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Impact of exogenous lactate on survival and radioresponse of carcinoma cells in vitro Janine Grotius a; Claudia Dittfeld a; Melanie Huether a; Wolfgang Mueller-Klieser b; Michael Baumann c; Leoni A. Kunz-Schughart a a Tumor Pathophysiology, OncoRay - Center for Radiation Research in Oncology, Faculty of Medicine Carl Gustav Carus, TU Dresden b Institute of Physiology and Pathophysiology, Johannes Gutenberg University Mainz, c OncoRay - Center for Radiation Research in Oncology, Faculty of Medicine Carl Gustav Carus, TU Dresden, Germany Online Publication Date: 01 November 2009

To cite this Article Grotius, Janine, Dittfeld, Claudia, Huether, Melanie, Mueller-Klieser, Wolfgang, Baumann, Michael and Kunz-

Schughart, Leoni A.(2009)'Impact of exogenous lactate on survival and radioresponse of carcinoma cells in vitro',International Journal of Radiation Biology,85:11,989 — 1001 To link to this Article: DOI: 10.3109/09553000903242156 URL: http://dx.doi.org/10.3109/09553000903242156

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Int. J. Radiat. Biol., Vol. 85, No. 11, November 2009, pp. 989–1001

Impact of exogenous lactate on survival and radioresponse of carcinoma cells in vitro

JANINE GROTIUS1, CLAUDIA DITTFELD1, MELANIE HUETHER1, WOLFGANG MUELLER-KLIESER2, MICHAEL BAUMANN3, & LEONI A. KUNZ-SCHUGHART1 Downloaded By: [SLUB Sachsische Landesbibliothek] At: 08:04 4 November 2009

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Tumor Pathophysiology, OncoRay – Center for Radiation Research in Oncology, Faculty of Medicine Carl Gustav Carus, TU Dresden, 2Institute of Physiology and Pathophysiology, Johannes Gutenberg University Mainz, and 3OncoRay – Center for Radiation Research in Oncology, Faculty of Medicine Carl Gustav Carus, TU Dresden, Germany (Received 8 April 2009; Revised 15 July 2009; Accepted 21 July 2009) Abstract Purpose: Tumour lactate levels have been shown to correlate with high radioresistance in tumour models in vivo. The study aimed to evaluate the impact of pathophysiological extracellular lactate concentrations and acidosis on the in vitro survival and radioresponse of various cancer cell lines. Materials and methods: HCT-116, HT29 (colorectal) and FaDu (HNSCC) carcinoma cells were studied. Lactate release rates were determined, and expression of the monocarboxylate transporter MCT1 and its cofactor CD147 were monitored by immunofluorescence and flow cytometry. Colony formation was compared for cells exposed to 20 mM exogenous lactate, acidosis (pH 6.4) and lactate plus acidosis relative to control and dose response curves (0.5–10 Gy) were documented. Results: All cell lines expressed MCT1 and CD147 and showed comparable lactate release rates. High lactate levels and acidosis slightly decreased HCT-116 colony forming capacity. This effect was neither additive nor did it affect radioresponse. Clonogenic survival of HT29 cells, however, was critically reduced in a lactate-enriched or acidic milieu and a synergistic effect was observed. Here, both conditions enhanced radiosensitivity. Exogenous lactate also impaired colony formation of FaDu cells but acidosis was ineffective. This cell line was more susceptible to irradiation under lactate exposure independent of pH. Conclusions: Tumour cell behaviour and radioresponse in a lactate environment is multifaceted. The consideration of lactate accumulation as a parameter affecting radiotherapeutic intervention and as a target for new therapeutic strategies is interesting but requires extended mechanistic studies.

Keywords: tumour pathophysiology, lactate, acidosis, cell survival, irradiation, radioresponse, MCT1

Introduction Many solid tumours are characterised by spatiotemporal heterologous supply conditions due to a histomorphological and functional chaos of their vascular network (Fukumura & Jain 2008, Fukumura & Jain 2007, Sivridis et al. 2003, Ribatti et al. 2007). Oxygen-deficiency as a pathophysiologic consequence is a well-established, yet still not entirely understood, direct radiation and chemotherapeutic resistance factor (Bertout et al. 2008, Ho¨ckel & Vaupel 2001, Tatum et al. 2006, Vaupel

2004, Vaupel et al. 2001) while other milieu conditions such as tissue acidosis and lactate accumulation are considered only as indirect modulators of tumour radioresponse. Enhanced levels of local and overall lactate in tumour tissues to some extent result from the pathologic vascular supply as oxygen deficiency leads to an increased cellular glycolytic flux (Pasteur effect) but disposal of waste products is impeded. In addition, genetic alterations as a consequence of malignant transformation are known to contribute to lactate accumulation under aerobic conditions in tumours.

