ABSTRACT
Manganese-52 is gaining interest as an isotope for PET imaging due to its desirable decay and chemical properties for radiopharmaceutical development. Somatostatin receptor 2 (SSTR2) is significantly overexpressed by neuroendocrine tumors (NETs) and is an important target for nuclear imaging and therapy. As an agonist, [68Ga]Ga-DOTATATE has demonstrated significant internalization upon interaction with receptor ligands, whereas [68Ga]Ga-DOTA-JR11(as an antagonist) exhibits limited internalization but better pharmacokinetics and increased tumor uptake. The goal of this study was to label both DOTATATE and DOTA-JR11 peptides with 52Mn in high radiochemical yields (RCY) and sufficient specific activity. A comparison of these two compounds was performed in in vitro and in vivo studies in animals with somatostatin receptor-positive xenografts to characterize differences in cell, tumor, and tissue uptake. Radiolabeling of DOTATATE and DOTA-JR11 was carried out by combining varying concentrations of the peptides with [52Mn]MnCl2. In vitro stability of the radiotracers was determined in mouse serum. In vitro cell uptake and internalization assays were performed in SSTR2 + AR42J cells and negative controls. In vivo biodistribution and longitudinal PET imaging was evaluated in mice bearing AR42J tumors. Both [52Mn]Mn-DOTATATE and [52Mn]Mn-DOTA-JR11showed affinity for SSTR2 in AR42J cells. However, the uptake of [52Mn]Mn-DOTATATE was higher (11.95 ± 0.71%/ mg) compared to [52Mn]Mn-DOTA-JR11 (7.31 ± 0.38%/ mg) after 2 h incubation. After 4 h incubation, 53.13 ± 1.83% of the total activity of [52Mn]Mn-DOTATATE was internalized, whereas only 20.85 ± 0.59% of the total activity of [52Mn]Mn-DOTA-JR11 was internalized. The PET images revealed similar biodistribution results, with [52Mn]Mn-DOTATATE showing a significant tumor uptake of 11.16 ± 2.97% ID/g, while [52Mn]Mn-DOTA-JR11 exhibited a lower tumor uptake of 2.11 ± 0.30% ID/g 4 h post-injection. The synthesis of both radiotracers was accomplished with high RCY and purity. The cell uptake and internalization of [52Mn]Mn-DOTATATE showed higher levels compared to [52Mn]Mn-DOTA-JR11. PET images of the radiotracers in AR42J tumor bearing mice demonstrated similar biodistribution in all organs except the tumor, with [52Mn]Mn-DOTATATE showing higher tumor uptake compared to [52Mn]Mn-DOTA-JR11. The variations in properties of these tracers could be used to guide further imaging and treatment studies.
ABSTRACT
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.
Subject(s)
Breast Neoplasms , CD4-Positive T-Lymphocytes , CD8-Positive T-Lymphocytes , Disease Models, Animal , Immune Checkpoint Inhibitors , Positron-Emission Tomography , Tumor Microenvironment , Animals , Tumor Microenvironment/immunology , Female , Mice , CD8-Positive T-Lymphocytes/immunology , Immune Checkpoint Inhibitors/therapeutic use , Immune Checkpoint Inhibitors/pharmacology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Positron-Emission Tomography/methods , Breast Neoplasms/diagnostic imaging , Breast Neoplasms/immunology , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Breast Neoplasms/metabolism , Breast Neoplasms/therapy , Humans , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Cell Line, Tumor , Zirconium , Radiopharmaceuticals , RadioisotopesABSTRACT
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.
Subject(s)
Immune Checkpoint Inhibitors , Misonidazole , Positron-Emission Tomography , Tumor Hypoxia , Animals , Positron-Emission Tomography/methods , Mice , Misonidazole/analogs & derivatives , Tumor Hypoxia/drug effects , Immune Checkpoint Inhibitors/therapeutic use , Immune Checkpoint Inhibitors/pharmacology , Cell Line, Tumor , Colorectal Neoplasms/diagnostic imaging , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/therapy , Female , Tumor MicroenvironmentABSTRACT
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.
