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Written by Subia Bano, Postdoc

subia.bano@elvesys.com

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Short review about microfluidic Tumor-on-Chip systems for breast cancer research

1. Introduction to microfluidic breast tumor-on-chip systems for breast cancer studies

Breast cancer research-tumor on chip-microfluidics-elveflow-startup-innovation

Breast cancer is one of the most frequent malignant tumors among women worldwide. In the early stage of breast cancer neoplastic epithelial cells accumulate in the lumen of mammary duct that form pre-invasive cancerous lesions known as ductal carcinoma in situ (DCIS). Ductal means that the cancer starts inside the milk ducts. Progression of invasive breast cancer occurs when the tumor cells in DICS penetrates the basement membrane and invade the surrounding tissues [1]. Transition from in situ ductal carcinoma to invasive ductal carcinoma brings aberrant changes in matrix remodeling, paracrine signaling and immune responses [2-4]. Metastatic breast cancer occurs when the cancer spreads from the breast to another part of the body such as lungs, liver, bone etc. The tumor microenvironment is comprised of a dynamic network of extracellular matrix (ECM) proteins associated with fibroblast cells, bone marrow-derived cells, endothelial cells, and infiltrating immune cells [5]. These stromal cells remodel the ECM and provide mechanical and chemical cues to the tumor cells. Symptoms and treatment of this stage of breast cancer are difficult, despite the extensive research and enormous efforts in drug discovery over the last few decades. This is partly due to the need of better understanding of tumor microenvironment and high cost for the development of a new anti-cancer drug [6,7].

Circulating tumor cells can likewise be studied using microfluidic approaches. Read here for more information.

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Cancer research typically depends on 2D in vitro studies and animal models to investigate the tumor mechanism, angiogenesis, invasion, and metastasis [8], although two- and three-dimensional in vitro models have been widely used for screening anti-cancer drugs, and studying cell signaling, proliferation, migration and drug responses including altered protein/gene expression [9]. These in vitro models may not be able to mimic the exact tumor microenvironment, and thus not fully suitable to study the effect of complex spatial organization and interaction of cells. However, these 3D tumor models create tumor microstructures and are capable of capturing cell-cell and cell-matrix interactions. Although 3D models have the benefit of recapitulating the tumor cell organization, they still lack the vasculature and fluid shear stress found in vivo [10]. On the other hand, animal models can provide essential in vivo information of tumor growth and response to drug molecules, but are very costly and results may vary between animals. In addition to the ethics surrounding in vivo model usage, serious concerns still exist over their biological relevance to humans [11]. To make significant improvements in cancer therapy, it is necessary to develop more effective approaches to screen anti-cancer drug candidates and to have a better understanding of tumor microenvironment using advanced technologies, including microfluidics and organ-on-chip technology.

2. Microfluidic tumor-on-a-chip systems

Tumor-on-chip is a microfluidic-based 3D system capable of recapitulating the biological activities, mechanical properties, and physiological responses of tumor cells [12]. Different from conventional 3D in vitro models, tumor-on-a-chip utilizes fluid dynamics in order to enhance long-term culturing capabilities under physiological conditions. Typically, tumor-on-chip incorporates a 3D culturing system with fluid tubing and channels to control the delivery of nutrients and the removal of waste from the system to mimic the microenvironments of tumor cells grown in vivo.

3. Breast cancer tumor-on-chip devices

To mimic the 3D structural organization of the human mammary duct, Choi et al., (2015) designed a microdevice consisting of an upper and a lower microchannel separated by a semi-permeable membrane that mimics the basement membrane. In this device the compartmentalized 3D microfluidic device was used for drug screening on cocultured breast tumor spheroids with human mammary duct epithelial cells and mammary fibroblast cells. In this design the upper microchannel represented the ductal lumen which allows continuous flow of culture media that is required for growing and maintaining the mammary epithelial cells and DCIS spheroids on the upper side of the Extracellular Matrix (ECM) membrane (Fig 1). A stromal layer impregnated with mammary fibroblast is formed on the lower side of ECM membrane and perfused with culture media through the lower microchannel to mimic the vascular compartment of capillaries in mammary stroma. The upper and lower microchannels were separated by a collagen membrane. In this microfabricated device the cocultured cells were maintained by continuous flow of culture media at the physiological rate of interstitial flow (2-70 µl/hr). They also studied the impact of the clinical chemotherapeutic anticancer drug Paclitaxel related to the size of Ductal Carcinoma in situ (DCIS). They were able to show that Paclitaxel is capable of arresting tumor cell proliferation and inhibits the growth of DCIS in the tumor microenvironment [1]. This micro-engineered disease model successfully represented the complexity of breast cancer pathophysiology and could be used as a tool to systematically study the invasiveness of DCIS in a breast cancer microenvironment similar to that found in the patient’s body.

