I. INTRODUCTION

Section:

ChooseTop of pageABSTRACTI. INTRODUCTION <<II. MATERIALS AND METHODSIV. RESULTS AND DISCUSSIO...V. CONCLUSIONSSUPPLEMENTARY MATERIALREFERENCESIn 2020, breast cancer surpassed lung cancer, becoming the most common cancer for women and the leading cause of cancer deaths among women worldwide.11. H. Sung, J. Ferlay, R. L. Siegel, M. Laversanne, I. Soerjomataram, A. Jemal, and F. Bray, CA Cancer J. Clin. 71(3), 209–249 (2021). https://doi.org/10.3322/caac.21660 In advanced breast cancer, spread of the cancer throughout the body is caused by the complex transendothelial migration behavior of cancer cells, making it difficult to cure cancer.22. N. Harbeck and M. Gnant, Lancet 389, 1134–1150 (2017). https://doi.org/10.1016/S0140-6736(16)31891-8 Therefore, the tumor microenvironment (TME) with complex microvascular networks has been identified as one of the driving factors of tumor progression.3–53. M. R. Junttila and F. J. de Sauvage, Nature 501, 346–354 (2013). https://doi.org/10.1038/nature126264. M. Nurmik, P. Ullmann, F. Rodriguez, S. Haan, and E. Letellier, Int. J. Cancer 146, 895–905 (2020). https://doi.org/10.1002/ijc.321935. N. Peela, T. Danh, H. Saini, H. Chu, S. Mashaghi, S. L. Ham, S. Singh, H. Tavana, B. Mosadegh, and M. Nikkhah, Biomaterials 133, 176–207 (2017). https://doi.org/10.1016/j.biomaterials.2017.04.017 As the most abundant component in TME,66. X. M. Chen and E. W. Song, Nat. Rev. Drug Discovery 18, 99–115 (2019). https://doi.org/10.1038/s41573-018-0004-1 cancer-associated fibroblasts (CAFs) not only produce many components of ECM and the basement membrane77. R. Kalluri, Nat. Rev. Cancer 16, 582–598 (2016). https://doi.org/10.1038/nrc.2016.73 but also produce a crucial angiogenesis agent–vascular endothelial growth factor (VEGF-A), promoting the development of cancer angiogenesis.8–108. M. De Palma, D. Biziato, and T. V. Petrova, Nat. Rev. Cancer 17, 457–474 (2017). https://doi.org/10.1038/nrc.2017.519. D. Hanahan and L. M. Coussens, Cancer Cell 21, 309–322 (2012). https://doi.org/10.1016/j.ccr.2012.02.02210. S. H. Lee, D. Jeong, Y. S. Han, and M. J. Baek, Ann. Surg. Treat. Res. 89, 1–8 (2015). https://doi.org/10.4174/astr.2015.89.1.1 Some researchers have also found that the CAF could enhance drug resistance of cancer cells.11,1211. S. Su, J. Chen, H. Yao, J. Liu, S. Yu, L. Lao, M. Wang, M. Luo, Y. Xing, F. Chen, D. Huang, J. Zhao, L. Yang, D. Liao, F. Su, M. Li, Q. Liu, and E. Song, Cell 172, 841–856 (2018). https://doi.org/10.1016/j.cell.2018.01.00912. Y. Shintani, A. Fujiwara, T. Kimura, T. Kawamura, S. Funaki, M. Minami, and M. Okumura, J. Thorac. Oncol. 