Long-term fluorescence hyperspectral imaging of on-chip treated co-culture tumour spheroids to follow clonal evolution

Abstract

Multicellular tumour spheroids are an ideal in vitro tumour model to study clonal heterogeneity and drug resistance in cancer research because different cell types can be mixed at will. However, measuring the individual response of each cell population over time is challenging: current methods are either destructive, such as flow cytometry, or cannot image throughout a spheroid, such as confocal microscopy. Our group previously developed a wide-field fluorescence hyperspectral imaging system to study spheroids formed and cultured in microfluidic chips. In the present study, two subclones of a single parental ovarian cancer cell line transfected to express different fluorophores were produced and co-culture spheroids were formed on-chip using ratios forming highly asymmetric subpopulations. We performed a 3D proliferation assay on each cell population forming the spheroids that matched the 2D growth behaviour. Response assays to PARP inhibitors and platinum-based drugs were also performed to follow the clonal evolution of mixed populations. Our experiments show that hyperspectral imaging can detect spheroid response before observing a decrease in spheroid diameter. Hyperspectral imaging and microfluidic-based spheroid assays provide a versatile solution to study clonal heterogeneity, able to measure response in subpopulations presenting as little as 10% of the initial spheroid.

Carboplatin sensitivity in epithelial ovarian cancer cell lines: The impact of model systems

“…In the particular case of ovarian cancer, 3D spheroids are clinically relevant models since in advanced disease EOC cells can spread to the peritoneum as aggregates/spheroids [52,53]. In the literature, several EOC cell lines have been studied by different spheroid generating methods, including hanging-droplets [16, 22, 27-29, 41, 54, 55], biomimetic hydrogels [23], plastic wells coated with matrix scaffolds [20,46,56], and more recently by microfluidics [33,34,37,38,42,44] and commercially available round-bottom ULA plates [18,45,50,57]. In the present study we first compared the ability of four EOC cell lines to form spheroids using these ULA 96-well plates (in the absence or presence of Matrigel) with that of hanging-droplets and PDMS-based microfluidic chips.…”

“…In the present study we first compared the ability of four EOC cell lines to form spheroids using these ULA 96-well plates (in the absence or presence of Matrigel) with that of hanging-droplets and PDMS-based microfluidic chips. For the latter, we used a recently developed [34,37,38] PDMS microfluidic device with a capacity of 120 spheroids per device. We showed that both Matrigel-assisted ULA plates and simple well-based PDMS microfluidic devices were the most robust and efficient methods to form spheroids.…”

“…Matrigel, consisting of collagen type IV, perlecan/heparin sulfate proteoglycan 2 and laminin, is one of the most commonly used ECM mimics [11,32] and has been shown to improve spheroid formation in ULA plates for cell lines that otherwise would not have the capacity to aggregate [18,19]. More recently, many bioengineered microfluidic devices have been developed and shown to be effective in forming spheroids [33][34][35][36][37][38][39]. In addition, the use of these microfluidic devices has gained popularity as a 3D model system for drug screening [12,[33][34][35][36][37][38][39][40][41][42][43][44].…”

“…More recently, many bioengineered microfluidic devices have been developed and shown to be effective in forming spheroids [33][34][35][36][37][38][39]. In addition, the use of these microfluidic devices has gained popularity as a 3D model system for drug screening [12,[33][34][35][36][37][38][39][40][41][42][43][44]. In ovarian cancer, our group has been a pioneer in the application of this technology to evaluate drug response in EOC cell lines [37,38,41].…”

Carboplatin response in preclinical models for ovarian cancer: comparison of 2D monolayers, spheroids, ex vivo tumors and in vivo models

“…Thereafter, spheroids were treated by adding 100 µL of three different concentrations of carboplatin (within 0 to 3000 µM final in-well concentration), whereas the control received 100 µL of complete OSE medium. Spheroids were treated for 24 h followed by a 24-h recovery period, based on literature suggesting that 24 h of drug exposure is required to penetrate the spheroid 25 – 27 . A 24-h recovery was chosen based on published in vitro studies demonstrating the effect of chemotherapy only after its removal 27 , 28 and to mimic the physiologic metabolism of the drug.…”