, HRAS, or HER2 cell conditioned medium, mean circumferential angles of ECs from 89, 102, or 117 rings were analyzed, respectively. (e) Schematic diagram of endothelial monolayer permeability measurement using a transwell model. (f) Permeability of the hUVEC monolayer with TC conditioned medium or TC physical contact. Data are presented as mean ± SD (n = 4). “*” in (d) and (f) indicates significant differences between the experimental group and control; “*” or “#” indicates P <0.05; “**” or “##” indicates P <0.01; and “***” or “###” indicates P <0.001 by one-way analyses of variance (ANOVAs) with the Tukey method between groups. “n.s.” stands for no significant difference.

The cell chirality is closely associated with the integrity of the EC monolayer. Its permeability increases with the weakening of CW chirality and peaks when ECs become completely non-chiral.3131. J. Fan et al., “ Cell chirality regulates intercellular junctions and endothelial permeability,” Sci. Adv. 4, eaat2111 (2018). https://doi.org/10.1126/sciadv.aat2111 The results from a permeability assay show that the TC conditioned medium is not sufficient to induce a significant change in EC permeability [Figs. 2(e) and 2(f)], suggesting the limited influence of TC secretions on the EC chirality. On the other hand, in the physical TC–EC co-culture groups where EC chirality was weakened, significant increases in EC permeability were also observed, as expected [Fig. 2(f)].

Physical contact with tumor cells disrupts the chiral alignments of endothelial cells

The integrity of cell–cell junctions plays an important role in the collective migration of planar cells to form multicellular chiral alignment on the micropattern.2525. K. E. Worley, D. Shieh, and L. Q. Wan, “ Inhibition of cell-cell adhesion impairs directional epithelial migration on micropatterned surfaces,” Integr. Biol. 7, 580–590 (2015). https://doi.org/10.1039/c5ib00073d Previously, we have found significant local misalignment of ECs occurred on the ring-shaped micropattern when they became non-chiral.3131. J. Fan et al., “ Cell chirality regulates intercellular junctions and endothelial permeability,” Sci. Adv. 4, eaat2111 (2018). https://doi.org/10.1126/sciadv.aat2111 In this study, it is shown in fluorescent images that the ECs form a well-arranged monolayer on the micropatterns with intact cell–cell junctions between adjacent cells under the control condition (Fig. 3). With only tumor secretion in TC–EC non-contact groups, the ECs kept relatively intact and continuous cell–cell junctions, while the endothelial monolayer is significantly interrupted with physical involvement of TCs, and misalignment of ECs occurs at the boundary of TCs, suggesting the ECs with direct contact with TCs may possess inconsistent cell chirality.

Physical contact of tumor cells compromises the left–right biases of endothelial cells

The individual cell chirality can be reflected by the biased positioning of cell organelles.24,27,29,3124. J. Xu et al., “ Polarity reveals intrinsic cell chirality,” Proc. Natl. Acad. Sci. U. S. A. 104, 9296–9300 (2007). https://doi.org/10.1073/pnas.070315310427. K. Taniguchi et al., “ Chirality in planar cell shape contributes to left-right asymmetric epithelial morphogenesis,” Science 333, 339–341 (2011). https://doi.org/10.1126/science.120094029. P. Ray et al., “ Intrinsic cellular chirality regulates left-right symmetry breaking during cardiac looping,” Proc. Natl. Acad. Sci. U. S. A. 115, E11568–E11577 (2018). https://doi.org/10.1073/pnas.180805211531. J. Fan et al., “ Cell chirality regulates intercellular junctions and endothelial permeability,” Sci. Adv. 4, eaat2111 (2018). https://doi.org/10.1126/sciadv.aat2111 We have demonstrated CW rings contain mainly right-biased cells, while CCW rings contain significantly more left-biased cells.2828. J. Fan, H. Zhang, T. Rahman, D. N. Stanton, and L. Q. Wan, “ Cell organelle-based analysis of cell chirality,” Commun. Integr. Biol. 12, 78–81 (2019). https://doi.org/10.1080/19420889.2019.1605277 Each fluorescent image of micropatterned EC monolayer was further segmented into cell borders, cell centroids, nuclear centroids, and centrosomes as described in Methods section [Figs. 4(a)–4(d)]. The left–right biases of individual ECs were then determined by the positional bias of the cell centroid relative to the nucleus-centrosome axis to study the role of TC physical contact in EC chirality [Figs. 4(e) and 4(f)]. The ECs spaced from TCs (without direct contact with TCs) show a significant right bias and a positive chiral factor which is consistent with their CW chirality [Figs. 4(g) and 4(h)]. A neighboring relationship with MCF10A cells did not affect the right bias of ECs; however, those ECs neighbored with HRAS or HER2 cells are lack of left–right bias, and their chiral factors change into negative values close to zero, indicating a slight reversal of the intrinsic chiral bias of ECs [Figs. 4(g) and 4(h)]. These results together suggest the chirality of ECs is significantly altered by the physical contact of malignant TCs with HRAS and HER2 overexpression.

