Pyruvate sensitizes pancreatic tumors to hypoxia-activated prodrug TH-302
- Jonathan W Wojtkowiak1Email author,
- Heather C Cornnell1,
- Shingo Matsumoto8,
- Keita Saito8,
- Yoichi Takakusagi8,
- Prasanta Dutta1,
- Munju Kim3,
- Xiaomeng Zhang1,
- Rafael Leos5, 7,
- Kate M Bailey1,
- Gary Martinez1,
- Mark C Lloyd4,
- Craig Weber5,
- James B Mitchell8,
- Ronald M Lynch5, 6,
- Amanda F Baker5, 7,
- Robert A Gatenby2, 3,
- Katarzyna A Rejniak3, 10,
- Charles Hart9,
- Murali C Krishna8 and
- Robert J Gillies1, 2
© Wojtkowiak et al.; licensee BioMed Central. 2015
Received: 21 August 2014
Accepted: 24 November 2014
Published: 29 January 2015
Hypoxic niches in solid tumors harbor therapy-resistant cells. Hypoxia-activated prodrugs (HAPs) have been designed to overcome this resistance and, to date, have begun to show clinical efficacy. However, clinical HAPs activity could be improved. In this study, we sought to identify non-pharmacological methods to acutely exacerbate tumor hypoxia to increase TH-302 activity in pancreatic ductal adenocarcinoma (PDAC) tumor models.
Three human PDAC cell lines with varying sensitivity to TH-302 (Hs766t > MiaPaCa-2 > SU.86.86) were used to establish PDAC xenograft models. PDAC cells were metabolically profiled in vitro and in vivo using the Seahorse XF system and hyperpolarized 13C pyruvate MRI, respectively, in addition to quantitative immunohistochemistry. The effect of exogenous pyruvate on tumor oxygenation was determined using electroparamagnetic resonance (EPR) oxygen imaging. Hs766t and MiaPaCa-2 cells exhibited a glycolytic phenotype in comparison to TH-302 resistant line SU.86.86. Supporting this observation is a higher lactate/pyruvate ratio in Hs766t and MiaPaCa xenografts as observed during hyperpolarized pyruvate MRI studies in vivo. Coincidentally, response to exogenous pyruvate both in vitro (Seahorse oxygen consumption) and in vivo (EPR oxygen imaging) was greatest in Hs766t and MiaPaCa models, possibly due to a higher mitochondrial reserve capacity. Changes in oxygen consumption and in vivo hypoxic status to pyruvate were limited in the SU.86.86 model. Combination therapy of pyruvate plus TH-302 in vivo significantly decreased tumor growth and increased survival in the MiaPaCa model and improved survival in Hs766t tumors.
Using metabolic profiling, functional imaging, and computational modeling, we show improved TH-302 activity by transiently increasing tumor hypoxia metabolically with exogenous pyruvate. Additionally, this work identified a set of biomarkers that may be used clinically to predict which tumors will be most responsive to pyruvate + TH-302 combination therapy. The results of this study support the concept that acute increases in tumor hypoxia can be beneficial for improving the clinical efficacy of HAPs and can positively impact the future treatment of PDAC and other cancers.
KeywordsHypoxia Hypoxia-activated prodrugs TH-302 Tumor microenvironment Metabolism Pancreatic cancer Functional imaging Computational modeling
Tumor hypoxia, a routinely observed phenotype, contributes to chemo- and radioresistance, causing major therapeutic concern . Due to extensive hypoxia, considerable effort has been devoted to harness its therapeutic potential. To exploit hypoxia, a class of hypoxia-activated prodrugs (HAPs) has been developed to selectively target tumor cells residing in hypoxic niches [2,3]. The most common class of HAPs is based on 2-nitroimidazoles, exemplified by TH-302, which is activated by cytochrome P450 reductase and contains alkylating nitrogen mustard, Br-isophosphoramide, as a cytotoxic effector . There are 12 clinical trials with TH-302 underway in a variety of cancers (e.g., pancreatic, sarcoma, breast, melanoma) (Additional file 1: Table S1).
