Evaluating the immunologically "cold" tumor microenvironment after treatment with immune checkpoint inhibitors utilizing PET imaging of CD4 + and CD8 + T cells in breast cancer mouse models.

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.

PET Imaging for Monitoring Cellular and Immunotherapy of Cancer.

ABSTRACT: Cancer immunotherapy, including checkpoint blockade and cellular therapy, has become a cornerstone in cancer treatment. However, understanding the factors driving patient response or resistance to these therapies remains challenging. The dynamic interplay between the immune system and tumors requires new approaches for characterization. Biopsies and blood tests provide valuable information, but their limitations have led to increased interest in positron emission tomography (PET)/computed tomography imaging to complement these strategies. The noninvasive nature of PET imaging makes it ideal for monitoring the dynamic tumor immune microenvironment. This review discusses various PET imaging approaches, including immune cell lineage markers, immune functional markers, immune cell metabolism, direct cell labeling, and reporter genes, highlighting their potential in targeted immunotherapies and cell-based approaches. Although PET imaging has limitations, its integration into diagnostic strategies holds promise for improving patient outcomes and accelerating drug development in cancer immunotherapy.

[18F]FMISO-PET imaging reveals the role of hypoxia severity in checkpoint blockade response.

CONTEXT: Hypoxia within the tumor microenvironment is a critical factor influencing the efficacy of immunotherapy, including immune checkpoint inhibition. Insufficient oxygen supply, characteristic of hypoxia, has been recognized as a central determinant in the progression of various cancers. The reemergence of evofosfamide, a hypoxia-activated prodrug, as a potential treatment strategy has sparked interest in addressing the role of hypoxia in immunotherapy response. This investigation sought to understand the kinetics and heterogeneity of tumor hypoxia and their implications in affecting responses to immunotherapeutic interventions with and without evofosfamide. PURPOSE: This study aimed to investigate the influence of hypoxia on immune checkpoint inhibition, evofosfamide monotherapy, and their combination on colorectal cancer (CRC). Employing positron emission tomography (PET) imaging, we developed novel analytical methods to quantify and characterize tumor hypoxia severity and distribution. PROCEDURES: Murine CRC models were longitudinally imaged with [18F]-fluoromisonidazole (FMISO)-PET to quantify tumor hypoxia during checkpoint blockade (anti-CTLA-4 + and anti-PD1 +/- evofosfamide). Metrics including maximum tumor [18F]FMISO uptake (FMISOmax) and mean tumor [18F]FMISO uptake (FMISOmean) were quantified and compared with normal muscle tissue (average muscle FMISO uptake (mAvg) and muscle standard deviation (mSD)). Histogram distributions were used to evaluate heterogeneity of tumor hypoxia. FINDINGS: Severe hypoxia significantly impeded immunotherapy effectiveness consistent with an immunosuppressive microenvironment. Hypoxia-specific PET imaging revealed a striking degree of spatial heterogeneity in tumor hypoxia, with some regions exhibiting significantly more severe hypoxia than others. The study identified FMISOmax as a robust predictor of immunotherapy response, emphasizing the impact of localized severe hypoxia on tumor volume control during therapy. Interestingly, evofosfamide did not directly reduce hypoxia but markedly improved the response to immunotherapy, uncovering an alternative mechanism for its efficacy. CONCLUSIONS: These results enhance our comprehension of the interplay between hypoxia and immune checkpoint inhibition within the tumor microenvironment, offering crucial insights for the development of personalized cancer treatment strategies. Non-invasive hypoxia quantification through molecular imaging evaluating hypoxia severity may be an effective tool in guiding treatment planning, predicting therapy response, and ultimately improving patient outcomes across diverse cancer types and tumor microenvironments. It sets the stage for the translation of these findings into clinical practice, facilitating the optimization of immunotherapy regimens by addressing tumor hypoxia and thereby enhancing the efficacy of cancer treatments.