3D bioprinting for reconstituting the cancer microenvironment.

The cancer microenvironment is known for its complexity, both in its content as well as its dynamic nature, which is difficult to study using two-dimensional (2D) cell culture models. Several advances in tissue engineering have allowed more physiologically relevant three-dimensional (3D) in vitro cancer models, such as spheroid cultures, biopolymer scaffolds, and cancer-on-a-chip devices. Although these models serve as powerful tools for dissecting the roles of various biochemical and biophysical cues in carcinoma initiation and progression, they lack the ability to control the organization of multiple cell types in a complex dynamic 3D architecture. By virtue of its ability to precisely define perfusable networks and position of various cell types in a high-throughput manner, 3D bioprinting has the potential to more closely recapitulate the cancer microenvironment, relative to current methods. In this review, we discuss the applications of 3D bioprinting in mimicking cancer microenvironment, their use in immunotherapy as prescreening tools, and overview of current bioprinted cancer models

Rebuilding the hematopoietic stem cell niche: Recent developments and future prospects

Hematopoietic stem cells (HSCs) have proven their clinical relevance in stem cell transplantation to cure patients with hematological disorders. Key to their regenerative potential is their natural microenvironment – their niche – in the bone marrow (BM). Developments in the field of biomaterials enable the recreation of such environments with increasing preciseness in the laboratory. Such artificial niches help to gain a fundamental understanding of the biophysical and biochemical processes underlying the interaction of HSCs with the materials in their environment and the disturbance of this interplay during diseases affecting the BM. Artificial niches also have the potential to multiply HSCs in vitro, to enable the targeted differentiation of HSCs into mature blood cells or to serve as drug-testing platforms. In this review, we will introduce the importance of artificial niches followed by the biology and biophysics of the natural archetype. We will outline how 2D biomaterials can be used to dissect the complexity of the natural niche into individual parameters for fundamental research and how 3D systems evolved from them. We will present commonly used biomaterials for HSC research and their applications. Finally, we will highlight two areas in the field of HSC research, which just started to unlock the possibilities provided by novel biomaterials, in vitro blood production and studying the pathophysiology of the niche in vitro. With these contents, the review aims to give a broad overview of the different biomaterials applied for HSC research and to discuss their potentials, challenges and future directions in the field. Statement of significance Hematopoietic stem cells (HSCs) are multipotent cells responsible for maintaining the turnover of all blood cells. They are routinely applied to treat patients with hematological diseases. This high clinical relevance explains the necessity of multiplication or differentiation of HSCs in the laboratory, which is hampered by the missing natural microenvironment – the so called niche. Biomaterials offer the possibility to mimic the niche and thus overcome this hurdle. The review introduces the HSC niche in the bone marrow and discusses the utility of biomaterials in creating artificial niches. It outlines how 2D systems evolved into sophisticated 3D platforms, which opened the gateway to applications such as, expansion of clinically relevant HSCs, in vitro blood production, studying niche pathologies and drug testing

In Vitro Modeling of Non-Solid Tumors: How Far Can Tissue Engineering Go?