The major predictor of TH-302 activity is the extent of tumor hypoxia . We therefore hypothesized that increasing tumor hypoxia could improve TH-302 activity. Experimental evidence show that the most effective approach to decrease tumor oxygenation (pO2) is increasing the oxygen consumption rate (OCR) . This was effective in RKO cells in which pharmacological inhibition of hypoxia-inducible factor 1-alpha (HIF-1α) with echinomycin increased OCR, decreased tumor pO2, and increased the activity of the HAP, tirapazamine . While HIF-1α inhibition was effective at increasing OCR, pharmacological inhibition may induce chronic hypoxia that could promote further adaptation and possibly increased HAP side effects. A separate study demonstrated that [1-13C]pyruvate infusion during hyperpolarized MRI studies substantially decreased tumor pO2 and increased hypoxia in squamous cell carcinoma (SCC) tumors . The effect was transient, with peak hypoxia observed at 30 min, returning to baseline within 5 h. Pyruvate also stimulates OCR across a panel of breast cancer cell lines , consistent with historical data that shows increased OCR in response to pyruvate (or lactate) in neurons and cardiac myocytes [10-12]. Because pyruvate 1) transiently decreases tumor pO2, 2) decreases tumor pO2 metabolically rather than pharmacologically, and 3) produces no detectable side effects, we hypothesize that pyruvate would be a successful adjuvant to enhance TH-302 efficacy.
A consequence of a desmoplastic stroma is poor perfusion, particularly in pancreatic ductal adenocarcinoma (PDAC). PDAC tumors are profoundly hypoxic in comparison to normal pancreatic tissue  which is a negative prognostic factor . TH-302 is highly effective in preclinical pancreatic models  and has been effective in phase I/II trials in combination with gemcitabine (GEM), increasing progression-free survival of late-stage cancers by 2 months . These encouraging results justified a phase III trial, which is ongoing (NCT01746979).
In the present study, we utilized PDAC models to demonstrate the ability of exogenous pyruvate to stimulate oxygen consumption in vitro and transiently decrease tumor pO2 in vivo. The decrease in tumor pO2 correlated with improved TH-302 efficacy, demonstrated by significant tumor volume decreases and increased survival. We also identified potential imaging and histological biomarkers that can be used to predict responsive tumor types. These data support the concept that temporarily increasing tumor hypoxia can improve the efficacy of HAPs, including TH-302, against PDAC and other cancer types in the clinic.
SU.86.86 and Hs766t cells were obtained from Threshold Pharmaceuticals (Redwood City, CA, USA), and MiaPaCa-2 cells were obtained from American Type Cell Collection (ATCC, Manassas, VA, USA). The cells were maintained in RPMI-1640 (Life Technologies) supplemented with 10% fetal bovine serum (FBS) (HyClone) (SU.86.86) and Dulbecco’s modified Eagle’s medium-F12 (DMEM-F12) (Life Technologies) supplemented with 10% FBS (Hs766t and MiaPaCa-2). All three cell lines were resuscitated from low passage with all experiments carried out with cells of passage numbers less than 10 (SU.86.86) and 20 (Hs766t and MiaPaCa-2).
All animal experiments were carried out at the Moffitt Cancer Center and National Cancer Institute in compliance with the Guide for the Care and Use of Laboratory Animal Resources . Experimental protocols conducted at Moffitt Cancer Center were approved by the Institutional Animal Care and Use Committee, University of South Florida. Experimental protocols performed at the National Cancer Institute were approved by the Institutional Animal Care and Use Committee, National Cancer Institute.
