Background: Immune-positron emission tomography (PET) imaging with tracers that target CD8 and granzyme B has shown promise in predicting the therapeutic response following immune checkpoint blockade (ICB) in immunologically “hot” tumors. However, immune dynamics in the low T-cell infiltrating “cold” tumor immune microenvironment during ICB remain poorly understood. This study uses molecular imaging to evaluate changes in CD4 + T cells and CD8 + T cells during ICB in breast cancer models and examines biomarkers of response. Methods: [⁸⁹Zr]Zr-DFO-CD4 and [⁸⁹Zr]Zr-DFO-CD8 radiotracers were used to quantify changes in intratumoral and splenic CD4 T cells and CD8 T cells in response to ICB treatment in 4T1 and MMTV-HER2 mouse models, which represent immunologically “cold” tumors. A correlation between PET quantification metrics and long-term anti-tumor response was observed. Further biological validation was obtained by autoradiography and immunofluorescence. Results: Following ICB treatment, an increase in the CD8-specific PET signal was observed within 6 days, and an increase in the CD4-specific PET signal was observed within 2 days in tumors that eventually responded to immunotherapy, while no significant differences in CD4 or CD8 were found at the baseline of treatment that differentiated responders from nonresponders. Furthermore, mice whose tumors responded to ICB had a lower CD8 PET signal in the spleen and a higher CD4 PET signal in the spleen compared to non-responders. Intratumoral spatial heterogeneity of the CD8 and CD4-specific PET signals was lower in responders compared to non-responders. Finally, PET imaging, autoradiography, and immunofluorescence signals were correlated when comparing in vivo imaging to ex vivo validations. Conclusions: CD4- and CD8-specific immuno-PET imaging can be used to characterize the in vivo distribution of CD4 + and CD8 + T cells in response to immune checkpoint blockade. Imaging metrics that describe the overall levels and distribution of CD8 + T cells and CD4 + T cells can provide insight into immunological alterations, predict biomarkers of response to immunotherapy, and guide clinical decision-making in those tumors where the kinetics of the response differ.

Rationale: Novel immune-activating therapeutics for the treatment of glioblastoma multiforme (GBM) have shown potential for tumor regression and increased survival over standard therapies. However, immunotherapy efficacy remains inconsistent with response assessment being complicated by early treatment-induced apparent radiological tumor progression and slow downstream effects. This inability to determine early immunotherapeutic benefit results in a drastically decreased window for alternative, and potentially more effective, treatment options. The objective of this study is to evaluate the effects of combination immunotherapy on early CD8⁺ cell infiltration and its association with long term response in orthotopic syngeneic glioblastoma models.

Our results from quantitative RT-PCR, Western blotting, immunohistochemistry, and the tissue microarray of medullary thyroid cancer (MTC) cell lines and patient specimens confirm that VGSC subtype Na_V1.7 is uniquely expressed in aggressive MTC and not expressed in normal thyroid cells and tissues. We establish the druggability of Na_V1.7 in MTC by identifying a novel inhibitor (SV188) and investigate its mode of binding and ability to inhibit I_Na current in Na_V1.7. The whole-cell patch-clamp studies of the SV188 in the Na_V1.7 channels expressed in HEK-293 cells show that SV188 inhibited the I_Na current in Na_V1.7 with an IC₅₀ value of 3.6 µM by a voltage- and use-dependent blockade mechanism, and the maximum inhibitory effect is observed when the channel is open. SV188 inhibited the viability of MTC cell lines, MZ-CRC-1 and TT, with IC₅₀ values of 8.47 μM and 9.32 μM, respectively, and significantly inhibited the invasion of MZ-CRC-1 cells by 35% and 52% at 3 μM and 6 μM, respectively. In contrast, SV188 had no effect on the invasion of TT cells derived from primary tumor, which have lower basal expression of Na_V1.7. In addition, SV188 at 3 μM significantly inhibited the migration of MZ-CRC-1 and TT cells by 27% and 57%, respectively.

Epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), and hypoxia are associated with radioresistance. The goal of this study is to study the synergy of anti-HER2, trastuzumab, and anti-EGFR, cetuximab, and characterize the tumor microenvironment components that may lead to increased radiation sensitivity with dual anti-HER2/EGFR therapy in head and neck squamous cell carcinoma (HNSCC). Positron emission tomography (PET) imaging ([⁸⁹Zr]-panitumumab and [⁸⁹Zr]-pertuzumab) was used to characterize EGFR and HER2 in HNSCC cell line tumors. HNSCC cells were treated with trastuzumab, cetuximab, or combination followed by radiation to assess for viability and radiosensitivity (colony forming assay, immunofluorescence, and flow cytometry). In vivo, [¹⁸F]-FMISO-PET imaging was used to quantify changes in oxygenation during treatment. Bliss Test of Synergy was used to identify combination treatment synergy. Quantifying EGFR and HER2 receptor expression revealed a 50% increase in heterogeneity of HER2 relative to EGFR. In vitro, dual trastuzumab-cetuximab therapy shows significant decreases in DNA damage response and increased response to radiation therapy (p < 0.05). In vivo, tumors treated with dual anti-HER2/EGFR demonstrated decreased tumor hypoxia, when compared to single agent therapies. Dual trastuzumab-cetuximab demonstrates synergy and can affect tumor oxygenation in HNSCC. Combination trastuzumab-cetuximab modulates the tumor microenvironment through reductions in tumor hypoxia and induces sustained treatment synergy.

The highly heterogeneous and invasive nature characteristic of high-grade gliomas (HGG) has historically limited the efficacy of standard-of-care approaches, resulting in poor prognosis and treatment outcomes. Novel immunotherapies have shown remarkable potential to promote antitumoral immune responses and allow for long-term tumor remission. However, the complexity of the HGG tumor microenvironment and the dynamic immunological changes associated with immunotherapy response can limit the diagnostic utility of conventional magnetic resonance imaging (MRI) and positron emission tomography (PET) approaches. Consequently, distinguishing true tumor progression from immunotherapy-related effects often requires prolonged clinical follow-up over several months. To address this, novel quantitative MRI and PET-based approaches are being evaluated in preclinical studies and clinical trials. These advanced imaging methods target key biological features of the tumor microenvironment, including vascularity, cellularity, intratumoral habitats, tracer pharmacokinetics and immune infiltration, and can provide metrics to stratify patient response at earlier timepoints to support clinical decision making and improve treatment outcomes. This review highlights key HGG biological characteristics, describes standard-of-care and emerging therapeutic strategies, and discusses both conventional and advanced imaging methods to characterize immunotherapeutic responses.