Use of a single hybrid imaging agent for integration of target validation with in vivo and ex vivo imaging of mouse tumor lesions resembling human DCIS

Abstract

Screening of biomarker expression levels in tumor biopsy samples not only provides an assessment of prognostic and predictive factors, but may also be used for selection of biomarker-specific imaging strategies. To assess the feasibility of using a biopsy specimen for a personalized selection of an imaging agent, the chemokine receptor 4 (CXCR4) was used as a reference biomarker.

Methods: A hybrid CXCR4 targeting peptide (MSAP-Ac-TZ14011) containing a fluorescent dye and a chelate for radioactive labeling was used to directly compare initial flow cytometry-based target validation in fresh tumor tissue to in vivo single photon emission computed tomography (SPECT) imaging and in vivo and ex vivo fluorescence imaging.

Results: Flow cytometric analysis of mouse tumor derived cell suspensions enabled discrimination between 4T1 control tumor lesions (with low levels of CXCR4 expression) and CXCR4 positive early, intermediate and late stage MIN-O lesions based on their CXCR4 expression levels; CXCR4(basal), CXCR4(+) and CXCR4(++) cell populations could be accurately discriminated. Mean fluorescent intensity ratios between expression in MIN-O and 4T1 tissue found with flow cytometry were comparable to ratios obtained with in vivo SPECT/CT and fluorescence imaging, ex vivo fluorescence evaluation and standard immunohistochemistry.

Conclusion: The hybrid nature of a targeting imaging agent like MSAP-Ac-TZ14011 enables integration of target selection, in vivo imaging and ex vivo validation using a single agent. The use of biopsy tissue for biomarker screening can readily be expanded to other targeting hybrid imaging agents and can possibly help increase the clinical applicability of tumor-specific imaging approaches.

Figure 1. Schematic representation of the integrated logistics made possible by using a targeting hybrid imaging agent.

A) Analysis of tumor biopsy samples using the fluorescent beacon of the imaging agent using flow cytometry. B) Non-invasive tumor visualization using SPECT/CT after radiolabeling of the hybrid agent. Fluorescence imaging enables intraoperative surgical guidance. C) Ex vivo evaluation of tracer distribution using fluorescence imaging and –microscopy after excision of the tumor.

Figure 2. Structure of MSAP-Ac-TZ14011.

Figure 3. CXCR4 staining using immunohistochemistry.

Membranous staining of fixed tumor tissue slices after incubation with the anti-CXCR4 antibody 2B11 in A) early, B) intermediate, C) late stage MIN-O tumor lesions and D) 4T1 control tumors (40× magnification).

Figure 4. Fluorescence based fresh tumor biopsy analysis after incubation with MSAP-Ac-TZ14011.

A) CXCR4 staining pattern in early (black), intermediate (red) and late stage (blue) MIN-O biopsy samples. B) CXCR4 staining pattern in 4T1 biopsy samples. Non-incubated control samples are depicted in grey. C–F) Analysis of CD45 expression in CXCR4 positive cells in early, intermediate and late stage MIN-O tumor lesions and 4T1 tumor lesions. For percentages of populations with different CXCR4 expression, see Table 2.

Figure 5. Evaluation of freshly isolated MIN-O tumor slices.

A) ex vivo incubation with MSAP-Ac-TZ14011 and B) after intravenous injection of MSAP-Ac-TZ14011 twenty-four hours prior to evaluation of the tissue. I: 10× magnification. II) 63× magnification. Signal emitted by MSAP-Ac-TZ14011 is depicted in magenta and DAPI (blue) was used to visualize the cell nucleus.

Figure 6. Non-invasive imaging.

A) SPECT/CT imaging after intravenous injection of ¹¹¹In-MSAP-Ac-TZ14011. B) in vivo and C) ex vivo fluorescence imaging. D) fluorescence microscopy (10× magnification). Intermediate stage MIN-O: top images. Late stage 4T1: bottom images.