11, 1482–1492 (2016). https://doi.org/10.1016/j.jtho.2016.05.025 In TME, tumor angiogenesis is the inevitable result of further development of the cancer.1313. M. Potente, H. Gerhardt, and P. Carmeliet, Cell 146, 873–887 (2011). https://doi.org/10.1016/j.cell.2011.08.039 New blood vessel structures are produced in this process, while also destroying the barrier function of the original blood vessel.14,1514. G. Bergers and L. E. Benjamin, Nat. Rev. Cancer 3, 401–410 (2003). https://doi.org/10.1038/nrc109315. P. Carmeliet and R. K. Jain, Nature 407, 249–257 (2000). https://doi.org/10.1038/35025220 Tumor angiogenesis is also a vital sign, suggesting that tumors turn from benign to malignant.1616. D. Hanahan and R. A. Weinberg, Cell 144, 646–674 (2011). https://doi.org/10.1016/j.cell.2011.02.013 Therefore, constructing an in vitro cancer model containing CAFs and self-assembled microvascular networks is critical in investigating the physiological behavior of cancer and testing the accurate preclinical effect of anti-cancer drugs.Most methods for evaluating cancer treatments were first verified in two-dimensional (2D) monolayer models and then in animal models.1717. L. S. Costard, R. R. Hosn, H. Ramanayake, F. J. O’Brien, and C. M. Curtin, Acta Biomater. 132(132), 360–378 (2021). https://doi.org/10.1016/j.actbio.2021.01.023 However, animal models cannot reflect the specific interaction between human cancer cells and blood vessels.1818. J. S. Jeon, S. Bersini, M. Gilardi, G. Dubini, J. L. Charest, M. Moretti, and R. D. Kamm, Proc. Natl. Acad. Sci. U.S.A. 112, 214–219 (2015). https://doi.org/10.1073/pnas.1417115112 2D culture lacks the physical cell–cell interactions of tumors,1919. S. I. Montanez-Sauri, D. J. Beebe, and K. E. Sung, Cell. Mol. Life Sci. 72, 237–249 (2015). https://doi.org/10.1007/s00018-014-1738-5 although it has an advantage in high-throughput drug screening due to its simple implementation.1717. L. S. Costard, R. R. Hosn, H. Ramanayake, F. J. O’Brien, and C. M. Curtin, Acta Biomater. 132(132), 360–378 (2021). https://doi.org/10.1016/j.actbio.2021.01.023 Scientists have gradually realized that simple 2D culture models or animal models are not sufficient enough for complicated tumor metastasis research.2020. S. Roberts, S. Peyman, and V. Speirs, Adv. Exp. Med. Biol. 1152, 413–427 (2019). https://doi.org/10.1007/978-3-030-20301-6_22 3D co-culture models have become important methods for complex vascular simulation2121. N. Reymond, B. B. d’Agua, and A. J. Ridley, Nat. Rev. Cancer 13, 858–870 (2013). https://doi.org/10.1038/nrc3628 and tumor invasion research,2222. S. Khuon, L. Liang, R. W. Dettman, P. H. S. Sporn, R. B. Wysolmerski, and T.-L. Chew, J. Cell Sci. 123, 431–440 (2010). https://doi.org/10.1242/jcs.053793 although their construction still has challenges. The rapid development of microfluidic chip technology has made it possible to build a 3D human organ model on a chip, and a new term, the organ chip, has been derived.2323. A. Sontheimer-Phelps, B. A. Hassell, and D. E. Ingber, Nat. Rev. Cancer 19, 65–81 (2019). https://doi.org/10.1038/s41568-018-0104-6 In the tumor microenvironment in vitro, the construction of a blood vessel structure is the key. There are two methods for constructing blood vessel structure on chips, one was simply attaching human umbilical vein endothelial cells (HUVECs) to the gel wall for growing,24,2524. D. H. T. Nguyen, E. Lee, S. Alimperti, R. J. Norgard, A. Wong, J. J. K. Lee, J. Eyckmans, B. Stanger, and C. S. Chen, Sci. Adv. 5, eaav6789 (2019). https://doi.org/10.1126/sciadv.aav678925. W. J. Polacheck, M. L. Kutys, J. B. Tefft, and C. S. Chen, Nat. Protoc. 