Correspondence: Prof. Dr. Leoni A. Kunz-Schughart, OncoRay – Center for Radiation Research in Oncology, Faculty of Medicine Carl Gustav Carus, TU Dresden, Fetscherstraße 74, PF 86, 01307 Dresden, Germany. Tel: þ49 351 4587405. E-mail: [email protected] ISSN 0955-3002 print/ISSN 1362-3095 online Ó 2009 Informa UK Ltd. DOI: 10.3109/09553000903242156

Downloaded By: [SLUB Sachsische Landesbibliothek] At: 08:04 4 November 2009

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This may be true for genes (i) which control the expression or activity of glycolytic enzymes and related transporters and its modulators and/or (ii) which lead to a truncated citric acid cycle and support glutaminolysis (DeBerardinis et al. 2007, Gatenby & Gillies 2004, Helmlinger et al. 2002, Hsu & Sabatini 2008, Mazurek & Eigenbrodt 2003). In various tumour entities including squamous cell carcinomas of head and neck and adenocarcinomas of the rectum high lactate concentrations (48–10 mM as compared to normal tissues with *2 mM) were shown to correlate with risk of metastasis and/or reduced recurrence-free as well as overall survival (Brizel et al. 2001, Schwickert et al. 1995, Walenta et al. 1997, Walenta et al. 2003, Walenta et al. 2004). Lactate accumulation not only is thought to reflect the degree of malignancy but to have numerous adverse effects on malignant and tumour-associated stromal cell types which may causally contribute to the phenomenon described above. Lactate for example affects fibroblast and endothelial cell proliferation, differentiation and maturation processes and may thus modulate angiogenesis in a complex manner organising locoregional harmoniously collaborating metabolic domains (Koukourakis et al. 2006, Kumar et al. 2007, Schmid et al. 2007). In addition, lactate was recently shown to suppress the activation and/ or differentiation of various immune cell populations such as dendritic and T cells and supposedly participates in tumour-associated immune escape (Fischer et al. 2007, Gottfried et al. 2006a, b). With respect to the impact of lactate on tumour cell behaviour, new in vitro data imply a migration stimulating activity in glioma and will clearly encourage extended investigations in cancer cell lines and models of various tumour entities (Baumann et al. 2008). The present study was, however, motivated by the observation of high lactate levels in a panel of subcutaneous xenograft models to positively and independent of hypoxia correlate with radioresistance indicating that lactate causally and/or directly relates to radioresponse (Quennet et al. 2006). The experiments were designed to evaluate the impact of pathophysiological, high extracellular lactate levels and reduced pH on tumour cell survival and radioresponse in a classical in vitro assay. As a first approach to gain mechanistic insight, we monitored the effect of exogenous lactate exposure in three different tumour cell types that all express the proton-dependent lactate transporter MCT1 (Monocarboxylate transporter 1) as well as its cofactor CD147 (Cluster of Differentiation 147; Basigin, also know as EMMPRIN) and that do not critically differ in lactate release under control conditions.

Materials and methods Routine cell culturing and handling Two colon carcinoma cell lines (HCT-116 - near diploid, microsatellite instable; HT29 – aneuploid, microsatellite stable) purchased from the ATCC (American Type Culture Collection, Manassas, USA) and one HNSCC (Head and neck squamous cell carcinoma) cell line [FaDu ¼ FaDuDD, a subline of FaDu-ATCC HTB-43 (Eicheler et al. 2002)] were studied. All cells were routinely grown in DMEM (Dulbecco’s Modified Eagle Medium, PAN Biotech GmbH, Aidenbach, Germany) containing antibiotics, phenolred, 25 mM HEPES (4(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 5 mM glucose, 1 mM sodium pyruvate and 4 mM L-glutamine and supplemented with 10% FCS (Fetal Calf Serum, PAN Biotech GmbH). Culturing was performed at 37 8C in a 5% CO2 in air, humidified atmosphere. All cell lines were free of mycoplasm and checked for correct genetic profile. For experimental series, monolayer cultures were dissociated by enzymatic and mechanic means using a 0.05% trypsin/0.02% EDTA (Ethylenediaminetetraacetic acid) in PBS (Phosphate buffered saline, PAN Biotech GmbH) solution. Cell numbers and volumes were routinely determined via the Casy1 cell analyser system (Schaerfe System GmbH, Reutlingen, Germany). Experimental design and colony forming assays (CFA) CFA were carried out using cell-line adapted routine protocols (seeding densities: 100 or 300 cells per well in 6-well plates; incubation intervals: 10–17 days). The standard experimental design to examine the impact of exogenous lactate on colony forming capacity included the following four culture conditions: (i) DMEM at pH 7.2 (control), (ii) DMEM at (low) pH 6.4, (iii) DMEM supplemented with 20 mM lactate at pH 7.2, and (iv) DMEM supplemented with 20 mM lactate at pH 6.4. Lactate solutions were freshly prepared for each experiment and day of treatment. For this purpose, appropriate amounts of crystalline lactic acid stored at 4 8C in an exsiccator were dissolved in supplemented DMEM to obtain pH re-adjusted (pH 7.2 + 0.1) or non-adjusted (pH 6.4 + 0.1) 20 mM lactate working concentrations for immediate usage. All solutions were filtered through 0.2 mm sterile filters. Culture condition (C) was obtained by adjusting the medium pH with 4 N HCl. Exponentially growing cells in T25 culture flasks were exposed for 20 h at 37 8C to one of the four conditions of interest (i–iv) by medium exchange. Then, cells were dissociated and single cell suspensions were prepared by resuspension in the


