Universität zu Köln

Pfeifer, Rita, Henze, Janina, Wittich, Katharina, Gosselink, Andre ORCID: 0000-0003-0181-456X, Kinkhabwala, Ali, Gremse, Felix, Bleilevens, Cathrin, Bigott, Kevin, Jungblut, Melanie, Hardt, Olaf ORCID: 0000-0003-0825-071X, Alves, Frauke and Al Rawashdeh, Wa'el (2022). A multimodal imaging workflow for monitoring CAR T cell therapy against solid tumor from whole-body to single-cell level. Theranostics, 12 (11). S. 4834 - 4851. LAKE HAVEN: IVYSPRING INT PUBL. ISSN 1838-7640

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Abstract

CAR T cell research in solid tumors often lacks spatiotemporal information and therefore, there is a need for a molecular tomography to facilitate high-throughput preclinical monitoring of CAR T cells. Furthermore, a gap exists between macro-and microlevel imaging data to better assess intratumor infiltration of therapeutic cells. We addressed this challenge by combining 3D mu Computer tomography bioluminescence tomography (mu CT/BLT), light-sheet fluorescence microscopy (LSFM) and cyclic immunofluorescence (IF) staining. Methods: NSG mice with subcutaneous AsPC1 xenograft tumors were treated with EGFR CAR T cell (+/- IL-2) or control BDCA-2 CAR T cell (+/- IL-2) (n = 7 each). Therapeutic T cells were genetically modified to co-express the CAR of interest and the luciferase CBR2opt. IL-2 was administered s.c. under the xenograft tumor on days 1, 3, 5 and 7 post-therapy-initiation at a dose of 25,000 Ill/mouse. CAR T cell distribution was measured in 2D BLI and 3D mu CT/BLT every 3-4 days. On day 6, 4 tumors were excised for cyclic IF where tumor sections were stained with a panel of 25 antibodies. On day 6 and 13, 8 tumors were excised from rhodamine lectin-preinjected mice, permeabilized, stained for CD3 and imaged by LSFM. Results: 3D mu CT/BLT revealed that CAR T cells pharmacokinetics is affected by antigen recognition, where CAR T cell tumor accumulation based on target-dependent infiltration was significantly increased in comparison to target-independent infiltration, and spleen accumulation was delayed. LSFM supported these findings and revealed higher T cell accumulation in target-positive groups at day 6, which also infiltrated the tumor deeper. Interestingly, LSFM showed that most CAR T cells accumulate at the tumor periphery and around vessels. Surprisingly, LSFM and cyclic IF revealed that local IL-2 application resulted in early-phase increased proliferation, but long-term overstimulation of CAR T cells, which halted the early added therapeutic effect. Conclusion: Overall, we demonstrated that 3D mu CT/BLT is a valuable non-isotope-based technology for whole-body cell therapy monitoring and investigating CAR T cell pharmacokinetics. We also presented combining LSFM and MICS for ex vivo 3D-and 2D-microscopy tissue analysis to assess intratumoral therapeutic cell distribution and status.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Pfeifer, Rita UNSPECIFIED UNSPECIFIED UNSPECIFIED Henze, Janina UNSPECIFIED UNSPECIFIED UNSPECIFIED Wittich, Katharina UNSPECIFIED UNSPECIFIED UNSPECIFIED Gosselink, Andre UNSPECIFIED orcid.org/0000-0003-0181-456X UNSPECIFIED Kinkhabwala, Ali UNSPECIFIED UNSPECIFIED UNSPECIFIED Gremse, Felix UNSPECIFIED UNSPECIFIED UNSPECIFIED Bleilevens, Cathrin UNSPECIFIED UNSPECIFIED UNSPECIFIED Bigott, Kevin UNSPECIFIED UNSPECIFIED UNSPECIFIED Jungblut, Melanie UNSPECIFIED UNSPECIFIED UNSPECIFIED Hardt, Olaf UNSPECIFIED orcid.org/0000-0003-0825-071X UNSPECIFIED Alves, Frauke UNSPECIFIED UNSPECIFIED UNSPECIFIED Al Rawashdeh, Wa'el UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-680129
DOI:	10.7150/thno.68966
Journal or Publication Title:	Theranostics
Volume:	12
Number:	11
Page Range:	S. 4834 - 4851
Date:	2022
Publisher:	IVYSPRING INT PUBL
Place of Publication:	LAKE HAVEN
ISSN:	1838-7640
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language ADOPTIVE IMMUNOTHERAPY; GLIOBLASTOMA; TIM-3; LAG-3; PD-1; EGFR; CD19 Multiple languages Medicine, Research & Experimental Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/68012