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In Vivo Evaluation of Near-Infrared Fluorescent Probe for TIM3 Targeting in Mouse Glioma

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Abstract

Purpose

Current checkpoint inhibitor immunotherapy strategies in glioblastoma are challenged by mechanisms of resistance including an immunosuppressive tumor microenvironment. T cell immunoglobulin domain and mucin domain 3 (TIM3) is a late-phase checkpoint receptor traditionally associated with T cell exhaustion. We apply fluorescent imaging techniques to explore feasibility of in vivo visualization of the immune state in a glioblastoma mouse model.

Procedures

TIM3 monoclonal antibody was conjugated to a near-infrared fluorescent dye, IRDye-800CW (800CW). The TIM3 experimental conjugate and isotype control were assessed for specificity with immunofluorescent staining and flow cytometry in murine cell lines (GL261 glioma and RAW264.7 macrophages). C57BL/6 mice with orthotopically implanted GL261 cells were imaged in vivo over 4 days after intravenous TIM3-800CW injection to assess tumor-specific uptake. Cell-specific uptake was then assessed on histologic sections.

Results

The experimental TIM3-800CW, but not its isotype control, bound to RAW264.7 macrophages in vitro. Specificity to RAW264.7 macrophages and not GL261 tumor cells was quantitatively confirmed with the corresponding clone of TIM3 on flow cytometry. In vivo fluorescence imaging of the 800CW signal was localized to the intracranial tumor and significantly higher for the TIM3-800CW cohort, relative to non-targeting isotype control, immediately after tail vein injection and for up to 48 h after injection. Resected organs of tumor bearing mice showed significantly higher uptake in the liver and spleen. TIM3-800CW was seen to co-stain with CD3 (13%), CD11b (29%), and CD206 (26%).

Conclusions

We propose fluorescent imaging of immune cell imaging as a potential strategy for monitoring and localizing immunologically relevant foci in the setting of brain tumors. Alternative markers and target validation will further clarify the temporal relationship of immunosuppressive effector cells throughout glioma resistance.

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Funding

MZ receives research funding from the National Institutes of Health (5T32CA009695-27, MPI).

Author information

Authors and Affiliations

  1. Department of Neurosurgery, Stanford University, Stanford, CA, USA

    Michael Zhang, Quan Zhou, Gordon Li & Michael Lim

  2. Department of Radiology, Stanford University, Stanford, CA, USA

    Michael Zhang, Chinghsin Huang, Carmel T. Chan, Wei Wu, Sanjiv S. Gambhir & Heike E. Daldrup-Link

  3. Department of Otolaryngology-Head and Neck Surgery, Stanford University, Stanford, CA, USA

    Quan Zhou

Authors
  1. Michael Zhang
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  2. Quan Zhou
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  3. Chinghsin Huang
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  4. Carmel T. Chan
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  5. Wei Wu
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  6. Gordon Li
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  7. Michael Lim
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  8. Sanjiv S. Gambhir
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  9. Heike E. Daldrup-Link
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Contributions

Conception and design: MZ, QZ, CTC, GL, ML, SSG, HED.

Acquisition of data/materials: MZ, QZ, CH, WW, CTC, SSG, HED.

Analysis of data: MZ, QZ, CH, CTC.

Drafting of manuscript: MZ.

Critical revision: QZ, CH, CTC, WW, GL, ML, SSG, HED.

Final approval: MZ, QZ, CH, CTC, WW, GL, ML, SSG, HED.

Agreement of accountability: MZ, QZ, CH, CTC, WW, GL, ML, SSG, HED.

Corresponding author

Correspondence to Heike E. Daldrup-Link.

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The authors declare that they have no conflict of interest.

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Zhang, M., Zhou, Q., Huang, C. et al. In Vivo Evaluation of Near-Infrared Fluorescent Probe for TIM3 Targeting in Mouse Glioma. Mol Imaging Biol 24, 280–287 (2022). https://doi.org/10.1007/s11307-021-01667-0

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  • DOI: https://doi.org/10.1007/s11307-021-01667-0

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