The existence of hypoxia in tumors and its implications for the failure of radiation therapy were predicted in 1955 by Thomlinson and Gray (6
) on the basis of observations that necrosis in human lung carcinomas occurred at approximately 150 μm, the calculated diffusion distance of oxygen, from the nearest capillary. Four decades later, hypoxia and hypoxia-inducible factors (HIF-1 and HIF-2) are being associated with the transcriptional activation of an ever-increasing number of genes that regulate several phenotypic characteristics of cancer (7
). Tumor hypoxia can be diffusion-limited or chronic, or can arise acutely from vascular collapse. Most treatments, especially radiation therapy, result in reoxygenation (8
HIF-1 is a heterodimeric protein that consists of a constitutively expressed β-subunit and an oxygen-dependent α-subunit that is rapidly degraded through polyubiquitination and proteasomal degradation under well-oxygenated conditions. Under hypoxic conditions, HIF-1α or HIF-2α undergo heterodimerization with HIF-1β and bind to hypoxia response elements (HREs) to activate the transcription of several target genes. These target genes control angiogenesis, cell survival, resistance to chemo- and radiation therapy, metabolism, pH regulation, invasion and metastasis, increased genetic instability and dedifferentiation (7
). Increased invasion of ECM by colon cancer cells under hypoxic conditions (11
) can be partly reversed by a HIF-specific small interfering RNA (siRNA). Antisense RNA also limits the invasion and metastasis of pancreatic cancer cells (12
). HIF stabilization has also been linked to the epithelial-to-mesenchymal transition observed in many cancer cells, as well as the loss of E-cadherin, an extracellular marker of differentiated epithelial cells (13
). HIF-1 has also been shown to promote lysyl oxidase-associated invasion and metastasis of cancer cells (14
). A detailed review on the causes, consequences and detection of hypoxia can be found in this issue.
Efforts to target hypoxia, initially to improve the outcome of radiation therapy through the use of radiation sensitizers, and by using hyperbaric oxygen, have not been successful and require further optimization (15
). As hypoxic areas are, by default, also poorly perfused, the limited success of radiation sensitizers and hyperbaric oxygen could be a result, in part, of the limited delivery of agents and oxygen in these poorly perfused regions. More recently, low-molecular-weight HIF-1 inhibitors have achieved better success. In addition, the discovery that several chemotherapeutic agents also inhibit HIF-1 is providing new opportunities for the targeting of HIF-1 (17
The development of a theranostic agent for HIF-1 requires the expression of an imaging reporter in the tumor under hypoxic conditions, and the delivery of a therapeutic payload only where hypoxia is present. HRE has been exploited in several studies to target hypoxia using either prodrug enzymes or suicide genes (18
), although, to date, this has not been combined with imaging. As mentioned earlier, the minimization of damage to normal tissues is one of the most sought-after goals in cancer treatment. One strategy is to deliver a drug-activating enzyme to the tumor, followed by the administration of a nontoxic prodrug administered systemically. Several prodrug–enzyme systems exist that can be easily incorporated for the theranostic imaging of hypoxia (20
). HRE-driven expression of the prodrug enzyme cytosine deaminase (CD) has already been demonstrated (19
). CD converts a nontoxic prodrug 5-fluorocytosine (5-FC) to cytotoxic 5-fluorouracil (5-FU). The conversion of 5-FC to 5-FU can be detected in vivo
F MRS, and such studies should be possible in the future, with a potential for clinical translation, using either viral vector, liposomal or nanoparticle delivery of cDNA encoding for HRE-driven CD expression. Image-guided delivery of carriers that deliver siRNA to directly downregulate HIF-1 would be another option to reduce the damaging effects of HIF-1 expression in tumors.
Another example of hypoxia-based theranostic imaging is to use hypoxia imaging to prescribe radiation dose delivery, employing selective subvolume boosting and dose painting for theranostic radiation therapy (22
). Flynn et al.
) have recently described the use of 61
Cu-ASTM) positron emission tomography (PET) imaging to boost the radiation dose using intensity-modulated X-ray or intensity-modulated proton therapy in a patient with head and neck squamous cell carcinoma (). In the future, the optimization of dose prescription with the radiotracer activity concentration will be required. Dose prescriptions may also be combined with other theranostic agents or prodrug–enzyme treatments.
Figure 2 Coronal images of a patient with a head and neck squamous cell carcinoma with an overlay of a pretreatment 61Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) (61Cu-ATSM) positron emission tomography (PET) image. 61Cu-ATSM retention was highest in the planning (more ...)
