longitudinal label free quantitative tracking of cell death and viability in a 3d tumor model with oct

www.nature.com/scientificreports OPEN received: 06 January 2016 accepted: 12 May 2016 Published: 01 June 2016 Longitudinal, label-free, quantitative tracking of cell death and viability in a 3D tumor model with OCT Yookyung Jung*, Oliver J. Klein*, Hequn Wang* & Conor L. Evans Three-dimensional in vitro tumor models are highly useful tools for studying tumor growth and treatment response of malignancies such as ovarian cancer Existing viability and treatment assessment assays, however, face shortcomings when applied to these large, complex, and heterogeneous culture systems Optical coherence tomography (OCT) is a noninvasive, label-free, optical imaging technique that can visualize live cells and tissues over time with subcellular resolution and millimeters of optical penetration depth Here, we show that OCT is capable of carrying out high-content, longitudinal assays of 3D culture treatment response We demonstrate the usage and capability of OCT for the dynamic monitoring of individual and combination therapeutic regimens in vitro, including both chemotherapy drugs and photodynamic therapy (PDT) for ovarian cancer OCT was validated against the standard LIVE/DEAD Viability/Cytotoxicity Assay in small tumor spheroid cultures, showing excellent correlation with existing standards Importantly, OCT was shown to be capable of evaluating 3D spheroid treatment response even when traditional viability assays failed OCT 3D viability imaging revealed synergy between PDT and the standard-of-care chemotherapeutic carboplatin that evolved over time We believe the efficacy and accuracy of OCT in vitro drug screening will greatly contribute to the field of cancer treatment and therapy evaluation Tumor heterogeneity is thought to be a major factor in poor cancer treatment response and the development of treatment-resistant disease1 In ovarian cancer, for example, although 85% of patients will initially experience a complete therapeutic response, a significant majority of patients eventually succumb to recurrent, treatment-resistant metastatic ovarian cancer2,3 This high rate of recurrence leads to poor quality of life and an overall low 5-year survival rate of 30% However, ovarian cancer is one of the most treatable malignancies when detected early, with Stage I patients having greater than a 95% survival rate4 The difficulty in finding lasting treatments for late-stage patients is thought to stem from the highly heterogeneous nature of metastatic ovarian cancer, which exhibits not only widespread intra-and intertumoral genetic diversity, but also phenotypic and microenvironmental diversity4 In particular, a subpopulation of ovarian cancer cells are thought to have tumor-initiating or stem-like properties that allow even a small set of surviving cells to repopulate a patient with tumors5,6 Much of this cellular heterogeneity is unfortunately lost when tumor cells are plated on standard plastic culture dishes, which have stiff surfaces and lack biologically-relevant cell-cell and cell-matrix interactions Three-dimensional in vitro tumor cultures restore many of these important variables, and have been shown to replicate many features of ovarian tumors found in vivo7 3D in vitro culture models are of particular significance in studies of therapeutic response in ovarian cancer as their size and complexity are similar to that of ovarian metastatic lesions Metastatic ovarian cancer studs the surfaces within the peritoneal cavity and is composed of small tumor nodules that range in size from small avascular lesions a few hundred microns in diameter to larger occult lesions several centimeters wide The standard-of-care surgical resection received by nearly all ovarian cancer patients is considered successful if the remaining metastatic lesions are less than 1 cm in diameter 3D in vitro ovarian cancer cultures that mimic these small residual and often avascular lesions are considered highly Wellman Center for Photomedicine, Harvard Medical School, Massachusetts General Hospital, CNY149-3, 13th St, Charlestown, MA USA *These authors contributed equally to this work Correspondence and requests for materials should be addressed to C.L.E (email: evans.conor@mgh.harvard.edu) Scientific Reports | 6:27017 | DOI: 10.1038/srep27017 www.nature.com/scientificreports/ important as they model the target tumor dimensions of interest for the majority of therapeutics currently under development Despite their strengths, 3D cultures can prove difficult to interrogate accurately; disaggregating 3D multicellular spheroid cultures into individual cells can enable high-throughput analysis, but eliminates important spatial information Traditional techniques that have been used to monitor treatment response include fluorescence imaging The most commonly used method is the LIVE/DEAD Viability/Cytotoxicity Assay, which labels the live and dead cell populations with different fluorophores in order to differentiate and quantify these two cellular states7–9 High-content imaging of 3D cultures with fluorescent markers can successfully map viability and treatment response in small (