Glioblastoma (GBM) is a primary neuroepithelial tumor of the central nervous system, characterized by an extremely aggressive clinical phenotype. Patients with GBM have a poor prognosis and only 3–5% of them survive for more than 5 years. The current GBM treatment standards include maximal resection followed by radiotherapy with concomitant and adjuvant therapies. Despite these aggressive therapeutic regimens, the majority of patients suffer recurrence due to molecular heterogeneity of GBM. Consequently, a number of potential diagnostic, prognostic, and predictive biomarkers have been investigated. Some of them, such as IDH mutations, 1p19q deletion, MGMT promoter methylation, and EGFRvIII amplification are frequently tested in routine clinical practice. With the development of sequencing technology, detailed characterization of GBM molecular signatures has facilitated a more personalized therapeutic approach and contributed to the development of a new generation of anti-GBM therapies such as molecular inhibitors targeting growth factor receptors, vaccines, antibody-based drug conjugates, and more recently inhibitors blocking the immune checkpoints. In this article, we review the exciting progress towards elucidating the potential of current and novel GBM biomarkers and discuss their implications for clinical practice.

Bioluminescence imaging (BLI) is ubiquitous in scientific research for the sensitive tracking of biological processes in small animal models. However, due to the attenuation of visible light by tissue, and the limited set of near-infrared bioluminescent enzymes, BLI is largely restricted to monitoring single processes in vivo. Here we show, that by combining stabilised colour mutants of firefly luciferase (FLuc) with the luciferin (LH2) analogue infraluciferin (iLH2), near-infrared dual BLI can be achieved in vivo. The X-ray crystal structure of FLuc with a high-energy intermediate analogue, 5’-O-[N-(dehydroinfraluciferyl)sulfamoyl] adenosine (iDLSA) provides insight into the FLuc-iLH2 reaction leading to near-infrared light emission. The spectral characterisation and unmixing validation studies reported here established that iLH2 is superior to LH2 for the spectral unmixing of bioluminescent signals in vivo; which led to this novel near-infrared dual BLI system being applied to monitor both tumour burden and CAR T cell therapy within a systemically induced mouse tumour model.

Glioblastomas (GBMs) are high‐grade brain tumors, differentially driven by alterations (amplification, deletion or missense mutations) in the epidermal growth factor receptor (EGFR), that carry a poor prognosis of just 12–15 months following standard therapy. A combination of interventions targeting tumor‐specific cell surface regulators along with convergent downstream signaling pathways may enhance treatment efficacy. Against this background, we investigated a novel photoimmunotherapy approach combining the cytotoxicity of photodynamic therapy with the specificity of immunotherapy. An EGFR‐specific affibody (ZEGFR:03115) was conjugated to the phthalocyanine dye, IR700DX, which when excited with near‐infrared light produces a cytotoxic response. ZEGFR:03115–IR700DX EGFR‐specific binding was confirmed by flow cytometry and confocal microscopy. The conjugate showed effective targeting of EGFR positive GBM cells in the brain. The therapeutic potential of the conjugate was assessed both in vitro, in GBM cell lines and spheroids by the CellTiter‐Glo® assay, and in vivo using subcutaneous U87‐MGvIII xenografts. In addition, mice were imaged pre‐ and post‐PIT using the IVIS/Spectrum/CT to monitor treatment response. Binding of the conjugate correlated to the level of EGFR expression in GBM cell lines. The cell proliferation assay revealed a receptor‐dependent response between the tested cell lines. Inhibition of EGFRvIII+ve tumor growth was observed following administration of the immunoconjugate and irradiation. Importantly, this response was not seen in control tumors. In conclusion, the ZEGFR:03115–IR700DX showed specific uptake in vitro and enabled imaging of EGFR expression in the orthotopic brain tumor model. Moreover, the proof‐of‐concept in vivo PIT study demonstrated therapeutic efficacy of the conjugate in subcutaneous glioma xenografts.