Lactate and cellular radiation response respective freshly prepared media (i–iv) to inoculate the defined cell numbers in 6-well plates. For irradiation, plates were incubated under culture conditions for 4 h to allow cell adherence prior to treatment with single doses ranging from 0.5–10 Gy (200kV X-Rays; 0.5 mm Cu filter; YxlonY.TU 320; Yxlon.international, Hamburg, Germany). After 10–17 days (cell-line specific), colonies were routinely stained with 0.5% crystal violet followed by manual microscopic monitoring using an AxioVert200 microscope (Carl Zeiss MicroImaging GmbH, Heidelberg, Germany). Colonies of 450 cells were counted. Plating efficiencies were documented and the surviving fraction at 2Gy (SF2Gy) was calculated from the dose response curves. All experiments were performed at least in triplicate (n ¼ 3–4) with a minimum of three individual wells per condition in each experiment. Mean values for individual experiments were calculated and then averaged to estimate the inter-experimental mean plus standard deviation for all experiments. Average values with inter-experimental standard deviation are shown in most graphs if not stated otherwise. Statistical analysis was performed with a two-tailed t test, with p 5 0.05 being considered a statistically significant difference between two populations. Determination of lactate release To determine lactate release rates, medium of exponentially growing and confluent monolayer cultures, respectively, was replaced by fresh standard DMEM. After an incubation of 24 h conditioned media were collected, aliquoted and frozen at 720 8C. Lactate content in cell culture supernatants was measured using a standardised enzymatic, lactateoxidase based assay kit with an extinction measured at 520 nm (SYNCHRON CX1-System LAC, Beckman Coulter; Fullerton, USA). After medium retrieval, cell numbers and volumes were analysed for each well using the Casy 1 cell analyser system. Parallel wells were used for the assessment of cell numbers at the onset of the 24 h experiment, i.e., before medium renewal. For exponentially growing monolayers, mean cell numbers per well over a period of 24 h were estimated from these data via exponential fitting. For confluent monolayer cultures only the cell number at supernatant sampling was used for the calculation of cellular and volume-related release rates. Immunfluorescence imaging and flow cytometry To visualise the expression of the monocarboxylate transporters MCT1, (1–1.5) 6 105 cells were seeded on sterile glass slides and cultured for 2–3 days in

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standard medium. Exponential cells were fixed in 100% methanol and permeabilised with 0.2% TritonX100 in PBS. After three washing steps with PBS, cells were incubated for 30 min with 1% BSA (Bovine serum albumin) in PBS to block nonspecific antibody binding. Cells were incubated with a primary polyclonal mouse-anti-human MCT1 antibody (1:250, working concentration: 4 mg/ml; abcam, Cambridge, USA) for 30 min. To verify specificity of MCT1 staining, a rabbit immunoglobulin fraction (Dako Denmark A/S, Glostrup, Denmark) was used as polyclonal antibody control at the respective concentration in parallel samples. In addition, a peptide consisting of amino acids 488–500 of MCT1 (abcam, USA) was provided at concentrations of 0.04–4 mg/ml to block the specific binding of the anti-MCT1 antibody and respective staining of MCT-1 in the cells. An Alexa Fluor 488-conjugated goat anti-mouse secondary antibody (1:500, working concentration: 4 mg/ml, Invitrogen, Carlsbad, USA) was then applied for 1 h. All incubation steps took place at room temperature. Nuclei were counterstained with DAPI (40 ,6-diamidino-2-phenylindole, working concentration: 1 mM). Preparations were covered with Dako Fluorescent Mounting Medium (Dako Denmark A/S, Denmark) and monitored via an AXIO Imager.M1 equipped with an AxioCam MRm camera using the AxioVision 4.5 software (all from Carl Zeiss MicroImaging GmbH, Heidelberg, Germany). CD147 cell surface expression in exponentially grown, dissociated single cell suspensions was analysed by flow cytometry following staining with a FITC (Fluorescein isothiocyanate)-conjugated, anti-human CD147 antibody relative to the respective isotype-control (both from dianova GmbH; Hamburg, Germany). Both antibodies were applied by incubating 16106 cells in 70 ml antibody staining solution (Ab dilution 1:3.5 in PBS containing 2 mM EDTA and 0.5% BSA) for 30 minutes at 4 8C according to the manufacturer’s instructions. Propidiumiodide (working concentration: 2 mg/ml) was applied to discriminate membrane-defect cells. A total of 2 6 104 events was recorded on a BD FACS Scan (BD Biosciences, San Jose, USA) and data were analysed with the FlowJo 7.2.4 software (TreeStar, Inc., Ashland, USA).