Subject(s)
Head and Neck Neoplasms , Humans , Cetuximab/pharmacology , Cetuximab/therapeutic use , Squamous Cell Carcinoma of Head and Neck/drug therapy , Trastuzumab/pharmacology , Trastuzumab/therapeutic use , Head and Neck Neoplasms/drug therapy , Cell Line, Tumor , Tumor Microenvironment , ErbB ReceptorsABSTRACT
Rare diseases are underrepresented in biomedical research, leading to insufficient awareness. Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is a rare disease caused by genetic alterations that result in heterozygous loss of function of SON. While patients with ZTTK syndrome live with numerous symptoms, the lack of model organisms hampers our understanding of SON and this complex syndrome. Here, we developed Son haploinsufficiency (Son+/-) mice as a model of ZTTK syndrome and identified the indispensable roles of Son in organ development and hematopoiesis. Son+/- mice recapitulated clinical symptoms of ZTTK syndrome, including growth retardation, cognitive impairment, skeletal abnormalities, and kidney agenesis. Furthermore, we identified hematopoietic abnormalities in Son+/- mice, including leukopenia and immunoglobulin deficiency, similar to those observed in human patients. Surface marker analyses and single-cell transcriptome profiling of hematopoietic stem and progenitor cells revealed that Son haploinsufficiency shifted cell fate more toward the myeloid lineage but compromised lymphoid lineage development by reducing genes required for lymphoid and B cell lineage specification. Additionally, Son haploinsufficiency caused inappropriate activation of erythroid genes and impaired erythropoiesis. These findings highlight the importance of the full gene expression of Son in multiple organs. Our model serves as an invaluable research tool for this rare disease and related disorders associated with SON dysfunction.
Subject(s)
Hematopoiesis , Rare Diseases , Animals , Humans , Mice , Gene Expression Profiling , Hematopoiesis/genetics , MutationABSTRACT
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. Methods: Luciferase positive GBM orthotopic mouse models (GSC005-luc) were imaged via [89Zr]-CD8 positron emission tomography (PET) one week following treatment with saline, anti-PD1, M002 oncolytic herpes simplex virus (oHSV) or combination immunotherapy. Subsequently, brains were excised, imaged via [89Zr]-CD8 ImmunoPET and evaluated though autoradiography and histology for H&E and CD8 immunohistochemistry. Longitudinal immunotherapeutic effects were evaluated through [89Zr]-CD8 PET imaging one- and three-weeks following treatment, with changes in tumor volume monitored on a three-day basis via bioluminescence imaging (BLI). Response classification was then performed based on long-term BLI signal changes. Statistical analysis was performed between groups using one-way ANOVA and two-sided unpaired T-test, with p < 0.05 considered significant. Correlations between imaging and biological validation were assessed via Pearson's correlation test. Results: [89Zr]-CD8 PET standardized uptake value (SUV) quantification was correlated with ex vivo SUV quantification (r = 0.61, p < 0.01), autoradiography (r = 0.46, p < 0.01), and IHC tumor CD8+ cell density (r = 0.55, p < 0.01). Classification of therapeutic responders, via bioluminescence signal, revealed a more homogeneous CD8+ immune cell distribution in responders (p < 0.05) one-week following immunotherapy. Conclusions: Assessment of early CD8+ cell infiltration and distribution in the tumor microenvironment provides potential imaging metrics for the characterization of oHSV and checkpoint blockade immunotherapy response in GBM. The combination therapies showed enhanced efficacy compared to single agent immunotherapies. Further development of immune-focused imaging methods can provide clinically relevant metrics associated with immune cell localization that can inform immunotherapeutic efficacy and subsequent treatment response in GBM patients.