14, 1425–1454 (2019). https://doi.org/10.1038/s41596-019-0144-8 and another was forming a blood vessel network through the self-assembly behavior of HUVECs.26–2926. M. B. Chen, J. A. Whisler, J. Froese, C. Yu, Y. J. Shin, and R. D. Kamm, Nat. Protoc. 12, 865–880 (2017). https://doi.org/10.1038/nprot.2017.01827. J. Paek, S. E. Park, Q. Z. Lu, K.-T. Park, M. Cho, J. M. Oh, K. W. Kwon, Y. S. Yi, J. W. Song, H. I. Edelstein, J. Ishibashi, W. L. Yang, J. W. Myerson, R. Y. Kiseleva, P. Aprelev, E. D. Hood, D. Stambolian, P. Seale, V. R. Muzykantov, and D. Huh, ACS Nano 13, 7627–7643 (2019). https://doi.org/10.1021/acsnano.9b0068628. S. Kim, M. Chung, J. Ahn, S. Lee, and N. L. Jeon, Lab Chip 16, 4189–4199 (2016). https://doi.org/10.1039/c6lc00910g29. C. Hajal, L. Ibrahim, J. C. Serrano, G. S. Offeddu, and R. D. Kamm, Biomaterials 265, 120470 (2021). https://doi.org/10.1016/j.biomaterials.2020.120470 The latter is the more natural morphogenesis of endothelial cells.3030. S. Kim, H. Lee, M. Chung, and N. L. Jeon, Lab Chip 13, 1489–1500 (2013). https://doi.org/10.1039/c3lc41320a Kamm et al. introduced several extravasation models of breast cancer containing self-assembled microvascular networks within a bone-mimicking microenvironment1818. J. S. Jeon, S. Bersini, M. Gilardi, G. Dubini, J. L. Charest, M. Moretti, and R. D. Kamm, Proc. Natl. Acad. Sci. U.S.A. 112, 214–219 (2015). https://doi.org/10.1073/pnas.1417115112 or within a physiological flow environment.2929. C. Hajal, L. Ibrahim, J. C. Serrano, G. S. Offeddu, and R. D. Kamm, Biomaterials 265, 120470 (2021). https://doi.org/10.1016/j.biomaterials.2020.120470 Although they provided a usable model for the extravasation study, the stage of tumor development before the formation of circulating tumor cells is more worthy of attention, starting from the perspective of timely treatment of cancer.21,2921. N. Reymond, B. B. d’Agua, and A. J. Ridley, Nat. Rev. Cancer 13, 858–870 (2013). https://doi.org/10.1038/nrc362829. C. Hajal, L. Ibrahim, J. C. Serrano, G. S. Offeddu, and R. D. Kamm, Biomaterials 265, 120470 (2021). https://doi.org/10.1016/j.biomaterials.2020.120470 Nagaraju et al. constructed a breast cancer cell invasion model containing a self-assembled microvascular network to simulate breast cancer cells’ invasion toward the matrix of the primary tumor.3131. S. Nagaraju, D. Truong, G. Mouneimne, and M. Nikkhah, Adv. Healthcare Mater. 7, 1701257 (2018). https://doi.org/10.1002/adhm.201701257 This model provided a potential to study the signal transduction in breast cancer cells and self-assembled vessels, but the vascular structure in this model could not be maintained for a long time (only 6 days) because of the lack of necessary stromal cells, such as fibroblasts.77. R. Kalluri, Nat. Rev. Cancer 16, 582–598 (2016). https://doi.org/10.1038/nrc.2016.73 In addition, this model also ignored the study of vascularization behavior in the process of tumor invasion.

