Physical Characterization of Colorectal Cancer Spheroids and Evaluation of NK Cell Infiltration Through a Flow-Based Analysis

Abstract

To improve pathogenetic studies in cancer development and reliable preclinical testing of anti-cancer treatments, three-dimensional (3D) cultures, including spheroids, have been widely recognized as more physiologically relevant in vitro models of in vivo tumor behavior. Currently, the generation of uniformly sized spheroids is still challenging: different 3D cell culture methods produce heterogeneous populations in dimensions and morphology, that may strongly influence readouts reliability correlated to tumor growth rate or antitumor natural killer (NK) cell-mediated cytotoxicity. In this context, an increasing consensus claims the integration of microfluidic technologies within 3D cell culture, as the physical characterization of tumor spheroids is unavoidably demanded to standardize protocols and assays for in vitro testing. In this paper, we employed a flow-based method specifically conceived to measure weight, size and focused onto mass density values of tumor spheroids. These measurements are combined with confocal and digital imaging of such samples. We tested the spheroids of four colorectal cancer (CRC) cell lines that exhibit statistically relevant differences in their physical characteristics, even though starting from the same cell seeding density. These variations are seemingly cell line-dependent and associated with the number of growing cells and the degree of spheroid compaction as well, supported by different adenosine-triphosphate contents. We also showed that this technology can estimate the NK cell killing efficacy by measuring the weight loss and diameter shrinkage of tumor spheroids, alongside with the commonly used cell viability in vitro test. As the activity of NK cells relies on their infiltration rate, the in vitro sensitivity of CRC spheroids proved to be exposure time- and cell line-dependent with direct correlation to the cell viability reduction. All these functional aspects can be measured by the system and are documented by digital image analysis. In conclusion, this flow-based method potentially paves the way towards standardization of 3D cell cultures and its early adoption in cancer research to test antitumor immune response and set up new immunotherapy strategies.

Three-Dimensional Culture Models to Study Innate Anti-Tumor Immune Response: Advantages and Disadvantages

“…This prompts to generate models to study patients' immune response variability and drug sensitivity, and to characterize the interaction of immune cells with 3D CRC-derived spheroids [119,120,[125][126][127]136]. Tumor cell spheroids can be obtained applying different culture methods [125][126][127][128][129][130][131][132][133] (Figure 3). CRC is a very heterogeneous inter and intra patient disease [142][143][144][145].…”

“…Spheroids can be derived from either established cell lines or primary isolated tumor cells [ 35 , 118 , 119 , 120 , 121 , 122 , 123 , 124 , 125 , 126 , 127 , 128 ]. Usually, spheroids are obtained culturing tumor cells in ultra-low adherent plasticware or by hanging drop methods, forcing tumor cells to aggregate and generate tumor sphere in static or microfluidic systems [ 15 , 16 , 126 , 127 , 128 , 129 , 130 , 131 , 132 , 133 , 134 , 135 , 136 ]. The spheroids can be composed of a single (homotypic) or, less frequently, different (heterotypic) cell types [ 15 , 16 , 125 , 126 ].…”

“…Usually, spheroids are obtained culturing tumor cells in ultra-low adherent plasticware or by hanging drop methods, forcing tumor cells to aggregate and generate tumor sphere in static or microfluidic systems [ 15 , 16 , 126 , 127 , 128 , 129 , 130 , 131 , 132 , 133 , 134 , 135 , 136 ]. The spheroids can be composed of a single (homotypic) or, less frequently, different (heterotypic) cell types [ 15 , 16 , 125 , 126 ]. Of note, it has been reported that spheroids can be obtained from patients’ tumor-specimens digestion, and self-immune cells can be challenged with self-spheroids to analyze the effect of different drugs and plan their application into the clinic [ 15 , 16 , 117 , 119 , 123 ].…”

Pediatric Tumors-Mediated Inhibitory Effect on NK Cells: The Case of Neuroblastoma and Wilms’ Tumors

“…On the other hand, cancer cells themselves drive immunosuppressive activity on effector cells, including NK cells, releasing immunoregulatory molecules such as transforming growth factor β (TGF-β), prostaglandin E2 (PGE2), adenosine, lactate and kynurenine [ 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ].…”

A new method for the study of biophysical and morphological parameters in 3D cell cultures: Evaluation in LoVo spheroids treated with crizotinib

“…One of the main limitations for a comprehensive characterization of 3D models is related to the mass density and its correlation to the volume. To address this issue, we recently developed a new device (W8, CellDynamics iSRL) and relative analytical method, for the accurate, simultaneous, and rapid measurement of mass density, weight, and size of spheroids generated from different cell types [ 24 , 25 ]. In general, the device is able to measure all types of spheroids derived from cancer and primary cells which meet the operative range between 50 and 500 μm in diameter.…”