Educational Appendix

Tumor Microenvironment

A hallmark of cancer is the reprogramming cellular energy metabolism to more effectively support neoplastic proliferation[1]. Cells convert glucose to pyruvate via glycolysis in the cytoplasm and then use pyruvate in oxygen-consuming mitochondria to produce carbon dioxide and ATP[2]. Under anaerobic conditions, pyruvate is primarily converted to lactic acid yielding only 6.25% of the ATP that complete metabolism of glucose would provide through both glycolysis (net 2 ATP) and mitochondrial oxidative phosphorylation (net 30 ATP). In the early 1900's Professor Otto Warburg first reported that cancer cells limit their energy metabolism largely to glycolysis, even in the presence of oxygen[3, 4]. This unique metabolic characteristic is one of the key components leading to a differentiated microenvironment around the tumor cell.

The Warburg effect is the observation of a high rate of glycolysis (generally associated with anaerobic metabolism) even in the presence of oxygen. Glycolysis provides most of the building blocks required for cell proliferation.[5] Evidence attributes some of the high aerobic glycolytic rates to an overexpressed form of mitochondrially-bound hexokinase.[6] For example, in some kidney cancers, this effect could be due to the presence of mutations in the von Hippel–Lindau tumor suppressor gene upregulating glycolytic enzymes, including the M2 splice isoform of pyruvate kinase (M2-PK).[7] M2-PK is produced in all rapidly dividing cells, and is responsible for enabling cancer cells to consume glucose at an accelerated rate. Upon forcing tumor cells to switch to pyruvate kinase's alternative form by inhibiting the production of tumor M2-PK, their growth was curbed. This enzyme form is not usually found in healthy tissue, though it is apparently necessary when cells need to multiply quickly, e.g. in healing wounds or hematopoiesis.[8][9] Several studies have shown a reduced level of metabolism via oxidative phosphorylation in these cancer cells, which may assist the tumor cells in avoiding apoptosis or programmed cell death.

With an increase in glycolysis and concomitant reduction in oxidative phosphorylation, lactic acid levels increase, so it is not surprising that the tumor microenvironment is frequently characterized by an acidic external pH (pHe).[10]. To maintain an intracellular pH (pHi) that is slightly alkaline (~pH 7.4), tumor cells upregulate several proton extrusion mechanisms such as the Na+/H+ Exchanger (NHE), HCO3 Transporter, Carbonic Anhydrase IX, Vacuolar-ATPase, and the H+/K+ ATPase[11, 12]. Excess protons are excreted into the extracellular matrix, causing the pHe of the tumor microenvironment to become acidic. In certain tumor types such as human MCF-7 mammary carcinoma, the pHe has been measured to be as low as pH 6.44[12]. Chronic exposure to acidic pHe has been reported to promote invasiveness and metastatic behavior in several tumor types[13, 14, 15]. A lower tumor pHe can also contribute to resistance to chemotherapies that act as weak bases, or enhance the efficacy of chemotherapies that act as weak acids[16, 17, 18].In addition low pH is associated with reduced efficacy of immune cells in the tumor microenvironment.

Breast-Cancer

pH of Human Breast Cancer

CREDIT: Courtesy of Professor Marty Pagel, Director of Contrast Agent
Molecular Engineering Lab (CAMEL)
, University of Arizona Cancer Center

Professor Marty Pagel and colleagues at the University of Arizona have developed methods to non-invasively measure the extracellular pH (pHe) of the tumor microenvironment in vivo using a magnetic resonance imaging (MRI) method known as chemical exchange saturation transfer (CEST)[4, 19]. To detect a CEST MRI contrast agent, radio frequency saturation is applied at a specific MR frequency of an exchangeable proton on the agent, which reduces the detectable magnetization from this proton. Rapid chemical exchange of this saturated proton with a proton on a near-by water molecule causes the reduced detectable magnetization to be transferred to the water signal. A standard MRI acquisition sequence can then be used to measure the amplitude of the water signal throughout the image. A CEST spectrum is obtained by iterating the saturation frequency and recording the normalized water signal amplitude. Because the average chemical exchange rates of some labile protons are pH-dependent, and because CEST effects from two labile protons can be selectively detected, a ratio of two CEST effects from the same agent can be used to measure pH in a manner that is independent of concentration, magnetic resonance properties of the endogenous tissue, and incomplete saturation.

Professor Pagel and colleagues have termed the use of in vivo CEST MRI for measuring tumor acidosis “acidoCEST MRI”[20, 21]. Their image acquisition method uses an in vivo CEST-FISP MRI protocol that rapidly detect in vivo CEST effects, which is critical for tracking exogenous contrast agents within in vivo tissues. Our image analysis method uses Lorentzian line shape fittings that improve the precision of in vivo CEST measurements. They detect the CEST signals of iopamidol (Isovue™, Bracco Diagnostics, Inc.) that is administered to a mouse tumor model, to create a method that can noninvasively measure tumor pHe. Together, these technological developments result in a measure precision of 0.07 pH units and an accuracy of 0.034 pH units.

To demonstrate the utility of acidoCEST MRI, they have for example treated a Raji xenograft tumor model of lymphoma with MIBG which reduces mitochondrial activity[22]. They performed acidoCEST MRI before treatment and 4 hours after treatment with MIBG and measured a drastic decrease in average tumor pHe, indicating that this mitochondrial poison caused metabolism of glucose to shift towards anaerobic glycolysis and presumably produce substantial amounts of lactic acid. Thus, acidoCEST MRI may be very useful in better understanding cancer cell metabolism and how to exploit it in cancer treatment.

Lung-Cancer

Human Lung Cancer

CREDIT: Courtesy of Professor Marty Pagel, Director of Contrast Agent Molecular Engineering Lab (CAMEL), University of Arizona Cancer Center

Breast-Cancer-2

Human Breast Cancer

CREDIT: Courtesy of Professor Marty Pagel, Director of Contrast Agent Molecular Engineering Lab (CAMEL), University of Arizona Cancer Center

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