Purpose

To assess the cell-specific, intracellular partial pressure of oxygen (Po₂) dynamics of both tumor and chimeric antigen receptor (CAR) T cells in a murine immunotherapy model.

Materials and methods

Human glioblastoma cells or human T cells were intracellularly labeled with perfluorocarbon nanoemulsion droplet sensors prior to in vivo injection in severe combined immunodeficient mice to measure Po₂ in the two cell types in response to treatment. Two main sets of experiments were performed: (a) mice were injected in the flank with perfluorocarbon-labeled human glioblastoma cells and were then inoculated with either CAR T cells or untransduced T cells or were untreated 5 days after tumor inoculation; and (b) mice with unlabeled glioblastoma tumors were inoculated with perfluorocarbon-labeled CAR T cells or untransduced T cells 5 days after tumor inoculation. Longitudinal fluorine 19 (¹⁹F) spin-lattice relaxation time measurements of the tumor mass were used to ascertain absolute Po₂ in vivo. Results were analyzed for significance using an analysis of variance, a linear mixed-effect model, and a Pearson correlation coefficient test, as appropriate.

Results

The intracellular tumor cell Po₂ temporal dynamics exhibited delayed, transient hyperoxia at 3 days after infusion of CAR T cells, commensurate with significant tumor cell killing and CAR T-cell infiltration, as observed by bioluminescence imaging and histologic findings. Conversely, no significant changes were detected in CAR or untransduced T-cell intracellular Po₂ over time in tumor using these same methods. Moreover, it was observed that the total ¹⁹F tumor cell signal quenches with treatment, consistent with rapid tissue clearance of probe from apoptotic tumor cells.

Conclusion

Cell-specific Po₂ measurements using perfluorocarbon probes can provide insights into effector cell function and tumor response in cellular immunotherapeutic cancer models.Keywords: Animal Studies, MR-Imaging, MR-Spectroscopy, Molecular Imaging-Cancer, Molecular Imaging-Immunotherapy Supplemental material is available for this article. © RSNA, 2021See also commentary by Bulte in this issue.

Inflammation is associated with a range of serious human conditions, including autoimmune and cardiovascular diseases and cancer. The ability to image active inflammatory processes greatly enhances our ability to diagnose and treat these diseases at an early stage. We describe molecular compositions enabling sensitive and precise imaging of inflammatory hotspots in vivo. Methods: A functionalized nanoemulsion with a fluorocarbon-encapsulated radiometal chelate (FERM) was developed to serve as a platform for multimodal imaging probe development. The ¹⁹F-containing FERM nanoemulsion encapsulates ⁸⁹Zr in the fluorous oil via a fluorinated hydroxamic acid chelate. Simple mixing of the radiometal with the preformed aqueous nanoemulsion before use yields FERM, a stable in vivo cell tracer, enabling whole-body ⁸⁹Zr PET and ¹⁹F MRI after a single intravenous injection. Results: The FERM nanoemulsion was intrinsically taken up by phagocytic immune cells, particularly macrophages, with high specificity. FERM stability was demonstrated by a high correlation between the ¹⁹F and ⁸⁹Zr content in the blood (correlation coefficient > 0.99). Image sensitivity at a low dose (37 kBq) was observed in a rodent model of acute infection. The versatility of FERM was further demonstrated in models of inflammatory bowel disease and 4T1 tumor. Conclusion: Multimodal detection using FERM yields robust whole-body lesion detection and leverages the strengths of combined PET and ¹⁹F MRI. The FERM nanoemulsion has scalable production and is potentially useful for precise diagnosis, stratification, and treatment monitoring of inflammatory diseases.

Background

Adoptive transfer of T cells is a burgeoning cancer therapeutic approach. However, the fate of the cells, once transferred, is most often unknown. We describe the first clinical experience with a non-invasive biomarker to assay the apoptotic cell fraction (ACF) after cell therapy infusion, tested in the setting of head and neck squamous cell carcinoma (HNSCC). A patient with HNSCC received autologous tumor-infiltrating lymphocytes (TILs) labeled with a perfluorocarbon (PFC) nanoemulsion cell tracer. Nanoemulsion, released from apoptotic cells, clears through the reticuloendothelial system, particularly the Kupffer cells of the liver, and fluorine-19 (¹⁹F) magnetic resonance spectroscopy (MRS) of the liver was used to non-invasively infer the ACF.

Methods

Autologous TILs were isolated from a patient in their late 50s with relapsed, refractory human papillomavirus-mediated squamous cell carcinoma of the right tonsil, metastatic to the lung. A lung metastasis was resected for T cell harvest and expansion using a rapid expansion protocol. The expanded TILs were intracellularly labeled with PFC nanoemulsion tracer by coincubation in the final 24 hours of culture, followed by a wash step. At 22 days after intravenous infusion of TILs, quantitative single-voxel liver ¹⁹F MRS was performed in vivo using a 3T MRI system. From these data, we model the apparent ACF of the initial cell inoculant.

Results

We show that it is feasible to PFC-label ~70×10¹⁰ TILs (F-TILs) in a single batch in a clinical cell processing facility, while maintaining >90% cell viability and standard flow cytometry-based release criteria for phenotype and function. Based on quantitative in vivo ¹⁹F MRS measurements in the liver, we estimate that ~30% cell equivalents of adoptively transferred F-TILs have become apoptotic by 22 days post-transfer.

Conclusions

Survival of the primary cell therapy product is likely to vary per patient. A non-invasive assay of ACF over time could potentially provide insight into the mechanisms of response and non-response, informing future clinical studies. This information may be useful to developers of cytotherapies and clinicians as it opens an avenue to quantify cellular product survival and engraftment.