[EN] In hematological malignancies, leukemias or myelomas, malignant cells present bone marrow (BM) homing, in which the niche contributes to tumor development and drug resistance. BM architecture, cellular and molecular composition and interactions define differential microenvironments that govern cell fate under physiological and pathological conditions and serve as a reference for the native biological landscape to be replicated in engineered platforms attempting to reproduce blood cancer behavior. This review summarizes the different models used to efficiently reproduce certain aspects of BM in vitro; however, they still lack the complexity of this tissue, which is relevant for fundamental aspects such as drug resistance development in multiple myeloma. Extracellular matrix composition, material topography, vascularization, cellular composition or stemness vs. differentiation balance are discussed as variables that could be rationally defined in tissue engineering approaches for achieving more relevant in vitro models. Fully humanized platforms closely resembling natural interactions still remain challenging and the question of to what extent accurate tissue complexity reproduction is essential to reliably predict drug responses is controversial. However, the contributions of these approaches to the fundamental knowledge of non-solid tumor biology, its regulation by niches, and the advance of personalized medicine are unquestionable.PROMETEO/2016/063 project is acknowledged. The CIBER-BBN initiative is funded by the VI National R&D&I Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program. CIBER actions are financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. This work was also supported by the Spanish Ministry of Science, Innovation and Universities through Grant FPU17/05810 awarded to Sandra Clara-Trujillo.Clara-Trujillo, S.; Gallego Ferrer, G.; Gómez Ribelles, JL. (2020). In Vitro Modeling of Non-Solid Tumors: How Far Can Tissue Engineering Go?. International Journal of Molecular Sciences. 21(16):1-31. https://doi.org/10.3390/ijms21165747S1312116Langer, R., & Vacanti, J. P. (1993). Tissue Engineering. Science, 260(5110), 920-926. doi:10.1126/science.8493529Kelm, J. M., Lal-Nag, M., Sittampalam, G. S., & Ferrer, M. (2019). Translational in vitro research: integrating 3D drug discovery and development processes into the drug development pipeline. Drug Discovery Today, 24(1), 26-30. doi:10.1016/j.drudis.2018.07.007Pradhan, S., Hassani, I., Clary, J. M., & Lipke, E. A. (2016). Polymeric Biomaterials for In Vitro Cancer Tissue Engineering and Drug Testing Applications. Tissue Engineering Part B: Reviews, 22(6), 470-484. doi:10.1089/ten.teb.2015.0567Khetani, S. R., & Bhatia, S. N. (2006). Engineering tissues for in vitro applications. Current Opinion in Biotechnology, 17(5), 524-531. doi:10.1016/j.copbio.2006.08.009Gomes, M. E., Rodrigues, M. T., Domingues, R. M. A., & Reis, R. L. (2017). Tissue Engineering and Regenerative Medicine: New Trends and Directions—A Year in Review. Tissue Engineering Part B: Reviews, 23(3), 211-224. doi:10.1089/ten.teb.2017.0081Wang, Z., Lee, S. J., Cheng, H.-J., Yoo, J. J., & Atala, A. (2018). 3D bioprinted functional and contractile cardiac tissue constructs. Acta Biomaterialia, 70, 48-56. doi:10.1016/j.actbio.2018.02.007Tsukamoto, Y., Akagi, T., & Akashi, M. (2020). Vascularized cardiac tissue construction with orientation by layer-by-layer method and 3D printer. Scientific Reports, 10(1). doi:10.1038/s41598-020-59371-yVan Grunsven, L. A. (2017). 3D in vitro models of liver fibrosis. Advanced Drug Delivery Reviews, 121, 133-146. doi:10.1016/j.addr.2017.07.004Griffith, L. G., & Swartz, M. A. (2006). Capturing complex 3D tissue physiology in vitro. Nature Reviews Molecular Cell Biology, 7(3), 211-224. doi:10.1038/nrm1858Schenke-Layland, K., & Nerem, R. M. (2011). In vitro human tissue models — moving towards personalized regenerative medicine. Advanced Drug Delivery Reviews, 63(4-5), 195-196. doi:10.1016/j.addr.2011.05.001Dagogo-Jack, I., & Shaw, A. T. (2017). Tumour heterogeneity and resistance to cancer therapies. Nature Reviews Clinical Oncology, 15(2), 81-94. doi:10.1038/nrclinonc.2017.166Wright, W. E., & Shay, J. W. (2000). Telomere dynamics in cancer progression and prevention: fundamental differences in human and mouse telomere biology. Nature Medicine, 6(8), 849-851. doi:10.1038/78592Brancato, V., Oliveira, J. M., Correlo, V. M., Reis, R. L., & Kundu, S. C. (2020). Could 3D models of cancer enhance drug screening? Biomaterials, 232, 119744. doi:10.1016/j.biomaterials.2019.119744Riedl, A., Schlederer, M., Pudelko, K., Stadler, M., Walter, S., Unterleuthner, D., … Dolznig, H. (2016). Comparison of cancer cells cultured in 2D vs 3D reveals differences in AKT/mTOR/S6-kinase signaling and drug response. Journal of Cell Science. doi:10.1242/jcs.188102Wu, T., & Dai, Y. (2017). Tumor microenvironment and therapeutic response. Cancer Letters, 387, 61-68. doi:10.1016/j.canlet.2016.01.043Håkanson, M., Cukierman, E., & Charnley, M. (2014). Miniaturized pre-clinical cancer models as research and diagnostic tools. Advanced Drug Delivery Reviews, 69-70, 52-66. doi:10.1016/j.addr.2013.11.010Radhakrishnan, J., Varadaraj, S., Dash, S. K., Sharma, A., & Verma, R. S. (2020). Organotypic cancer tissue models for drug screening: 3D constructs, bioprinting and microfluidic chips. Drug