Tumor development and treatment
Surgically removed pancreatic xenografts were formalin fixed and paraffin embedded (FFPE). Untreated xenograft tissue cross sections were stained for carbonic anhydrase IX (anti-rabbit AB15086; Abcam, Cambridge, MA, USA), lactate dehydrogenase V (anti-rabbit AB9002; Abcam), monocarboxylate transporter 1 (anti-rabbit SC-50324; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and CD31 (anti-rabbit AB28364; Abcam). PDAC xenografts treated with TH-302 (50 mg/kg) for 48 h were formalin fixed, paraffin embedded, and stained for gamma-H2AX. Pimonidazole hydrochloride (60 mg/kg; Hypoxyprobe Inc.) was injected I.P. 1 h prior to tumor removal. Pimonidazole-positive tissue was detected using rabbit anti-sera against pimonidazole hydrochloride (2,627; Hypoxyprobe Inc.). A tissue microarray (TMA) containing FFPE human pancreatic adenocarcinoma specimen, acquired from Moffitt Cancer Center Total Cancer Care tissue database, was stained with antibodies against monocarboxylate transporter 1, monocarboxylate transporter 4 (anti-rabbit SC-376140; Santa Cruz Biotechnology), and pyruvate kinase isoform M2 (anti-mouse #3198; Cell Signaling Technology Inc., Danvers, MA, USA). The Ventana OmniMap anti-rabbit or anti-mouse secondary was used to detect primary antibodies. The detection system used was the Ventana ChromoMap Kit, and slides were then counterstained with hematoxylin. Histology stained slides were scanned using the Aperio ScanScope XT.
Seahorse XF metabolic assays
In vitro metabolic analysis using the XF96 (Seahorse Bioscience, Chicopee, MA, USA) was previously described . Briefly, real-time OC (pMole/min) and proton production rates (PPR, pMole/min) are determined by measuring oxygen and free protons in the media over a monolayer of cells. All collected data are normalized to protein using a standard BCA protein assay. Data are presented as the normalized mean ± standard deviation (S.D.)/mg protein.
Mitochondrial stress test
ATP-linked OCR and mitochondrial reserve capacity were determined following the sequential injections of oligomycin (1 μM), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (1 μM), and rotenone plus antimycin A (1 μM). ATP-linked oxygen consumption is determined by the difference between basal OCR and OCR following addition of oligomycin. Mitochondrial reserve capacity is defined as the difference of basal OCR and OCR following the addition of FCCP.
Glycolysis stress test
Glycolysis and glycolytic capacity were determined following sequential injections of D-glucose (2 g/L), oligomycin (1 μM), and 2-deoxyglucose (100 mM). Initially, the cells are glucose starved for 2 h prior to experiment start. Glycolysis was defined as PPR following the addition of D-glucose, and maximum glycolytic capacity was defined as PPR following the addition of oligomycin. PPR following treatment with 2-deoxyglucose is associated with non-glycolytic activity.
Glucose and pyruvate uptake assay
Glucose (K676) and pyruvate (K609) uptake from culture media was measured using colometric from BioVision Inc. The PDAC cells were seeded in standard growth media (see “Cell culture” section) and allowed to adhere overnight. At T = 0, the cells were rinsed twice with PBS and provided DMEM supplemented with 5.5 mM D-glucose, 1 mM L-glutamine, 2 mM sodium pyruvate, and 0.2% FBS. Following 12 h of culture time, the media were collected, centrifuged to remove cell debris, and immediately tested for glucose and pyruvate following BioVision assay kit protocols and compared to starting media conditions. Absorbance was quantified at 570 nM. Data are presented as percent substrate uptake (mean ± S.D.) relative to T = 0 culture media concentrations.
