Improved Methods to Generate Spheroid Cultures from Tumor Cells, Tumor Cells & Fibroblasts or Tumor-Fragments: Microenvironment, Microvesicles and MiRNA

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.

Modeling neoplastic disease with spheroids and organoids

“…Non-adherent conditions Physiological ratio cancer:stromal/ immune cells to mimic clinical tumors [105][106][107] fairly common [123], and medium without WNT and Rspondins is needed to obtain WNT-mutated tumoroids [7]. Patient-derived organoids can also be used to detect epigenetic and/or genetic alterations underlying drug resistance ( Fig.…”

Analysis of radiation effects in two irradiated tumor spheroid models

“…Three-dimensional cell culture has therefore been developed and optimized during the past forty years to create a feasible cellular model that surrogates non-vascularized micrometastases in vivo ( 3 ). Three-dimensional spheroids can be generated through a number of different methods either by allowing the cells to cluster through altering the adhesive properties of surface of cell culture dishes or environment, or by preventing aggregation through continuous agitation ( 4 , 5 ). The latter, most commonly through the use of spinner flasks or gyrating vessels, form spheroids in variable sizes as they are formed from an uncertain and indecipherable number of cells in an uncontrolled manner ( 4 ).…”

The Current Landscape of 3D In Vitro Tumor Models: What Cancer Hallmarks Are Accessible for Drug Discovery?

“…Spheroids can be generated from a number of cell sources such as cell lines or from patient, or xenograft material for increased model relevance. The spheroid model has been the gold standard for in vitro cancer research and drug screening for decades because of its simple workflow and high‐throughput capabilities, with additional flexibility to incorporate varying levels of microenvironmental complexity including oxygen and nutrient gradients by varying spheroid size or by adding additional cell populations including fibroblast cells, endothelial cells, or immune cells, or by embedding spheroids within a hydrogel ECM, traditionally collagen or Matrigel, that may contain additional cell types. The synthesis of novel hydrogels to enable reproducible alternatives to Matrigel, which potentially mimic important biochemical and physical properties of the tumor microenvironment, is an active area of study and is discussed in more detail in Section .…”