Cells regulate pHe and intracellular pH (pHi) using an array of transporters, including Na+
pumps, monocarboxylate transporters (MCTs) 1–4 and carbonic anhydrases (CAs) I–XIII (23
). Cells require these pumps to balance the excess of H+
produced by cellular functions, including glucose metabolism.
pHe of tumors is usually acidic, whereas pHi is neutral to alkaline (24
). Increased glycolysis, poor blood flow and hypoxia contribute to the acidic pHe observed in tumors. Cancer cells exhibit high glycolytic activity, even in the presence of oxygen, a phenomenon called the `Warburg effect' after observations made by Otto Warburg in 1930. The molecular mechanisms underlying this aerobic glycolysis are mediated, in part, through the stabilization of HIF-1α even under oxygenated conditions in cancer cells (25
). HIF-1α expression mediates the switch in glucose metabolism through the induction of lactate dehydrogenase, which converts pyruvate to lactate, and the inactivation of pyruvate dehydrogenase, the enzyme responsible for the conversion of pyruvate to acetyl-coenzyme. Poor blood flow in tumors and the resulting hypoxia also contribute towards increased anaerobic glycolysis (25
Excessive glycolysis in cancer cells results in large amounts of lactate production and secretion. Increased inflammation and hypoxia have been linked to enhanced transport of intracellular H+
through the actions of MCT-2 and CAIX and CAXII, respectively (26
). A detailed review on the causes, consequences and detection of tumor pH can be found in this issue.
Unlike hypoxia, where HRE can be exploited to drive the expression of imaging reporters and therapeutic agents, in the absence of a clearly identifiable pH response element, theranostic imaging of tumor pH will mostly rely on the delivery of a therapeutic payload that is only activated at low pHe. Innovative advances have been made in the design of polymers that are pH responsive (28
). Koo et al.
) have combined the delivery of photosensitizers using pH-responsive polymeric micelles with photodynamic therapy to target acidic environments. These pH-responsive micelles show pH-dependent demicellization at pH values below 6.5. The photosensitizer released as a result of demicellization produces fluorescence and singlet oxygen following laser excitation, which can be used for diagnosis and therapy (28
). Although optical imaging has limitations for the detection of signal from tissue at depths greater than a few millimeters, this advance would be useful for surface tumors or intra-operative treatments. Recently, Kato and Artemov (31
) have published a novel dual-contrast MRI technique to detect the release of agents from nanocarriers using simultaneous encapsulation of superparamagnetic iron oxide (SPIO) nanoparticles and a gadolinium-based paramagnetic contrast agent, gadolinium diethylenetriaminepentaacetic acid bismethylamide (GdDTPA-BMA). This technique may have wide-ranging applications as an MRI-based theranostic agent. As a result of their widely different molecular sizes, SPIO and GdDTPA-BMA have very different diffusion properties. When the nanocarrier is intact, SPIO and GdDTPA-BMA are in close proximity, and a strong negative signal enhancement caused by the T2
effects of SPIO dominates the positive T1
contrast generated by GdDTPA-BMA. Once the nanocarrier has released its cargo, a positive T1
contrast is observed once the distance between GdDTPA-BMA and SPIO molecules is beyond the T2
enhancement range. A combination of this dual-contrast MR technique with pH-responsive polymeric micelles or liposomes could extend this pH-based theranostic approach to deep-seated tissues, and for clinical translation.
Recently, Li et al.
) have developed a novel pH-activated near-infrared (NIR) fluorescence nanoprobe, in which a high payload of the NIR fluorophore IR783 is conjugated with biodegradable dextran via pH-labile hydrazone bonds. Self-quenching between spatially neighboring IR783 fluorophores results in a low background signal in normal tissue. However, cleavage of the fluorophores from the nanoprobe in the acidic TME results in significant fluorescence enhancement. This prototype nanoprobe can be used to deliver a therapeutic payload in acidic TMEs and to detect pH.