A pulsed electroparamagnetic resonance (EPR) scanner with a parallel coil resonator tuned to 300 MHz was used for oxygen imaging. After the mouse was placed in the resonator, triarylmethyl EPR oxygen tracer OX063 (1.125 mmol/kg bolus followed by 0.12 mmol/min of continuous infusion) was administrated via tail veil cannula. Single-point imaging with a three-gradient set was used to generate T2 * map, i.e., EPR line width map, which linearly correlates with local concentration of oxygen and allows pixelwise estimation of pO2. In total, four pO2 maps were obtained for each mouse before, 10, 30, and 60 min after IV injection of pyruvate (1.15 mmol/kg, bolus). After EPR study, the mice were transferred to a 7T MRI scanner (Bruker Bio-Spin MRI GmbH) for anatomic imaging.
The previously developed mathematical model microPD , which couples the reaction-diffusion/convection equations with the fluid-structure interaction methods on spatially explicit tissue structure, has been calibrated to match the histology of MiaPaCa-2 mouse xenograft, the PK/PD properties of oxygen, and both inactive and active TH-302 (Additional file 1: Methods). The simulations were executed, visualized, and analyzed using a suite of in-house Matlab routines. Tissue histology was digitized using ImageJ software and in-house Matlab routines.
A two-tailed unpaired Student’s t-test was used to determine statistical significance. The significance level was set at p < 0.05.
Identifying baseline TH-302 sensitivity in human PDAC tumor models
Three human PDAC cell lines (Hs766t, MiaPaCa-2, and SU.86.86) were used to evaluate the effect of exogenous pyruvate on tumor hypoxia and TH-302 efficacy. Subcutaneous tumors were shown to be histologically (H&E) similar to pancreatic orthotopic tumors (Additional file 1: Figure S1). These were used to accommodate imaging modalities used to monitor pyruvate metabolism and tumor oxygenation, which cannot currently be made with orthotopic PDAC tumors. TH-302 monotherapy studies were carried out against each tumor type. The greatest anti-tumor effect occurred in Hs766t tumors. MiaPaCa-2 tumors had an intermediate response, and SU.86.86 tumors displayed no sensitivity to TH-302 (Figure 1a–c). As hypoxia is the main determinant for TH-302 activity, immunohistochemistry (IHC) detecting pimonidazole and carbonic anhydrase IX, respectively, markers for physical  and biological  hypoxia, confirmed the greatest hypoxia in Hs766t tumors (Figure 1d,e). Hs766t cells also carry a nonsense mutation in FANCG  rendering them deficient in DNA repair and hence sensitizing them to alkylating agents, such as Br-ifosphoramide, the effector agent in TH-302. To confirm sensitivity to such agents, all three lines were treated with DNA cross-linking agent, mitomycin C (MMC), under normoxic conditions. Hs766t cells exhibited extreme sensitivity to low nanomolar concentrations of MMC (IC50 = 2.8 nM) in comparison to MiaPaCa-2 (IC50 = 15.1 nM) and SU.86.86 (IC50 = 29.7 nM) cells which carry no detectable DNA repair defects (Additional file 1: Figure S2a). Negligible TH-302 activity in SU.86.86 may be attributed to a well-oxygenated tumor environment as evidence by low CA9 expression and reduced sensitivity to DNA cross-linking agents. In vivo tumor doubling times also correlated with TH-302 sensitivity, as faster growing tumors (Hs766t) were more hypoxic and sensitive to TH-302 than slower growing tumors (SU.86.86), which we interpret as a higher propensity to outgrow existing blood vessels (Additional file 1: Figure S2b). Quantification of vascular density based on CD31 staining supports this observation as Hs766t (p = 0.0526) and MiaPaCa-2 (p = 0.0189) tumors had less vascular density than SU.86.86 tumors (Additional file 1: Figure S3). Th-302 toxicity was validated by quantification of γ-H2AX, a DNA damage response reporter  (Additional file 1: Figure S2d). γ-H2AX increased in treated Hs766t (p = 0.004) and MiaPaCa-2 (p = 0.061) tumors with no change in SU.86.86 tumors (p = 0.669) (Figure 1f,g).