DCIS-tumor on chip-breast cancer research-microfluidics-elveflow-startup-innovation

Figure 1 (A) DCIS is embedded in the mammary duct which consists of the mammary epithelium and a basement membrane surrounded by stromal tissue that contains fibroblasts. (B) The microarchitecture of DCIS and the surrounding tissue layers is reproduced in the breast cancer-on-a-chip microdevice comprised of the upper and lower cell culture chambers separated by an ECM-derived membrane. DCIS spheroids are embedded in the mammary epithelium formed in the upper channel, and the fibroblast-containing stromal layer is created on the other side of the membrane. Ref: Choi et al (2015)  Lab Chip. 2015 ;15(16): 3350–3357.

Paracrine signaling and extracellular matrix activation in breast cancer research

Similar to the above described approach, Gioiella et al., (2016), also used a microfluidic platform to simulate breast cancer on a chip nd to replicate epithelial-stroma interactions (cell phenotype, extracellular matrix composition and organization, macromolecular transport) that occur in the healthy stroma during the invasion of malignant epithelial breast cells. In this device, both stroma and epithelial tumor tissue were separated by an interface that allows physical contact. In this device, normal and cancer activated fibroblast cells were used to produce cancer microtissues. When these cells are cocultured together, normal fibroblast cells differentiated into myofibroblasts after physical contact with cancer cells in the microfluidic device. The group likewise investigated the expression of Matrix Metalloproteinase (MMPs) during cancer invasion. The paracrine signaling among cancer cells and fibroblast cells induced the production of MMPs (MMP2 and MMP9) that activated the migration of cancer cells in the 3D environment. These MMPs can degrade collagen IV and weaken the basement membrane [13]. The degradation products of the extracellular matrix, including collagen IV fragments, in turn, provide signaling cues to regulate the tumor cell motility.

The effects of different ECM components (collagen, hyaluronic acid (HA) and fibronectin) on tumor interactions were likewise evaluated in the tumor microenvironment. A significant number of studies reports that deposition of HA and fibronection increases in various types of cancer tissues including breast cancer tissues [14]. The rate of HA synthesis is much higher in cancer tissue compared to normal tissue. Increased HA levels in cancerous tissue promote cancer cell invasion, migration, metastasis and tumor-stroma interactions [13]. HA levels increase from in situ ductal carcinoma to later stages of invasive cancer sites.

Co-culture systems for breast cancer migratory analysis

Mi et al., (2016) have presented a novel microfluidic system where they establish an in vitro co-culture model of breast cancer cells and human mammary epithelial cells (HMEpiC) that mimics different regions of a metastatic breast tumor (Fig 2). This breast tumor model was used to study the effects of the anti-cancer drugs Paclitaxel and Tamoxifen on tumor migration. Drug screening using microfluidics is an innovative and promising approach and the results showed that migration of cancer cells (MDA-MB-231) was increased after coculture with human mammary epithelial cells. The secretion of proinflammatory cytokines was increased when breast cancer cells were cocultured with breast epithelial cells, which altered their cell morphology and modulated cell migration. The co-culture also led to decreased Cytokeratin-14 (a characteristic protein of epithelial cells) secretion and morphological changes in HMEpiC cells [15]. The significant inhibition of migration of the cancer cells was observed after addition of different concentrations of Paclitaxel and Tamoxifen.

