Photoconvertible tracking strategies assess the dynamic migration of cell populations. Here we develop phototruncation-assisted cell tracking (PACT) and apply it to evaluate the migration of immune cells into tumor-draining lymphatics. This method is enabled by a recently discovered cyanine photoconversion reaction that leads to the two-carbon truncation and consequent blue-shift of these commonly used probes. By examining substituent effects on the heptamethine cyanine chromophore, we find that introduction of a single methoxy group increases the yield of the phototruncation reaction in neutral buffer by almost 8-fold. When converted to a membrane-bound cell-tracking variant, this probe can be applied in a series of in vitro and in vivo experiments. These include quantitative, time-dependent measurements of the migration of immune cells from tumors to tumor-draining lymph nodes. Unlike previously reported cellular photoconversion approaches, this method does not require genetic engineering and uses near-infrared (NIR) wavelengths. Overall, PACT provides a straightforward approach to label cell populations with spatiotemporal control.

Chemical Biology Laboratory Center for Cancer Research National Cancer Institute National Institutes of Health Frederick Maryland 21702 United States

Department of Chemistry Faculty of Science Masaryk University Kamenice 5 625 00 Brno Czech Republic

Molecular Imaging Branch Center for Cancer Research National Cancer Institute National Institutes of Health Bethesda Maryland 20892 United States

RECETOX Faculty of Science Masaryk University Kamenice 5 625 00 Brno Czech Republic

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Luster AD; Alon R; von Andrian UH, Immune cell migration in inflammation: present and future therapeutic targets. Nat. Immunol. 2005, 6 (12), 1182–1190. PubMed

Liu J; Zhang X; Cheng Y; Cao X, Dendritic cell migration in inflammation and immunity. Cell. Mol. Immunol. 2021, 18 (11), 2461–2471. PubMed PMC

Fransen MF; van Hall T; Ossendorp F, Immune Checkpoint Therapy: Tumor Draining Lymph Nodes in the Spotlights. Int. J. Mol. Sci. 2021, 22 (17), 9401. PubMed PMC

Marzo AL; Lake RA; Lo D; Sherman L; McWilliam A; Nelson D; Robinson BW; Scott B, Tumor antigens are constitutively presented in the draining lymph nodes. J. Immunol. 1999, 162 (10), 5838–5845. PubMed

Goode EF; Roussos Torres ET; Irshad S, Lymph Node Immune Profiles as Predictive Biomarkers for Immune Checkpoint Inhibitor Response. Front. Mol. Biosci. 2021, 8, 382. PubMed PMC

Riedel A; Shorthouse D; Haas L; Hall BA; Shields J, Tumor-induced stromal reprogramming drives lymph node transformation. Nat. Immunol. 2016, 17 (9), 1118–1127. PubMed PMC

Gray EE; Cyster JG, Lymph node macrophages. J. Innate Immun. 2012, 4 (5–6), 424–436. PubMed PMC

Munn DH; Mellor AL, The tumor-draining lymph node as an immune-privileged site. Immunol. Rev. 2006, 213 (1), 146–158. PubMed

Hampton HR; Chtanova T, Lymphatic migration of immune cells. Front. immunol. 2019, 10, 1168. PubMed PMC

Hatta K; Tsujii H; Omura T, Cell tracking using a photoconvertible fluorescent protein. Nat. Protoc. 2006, 1 (2), 960–967. PubMed

Nienhaus GU; Nienhaus K; Hölzle A; Ivanchenko S; Renzi F; Oswald F; Wolff M; Schmitt F; Röcker C; Vallone B, Photoconvertible fluorescent protein EosFP: biophysical properties and cell biology applications. Photochem. Photobiol. 2006, 82 (2), 351–358. PubMed

Tomura M; Hata A; Matsuoka S; Shand FH; Nakanishi Y; Ikebuchi R; Ueha S; Tsutsui H; Inaba K; Matsushima K, Tracking and quantification of dendritic cell migration and antigen trafficking between the skin and lymph nodes. Sci. Rep. 2014, 4 (1), 1–11. PubMed PMC

Progatzky F; Dallman MJ; Lo Celso C, From seeing to believing: labelling strategies for in vivo cell-tracking experiments. Interface Focus 2013, 3 (3), 20130001. PubMed PMC

Kedrin D; Gligorijevic B; Wyckoff J; Verkhusha VV; Condeelis J; Segall JE; Van Rheenen J, Intravital imaging of metastatic behavior through a mammary imaging window. Nat. Methods 2008, 5 (12), 1019–1021. PubMed PMC

Mellott AJ; Shinogle HE; Moore DS; Detamore MS, Fluorescent Photo-conversion: A second chance to label unique cells. Cell. Mol. Bioeng. 2015, 8 (1), 187–196. PubMed PMC

Hampton HR; Bailey J; Tomura M; Brink R; Chtanova T, Microbe-dependent lymphatic migration of neutrophils modulates lymphocyte proliferation in lymph nodes. Nat. Commun. 2015, 6 (1), 1–11. PubMed PMC

Tomura M; Yoshida N; Tanaka J; Karasawa S; Miwa Y; Miyawaki A; Kanagawa O, Monitoring cellular movement in vivo with photoconvertible fluorescence protein “Kaede” transgenic mice. PNAS 2008, 105 (31), 10871–10876. PubMed PMC

Torcellan T; Hampton HR; Bailey J; Tomura M; Brink R; Chtanova T, In vivo photolabeling of tumor-infiltrating cells reveals highly regulated egress of T-cell subsets from tumors. PNAS 2017, 114 (22), 5677–5682. PubMed PMC

