TITLE : 1 Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids 2 using Optical Coherence Tomography 3 4

46 Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer 47 research and anti-cancer drug discovery. However, currently, high-throughput imaging 48 modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D 49 structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes 50 and depth-resolvability. Recently, our lab demonstrated the use of optical coherence 51 tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal 52 characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 53 3D morphological and physiological information of tumor spheroids growing up to about 600 μm 54 in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that 55 scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We 56 describe the details of the HT-OCT system and construction guidelines in the protocol. From the 57 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and 58 orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the 59 morphological information of size and volume, and monitor the growth of the dead-cell regions 60 in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can 61 be used as a high-throughput imaging modality for drug screening as well as characterizing 62 biofabricated samples. 63 64 INTRODUCTION: 65 Cancer is the second leading cause of death in the world1. Developing drugs targeting cancer is 66 of crucial importance for patients. However, it is estimated that more than 90% of new anti67 cancer drugs fail in the development phase because of a lack of efficacy and unexpected toxicity 68 in clinical trials2. Part of the reason can be attributed to the use of simple two-dimensional (2D) 69 cell culture models for compound screening, which provide results with limited predictive values 70 of compound efficacy and toxicity for the following stages of drug discovery2-4. Recently, three71 dimensional (3D) tumor spheroid models have been developed to provide clinically relevant 72 physiological and pharmacological data for anti-cancer drug discovery3-25. Since these spheroids 73 can mimic tissue-specific properties of tumors in vivo, such as nutrient and oxygen gradient, 74 hypoxic core as well as drug resistance19, the use of these models can potentially shorten drug 75 discovery timelines, reduce costs of investment, and bring new medicines to patients more 76 effectively. One critical approach to evaluating compound efficacy in 3D tumor spheroid 77 development is to monitor the spheroid growth and recurrence under treatments9,26. To do this, 78 quantitative characterizations of the tumor morphology, involving its diameter and volume, with 79 high-resolution imaging modalities, are imperative. 80 81 Conventional imaging modalities, such as bright-field, phase contrast7,9,22,24, and fluorescence 82 microscopy8,9,16,18,22 can provide a measurement of the spheroid’s diameter but cannot resolve 83 the overall structure of the spheroid in 3D space. Many factors contribute to these limitations, 84 including penetration of the probing light in the spheroid; diffusion of the fluorescent dyes into 85 the spheroid; emitting fluorescent signals from excited fluorescent dyes inside or on the opposite 86 surface of the spheroid due to strong absorption and scattering; and depth-resolvability of these 87 imaging modalities. This often leads to an inaccurate volume measurement. Development of the 88 necrotic core in spheroids mimics necrosis in in vivo tumors6,10,15,19,25. This pathological feature is 89 unlikely reproduced in 2D cell cultures19,25,27,28. With a spheroid size larger than 500 μm in 90 diameter, a three-layer concentric structure, including an outer layer of proliferating cells, a 91 middle layer of quiescent cells, and a necrotic core, can be observed in the spheroid6,10,15,19,25, 92 due to lack of oxygen and nutrients. Live and dead cell fluorescence imaging is the standard 93 approach to label the boundary of the necrotic core. However, again, penetrations of both these 94 fluorescent dyes and visible light hinder the potential to probe into the necrotic core to monitor 95 its development in its actual shape. 96 97 An alternative 3D imaging modality, optical coherence tomography (OCT) is introduced to 98 characterize the tumor spheroids. OCT is a biomedical imaging technique that is capable of 99 acquiring label-free, non-destructive 3D data from up to 1‒2 mm depths in biological tissues29-34. 100 OCT employs low-coherence interferometry to detect back-scattered signals from different 101 depths of the sample and provides reconstructed depth-resolved images at micron-level spatial 102 resolutions in both lateral and vertical directions. OCT has been widely adopted in 103 ophthalmology35-37 and angiography38,39. Previous studies have used OCT to observe the 104 morphology of in vitro tumor spheroids in basement membrane matrix (e.g., Matrigel) and 105 evaluate their responses to photodynamic therapy40,41. Recently, our group established a high106 throughput OCT imaging platform to systematically monitor and quantify the growth kinetics of 107 3D tumor spheroids in multi-well plates42. Precise volumetric quantification of 3D tumor 108 spheroids using a voxel counting approach and label-free necrotic tissue detection in the 109 spheroids based on intrinsic optical attenuation contrast were demonstrated. This paper 110 describes the details of how the OCT imaging platform was constructed and employed to obtain 111 high-resolution 3D images of tumor spheroids. The step-by-step quantitative analyses of the 112 growth kinetics of 3D tumor spheroids, including accurate measurements of spheroid diameter 113 and volumes, is described. Also, the method of the non-destructive detection of necrotic tissue 114 regions using OCT, based on the intrinsic optical attenuation contrast is presented. 115 116 PROTOCOL: 117 118 1. Preparation of Cells 119 120 1.1) Obtain cell lines from a qualified supplier. 121 122 NOTE: Verify that cells from the cell lines of interest can form spheroid in the culture media or 123 with the help of a substrate (basement membrane matrix like Matrigel). Look into the literature9 124 or perform one round of a pre-experiment for a check. 125 126 1.2) Thaw the frozen cells following the specific procedure provided by the cell-line supplier. A 127 general procedure can be found elsewhere43. 128 129 1.3) Culture the cells for 1-2 passages in 25 cm2 culture flasks. The cells are then ready to use for 130 3D cell culture. 131 132 1.4) Monitor the health status of the cells every day and maintain them in an incubator under 133 standard conditions (37 °C, 5% CO2, 95% humidity). Refresh the media as needed. 134 135 NOTE: The culture medium consists of DMEM (4.5 g/L glucose), 1% antibiotic-antimycotic, 10% 136 fetal bovine serum. Subculture cells before they reach confluence in the culture flask. Follow the 137 cell culture guideline provided by the supplier. A general procedure can be found elsehwhere44. 138 139 1.5) Perform 3D cell culture in multi-well plates based on the following general protocol9. 140 141 1.5.1) Remove the culture media from the culture flask and wash it with sterilized phosphate 142 buffered saline (PBS, heated to 37 °C). 143 144 1.5.2) Resuspend the cells by adding 1 mL of trypsin ethylenediaminetetraacetic acid (EDTA, 145 0.5%) into the flask for 3 min. Then, add culture media to dilute the trypsin. 146 147 1.5.3) Transfer the cell suspension into a 15 mL centrifuge tube and centrifuge for 5 min at 500 x 148 g and room temperature. 149 150 1.5.4) Remove the supernatant and resuspend cells with 4 mL of pre-warmed culture medium. 151 Pipette one drop of sample onto a hemocytometer for cell counting to determine cell 152 concentration. Dilute the cells to appropriate concentration for seeding (e.g., 3000 cells/mL). 153 154 NOTE: Optimize the initial cell concentration of the spheroid for each cell-line and each type of 155 multi-well plate (96-well, 384-well or 1536-well). 156 157 1.5.5) Seed cells into an ultra-low attachment (ULA) round-bottomed multi-well plate. Add 200 158 μL of cells suspension into each well at the concentration of 3,000 cells/mL so that each well has 159 about 600 cells. 160 161 1.5.6) At RT, centrifuge the whole plate using a plate adapter for 7 min, right after seeding, at a 162 speed of 350 x g or the lowest speed available. 163 164 NOTE: The centrifuge helps gather cells to the center of the well to facilitate forming a single, 165 uniform spheroid. The centrifuge step is performed only once at the beginning to form the tumor 166 spheroids. It will not be repeated when the tumor spheroids start growing. 167