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Sodium Multivitamin Transporter-Targeted Fluorochrome Facilitates Enhanced Metabolic Evaluation of Tumors Through Coenzyme-R Dependent Intracellular Signaling Pathways

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Abstract

Background

Intraoperative molecular imaging (IMI)-guided resections have been shown to improve oncologic outcomes for patients undergoing surgery for solid malignancies. The technology utilizes fluorescent tracers targeting cancer cells without the use of any ionizing radiation. However, currently available targeted IMI tracers are effective only for tumors with a highly specific receptor expression profile, and there is an unmet need for IMI tracers to label a broader range of tumor types. Here, we describe the development and testing of a novel tracer (CR)-S0456) targeted to the sodium multivitamin transporter (SMVT).

Methods

Preclinical models of fibrosarcoma (HT-1080), lung (A549), breast (4T1), and renal cancers (HEK-293 T) in vitro and in vivo were used for assessment of (CR)-S0456 specific tumor labeling via sodium-mediated SMVT uptake in dipotassium phosphate or choline chloride-containing media buffer. Additionally, pharmacologic inhibition of multiple intracellular coenzyme-R obligate signaling pathways, including holocarboxylase synthetase (sulconazole nitrate), PI3K/AKT/mTOR (omipalisib), and calmodulin-dependent phosphatase (calmidazolium), were investigated to assess (CR)-S0456 uptake kinetics. Human fibrosarcoma-bearing xenografts in athymic nude mice were used for tumor and metabolic-specific labeling. Novel NIR needle confocal laser endomicroscopic (nCLE) intratumoral sampling was performed to demonstrate single-cell specific labeling by CR-S0456.

Results

CR-S0456 localization in vitro correlated with highly proliferative cell lines (MTT) and doubling time (p < 0.05) with the highest microscopic fluorescence detected in aggressive human fibrosarcomas (HT-1080). Coenzyme-R-specific localization was demonstrated to be SMVT-specific after competitive inhibition of internal localization with excess administration of pantothenic acid. Inhibiting the activity of SMVT by affecting sodium ion hemostasis prevented the complete uptake of CR-S0456. In vivo validation demonstrated (CR)-S0456 localization to xenograft models with accurate identification of primary tumors as well as margin assessment down to 1 mm3 tumor volume. Systemic treatment of xenograft-bearing mice with a dual PI3K/mTOR inhibitor suppressed intratumoral cell signaling and (CR)-S0456 uptake via a reduction in SMVT expression. Novel analysis of in vivo intratumoral cytologic fluorescence using near-infrared confocal laser endomicroscopy demonstrated the absence of coenzyme-R-mediated NIR fluorescence but not fibroblast activation protein (FAP)-conjugated fluorochrome, indicating specific intracellular inhibition of coenzyme-R obligate pathways.

Conclusion

These findings suggest that a SMVT-targeted NIR contrast agent can be a suitable tracer for imaging a wide range of malignancies as well as evaluating metabolic response to systemic therapies, similar to PET imaging with immune checkpoint inhibitors.

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Data Availability

The data generated in this study are displayed in the manuscript and supplemental files. Data is available upon request from the corresponding author.

Material Availability

The data generated in this study are displayed in the manuscript and supplemental files. Data is available upon request from the corresponding author.

Abbreviations

IMI:

Intraoperative molecular imaging

CR:

Coenzyme R

SMVT:

Sodium dependent multivitamin transporter

NIR:

Near infrared

HLCS:

Holocarboxylase synthetase

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Acknowledgements

We would like to thank Drs. Steven Albelda and Philip Low for material and cell line support.

Funding

Dr. Azari was supported by the training grant in surgical oncology from the National Institutes of Health (T32CA251063-01), the Society of Thoracic Surgeons Thoracic Surgery Foundation Research Award, and the Stephen C. Cheung Award in Surgical Oncology. Dr. Singhal was supported by the National Institutes of Health (R01 CA193556) and the State of Pennsylvania Health Research Formula Fund. Dr. Kennedy was supported by the American Philosophical Society and the National Institutes of Health (F32 CA254210-01). Dr. Sullivan was supported by NIH T32 postdoctoral fellowship (NIH T32 CA009140).

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Authors and Affiliations

  1. Department of Thoracic Surgery, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA

    Feredun Azari, Gregory T. Kennedy, Ashley Chang, Bilal Nadeem, Patrick Bou-Samra, Austin Chang, Alix Segil & Sunil Singhal

  2. University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA

    Feredun Azari, Gregory T. Kennedy, Ashley Chang, Bilal Nadeem, Patrick Bou-Samra, Austin Chang, Alix Segil, Elizabeth Bernstein, Neil T. Sullivan, Evgeniy Eruslanov, James Delikatny & Sunil Singhal

  3. Department of Thoracic Surgery, Hospital of the University of Pennsylvania, 3400 Civic Center Boulevard, 14th floor, South Pavilion, Philadelphia, PA, 19104, USA

    Feredun Azari

  4. Department of Radiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA

    Elizabeth Bernstein, Neil T. Sullivan, Evgeniy Eruslanov & James Delikatny

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  1. Feredun Azari
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Contributions

FA, GTK, JD, and SS: study design, data validation, manuscript draft, manuscript finalization, and review. AC, NTS, AS, EB, BA, and EE: data acquisition and experiment conduction.

Corresponding author

Correspondence to Feredun Azari.

Ethics declarations

Ethics Approval and Consent to Participate

All experiments have been approved by the University of Pennsylvania Institutional Review Board. Animal studies were performed according to protocols approved by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) in accordance with the National Institutes of Health guidelines.

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N/A

Competing Interests

S.S. has been named a co-inventor and submitted a patent to the USPTO regarding the fluorochrome. In order to protect intellectual property while the patent application process is pending, the structure is omitted from the manuscript. The structure is readily available by contacting the corresponding author and the University of Pennsylvania Intellectual Protection Office.

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Azari, F., Kennedy, G.T., Chang, A. et al. Sodium Multivitamin Transporter-Targeted Fluorochrome Facilitates Enhanced Metabolic Evaluation of Tumors Through Coenzyme-R Dependent Intracellular Signaling Pathways. Mol Imaging Biol 25, 569–585 (2023). https://doi.org/10.1007/s11307-022-01792-4

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