It is widely accepted that altered metabolism contributes to cancer growth and has been described as a hallmark of cancer. Our view and understanding of cancer metabolism has expanded at a rapid pace, however, there remains a need to study metabolic dependencies of human cancer
The seminal work by Otto Warburg defined glycolysis under aerobic conditions as the basis of cancer metabolism (Warburg, 1956a). His primary basis for excess glucose metabolism was postulated to be a result of impaired mitochondria metabolism. While correct in the context of his working models, Warburg dismissed parallel studies from Sidney Weinhouse that accurately described oxidation of carbohydrates and fatty acids within tumors (Weinhouse
Our view of tumor metabolism defined by Otto Warburg (Warburg
In this review we will describe the use of multi-modality imaging (MMI) techniques that have been combinatorially used to quantitatively measure and assess tumor metabolism
Positron Emission Tomography (PET) imaging is a widely used imaging modality employed in both clinical and basic research settings. PET imaging works by detection of gamma rays from positron emitting radionuclides that have been injected into a patient or animal. The most commonly used radionuclide is 18F but there is a wide range of radionuclides available - the more commonly used isotopes include 18F, 11C and 15O. The high consumption of glucose by advanced tumors makes PET imaging with [18F]fluro-2-deoxyglucose (18F-FDG) an ideal probe to detect glycolytic tumors. 18F-FDG works by entering the cell through glucose transporters (GLUTs) (Fig. 1) where it is rapidly phosphorylated by hexokinase into 18F-FDG-6-phosphate where it can no longer be metabolized. Here the radiotracer remains trapped in the early stages of glycolysis allowing the detection of emitted gamma rays as the probe decays. Importantly, the 3 dimensional detection of gamma emission from a single source enables 3 dimensional reconstruction of the tumor. 18F-FDG is used to successfully diagnose a broad range of tumors that include cancers of the lung, liver, bone and soft tissue (Minn
18F-FDG PET is particularly useful for metabolic studies because it measures glucose flux into the tumor and activity in early steps of glycolysis. While it also serves to detect a tumor mass, importantly it provides valuable functional information about the metabolic needs of the tumor. Moreover, when imaging cancer metabolism, it is critical to identify probes and modalities that provide, in addition to anatomical registration, functional information about the metabolic activity within the tumor(s). The noninvasive nature of PET imaging allows for repeat scans to be performed on patients during the course of treatment. The PET Response Criteria in Solid Tumors or PERCIST is a set of criteria that utilizes PET imaging with 18F-FDG to determine therapeutic response in patients. The efficacy of PERCIST criteria was demonstrated in a clinical trial, which used 18F-FDG uptake in tumors to evaluate breast tumor response to the PI3K inhibitor Buparlisib (Mayer
Tumors do not solely rely on glucose. Therefore radiolabeling of additional metabolites such as acetate, choline, methionine and glutamine with either 18F or 11C provide opportunities to perform broad profiling of cancer metabolism with PET imaging. 11C-acetate is converted to acetyl-CoA and used in mitochondria in TCA cycle or incorporated into cell membranes (Vavere
Computed tomography (CT) imaging utilizes multi-positional X-ray imaging to generate a 3 dimensional view of the imaged area. Tomographic reconstruction of the X-ray images provides detailed anatomical information of the imaged patient or animal. CT imaging can be performed with contrast agents that register vasculature within the tumor and perfusion within the tumor(s). CT imaging with iodine based contrast agents such as iohexol, iodixanol and ioversol are advantageous because it renders a quantitative measure of blood vessels supplying the tumors (Kao
Tumor vasculature directly impacts the nutrients accessible to the tumors and can be used to differentiate well-perfused regions from hypoxic regions. Recent study has evaluated contrast-enhanced CT with HX4-PET, PET probe specific for hypoxia. This study showed that in lung cancer patients both modalities were able to classify tumors as normoxic or hypoxic (Even
Magnetic Resonance Imaging (MRI) is based on the physical phenomenon called nuclear magnetic resonance (NMR). NMR is based on quantifying changes in nuclear spin, a property of atomic nuclei, in response to a strong external magnetic field. NMR signal is detected upon relaxation of nuclear spins. For imaging metabolism most useful nuclei are 1H, 13C and 31P. Like CT imaging, MR imaging is most frequently used to determine anatomical registration of the tumor. In addition, MRI can be performed with contrast agents to determine perfusion within the tumor (Yankeelov and Gore, 2009).
