Hypoxia is a main factor in treatment outcome and prognosis in different cancers, including glioma (54). Multiple radiotracers can be used to visualize hypoxia or sense oxygen levels. The tracer ¹⁸F-Fluoromisoinodazole (¹⁸F-FMISO) was developed for glioma imaging; however, its success is limited due to low sensitivity for differentiating normoxic from hypoxic tissue as well as a low blood brain barrier permeability (55, 56). Nevertheless, some results such as immunohistochemistry (IHC) findings, suggest that additional clinical studies are needed to evaluate ¹⁸F-FMISO as an imaging tool for hypoxia (57, 58). Moreover, the use of multiple PET radiopharmaceuticals is suggested to improve the planning for patient treatment and monitoring due to enhancing tumor detection and metabolic and genetic characterization. However, further research is required to eliminate the technical and logical issues of this technique and develop the concept of simultaneous multiplexing of ¹⁸F-FMISO with other PET oncology tracers (54). ¹⁸F-labeled flouroazomycin arabinoside (¹⁸F-FAZA) has the potential to be a promising alternative to ¹⁸F-FMISO offering an improved tumor-to-background ratio regarding faster blood clearance (59, 60). There is preliminary evidence that ¹⁸F-FAZA PET may be useful for assessing radiotherapy response in glioblastoma patients (61).

Like peripheral cancers, brain cancer cells trigger inflammation as part of the immune response. A wide range of neuroinflammatory conditions, including high-grade GBM, have been studied with translocator protein 18 kDa (TSPO) PET imaging (62–66). The TSPO, previously known as the peripheral benzodiazepine receptor, is highly expressed in GBM and plays a critical role in essential mitochondria-based physiological processes (67, 68). According to several studies, there is a positive correlation between TSPO expression and the grade of malignancy and glioma cell proliferation; however, it has a negative correlation with survival in patients with glioma (69–71).

3′-deoxy-3′-fluorothymidine (FLT) was used as an antiviral compound in patients with HIV for the first time (72). It's absorbed by cells and is subsequently phosphorylated by thymidine kinase 1 (TK1), which causes intracellular trapping within the cell. In this way, FLT retention within the cell provides a measure of TK activity, a key enzyme for cell proliferation (73). The increase in TK1 activity in normal cells occurs only during the DNA synthetic phase (72), while it occurs more and permanently in malignant cells. ¹⁸F-FLT uptake has a strong correlation between SUV of ¹⁸F-FLT and the proliferative status in lung cancer, colorectal carcinoma, non-Hodgkin's lymphoma, and melanoma (74–77). ¹⁸F-FLT is also considered to be an attractive PET tracer for the imaging of brain tumors due to the low uptake of ¹⁸F-FLT in the intact brain tissue and providing a low-background cerebral image (78). Furthermore, unlike ¹⁸F-FET, ¹⁸F-FLT requires a breakdown of the blood-brain barrier for it to be absorbed (79). Based on the results of a preliminary study in a low number patients with newly diagnosed gliomas, FLT uptake heterogeneity using textural features seemed to be useful for the assessment of proliferation and potential prediction of survival in these patients (80).

¹⁸F-FLT PET as a marker of cellular proliferation was compared with ¹⁸F-FDG in 25 patients with newly diagnosed or previously treated glioma. The results showed that ¹⁸F-FLT was more sensitive than ¹⁸F-FDG, correlated better with Ki-67 values, and appeared to be a promising tracer as a substitute marker of proliferation in these patients (81). For recurrent malignant glioma patients treated with bevacizumab and irinotecan, kinetic parameters from FLT provided sufficient information to predict the overall survival compared to those from FDOPA (82). A meta-analysis study indicated that ¹⁸F-FLT PET had a moderately better overall accuracy for diagnosing glioma recurrence compared with ¹⁸F-FDG (83).

