Validation of a combined ultrasound and bioluminescence imaging system with magnetic resonance imaging in orthotopic pancreatic murine tumors

Preclinical mouse solid tumor models are widely used to evaluate efficacy of novel cancer therapeutics. Recent reports have highlighted the need for utilizing orthotopic implantation to represent clinical disease more accurately, however the deep tissue location of these tumors makes longitudinal assessment challenging without the use of imaging techniques. The purpose of this study was to evaluate the performance of a new multi-modality high-throughput in vivo imaging system that combines bioluminescence imaging (BLI) with robotic, hands-free ultrasound (US) for evaluating orthotopic mouse models. Long utilized in cancer research as independent modalities, we hypothesized that the combination of BLI and US would offer complementary advantages of detection sensitivity and quantification accuracy, while mitigating individual technological weaknesses. Bioluminescent pancreatic tumor cells were injected into the pancreas tail of C57BL/6 mice and imaged weekly with the combination system and magnetic resonance imaging (MRI) to serve as a gold standard. BLI photon flux was quantified to assess tumor activity and distribution, and US and MRI datasets were manually segmented for gross tumor volume. Robotic US and MRI demonstrated a strong agreement (R2 = 0.94) for tumor volume measurement. BLI showed a weak overall agreement with MRI (R2 = 0.21), however, it offered the greatest sensitivity to detecting the presence of tumors. We conclude that combining BLI with robotic US offers an efficient screening tool for orthotopic tumor models.

Translational imaging of the fibroblast activation protein (FAP) using the new ligand [68Ga]Ga-OncoFAP-DOTAGA

Purpose The fibroblast activation protein (FAP) is an emerging target for molecular imaging and therapy in cancer. OncoFAP is a novel small organic ligand for FAP with very high affinity. In this translational study, we establish [68Ga]Ga-OncoFAP-DOTAGA (68Ga-OncoFAP) radiolabeling, benchmark its properties in preclinical imaging, and evaluate its application in clinical PET scanning. Methods 68Ga-OncoFAP was synthesized in a cassette-based fully automated labeling module. Lipophilicity, affinity, and serum stability of 68Ga-OncoFAP were assessed by determining logD7.4, IC50 values, and radiochemical purity. 68Ga-OncoFAP tumor uptake and imaging properties were assessed in preclinical dynamic PET/MRI in murine subcutaneous tumor models. Finally, biodistribution and uptake in a variety of tumor types were analyzed in 12 patients based on individual clinical indications that received 163 ± 50 MBq 68Ga-OncoFAP combined with PET/CT and PET/MRI. Results 68Ga-OncoFAP radiosynthesis was accomplished with high radiochemical yields. Affinity for FAP, lipophilicity, and stability of 68Ga-OncoFAP measured are ideally suited for PET imaging. PET and gamma counting–based biodistribution demonstrated beneficial tracer kinetics and high uptake in murine FAP-expressing tumor models with high tumor-to-blood ratios of 8.6 ± 5.1 at 1 h and 38.1 ± 33.1 at 3 h p.i. Clinical 68Ga-OncoFAP-PET/CT and PET/MRI demonstrated favorable biodistribution and kinetics with high and reliable uptake in primary cancers (SUVmax 12.3 ± 2.3), lymph nodes (SUVmax 9.7 ± 8.3), and distant metastases (SUVmax up to 20.0). Conclusion Favorable radiochemical properties, rapid clearance from organs and soft tissues, and intense tumor uptake validate 68Ga-OncoFAP as a powerful alternative to currently available FAP tracers.

Gadolinium-Based Nanoparticles Combined With Checkpoint Blockade Amplify Radiation-Induced Abscopal Effect And Cancer Immunotherapy

Both immunotherapy checkpoint blocking and radiotherapy used alone in solid tumors show limited anticancer effect, due to the insufficient T cells infiltration and rarely elicited systemic immune responses. Methods: AGuIX has recently been developed for magnetic resonance imaging-guided radiotherapy and proven to act as an efficient radiosensitizer. In order to further improve the efficiency of tumor treatment, a unique synergistic strategy based on Gadolinium-based nanoparticles-AGuIX mediated radiotherapy and immunotherapy checkpoint blocking was developed for B16 tumor therapy in the present study. Clone formation, cell apoptosis and immunofluorescence were performed to detect the radiosensitization effect of AGuIX nanoparticles. The therapeutic effect was validated in both abscopal and metastasis tumor models, and analyzed the synergistic mechanism in vivo. Results: AGuIX as a radiosensitizer exacerbated radiation-induced DNA damage, cell cycle arrest and apoptosis on B16 cells. More importantly, it could efficiently induce the immunogenic cell death (ICD) of irradiated B16 tumor cells, and consequently triggered the maturation of dendritic cells (DCs) and activated systemic T-cell responses. Combining AGuIX-mediated radiotherapy with programmed cell death protein 1 (PD1) blocking demonstrated excellent synergistic therapeutic effects in both abscopal and metastasis tumor models by a significant increase in the infiltration of effector CD8+ T cells and effectively alleviating the immunosuppressive microenvironment, including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) in tumors.Conclusion: Our findings indicate that this combination therapy provided a new and powerful immunotherapy model to achieve durable anti-tumor responses, which is likely to be a promising comprehensive treatment strategy for cancer treatment.

A three-dimensional immune-oncology model for studying in vitro human NK cell cytotoxic activity v1

Immunotherapy has emerged as a promising therapeutic approach for treating several forms of cancer. Adoptive cell transfer of immune cells, such as natural killer (NK) cells, provides a powerful therapeutic potential against tumor cells. In the past decades, two-dimensional (2D) tumor models have been used to investigate the effectiveness of immune cell killing. However, the 2D tumor models exhibit less structural complexity and cannot recapitulate the physiological condition of the tumor microenvironment. Thus, the effectiveness of immune cells against tumor cells using these models cannot fully be translated to clinical studies. In order to gain a deeper insight into immune cell-tumor interaction, more physiologically relevant in vivo-like three-dimensional (3D) tumor models have been developed. These 3D tumor models can mimic the dynamic cellular activities, making them much closer to the in vivo tumor profiles. Here, we describe a simple and effective protocol to study the cytotoxic activity of primary human NK cells toward the 3D tumor spheroids. Our protocol includes isolation and expansion of human NK cells, labeling and formation of tumor spheroids, co-culture of NK cells and tumor spheroids, and evaluation of cytotoxic activity using a confocal microscope. This protocol is also applicable to other types of tumors and immune cells.