Subject(s)
Glioblastoma , Animals , Mice , Humans , Glioblastoma/diagnostic imaging , Glioblastoma/therapy , Tomography, X-Ray Computed , Immunotherapy , Positron-Emission Tomography , CD8-Positive T-Lymphocytes , Tumor MicroenvironmentABSTRACT
The goal of this study is to develop a mathematical model that captures the interaction between evofosfamide, immunotherapy, and the hypoxic landscape of the tumor in the treatment of tumors. Recently, we showed that evofosfamide, a hypoxia-activated prodrug, can synergistically improve treatment outcomes when combined with immunotherapy, while evofosfamide alone showed no effects in an in vivo syngeneic model of colorectal cancer. However, the mechanisms behind the interaction between the tumor microenvironment in the context of oxygenation (hypoxic, normoxic), immunotherapy, and tumor cells are not fully understood. To begin to understand this issue, we develop a system of ordinary differential equations to simulate the growth and decline of tumors and their vascularization (oxygenation) in response to treatment with evofosfamide and immunotherapy (6 combinations of scenarios). The model is calibrated to data from in vivo experiments on mice implanted with colon adenocarcinoma cells and longitudinally imaged with [18F]-fluoromisonidazole ([18F]FMISO) positron emission tomography (PET) to quantify hypoxia. The results show that evofosfamide is able to rescue the immune response and sensitize hypoxic tumors to immunotherapy. In the hypoxic scenario, evofosfamide reduces tumor burden by $ 45.07 \pm 2.55 $%, compared to immunotherapy alone, as measured by tumor volume. The model accurately predicts the temporal evolution of five different treatment scenarios, including control, hypoxic tumors that received immunotherapy, normoxic tumors that received immunotherapy, evofosfamide alone, and hypoxic tumors that received combination immunotherapy and evofosfamide. The average concordance correlation coefficient (CCC) between predicted and observed tumor volume is $ 0.86 \pm 0.05 $. Interestingly, the model values to fit those five treatment arms was unable to accurately predict the response of normoxic tumors to combination evofosfamide and immunotherapy (CCC = $ -0.064 \pm 0.003 $). However, guided by the sensitivity analysis to rank the most influential parameters on the tumor volume, we found that increasing the tumor death rate due to immunotherapy by a factor of $ 18.6 \pm 9.3 $ increases CCC of $ 0.981 \pm 0.001 $. To the best of our knowledge, this is the first study to mathematically predict and describe the increased efficacy of immunotherapy following evofosfamide.
Subject(s)
Adenocarcinoma , Colonic Neoplasms , Mice , Animals , Colonic Neoplasms/diagnostic imaging , Colonic Neoplasms/therapy , Cell Hypoxia , Adenocarcinoma/diagnostic imaging , Adenocarcinoma/therapy , Disease Models, Animal , Cell Line, Tumor , Hypoxia/therapy , Immunotherapy , Tumor MicroenvironmentABSTRACT
Immunotherapies such as checkpoint blockade to PD1 and CTLA4 can have varied effects on individual tumors. To quantify the successes and failures of these therapeutics, we developed a stepwise mathematical modeling strategy and applied it to mouse models of colorectal and breast cancer that displayed a range of therapeutic responses. Using longitudinal tumor volume data, an exponential growth model was utilized to designate response groups for each tumor type. The exponential growth model was then extended to describe the dynamics of the quality of vasculature in the tumors via [18F] fluoromisonidazole (FMISO)-positron emission tomography (PET) data estimating tumor hypoxia over time. By calibrating the mathematical system to the PET data, several biological drivers of the observed deterioration of the vasculature were quantified. The mathematical model was then further expanded to explicitly include both the immune response and drug dosing, so that model simulations are able to systematically investigate biological hypotheses about immunotherapy failure and to generate experimentally testable predictions of immune response. The modeling results suggest elevated immune response fractions (> 30 %) in tumors unresponsive to immunotherapy is due to a functional immune response that wanes over time. This experimental-mathematical approach provides a means to evaluate dynamics of the system that could not have been explored using the data alone, including tumor aggressiveness, immune exhaustion, and immune cell functionality.