Breast tumor on chip-breast cancer research-microfluidics-elveflow-startup-innovation

Figure 2 (A) Schematic representation of a metastatic breast tumor, (B) Steps for loading of cells and realization of co-culture, (C) Drug treatment on chip. Ref: Mai et al (2016). Sci Rep. 2016; 6: 35544.

4.      Future prospective of breast-tumor-on-chip systems

Ultimately, the key advantage of breast cancer-on-chip is to grow artificial breast tissues on-chip. A large number of therapeutic approaches can be tested faster and less expensive than using animal models or human subjects. The tumor-on-chip technology also holds the key to reducing the use of animals in pharmacodynamic and pharmacokinetic investigations, as well as toxicology studies. The devices will allow the design and testing of a larger number of preventive interventions that may lead to break-through advances in breast cancer prevention and treatment.

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References

[1] Choi Y, Hyun E, Seo J, Blundell C, Kim CH, Lee E, Lee S, Moon A, Hun D. A microengineered pathophysiological model of early-stage breast cancer. Lab Chip. 2015; 15(16): 3350–3357.

[2] Place AE, Jin Huh S, Polyak K. The microenvironment in breast cancer progression: biology and implications for treatment. Breast Cancer Res. 2011; 13(6):227.

[3] Khurana A, McKean H, Kim H, Kim SH, Mcguire J, Roberts LR, Goetz MP, Shridhar V. Silencing of HSulf-2 expression in MCF10DCIS.com cells attenuate ductal carcinoma in situ progression to invasive ductal carcinoma in vivo. Breast Cancer Res. 2012; 14(2): R43.

[4] Lyons TR, O’Brien J, Borges VF, Conklin MW, Keely PJ, Eliceiri KW, Marusyk A, Tan AC, Schedin P. Postpartum mammary gland involution drives progression of ductal carcinoma in situ through collagen and COX-2. Nat Med. 2011; 17(9):1109-15.

[5] Bonnans C,  Chou J, and  Werb Z. Remodelling the extracellular matrix in development and disease. Nat Rev Mol Cell Biol. 2014; 15(12): 786–801.

[6] Tsai HF, Trubelja A,  Shen AQ, and Bao G. Tumor-on-a-chip: microfluidic models of tumour morphology, growth and microenvironment. J R Soc Interface. 2017 Jun; 14(131): 20170137.

[7] Langhans SA. Three-Dimensional in Vitro Cell Culture Models in Drug Discovery and Drug Repositioning. Front Pharmacol. 2018; 9: 6

[8] Subia B, Dey T, S Sharma, Kundu SC. Target specific delivery of anticancer drug in silk fibroin based 3D distribution model of bone–breast cancer cells. ACS applied materials & interfaces 7 (4), 2269-2279.

[9] Jo Y,   Choi N,  Kim K,   Koob HJ,  Choi J,  Kim  HN. Chemoresistance of Cancer Cells: Requirements of Tumor Microenvironment-mimicking In Vitro Models in Anti-Cancer Drug Development. Theranostics. 2018; 8(19): 5259–5275.

[10] Fang Y and  Eglen RM. Three-Dimensional Cell Cultures in Drug Discovery and Development, SLAS Discov. 2017;22(5): 456–472.

[11] Festing S and  Wilkinson R. The ethics of animal research. Talking Point on the use of animals in scientific research. EMBO Rep. 2007; 8(6): 526–530.

[12] Shang M,   Soon RH,    Lim CT,  Khoo BL, and   Han I. Microfluidic modelling of the tumor microenvironment for anti-cancer drug development. Lab Chip, 2019;19, 369-386.

[13] Gioiella F, Urciuolo F, Imparato G, Brancato V, & Netti PA. An Engineered Breast Cancer Model on a Chip to Replicate ECM-Activation In Vitro during Tumor Progression. Advanced Healthcare Materials, 2016; 5(23), 3074–3084.

[14] Lijima J, Konno K. Inflammatory Alterations of the Extracellular Matrix in the Tumor Microenvironment. Cancers 2011; 3(3), 3189-3205.

[15] Mi S, Du Z, Xu Y, Wu Z, Quin X, Zhang M, Sun W. Microfluidic co-culture system for cancer migratory analysis and anti-metastatic drugs screening. Sci Rep. 2016; 6: 35544.

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