FIG. 4. (a) Immunofluorescence of ECs on a ring-shaped micropattern showing cell adherens junctions (VE-cad, red), cell nuclei (DAPI, blue), and centrosomes (pericentrin, green). (b) Cell borders segmented from the red channel in (a), shown with the calculated cell centroids (yellow). (c) Cell nuclei (blue) segmented from the blue channel in (a), shown with nuclear centroids (cyan). (d) Merged image for cell bias analysis, including cell borders (red), centrosomes (green), nuclear centroids (blue), and cell centroids (yellow). (e) A schematic diagram of determination of the left (L) or right (R) cell bias according to the positioning of the cell centroid relative to the nucleus-centrosome vector. (f) Color-coded cells by their biases on the micropattern. (g) and (h) L–R biases and the corresponding chiral factors of ECs with or without direct TC contact. Data are represented as mean ± SE. In the MCF10A-WT group, 16 rings and 1222 TC-spaced ECs were analyzed; 16 rings and 382 TC-neighbored ECs were analyzed; in the MCF10A-HRAS group, 15 rings and 1094 TC-spaced ECs were analyzed; 15 rings and 327 TC-neighbored ECs were analyzed; in MCF10A-HER2 group, 15 rings and 1417 TC-spaced ECs were analyzed; 15 rings and 325 TC-neighbored ECs were analyzed. “*” indicates P <0.05; “**” indicates P <0.01; and “***” indicates P <0.001 by student t-test. “n.s.” stands for no significant difference. Scale bar in (a): 100 μm.

TC–EC physical contact promotes the binding of CD44 and E-selectin, activates PKCα, and induces pseudopodial movement of EC toward TC

CD44 is a multifunctional cell surface adhesion receptor that is commonly expressed in MCF10A cell series.37–4037. L. T. Senbanjo and M. A. Chellaiah, “ CD44: A multifunctional cell surface adhesion receptor is a regulator of progression and metastasis of cancer cells,” Front. Cell Dev. Biol. 5, 00018 (2017). https://doi.org/10.3389/fcell.2017.0001838. C. Sheridan et al., “ CD44+/CD24− breast cancer cells exhibit enhanced invasive properties: An early step necessary for metastasis,” Breast Cancer Res. 8, R59 (2006). https://doi.org/10.1186/bcr161039. C. M. Fillmore and C. Kuperwasser, “ Human breast cancer cell lines contain stem-like cells that self-renew, give rise to phenotypically diverse progeny and survive chemotherapy,” Breast Cancer Res. 10, R25 (2008). https://doi.org/10.1186/bcr198240. J. Y. So, H. J. Lee, P. Kramata, A. Minden, and N. Suh, “ Differential expression of key signaling proteins in MCF10 cell lines, a human breast cancer progression model,” Mol. Cell. Pharmacol. 4, 31–40 (2012). It has been reported that CD44 binding to its EC surface ligands, E-selectin, activates the PKCα signaling in ECs.3232. P. Zhang, C. Goodrich, C. Fu, and C. Dong, “ Melanoma upregulates ICAM-1 expression on endothelial cells through engagement of tumor CD44 with endothelial E-selectin and activation of a PKCα–p38-SP-1 pathway,” FASEB J. 28, 4591–4609 (2014). https://doi.org/10.1096/fj.11-202747 We have demonstrated previously that PKCα signaling is involved in EC chirality regulation.3131. J. Fan et al., “ Cell chirality regulates intercellular junctions and endothelial permeability,” Sci. Adv. 4, eaat2111 (2018). https://doi.org/10.1126/sciadv.aat2111 In this study, we found a significant elevation of phosphorylated PKCα in ECs when co-cultured with HRAS or HER2 overexpressed TCs rather than the wild type, but the PKCα was inactivated by TC secretions, suggested by undetectable pPKCα bands in any groups of the TC conditioned medium [Fig. 5(a)]. Thus, PKCα activation in ECs requires the physical contact of TCs during the interactions.In most of the co-cultured models, each cell was physically communicating with multiple neighboring cells; therefore, the left–right bias is an integrated result of the physical stimulations from all surrounding cells. To further study the physical TC–EC interaction within one pair of TC and EC without the interference of other cells, we generated a cell doublet model by patterning single TCs and ECs on the “eight”-shaped micropatterns [Fig. 5(b)]. Interestingly, we found the EC moved its pseudopodia toward TC and formed E-selectin-CD44 binding, rather than the TC invading into the EC [Figs. 5(b) and 5(c)]. The malignant HRAS and HER2 oncogenes increased the physical interaction between the TC and EC, shown by the significantly higher colocalization of E-selectin/CD44 in these groups [Fig. 5(d)]. Furthermore, compared with the MCF10A-WT, the HRAS and HER2 overexpressed TCs induced significant increases in EC pseudopodial movement toward the TC [Fig. 5(e)], which could potentially interfere with the chiral alignment of ECs in physical TC–EC interaction.