Discovery Today, 25(5), 879-890. doi:10.1016/j.drudis.2020.03.002Broutier, L., Mastrogiovanni, G., Verstegen, M. M., Francies, H. E., Gavarró, L. M., Bradshaw, C. R., … Huch, M. (2017). Human primary liver cancer–derived organoid cultures for disease modeling and drug screening. Nature Medicine, 23(12), 1424-1435. doi:10.1038/nm.4438Drost, J., & Clevers, H. (2018). Organoids in cancer research. Nature Reviews Cancer, 18(7), 407-418. doi:10.1038/s41568-018-0007-6Angeloni, V., Contessi, N., De Marco, C., Bertoldi, S., Tanzi, M. C., Daidone, M. G., & Farè, S. (2017). Polyurethane foam scaffold as in vitro model for breast cancer bone metastasis. Acta Biomaterialia, 63, 306-316. doi:10.1016/j.actbio.2017.09.017Kim, M. J., Chi, B. H., Yoo, J. J., Ju, Y. M., Whang, Y. M., & Chang, I. H. (2019). Structure establishment of three-dimensional (3D) cell culture printing model for bladder cancer. PLOS ONE, 14(10), e0223689. doi:10.1371/journal.pone.0223689Carvalho, M. R., Barata, D., Teixeira, L. M., Giselbrecht, S., Reis, R. L., Oliveira, J. M., … Habibovic, P. (2019). Colorectal tumor-on-a-chip system: A 3D tool for precision onco-nanomedicine. Science Advances, 5(5). doi:10.1126/sciadv.aaw1317Paolillo, Colombo, Serra, Belvisi, Papetti, Ciusani, … Schinelli. (2019). Stem-like Cancer Cells in a Dynamic 3D Culture System: A Model to Study Metastatic Cell Adhesion and Anti-cancer Drugs. Cells, 8(11), 1434. doi:10.3390/cells8111434Lichtman, M. A. (2008). Battling the Hematological Malignancies: The 200 Years’ War. The Oncologist, 13(2), 126-138. doi:10.1634/theoncologist.2007-0228Jagannathan-Bogdan, M., & Zon, L. I. (2013). Hematopoiesis. Development, 140(12), 2463-2467. doi:10.1242/dev.083147Rieger, M. A., & Schroeder, T. (2012). Hematopoiesis. Cold Spring Harbor Perspectives in Biology, 4(12), a008250-a008250. doi:10.1101/cshperspect.a008250Harris, N. L., Jaffe, E. S., Diebold, J., Flandrin, G., Muller-Hermelink, H. K., Vardiman, J., … Bloomfield, C. D. (2000). The World Health Organization Classification of Neoplasms of the Hematopoietic and Lymphoid Tissues: Report of the Clinical Advisory Committee Meeting – Airlie House, Virginia, November, 1997. The Hematology Journal, 1(1), 53-66. doi:10.1038/sj.thj.6200013Arber, D. A., Orazi, A., Hasserjian, R., Thiele, J., Borowitz, M. J., Le Beau, M. M., … Vardiman, J. W. (2016). The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood, 127(20), 2391-2405. doi:10.1182/blood-2016-03-643544Palumbo, A., & Anderson, K. (2011). Multiple Myeloma. New England Journal of Medicine, 364(11), 1046-1060. doi:10.1056/nejmra1011442Méndez-Ferrer, S., Bonnet, D., Steensma, D. P., Hasserjian, R. P., Ghobrial, I. M., Gribben, J. G., … Krause, D. S. (2020). Bone marrow niches in haematological malignancies. Nature Reviews Cancer, 20(5), 285-298. doi:10.1038/s41568-020-0245-2Kumar, R., Godavarthy, P. S., & Krause, D. S. (2018). The bone marrow microenvironment in health and disease at a glance. Journal of Cell Science, 131(4). doi:10.1242/jcs.201707Galán-Díez, M., Cuesta-Domínguez, Á., & Kousteni, S. (2017). The Bone Marrow Microenvironment in Health and Myeloid Malignancy. Cold Spring Harbor Perspectives in Medicine, 8(7), a031328. doi:10.1101/cshperspect.a031328Itkin, T., Gur-Cohen, S., Spencer, J. A., Schajnovitz, A., Ramasamy, S. K., Kusumbe, A. P., … Lapidot, T. (2016). Distinct bone marrow blood vessels differentially regulate haematopoiesis. Nature, 532(7599), 323-328. doi:10.1038/nature17624Morikawa, T., & Takubo, K. (2017). Use of Imaging Techniques to Illuminate Dynamics of Hematopoietic Stem Cells and Their Niches. Frontiers in Cell and Developmental Biology, 5. doi:10.3389/fcell.2017.00062Galán-Díez, M., & Kousteni, S. (2017). The Osteoblastic Niche in Hematopoiesis and Hematological Myeloid Malignancies. Current Molecular Biology Reports, 3(2), 53-62. doi:10.1007/s40610-017-0055-9Klamer, S., & Voermans, C. (2014). The role of novel and known extracellular matrix and adhesion molecules in the homeostatic and regenerative bone marrow microenvironment. Cell Adhesion & Migration, 8(6), 563-577. doi:10.4161/19336918.2014.968501Walkley, C. R., Shea, J. M., Sims, N. A., Purton, L. E., & Orkin, S. H. (2007). Rb Regulates Interactions between Hematopoietic Stem Cells and Their Bone Marrow Microenvironment. Cell, 129(6), 1081-1095. doi:10.1016/j.cell.2007.03.055Walkley, C. R., Olsen, G. H., Dworkin, S., Fabb, S. A., Swann, J., McArthur, G. A., … Purton, L. E. (2007). A Microenvironment-Induced Myeloproliferative Syndrome Caused by Retinoic Acid Receptor γ Deficiency. Cell, 129(6), 1097-1110. doi:10.1016/j.cell.2007.05.014Xie, M., Lu, C., Wang, J., McLellan, M. D., Johnson, K. J., Wendl, M. C., … Ding, L. (2014). Age-related mutations associated with clonal hematopoietic expansion and malignancies. Nature Medicine, 20(12), 1472-1478. doi:10.1038/nm.3733Jaiswal, S., Fontanillas, P., Flannick, J., Manning, A., Grauman, P. V., Mar, B. G., … Ebert, B. L. (2014). Age-Related Clonal Hematopoiesis Associated with Adverse Outcomes. New England Journal of Medicine, 371(26), 2488-2498. doi:10.1056/nejmoa1408617Genovese, G., Kähler, A. K., Handsaker, R. E., Lindberg, J., Rose, S. A., Bakhoum, S. F., … McCarroll, S. A. (2014). Clonal Hematopoiesis and Blood-Cancer Risk Inferred from Blood DNA Sequence. New England Journal of Medicine, 371(26), 