3D ex vivo Microfluidics Platform Beyond Limitations: New Microfluidics Platform for Cancer Drug Screening and Profiling

High-attrition rates during clinical development of new cancer therapies still persist. A new 3D ex vivo microfluidics platform shall now enhance cancer drug screening and early development process by creating cancer models which predict clinical outcomes with significantly increased reliability.

Cancer is one of the leading causes of mortality. Specific cancer types, such as glioma and pancreatic, remain intractable to new therapies and advanced cancers represent critical areas of unmet therapeutic need. The challenges in developing successful cancer therapeutics is not just limited to the discovery of new drug therapies, but also to the availability of robust preclinical ex vivo models. Recent technological advances in high-throughput and content phenotypic screening, as well as 3D multicellular assay methods, have opened doors for reshaping several key processes during drug discovery and development.

Challenges in cancer therapeutics

Despite increased research investments and ongoing efforts toward finding novel modalities for the treatment of the most challenging cancers, translation into patient benefit has been slow. Progress from preclinical drug discovery to positive clinical outcomes is limited by the fact that translational cancer research from early drug discovery to late stage drug development and assessment in clinical trials is a long process. Moreover, traditional drug discovery and discovery medicine activities have relied upon rudimentary 2D cell monolayer models , 3D clonogenic assay platforms and small animal models, mostly utilizing established cancer cell lines.

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It is now apparent that such preclinical models do not accurately recapitulate the complex pathophysiology of cancers found in patients and poorly predict clinical response. Therefore, development of more predictive, patient-specific models of human cancer are pivotal in the quest for profiling of novel anticancer drugs, either as single entities or as drug combinations. Oncologists have recognized the biological similarities of cells grown ex vivo in 3D culture systems with avascular tumors in vivo several decades ago [1, 2). The better the in-vitro models reflect the function and structure of their in-vivo counter parts, the more predictive the cell-based assays becomes. In this regard, several advances have been made in developing robust ex vivo 3D cell culture platforms using microtiter plate formats, 3D perfusion systems and 3D microfluidics formats with special focus on multicellular tumor spheroids (MCTS) systems. The emergence of these technologies have made it possible to conduct drug screening and early development programs in cost effective and efficient ways using clinically relevant cell types derived from multiple cancers.

Currently, the use of ex vivo 3D assay platforms is being pursued by researchers as a tool for drug activity response and monitoring potential changes at subcellular levels to identify downstream targets post drug treatment. Additionally, these platforms are extensively being used as a drug development tool as it has been shown to closely simulate the tumour microenvironment within the in vitro milieu [3-6]. It has previously been reported that the drug response of cancer cells is not just determined by the inherent characteristics of the epithelial tumor cells, but, are also controlled by signals derived from other cells (stromal/fibroblasts/distinct immune cell compartments) within the tumor microenvironment [7-10].

Harris AL (2002) [11] and Mellor & Callaghan (2008) [12] have reported that the abnormal vascularization of solid tumors leads to the generation of tumor microenvironments that are chronically starved of oxygen and nutrients. As a result, cells residing in such environment demonstrate altered phenotypic characteristics when compared to the cells located in more vascularized regions around the outer core [13-15]. Therefore, there has been immense drive towards developing methods which can exploit the altered phenotype of tumors.

One of the earliest 3D-related effects, which were correlated to in-vivo observations, included multicellular drug resistance (MDR). Cancer cells grown as 3D spheroids generally display reduced drug sensitivity compared to 2D monolayer cultures [16]. These observations indicate that the use of 3D spherical microtissues may enable improved discrimination of the most active anti-cancer drugs with improved therapeutic index.

Need for complex and relevant model systems

As the understanding of complex biological and pathological pathways leading to disease is increasing, so has the need for the complex and relevant model systems arisen to study the mechanisms involved in various disease aspects. Yin X, et al (2016) [19] have aptly described how the model systems (see Figure 2) have been developed across the organismal hierarchy to address specific questions within the realm of biology and medicine.

Within the context of three-dimensional ex vivo platforms, microfluidic technologies have emerged as potentially highly relevant ex vivo tools in cancer research [17, 18]. This rapidly expanding technology relies on the use of small channels equipped to handling small fluids and, therefore, small amount of chemicals and cell material. These platforms have shown to represent physiological relevance over other in vitro technologies, allowing precise control of the cellular, physical and biochemical environment, ability to study cancer-immune interactions, making them a complementary platform for preclinical in vivo models [20].

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3D ex vivo microfluidics platform

The microfluidics system developed by Amsbio in collaboration with researchers at the University of Strathclyde/UK, a miniaturized and high-throughput 3D ex-vivo assay platform, provides a robust, customisable and cost-effective screening system [Mullholland et al, publication under review]. The novel characteristics of this platform enable rapid decision making to prioritize the most promising drug candidates, biomarkers and drug combination strategies for preclinical drug discovery and development.

An advantage of the platform is the ability of cells to form spheroid without the presence of exogenously added matrices/scaffolds to help cells polarize into formation of spheroids. We believe that the endogenously produced ECMs (extracellular matrix, ECM) by cells help polarization and induce cell-contact. This, together with precise fluid control, enables long term culture of spheroids, especially cells derived from patient biopsies or PDXs (Patient derived xenograft, PDX). It has been observed that patient derived primary cancer cells require longer time to form compact spheroids using a standard scaffold free 3D ex vivo technology. However, in our platform we have observed that primary cells form compact spheroids already in less than five days. Therefore, the system enables long-term culture as well as fractionated chemo- and targeted-therapy to study drug efficacy, as well as acquired drug resistance.