Results Lactate release, MCT1 and CD147 expression Before the onset of our experimental series, we verified that the cell lines of primary interest (colorectal HCT-116 and HT29 cells), both at exponential and confluent growth phases do not essentially differ in their cellular and volume-related


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lactate release rates. This was to guarantee that extracellular lactate levels are primarily determined by the addition of the 20 mM exogenous lactate. The cell lines were also checked for their expression of the ubiquitous, proton-dependent monocarboxylate transporter MCT1 and its cofactor CD147. Immunofluorescent staining revealed that MCT1 is expressed by both cell lines and located on the cell membrane. Flow cytometric analyses verified CD147 positivity. The same is true for the cell line FaDu which was included in the study later (Figure 1). Semiquantitative determination of CD147 in three independent flow cytometric experiments implies a marginally lower expression of CD147 in exponential HT29 cells as compared with HCT-116 and FaDu cells (relative mean: HCT-116: 11.0 + 1.2; HT29: 7.4 + 0.3; FaDu 10.1 + 1.9; Figure 1). The volume-related lactate release rates of exponentially growing cells under standard culture conditions ranged between 0.8–1.0 g/ml cell in 24 h for all three cell lines studied.

Impact of high lactate levels and acidosis on HCT-116 and HT29 cell survival The initial experimental series were performed to examine the effect of pathophysiological lactate levels and acidosis on the clonogenic survival of two colorectal cancer cell lines. It is noted that the experimental set-up was not designed to obtain longterm acidic conditions but only to provide protons at the onset of lactate exposition. The pH normalised within two hours after transfer into a 37 8C, humidified 5% CO2 in air atmosphere. Exponential cells were pre-incubated for 20 h using the various media conditions (i-iv) as described in the Materials and Methods section, i.e., DMEM containing 20 mM exogenous lactate with (iv) and without (iii) acidosis or acidosis alone (ii) in comparison to standard medium (i). Cultures were then dissociated and seeded in the respective media for the evaluation of colony forming capacity. If not stated otherwise, graphs show the averaged mean values (þ standard deviation) of 3–4 independent experiments.

Figure 1. Cell lines of interest express MCT1 and its co-factor CD147. (A) Cell surface localisation of the monocarboxylate transporter MCT1 (green) in exponentially growing HCT-116, HT29 and FaDu cells is shown in the upper panel. A two-step immunofluorescence staining was carried out with a polyclonal anti-human MCT1 and an Alexa Fluor 488-conjugated secondary antibody, DAPI (blue) counterstaining was used to visualise all nuclei. Staining specificity was verified in various control experiments as documented for FaDu cells in the lower panel, i.e., by using secondary antibody only, by application of a rabbit immunoglobulin fraction as polyclonal primary antibody control and by positive concentration–dependent blockade of binding and staining, respectively, by an MCT1 peptide (aa 488-500). (B) Flow cytometric histograms in the lower panel show the expression of the MCT1 co-factor CD147 in exponentially grown, dissociated HCT-116, HT29 and FaDu cells stained with a FITC-conjugated anti-human CD147 antibody (solid line) or the respective isotype-control (dotted line).


Lactate and cellular radiation response We first observed that the majority of HCT-116 colonies generated under pathophysiological conditions were smaller than those in controls (Figure 2A) and the viability/survival of HCT-116 cells was slightly but insignificantly reduced by both pH reduction from 7.2 to 6.4 and exposure to 20 mM exogenous lactate. This minor effect was not additive (Figure 2B). Reduction of colony size in CFA experiments under pathophysiological conditions were even more pronounced in HT29 cells (Figure 3A). Accordingly, clonogenic survival of HT29 cells was significantly reduced under high exogenous lactate conditions and under acidosis. Here, 20 mM lactate at low pH (6.4) reproducibly resulted in a synergistic effect (Figure 3B). Due to this observation, the impact of combined (lactate þ acidosis) exposure on radioresponse in HT29 cells could not be evaluated using the standard combination conditions (20 mM lactate, pH 6.4, pre-treatment for 20 h).

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Two experimental strategies were tested to allow further examination of this phenomenon. First, we reduced lactate concentration for exposure and found 10 mM lactate and a respective pH of 6.8 to not affect plating efficiency at all, while 15 mM and a pH of 6.6 induced intermediate, additive but not synergistic effects in HT29 cells (Figure 4A/B). Thus, clonogenic survival of HT29 is reduced in a pH and lactate concentration dependent manner. In a second approach the interval of exposure to 20 mM lactate and acidosis (pH 6.4) was reduced. Here, cells were not pre-incubated but only resuspended in the respective media when seeded for CFA. With this setting, comparable but reduced effects of lactate exposure and acidosis on HT29 cell survival were observed (Figure 4C). According to the 20 h pre-treatment approach (Figure 3B) and opposed to the 15 mM plus pH 6.6 condition (Figure 4B), 20 mM lactate at pH 6.4 again resulted

