(H-317) Optical Imaging of Immune-Targeted Rare Earth Nanoparticles for Prognostication of Metastasis in a Syngeneic Model of Breast Cancer

Introduction:: Over the past several decades the importance of distinct immune subsets in cancer progression, promoting cancer metastasis, and therapy evasion has become evident and these subsets can be potential biomarkers for real-time disease monitoring and prognostication ^1,2. For breast cancer patients though outcomes have improved, metastatic disease remains difficult to treat and are responsible for 90% of breast cancer deaths, and 60% of patients who die have evidence of lung metastases ³. Novel approaches are needed to identify patients at high risk of metastatic disease prior to tumor colonization at distant sites.

One immune biomarker that has been identified as a key regulator for priming the pre-metastatic niche is the myeloid-derived suppressor cell (MDSC) ^1,4. MDSCs are an immunosuppressive cell type derived from erroneously differentiated myeloid cells that are recruited in cancer and are known to prepare the pre-metastatic niche for tumor cells through the release of factors that mediate immune suppression, increased vascular permeability, inflammation, and tumor cell colonization ^1,5. Furthermore evidence has found a direct link between MDSC lung burden and increased number of lung metastases ⁴.

Previous work within our research group has established tumor-targeted shortwave infrared (SWIR) emitting nanoprobes as optical contrast agents for imaging of tumor metastases and as theranostic probes for treating and tracking lung metastases in mouse models ^6–9. In this project we engineer these SWIR emitting nanoprobes with MDSC targeted antibodies for in vivo monitoring of MDSC recruitment to the lung pre-metastatic niche in a spontaneous metastasis model of breast cancer.


Materials and Methods:: In Vitro Characterization

SWIR-emitting rare earth nanoprobes (REs) were encapsulated in albumin through controlled coacervation to produce rare earth encapsulated albumin nanocomposites (ReANCs). IgG antibody targeting mouse Gr-1 (Ly6G/Ly6C) or an isotype control (Iso) antibody are crosslinked to the albumin shell of the ReANC using sulfosuccinimidyl-4-( N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) chemistry to produce Gr-ReANC and Iso-ReANC. Antibody loading was assessed by ELISA assays on the functionalized nanoprobes. Targeting specificity was validated through in vitro uptake assays by incubating fluorescent Gr-ReANCs and Iso-ReANCs with MDSCs isolated from the spleens of tumor bearing mice. Target specificity was determined, using flow cytometry, as a measure of changes in fluorescence intensity .

In Vivo Validation

In vivo studies were performed with two syngeneic models of breast cancer, murine 4T1 and EMT6 mammary cell lines in both athymic and Balb/c mice. Either 4T1 or EMT6 cells were seeded orthotopically at a density of 20,000 cells per mouse in the mammary fat pad. On day 14 and day 21 post-inoculation mice received a tail vein injection of either Gr-ReANC or Iso-ReANC. Six hours post-nanoprobe injection mice were imaged with a 980nm laser and nanoprobe emissions are measured at 1525nm. Mean pixel intensities in the region of interest were compared between the two models in mice treated with Gr-ReANCs and significance determined using ANOVA. Ex vivo SWIR imaging of lungs was performed prior to either fixation for immunohistochemistry or isolation of cells from processed lungs to assess MDSC burden by Gr-1 and CD11b co- staining using flow cytometry.

Results, Conclusions, and Discussions:: Flow cytometry performed on lung tissue from mice bearing 4T1 and EMT6 tumors demonstrates that there is a significantly higher MDSC lung burden in mice with the highly metastatic 4T1 tumors compared to less invasive EMT6 tumors. This is validated by increased macroscopic lung metastases and increased MDSC infiltration measured by immunohistochemical staining of 4T1 tumors compared to EMT6 tumors on days 21 and 28 post-inoculation. These results support the fact that MDSCs are correlated with metastatic risk and rates of metastases within these breast cancer models.

For detection of MDSCs within the pre-metastatic niche, ReANCs with anti-Gr-1 IgG (14.3-28.6 ug/mg albumin) were synthesized. Targeting specificity was determined through significant increase in cellular uptake of Gr1-ReANCs compared to Iso-ReANCs as determined by FACS analysis. In vivo imaging at day 14 demonstrates that 4T1 mice receiving Gr-ReANC had higher SWIR emissions from the lungs compared to 4T1 mice receiving Iso-ReANC and EMT6 mice receiving Gr-ReANC. There was no appreciable difference between EMT6 mice receiving Gr-ReANC and Iso-ReANC. Ex vivo SWIR imaging further validated the increased accumulation of Gr-ReANCs within the lungs of 4T1 tumor-bearing mice compared to Iso-ReANCs and EMT6 tumor-bearing mice.

The results of this study demonstrate that SWIR emitting nanoprobes functionalized with a MDSC targeted antibody are capable of distinguishing between mouse breast cancer models with distinct metastatic rates based on their lung MDSC burdens alone. Additionally, results indicate that initial MDSC imaging timepoints occur prior to macro-metastases formation with few metastases present. This represents a novel approach to tracking disease progression and prognosticating lung metastases in a low cost, non-invasive manner.

Due to the various roles of MDSCs in cancer progression and immunotherapy evasion, this technology lends itself to several future applications. In addition to MDSC burden assessment to determine metastatic index, SWIR imaging can be used for prognosticating immunotherapy responsiveness by whole tumor assessment of the immunosuppressive MDSC burden in correlation to the immunogenic CD8 burden. Future studies will also address the nanoprobes as potential theranostics to reduce MDSC burden and shift the tumor immune microenvironment from immunosuppressive to an immune-stimulatory environment.


Acknowledgements (Optional): : This research was funded by the National Institute of Biomedical Imaging and Bioengineering (R01 EB0183878), National Institute of General Medical Sciences (T32 GM135141) and the New Jersey Commission on Cancer Research (DCHS20PPC036), We would like to acknowledge the Rutgers Molecular Imaging Core, the Rutgers Biomedical Engineering High Resolution Microscopy Core, and the Rutgers Flow Cytometry/Cell Sorting & Confocal Microscopy Core Facility

References (Optional): : 1. Wang, Y., Ding, Y., Guo, N. & Wang, S. MDSCs: Key Criminals of Tumor Pre-metastatic Niche Formation. Front Immunol 10, (2019).

2. Fultang, L. et al. MDSC targeting with Gemtuzumab ozogamicin restores T cell immunity and immunotherapy against cancers. EBioMedicine 47, 235–246 (2019).

3. Medeiros, B. & Allan, A. L. Molecular Mechanisms of Breast Cancer Metastasis to the Lung: Clinical and Experimental Perspectives. Int J Mol Sci 20, 2272 (2019).

4. Oh, K. et al. A mutual activation loop between breast cancer cells and myeloid-derived suppressor cells facilitates spontaneous metastasis through IL-6 trans-signaling in a murine model. Breast Cancer Research 15, R79 (2013).

5. Cui, W., Wang, Z., Lv, J., Qin, Y. & Shi, H. MDSCs: The Key Players in the Formation of Pre-Metastatic Niche. FBL 28, 58 (2023).

6. Shah, J. V. et al. Shortwave Infrared-Emitting Theranostics for Breast Cancer Therapy Response Monitoring. Frontiers in Molecular Biosciences 7, (2020).

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9. Zevon, M. et al. CXCR-4 Targeted, Short Wave Infrared (SWIR) Emitting Nanoprobes for Enhanced Deep Tissue Imaging and Micrometastatic Lesion Detection. Small 11, 6347–6357 (2015).