In this study, a mimicking breast cancer invasion model based on microfluidic technology was constructed to study the role of the vascular structure of TME in the process of tumor metastasis. First, fibroblast cells were involved in the TME to explore suitable co-culture strategies to develop and maintain the microvascular networks in vitro. Then, based on the self-assembled microvascular networks, a breast cancer invasion model was constructed to analyze and evaluate tumor metastasis behavior from multiple perspectives, such as cancer cell invasion, tumor angiogenesis, and cancer cell intravasation. Finally, the drug tests were carried out to evaluate the drug resistance differences of the anti-cancer drugs 5-Fluorouracil (5-FU) and Doxorubicin (DOX) between our model and the 2D model. Meanwhile, we also analyzed the effect of the combination of anti-cancer drugs (5-FU and DOX) and anti-angiogenic drug Apatinib (VEGFR inhibitor) on tumor invasion behavior to illustrate the critical role of anti-cancer drug combination strategies in tumor treatment.

II. MATERIALS AND METHODS

Section:

ChooseTop of pageABSTRACTI. INTRODUCTIONII. MATERIALS AND METHODS <<IV. RESULTS AND DISCUSSIO...V. CONCLUSIONSSUPPLEMENTARY MATERIALREFERENCES

A. Device fabrication

The chip structure pattern was designed using Auto CAD software and then using it for manufacturing the mask. According to the standard process, SU-8-2100 (Microchem) was poured on the 3-in. silicon wafer to form a lithography layer (120 μm) at 2500 r/min. Then, the ultraviolet light was shined on the silicon wafer through the mask after the soft bake. The silicon wafer became the mold after postexposure bake, development, rinse, and dry, according to the standard process. Then, polydimethylsiloxane (PDMS, SYLGARD184, Dow Corning) was poured on the mold and was solidified at 80 °C for 1 h. The cured PDMS was cut and punched, and then invisible tapes were used to clean the surface of the PDMS structure, which needed to be bonded. Then, the PDMS structure was bonded to a 24 × 60 mm2 glass sheet with a thickness of 0.2 mm to form the microfluidic chip. After that, the chip was ultrasonically cleaned with 75% alcohol for 10 min and was sterilized by the high temperature and pressure sterilization and the UV irradiation, respectively.

B. Cell culture

Primary human umbilical vein endothelial cells (HUVECs, 8000, ScienCell, <p8) were transduced to stably express fluorescent protein by lentivirus. The HUVECs were cultured in the endothelial growth medium (EGM, CC3162, Lonza) supplemented with 5% fetal bovine serum (10100147, Gibco). The breast cancer cell line MDA-MB-231 cells that were obtained from the Shanghai cell bank of the Chinese Academy of Sciences were cultured in DMEM high glucose medium (C11995500BT, Gibco) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (15140122, Gibco). MDA-MB-231 cells were also infected by lentivirus to express the fluorescent protein for observation of the cell morphology and migration. Primary human mammary cancer-associated fibroblasts (HMCAF, HUM-iCELL-f028, iCELL, <p9) were cultured in a special medium for fibroblasts (PriMed-iCELL-003, iCELL). The above cells were cultured in an incubator at 37 °C, 5% CO2. In the process of cell passaging, cells were trypsinized with 0.25% Trypsin-EDTA (25200072, Gibco) to obtain the cell suspension.

C. Forming pre-existing blood vessels through vasculogenesis

Human fibrinogen (F3879, Sigma) was dissolved in Ca2+ and Mg2+ free DPBS (14190144, Gibco) to prepare a working solution at 8 mg/ml. Thrombin (T4648, Sigma) was dissolved in Ca2+ and Mg2+ free DPBS to obtain a stock solution of 100 U/ml, and then the stock solution was diluted with EGM to make a working solution at 4 U/ml. When loading the cells, HMCAFs were resuspended in thrombin working solution at a cell density of 3 × 106 cells/ml, and then the cell suspension was mixed with a fibrinogen working solution at a ratio of 1:1. The mixture was immediately perfused into the matrix channel of the chip (Fig. 1, Ch2). Waiting for 10 min could ensure the formation of the stable gel in Ch2 and effectively prevent the gel structure in CH2 from causing any damage to the perfusion pressure when loading the HUVERC cells into Ch1. The same method was performed to obtain the stable HUVECs gel in the vessel area of the chip (Fig. 1, Ch1). The fresh medium was replaced into the chips every 24 h.

D. Seeding MDA-MB-231 gel into the self-assembled blood vessel model

At day 3 of developing the self-assembled microvascular network, the medium in the chip was gently aspirated. The MDA-MB-231 cell-thrombin suspensions were mixed with the prepared fibrinogen working solution, and then the mixture was immediately added into the cancer cell channel (Fig. 1, Ch3) to form 3D tumor tissue. The final cell density of MDA-MB-231 cells was 1 × 107 cells/ml, and this vascularized breast cancer model was cultured for 9–11 days. During this period, the growth behaviors of MDA-MB-231 cells, such as cancer cell invasion, intravasation, angiogenesis, were recorded by the microscope.

