Direct and Indirect Chimeric Antigen Receptor T-Cell Imaging with PET/MRI in a Tumor Xenograft Model.

Background Chimeric antigen receptor (CAR) T cells are a promising cancer therapy; however, reliable and repeatable methods for tracking and monitoring CAR T cells in vivo remain underexplored. Purpose To investigate direct and indirect imaging strategies for tracking the biodistribution of CAR T cells and monitoring their therapeutic effect in target tumors. Materials and Methods CAR T cells co-expressing a tumor-targeting gene (anti-CD19 CAR) and a human somatostatin receptor subtype 2 (hSSTr2) reporter gene were generated from human peripheral blood mononuclear cells. After direct labeling with zirconium 89 (89Zr)-p-isothiocyanatobenzyl-desferrioxamine (DFO), CAR T cells were intravenously injected into immunodeficient mice with a CD19-positive and CD19-negative human tumor xenograft on the left and right flank, respectively. PET/MRI was used for direct in vivo imaging of 89Zr-DFO-labeled CAR T cells on days 0, 1, 3, and 7 and for indirect cell imaging with the radiolabeled somatostatin receptor-targeted ligand gallium 68 (68Ga)-DOTA-Tyr3-octreotide (DOTATOC) on days 6, 9, and 13. On day 13, mice were euthanized, and tissues and tumors were excised. Results The 89Zr-DFO-labeled CAR T cells were observed on PET/MRI scans in the liver and lungs of mice (n = 4) at all time points assessed. However, they were not visualized in CD19-positive or CD19-negative tumors, even on day 7. Serial 68Ga-DOTATOC PET/MRI showed CAR T cell accumulation in CD19-positive tumors but not in CD19-negative tumors from days 6 to 13. Notably, 68Ga-DOTATOC accumulation in CD19-positive tumors was highest on day 9 (mean percentage injected dose [%ID], 3.7% ± 1.0 [SD]) and decreased on day 13 (mean %ID, 2.6% ± 0.7) in parallel with a decrease in tumor volume (day 9: mean, 195 mm3 ± 27; day 13: mean, 127 mm3 ± 43) in the group with tumor growth inhibition. Enhanced immunohistochemistry staining of cluster of differentiation 3 (CD3) and hSSTr2 was also observed in excised CD19-positive tumor tissues. Conclusion Direct and indirect cell imaging with PET/MRI enabled in vivo tracking and monitoring of CAR T cells in an animal model. © RSNA, 2024 Supplemental material is available for this article. See also the editorial by Bulte in this issue.

Feasibility of real-time in vivo 89Zr-DFO-labeled CAR T-cell trafficking using PET imaging.

INTRODUCTION: Chimeric antigen receptor (CAR) T-cells have been recently developed and are producing impressive outcomes in patients with hematologic malignancies. However, there is no standardized method for cell trafficking and in vivo CAR T-cell monitoring. We assessed the feasibility of real-time in vivo 89Zr-p-Isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS, DFO) labeled CAR T-cell trafficking using positron emission tomography (PET). RESULTS: The 89Zr-DFO radiolabeling efficiency of Jurkat/CAR and human peripheral blood mononuclear cells (hPBMC)/CAR T-cells was 70%-79%, and cell radiolabeling activity was 98.1-103.6 kBq/106 cells. Cell viability after radiolabeling was >95%. Cell proliferation was not significantly different during the early period after radiolabeling, compared with unlabeled cells; however, the proliferative capacity decreased over time (day 7 after labeling). IL-2 or IFN-γ secretion was not significantly different between unlabeled and labeled CAR T-cells. PET/magnetic resonance imaging in the xenograft model showed that most of the 89Zr-DFO-labeled Jurkat/CAR T-cells were distributed in the lung (24.4% ± 3.4%ID) and liver (22.9% ± 5.6%ID) by one hour after injection. The cells gradually migrated from the lung to the liver and spleen by day 1, and remained stable in these sites until day 7 (on day 7: lung 3.9% ± 0.3%ID, liver 36.4% ± 2.7%ID, spleen 1.4% ± 0.3%ID). No significant accumulation of labeled cells was identified in tumors. A similar pattern was observed in ex vivo biodistributions on day 7 (lung 3.0% ± 1.0%ID, liver 19.8% ± 2.2%ID, spleen 2.3% ± 1.7%ID). 89Zr-DFO-labeled hPBMC/CAR T-cells showed a similar distribution, compared with Jurkat/CAR T-cells, on serial PET images. CAR T cell distribution was cross-confirmed by flow cytometry, Alu polymerase chain reaction, and immunohistochemistry. CONCLUSION: Real-time in vivo cell trafficking is feasible using PET imaging of 89Zr-DFO-labeled CAR T-cells. This can be used to investigate cellular kinetics, initial in vivo biodistribution, and safety profiles in future CAR T-cell development.

