Near-Infrared Imaging of Adoptive Immune Cell Therapy in Breast Cancer Model Using Cell Membrane Labeling

Abstract

The overall objective of this study is to non-invasively image and assess tumor targeting and retention of directly labeled T-lymphocytes following their adoptive transfer in mice. T-lymphocytes obtained from draining lymph nodes of 4T1 (murine breast cancer cell) sensitized BALB/C mice were activated in-vitro with Bryostatin/Ionomycin for 18 hours, and were grown in the presence of Interleukin-2 for 6 days. T-lymphocytes were then directly labeled with 1,1-dioctadecyltetramethyl indotricarbocyanine Iodide (DiR), a lipophilic near infrared fluorescent dye that labels the cell membrane. Assays for viability, proliferation, and function of labeled T-lymphocytes showed that they were unaffected by DiR labeling. The DiR labeled cells were injected via tail vein in mice bearing 4T1 tumors in the flank. In some cases labeled 4T1 specific T-lymphocytes were injected a week before 4T1 tumor cell implantation. Multi-spectral in vivo fluorescence imaging was done to subtract the autofluorescence and isolate the near infrared signal carried by the T-lymphocytes. In recipient mice with established 4T1 tumors, labeled 4T1 specific T-lymphocytes showed marked tumor retention, which peaked 6 days post infusion and persisted at the tumor site for up to 3 weeks. When 4T1 tumor cells were implanted 1-week post-infusion of labeled T-lymphocytes, T-lymphocytes responded to the immunologic challenge and accumulated at the site of 4T1 cell implantation within two hours and the signal persisted for 2 more weeks. Tumor accumulation of labeled 4T1 specific T-lymphocytes was absent in mice bearing Meth A sarcoma tumors. When lysate of 4T1 specific labeled T-lymphocytes was injected into 4T1 tumor bearing mice the near infrared signal was not detected at the tumor site. In conclusion, our validated results confirm that the near infrared signal detected at the tumor site represents the DiR labeled 4T1 specific viable T-lymphocytes and their response to immunologic challenge can be imaged in vivo.

Rapamycin/DiR loaded lipid-polyaniline nanoparticles for dual-modal imaging guided enhanced photothermal and antiangiogenic combination therapy

“…Recently, great interests have been devoted to the NIR fluorescent dyes and their nanostructures for imaging guided PTT applications [35][36][37][38][39][40][41]. One representative example is the 1,1-dioctadecyl-3,3,3,3-tetramethylindotri-carbocyanine iodide (DiR) that is a lipophilic fluorescent probe (a subclass of cyanine dyes) with strong absorption in the NIR region and negligible cytotoxicity at low concentration suitable for both in vivo fluorescence imaging and photothermal treatment [42][43][44]. DiR has been developed as a particularly promising photothermal candidate because it can A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT 5 convert absorbed fluorescent energy into heat via the non-radiative transition under the NIR laser irradiation.…”

Environment-responsive aza-BODIPY dyes quenching in water as potential probes to visualize the in vivo fate of lipid-based nanocarriers

“…To highlight the superiority of the water-quenching dyes, a non-water-quenching commercial dye DiR iodide ( Figure S10), which was also highly hydrophobic and usually used for cell membrane labelling, 35 was compared with the water-quenching P2 dye by introducing it into the lipid matrix of SLNs and studying the behavior of the vehicles both in vitro and in vivo. In contrary to P2-labelled SLNs, the ARE of DiR-labelled SLNs remained at relatively high levels (80-100%) for at least 60 min…”

Surface biotinylation of cytotoxic T lymphocytes for in vivo tracking of tumor immunotherapy in murine models

“…This cell surface labeling assures even distribution of dyes, which makes quantification of cell accumulation more accurate. Youniss et al and Du et al also tried labeling cells directly on cell surface by interacting positively charged fluorescent dye with the negatively charged cell surface [11, 28], but this method was limited to fluorescent imaging. A key advantage to our method is in its versatility.…”