DISCUSSION

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ChooseTop of pageABSTRACTINTRODUCTIONRESULTSDISCUSSION <<METHODSSUPPLEMENTARY MATERIALDuring metastasis, close interaction between TCs and ECs occurs during the intravasation and extravasation, eliciting the endothelial barrier remodeling to enable the transmigration of metastatic TCs.4141. I. K. Zervantonakis et al., “ Three-dimensional microfluidic model for tumor cell intravasation and endothelial barrier function,” Proc. Natl. Acad. Sci. U. S. A. 109, 13515–13520 (2012). https://doi.org/10.1073/pnas.1210182109 In tumor blood vessels, the endothelial barrier is modulated by tumor secretions, but their role in cell chirality is limited. It is consistent with our previous reports that common growth factors or inflammatory factors do not change the EC chirality since ECs are constantly exposed to various cytokines in the bloodstream. However, it is also possible that accumulated secretions from HER2-positive tumors at the local microenvironment of interstitial space or micro blood circulation may induce non-chiral ECs on the blood vessels. Compared with paracrine signaling, the physical contact between the EC and TC could significantly weaken the intrinsic CW chirality of ECs by binding the tumor surface adhesion molecules to their ligands on the EC surface.The non-contacting TC–EC co-culture results in more complicated communications between the two cell types, than the monoculture of ECs with the TC conditioned medium. In the case of non-contacting co-culture, the communication between TCs and ECs is bi-directional and continuous during the period when the TCs have paracrine effects on ECs at the same time when the ECs exert paracrine effects on TCs. It allows both cell types to alter their paracrine signaling and reactions constantly to the stimuli along with culture time. In the case of monoculture of ECs with the TC conditioned medium, the communication is unidirectional and only one-shot with the inherent tumor secretions from TCs to ECs without potential adjustments over time. This phenomenon can be reflected by the results from Figs. 1(d) and 2(b) that the TCs with HER2 overexpression results in a decrease in CW chirality of ECs under the non-contact co-culture condition, but not under any TC conditioned medium, which suggests the importance of bi-directionality and continuity in TC–EC communication in this process.8,42,438. J. A. Joyce and J. W. Pollard, “ Microenvironmental regulation of metastasis,” Nat. Rev. Cancer 9, 239–252 (2009). https://doi.org/10.1038/nrc261842. N. Maishi and K. Hida, “ Tumor endothelial cells accelerate tumor metastasis,” Cancer Sci. 108, 1921–1926 (2017). https://doi.org/10.1111/cas.1333643. H. Choi and A. Moon, “ Crosstalk between cancer cells and endothelial cells: Implications for tumor progression and intervention,” Arch. Pharm. Res. 41, 711–724 (2018). https://doi.org/10.1007/s12272-018-1051-1 Compared to HER2, a transmembrane glycoprotein receptor sensing various paracrine signals, the HRAS is a GTPase enzyme completely enclosed in the TC cytoplasm. The HER2 overexpression in TCs may, therefore, involve more active communication with ECs and induce more significant alteration in chirality.PKC signaling pathway is known as the chirality regulatory mechanism in ECs. Activation of PKC at an intermediate level changed the EC chirality from CW to NC, resulting in the endothelial leakage; while hyperactivation of PKC may further induce a CCW chirality which may re-align the cells in a reversed pattern and re-enforce the endothelial barrier.