Fernandez et al. determined the probability of combining ¹⁸F-FLT PET and MRI to improve the detection of tumoral tissue compared to MRI alone in 13 patients with newly diagnosed glioma. They also evaluated whether ¹⁸F-FLT uptake had a prognostic value by studying its association with histopathological features. According to the results, a combination of MRI and ¹⁸F-FLT PET detected additional tumor tissue, which might lead to a more complete surgical resection. The negative predictive value of a negative PET was also increased when combined with a negative MRI. Nevertheless, ¹⁸F-FLT underestimated the margins of the lesion and did not correlate with histopathological findings (84).

According to Nikaki et al. ¹⁸F-FLT accumulates in metastatic lesions, and it is highly sensitive and accurate. They reported that it could be used as a complementary tool to other imaging procedures. The tumor-to-background ratio and SUVmax provided the best diagnostic accuracy, while PV50% (proliferation × volume) and PV75% were of less diagnostic significance (85).

One of the most abundant isoforms of PARP enzymes is nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP-1) (86). Among the 18 members of the PARP family, PARP-1 is the founder and major isoform with diverse regional expression, subcellular distribution, and domain composition with mono (ADP-ribosylation) and poly (ADP-ribosylation) activities (87).

Several physiological functions of Poly(ADP-ribosyl)ation (PARylation) have been implicated, including DNA repair, gene transcription, cell cycle progression, cell death, chromatin function, and genomic stability (88). Since PARP becomes activated in response to DNA breaks, understanding how various endogenous species can induce DNA strand breaks, and thus activate PARP, in various diseases has become increasingly important. Several studies have demonstrated that neutralizing peroxynitrite and/or inhibiting PARP pharmacologically or genetically is effective in treating cardiovascular, inflammatory, vascular, and neurodegenerative diseases by preventing cell death as well as down-regulating multiple inflammatory pathways (89–91). Despite PARP1's therapeutic potential, it has also attracted attention as a target for imaging agents. Reiner et al. developed a PARP agent for PET/CT imaging based on the Olaparib scaffold using rapid bioorthogonal conjugation chemistries (¹⁸F-BO) (92). ¹⁸F-BO (biorthogonal olaparib) was produced as the first inhibitor for PET imaging. Another specific target, ¹⁸F-PARPi, has been successfully used in GBM cell lines with high binding to peripheral tumors. It has been shown that the biodistribution and uptake of this tracer in the brain is very low around 2 h post-injection. In comparison to previous amino acid-based tracers, PARPi has a lower uptake in brain tumors with a lower background uptake in the cortex (93). As an important point, small PARPi molecules are needed for penetrating through the BBB as a major obstacle for any successful radiotracer targeting GBM. This limitation is mostly related to poor BBB penetration and high chemoresistance of GBM cells (94). With a high precision and good signal/noise ratio, ¹⁸F-PARPi can be used to visualize glioblastoma in xenograft and orthotopic mouse models. It offers new opportunities to image tumor growth and monitor interventions non-invasively (95).

¹⁸F-PARPi-FL, as a bimodal fluorescent/PET agent, is used for PARP1 imaging in glioblastoma cells. A study found that PARPi-FL could be radiolabeled and used to augment the limitations of the “fluorescence only” intraoperative imaging molecule (96). For this purpose, PET and SPECT imaging were performed in orthotopic glioblastoma models with ¹²⁴I- and ¹³¹I-I2-PARP inhibitor (Olaparib) (97). The results showed the specific binding of the I2-PARPi tracer to PARP1 indicating the potential of this tracer for glioblastoma detection. Moreover, the results of an important preclinical study by Pirovano et al. provided the first full characterization of an auger-emitting PARP inhibitor, which showed a survival benefit in mouse models of GBM and verified the high potential of ¹²³I-MAPi for clinical translation (98). With their short range of action, auger electrons possess high biological efficacy, which allows them to inflict DNA damage and cell death precisely. In addition to imaging tumor progression and monitoring therapy response, ¹²³I-MAPi can also be used to calculate patient dosimetry.