Subject(s)
Neoplasms , Mice , Animals , Neoplasms/therapy , Neoplasms/pathology , Positron-Emission Tomography/methods , Disease Models, Animal , ImmunotherapyABSTRACT
Rare diseases are underrepresented in biomedical research, leading to insufficient awareness. Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is a rare disease caused by genetic alterations that result in heterozygous loss-of-function of SON. While ZTTK syndrome patients suffer from numerous symptoms, the lack of model organisms hamper our understanding of both SON and this complex syndrome. Here, we developed Son haploinsufficiency (Son+/-) mice as a model of ZTTK syndrome and identified the indispensable roles of Son in organ development and hematopoiesis. Son+/- mice recapitulated clinical symptoms of ZTTK syndrome, including growth retardation, cognitive impairment, skeletal abnormalities, and kidney agenesis. Furthermore, we identified hematopoietic abnormalities in Son+/- mice, similar to those observed in human patients. Surface marker analyses and single-cell transcriptome profiling of hematopoietic stem and progenitor cells revealed that Son haploinsufficiency inclines cell fate toward the myeloid lineage but compromises lymphoid lineage development by reducing key genes required for lymphoid and B cell lineage specification. Additionally, Son haploinsufficiency causes inappropriate activation of erythroid genes and impaired erythroid maturation. These findings highlight the importance of the full gene dosage of Son in organ development and hematopoiesis. Our model serves as an invaluable research tool for this rare disease and related disorders associated with SON dysfunction.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Breast Neoplasms , Positron-Emission Tomography , Female , Humans , Breast Neoplasms/diagnostic imaging , Breast Neoplasms/metabolism , Positron-Emission Tomography/methods , Receptor, ErbB-2 , Receptor, ErbB-3ABSTRACT
Background: Axial pattern flaps are a common reconstructive option following resection of soft tissue malignancies. We determine the early dependence of an axial flap on wound bed vasculature by isolating the underlying wound bed and depriving contact with the overlying flap. Materials and Methods: Mice were divided into 5 groups: No silicone (n = 7), silicone in the proximal 50% of the wound bed (n = 8), silicone in the distal 50% of the wound bed (n = 5), silicone over the full length of the wound bed with pedicle preservation (n = 5), and silicone over the full length of the wound bed with pedicle sacrifice (n = 5). The pedicle was the lateral thoracic artery. Daily photographs were taken, and the percent of viable flap was determined using ImageJ© software (public domain JAVA image processing program, National Institute of Health, Bethesda, MA). Percent flap viability for each group was compared to the no silicone group, which acted as the reference. Results: Mean differences in percent flap necrotic area (with 95% confidence interval) compared to the no silicone group were -0.15% (-15.09 to 14.09), 2.07% (-5.26 to 9.39), 2.98% (-10.98 to 16.94), and 14.21% (0.48 to 27.94) for the full-length silicone with preserved pedicle, proximal silicone, distal silicone, and full-length silicone with sacrificed pedicle groups, respectively. The full-length silicone with sacrificed pedicle group had a significant difference in flap viability (P = .045) compared to the no silicone group. Conclusion: We investigate the role of the wound bed vasculature in a murine axial flap model and demonstrate that the wound bed vasculature is not essential for early distal flap survival.