[18F]fluorothymidine PET Informs the Synergistic Efficacy of Capecitabine and Trifluridine/Tipiracil in Colon Cancer.

In cancer therapy, enhanced thymidine uptake by the salvage pathway can bypass dTMP depletion, thereby conferring resistance to thymidylate synthase inhibition. We investigated whether sequential combination therapy of capecitabine and trifluridine/tipiracil (TAS-102) could synergistically enhance antitumor efficacy in colon cancer xenograft models. We also examined 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) PET as a means to predict therapeutic response to a sequential combination of capecitabine and trifluridine/tipiracil. [3H]FLT uptake after 5-fluorouracil treatment in vitro and [18F]FLT uptake after capecitabine (360 mg/kg/day) in athymic nude mice (Balb/c-nu) with xenografts (n = 10-12 per group) were measured using eight human colon cancer cell lines. We determined the synergistic effects of sequential combinations of 5-fluorouracil and trifluridine in vitro as well as the sequential combination of oral capecitabine (30-360 mg/kg) and trifluridine/tipiracil (trifluridine 75 or 150 mg/kg with tipiracil) in six xenograft models (n = 6-10 per group). We observed significant increases in [3H]FLT uptake in all cell lines and [18F]FLT uptake in five xenograft models after 5-fluorouracil and capecitabine treatment, respectively. Increased [18F]FLT uptake after capecitabine followed by extinction of uptake correlated strongly with tumor growth inhibition (ρ = -0.81, P = 0.02). The effects of these combinations were synergistic in vitro A synergy for sequential capecitabine and trifluridine/tipiracil was found only in mouse xenograft models showing increased [18F]FLT uptake after capecitabine. Our results suggest that the sequential combination of capecitabine and trifluridine/tipiracil is synergistic in tumors with an activated salvage pathway after capecitabine treatment in mice, and [18F]FLT PET imaging may predict the response to capecitabine and the synergistic antitumor efficacy of a sequential combination of capecitabine and trifluridine/tipiracil. Cancer Res; 77(24); 7120-30. ©2017 AACR.

TC1(C8orf4) regulates hematopoietic stem/progenitor cells and hematopoiesis.

Hematopoiesis is a complex process requiring multiple regulators for hematopoietic stem/progenitor cells (HSPC) and differentiation to multi-lineage blood cells. TC1(C8orf4) is implicated in cancers, hematological malignancies and inflammatory activation. Here, we report that Tc1 regulates hematopoiesis in mice. Myeloid and lymphoid cells are increased markedly in peripheral blood of Tc1-deleted mice compared to wild type controls. Red blood cells are small-sized but increased in number. The bone marrow of Tc1-/- mice is normocellular histologically. However, Lin-Sca-1+c-Kit+ (LSK) cells are expanded in Tc1-/- mice compared to wild type controls. The expanded population mostly consists of CD150-CD48+ cells, suggesting the expansion of lineage-restricted hematopoietic progenitor cells. Colony forming units (CFU) are increased in Tc1-/- mice bone marrow cells compared to controls. In wild type mice bone marrow, Tc1 is expressed in a limited population of HSPC but not in differentiated cells. Major myeloid transcriptional regulators such as Pu.1 and Cebpα are not up-regulated in Tc1-/- mice bone marrow. Our findings indicate that TC1 is a novel hematopoietic regulator. The mechanisms of TC1-dependent HSPC regulation and lineage determination are unknown.