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

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.

Keywords: Checkpoint inhibitor; Fluorescence; Glioblastoma; Immune suppression; TIM3.

Figure 1.

Immunofluorescence microscopy of RAW 264.7 macrophages following conjugate labeling with (a) TIM3-800CW. Controls were labeled with (b) IgG-800CW and (c) DAPI-only. Images obtained with objective 40, numerical aperture 0.8. (d) Quantification of mean fluorescence intensity of 800CW from single cells segmentations (N = 10 per group). Intensity was measured by the calculated total cell fluorescence (CTCF), calculated as: mean integrated density – (area * mean background).

Figure 2.

(a) Interval IVIS Spectrum images obtained at excitation and emission of 745 and 800 nm, small binning, f-stop 2, and 3 seconds of exposure. (b) Line graph of fluorescent signal in vivo for mice injected with TIM3-800CW and IgG-800CW conjugate. Mean fluorescent signal was measured by ratio of the brain tumor region of interest and normal tissue. *p < 0.05

Figure 3.

Representative ex vivo (a) fluorescent and (b) brightfield imaging of organs from mice treated with either TIM3-800CW (N=4) or IgG-800CW (N=4). (c) Dot plot summarizing the average mean fluorescent intensities of organs and their respective standard errors. Organs were collected from Day 3 (cyan dot) and 4 (black dot) of the in vivo trial. *p < 0.05

Figure 4.

Histologic mouse brain sections from a mouse treated with (a-c) TIM3-800CW and (d-f) IgG-800CW conjugates. Low power view using near infrared imaging (b, e). of tumor area for TIM3-800CW versus IgG-800CW (excitation/emission 785/820 nm, resolution = 21 μm). Signal intensities were measured based on segmentations of the corresponding areas of hematoxylin and eosin. These were normalized to contralateral brain (yellow circles). High power view using near infrared imaging (c, f) with DAPI counterstain depicts cellular uptake of TIM3-800CW in tumor. Additional high-power field of controls of (h) contralateral brain (red square), and (i) liver for a mouse treated with TIM3-800CW. Images obtain with objective 20, numerical aperture 0.75.