Method for estimation of apoptotic cell fraction of cytotherapy using in vivo fluorine-19 magnetic resonance: pilot study in a patient with head and neck carcinoma receiving tumor-infiltrating lymphocytes labeled with perfluorocarbon nanoemulsion

J Immunother Cancer. 2023 Jun;11(6):e007015. doi: 10.1136/jitc-2023-007015.


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.

Keywords: Immunologic Techniques; Immunotherapy; Lymphocytes, Tumor-Infiltrating; T-Lymphocytes.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Apoptosis
  • Carcinoma, Squamous Cell* / pathology
  • Fluorine
  • Fluorocarbons*
  • Head and Neck Neoplasms* / diagnostic imaging
  • Head and Neck Neoplasms* / pathology
  • Head and Neck Neoplasms* / therapy
  • Humans
  • Lymphocytes, Tumor-Infiltrating / pathology
  • Magnetic Resonance Imaging
  • Magnetic Resonance Spectroscopy
  • Pilot Projects
  • Squamous Cell Carcinoma of Head and Neck / diagnostic imaging
  • Squamous Cell Carcinoma of Head and Neck / pathology
  • Squamous Cell Carcinoma of Head and Neck / therapy


  • Fluorine
  • Fluorocarbons