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.

Dual anti-HER2/EGFR inhibition synergistically increases therapeutic effects and alters tumor oxygenation in HNSCC.

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.

Combination Therapy with Trastuzumab and Niraparib: Quantifying Early Proliferative Alterations in HER2+ Breast Cancer Models.

HER2-targeted treatments have improved survival rates in HER2+ breast cancer patients, yet poor responsiveness remains a major clinical obstacle. Recently, HER2+ breast cancer cells, both resistant and responsive to HER2-targeted therapies, have demonstrated sensitivity to poly-(ADP-ribose) polymerase (PARP) inhibition, independent of DNA repair deficiencies. This study seeks to describe biological factors that precede cell viability changes in response to the combination of trastuzumab and PARP inhibition. Treatment response was evaluated in HER2+ and HER2- breast cancer cells. Further, we evaluated the utility of 3'-Deoxy-3'-[18F]-fluorothymidine positron emission tomography ([18F]FLT-PET) imaging for early response assessment in a HER2+ patient derived xenograft (PDX) model of breast cancer. In vitro, we observed decreased cell viability. In vivo, we observed decreased inhibition in tumor growth in combination therapies, compared to vehicle and monotherapy-treated cohorts. Early assessment of cellular proliferation corresponds to endpoint cell viability. Standard summary statistics of [18F]FLT uptake from PET were insensitive to early proliferative changes. Meanwhile, histogram analysis of [18F]FLT uptake indicated the potential translatability of imaging proliferation biomarkers. This study highlights the potential of combined trastuzumab and PARP inhibition in HER2+ breast cancer, while demonstrating a need for optimization of [18F]FLT-PET quantification in heterogeneous models of HER2+ breast cancer.

[89Zr]-Atezolizumab-PET Imaging Reveals Longitudinal Alterations in PDL1 during Therapy in TNBC Preclinical Models.

Triple-negative breast cancers (TNBCs) currently have limited treatment options; however, PD-L1 is an indicator of susceptibility to immunotherapy. Currently, assessment of PD-L1 is limited to biopsy samples. These limitations may be overcome with molecular imaging. In this work, we describe chemistry development and optimization, in vitro, in vivo, and dosimetry of [89Zr]-Atezolizumab for PD-L1 imaging. Atezolizumab was conjugated to DFO and radiolabeled with 89Zr. Tumor uptake and heterogeneity in TNBC xenograft and patient-derived xenograft (PDX) mouse models were quantified following [89Zr]-Atezolizumab-PET imaging. PD-L1 expression in TNBC PDX models undergoing therapy and immunohistochemistry (IHC) was used to validate imaging. SUV from PET imaging was quantified and used to identify heterogeneity. PET/CT imaging using [89Zr]-Atezolizumab identified a significant increase in tumor:muscle SUVmean 1 and 4 days after niraparib therapy and revealed an increased trend in PD-L1 expression following other cytotoxic therapies. A preliminary dosimetry study indicated the organs that will receive a higher dose are the spleen, adrenals, kidneys, and liver. [89Zr]-Atezolizumab PET/CT imaging reveals potential for the noninvasive detection of PD-L1-positive TNBC tumors and allows for quantitative and longitudinal assessment. This has potential significance for understanding tumor heterogeneity and monitoring early expression changes in PD-L1 induced by therapy.

Human Epidermal Growth Factor Receptor 2/Human Epidermal Growth Factor Receptor 3 PET Imaging: Challenges and Opportunities.

Human epidermal growth factor receptor 2 (HER2) and HER3 provide actionable targets for both therapy and imaging in breast cancer. Further, clinical trials have shown the prognostic impact of receptor status discordance in breast cancer. Intra- and intertumoral heterogeneity of both HER and hormone receptor expression contributes to inherent errors in tissue sampling, and single biopsies are incapable of identifying discordance in biomarker expression. Numerous PET radiopharmaceuticals have been developed to evaluate (or target for therapy) HER2 and HER3 expression. This review seeks to inform on challenges and opportunities in HER2 and HER3 PET imaging in both clinical and preclinical settings.

Modulation of the Tumor Microenvironment with Trastuzumab Enables Radiosensitization in HER2+ Breast Cancer.

DNA damage repair and tumor hypoxia contribute to intratumoral cellular and molecular heterogeneity and affect radiation response. The goal of this study is to investigate anti-HER2-induced radiosensitization of the tumor microenvironment to enhance fractionated radiotherapy in models of HER2+ breast cancer. This is monitored through in vitro and in vivo studies of phosphorylated γ-H2AX, [18F]-fluoromisonidazole (FMISO)-PET, and transcriptomic analysis. In vitro, HER2+ breast cancer cell lines were treated with trastuzumab prior to radiation and DNA double-strand breaks (DSB) were quantified. In vivo, HER2+ human cell line or patient-derived xenograft models were treated with trastuzumab, fractionated radiation, or a combination and monitored longitudinally with [18F]-FMISO-PET. In vitro DSB analysis revealed that trastuzumab administered prior to fractionated radiation increased DSB. In vivo, trastuzumab prior to fractionated radiation significantly reduced hypoxia, as detected through decreased [18F]-FMISO SUV, synergistically improving long-term tumor response. Significant changes in IL-2, IFN-gamma, and THBS-4 were observed in combination-treated tumors. Trastuzumab prior to fractionated radiation synergistically increases radiotherapy in vitro and in vivo in HER2+ breast cancer which is independent of anti-HER2 response alone. Modulation of the tumor microenvironment, through increased tumor oxygenation and decreased DNA damage response, can be translated to other cancers with first-line radiation therapy.