2477-2487. doi:10.1056/nejmoa1409405Sala-Torra, O., Hanna, C., Loken, M. R., Flowers, M. E. D., Maris, M., Ladne, P. A., … Radich, J. P. (2006). Evidence of Donor-Derived Hematologic Malignancies after Hematopoietic Stem Cell Transplantation. Biology of Blood and Marrow Transplantation, 12(5), 511-517. doi:10.1016/j.bbmt.2006.01.006Ghosh, A. K., Secreto, C. R., Knox, T. R., Ding, W., Mukhopadhyay, D., & Kay, N. E. (2010). Circulating microvesicles in B-cell chronic lymphocytic leukemia can stimulate marrow stromal cells: implications for disease progression. Blood, 115(9), 1755-1764. doi:10.1182/blood-2009-09-242719Zhang, B., Chu, S., Agarwal, P., Campbell, V. L., Hopcroft, L., Jørgensen, H. G., … Bhatia, R. (2016). Inhibition of interleukin-1 signaling enhances elimination of tyrosine kinase inhibitor–treated CML stem cells. Blood, 128(23), 2671-2682. doi:10.1182/blood-2015-11-679928Schepers, K., Pietras, E. M., Reynaud, D., Flach, J., Binnewies, M., Garg, T., … Passegué, E. (2013). Myeloproliferative Neoplasia Remodels the Endosteal Bone Marrow Niche into a Self-Reinforcing Leukemic Niche. Cell Stem Cell, 13(3), 285-299. doi:10.1016/j.stem.2013.06.009Hawkins, E. D., Duarte, D., Akinduro, O., Khorshed, R. A., Passaro, D., Nowicka, M., … Lo Celso, C. (2016). T-cell acute leukaemia exhibits dynamic interactions with bone marrow microenvironments. Nature, 538(7626), 518-522. doi:10.1038/nature19801Paggetti, J., Haderk, F., Seiffert, M., Janji, B., Distler, U., Ammerlaan, W., … Moussay, E. (2015). Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts. Blood, 126(9), 1106-1117. doi:10.1182/blood-2014-12-618025Arranz, L., Sánchez-Aguilera, A., Martín-Pérez, D., Isern, J., Langa, X., Tzankov, A., … Méndez-Ferrer, S. (2014). Neuropathy of haematopoietic stem cell niche is essential for myeloproliferative neoplasms. Nature, 512(7512), 78-81. doi:10.1038/nature13383Dias, S., Hattori, K., Zhu, Z., Heissig, B., Choy, M., Lane, W., … Rafii, S. (2000). Autocrine stimulation of VEGFR-2 activates human leukemic cell growth and migration. Journal of Clinical Investigation, 106(4), 511-521. doi:10.1172/jci8978Warburg, O. (1956). On the Origin of Cancer Cells. Science, 123(3191), 309-314. doi:10.1126/science.123.3191.309Kreitz, J., Schönfeld, C., Seibert, M., Stolp, V., Alshamleh, I., Oellerich, T., … Serve, H. (2019). Metabolic Plasticity of Acute Myeloid Leukemia. Cells, 8(8), 805. doi:10.3390/cells8080805Lagadinou, E. D., Sach, A., Callahan, K. P., Rossi, R. M., Neering, S., Pei, S., … Jordan, C. T. (2012). Bcl-2 Inhibitor ABT-263 Targets Oxidative Phosphorylation and Selectively Eradicates Quiescent Human Leukemia Stem Cells. Blood, 120(21), 206-206. doi:10.1182/blood.v120.21.206.206Lutzny, G., Kocher, T., Schmidt-Supprian, M., Rudelius, M., Klein-Hitpass, L., Finch, A. J., … Ringshausen, I. (2013). Protein Kinase C-β-Dependent Activation of NF-κB in Stromal Cells Is Indispensable for the Survival of Chronic Lymphocytic Leukemia B Cells In Vivo. Cancer Cell, 23(1), 77-92. doi:10.1016/j.ccr.2012.12.003Yao, J.-C., & Link, D. C. (2016). Concise Review: The Malignant Hematopoietic Stem Cell Niche. STEM CELLS, 35(1), 3-8. doi:10.1002/stem.2487Spaggiari, G. M., Capobianco, A., Abdelrazik, H., Becchetti, F., Mingari, M. C., & Moretta, L. (2008). Mesenchymal stem cells inhibit natural killer–cell proliferation, cytotoxicity, and cytokine production: role of indoleamine 2,3-dioxygenase and prostaglandin E2. Blood, 111(3), 1327-1333. doi:10.1182/blood-2007-02-074997Jin, L., Hope, K. J., Zhai, Q., Smadja-Joffe, F., & Dick, J. E. (2006). Targeting of CD44 eradicates human acute myeloid leukemic stem cells. Nature Medicine, 12(10), 1167-1174. doi:10.1038/nm1483Krause, D. S., Lazarides, K., von Andrian, U. H., & Van Etten, R. A. (2006). Requirement for CD44 in homing and engraftment of BCR-ABL–expressing leukemic stem cells. Nature Medicine, 12(10), 1175-1180. doi:10.1038/nm1489Azab, A. K., Runnels, J. M., Pitsillides, C., Moreau, A.-S., Azab, F., Leleu, X., … Ghobrial, I. M. (2009). CXCR4 inhibitor AMD3100 disrupts the interaction of multiple myeloma cells with the bone marrow microenvironment and enhances their sensitivity to therapy. Blood, 113(18), 4341-4351. doi:10.1182/blood-2008-10-186668Jacamo, R., Chen, Y., Wang, Z., Ma, W., Zhang, M., Spaeth, E. L., … Andreeff, M. (2014). Reciprocal leukemia-stroma VCAM-1/VLA-4-dependent activation of NF-κB mediates chemoresistance. Blood, 123(17), 2691-2702. doi:10.1182/blood-2013-06-511527Hatano, K., Kikuchi, J., Takatoku, M., Shimizu, R., Wada, T., Ueda, M., … Ozawa, K. (2008). Bortezomib overcomes cell adhesion-mediated drug resistance through downregulation of VLA-4 expression in multiple myeloma. Oncogene, 28(2), 231-242. doi:10.1038/onc.2008.385Bourgine, P. E., Martin, I., & Schroeder, T. (2018). Engineering Human Bone Marrow Proxies. Cell Stem Cell, 22(3), 298-301. doi:10.1016/j.stem.2018.01.002Chramiec, A., & Vunjak-Novakovic, G. (2019). Tissue engineered models of healthy and malignant human bone marrow. Advanced Drug Delivery Reviews, 140, 78-92. doi:10.1016/j.addr.2019.04.003Tavakol, D. N., Tratwal, J., Bonini, F., Genta, M., Campos, V., Burch, P., … Braschler, T. (2020). Injectable, scalable 3D tissue-engineered model of marrow hematopoiesis. Biomaterials, 232, 119665. doi:10.1016/j.biomaterials.2019.119665Isern, J., Martín-Antonio, B., Ghazanfari, R., Martín, A. M., López, J. A., del Toro, R., … Méndez-Ferrer, S. (2013). Self-Renewing Human Bone Marrow Mesenspheres Promote Hematopoietic Stem Cell Expansion. Cell Reports, 3(5), 1714-1724. doi:10.1016/j.celrep.2013.03.041Jing, D., Fonseca, A. V., Alakel, N., Fierro, F. A., Muller, K., Bornhauser, M., … Ordemann, R. (2010). Hematopoietic stem cells in co-culture with mesenchymal stromal cells - modeling the niche compartments in vitro. Haematologica, 95(4), 542-550. doi:10.3324/haematol.2009.010736Butler, J. M., Gars, E. J., James, D. J., Nolan, D. J., Scandura, J. M., & Rafii, S. (2012). Development of a vascular niche platform for expansion of repopulating human cord blood stem and progenitor cells. Blood, 120(6), 1344-1347. doi:10.1182/blood-2011-12-398115Leisten, I., Kramann, R., Ventura Ferreira, M. S., Bovi, M., Neuss, S., Ziegler, P., … Schneider, R. K. (2012). 