Human tumor cell lines including C6 glioma, neuroblastoma, and NG108-15 neuroblastoma-glioma hybrid express sigma 1 and sigma 2 receptors. Sigma 1 receptors are associated with invasiveness, while sigma 2 receptors are associated with proliferation. These two receptors are interesting targets for PET imaging in glioblastomas (99). Initially, ¹¹C-SA4503 sigma-1 based tracer was prepared as the first PET ligand for GBM (100). Moreover, fluorinated sigma-1 receptor ligands were developed such as ¹⁸F-(S)-Fluspidine, ¹⁸F-FTC-146 and ¹⁸F-FPS with high binding affinity. Further efforts by radiopharmacists led to ¹²⁵I- PIMBA and ¹⁸F-RHM-4 with high background binding to S2R (101, 102).

Several malignancies have been associated with the human epidermal growth factor receptor (EGFR) family. All four members of this family, including HER1 (EGFR, ErbB1), HER2 (Neu, ErbB2), HER3 (ErbB3), and HER4 (ErbB4), play an important role in cell growth regulation, proliferation, and tumor migration (103). Epidermal growth factor receptor overexpression and activation of downstream signaling pathways, including RASYRAFYMAPK and PI3K/AKT, is considered to play a critical role in developing and progressing malignant gliomas (104, 105). Therefore, it is imperative to develop various treatment options, including tyrosine kinase inhibitors and monoclonal antibodies targeting EGFR, HER2, or both. Additionally, molecular imaging radiopharmaceuticals targeting EGFR family can improve the sensitivity and specificity of glioma detection (106). Following the use of ⁶⁴Cu-DOTA-trastuzumab PET in breast cancer patients, it was found to be effective in detecting brain metastases (107). As part of a pilot study conducted in 2014, 11 patients with histopathologically proven GBM underwent PET/CT examination before surgery using ¹¹C-PD153035. Six of the 8 patients with GBM were clearly visualized by ¹¹C-PD153035 PET/CT, while 2 patients with GBM, 1 with anaplastic astrocytoma, and 2 with oligodendroglioma did not show any significant uptake. According to the results, ¹¹C-PD153035 PET/CT was a promising tool for EGFR-targeted molecular imaging of GBM, which could translate into clinical applications to select patients suitable for EGFR-targeted therapies and to assess the early response of malignant gliomas to such therapies (108). A recent study evaluated the potential use of ⁸⁹Zr-DFO-nimotuzumab in assessing the EGFR status in glioma (109).

Integrins are heterodimeric transmembrane glycoproteins containing 24 αβ (18 different α and 8 β) subunits that regulate cell survival, proliferation, and differentiation through their ability to transduce signals and interact with other cellular receptors [Table 1] (110).

Among them, the pro-angiogenic αvβ3 has been found to be highly expressed in high-grade brain tumors (112). This integrin belongs to the subclass of integrins that recognize the tripeptide sequence Arg-Gly-Asp (RGD) and is found in numerous extracellular matrix proteins, including fibronectin. While αvβ3 integrins were first introduced as a marker of tumor progression and angiogenesis, specifically on both tumor-associated EC and GB cells, αvβ5 is also used as a complementary attractive therapeutic target in GB (113, 114, 119, 127–129). Numerous studies confirmed the pro-tumorigenic and pro-angiogenic activity of αvβ3/αvβ5 and most recently α6β1 in GBM (130, 131), which makes them suitable for anticancer therapy (132). In fact, comparative IHC of integrins in GB has shown an overexpression of α2, α3, α4, α5, α6 and β1 (133) as well as β3/αvβ3, αvβ5, and αvβ8 (134). Moreover, αvβ3 is preferentially expressed at the invasion front of GBM. Integrins, as the main link between a cell and extracellular matrix, play an essential role in tumor invasion in the complex regulatory network of tumor progression (135). Due to its inherent safety and ability to target both tumor and endothelial cells, RGD peptide can be used as a selective carrier for delivering anti-cancer drugs. In this regard, a number of RGD peptide drug conjugates have been proposed as targeted drug delivery systems (136, 137). Radiolabeled cyclic RGD peptides in combination with radio-immunotherapy enhance therapeutic regimens with rapid and accurate detection of tumor growth and metastasis (138, 139).

