Cell culture

Cell lines were chosen based on published results on metabolism [20, 21], and spheroid production [22,23,24,25,26,27,28,29]. The goal was to achieve some metabolic variation among the cell lines, so lines were chosen that were purported to differ in their dependence on glycolysis and oxidative phosphorylation. Since 3D culture was essential to this study, only cell lines that had already been confirmed to produce spheroids were considered. This led to the selection of SW948 and HCT116, colorectal cancer (CRC) lines, and Panc1 and MIA-Pa-Ca-2, pancreatic ductal adenocarcinoma (PDAC) cell lines.

SW948 and MIA Pa-Ca-2 were purchased from European Collection of Authenticated Cell Cultures (ECACC), Panc1 and HCT116 cell lines were generously provided by collaborators at the Stavanger University Hospital Molecular Biology Lab. All cell lines were cultured in DMEM (Corning, Corning, USA) supplemented with 10% fetal bovine serum (FBS) (BioWest, Nuaillé, France), 5 mM glucose (Sigma-Aldrich, St. Louis, USA), 2 mM l-glutamine (Corning, Corning, USA), penicillin (100 U/ml), streptomycin (100 μg/ml) (Merck Millipore Corporation, Burlington, USA) in a humidified incubator at 37 °C with 5% CO2 infusion. Cells were grown in 2D adherent culture conditions, from which spheroids were prepared before each experiment. Spheroids were formed from a 40-μl volume of detached single-cell suspensions with 5000 cells, either in hanging drops in a dish, or in CELLSTAR cell repellent U-bottom plates (Greiner Bio-One, Kremsmünster, Austria). Spheroids were grown for 3 days (CRC) or 4 days (PDAC) before conducting metabolic assays.

2D doubling time

Cells were seeded in flat-bottom 96-well plate at a density of 5000 cells/well. At each timepoint, 3 wells were stained with Hoechst and the entire well imaged using Leica SP8 confocal microscope (Leica Microsystems, Mannheim, Germany) for direct cell detection and counting in the LASX software.

Spheroid growth

Spheroids were cultured in U-bottom plates as described above, but in densities from 200 cells/well to 10,000 cells/well. Transmitted light images of the spheroids were captured on days 3, 4, 6, and 8 using the Leica SP8 confocal microscope. Images were analyzed in ImageJ to obtain the cross-sectional area of the spheroid.

Metabolic flux assays

Mitochondrial respiration and glycolysis were measured using the Seahorse XF96e and XFp flux analyzers (Agilent). For mitochondrial oxygen measurements, assay media consisted of unbuffered, serum-free DMEM 8.3 g/L (D5030, Sigma-Aldrich, St. Louis, USA), NaCl 1.85 g/L, 2 mM l-glutamine, and 5 mM glucose adjusted pH to 7.4 before running the experiment. Before running mitochondrial and glycolysis assays, titration of CCCP over a range of concentrations was performed as per the Seahorse cell characterization procedure. The CCCP concentration chosen was that which yielded the maximum OCR value, for each cell line and culture method (Table S2).

2D assays

One day prior to assay analysis, 10,000 cells were seeded in each well of a XF96e cell culture plate using culture media as described above. Approximately 1 h before the assays, culture media was exchanged for 180 μl assay media. The plates were then incubated at 37 °C in a CO2-free incubator for 45 min–1 h prior to running the assay. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured over 90 min (15 mix and measure cycles), with compounds being injected every 3 cycles. For the mitochondrial respiration assays, the following compounds were injected sequentially (final concentrations in the wells): oligomycin (3 μM), CCCP (0.5 μM), rotenone (1 μM), and antimycin A (AMA, 1 μM) (all compound reagents from Sigma-Aldrich, St. Louis, USA). For the glycolysis assays, the assay media was not supplemented with glucose and the following compounds were injected sequentially (final concentrations in the wells): glucose (10 mM), oligomycin (3 μM), and 2-deoxy-D-glucose (100 mM). Protein concentration was measured in each well for normalization using standard BCA assay (PanReac AppliChem, Darmstadt, Germany) according to manufacturer’s instructions.