3D co-culture of hematopoietic stem and progenitor cells and mesenchymal stem cells in collagen scaffolds as a model of the hematopoietic niche. Biomaterials, 33(6), 1736-1747. doi:10.1016/j.biomaterials.2011.11.034Raic, A., Rödling, L., Kalbacher, H., & Lee-Thedieck, C. (2014). Biomimetic macroporous PEG hydrogels as 3D scaffolds for the multiplication of human hematopoietic stem and progenitor cells. Biomaterials, 35(3), 929-940. doi:10.1016/j.biomaterials.2013.10.038Severn, C. E., Macedo, H., Eagle, M. J., Rooney, P., Mantalaris, A., & Toye, A. M. (2016). Polyurethane scaffolds seeded with CD34+ cells maintain early stem cells whilst also facilitating prolonged egress of haematopoietic progenitors. Scientific Reports, 6(1). doi:10.1038/srep32149Mahadik, B. P., Bharadwaj, N. A. K., Ewoldt, R. H., & Harley, B. A. C. (2017). Regulating dynamic signaling between hematopoietic stem cells and niche cells via a hydrogel matrix. Biomaterials, 125, 54-64. doi:10.1016/j.biomaterials.2017.02.013Wilkinson, A. C., Ishida, R., Kikuchi, M., Sudo, K., Morita, M., Crisostomo, R. V., … Yamazaki, S. (2019). Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation. Nature, 571(7763), 117-121. doi:10.1038/s41586-019-1244-xSieber, S., Wirth, L., Cavak, N., Koenigsmark, M., Marx, U., Lauster, R., & Rosowski, M. (2017). Bone marrow‐on‐a‐chip: Long‐term culture of human haematopoietic stem cells in a three‐dimensional microfluidic environment. Journal of Tissue Engineering and Regenerative Medicine, 12(2), 479-489. doi:10.1002/term.2507Bourgine, P. E., Klein, T., Paczulla, A. M., Shimizu, T., Kunz, L., Kokkaliaris, K. D., … Martin, I. (2018). In vitro biomimetic engineering of a human hematopoietic niche with functional properties. Proceedings of the National Academy of Sciences, 115(25), E5688-E5695. doi:10.1073/pnas.1805440115De la Puente, P., Muz, B., Gilson, R. C., Azab, F., Luderer, M., King, J., … Azab, A. K. (2015). 3D tissue-engineered bone marrow as a novel model to study pathophysiology and drug resistance in multiple myeloma. Biomaterials, 73, 70-84. doi:10.1016/j.biomaterials.2015.09.017Torisawa, Y., Spina, C. S., Mammoto, T., Mammoto, A., Weaver, J. C., Tat, T., … Ingber, D. E. (2014). Bone marrow–on–a–chip replicates hematopoietic niche physiology in vitro. Nature Methods, 11(6), 663-669. doi:10.1038/nmeth.2938Reinisch, A., Hernandez, D. C., Schallmoser, K., & Majeti, R. (2017). Generation and use of a humanized bone-marrow-ossicle niche for hematopoietic xenotransplantation into mice. Nature Protocols, 12(10), 2169-2188. doi:10.1038/nprot.2017.088Theocharides, A. P. A., Rongvaux, A., Fritsch, K., Flavell, R. A., & Manz, M. G. (2015). Humanized hemato-lymphoid system mice. Haematologica, 101(1), 5-19. doi:10.3324/haematol.2014.115212Abarrategi, A., Mian, S. A., Passaro, D., Rouault-Pierre, K., Grey, W., & Bonnet, D. (2018). Modeling the human bone marrow niche in mice: From host bone marrow engraftment to bioengineering approaches. Journal of Experimental Medicine, 215(3), 729-743. doi:10.1084/jem.20172139Rose-Zerilli, M. J. J., Gibson, J., Wang, J., Tapper, W., Davis, Z., Parker, H., … Strefford, J. C. (2016). Longitudinal copy number, whole exome and targeted deep sequencing of «good risk» IGHV-mutated CLL patients with progressive disease. Leukemia, 30(6), 1301-1310. doi:10.1038/leu.2016.10Reinisch, A., Thomas, D., Corces, M. R., Zhang, X., Gratzinger, D., Hong, W.-J., … Majeti, R. (2016). A humanized bone marrow ossicle xenotransplantation model enables improved engraftment of healthy and leukemic human hematopoietic cells. Nature Medicine, 22(7), 812-821. doi:10.1038/nm.4103Vaiselbuh, S. R., Edelman, M., Lipton, J. M., & Liu, J. M. (2010). Ectopic Human Mesenchymal Stem Cell-Coated Scaffolds in NOD/SCID Mice: An In Vivo Model of the Leukemia Niche. Tissue Engineering Part C: Methods, 16(6), 1523-1531. doi:10.1089/ten.tec.2010.0179Groen, R. W. J., Noort, W. A., Raymakers, R. A., Prins, H.-J., Aalders, L., Hofhuis, F. M., … Martens, A. C. M. (2012). Reconstructing the human hematopoietic niche in immunodeficient mice: opportunities for studying primary multiple myeloma. Blood, 120(3), e9-e16. doi:10.1182/blood-2012-03-414920Chen, Y., Jacamo, R., Shi, Y., Wang, R., Battula, V. L., Konoplev, S., … Andreeff, M. (2012). Human extramedullary bone marrow in mice: a novel in vivo model of genetically controlled hematopoietic microenvironment. Blood, 119(21), 4971-4980. doi:10.1182/blood-2011-11-389957Holzapfel, B. M., Hutmacher, D. W., Nowlan, B., Barbier, V., Thibaudeau, L., Theodoropoulos, C., … Levesque, J.-P. (2015). Tissue enginee