Over the past decades, several radiotracers targeting αvβ3 have been developed and investigated for clinical applications. Many of them are based on tripeptide RGD due to its high affinity and specificity for integrin αvβ3. All of the investigated RGD peptides have shown very similar in vivo distribution with high uptake in the urinary tract, due to the urinary elimination of the tracer, as well as moderate liver and intestine uptake. In one study, a sugar amino acid was conjugated to the cyclic peptide c(RGDfK) to design ¹⁸F-galacto-RGD PET tracer (140). In ¹⁸F-Galacto-RGD PET images, the tracer uptake significantly correlated with the amount of integrin 3 expressed in tumor samples based on IHC examination. The data suggested that ¹⁸F-Galacto-RGD PET might be a promising tool for planning and monitoring individualized cancer therapies targeting this integrin. A first-in-human study of ⁶⁴Cu-labeled long-acting integrin αvβ3 targeting molecule, ⁶⁴Cu-NOTA-EB-RGD, indicated the safety, favorable dosimetry profile, and pharmacokinetic properties. In three GBM patients, the EB-derived albumin-binding moiety present in ⁶⁴Cu-NOTA-EB-RGD resulted in more efficient pharmacokinetics and a higher contrast. Theranostic applications for ⁶⁴Cu-labeled EB-RGD such as ⁶⁷Cu, ¹⁷⁷Lu, ⁹⁰Y or ²²⁵Ac were recommended (141). Recently, the in vitro and in vivo characteristics of a ⁶⁸Ga-radiolabeled dimeric RGD, ⁶⁸Ga-NODAGA-c(RGDyK)2, were investigated in a glioblastoma multiforme tumor model. ⁶⁸Ga-RGD peptides showed better imaging properties than ¹⁸F-FDG and ¹⁸F-FLT in a glioblastoma tumor model in mice. Except in the excretory organs, ⁶⁸Ga-RGD peptides showed higher tumor-to-background ratios (142). A study evaluated the efficacy of ⁶⁸Ga-NOTA-RGD dimer for noninvasive integrin αvβ3 imaging in preoperative patients with different grades of glioma. According to the results, it could evaluate glioma demarcation more precisely compared to ¹⁸F-FDG PET/CT because of being specific to brain tumors, but not to brain parenchyma, except for the choroid plexus. Moreover, ⁶⁸Ga-RGD SUVmax had a significant correlation with glioma grade (143). Lo et al. developed a long-circulation integrin-targeted molecule consisting of a DOTA chelator, a truncated Evans blue dye (EB), a modified linker, and cRGDfK peptide. ¹¹¹In-DOTA-EB-cRGDfK showed significant tumor accumulation with a high tumor-to-background ratio at 24 h post-injection in the preclinical phase. The data suggested that the ability to specifically bind to α_vβ₃-expressing lesions present in the radiolabeled DOTA-EB-cRGDfK could be considered as a promising factor for glioblastoma tumor imaging indicating its potential for use as a theranostic radiopharmaceutical (144).

Chemokine receptor-4 (CXCR4) was introduced as a member of the G-protein-coupled chemokine receptor family. It affects embryogenesis, neo-angiogenesis, hematopoiesis, and inflammation. In addition to hematological malignancies, solid neoplasias express CXCR4 chemokine receptors. The activation of CXCR4 by CXCL12 induces migration and/or survival of neoplastic cells, such as tumor cells from brain tumors (neuronal and glial tumors), colorectal cancers, prostate cancer, melanoma, renal cell cancer, neuroblastoma cells, and ovarian cancer (145). One study found that in human glioma cell lines and primary tumors, CXCR4 expression correlated directly with malignancy. CXCR4 activation induced tumor cell chemotaxis and increased the production of vascular endothelial growth factor. Additionally, glioma cells expressing higher levels of CXCR4 formed more rapidly growing and lethal tumors in nude mice (146). A poorer prognosis was observed after surgery in patients with CXCR4-positive gliomas (137).