ABSTRACT
Tumor-associated macrophages (TAMs) are large phagocytic cells that play numerous roles in cancer biology and are an important component of the relationship between immune system response and tumor progression. The peptide, RP832c, targets the Mannose Receptor (CD206) expressed on M2-like macrophages and is cross-reactive to both human and murine CD206. Additionally, it exhibits therapeutic properties through its ability to shift the population of TAMs from an M2-like (protumor) toward an M1-like phenotype (antitumor) and has demonstrated promise in inhibiting tumor resistance in PD-L1 unresponsive melanoma murine models. In addition, it has shown inhibition in bleomycin-induced pulmonary fibrosis through interactions with CD206 macrophages.1,2 Our work aims to develop a novel CD206 positron emission tomography (PET) imaging probe based on RP832c (Kd = 5.64 µM) as a direct, noninvasive method for the assessment of TAMs in mouse models of cancer. We adapted RP832c to incorporate the chelator DOTA to allow for radiolabeling with the PET isotope 68Ga (t1/2 = 68 min; ß+ = 89%). In vitro stability studies were conducted in mouse serum up to 3 h. The in vitro binding characteristics of [68Ga]RP832c to CD206 were determined by a protein plate binding assay and Surface Plasmon Resonance (SPR). PET imaging and biodistribution studies were conducted in syngeneic tumor models. Stability studies in mouse serum demonstrated that 68Ga remained complexed up to 3 h (less than 1% free 68Ga). Binding affinity studies demonstrated high binding of [68Ga]RP832c to mouse CD206 protein and that the binding of the tracer was able to be blocked significantly when incubated with a blocking solution of native RP832c. PET imaging and biodistribution studies in syngeneic tumor models demonstrated uptake in tumor and CD206 expressing organs of [68Ga]RP832c. A significant correlation was found between the percentage of CD206 present in each tumor imaged with [68Ga]RP832c and PET imaging mean standardized uptake values in a CT26 mouse model of cancer. The data shows that [68Ga]RP832c represents a promising candidate for macrophage imaging in cancer and other diseases.
Subject(s)
Gallium Radioisotopes , Neoplasms , Animals , Humans , Mice , Cell Line, Tumor , Gallium Radioisotopes/chemistry , Macrophages/metabolism , Neoplasms/metabolism , Peptides/metabolism , Positron-Emission Tomography/methods , Tissue Distribution , Mannose Receptor/metabolismABSTRACT
Hypoxia is a common feature of the tumor microenvironment, including that of triple-negative breast cancer (TNBC), an aggressive breast cancer subtype with a high five-year mortality rate. Using [18F]-fluoromisonidazole (FMISO) positron emission tomography (PET) imaging, we aimed to monitor changes in response to immunotherapy (IMT) with chemotherapy in TNBC. TNBC-tumor-bearing mice received paclitaxel (PTX) ± immune checkpoint inhibitors anti-programmed death 1 and anti-cytotoxic T-lymphocyte 4. FMISO-PET imaging was performed on treatment days 0, 6, and 12. Max and mean standard uptake values (SUVmax and SUVmean, respectively), histological analyses, and flow cytometry results were compared. FMISO-PET imaging revealed differences in tumor biology between treatment groups prior to tumor volume changes. 4T1 responders showed SUVmean 1.6-fold lower (p = 0.02) and 1.8-fold lower (p = 0.02) than non-responders on days 6 and 12, respectively. E0771 responders showed SUVmean 3.6-fold lower (p = 0.001) and 2.7-fold lower (p = 0.03) than non-responders on days 6 and 12, respectively. Immunohistochemical analyses revealed IMT plus PTX decreased hypoxia and proliferation and increased vascularity compared to control. Combination IMT/PTX recovered the loss of CD4+ T-cells observed with single-agent therapies. PET imaging can provide timely, longitudinal data on the TNBC tumor microenvironment, specifically intratumoral hypoxia, predicting therapeutic response to IMT plus chemotherapy.