E. Perfusibility measurement of the self-assembled blood vessel

In order to test the perfusion performance of the self-assembled blood vessel, 70 kDa FITC-Dextran solution (50 μg/ml, 46945, Sigma) was used. A fluorescence microscope (Dmi8, Leica) was used for continuous observation of the tracers. The tip of a pipet was cut and then was inserted into the one side opening module of the chip. Solid acrylic columns were used to block the other outlets on the chip, except for the opening module on the other side. The prepared solution mixed with tracers was loaded into the tip through a syringe to generate a hydrostatic pressure gradient along the vertical line of the blood vessel development area, providing a driving force for the tracer flowing through the blood vessel.

F. Quantitative analysis of microvascular networks and vascular sprouting

The software of Angiotool was used to quantitatively analyze the three development indicators of blood vessels under the same microscope magnification: vessel percentage area, total vessel length, and the total number of junctions.3232. E. Zudaire, L. Gambardella, C. Kurcz, and S. Vermeren, PLoS One 6, e27385 (2011). https://doi.org/10.1371/journal.pone.0027385 Vessel percentage area was the percentage of the blood vessel area in the total area of the culture region, representing the blood vessel density. The total vessel length and the total number of junctions, respectively, referred to the sum of the vessel length and the sum of the vessel branches in the blood vessel network.32,3332. E. Zudaire, L. Gambardella, C. Kurcz, and S. Vermeren, PLoS One 6, e27385 (2011). https://doi.org/10.1371/journal.pone.002738533. M. Segarra, M. R. Aburto, F. Cop, C. Llao-Cid, R. Hartl, M. Damm, I. Bethani, M. Parrilla, D. Husainie, A. Schanzer, H. Schlierbach, T. Acker, L. Mohr, L. Torres-Masjoan, M. Ritter, and A. Acker-Palmer, Science 361, 15 (2018). https://doi.org/10.1126/science.aao2861 S1 in the supplementary material is a schematic diagram of the analysis of blood vessel network pictures using Angiotool in this article. The solid yellow line represents the outline of the blood vessel, and it could be used to calculate the total coverage area of the blood vessel. The green line represents the skeleton of the blood vessel, and it can be used to calculate the total length of the blood vessel. The blue points represent the vessel branches, and it can be used to analyze the total number of junctions. The values of the vessels’ percentage area under different conditions were divided by the valve of the vessels’ percentage area under the control condition at day 2 to get the relative vessels’ percentage area, facilitating a comparison between the values of different conditions. The relative total vessel length and the relative total number of junctions were obtained under the same procedure.Software ImageJ was used to quantify the blood vessel diameter, blood vessel sprout area, and average sprout length under the same microscope magnification.3434. C. A. Schneider, W. S. Rasband, and K. W. Eliceiri, Nat. Methods 9, 671–675 (2012). https://doi.org/10.1038/nmeth.2089 S2(A) in the supplementary material was a schematic diagram for calculating the angiogenic sprout length of blood vessels using ImageJ. The length of all the dashed green lines was averaged, representing the average length of sprout blood vessels. S2(B) in the supplementary material is a schematic diagram for calculating the angiogenic sprout area of the sprout blood vessel in the yellow dashed box using ImageJ.

G. Immunofluorescence

All liquids are loaded into the chip medium channel. First, excess PBS solution was added into the chips to wash the cells, and then 4% paraformaldehyde was used to perform the fixing process for 20 min at 4 °C. Then, the cells were washed with PBS. After 30 min of permeabilization with 0.1% Triton X-100 solution, the cells were washed again with PBS. A blocking step was then performed using 5% BSA solution (or 4% goat serum solution) for 3 h at room temperature (RT), and then the primary antibody working solution was added into the chip, incubating cells overnight at 4 °C. For vascular barrier markers, mouse monoclonal anti-CD31 (1:100, Ab24590, Abcam) and mouse monoclonal anti-VE-Cadherin (5 μg/ml, 14-1449-82, eBioscience™) were used to detect the expressions of the corresponding proteins. Rabbit monoclonal to anti-FAP (1:100, 66562S, CST) were used to analyze the expression of HMCAF markers. For the cytoskeleton, we used Phalloidin-iFluor 555 conjugate (1:1000, ab176755, Abcam) to incubate cells at RT for 1 h. After incubating cells with the primary antibody, the cells were washed with PBS and were incubated with the corresponding appropriate secondary antibody (A 32731, ThermoFisher; or A 32727, ThermoFisher; or Ab 150113, Abcam) for 1 h according to the added primary antibody species. Then, the cells were washed again with PBS and were incubated with for DAPI (1:500, C1002, Beyotime) for 20 min. Finally, the cells were washed with PBS and were observed under a fluorescence microscope for taking pictures.

