The tumor vascular system is the main factor determining the internal microenvironment of a tumor during its growth and is responsible for the exchange of oxygen, metabolites, and energy information between blood and tissue fluids. However, the vasculature of tumors differs from that of normal tissues. This structural difference increases the diffusion distance between the supplying vessels and tumor cells, forming a diffusion gradient and resulting in insufficient oxygen supply to the tumor cells that are away from the vessels, eventually leading to tissue hypoxia (1). As the volume of solid tumors continues to increase, especially when larger than 180–200 microns, coupled with the uncontrolled growth of tumor cells that consume high levels of oxygen and nutrients, tumor tissues will have insufficient local tissue perfusion or oxygen supply and lack of nutrients, resulting in the production of a series of acidic wastes and leading to extracellular acidosis. Collectively, these events ultimately result in tumor cells having a hypoxic microenvironment (2).

Hypoxia-inducible factor (HIF)-1 is a nuclear transcription factor that is widely present in mammalian cells. HIF-1 activates selected genes by transcription. These genes increase cell survival and proliferation to help cancer cells adapt to hypoxia by increasing angiogenesis, altering metabolism, degrading the extracellular matrix, inducing tumor cell dedifferentiation, and arresting the cell cycle (3–5). When the oxygen concentration is normal, HIF-1α is ubiquitinated and can be rapidly hydrolyzed by the ubiquitin-proteasome system. Therefore, HIF expression in normoxic cells is low (6). However, hypoxia prevents the ubiquitination of HIF-1α and induces an increase in the expression of HIF-1α (7). The transcriptional activity of HIF-1 also increases with the degree of hypoxia, resulting in activation of the p53 gene and expression of the cyclin-dependent kinase inhibitor (CKI) (8). Simultaneously, HIF-1α can also directly bind to the tumor suppressor gene p53 to promote p53-dependent cell apoptosis (9). In hypoxic conditions, the inhibition of prolyl hydroxylase stabilizes HIF-1α, followed by its binding to HIF-1β and transportation to the nucleus. The transcriptional activator protein p300/CBP is recruited through a transcriptional activation region of subunit a, and then interacts with the hypoxia response element (HRE), composed of the core sequence 5′-RCGTG-3′ of the promoter region of the target gene, such as carbonic anhydrase-IX (CA-IX). The downstream factors or enzymes are regulated and can then play a regulatory role (10) ( Figure 1 ).

HIF can regulate vascular endothelial growth factor (VEGF), transforming growth factor (TGF), tumor necrosis factor (TNF), and angiopoietin-2 (ANG-2) receptor, among others (11), to induce the proliferation of vascular endothelial cells. While inducing new blood vessels, HIF also regulates the vascular system of the host for the metastasis of tumor cells (12). Thus, the tumor can not only obtain nutrients and oxygen from the host through the new tumor blood vessels, but can also continuously deliver metastatic cells to the host through these blood vessels, while continuing to grow and inducing blood-vessel formation in other parts of the body, ultimately leading to tumor invasion and migration (13, 14). It is noteworthy that the formation of new blood vessels may lead to reoxygenation of hypoxic areas. Using current standard radiotherapy, hypoxic cells can also gradually become reoxygenated (REOX) during the treatment process due to the killing of the radiosensitive oxygen-consuming cells near the capillaries, as the remaining cells can obtain more nutrients and oxygen (15). Activated HIF can also induce the formation of tumor cell stemness and screen out more aggressive cells.

Additionally, increasing experimental evidence suggests that CA-IX plays a direct role in many tumor phenotypic characteristics caused by hypoxia and acidosis, including increased local adhesion during cell proliferation, unstable cell contact, tumor interstitial crosstalk, and maintaining the stem cell phenotype, signal transduction, and other cancer-related phenomena (16). Importantly, CA-IX is not expressed in most normal tissues except in the stomach and gallbladder epithelium (17). However, it is usually overexpressed in tumors and supports tumor cell migration and invasion. Inhibiting the catalytic activity of CA-IX can significantly improve chemotherapeutic sensitivity or radiosensitivity. Similarly, inhibiting CA-IX can enhance the anti-angiogenic effect of anti-VEGF antibodies (18).