⁶⁸Ga-Pentixafor was developed as a radiolabeled cyclic pentapeptide with high affinity to CXCR4 (147). The researchers found that ⁶⁸Ga-Pentixafor PET imaging would be a promising radiotracer for the management of treatment planning in high-grade gliomas. Lapa et al. demonstrated that non-invasive imaging of CXCR4 in human malignant glioma using ⁶⁸Ga-Pentixafor was feasible (147). They showed that ⁶⁸Ga-Pentixafor PET in high-grade glioma yielded positive and receptor expression of malignant cells could be confirmed by immunohistochemical workup. Due to the extremely low background activity uptake of ⁶⁸Ga-Pentixafor in the brain, it is very favorable and has a significantly higher tumor uptake compared to amino acid tracers such as ¹⁸F-FET, which confirms the high specificity of the tracer. In this regard, radioligand agents such as ¹⁷⁷Lu in combination with Pentixafor seem to be a promising new approach for glioblastoma therapy. CXCR4-directed therapy alone or with temozolomide has already yielded promising results.

Figure 2 shows a 62-year-old man with high-grade glioma who underwent ⁶⁸Ga-CXCR4 PET/CT. A large hypo-dense cerebral lesion with good tumor delineation was observed in the right parieto-occipital lobe. Figure 3 demonstrates ⁶⁸Ga-Pentixather and ⁶⁸Ga-FAPI imaging in a 55-year-old patient with a history of GBM in the right frontotemporal lobe that underwent surgery and external beam radiation therapy and received ¹⁷⁷Lu-DOTATATE.

As a cyclic neuropeptide, somatostatin is well known in nuclear medicine for diagnosis and treatment of neuroendocrine tumors (148). The majority of the human tumors overexpress one or more of the five distinct human SSTR subtype receptors (149). Based on preliminary results from the MAURITIUS trial, of 43 glioblastoma patients treated with 400 to 3,700 MBq of ⁹⁰Y-DOTA-lanreotide in fractionated one-to-six cycles, 5 cases showed regressive disease (reduction of more than 25% of tumor size), 14 cases showed stable disease, and 24 patients had progressive disease (increase of more than 25% of tumor size). In addition, 5 patients reported a subjective improvement in the quality of life measures (150). The authors reported that preclinical data and clinical studies confirmed the promise usefulness of radiolabeled lanreotide for tumor diagnosis and therapy. A case study reported that locally injected ⁹⁰Y-DOTATOC in three patients with grade IV recurrent glioblastoma was feasible and well tolerated. An epileptic seizure recurred in one patient and a transient mild headache occurred on the day of the radiopharmaceutical application in another patient. This approach showed an attractive strategy for treating locally recurring or progressing glioblastoma (151).

PSMA is a type II transmembrane glycoprotein. Its overexpression is positively associated with higher tumor grades and stages in prostate cancer. PSMA expression is not limited to prostate cancers. Some tissues, such as salivary glands, express PSMA physiologically, and monoclonal antibodies to the extracellular domain of PSMA are also highly reactive with tumor vasculature in a variety of cancers, such as lung, breast, and colon carcinoma (152, 153). In one study, a single case of a glioblastoma patient was analyzed in whom the tumor also expressed PSMA on its neo-vasculature (154). Multiple immunohistopathological studies have confirmed PSMA expression in the tumoral vessels of high-grade gliomas (155–159).

With the development of ⁶⁸Ga-labeled PSMA, a PSMA-targeted small molecule labeled with ⁶⁸Ga, the researchers also use it as a therapeutic target with different radionuclides, e.g., ¹⁷⁷Lu. ⁶⁸Ga-PSMA is increasingly used in the routine clinical assessment of prostate cancer patients in the initial staging of intermediate or high-risk prostate cancers and in restaging after biochemical clue of tumor recurrence. With its extremely low background uptake in the normal brain tissue and a high tumor-to-brain ratio, ⁶⁸Ga-PSMA-11 PET/CT is highly promising for the diagnosis of recurrent GBM (160). Nevertheless, a low tumor-to-liver ratio observed in a study by Kunikowska et al. suggested that radiolabeled PSMA ligands may have a limited role in targeted radionuclide therapy of recurrent GBM (160). In 2021, a re-resection of a recurrent tumor following radiochemotherapy and subsequent chemotherapy was performed in 16 patients with de-novo glioblastoma (161). The results showed that both the initial diagnosis and recurrence of glioblastoma exhibited PSMA expression. The authors argued that high vascular PSMA expression upon recurrence appeared to be associated with a poor prognosis; therefore, PSMA expression in recurrent GBM might serve as a promising target for theranostic approaches. The authors recommended further studies on PSMA PET imaging and PSMA-directed radioligand therapy in patients with brain tumors. PSMA PET imaging was used to assess the potential of PMSA radioligand therapy in syngeneic GL261 GBM models in one study (162). The results showed that although ¹⁸F-PSMA-1007 PET imaging of GL261 tumor-bearing mice was feasible and produced high tumor-to-background ratio, absolute tumor uptake values remained low, suggesting the limited application of the GL261 model for PSMA-directed therapy studies.