Pyruvate stimulates oxygen consumption in glycolytic cells
Pyruvate was administered to cells in increasing doses (0–2 mM) with expectations that respiration would increase. 2 mM pyruvate stimulated the largest OCR response (Figure 2f–h). Hs766t cells exhibited the largest relative increase and SU.86.86 cells the lowest, despite the fact that they had the largest oxidative reserve (Additional file 1: Figure S5e). No significant difference of pyruvate consumption was observed between cell lines, suggesting that intracellular pyruvate utilization may differ between cell lines (Figure 2j). PPR response to pyruvate was inversely related to OCR, presumably due to a reduction in net acid production (i.e., lactate derived from pyruvate rather than glucose), as well as a greater concentration of pyruvate entering the tricarboxylic acid cycle (TCA) cycle (Additional file 1: Figure S5a–d). Mitochondrial membrane potential increased following administration of 4 mM pyruvate in SU.86.86 cells as determined using tetramethylrhodamine ethyl ester (TMRE), confirming stimulation of the TCA cycle (Additional file 1: Figure S5f). These data indicate that cells with a glycolytic phenotype are more susceptible to pyruvate-stimulated OCR than oxidative SU.86.86 cells.
Pyruvate transiently decreases tumor oxygenation
To investigate underlying mechanisms for pyruvate sensitivity in vivo, untreated pancreatic tumors were stained for monocarboxylate transporter 1 (MCT-1), a high affinity Pyr/Lac membrane transporter (Km = 0.7 mM/3.5 mM)  (Figure 4f). Expression was higher in Hs766t and MiaPaCa-2 tumors, compared to SU.86.86. MCT-1 was quantified per cell, shown as a heat map in the lower panels of Figure 4f. Expression was heterogeneous with relatively small populations of high MCT-1 expressing cells (red) surrounded by low expressing cells (yellow or blue). To determine if this histological observation was clinically relevant, we stained a human PDAC tissue microarray for MCT-1 and additional glycolytic markers, PKM2 and MCT-4  (Figure 4g–j). All markers exhibited strong staining patterns in poorly differentiated ductal epithelial cells, as well as in stromal fibroblasts, indicating that stromal cells have altered metabolic activity, similar to the cancer cells. Stroma exhibiting such staining patterns may also contribute to hypoxic exacerbation by increasing respiration in their compartment, and this is under active investigation.
Effect of pyruvate on TH-302 efficacy
Because hypoxic tumors are poorly prognostic and resistant to chemo- and radiotherapies, there has been intense interest in agents that will target hypoxic volumes. HAPs are a new class of anti-cancer therapeutics, and TH-302 is a leading candidate in clinical trials. The pharmacodynamic effects of these agents are complex and depend on vascular delivery, tissue penetration-to-hypoxic volumes, activation, and bystander killing. This is made even more complex by the short plasma half-lives of these agents. We thus contend that increased efficacy can be achieved through transient exacerbation of hypoxia in combination with advanced pharmacodynamics modeling. We have shown that exogenous pyruvate transiently exacerbates tumor hypoxia by increasing the rate of cellular OCR leading to improved TH-302 anti-tumor efficacy. This work was performed in three PDAC models: Hs766t, MiaPaCa-2, and SU.86.86 cells that have high, moderate, and low sensitivity to TH-302 monotherapy, respectively. Molecular and metabolic characterizations of these cells and tumors were consistent with their responses, as were their responses to pyruvate in vitro and in vivo. As predicted, pyruvate pretreatment caused a significant therapeutic benefit to the intermediately sensitive MiaPaCa-2 tumors exclusively. However, there were dramatic increases in survival for Hs766t tumors, which are highly sensitive to TH-302 monotherapy. Nonetheless, in contrast to the strong in vitro and in vivo effects of pyruvate on OCR and oxygen levels, the therapeutic benefits of combining pyruvate with HAP therapy, albeit significant, were only modest. Hence, we propose that additional optimization of timing and dose, using in silico models, can lead iteratively to improved therapeutic outcome of this novel combination therapy.