H. CCK8 assay and live/dead assay

MDA-MB-231 cells were treated with various concentrations of 5-FU and DOX for 24 or 48 h in a 96-well plate. Then, the cells were incubated in a serum-free medium supplemented with 10% CCK8 solution (Abs 50003, Absin) for 2 h at 37 °C. After shaking the 96-well plate for 5 min, the optical density was measured at 450 nm with a microplate reader.

The Live/Dead Cell Imaging Kit (Molecular Probes, Life Technologies Corporation) was used to evaluate the cell viability of MDA-MB-231 cells treated with 5-FU and DOX for 24 or 48 h on a chip. First, PBS was used to wash the chip for 1–3 min. Then, the cells were incubated with the Live/Dead Cell Imaging Kit for 15–30 min at 37 °C. Finally, we used PBS again to wash out the reagent for 3–5 min and observed the chip under a fluorescent microscope.

I. Statistical analysis

For statistical analysis, all values were obtained from three independent experiments (devices), and the data were expressed as mean ± standard deviation. Values, such as cell number, migration distance, and vessel diameter, were obtained from the fluorescent images by using the image processing and data statistics functions of the ImageJ software and the Angiotool software. Origin software was used to evaluate significant differences by performing unpaired two-tailed t-tests, * means p < 0.05, ** means p < 0.01, *** means p < 0.001, **** means p < 0.0001, and n means the number of independent replicate experiments.

V. CONCLUSIONS

Section:

ChooseTop of pageABSTRACTI. INTRODUCTIONII. MATERIALS AND METHODSIV. RESULTS AND DISCUSSIO...V. CONCLUSIONS <<SUPPLEMENTARY MATERIALREFERENCES

In this work, we developed a biomimetic breast cancer model containing self-assembled microvascular networks and the HMCAF matrix. The decisive role of primary vascular endothelial cells in the development of microvascular networks in vitro has been proved, and the HMCAF cells played an important role in maintaining the structure and function of the microvascular networks for a long time. Therefore, the blood vessel network developed in our work has achieved the morphology and function maintenance for up to 13 days, which was much higher than the reported time of the existing studies. In our self-assembled vascularized breast cancer model, the invasion behavior of tumor cells and the phenomenon of tumor vascularization occurred at the same time, and the results of related quantitative analysis also showed that the two promote each other. We also observed that the intravasation behavior already existed in the early stage of tumor invasion. This suggests that, although these intravasation cancer cells were unable to enter the main blood vessel channel, due to the sufficient blood nutrient supply, their proliferation behavior in blood vessels may play an important role in the process of tumor metastasis. In the drug test, the drug effects were assessed from the aspects of the cancer cell invasion behavior, the vascular endothelial behavior, and cell viability. The results showed that this cancer model had more consistent drug resistance with the human body, compared with the 2D models. Meanwhile, the anti-angiogenic drug Apatinib enhanced the treatment of DOX on breast cancer cells, instead indicating that the combination strategy of anti-cancer drugs and anti-angiogenic drugs can become an important means of cancer treatment. In general, our research provided a more bionic and quantitatively analyzable model for the construction of in vitro breast cancer models and the testing of anti-cancer drugs.

ACKNOWLEDGMENTS

This work was funded by the Project of Basic Research of Shenzhen, China (Nos. JCYJ20180507183655307 and JCYJ20190813143221901). Shengli Mi received the funding. The funders had no role in the study design, data collection, and analysis and in the decision on the publication or preparation of the manuscript.