Under hypoxic conditions, up-regulated HIF can regulate a variety of cancer phenotypes, causing various adverse changes in tumors and tissues ( Figure 2 ).

Hypoxia can cause genetic damage. The rapid change in oxygen concentration produces free radicals, which act on tissue cells and cause DNA damage (19). Moreover, hypoxia may lead to the overexpression of tumor suppressor genes, pro-apoptotic factors, and anti-apoptotic factors to salvage damaged cells, but may make these cells polyploid. A study by Nelson et al. (20) shows that these cells have certain defects in their inherent apoptotic pathways, which are important signs of cancer and precancer.

The most important biological effect caused by hypoxia is resistance to radiotherapy and chemotherapy, which is directly related to disease prognosis. Molecular oxygen is very important for the sensitivity of radiotherapy. In conventional photon-based radiotherapy or light ion therapy, such as those using protons, oxygen has a high electron affinity and plays a central role in the fixation process (21, 22). Hypoxia reduces the ability of the drug to produce free oxygen radicals, resulting in hypoxic cells that are less sensitive to chemotherapy.

Another interesting phenomenon is that when the hypoxic state causes cell death, the migration of tumor-associated macrophages (TAM) to the hypoxic area is inhibited, thereby preventing TAM from removing dead and damaged cells. Other effects of TAM include inhibition of the immune response of the body to tumor cells and inhibition of the activation of adjacent macrophages. These phenomena promote angiogenesis and lymphangiogenesis, thereby promoting tumor growth, migration, and transformation (23, 24). Hypoxia-related TAM can upregulate HIF-1 and HIF-2 expression both in vivo and in vitro (25).

E-cadherin is an important intercellular adhesion molecule that mainly maintains the integrity and polarity of epithelial cells. Loss of E-cadherin is a sign of tumor epithelial-to-mesenchymal transition (EMT) in tumor cells and is related to hypoxia-induced HIF activation.

There are considerable differences in the degree of hypoxia among different tumor types; thus, it is impossible to propose a definite value to define hypoxia. The currently recognized “gold standard” is to measure the distribution of the partial pressure of oxygen (pO2) using polarographic electrodes, but this method is invasive and greatly limits its use in tumor determination. As the oxygen requirement of tissues is met by blood, a method of responding to hypoxia by measuring the vascular supply was proposed. Cryophotometry or magnetic resonance imaging (MRI) to measure blood oxygen saturation, or MRI, computed tomography (CT), or positron emission tomography (PET) to measure tumor perfusion have been widely used ( Table 1 ). However, vascular supply is not the only factor that causes hypoxia in cells and tissues; thus, monitoring of the vascular supply alone cannot completely determine hypoxia (50). Later, it was discovered that the expression of certain genes and proteins changes during hypoxia. Therefore, it was proposed that the state of hypoxia could be determined by measuring the levels of the endogenous markers. These principal markers include HIF-1, CA-IX, glucose transporter (GLUT)-1 and -3, and osteopontin (OPN). However, some reactions that are not related to hypoxia can also lead to upregulation of the endogenous markers. Some studies have reported that reactive oxygen species, cancer-specific mutations, and changes in signal transduction pathways can cause these changes even in normoxic conditions (51).

However, the most widely used method for detecting hypoxia is based on PET/SPECT imaging. For example, foreign substances (imidazole drugs, cytarabine, or other substances) are labeled with radionuclides or fluorescent probes and used as tracers with specific chemical properties to target hypoxic areas within the tumor.

To summarize, pre-monitoring of hypoxic information before tumor diagnosis or treatment is extremely important in selecting accurate and effective individualized treatments and monitoring the efficacy and prognosis. As PET is more commonly used in a clinical setting for tumor diagnosis, in this review, we have focused and conducted a thorough evaluation on several methods that use markers in PET imaging to detect hypoxia. This study sheds light on the timely intensive treatment methodologies to overcome the resistance caused by hypoxia and prevent local recurrence.

Janet Rasey and her colleagues at the University of Washington first proposed the noninvasive imaging of hypoxia by PET (54). They synthesized 18F-fluoromisonidazole and then verified its feasibility as a PET hypoxia tracer in several models of human tumors. Since then, researchers have extensively explored the possibility of using ¹⁸F-labeled compounds as hypoxia tracers ( Figure 3 ).

