September 15, 2014 (Vol. 34, No. 16)

Brad Larson principal scientist BioTek Instruments
Nicky Slawny Ph.D. Applications Director 3D Biomatrix
Wini Luty product manager Enzo Life Sciences
Peter Banks Ph.D. Scientific Director BioTek Instruments

New System Combines 3D Culture Platform, Instruments, and Hypoxia-Monitoring Reagent

Innovative solid-tumor therapies will require a deeper understanding of the heterogeneous cancer microenvironment. The Warburg effect is a metabolic shift from oxidative phosphorylation to aerobic glycolysis that occurs, perhaps, even before the inevitable hypoxia as the tumor outgrows its insufficient and often irregular vasculature.

The culmination of metabolic changes in cancer cells contributes significantly to increased metastasis and drug resistance, aspects which increase patient mortality. Based on these observations, proteins that mediate metabolism and directly target hypoxic cells in tumors are attractive targets for therapeutic intervention.

Spheroids, self-assembled microscale aggregates of cells, generated in Perfecta3D® Hanging Drop Plates (HDPs, 3D Biomatrix) are a superior model of avascular microtumors. Because of their 3D structure, spheroids contain mass-transfer gradients of oxygen, nutrients, wastes, and even test compounds that are comparable to what occurs in tumors within the human body.

The metabolic gradients drive proliferation gradients and spheroids contain quiescent cells hypothesized to mimic drug-resistant populations within tumors. Given sufficient cell numbers and time in culture, spheroids develop hypoxic cores that progress to necrosis, closely mimicking what is observed in vivo.

This article illustrates a method to interrogate compounds that specifically target the hypoxic microenvironment of tumors. Fibroblasts can influence tumor cell behavior and condition the microenvironment; therefore, co-culture spheroids were created for a more physiological 3D model.

Colon carcinoma cells (HCT116) and human neonatal dermal fibroblasts were seeded in HDPs with the MultiFlo™ FX Microplate Dispenser (BioTek). The hypoxic cores of the resulting spheroids were imaged with the Cytation™ 3 (BioTek) using Cyto-ID® Hypoxia Red Detection Reagent (HRDR, Enzo Life Sciences), a cell-permeable dye that utilizes the nitroreductase activity present in hypoxic cells to generate a fluorescent signal.

The carbonic anhydrase inhibitor U 104 (R&D Systems) was used as a proof-of-concept test compound to target hypoxic cells. Carbonic anhydrase IX (CA IX) is a hypoxia-inducible factor 1-alpha regulated protein that functions to maintain intracellular pH. The U 104 family of inhibitors has been shown to reduce cancer cell growth and metastasis. The combination of 3D culture platform, instruments, and hypoxia-monitoring reagent is a robust system to identify effects of lead molecules.

Detailed experimental procedures and instrument parameters for these experiments can be found in the application note Inhibition of Hypoxic Tumor Cells using a Three-Dimensional Spheroid Model available at

Detecting Hypoxia Red Reagent Signal in Spheroids Containing RFP-Expressing Cells

When utilizing co-cultures for drug discovery, it is important to determine which cell populations are susceptible to each test compound. Unlabeled HCT116 and red fluorescent protein-expressing (RFP) fibroblast cells were harvested, combined in equal parts, and dispensed with the MultiFlo FX in 40 µL drops with 5,000 cells into each well.

The HDPs were incubated at 37°C and 5% CO2 and observed every 24 hours to monitor spheroid formation. For imaging, the entire HDP assembly was placed into the Cytation 3 since the imager can focus through the clear bottom tray. Resulting spheroids were used for experimentation as described below.

The HRDR was diluted in media and added to spheroid-containing wells followed by incubation for two, four, or six hours. To facilitate reduced background, the HRDR-containing media was washed out by removal of 10 µL of media and replacement with 10 µL of Dulbecco’s phosphate buffered saline. This was repeated 5–6 times to ensure a complete exchange. Figures 1A–1C illustrates that a six-hour incubation time generated the optimal signal-to-noise ratio, which became the incubation period for all subsequent experiments.

