(T1330-03-19) Targeting T-Cell Exhaustion in 3D-Patient-Derived Triple Negative Breast Cancer Organoids and PDx Mice Models Using Novel Calcium Nanoparticles

Purpose: Despite recent advancements in breast cancer research, patients with Triple Negative Breast Cancer (TNBC) remain challenged with limited treatment options, worse disease prognosis, and poor overall survival[1]. Exhausted Tumor-Infiltrating Human Lymphocytes (TILs) have been identified as an unresolved contributor to immunosuppression and impaired response to available immunotherapy options in patients with TNBC. T-cell exhaustion arises from multiple factors, including genetic variability and complex interactions among tumor cells, immune cells, and microbes within the Tumor Microenvironment (TME)[2]. Although immunotherapy has revolutionized the treatment landscape of TNBC, only about 5-25% of patients show sensitivity to immunotherapy, most of whom eventually acquire resistance[3]. Given that the effectiveness of immune checkpoint inhibitors (ICIs) depends on tumor infiltration by precursor-exhausted CD8+ T-cells, our research aims to reinvigorate exhausted TILs to restore their anti-tumor activity and sensitivity to immunotherapy.

Methods: We utilize clinically relevant 3D-Patient-Derived Tumor Organoids (3D-PDTOs) to recapitulate complex TME interactions, surpassing critical limitations of traditional 2D cell cultures. Our collaborators have developed novel anti-human PD-1 antibody-conjugated Calcium Nanoparticles (CaNPs) to preferentially activate human PD-1+ T-cells via the NF-κB and NFAT pathways. Given the elevated PD-1 expression on exhausted human T-cells, we leverage these CaNPs to restore anti-tumor functions in TILs. The results from our ex vivo 3D-tumor-immune co-culture systems were further validated in Humanized patient-derived xenograft (PDX) mice models. Single-cell analyses, flow cytometry, confocal microscopy, continuous 3D-live organoid imaging, and 3D bioluminescence assays are employed to rigorously evaluate the efficacy of CaNPs in reinvigorating T-cell exhaustion in Triple Negative Breast Cancer.

Results: Our results show the successful establishment of our bi-partite 3D-patient derived TNBC organoid and human CD8+ve cytotoxic T-cells as a robust platform to recapitulate the human tumor-immune microenvironment (TIME). Treatment of the 3D-Patient derived Triple Negative Breast Cancer Organoids and Humanized mice models with the novel PD-1-CaNPs significantly decreases the progression and viability of Triple Negative Breast Cancer in 3D-Patient derived TNBC Organoids and Humanized mice models. Immunoblotting, Confocal microscopy, and flow cytometry results show that the novel PD-1-linked calcium nanoparticles significantly increase the activation and infiltration of human CD8+ cytotoxic T-cells via the NFKB and NFAT signaling pathways.

Conclusion: Novel PD-1-linked calcium nanoparticles improve anti-tumor function in human TILs by intracellular calcium to activate cytotoxic T-cells via the NF-kB and NFAT signaling pathways. The improved T-cell activation translated to a significant decrease in the viability of 3D-patient-derived organoids and in vivo tumor growth in humanized mice models. These findings support a viable strategy to overcome resistance and improve response to immunotherapy which are some of the most pressing bottlenecks challenging almost all forms of immunotherapy. Elucidating the interactions within the TME and overturning exhausted T-cells ensures immunotherapy remains an effective treatment option for patients with TNBC.

References: Yi B., Chang H., Ma R., Feng X., Li W., Piazza G. A., Xi Y. Inhibition of breast cancer cell motility with a non-cyclooxygenase inhibitory derivative of sulindac by suppressing TGFβ/miR-21 signaling. Oncotarget. 2016; 7: 7979-7992.
Blandino, G., Satchi-Fainaro, R., Tinhofer, I., Tonon, G., Heilshorn, S. C., Kwon, Y. J., Pestana, A., Frascolla, C., Pompili, L., Puce, A., Iachettini, S., Tocci, A., Karkampouna, S., Kruithof-de Julio, M., Tocci, P., Porciello, N., Maccaroni, K., Rutigliano, D., Shen, X., & Ciliberto, G. (2024). Cancer Organoids as reliable disease models to drive clinical development of novel therapies. Journal of experimental & clinical cancer research: CR, 43(1), 334.
Shi, Y., Yu, Y., Zhao, J., Huang, L., Wang, Q., Sun, Q., Liu, L., & Sun, C. (2025). Development of a prognostic model based on four genes related to exhausted CD8+ T cells in triple-negative breast cancer patients: a comprehensive analysis integrating scRNA-seq and bulk RNA-seq. Discover oncology, 16(1), 114.

PD-1 linked Calcium Nanoparticles significantly increase the activation of Human Cytotoxic T-cells compared to other treatment groups

PD-1 linked Calcium Nanoparticles significantly increase Human Cytotoxic TILs Infiltration in 3D-Patient Derived TNBC Organoids compared to other treatment groups

PD-1 linked Calcium Nanoparticles significantly decreases the viability of 3D-Patient derived TNBC Organoids compared to other treatment groups