Generation of Confrontation Cultures From Embryoid Bodies and Multicellular Tumor Spheroids

Confrontation cultures of embryoid bodies and multicellular tumor spheroids are ideally suited to investigate tumor-induced angiogenesis. To discriminate the tumor tissue from the embryoid body-derived tissue, tumor spheroids have to be stained either by reporter gene transfection based on fluorescent reporters (GFP, YFP, RFP) or incubation with long term cell tracker dyes (e.g., CellTracker Green CMFDA, Orange-fluorescent tetramethylrhodamine CellTracker Orange CMTMR, Red-fluorescent CellTracker Red CMTPX). On cultivation in hanging drops tumor spheroids and embryoid bodies come into intimate contact and coalesce. This allows the subsequent invasion of capillary structures from the embryoid body (host tissue) to the tumor spheroid (cancer tissue). The penetration of capillaries from the embryoid body towards the tumor spheroid is accompanied by dramatic changes in the pericellular oxygen tension of the tumor spheroid. The improved supply with nutrients results in excessive growth of the tumor spheroids with concomitant increased oxygen consumption (12). Furthermore, during tumor-induced angiogenesis, changes in angiogenesis-related genes can be investigated. In this respect, we have demonstrated that VEGF protein is upregulated in the tumor tissue. We also demonstrated that tumor-induced angiogenesis resulted in increased P-glycoprotein-mediated drug resistance, which may be related to the decreased pericellular oxygen tension in the tumor tissue (12). The latter finding is of primordial interest for investigations on the capacity of antiangiogenic therapy in cancer treatment since our data suggest that this promising cancer therapy may add to circumvention of a MDR phenotype.

3.3.1. Preparation of Hanging Drops

For the differentiation of embryoid bodies in distinct cell phenotypes, cells must be cultivated in three-dimensional embryoid bodies that allows for the generation of confrontation cultures with equal-sized multicellular tumor spheroids.

1. Grow embryoid bodies as well as multicellular tumor spheroids to a size of 350-450 |im, which is achieved in human embryoid bodies after approx 21 d of culture (14 d when using in vitro fertilization dishes, 7 d in 60-mm bacteriological Petri dishes, and multicellular tumor spheroids after 18 d of culture).

2. Add 15 mL PBS to a 100-mm bacteriological Petri dish.

3. Place 20 |L drops (N = 30-50) of stem cell differentiation medium on the lid of the bacteriological Petri dish prepared in step 2.

4. Transfer one embryoid body and one tumor spheroid into each of the drops by using a 1000 ||L (blue tip) Gilson microliter pipet (see Note 3).

5. Cover the Petri dish prepared in step 2 with the lid prepared in step 3; the embryoid body and the tumor spheroid will come into intimate contact at the tip of the hanging drop and will coalesce.

6. After 48 h, remove the confrontation cultures from the hanging drops by using a 1000-|L (blue tip) Gilson microliter pipette (see Note 3). Transfer confrontation cultures to a 100-mm bacteriological Petri dish filled with 15 mL of stem cell differentiation medium.

7. Plate confrontation cultures to cover slips coated with 0.1% gelatin in PBS.

8. Cultivate confrontation cultures for different times up to 10 d with medium change every day.

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