Figure 2. Lactate marginally affects cell survival but not radioresponse of HCT-116 cells. Colony formation assays (CFA) were performed _ for HCT116 cells pre-exposed for 20 h to the following four different milieu conditions: (i) DMEM at pH 7.2 (control), (ii) DMEM at (low) pH 6.4, (iii) DMEM supplemented with 20 mM lactate at pH 7.2, and (iv) DMEM supplemented with 20 mM lactate at pH 6.4. CFA setup: 100 cells/well; culturing in respective media for 10 days after 20 h pre-exposure to the milieu conditions (i–iv). (A) Representative images (10 6 objective) of colonies formed under pathophysiological milieu conditions. A slight decrease in colony size is observed under high lactate and/or acidic conditions. (B) Average plating efficiencies (þSD) from n ¼ 3 independent experiments reveal a slight but not additive impact of exogenous lactate and acidosis on clonogenic cell survival. (C) Representative dose response curves of clonogenic HCT-116 cell survival in the milieu conditions (i–iv) as mentioned with a single dose irradiation regime (0.5–10 Gy) 4 hours after plating. Clonogenic survival at 0 Gy (control) for each condition was set to 100% for normalisation. Symbols represent mean values + intra-experimental SD. (D) Average clonogenic survival at 2 Gy (SF2Gy þ inter-experimental SD) determined from n ¼ 3 independent experiments according to (C). Neither exogenous lactate nor acidosis affect radioresponse of this cell line.


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Figure 3. Lactate significantly reduces cell survival and radioresistance of HT29 cells. Colony formation assays (CFA) were performed for HT29 cells pre-exposed for 20 h to the different milieu conditions depicted in Figure 2 legend; CFA set-up: 100 cells/well; culturing in respective media for 17 days after 20 h pre-exposure to the milieu conditions (i–iv). (A) Representative images (10 6 objective) of colonies formed under pathophysiological milieu conditions. A massive decrease in colony size is observed under high lactate and acidic conditions. (B) Average plating efficiencies (þinter-experimental SD) from n ¼ 3 independent experiments indicate a significant loss of clonogenic survival of HT29 cells in a lactate-enriched milieu and a synergistic effect when combined with acidosis. (C) Representative dose response curves of clonogenic HT29 cell survival in the milieu conditions (i–iv) as mentioned with a single dose irradiation regime (0.5–10 Gy) 4 h after plating. Clonogenic survival at 0 Gy (control) for each condition was set to 100% for normalisation. Symbols represent mean values + intra-experimental SD. (D) Average clonogenic survival at 2 Gy (SF2Gy þ inter-experimental SD) determined from n ¼ 3 independent experiments according to (C). Lactate significantly enhances radiosensitivity of HT29 cells. *p 5 0.05; **p 5 0.005.

in a synergistic decrease in clonogenic HT29 cell survival. This observation in addition to the clear advantage of the latter experimental set-up with only one preparation of single cells required to study all four conditions in respective CFA series motivated us to proceed with this approach for evaluating radioresponse in HT29 cells under combined pathophysiological conditions. Impact of high lactate and acidosis on radioresponse of HCT-116 and HT29 Experiments were designed according to the clonogenic cell survival assessment under pathophysiologic conditions with a 20 h pre-incubation interval. Yet, CFA were irradiated with single doses of 0.5–10 Gy

4 h after single cell seeding to guarantee cell attachment, which was routinely monitored. Accordingly, cells were exposed to the four different media conditions (i–iv) for a total of 24 h before irradiation. With this setting, 20 mM lactate neither without nor with acidosis (pH 6.4) affected radioresponse of HCT-116 cells. This is documented in Figure 2C showing the dose-effect curves of a representative single experiment and in Figure 2D which summarises the survival fraction at 2Gy (SF2Gy) as calculated and averaged for the four different conditions from three independent experiments. In HT29 cultures both lactate exposure and acidosis led to a reproducible but only slight enhancement in radiosensitivity as indicated in the dose-effect curves of a representative experiment in

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Figure 4. HT29 cell survival is affected by lactate and pH in a dose-dependent manner. Lactate and acidosis do not synergistically affect radioresponse of HT29 cells in vitro. Colony formation assays (CFA) were performed for HT29 cells pre-exposed for 20 h to different concentrations of lactate (A/B) or were seeded without pre-exposure in media with the different milieu conditions depicted in Figure 2 legend; CFA set-up was according to Figure 3. Average plating efficiencies (þinter-experimental SD) from n ¼ 3 independent experiments for each setting indicate that (A) 10 mM lactate and a pH of 6.8 do not affect clonogenic HT29 cell survival while (B) 15 mM lactate and a pH of 6.6 reduce the formation of HT29 colonies showing an additive effect, and (C) 20 mM and a pH of 6.4 provided only when seeding the cells but without pre-exposure leads to a pronounced synergistic effects on HT29 cell survival. (D) Representative dose response curves of clonogenic HT29 cell survival seeded in the milieu conditions according to (C) with a single dose irradiation regime (0.5–10 Gy) 4 h after plating. Clonogenic survival at 0 Gy (control) for each condition was set to 100% for normalisation. Symbols represent mean values + intraexperimental SD. (E) Average clonogenic survival at 2 Gy (SF2Gy þ inter-experimental SD) determined from n ¼ 3 independent experiments according to (D). *p 5 0.05; **p 5 0.005.