⁶⁸Ga (t_{1/2 =} 68 _min, 89% b+, Eb+max= 1.92 MeV, 11% EC) is a generator-produced radionuclide that offers excellent coordination chemistry with several bifunctional chelating agents and provides rapid radiolabeling over a range of pH (191–193). Therefore, ⁶⁸Ga has been preferentially studied in hypoxic PET. For example, Sudhakara et al. developed ⁶⁸Ga-labeled agents based on mono-, bis-, and tris-nitroimidazole conjugates with the chelating agent 1,4,7-triazacyclononane-1,4,7-tris[methyl(2-carboxyethyl)phosphinic acid] (TRAP), to obtain a series of ⁶⁸Ga-labeled compounds. The radiochemical yields were extremely high and the uptake of tumor models in in vivo experiments revealed its remarkable ability to target hypoxic areas (194). Recently, Yoichi Shimizu et al. also confirmed that the ⁶⁸Ga marker could help in the visualization of tumor tissues and hypoxic areas within 2 h of intravenous injection (195). In short, studies on lung (196) and colon cancers (197, 198) show that on the basis of the ease in production, ⁶⁸Ga-labeled hypoxia tracers are comparable to traditional ¹⁸F-labeled tracers.

Radioactive iodine is a commonly used radiotherapy agent for the diagnosis and treatment of thyroid diseases. The characteristics of its radiation make it well-sought after for radioimmunotherapy (199). Radioactive iodine isotopes play a role in the field of hypoxia imaging as they can be used to label various substrates that can target hypoxic regions. For example, ¹²³I-IAZA is the most common radiotracer with a chemical structure similar to that of ¹⁸F-FAZA. ¹²⁴I is a popular choice owing to its longer half-life. The distribution of ¹³¹I-IAZGP (200) is similar to that of pimonidazole, and has a similar CA-IX expression profile in colorectal cancer models. ¹²⁴I-IAZGP (201) can be used to image liver tumors within 6 h (202); however, there are insufficient tumor-absorption studies that support the clinical role of ¹²⁴I-IAZGP PET in patients with colorectal cancer and head and neck cancer. ¹²⁴I-FIAU (203) can be used to evaluate the expression of HSV1-tkeGFP fusion gene that is associated with hypoxia. The area where ¹²⁵I-IPOS accumulates is positively correlated with the HIF-1α-positive area (204). ¹²⁵I-M75 specifically accumulates in colorectal cancer xenografts within 48 h of administration and delineates hypoxia by targeting CA-IX (205). However, several radioactive iodine isotopes have long half-lives. Although these isotopes play a unique role in radioimmunotherapy, their use as imaging agents should be carefully assessed.

The ^99mTc-labeled complex is one of the earliest non-nitroimidazole contrast agents used to determine hypoxia in cells. ^99mTc-labeled 2-nitroimidazole was also developed, with BMS181321 being the first. However, these agents are unstable and lipophilic, which limits their application (206). Subsequently, other tracers were developed to overcome these shortcomings (207). By choosing different cores and ligands, the tracer was designed to exhibit different characteristics to optimize the selection. The [^99mTc(CO)₃ (H₂O)]⁺ nucleus is well studied for the labeling of nitroimidazole compounds (208). Recently, more focus has been placed on the introduction of tumor-targeting moieties (e.g., RGD). The ^99mTc nitroimidazole complex is retained longer in hypoxic tumor cells.

In addition to the radionuclides discussed above, several others have been used for hypoxic PET. For example, ¹¹¹In is a promising candidate for imaging hypoxic areas to study head and neck tumors (209), prostate cancer (210), and colorectal cancer (211). The nitroimidazole and gastrin-releasing peptide receptor (BB2R) conjugate was labeled with ¹⁷⁷Lu (212); the CA-IX antibody G250-F(ab′)2 was labeled with ⁸⁹Zr (213) and some promising results were obtained. Several nuclides are used in radiotherapy although their imaging effects are unsatisfactory or there are many factors worthy of improvement; however, these studies could help develop new ideas and research methodologies.