2 Translational RelevanceCurrently, inter-and intra-tumor heterogeneity is a clinical challenge, as histological techniques can fail to provide a representative indication of molecular variation, due to dependence on the section of tumor that is chosen for sampling. This underscores the need to introduce novel imaging biomarkers that allow the examination of the whole tumor mass and may significantly help to better understand and treat cancer. Therefore, we have developed a novel PET radiotracer that will provide information on heterogeneous HER3 expression and receptor expression changes due to downstream signaling inhibition, which is increasingly being recognized as a key player in therapeutic resistance. This would aid in the selection of patients for novel HER3 targeted therapies and potentially enable patients to be spared ineffective therapies, and, if necessary, being switched sooner to more effective therapeutic regimens.Research. Zr-DFO-Z HER3:8698 can track changes in receptor expression in HER3-positive xenograft models and monitor the outcome of AUY922 treatment. Our in vitro findings showed that MCF-7 cells, which are phenotypically different from BT474, develop resistance to AUY922 through HER3/IGF-1R-mediated signaling. Of note, the lack of response in vitro due to HER3 recovery was confirmed in vivo using 89 Zr-DFO-Z HER3:8698 -based imaging. Upon AUY922 treatment, higher radioconjugate uptake was detected in treated MCF-7 xenografts, correlating with an AUY922-induced HER3 up-regulation concomitant with an increase in IGF-1R expression. Conclusion: These data underline the potential of HER3-based PET imaging to noninvasively provide information about HER3 expression and to identify patients not-responding to targeted therapies due to HER3 recovery.

In head and neck squamous cell carcinoma (HNSCC), the human epidermal growth factor receptor 1 (EGFR) is the dominant signaling molecule among all members of the family. So far, cetuximab is the only approved anti-EGFR mAb used for the treatment of HNSCC, but despite the benefits of adding it to standard treatment regimens, attempts to define a predictive biomarker to stratify patients for cetuximab treatment have been unsuccessful. We hypothesized that imaging with EGFR-specific radioligands may facilitate non-invasive measurement of EGFR expression across the entire tumor burden and also allow for dynamic monitoring of cetuximab-mediated changes in receptor expression. EGFR-specific Affibody molecule (ZEGFR:03115) was radiolabeled with zirconium-89 (Zr) and fluorine-18 (F). The radioligands were characterized in vitro and in mice bearing subcutaneous tumors with varying levels of EGFR expression. The protein dose for imaging studies was assessed by injecting Zr-DFO-ZEGFR:03115 (2.4-3.6 MBq, 2 µg) either together with or 30 mins after increasing amounts of unlabeled ZEGFR:03115 (1, 5, 10, 15 and 20 µg). PET images were acquired at 3, 24 and 48 h post-injection and the image quantification data were correlated with the biodistribution results. The EGFR expression and biodistribution of the tracer were assessed ex vivo by immunohistochemistry, Western blot and autoradiography. To downregulate the EGFR level, treatment with cetuximab was performed andF-AlF-NOTA-ZEGFR:03115 (12 µg, 1.5-2 MBq/mouse) used to monitor receptor changes. In vivo studies demonstrated that co-injecting 10 µg of non-labeled molecules withZr-DFO-ZEGFR:03115 allows for clear tumor visualization 3 h post-injection. The radioconjugate tumor accumulation was EGFR-specific and PET imaging data showed a clear differentiation between xenografts with varying EGFR expression levels. A strong correlation was observed between PET analysis, ex vivo estimates of tracer concentration and receptor expression in tumor tissues. Additionally, F-AlF-NOTA-ZEGFR:03115 could measure receptor downregulation in response to EGFR inhibition. ZEGFR03115-based radioconjugates can assess different levels of EGFR level in vivo and measure receptor expression changes in response to cetuximab, indicating a potential for assessment of adequate treatment dosing with anti-EGFR antibodies.