ABSTRACT
Triple-negative breast cancer (TNBC) is a heterogenous disease that is defined by its lack of targetable receptors, thus limiting treatment options and resulting in higher rates of metastasis and recurrence. Combination chemotherapy treatments, which inhibit tumor cell proliferation and regeneration, are a major component of standard-of-care treatment of TNBC. In this manuscript, we build a coupled ordinary differential equation model of TNBC with compartments that represent tumor proliferation, necrosis, apoptosis, and immune response to computationally describe the biological tumor affect to a combination of chemotherapies, doxorubicin (DRB) and paclitaxel (PTX). This model is parameterized using longitudinal [18F]-fluorothymidine positron emission tomography (FLT-PET) imaging data which allows for a noninvasive molecular imaging approach to quantify the tumor proliferation and tumor volume measurements for two murine models of TNBC. Animal models include a human cell line xenograft model, MDA-MB-231, and a syngeneic 4T1 mammary carcinoma model. The mathematical models are parameterized and the percent necrosis at the end time point is predicted and validated using histological hematoxylin and eosin (H&E) data. Global Sobol' sensitivity analysis is conducted to further understand the role each parameter plays in the model's goodness of fit to the data. In both the MDA-MB-231 and the 4T1 tumor models, the designed mathematical model can accurately describe both tumor volume changes and final necrosis volume. This can give insight into the ordering, dosing, and timing of DRB and PTX treatment. More importantly, this model can also give insight into future novel combinations of therapies and how the immune system plays a role in therapeutic response to TNBC, due to its calibration to two types of TNBC murine models.
Subject(s)
Triple Negative Breast Neoplasms , Humans , Animals , Mice , Triple Negative Breast Neoplasms/diagnostic imaging , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/metabolism , Cell Line, Tumor , Mathematical Concepts , Models, Biological , Paclitaxel/pharmacology , Paclitaxel/therapeutic use , Cell Proliferation , Drug Therapy, Combination , Necrosis/drug therapy , ApoptosisABSTRACT
Background: Trastuzumab induces cell cycle arrest in HER2-overexpressing cells and demonstrates potential in radiosensitizing cancer cells. The purpose of this study is to quantify combination trastuzumab and radiotherapy to determine their synergy. Methods: In vitro, HER2+ cancer cells were treated with trastuzumab, radiation, or their combination, and imaged to evaluate treatment kinetics. In vivo, HER2+ tumor-bearing mice were treated with trastuzumab and radiation, and assessed longitudinally. An additional cohort was treated and sacrificed to quantify CD45, CD31, α-SMA, and hypoxia. Results: The interaction index revealed the additive effects of trastuzumab and radiation in vitro in HER2+ cell lines. Furthermore, the results revealed significant differences in tumor response when treated with radiation (p < 0.001); however, no difference was seen in the combination groups when trastuzumab was added to radiotherapy (p = 0.56). Histology revealed increases in CD45 staining in tumors receiving trastuzumab (p < 0.05), indicating potential increases in immune infiltration. Conclusions: The in vitro results showed the additive effect of combination trastuzumab and radiotherapy. The in vivo results showed the potential to achieve similar efficacy of radiotherapy with a reduced dose when combined with trastuzumab. If trastuzumab and low-dose radiotherapy induce greater tumor kill than a higher dose of radiotherapy, combination therapy can achieve a similar reduction in tumor burden.
ABSTRACT
Immune checkpoint inhibitors (ICI) are promising in adjuvant settings for solid tumors and hematologic malignancies. They are currently used in the treatment as mAbs in high concentrations, raising concerns of toxicity and adverse side effects. Among various checkpoint molecules, targeting the programmed cell death protein-1 (PD-1)-programmed death-ligand 1 (PD-L1) axis has garnered more clinical utility than others have. To develop a physiologically relevant and systemically stable level of ICIs from a one-time application by genetic antibody engineering, we endeavored using a nonpathogenic, replication-deficient recombinant adeno-associated vector (rAAV) expressing single-chain variable fragments (scFv) of PD-L1 antibody and tested in syngeneic mouse therapy models of MC38 colorectal and EMT6 breast tumors. Results of this study indicated a significant protection against PD-L1-mediated inhibition of CD8+ T-cell function, against the growth of primary and secondary tumors, and durable antitumor CTLs activity by adoptive CD8+ T-cell transfer. Stable maintenance of PD-L1 scFv in vivo resulted in an increase in PD-1- CD8+ T cells and a concomitant decrease in regulatory T cells, M2 macrophages, and myeloid-derived suppressor cells in the tumor microenvironment. Overall, these data demonstrate the potential of rAAV-PD-L1-scFv as an alternative to mAb targeting of PD-L1 for tumor therapy.