Tuneable 3D biocompatible scaffolds for biological and biophysical solid-tumour microenvironment studies; applications in Ovarian Cancer

Recently, three-dimensional (3D) tumour models mimicking the tumour microenvironment and reducing the use of experimental animals have been developed generating great interest to appraise tumour response to treatment strategies in cancer therapy. As tumours have distinct mechanics compared to normal tissues, biomaterials have also been utilized in 3D culture to model the mechanical properties of the tumour microenvironment, and to study the effects of extracellular matrix (ECM) mechanics on tumour development and progression. Mechanical cues regulate various cell behaviours through mechanotransduction, including proliferation, migration, and differentiation. In the context of cancer, both stromal cells (cancer associated fibroblasts) and tumour cells remodel the ECM and change its mechanical properties, and the altered mechanical niche in turn is likely to influence tumour progression. In this study, bovine derived collagen type I and Jellyfish derived marine collagen sources, were tested as biomaterial candidates for cancer studies, moulded to porous scaffolds with tuneable mechanical properties. The resulting interconnected network of collagen fibre constructs, fabricated using lyophilisation provide good control of scaffolding architecture, pore sizes range, high porosity levels, high level of cell viability and low production cost. Importantly these sponge scaffolds were, in the form of 3D models, compatible with a host of cellular and molecular biology assays used to investigate mechanical and biological effects of collagen crosslinking and (hyaluronic acid) HA inclusion on both fibroblasts and ovarian cancer cells. Stromal cells and cancer cells respond differently to the altered stiffness of their local microenvironment. Fibroblasts, once activated with TGF1, converge toward a ‘senescent-like phenotype’, blocking migration and matrix remodelling and promote tumour progression, probably through the secretion of tumour-promoting signals, in stiffer mechanical environments. Cancer cells, of both epithelial and mesenchymal phenotype, respond to increased local matrix stiffness by increasing proliferation while, at the same time, becoming more susceptible to treatment. Mechanically informative scaffolds resemble the physical characteristics of both normal and pathological ovarian tissue mechanics, where ovarian cancer originates. Physical changes observed in the later stage of ovarian cancer disease progression may therefore be fundamental for the increased cancer proliferation that drives metastatic progression, however opening an interesting window for cancer treatment. Bio-physical inclusive models not only lead the path to unveil complex interactions of biophysical and biological signals in the tumour microenvironment, but they represent a highly informative and effective platform to test novel target therapies with effective costs and high throughput. They can accommodate coculture systems and potentially patients-derived cell cultures, providing a platform to test current and new drugs and to evaluate drug efficacy following a precision medicine approach