While the effect of exogenous pyruvate on metabolism has only recently been explored in the context of cancer [8,9], pyruvate has been thoroughly investigated and identified in neuronal models as the predominant mitochondrial substrate . We therefore hypothesized that reduced pO2 in pancreatic tumors by exogenous pyruvate was a result of elevated OCR. However, this does not explain why exogenous pyruvate alone would stimulate oxygen consumption in cancer cells. MiaPaCa-2 and Hs766t are glycolytic, implying that the pyruvate flux should be substantial and not limiting for mitochondrial consumption. However, the OCRs of these two tumor types were the most strongly affected by pyruvate. One possible explanation is that PKM2, the enzyme that catalyzes the dephosphorylation of phosphoenolpyruvate to pyruvate, is located adjacent to the plasma membrane, whereas the mitochondria are peri-nuclear [29,30]. The rate of pyruvate production is regulated by PKM2 and expression is consistently observed to be upregulated in transformed cells exhibiting glycolytic phenotypes. Since enzymes of the lower half of glycolysis are localized near the plasma membrane, we hypothesize that PKM2 is closely associated spatially with these enzymes, making the fate of pyruvate a readily available substrate for lactate dehydrogenase, rather than transport into the mitochondria . Hence, PKM2-produced pyruvate must traverse several microns of intracellular space, with diffusion gradients and intervening bioconversions, before it can be used to fuel respiration. With these distances, reaction-diffusion models predict that the local concentration of pyruvate at PKM2 would have to be at least 100 times higher than the Km for mitochondrial consumption for the mitochondrial pyruvate dehydrogenase to become saturated .
In contrast, exogenous pyruvate may have a different metabolic fate than pyruvate generated via glycolysis. Following uptake, the process of converting pyruvate to lactate continues as shown by hyperpolarized 13C pyruvate MR studies. However, since exogenous pyruvate can bypass PKM2, the available pyruvate concentration for transport into the mitochondria increases, stimulating respiration. Since pyruvate is delivered as a single bolus, exogenous pyruvate is quickly metabolized and the effect on respiration only transient; hence, the approximately 30-min decrease of tumor pO2 in our pancreatic tumor model (Figure 4).
Finally, chronic hypoxia has been shown to select for cells that are resistant to apoptosis [32,33]. Hence, there may be concern that exacerbating hypoxia may lead to enhanced progression or therapy resistance. In contrast to previous studies that induced hypoxia pharmacologically over many days , the current metabolic approach lasts <1 h, which is consistent with the blood half-life of TH-302. We contend that acute changes in pO2 will not lead to enhanced proliferation or resistance, but this will have to be investigated. Additionally, as pyruvate has also been shown to increase OCR in neurons, glia, and cardiac muscle [10-12], it is possible that this treatment may lead to hypoxia in normal tissues and increase TH-302 off-target effects. No increased toxicity of TH-302 was observed in our studies, and we reason that active maintenance of vessel tone in normal tissues, in contrast to tumors, exerts a protective effect by increasing blood flow to volumes with higher respiration. Nonetheless, further studies are warranted to investigate potential synergism between compounds that exacerbate tumor hypoxia and hypoxia-activated prodrugs.
PDAC has one of the worst prognoses of all cancers due to limited efficacy of current therapies. The hypoxia-activated prodrug, TH-302, has shown promise clinically but advances are needed to improve its efficacy against late stage pancreatic cancer. This study supports the concept that temporarily increasing tumor hypoxia with pyruvate can improve the efficacy of HAPs, including TH-302, against PDAC and other cancer types in the clinic. While the effect of pyruvate on TH-302 activity in vivo was significant, future studies are necessary to optimize timing and dose, using in silico models, iteratively leading to improved therapeutic outcome of this novel combination therapy.
This work is supported by U.S. National Cancer Institute grants R01CA077575, R01CA125627, and U54CA143970 (RJG).
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