Ando R; Hama H; Yamamoto-Hino M; Mizuno H; Miyawaki A, An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. PNAS 2002, 99 (20), 12651–12656. PubMed PMC

Zimmer M, GFP: from jellyfish to the Nobel prize and beyond. Chem. Soc. Rev. 2009, 38 (10), 2823–2832. PubMed

Day RN; Davidson MW, The fluorescent protein palette: tools for cellular imaging. Chem. Soc. Rev. 2009, 38 (10), 2887–2921. PubMed PMC

Bresser K; Dijkgraaf FE; Pritchard CE; Huijbers IJ; Song J-Y; Rohr JC; Scheeren FA; Schumacher TN, A mouse model that is immunologically tolerant to reporter and modifier proteins. Commun. Biol. 2020, 3 (1), 1–6. PubMed PMC

Gambotto A; Dworacki G; Cicinnati V; Kenniston T; Steitz J; Tüting T; Robbins P; DeLeo A, Immunogenicity of enhanced green fluorescent protein (EGFP) in BALB/c mice: identification of an H2-K d-restricted CTL epitope. Gene Ther. 2000, 7 (23), 2036–2040. PubMed

Han W; Unger W; Wauben M, Identification of the immunodominant CTL epitope of EGFP in C57BL/6 mice. Gene Ther. 2008, 15 (9), 700–701. PubMed

Limberis M; Bell C; Wilson J, Identification of the murine firefly luciferase-specific CD8 T-cell epitopes. Gene Ther. 2009, 16 (3), 441–447. PubMed PMC

Lippincott-Schwartz J; Patterson GH, Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging. Trends Cell Biol. 2009, 19 (11), 555–565. PubMed PMC

Shcherbakova DM; Subach OM; Verkhusha VV, Red fluorescent proteins: advanced imaging applications and future design. Angew. Chem. Int. Ed. 2012, 51 (43), 10724–10738. PubMed PMC

Gorka AP; Nani RR; Schnermann MJ, Cyanine polyene reactivity: scope and biomedical applications. Org. Biomol. Chem. 2015, 13 (28), 7584–98. PubMed PMC

Gorka AP; Nani RR; Schnermann MJ, Harnessing cyanine reactivity for optical imaging and drug delivery. Acc. Chem. Res. 2018, 51 (12), 3226–3235. PubMed

Jradi FM; Lavis LD, Chemistry of Photosensitive Fluorophores for Single-Molecule Localization Microscopy. ACS Chem. Biol. 2019, 14 (6), 1077–1090. PubMed

Eiring P; McLaughlin R; Matikonda SS; Han Z; Grabenhorst L; Helmerich DA; Meub M; Beliu G; Luciano M; Bandi V; Zijlstra N; Shi Z-D; Tarasov SG; Swenson R; Tinnefeld P; Glembockyte V; Cordes T; Sauer M; Schnermann MJ, Targetable Conformationally Restricted Cyanines Enable Photon-Count-Limited Applications. Angew. Chem. Int. Ed. 2021, 60 (51), 26685–26693. PubMed PMC

Helmerich DA; Beliu G; Matikonda SS; Schnermann MJ; Sauer M, Photoblueing of organic dyes can cause artifacts in super-resolution microscopy. Nat. Methods 2021, 1–5. PubMed PMC

Matikonda SS; Helmerich DA; Meub M; Beliu G; Kollmannsberger P; Greer A; Sauer M; Schnermann MJ, Defining the Basis of Cyanine Phototruncation Enables a New Approach to Single-Molecule Localization Microscopy. ACS Cent. Sci. 2021. PubMed PMC

Štacková L; Muchová E; Russo M; Slavíček P; Štacko P; Klán P, Deciphering the structure–property relations in substituted heptamethine cyanines. J. Org. Chem. 2020, 85 (15), 9776–9790. PubMed

Štacková L; Štacko P; Klán P, Approach to a substituted heptamethine cyanine chain by the ring opening of zincke salts. J. Am. Chem. Soc. 2019, 141 (17), 7155–7162. PubMed

Nani RR; Gorka AP; Nagaya T; Yamamoto T; Ivanic J; Kobayashi H; Schnermann MJ, In Vivo Activation of Duocarmycin-Antibody Conjugates by Near-Infrared Light. Acs Central Sci 2017, 3 (4), 329–337. PubMed PMC

Celso CL; Fleming HE; Wu JW; Zhao CX; Miake-Lye S; Fujisaki J; Côté D; Rowe DW; Lin CP; Scadden DT, Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche. Nature 2009, 457 (7225), 92–96. PubMed PMC

Kalchenko V; Shivtiel S; Malina V; Lapid K; Haramati S; Lapidot T; Brill AG; Harmelin A, Use of lipophilic near-infrared dye in whole-body optical imaging of hematopoietic cell homing. J. Biomed. Opt. 2006, 11 (5), 050507. PubMed

Carlson AL; Fujisaki J; Wu J; Runnels JM; Turcotte R; Celso CL; Scadden DT; Strom TB; Lin CP, Tracking single cells in live animals using a photoconvertible near-infrared cell membrane label. PLoS One 2013, 8 (8), e69257. PubMed PMC

Osseiran S; Austin LA; Cannon TM; Yan C; Langenau DM; Evans CL, Longitudinal monitoring of cancer cell subpopulations in monolayers, 3D spheroids, and xenografts using the photoconvertible dye DiR. Sci. Rep. 2019, 9 (1), 1–10. PubMed PMC

Thermal Truncation of Heptamethine Cyanine Dyes