Figure 3C as well as in Figure 3D showing the averaged SF2Gy values. As a consequence of the synergistic effect of lactate and acidosis on the cell survival of HT29 (Figure 3B), radioresponse could not be determined under combined high-lactate, low pH pre-treatment conditions. The additional strat-

egy to exclusively seed HT29 cells for CFA in the four different conditions and irradiate them after a short pre-incubation of only 4 h confirms that exogenous lactate leads to a slight increase in radioresponse. The pH effect, however, is not detectable any longer. In spite of this, dose-response


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Figure 5. Lactate significantly reduces cell survival and radioresistance of FaDu cells. Colony formation assays (CFA) were performed for FaDu cells pre-exposed for 20 h to the different milieu conditions depicted in Figure 2 legend; CFA set-up: 300 cells/well; culturing in respective media for 14 days after 20 h pre-exposure to the milieu conditions (i–iv). (A) Representative images (5 6 objective) of colonies formed under pathophysiological milieu conditions. A decrease in colony size is observed under high lactate but not acidic conditions. (B) Average plating efficiencies (þSD) from n ¼ 3 independent experiments indicate a significant loss of clonogenic survival of FaDu cells in a lactate-enriched but not in the acidic milieu; an additive or synergistic effect is absent. (C) Representative dose response curves of clonogenic FaDu cell survival in the milieu conditions (i–iv) as mentioned with a single dose irradiation regime (0.5–10 Gy) 4 h after plating. Clonogenic survival at 0 Gy (control) for each condition was set to 100% for normalisation. Symbols represent mean values + intraexperimental SD. (D) Mean clonogenic survival at 2 Gy (SF2Gy þ inter-experimental SD) determined from n ¼ 3 independent experiments according to (C). Lactate but not acidosis enhances radiosensitivity of FaDu cells. *p 5 0.05; **p 5 0.005.

curves and SF2Gy values imply a radiosensitisation of HT29 cells in an acidic 20 mM lactate milieu when compared to control conditions (Figure 4D/E). Impact of high lactate and acidosis on cell survival and radioresponse of FaDu The data recorded for the colorectal carcinoma cell lines do not support the hypothesis of exogenous lactate as a direct, general or hypoxia-independent radioresistance factor in vitro. However, lactate concentrations correlated with radioresistance in a panel of human squamous cell carcinoma xenograft mouse models in vivo (Quennet et al. 2006). We therefore included the HNSCC cell line FaDu in our in vitro experimental series as a potentially better model. The milieu conditions and experimental design was according to the standard setting with 20 h pre-exposure. Figure 5A depicts the reproducible observation of reduced sizes of FaDu colonies

in a 20 mM lactate but not acidic environment. Colony counting revealed that exogenous lactate leads to a reduced colony formation of FaDu while acidosis alone or in combination with lactate does not alter clonogenic survival (Figure 5B). With respect to radioresponse, high exogenous lactate levels were shown to significantly sensitise FaDu cells whereas neither an impact of acidosis alone nor an additive effect of acidosis with 20 mM exogenous lactate was recorded as documented in Figures 5C,D. Relevance of glucose availability on lactate induced effects in FaDu In many laboratories HNSCC cell cultures are routinely grown in glucose-enriched media to promote cell survival and proliferative activity, whereas in our laboratory such cells are routinely grown in a physiological glucose concentration of 5 mM.


Lactate and cellular radiation response Consequently, we tested, whether high glucose concentrations of 25 mM would lead to an enhanced clonogenic survival or would modify the impact of exogenous lactate on cell survival and radioresponse of FaDu cells. In triplicate experiments with the standard 20 h pre-treatment setting, plating efficiencies in 5 mM vs. 25 mM glucose containing media did not significantly differ and the decrease of cell survival of FaDu cells in CFA after addition of exogenous lactate was comparable (Figure 6A, B). There was no impact of acidosis and no additive effect of acidosis plus 20 mM lactate neither at 5 mM nor at 25 mM glucose (Figure 6A vs. 5B). However, the radiosensitising effect of high exogenous lactate in FaDu cells was attenuated or compensated when grown in 25 mM glucose (Figure 6B vs. 5D). Discussion The aim of the present study was to evaluate the direct impact of exogenous lactate on cell survival and radioresponse of tumour cells in vitro. The initial experiments focused on the two colorectal cell lines HCT-116, which is near diploid, microsatellite instable (MSI), and HT29, an aneuploid chromosomal instable (CIN) cell line, as they reflect the two different pathways of colorectal cancer carcinogenesis (Kleivi et al. 2004). These studies were extended to the cell line FaDu which is known to produce xenograft tumours with an intermediate lactate accumulation (Quennet et al. 2006). All cell lines were analysed in conventional CFA under high lactate and/or transient acidic milieu conditions. Two aspects of our observations shall be particularly discussed. The first part of the discussion focuses on