Microfluidic studies for monitoring the metastatic cascade and cancer-immune cells interaction

Metastases are the primary cause of death in cancer patients. Small animal models are helping dissecting some key features of the metastatic cascade but many bio-mechanic details remains difficult to analyze in vivo. For this reason a series of tools for performing systematic analysis of vascular permeability, tissue architecture, blood flow, biochemical stimuli and inflammation were produced in the last decade. Particularly relevant for this field is the use of microfluidic chips allowing to include in vitro models a vascular component. During my PhD, I applied this novel technologies to replicate in vitro key steps in the metastatic cascade and cancer-immune cell interaction with a focus on the establishment of microfluidics for metastasis. More specifically I used 3 different microfluidic chips: i) a single-channel microfluidic chip allowing to study CTCs adhesion and rolling inside a small capillary; ii) a double-channel microfluidic chip, composed by an upper and a lower channels mimicking the vascular and extravascular compartments; the channels are laterally connected by an array of micro pillars acting as a vascular membrane; iii) a three channel device composed by a central 3D culture of tumor cells embedded into a collagen matrix flanked by 2 channels connected to the former by a series of trapezoidal pillars. The two lateral compartments are used to simulate the vascular and stromal environment respectively. In the text we show how the aforementioned microfluidic devices can efficiently recapitulate in vitro multiple key steps of cancer metastatic cascade and some of the most important interactions between immune-cancer cell interactions