Neutralization of the acidic TME has been shown to reduce metastasis (33
). It may be possible to design pH-targeting theranostic agents to deliver chemicals at acidic pH that would absorb protons. Strategies to exploit the existing pH gradient in tumors can also be used to improve the uptake of weakly basic chemotherapeutic agents (34
More than a decade ago, Dvorak (36
) postulated that tumors are `wounds that do not heal'. The similarities between wounds and solid tumors, such as hypoxia, high lactate and secretion of proinflammatory molecules, are remarkable. A characteristic response of living vascularized tissue to injury is inflammation, which induces the formation of eicosanoids. Three well-known classes of phospholipase, phospholipase A2
), phospholipase C and phospholipase D, participate in the formation of free arachidonic acid (AA) from membrane phospholipids in response to mechanical, chemical and physical stimuli (37
). As AA is derived from membrane phospholipids, its production and utilization in the formation of eicosanoids is closely coupled to membrane choline phospholipid metabolism (38
). AA is converted to various eicosanoids by the action of lipoxygenases and cyclooxygenases (COXs) (37
). These eicosanoids impact on cell motility, invasion, vascular characteristics and metastatic dissemination (40
). Several tumors exhibit inflammatory properties, characterized by increased levels of prostaglandins and other proinflammatory molecules that are secreted by tumor cells, stromal cells and specialized immune cells during inflammation (43
COX-1 and COX-2 are cytoplasmic enzymes that convert PLA2
-mobilized AA into the lipid signal transduction molecules prostaglandins and thromboxanes (44
). One major product of the COX-2-catalyzed reaction is prostaglandin E2
), an inflammatory mediator participating in several biological processes, including development, pain, immunity, angiogenesis and cancer (45
). COX-2 function has been the target of pharmaceutical intervention in several different cancers, such as gastric, lung, breast and colon cancer (42
). Our studies have shown that silencing of COX-2 in highly metastatic breast cancer cells delays orthotopic tumor growth and inhibits extrapulmonary colonization by these cells (53
has also been shown to promote the expression of genes associated with increased angiogenesis, such as vascular endothelial growth factor (VEGF), in the mammary fat pad of mice (54
). COX-2 (and PGE2
) also result in HIF-1 stabilization (55
), suggesting that COX-2 function may be mediated, in part, through the HIF-1 axis, which includes drug resistance, increased invasion, increase in choline kinase (57
) and the emergence of an aggressive phenotype, even under well-oxygenated conditions.
The development of COX-2 imaging agents is of major interest for several diseases including cancer. Although PET and single photon emission computed tomography (SPECT) imaging agents derived from COX-2 inhibitors have been described previously (58
), the selective binding of these agents has not been clearly validated in vivo
. A recent study demonstrated the feasibility of using indomethacin-derived fluorescent COX-2 imaging agents in vivo
). In these studies, the retention of the probe in the tumor was found to be dependent on COX-2 activity.
Downregulation of COX-2 can be achieved by the delivery of siRNA loaded in cationic multifunctional liposomes that are decorated with imaging reporters for MRI-guided delivery of COX-2 siRNA in tumors (61
). COX-2 siRNA can be loaded directly into cationic liposomes without changing the functionality of siRNA. The incorporation of MR contrast agents within liposomes creates the possibility of imaging siRNA delivery in vivo
with MRI with a clear path of clinical translatability.
Ideally, a theranostic agent for COX-2 would report on its expression and activity, and an agent for PGE2 would report on its levels in the microenvironment or its binding to specific receptors (EP1–4). These agents would contain a payload, the delivery of which would be activated by COX-2 and PGE2 expression or function. Although such an agent is currently not available, a molecular theranostic agent, driven by a promoter strongly activated by PGE2, may be a candidate.
Interactions between cancer cells and the TME, which often result in inflammatory signaling, invasion and metastasis, are mediated by soluble messengers, known as chemokines, that bind to specialized chemokine receptors (62
). Chemokines are small (8–17-kDa) proteins, which, like their G-protein-coupled receptors, can be categorized into four subfamilies, depending on the number and spacing of cysteine residues. One widely researched chemokine interaction in the TME involves the binding of CXCL12 (cysteine-X-cysteine ligand 12; also known as SDF-1) to CXCR4 (cysteine-X-cysteine receptor 4). CXCR4 receptor expression is regulated by hypoxia (63
) and by COX-2 (27
), and its expression is increased at the sites of metastasis. CXCR4 inhibitors have also been shown to reduce the incidence of metastasis (64
). CXCR4 receptor imaging in preclinical studies has been performed with 125
I-labeled antibodies using SPECT imaging. More recently, Nimmagadda et al
) have developed a PET imaging agent using [64
Cu]AMD3100, a positron-emitting analog of the stem cell mobilizing agent plerixafor. The CXCR4 receptor may be a good candidate for the development of a theranostic agent for metastatic cancer. However, the payload must target pathways specific to cancer, as CXCR4 is also expressed on immune cells and CD34+
-expressing hematopoietic progenitor cells, as it mediates leukocyte homing and bone marrow homeostasis (66