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the large heterogeneity in cellular response to the pathophysiological milieu conditions in vitro while the second part highlights and discusses the potential role of lactate accumulation in radioresponse. Cell survival under pathophysiological milieu conditions in vitro The three cell lines studied show three different pattern of behaviour when exposed to exogenous lactate and transient acidosis: (i) HCT-116 cell survival is hardly influenced by exogenous lactate levels and/or acidosis, (ii) FaDu cells are sensitive to high lactate independent of the presence of protons and (iii) HT29 are most sensitive with lactate reducing cell survival in a concentration dependent and pH synergistic manner. A relationship to transport mechanisms, functionality and quantity of transporters, such as the MCT1, is likely to play a role in HT29 susceptibility since lactate transport via monocarboxylate transporter is associated with cotransport of protons (Cheeti et al. 2006). Since acidosis at the onset of exposure to lactate did not modulate lactate efficacy in FaDu cells, it is speculated that monocarboxylate transporters are either less relevant, and/or other functional aspects are to be considered in this cell line. The monocarboxylate transporter MCT1, as well as its cofactor CD147 which is essential for its function (Kirk et al. 2000, Wilson et al. 2002), is expressed in all cell lines of interest. However, MCT1 expression and activity have to be quantified in further studies. Similarly, other functional transporter isoforms which differ in their tissue distribution and biochemical properties should also be involved in future analyses (Halestrap & Meredith

Figure 6. Glucose availability does not affect lactate dependent loss of FaDu cell survival but abrogates the radiosensitising effect in the surviving cell population. Colony formation assays (CFA) were performed for FaDu cells pre-exposed for 20 h to the different milieu conditions (i–iv) depicted in Figure 2 legend. The CFA set-up was according to Figure 3 but DMEM containing high (25 mM) glucose was used for pre-exposure and cell culturing. (A) Average plating efficiencies (þinter-experimental SD) from n ¼ 4 independent experiments. Glucose content does not affect plating efficiency nor does it alter the significant and pH independent loss of clonogenic survival of FaDu cells in the 20 mM lactate-enriched milieu. (B) Average clonogenic survival at 2 Gy (SF2Gy þ inter-experimental SD) determined from n ¼ 4 independent experiments with the 0 Gy control for each condition set to 100% for normalisation.


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2004, Halestrap & Price 1999). One of these is MCT4 (Monocarboxylate transporter 4) which has in addition to MCT1 been shown to be overexpressed in primary colorectal carcinomas (Pinheiro et al. 2008). While MCT1 is ubiquitously expressed, highly affine for lactate and supposedly contributes to the uptake of lactate in various oxygenated cell types to fuel respiration, MCT4 possesses a lower affinity, is expressed in cells with a high glycolytic activity and/or under hypoxia and seems to play a major role in cellular lactate disposal (Dimmer et al. 2000, Sonveaux et al. 2008, Ullah et al. 2006). To gain mechanistic insight into lactate transport processes in the cells of interest, the aspect of oxygen deficiency as well as the application of MCT inhibitors such as CHC (a-cyano-4-hydroxycinnamate), according to the study by Sonveaux et al. (2008), are envisioned to complete the phenomenological data presented herein. It will then also be important to modulate other conventional media supplements in concert with lactate, acidosis and oxygen availability. This includes for example pyruvate, glutamine and glucose which in relevant physiologic concentrations may stimulate, convey or lead to a switch to certain metabolic pathways in particular in tumour cells as a consequence of different malignant transformation. Various genetic alterations in tumour cells are associated with differences in lactate-related metabolic pathways, as they result in an increased glycolytic flux according to the Warburg effect, metabolisation of lactate or enhanced aerobic glutaminolysis. Beside the changes in expression and activity of relevant transporters, such as the MCTs or the glucose transporters, e.g., GLUT1, variances in activity and expression of diverse glycolytic enzymes as well as a truncated citric acid cycle are described (DeBerardinis et al. 2007, Ganapathy et al. 2009, Mazurek et al. 2005, Walenta & Mueller-Klieser 2004). Some of the transporters and enzymes were shown to be modulated by Ras oncoproteins, and ectopic expression of oncogenic Ras in various cell models was accompanied by enhanced glycolytic flux and increased sensitivity to glycolytic inhibitors (Chesney 2006, Gatenby & Gillies 2004, Mazurek et al. 2001, Telang et al. 2007). K-ras (Kirsten-ras) mutations as an early and frequent event in spontaneous colorectal carcinogenesis may thus contribute to a different metabolic behaviour of the colorectal tumour cell lines since HCT-116 cells carry an activating K-ras mutation but the lactatesensitive HT29 cells are K-ras wt (Gayet et al. 2001). This is consistent for the K-ras wt HNSCC cell line FaDu (Toulany et al. 2005) which was also sensitive to lactate. Furthermore, the p53 tumour suppressor status of the cells deserves close attention, since p53, a sentinel in colorectal carcinogenesis, appears to