During recent decades, numerous radiotracers and their application in neuro-oncology have been investigated in gliomas. Functional imaging techniques, as complementary tools to structural MRI, are highly needed for tumor entities such as anaplastic astrocytoma and GBM (163). As discussed earlier, while amino acid-based tracers such as FET have shown good results in comparison with FDG, other promising radiotracers such as fibroblast activated protein inhibitors (FAPIs) can also detect tumors based on the expression of fibroblast activation protein in the tumor stroma in cancer-activated fibroblasts of GBM.

Various types of FAPI have been already established such as FAPI-02/04-46/74 and their biodistribution in GBM patients is currently under investigation (164, 165). As a pioneer researcher in the production of FAP, Giesel et al. assessed the biodistribution of ⁶⁸Ga-FAPI-02/04 in eight patients with head and neck cancer (166). The results showed that tumor-to-background ratios of FAP-specific PET were almost similar to ¹⁸F-FDG-PET. Röhrich et al. showed increased FAPI-02/04 uptake in 13 patients with grade III/IV gliomas and high-grade glioblastomas (167). Additionally, Chen et al. compared ⁶⁸Ga-DOTA-FAPI-04 and ¹⁸F-FDG PET/CT for the diagnosis of primary and metastatic lesions in patients with various types of cancer including four glioma patients (two GBM, one grade II glioma, and one grade III glioma). However, the absolute uptake of FAP for tumor was low and the tumor-to-background ratio was high, lower uptake of FAP with a higher tumor-to-background ratio were observed in gliomas in other brain studies (168–170).

A case report study by Ballal1 et al. showed the possibility of a theranostic approach of ⁶⁸Ga-DOTA.SA.FAPi PET/CT-guided ¹⁷⁷Lu-DOTA.SA.FAPi radionuclide therapy in an end-stage breast cancer patient with confirmed brain metastasis (171). The patient received a single cycle of ¹⁷⁷Lu-DOTA.SA.FAPi. There was physiological uptake of ¹⁷⁷Lu-DOTA.SA.FAPi in the liver, kidneys, pancreas, and background muscle, and an intense accumulation of radiotracer was noted in all lesions in agreement with ⁶⁸Ga-DOTA.SA.FAPi PET/CT scans. The authors declared that in patients not responding to conventional treatment options, ⁶⁸Ga-DOTA.SA.FAPi PET/CT-guided ¹⁷⁷Lu-DOTA.SA.FAPi therapy might offer a new opportunity for a theranostic approach in breast cancer therapy.

Moon et al. reported better tumor retention by introducing squaric acid (SA) as a linker into the FAP inhibitor yielding DATA5m.SA.FAPi and DOTA.SA.FAPi (172). Wang et al. demonstrated that ⁶⁸Ga-DOTA.SA.FAPi PET/CT and ¹⁸F-FDG PET/CT had similarly high SUL uptake values in the primary site of head and neck cancers (173). In another study by Ballal et al. ⁶⁸Ga-DOTA.SA.FAPi identified additional lesions in the brain that could not be detected on ¹⁸F-FDG PET/CT (174). According to the results, lower cortex uptake of ⁶⁸Ga-DOTA.SA.FAPi in the brain makes it an ideal radiotracer where ¹⁸F-FDG PET/CT fails to detect brain tumors.

Figure 4. A 55-year-old man with recurrent high-grade glioma tumor undergoing ⁶⁸Ga-FAPI PET/CT (left side) and one cycle of ¹⁷⁷Lu-FAPI (right side).