Abstract
Diagnostic and prognostic indicators are key components to achieve the goal of personalized cancer therapy. Two distinct approaches to this goal include predicting response by genetic analysis and direct testing of possible therapies using cultures derived from biopsy specimens. Optimally, the latter method requires a rapid assessment, but growing xenograft tumors or developing patient-derived cell lines can involve a great deal of time and expense. Furthermore, tumor cells have much different responses when grown in 2D versus 3D tissue environments. Using a modification of existing methods, we show that it is possible to make tumor-fragment (TF) spheroids in only 2–3 days. TF spheroids appear to closely model characteristics of the original tumor and may be used to assess critical therapy-modulating features of the microenvironment such as hypoxia. A similar method allows the reproducible development of spheroids from mixed tumor cells and fibroblasts (mixed-cell spheroids). Prior literature reports have shown highly variable development and properties of mixed-cell spheroids and this has hampered the detailed study of how individual tumor-cell components interact. In this study, we illustrate this approach and describe similarities and differences using two tumor models (U87 glioma and SQ20B squamous-cell carcinoma) with supporting data from additional cell lines. We show that U87 and SQ20B spheroids predict a key microenvironmental factor in tumors (hypoxia) and that SQ20B cells and spheroids generate similar numbers of microvesicles. We also present pilot data for miRNA expression under conditions of cells, tumors, and TF spheroids.
Highlights
Modeling the 3D environment of tumors using cells in tissue culture is known to be challenging
Fibroblasts are not generally required [21] since U87 glioblastoma and Panc1 adenocarcinoma cells made very large spheroids even without the addition of stromal cells (Table 1) which is consistent with the general use of the liquid overlay technique
In contrast to the above complexities, SQ20B tumor fragments placed into the liquid overlay culture quickly (~2 days) formed large TF spheroids that remained viable for two to three weeks in culture (Figs 1 and 2)
Summary
Modeling the 3D environment of tumors using cells in tissue culture is known to be challenging. Use of high-density cultures, allowing 2D contact, has demonstrated modifications in radiation response that were subsequently confirmed in tumors (e.g. potentially-lethal-damage repair and modified rate of sublethal damage repair; [1, 2]). Such cultures require frequent feeding to prevent nutrient depletion, and continue to cycle, unlike the non-cycling state commonly found for cells (usually the majority) in tumors [3]. Several of the many innovations provided by this model included 3D cell-contact effects that were shown to modify therapy response and growth properties of the cells, demonstration of drug and nutrient diffusion limitations (in common with tumors) and development of central hypoxia and necrosis [4, 5, 7]
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