3D assays

Spheroid cell culture plates were used for the XF96e assays. Each well was coated with 25 μl CellTak (Corning, Corning, USA) at a concentration of 33 μg/ml. Spheroids were transferred from hanging drops and placed in the centers of wells in the spheroid assay, which contained 160 μl assay media. Plates were then incubated at 37 °C in a CO2-free incubator for 45 min–1 h prior to running the assay. OCR and ECAR were measured over 150 min (23 mix and measure cycles). For the mitochondrial respiration assays, the following compounds were injected sequentially (final concentrations in the wells): oligomycin after cycle 3 (3 μM), CCCP after cycle 9 (cell line dependent, see Table S2), rotenone after cycle 15 (1 μM), and antimycin A after cycle 19 (1 μM). For the glycolysis assays, the assay media consisted of unbuffered, serum-free DMEM 8.3 g/L, NaCl 1.85 g/L, 2 mM l-glutamine, adjusted pH to 7.4 before running the experiment and the following compounds were injected sequentially (final concentrations in the wells): glucose after cycle 3 (10 mM), oligomycin after cycle 9 (3 μM), and 2-deoxy-D-glucose after cycle 15 (100 mM). To use the data for comparison of metabolic phenotypes across cell models regardless of absolute metabolic activity, OCR and ECAR were normalized to basal OCR levels (MST) and ECAR level after glucose injection (GST). Statistical significance was determined in GraphPad using ordinary one-way ANOVA, correction for multiple comparisons by the Sidak method, with alpha = 0.05.

ATP production calculations

Buffer Factor of assay media was calculated using the Agilent Seahorse XF Buffer Factor Protocol. This number (1.70 mM/pH) was then used for the calculation of the ATP production rate after each injection following the information contained in the Agilent White Paper: “Quantifying Cellular ATP Production Rate Using Agilent Seahorse XF Technology” and Mookerjee et al .[30], according to the following equations:

$${mathrm{PPR}}_{mathrm{total}}=mathrm{ECAR}/mathrm{BP}$$

(1)

$${mathrm{PPR}}_{mathrm{resp}}=left({10}^{mathrm{pH}-{mathrm{pK}}_{mathrm{CO}2to mathrm{HCO}3}}/left(1+{10}^{mathrm{pH}-{mathrm{pK}}_{mathrm{CO}2to mathrm{HCO}3}}right)right)bullet {mathrm{OCR}}_{mathrm{mito}}$$

(2)

$${mathrm{PPR}}_{mathrm{glyc}}={mathrm{PPR}}_{mathrm{total}}-{mathrm{PPR}}_{mathrm{resp}}$$

(3)

$${mathrm{ATP}}_{mathrm{glyc}}=left[{mathrm{PPR}}_{mathrm{glyc}}bullet mathrm{ATP}/mathrm{lactate}right]+left[{mathrm{OCR}}_{mathrm{mito}}cdot 2mathrm{P}/mathrm{O}right]$$

(4)

$${mathrm{ATP}}_{mathrm{ox}}=left[{mathrm{OCR}}_{mathrm{coupled}}bullet 2mathrm{P}/mathrm{O}right]+left[{mathrm{OCR}}_{mathrm{mito}}cdot 2mathrm{P}/mathrm{O}right]$$

(5)

$${mathrm{ATP}}_{mathrm{total}}={mathrm{ATP}}_{mathrm{glyc}}+{mathrm{ATP}}_{mathrm{ox}}$$

(6)

The buffer factor was converted to a buffering power (BP) of 0.258 mpH/pmol H+ and combined with data from the glycolysis assays and residual OCR after AMA from mitochondrial stress test, run in parallel to GST. The total proton production rate (PPR) is found by dividing ECAR by BP (Eq. 1) and can be broken down into PPRresp and PPRglyc, with the PPRresp being from all mitochondrial oxygen consumption (non-mitochondrial is subtracted) (Eq. 2) where pH = 7.4 and pKCO2→HCO3 = 6.093. PPRglyc is the PPRtotal minus PPRresp (Eq. 3). The PPR is converted to ATP by calculations using known values for mol ATP yielded per mol oxygen consumed (P/O ratio). The result is ATPglyc (Eq. 4) that incorporates all ATP produced through glycolysis, including that which ends in lactate production or pyruvate that is shuttled to mitochondria resulting in production of CO2 (conversion of CO2 to bicarbonate is a major source of extracellular acidification in Seahorse assays [31] due to the use of unbuffered media). In the last case, this may or may not be coupled to ATP production via OXPHOS. Metabolic phenotypes are described using the bioenergetic indices also described in Mookerjee et al. [30].

$$mathrm{GI}=100 left({mathrm{ATP}}_{mathrm{glyc}}/{mathrm{ATP}}_{mathrm{total}}right)$$

(7)

$$mathrm{CI}={mathrm{GI}mathrm{g}}_{mathrm{lucose}}-{mathrm{GI}}_{mathrm{basal}}$$