Most commonly Magnetic Resonance Spectroscopy (MRS) has been used to evaluate endogenous 1H signals from choline-containing molecules. Signal intensity of the 1H MRS peak has correlated with proliferation in brain, prostate, breast, colon and cervical cancers (Nelson
Hyperpolarized [1-13C]-pyruvate has been used to detect levels of lactate and alanine as well as total amount of hyperpolarized 13C in preclinical models of prostate cancer (Albers
As with other modalities of non-invasive imaging, monitoring response to therapy is one of the most promising aspects of MRS imaging. In a pre-clinical model of lymphoma, 13C-pyruvate was used to show that loss of flux from pyruvate to lactate correlated with response to chemotherapy (Day
Optical imaging is a widely used modality in basic research laboratories that measures and quantifies emission of visible light and wavelengths in near infrared spectrum. Optical imaging can be done in both fluorescent and bioluminescent models. Fluorescence imaging of dyes and proteins such as green fluorescent protein (GFP) has a low signal to noise ratio, due to the emission of light within the visible spectrum by cellular proteins and DNA. This has been improved upon with the development of fluorescent probes with emission in the near infrared spectrum (Zhang
Bioluminescence is a natural phenomenon observed across numerous species from bacteria, to worms to beetles to fireflies in which the luciferase enzyme catalyzes a reaction that releases photons of light. First taking advantage of the luciferase enzyme from fireflies, researchers were able to clone luciferase and introduce it as a reporter in cell lines and transgenic animals (Fan and Wood, 2007; Woolfenden
The sensitivity of BLI enables small tumor lesions to be detected at early stages of tumorigenesis that fall below the level of detection for CT, MRI and PET imaging (Shackelford
Imaging with D-luciferin can detect the presence of tumors, but can BLI be used to study functional metabolism? BLI using luciferase can be used to measure ATP levels, allowing for the estimation of the energetic charge within the cell. This is based on the fact that reaction catalyzed by luciferase requires ATP, thus emitted light can be correlated to the amount of ATP available (Kimmich
Metabolomics refers to the global study of metabolism and can be examined using multiple techniques. While technologies such as gene set enrichment analysis (GSEA) and protein expression can be used to profile the tumor’s global metabolic profile (Nielsen, 2017), this review will focus on direct analysis of metabolites using Stable Isotope Resolved Metabolomics (SIRM). This technique utilizes liquid chromatography mass spectrometry (LC-MS) or gas chromatography mass spectrometry (GC-MS) as a direct means to measure the distribution of labeled metabolites. It takes advantage of low abundance in the cells and tissues of 13C and 15N isotopes compared to high abundance of natural isotopes 12C and 14N. This selectivity is coupled to extremely high sensitivity of detection of stable 13C and 15N isotopes offered by mass spectrometry. Employing SIRM allows tracing of the metabolic fate of the individual atoms from the labeled molecule. This means that as labeled molecule undergoes transformation in a metabolic pathway its contribution to each step of the pathway can be accurately quantified. A number of 13C- and 15N-labeled molecules are commercially available (for example 13C-glucose, 13C- and 15N-glutamine, 13C-palmitate) making it possible to study the fate of an individual metabolite as it is processed through multiple enzymatic steps in the cell.
The Fan laboratory has published a number of pioneering studies using SIRM technology in lung cancer cell lines (Fan
Additional mass spectrometry based approaches have taken advantage of ‘click’ chemistry to design probes such as MitoClick that measure mitochondrial membrane potential (Logan
The use of bright field microscopy has been a staple of histological analysis of tumor tissue for decades and is widely used by clinical pathology as well as basic research laboratories. Quantitative immunohistochemical (qIHC) staining of key signaling proteins and metabolic enzymes that regulate metabolism can readily be performed on formalin fixed paraffin embedded (FFPE) tumor sections in both human and mouse tumors. Fig. 1 represents signaling kinases within pathways such as the PI3K/AKT/mTOR signaling axis that function as critical regulators of glycolysis (Elstrom
To quantify IHC staining, slides are digitally scanned and staining intensity can be quantified using morphometric software. Next, druggable targets can be identified and treated with targeted therapies. Fig. 2B shows IHC staining of the mTORC1 substrate phospho-S6 (P-S6) is elevated in lung tumors isolated from Kras/Lkb1 mutant GEMMs. The signaling pathway and several druggable targets are summarized in Fig. 2C. Our laboratory has performed pre-clinical
While noninvasive 18F-FDG PET imaging provides both spatial and temporal assessment of glycolysis at different stages of tumorigenesis, this modality does not inform us of the fate of glucose beyond early steps of glycolysis. Therefore, by coupling 18F-FDG PET imaging to SIRM analysis using LC-MS of 13C-glucose one can attain a comprehensive picture of glucose uptake and glucose utilization within the cell. Examples of this type of approach are highlighted in a study examining the role of Kras dependent metabolism in pancreatic ductal adenocarcinoma (PDAC) (Ying
Imaging mice with two different PET probes on consecutive days was performed using 18F-FDG and 18F-Glutamine in order to evaluate glucose and glutamine uptake in tumors. Venetti
Hyperpolarized 13C-Pyruvate has been used in multiple preclinical studies of lymphoma (Day