The platform has been validated by assessing the efficacy of standard of care compounds against multiple cancer types including glioblastoma, prostate, breast cancer, lung and pancreatic. This platform offers the ability to perform multiparametric end point measurements that include (but are not limited to) viability measurement, changes in spheroid size and shape, assessing the temporal evolution of spheroid response post drug treatment. The system also allows researchers to retrieve spheroids for proteomics and transcriptomics analysis, as well as changes in the spheroid biomarker status using immunohistochemistry techniques (see Figure 3).

In conclusion, Amsbio 3D ex vivo microfluidics platform is well suited for cancer drug discovery, early development and profiling of broad spectrum of molecules including small, targeted and biologics tested against various cancer models.

References

[1] Hirschhaeuser F. Menne H. Dittfeld C. et al. (2010). Multicellular tumor spheroids: an underestimated tool is catching up again. Jour of Biotechno, 148(1), 3–15.

[2] Durand RE (1984). Growth and cellular characteristics of multicell spheroids. Recent Results Can Res. 95:24-49.

[3] Cukierman E, Pankov R, Stevens DR, Yamada KM (2001). Taking cell-matrix adhesions to be the third dimension. Science. 294:1708-12.

[4] Mizushima H, Wang X, Miyamoto S, Mekada E (2009). Integrin signal masks growth promoting activity of HB-EGF in monolayer cell cultures. J Cell Sci. 122:4277-86.

[5] Kondo J, Endo H, Okuyama H, Ishikawa O. et al. (2011). Retaining cell-cell contact enables preparation and culture of spheroids composed of pure primary cancer cells from colorectal cancer. Proc Natl Acad Sci . Apr 12;108 (15):6235-40.

[6] Yoshii Y, Furukawa T, Waki A, Okuyama H, Inuoue M, Itoh M et al. (2015). High-throughput screening with nanoimprinting 3D culture for efficient drug development by mimicking the tumor environment. Biomaterials 5:278-89.

[7] Sun Y, Campisi J, Higano, C. Beer TM. et al. (2012). Treatment-induced damage to the tumor microenvironment promotes prostate cancer therapy resistance through WNT16B. Sep;18 (9):1359-68.

[8] Morikawa, T. Shee, K. et al. (2012). Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion. Jul 26;487 (7408):500-4.

[9] Wilson TR, Fridlyand J, Yan Y, Burton L. et al. (2012). Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors. Jul 26; 487(7408):505-9.

[10] Ostman A (2012). The tumor microenvironment controls drug sensitivity. Sep;18 (9):1332-4.

[11] Harris AL (2002). Hypoxia-a key regulatory factor in tumor growth. Nat. Rev. Cancer 2, 38-47.

[12] Mellor HR & Callaghan R (2008). Resistance to chemotherapy in cancer: a complex and integrated cellular response. Pharmacology, 81, 275-300.

[13] Sahin AA, R JY, el-Naggar AK, Wilson PL et al. (1991). Tumor proliferative fraction in solid malignant neoplasms. A comparative study of ki67 immunostaining and flow cytometric determination. Am J Clin Pathol. 96, 512-519.

[14] St Croix B, Flørenes VA, Rak JW, Flanagan M, Bhattacharya N et al.(1996). Impact of the cclin-dependent kinase inhibitor p27Kip1 on resistance of tumor cells to anticancer agents. Nat. Med. 2, 1204-1210.

[15] Gardener L.B. et al. (2001). Hypoxia inhibitsG1/S transition through regulation of p27expression. J. Biol. Chem. 276, 7919-7926.

[16] Desoize B, Jardillier J. (2000). Multicellular resistance: a paradigm for clinical resistance? Cri Rev Oncol Hematol, Nov-Dec; 36 (2-3): 193-197.

[17] Terrell-Hall TB, Nounou MI, El-Amrawy F, Griffith JIG, Lockman PR. (2017).Trastuzumab distribution in an in-vivo and in-vitro model of brain metastases of breast cancer. Oncotarget. Jul 26;8(48): 83734-83744.

[18] Lanz HL, Saleh A, Kramer B, Cairns J, Ng CP, Yu J, Trietsch SJ, Hankemeier T, Joore J, Vulto P, Weinshilboum R, Wang L. (2017). Therapy response testing of breast cancer in a 3D high-throughput perfused microfluidic platform. BMC Cancer. Nov 2;17(1):709.

[19] Yin X, Mead, E.B, Safaee H, Langer R, Karp JM. (2016). Engineering Stem Cell Organoids. Cell Stem Cell, January 7 (18): 25-38.

[20] Boussomier-Calleja A, Li R, Chen MB, Wong SC and Kamm RD. (2016). Microfluidics: A new tool for modelling cancer.immune interactions. Trends Cancer. January 1;2(1): 6-19.

* S. Dhar, A. Sim: AMS Biotechnology Ltd, Abingdon, OX14 4SE, United Kingdom

* * T. Mullholand, G. Robertson, M. Zagnoni: Center for Microsystems & Photonics, Dept. Electronic and Electric Engineering, University of Strathclyde, Royal College Building, Glasgow G1 1XW, United Kingdom

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