Imaging Expression of the Human Somatostatin Receptor Subtype-2 Reporter Gene with ⁶⁸Ga-DOTATOC

Abstract

The human somatostatin receptor subtype 2 (hSSTr2)-68 Ga-DOTATOC reporter system has several attractive features for potential translation to human studies. These include a low expression of hSSTr2 in most organs, a rapid internalized accumulation of 68 Ga-DOTATOC in the SSTr2-expressing cells, and a rapid excretion of unbound radioligand by the renal system. We performed a series of in vitro and in vivo validation studies of this reporter system. Methods: A retroviral vector containing a dual reporter, pQCXhSSTr2-IRES-GFP (IRES: internal ribosome entry site; GFP: green fluorescent protein), was constructed and transduced into Jurkat, C6, and U87 cells. Stably transduced reporter cells were characterized in vitro using optical and radiometric methods. Multiple tumor-bearing mice were evaluated with 68 Ga-DOTATOC PET studies. Results: The dual-reporter genes were incorporated into all tumor cell lines, and their expression levels were confirmed by fluorescence-activated cell sorting (FACS), GFP visualization, and reverse-transcriptase polymerase chain reaction (RT-PCR) analysis for hSSTr2. In vitro, hSSTr2 cell membrane expression was 36,000, 280,000, and 1,250,000 copies per cell for the SSTR2-transfected Jurkat, U87, and C6 cell lines. Small-animal PET of 68 Ga-DOTATOC in tumor-bearing mice demonstrated that the in vivo uptake of this radioligand was directly proportional to the in vitro expression of hSSTr2. The in vivo uptake of 68 Ga-DOTATOC, at 2 h after injection, was low in all organs except the kidneys (7.8 percentage of injected dose per gram [%ID/g]) and as high as 15.2 %ID/g in transduced C6 tumors. The corresponding transduced-to-nontransduced tumor uptake ratio was 64, and the tumor-to-muscle uptake ratio was around 500. Conclusion: 68 Ga-DOTATOC is an excellent specific ligand for this hSSTr2 reporter system and for hSSTr2 reporter gene PET. Because DOTATOC has undergone extensive clinical testing, this human reporter system has the potential for translation to human studies.

Longitudinal PET imaging demonstrates biphasic CAR T cell responses in survivors

“…Over the course of tumor growth, DOTATOC uptake was higher in tumors comprising increasing percentages of SSTR2 + cells ( Figure 2B). PET/CT images showed DOTATOC uptake by tumors and uniformly higher uptake by the kidneys and bladder -consistent with its known biodistribution and renal clearance ( Figure 2C) (19). DOTATOC uptake values, as measured by %ID/cm 3 , agreed with visual assessments of PET/CT images over the course of tumor growth and correlated with increasing SSTR2 + percentages within the tumors ( Figure 2, B and C, and Supplemental Figure 2).…”

“…Therefore, to approximate the DOTATOC-based detection limit of tumor-infiltrating SSTR2-transduced T cells, we utilized mosaic tumor xenografts of Jurkat T cells with increasing ratios of SSTR2 expression to create a standard by which quantitative PET signals can be related to T cells of known density within tumors. Using the lentivirus developed in this study, the surface expression of SSTR2 in transduced Jurkat T cells was in the range of several million per cell, a level significantly higher than previously published (19) and therefore likely to extend the lower limit of SSTR2 + T cell detection. We observed that with a known threshold of radiotracer uptake, one can detect tumor-infiltrating T cells down to a minimum density of 0.8% or ~4 × 10 6 cells/cm 3 , with 95% specificity and 87% sensitivity.…”

“…SSTR2 belongs to a family of G protein-coupled receptors, with expression restricted to the cerebrum and kidneys, and at low levels in the gastrointestinal tract (23). It is a potentially ideal reporter marker for monitoring ACT owing to its limited basal expression throughout the body and the availability of clinically approved radiotracers for SSTR2, for example, gallium-68-chelated octreotide analogues ( 68 Ga-DOTATOC) for the detection of SSTR2-overexpressing neuroendocrine tumors (19,(24)(25)(26). To determine the detection limit of T cells within a solid tumor mass, we introduced SSTR2 into Jurkat T cells and titrated these with wild-type Jurkats to produce subcutaneous Jurkat tumor xenografts, each with defined ratios of SSTR2-expressing cells ranging from 0% to 100%.…”

“…Additionally, as only live cells are capable of continually expressing the reporter gene, observed signals are limited to these cells only. Current reporter genes used in preclinical and clinical studies are based on both intracellular enzymes, e.g., herpes simplex virus type-1 thymidine kinase (HSV1-tk) and human deoxycytidine kinase (14)(15)(16), and surface receptors, e.g., sodium iodide symporter (NIS) (17), prostate-specific membrane antigen (PSMA) (18), and SSTR2 (19). However, previous imaging studies have failed to demonstrate quantitative monitoring of critical T cell efficacy parameters, namely, whole-body, longitudinal visualization of T cell dynamics spanning initial localization, expansion, and subsequent contraction at primary and/or metastatic tumor sites, and the relationship between these parameters and tumor killing (5,13,(20)(21)(22).…”

68Ga-DOTATOC and FDG PET Imaging of Preclinical Neuroblastoma Models

“…Concerning the biodistribution of 68 Ga-DOTATOC in normal organs of nude mice, our data are consistent with those reported by other teams (21,22), although the engrafted tumours in their studies were derived from the pancreatic tumour cell line AR4-2J, with low %ID/g in all normal organs except in the kidneys. In humans, 68 Ga-DOTATOC PET is significantly taken up in the pancreas, which we and another team did not observe in nude mice (23).…”