Multi-modality imaging in preclinical mouse studies has routinely combined PET/CT with BLI bringing together sensitivity of PET to detect metabolic activity within tumors with sensitivity of BLI for detection of small number of cells. Pioneering studies by the Gambhir lab, demonstrated that combining 18F-FDG/CT imaging with BLI was an effective and sensitive method to study tumor metabolism (Deroose
Optical imaging represents a cost effective alternative to more costly approaches such as PET and MRS imaging. Moreover, recent studies have demonstrated that caged luciferin probes can be designed to detect metabolite or enzyme activity (Wehrman
Is it possible to translate MMI imaging from pre-clinical studies in mice to cancer patients? Yes. This was recently demonstrated in a seminal study by the DeBerardinis laboratory in which they profiled glucose metabolism in human lung cancer patients using MMI coupled to detailed metabolic and molecular analysis (Hensley
Can profiling tumor metabolism improve cancer treatment? Tumor metabolism is recognized as a hallmark of cancer therefore characterizing the metabolic phenotype of the tumor is an important first step in diagnosis and treatment. Tumor metabolism informs us of the anabolic growth rate of the tumor and its capacity for continued growth. Importantly, metabolic profiling provides functional analysis of the tumor. Furthermore, coordinating PET with LC-MS and qIHC will serve to identify metabolic dependencies and druggable proteins within tumors that can be inhibited with targeted therapies or even combined with immune based therapies. Metabolic profiling performed on human NSCLC revealed tumor metabolism is heterogeneous, suggesting that combination treatments will be required. However, in order for metabolic therapies to successful target a tumor, first the metabolic dependencies must be identified. If a tumor uses a multitude of metabolites as its fuel source then a treatment strategy that inhibits only one metabolite such as glucose is predicted to fail. By profiling tumors noninvasively with MMI and validating with metabolomics, researchers and clinicians will be able to take a snap shot of the tumor’s metabolic dependencies. This will allow for design of precise therapies that inhibit key metabolic nodes. The noninvasive nature of PET and MRS imaging means that repeat imaging will be possible with the goal of this imaging informing oncologists and radiologists of treatment efficacy and course of action.
This was demonstrated recently in KrasG12D driven GEMMs of lung cancer treated with the glutaminase (GLS) inhibitor CB-839. As a single therapy, CB-839 failed to have any impact on tumor growth because these tumors are not dependent upon glutamine metabolism (Davidson
Distinct populations of tumors such as lung adenocarcinomas driven by EGFR mutations or triple negative breast cancer have shown dependency on glutamine metabolism
The importance of PET imaging in determining response to therapy was elegantly demonstrated using 18F-FAC, a deoxycytidine kinase analog that is phosphorylated by deoxycytidine kinase (dCK) and incorporated in DNA synthesis pathway (Radu
Both PET and MRS imaging are costly ventures and often are not feasible on an academic budget. Can we identify and develop cost effective strategies to expand imaging in cancer metabolism in both pre-clinical and clinical studies? Development of optical imaging probes may provide a cost effective alternative to measure metabolism in tumors. However, development and careful validation of these probes will be required. Since caged luciferin probes rely on reporter-based systems their applications are restricted to pre-clinical studies. Combining multiple imaging platforms such as PET and MRI into single imaging units will likely increase the demand and throughput for pre-clinical imaging. Lastly, the availability of affordable radiotracers and hyperpolarized probes as well as micro-PET and MRS scanners hold promise to expand probe development and usage of MMI in the field of cancer metabolism. Miniaturized radiotracer development platforms such as ELIXYS may provide critical first steps towards wider availability of probes for basic and clinical research (Lazari
Regardless of the imaging modality used, imaging of bulk tumors reflects a total sum from all cells in the tumor. Quantitative analysis within a heterogeneous tumor microenviroment presents a cadre of challenges. For example, what are the contributions of probe uptake for tumor supportive cells, such as stroma and tumor infiltrating leukocytes (TILs)? The recent success of immune based therapies in cancer have ushered in a wave of studies that have begun exploring the intersection of metabolism between the tumor and its microenvironment. It will be important to consider what proportion of the imaging signal is contributed by the tumor microenvironment contribute when imaging tumors with PET or MRS and validating with SIRM. Comparing the signal from bulk tumor to that of isolated tumor and immune cells may provide answers to these important questions.
Metabolism is a fundamental cornerstone of PET and MRS imaging and represents a powerful tool to study tumor metabolism in basic research and clinical settings. Tumors are heterogeneous by nature and their metabolism reflects this. Since tumors utilize a host of metabolites beyond glucose it is conceivable that the future of PET and MRS imaging in patients will utilize a multitude of radiotracers and 13C labeled metabolites to provide a detailed and real time signature of the tumor’s metabolic profile. This signature promises to provide valuable information to guide precise and personalized treatments. The likely future of cancer treatment is to manage the disease chronically with cocktail therapies similar to what has been done for HIV patients. Here, MMI guided metabolic profiling would provide oncologists, radiologists and pathologists with a roadmap to how tumors adapt and how we can stay one step ahead with effective therapies.