Designing stem cell niches for differentiation and self-renewal

Mesenchymal stem cells, characterized by their ability to differentiate into skeletal tissues and self-renew, hold great promise for both regenerative medicine and novel therapeutic discovery. However, their regenerative capacity is retained only when in contact with their specialized microenvironment, termed the stem cell niche. Niches provide structural and functional cues that are both biochemical and biophysical, stem cells integrate this complex array of signals with intrinsic regulatory networks to meet physiological demands. Although, some of these regulatory mechanisms remain poorly understood or difficult to harness with traditional culture systems. Biomaterial strategies are being developed that aim to recapitulate stem cell niches, by engineering microenvironments with physiological-like niche properties that aim to elucidate stem cell-regulatory mechanisms, and to harness their regenerative capacity in vitro. In the future, engineered niches will prove important tools for both regenerative medicine and therapeutic discoveries

Collagen-Based Biomimetic Systems to Study the Biophysical Tumour Microenvironment

The extracellular matrix (ECM) is a pericellular network of proteins and other molecules that provides mechanical support to organs and tissues. ECM biophysical properties such as topography, elasticity and porosity strongly influence cell proliferation, differentiation and migration. The cell’s perception of the biophysical microenvironment (mechanosensing) leads to altered gene expression or contractility status (mechanotransduction). Mechanosensing and mechanotransduction have profound implications in both tissue homeostasis and cancer. Many solid tumours are surrounded by a dense and aberrant ECM that disturbs normal cell functions and makes certain areas of the tumour inaccessible to therapeutic drugs. Understanding the cell-ECM interplay may therefore lead to novel and more effective therapies. Controllable and reproducible cell culturing systems mimicking the ECM enable detailed investigation of mechanosensing and mechanotransduction pathways. Here, we discuss ECM biomimetic systems. Mainly focusing on collagen, we compare and contrast structural and molecular complexity as well as biophysical properties of simple 2D substrates, 3D fibrillar collagen gels, cell-derived matrices and complex decellularized organs. Finally, we emphasize how the integration of advanced methodologies and computational methods with collagen-based biomimetics will improve the design of novel therapies aimed at targeting the biophysical and mechanical features of the tumour ECM to increase therapy efficacy

Mechanical Studies of the Third Dimension in Cancer: From 2D to 3D Model

From the development of self-aggregating, scaffold-free multicellular spheroids to the inclusion of scaffold systems, 3D models have progressively increased in complexity to better mimic native tissues. The inclusion of a third dimension in cancer models allows researchers to zoom out from a significant but limited cancer cell research approach to a wider investigation of the tumor microenvironment. This model can include multiple cell types and many elements from the extracellular matrix (ECM), which provides mechanical support for the tissue, mediates cell-microenvironment interactions, and plays a key role in cancer cell invasion. Both biochemical and biophysical signals from the extracellular space strongly influence cell fate, the epigenetic landscape, and gene expression. Specifically, a detailed mechanistic understanding of tumor cell-ECM interactions, especially during cancer invasion, is lacking. In this review, we focus on the latest achievements in the study of ECM biomechanics and mechanosensing in cancer on 3D scaffold-based and scaffold-free models, focusing on each platform's level of complexity, up-to-date mechanical tests performed, limitations, and potential for further improvements

Sensing the difference: the influence of anisotropic cues on cell behavior

From tissue morphogenesis to homeostasis, cells continuously experience and respond to physical, chemical and biological cues commonly presented in gradients. In this article we focus our discussion on the importance of nano/micro topographic cues on cell activity, and the role of anisotropic milieus play on cell behavior, mostly adhesion and migration. We present the need to study physiological gradients in vitro. To do this, we review different cell migration mechanisms and how adherent cells react to the presence of complex tissue-like environments and cell-surface stimulation in 2D and 3D (e.g. ventral/dorsal anisotropy)