regulate a number of key enzymes along the glycolytic pathway and to affect mitochondrial respiration in a multifactorial manner (Bensaad & Vousden 2007, Ma et al. 2007, Yeung et al. 2008). Interestingly, HCT-116 cells which are rather insensitive to exogenous lactate are p53 wt, while the more sensitive cell line HT29 as well as the FaDu cells applied herein do not express functional p53 protein (Eicheler et al. 2002, Gayet et al. 2001). Lactate accumulation and radioresponse Taking advantage of the high variability, we used the three cell lines to examine whether exogenous lactate directly affects radioresponse of cells with different genetic background. The study was motivated specifically by the observation of a positive correlation between lactate levels and radioresponse in a panel of in vivo squamous cell carcinoma models (Quennet et al. 2006). However, in the conventional in vitro assay lactate-enriched medium neither in absence nor presence of protons induced radioresistance. The results rather indicate a moderate radiosensitising effect of exogenous lactate. This apparent conflict of in vitro and in vivo results may have been caused by a variety of mechanisms which deserve further investigation. This seems important for addressing the potential of pretherapeutic tumour lactate determinations as prognostic or predictive parameter for individualising radiotherapy or combined modality treatments. One important aspect is that the correlation of tumour lactate concentration and results of radiotherapy in the tumour xenografts in vivo was based on the comparison of the average values per tumour line with TCD50 (tumour control dose 50%) values determined in parallel for the same tumour line (Quennet et al. 2006, Yaromina et al. 2007). Thus, these experiments detected intertumoural (or inter tumour line) differences in lactate level as a prognostic factor for the outcome of radiotherapy. Differences of the lactate concentration between individual tumours of the same line and the chance for local tumour control were not investigated in that study but would be expected to be closer to the in vitro observations of cell-line specific lactate-induced changes in the CFA reported here. An experimental design for in vivo verification could be based either on biopsies or MRI (Magnetic Resonance Imaging) technique before treatment and follow-up of exactly the same individual tumour for local tumour control after irradiation. Such an approach to evaluating the predictive impact of parameters of the microenvironment measured by PET (Positron Emission Tomography) has been reported recently (Schu¨tze et al. 2007). Another important difference of the in vivo and in vitro experiments that needs to be addressed in

Lactate and cellular radiation response further studies is that the in vivo lactate concentration may be a surrogate marker for other parameters of the tumour micromilieu which could be associated with radioresistance, e.g., perfusion (Yaromina et al. 2009). Such associations would not be reflected in the in vitro experiments reported herein.


Perspectives We conclude that tumour cell behaviour in a lactate environment is multifaceted and that the correlation of radioresponse with lactate level in vivo is not caused by a direct impact of exogenous lactate on tumour cells. We rather hypothesise that it either reflects an intrinsic metabolic switch that needs to be elucidated to identify the relevant radioresponse modifier(s), is associated with a complex regulation of monocarboxylate transporters and metabolic enzymes, is caused by differences in the experimental designs reflecting either intratumoural or intertumoural differences or is due to other factors such as surrogate association between different parameters of the tumour micromilieu. The contribution of high lactate conditions to malignant progression of tumour cells (Walenta & Mueller-Klieser 2004), but also its adverse impact on various stromal cell types such as fibroblasts, endothelial and immune cells (Fischer et al. 2007, Gottfried et al. 2006b, Koukourakis et al. 2006, Schmid et al. 2007) that are present in tumour tissue in vivo may be assessed by sophisticated in vitro techniques. Such in vitro approaches, in addition to detailed further in vivo studies, are envisioned to shed more light on this phenomenon. The idea of lactate accumulation as parameter reflecting and affecting radiotherapeutic intervention and as target for new therapeutic strategies is interesting and potentially highly important for clinical radiotherapy which clearly calls for such extended mechanistic studies. Acknowledgements The authors gratefully acknowledge the excellent technical assistance of Ms Melanie Huether. We thank the Institute of Clinical Chemistry and Laboratory Medicine (TU Dresden, Germany) for determination of lactate concentrations in cell culture supernatants. This work was supported by the German Federal Ministry of Education and Research (BMBF) through grant 01ZZ0502 (LAKS), by the Faculty of Medicine Carl Gustav Carus, TU Dresden (MedDrive - CD) and by the German Research Foundation (DFG) through grants Ba 1433/5 (MB) and Mu 576/14 (WMK). OncoRay is funded by the BMBF in the program

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‘Center for Innovation Competence’. Dr. Grotius and Dr. Dittfeld contributed equally to this article. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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