(8)

$$mathrm{PI}={mathrm{GI}}_{mathrm{oligo}}-{mathrm{GI}}_{mathrm{glucose}}$$

(9)

The Glycolytic Index (GI) is a way to normalize between samples, allowing comparison of phenotype and contribution of glycolysis to ATP production regardless of changes in absolute ATP production rates (Eq. 7). The Crabtree Index (CI) quantifies the shift away from OXPHOS upon addition of glucose (Eq. 8) and The Pasteur Index (PI) quantifies the shift to OXPHOS upon “removal” of mitochondrial inhibitor oligomycin (Eq. 9). Statistical significance was determined in GraphPad using multiple t tests, correction for multiple comparisons by the Holm-Sidak method, with alpha = 0.05.

Metabolite assays

Media was collected from spheroids grown in ULA round-bottom 96-well plates and 2D cultures from flat 96-well plates, at days 0, 2, and 4, with no refeeding of media during this period. Day 0 for spheroids is culture day 3 for CRC spheroids and day 4 for PDAC spheroids and on this day they received full media exchange. The media from each timepoint was then used in the metabolite assays. Glucose concentration was assayed using the GlucCell glucose monitoring system (Cesco Bioengineering, Taichungy, Taiwan) according to the manufacturer’s instructions. Lactate was assayed using the l-Lactate Assay Kit (MAK329, Sigma-Aldrich, St. Louis, USA) according to the manufacturer’s instructions. Glutamine was assayed using the Glutamine/Glutamate Determination Kit (GLN1, Sigma-Aldrich, St. Louis, USA), adjusted for a low volume assay in a microplate. These results include both glutamine and endogenous glutamate. All values are presented normalized to surface area of cultures grown for equivalent period as that in the. Surface area in 2D was obtained from area measured in images on day 4 from the proliferation experiments. Surface area in 3D was estimated from area of spheroids in growth experiments after the same time in culture and extrapolated to the surface area of sphere, A = 4·π·r2.

Protein expression

Expression of metabolic proteins (MCT1, MCT4, GLUT1, UCP2, TOMM20) was measured by flow cytometry. Spheroids (approximately n = 600) comprised of 10,000 cells were produced and grown for 3–4 days before collection and dissociation using Accutase (Innovative Cell Technologies, Inc., San Diego). The spheroids were collected by rinsing plates with PBS and placed in a centrifuge tube. After centrifuging 5 min at 100 RCF, the supernatant was discarded and the pellet was resuspended in 5 ml Accutase. The tubes were placed on a rocker at room temperature and resuspended every 10 min using a P1000 pipet to gently disrupt the spheroids. Adherent cells were collected on the same day as the spheroids, also detached using Accutase. The cell lines varied considerably in incubation time needed for complete detachment and dissociation. Cell suspensions were counted using a Muse cell analyzer and Muse count and viability assay (Luminex, Austin, USA). The cell suspensions were then fixed with 3.7% PFA for 30 min. Cells were kept in PBS until staining. 5 × 105 cells were used for each staining reaction (at 1 × 106 cells/ml). Each tube was incubated in blocking/permeabilization buffer (PBS, 20% FCS, 0.05% Tween 20) for 1 h at room temp, rinsed with PBS before adding primary antibodies (single stain per tube) and incubated overnight at 4 °C, rocking. The next day, they were rinsed with PBS and the secondary antibodies were added, with the exception of unstained control and the tubes stained with conjugated TOMM20 antibody. Finally, cells were rinsed and resuspended in PBS containing 0.5% BSA for analysis in Bio-Rad S3e (Bio-Rad Laboratories, Hercules, USA) (experimental replicates 1 and 2) and CytoFlex (Beckman Coulter, Brea, USA) (experimental replicate 3). Primary antibodies: Rabbit Anti-Glucose Transporter GLUT1 antibody, EPR3915 (Abcam, Amsterdam, Netherlands); Mouse MCT1 Antibody SC-365501 and Mouse MCT4 Antibody SC-376140 (Santa Cruz Biotechnology, Dallas, USA); Rabbit UCP2 antibody (Bioss Antibodies Inc., Woburn, USA); Llama anti-rabbit IgG polyclonal antibody, CF™ 488A and anti-mouse IgG polyclonal antibody, CF™ 488A (Biotium, Fremont, USA). Datasets were analyzed using FCS Express (De Novo Software, Pasadena, USA), gated to singlet populations, and the median values were compared in ratios of 3D to 2D.

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