Subject(s)
B7-H1 Antigen , Neoplasms , Mice , Animals , Programmed Cell Death 1 Receptor , Immunotherapy/methods , Neoplasms/pathology , Antibodies, Monoclonal/pharmacology , Tumor MicroenvironmentABSTRACT
PURPOSE: The primary goal of this study is to evaluate the accuracy of the fluorescence ubiquitination cell cycle indicator (FUCCI) system with fluorescence in vivo imaging compared to 3'-deoxy-3'-[18F]fluorothymidine ([18F]-FLT) positron emission tomography (PET)/computed tomography (CT) and biological validation through histology. Imaging with [18F]-FLT PET/CT can be used to noninvasively assess cancer cell proliferation and has been utilized in both preclinical and clinical studies. However, a cost-effective and straightforward method for in vivo, cell cycle targeted cancer drug screening is needed prior to moving towards translational imaging methods such as PET/CT. PROCEDURES: In this study, fluorescent MDA-MB-231-FUCCI tumor growth was monitored weekly with caliper measurements and fluorescent imaging. Seven weeks post-injection, [18F]-FLT PET/CT was performed with a preclinical PET/CT, and tumors samples were harvested for histological analysis. RESULTS: RFP fluorescent signal significantly correlated with tumor volume (r = 0.8153, p < 0.0001). Cell proliferation measured by GFP fluorescent imaging was correlated with tumor growth rate (r = 0.6497, p < 0.001). Also, GFP+ cells and [18F]-FLT regions of high uptake were both spatially located in the tumor borders, indicating that the FUCCI-IVIS method may provide an accurate assessment of tumor heterogeneity of cell proliferation. The quantification of total GFP signal was correlated with the sum of tumor [18F]-FLT standard uptake value (SUV) (r = 0.5361, p = 0.0724). Finally, histological analysis confirmed viable cells in the tumor and the correlation of GFP + and Ki67 + cells (r = 0.6368, p = 0.0477). CONCLUSION: Fluorescent imaging of the cell cycle provides a noninvasive accurate depiction of tumor progression and response to therapy, which may benefit in vivo testing of novel cancer therapeutics that target the cell cycle.
Subject(s)
Dideoxynucleosides , Neoplasms , Humans , Positron-Emission Tomography , Positron Emission Tomography Computed Tomography , Neoplasms/diagnostic imaging , Cell Proliferation , Cell Cycle , Ubiquitination , Radiopharmaceuticals , Fluorodeoxyglucose F18ABSTRACT
This study identifies physiological habitats using quantitative magnetic resonance imaging (MRI) to elucidate intertumoral differences and characterize microenvironmental response to targeted and cytotoxic therapy. BT-474 human epidermal growth factor receptor 2 (HER2+) breast tumors were imaged before and during treatment (trastuzumab, paclitaxel) with diffusion-weighted MRI and dynamic contrast-enhanced MRI to measure tumor cellularity and vascularity, respectively. Tumors were stained for anti-CD31, anti-ÉSMA, anti-CD45, anti-F4/80, anti-pimonidazole, and H&E. MRI data was clustered to identify and label each habitat in terms of vascularity and cellularity. Pre-treatment habitat composition was used stratify tumors into two "tumor imaging phenotypes" (Type 1, Type 2). Type 1 tumors showed significantly higher percent tumor volume of the high-vascularity high-cellularity (HV-HC) habitat compared to Type 2 tumors, and significantly lower volume of low-vascularity high-cellularity (LV-HC) and low-vascularity low-cellularity (LV-LC) habitats. Tumor phenotypes showed significant differences in treatment response, in both changes in tumor volume and physiological composition. Significant positive correlations were found between histological stains and tumor habitats. These findings suggest that the differential baseline imaging phenotypes can predict response to therapy. Specifically, the Type 1 phenotype indicates increased sensitivity to targeted or cytotoxic therapy compared to Type 2 tumors.