As both RFP used to mark the fibroblasts and the HRDR fluoresce in the red spectrum, brightfield imaging with the RFP filter versus the Texas Red filter (HRDR signal) was used to verify that the signals could be clearly separated. Overlaid brightfield and RFP images illustrate the spheroid boundary containing fibroblasts. Figure 1D shows the same brightfield image with the signal from the HRDR. When comparing the fluorescent signal in Figures 1D & 1E with the overlaid image in Figure 1F, it is evident that RFP fluorescence is not captured with the Texas Red filter.

Figure 1. Multichannel HCT116/fibroblast images following Hypoxia Red Reagent incubation. 4× images of Hypoxia Red Detection Reagent signal captured after (A) 2; (B) 4; and (C) 6 hours of incubation with the spheroid. Overlaid 4× images captured using (D) brightfield and RFP; (E) brightfield and Texas Red; and (F) all three imaging filter cubes.

Analysis of Carbonic Anhydrase Inhibition on Spheroid Hypoxia

U 104 was added to spheroid cultures by removal of 10 µL of media and replacement with 10 µL of media containing the test compound; repeated 5–6 times to ensure a complete exchange. Dosing was repeated daily for two weeks. HRDR was added and imaged on 1, 4, 7, and 14 days of compound treatment as described above. The Cellular Analysis feature of the Gen5 Data Analysis Software was employed to analyze the fluorescent signal only from the cells within a spheroid, thereby reducing background signal.

Figure 2 illustrates the effect of U 104 at a 10 µM concentration compared to the negative control. The raw fluorescent signal generated from active hypoxic cells within each spheroid was plotted for each compound concentration tested over the two-week dosing period (Figure 3A). Comparison of fluorescent units from day-one analysis to fluorescent units from subsequent day analyses was used for normalization and expressed as a percentage (Figure 3B).

U 104 specifically affects hypoxic tumor cells, as demonstrated by the dose-dependent decrease in nitroreductase activity over the 14-day dosing period. We hypothesize that the reduction in HRDR signal is due to increased cell death of hypoxic cells. This agrees with previously published results, that therapeutic treatment of tumor cells by inhibition of CA IX is accomplished, in part, by decreasing adaption of cells to the low extracellular pH found in hypoxic regions of primary tumors.

Figure 2. 4× brightfield and Texas Red overlaid images of HCT116/fibroblast spheroids, following Hypoxia Red Detection Reagent addition, incubation, and imaging.


Co-cultured spheroids were used successfully to detect the effects of a CA IX inhibitor on hypoxic tumor cells. The MultiFlo FX rapidly and efficiently dispensed cells, media, reagents, and test compounds into the Perfecta3D Hanging Drop Plates where spheroid formation and real-time imaging with the Cytation 3 occurred. Nitroreductase enzyme activity, as a measure of hypoxic cell health, was easily monitored with the incorporation of the Cyto-ID Hypoxia Red Detection Reagent.

This combination of 3D culture platform, instruments, and hypoxia-monitoring reagent can quickly and inexpensively provide critical preclinical data about the effects of test compounds on potentially drug resistant and metastatic cells found within a physiologically relevant in vitro model system.

Figure 3. U 104-induced reduction of hypoxic cell nitroreductase activity. (A) Mean raw fluorescence values from Hypoxia Red Detecting Reagent using a 4× objective and the Texas Red imaging cube; (B) Normalization of fluorescent signal calculated by the following formula: (Mean RFUDay X / Mean RFUDay 1).

Brad Larson is principal scientist at BioTek Instruments, Nicky Slawny ( is applications director at 3D Biomatrix, Wini Luty is product manager at Enzo Life Sciences, and Peter Banks is scientific director at BioTek Instruments.