Hyperintensity of integrin-targeted fluorescence agent IntegriSense750 accurately predicts flap necrosis compared to Indocyanine green.

BACKGROUND: Flap necrosis is a feared complication of reconstructive surgery. Current methods of prediction using Indocyanine green (ICG) lack specificity. IntegriSense750 is a fluorescence agent that binds sites of vascular remodeling. We hypothesized that IntegriSense750 better predicts flap compromise compared to ICG. METHODS: Fifteen mice underwent lateral thoracic artery axial flap harvest. Mice received an injection of ICG (n = 7) or IntegriSense750 (n = 8) daily from postoperative days (POD) 0-3 and were imaged daily. Mean signal-to-background ratios quantified the change in fluorescence as necrosis progressed. RESULTS: Mean signal-to-background ratio was significantly higher for IntegriSense750 compared to ICG on POD0 (1.47 ± 0.17 vs. 0.86 ± 0.21, p = 0.01) and daily through POD3 (2.12 ± 0.70 vs. 0.96 ± 0.29, p < 0.001). CONCLUSIONS: IntegriSense750 demonstrates increased signal-to-background ratio at areas of flap distress compared to ICG which may increase identification of flap necrosis and improve patient outcomes.

[89Zr]-Pertuzumab PET Imaging Reveals Paclitaxel Treatment Efficacy Is Positively Correlated with HER2 Expression in Human Breast Cancer Xenograft Mouse Models.

Paclitaxel (PTX) treatment efficacy varies in breast cancer, yet the underlying mechanism for variable response remains unclear. This study evaluates whether human epidermal growth factor receptor 2 (HER2) expression level utilizing advanced molecular positron emission tomography (PET) imaging is correlated with PTX treatment efficacy in preclinical mouse models of HER2+ breast cancer. HER2 positive (BT474, MDA-MB-361), or HER2 negative (MDA-MB-231) breast cancer cells were subcutaneously injected into athymic nude mice and PTX (15 mg/kg) was administrated. In vivo HER2 expression was quantified through [89Zr]-pertuzumab PET/CT imaging. PTX treatment response was quantified by [18F]-fluorodeoxyglucose ([18F]-FDG) PET/CT imaging. Spearman's correlation, Kendall's tau, Kolmogorov-Smirnov test, and ANOVA were used for statistical analysis. [89Zr]-pertuzumab mean standard uptake values (SUVmean) of BT474 tumors were 4.9 ± 1.5, MDA-MB-361 tumors were 1.4 ± 0.2, and MDA-MB-231 (HER2-) tumors were 1.1 ± 0.4. [18F]-FDG SUVmean changes were negatively correlated with [89Zr]-pertuzumab SUVmean (r = -0.5887, p = 0.0030). The baseline [18F]-FDG SUVmean was negatively correlated with initial [89Zr]-pertuzumab SUVmean (r = -0.6852, p = 0.0002). This study shows PTX treatment efficacy is positively correlated with HER2 expression level in human breast cancer mouse models. Molecular imaging provides a non-invasive approach to quantify biological interactions, which will help in identifying chemotherapy responders and potentially enhance clinical decision-making.

CD4 T-cell immune stimulation of HER2 + breast cancer cells alters response to trastuzumab in vitro.

INTRODUCTION: The HER2 + tumor immune microenvironment is composed of macrophages, natural killer cells, and tumor infiltrating lymphocytes, which produce pro-inflammatory cytokines. Determining the effect of T-cells on HER2 + cancer cells during therapy could guide immunogenic therapies that trigger antibody-dependent cellular cytotoxicity. This study utilized longitudinal in vitro time-resolved microscopy to measure T-cell influence on trastuzumab in HER2 + breast cancer. METHODS: Fluorescently-labeled breast cancer cells (BT474, SKBR3, MDA-MB-453, and MDA-MB-231) were co-cultured with CD4 + T-cells (Jurkat cell line) and longitudinally imaged to quantify cancer cell viability when treated with or without trastuzumab (10, 25, 50 and 100 µg/mL). The presence and timing of T-cell co-culturing was manipulated to determine immune stimulation of trastuzumab-treated HER2 + breast cancer. HER2 and TNF-α expression were evaluated with western blot and ELISA, respectively. Significance was calculated using a two-tailed parametric t-test. RESULTS: The viability of HER2 + cancer cells significantly decreased when exposed to 25 µg/mL trastuzumab and T-cells, compared to cancer cells exposed to trastuzumab without T-cells (p = 0.01). The presence of T-cells significantly increased TNF-α expression in trastuzumab-treated cancer cells (p = 0.02). Conversely, cancer cells treated with TNF-α and trastuzumab had a similar decrease in viability as trastuzumab-treated cancer cells co-cultured with T-cells (p = 0.32). CONCLUSIONS: The presence of T-cells significantly increases the efficacy of targeted therapies and suggests trastuzumab may trigger immune mediated cytotoxicity. Increased TNF-α receptor expression suggest cytokines may interact with trastuzumab to create a state of enhanced response to therapy in HER2 + breast cancer, which has potential to reducing tumor burden.