(684g) Exploring (some of) the Empowerment Mechanisms of Alpha-Particle Radiopharmaceutical Therapy By Low-Dose Chemotherapy in Breast Cancer Brain Metastasis

Breast cancer brain metastases (BCBM) remain incurable [1]. Despite multi-modal therapies, the location of the tumor makes it difficult to treat aggressively due to adverse consequences to peripheral brain, resulting in delivery heterogeneities, since not all cancer cells receive lethal doses. Additionally, patients may exhibit resistance to therapies due to tumor-cell intrinsic heterogeneities.

We unexpectedly found that when we combine alpha-particle RadioPharmaceutical Therapy (αRPT) with low, non-lethal doses of standard-of-care DNA-damaging chemotherapy, we observe improved cell killing and remarkably prolonged survival in animals bearing TNBC tumors in the brain, compared to when treated with each modality alone. This is surprising, because αRPT is orders-of-magnitude more powerful than any chemotherapy and/or other radiotherapies. Currently, we're probing the mechanism(s) underlying the enhanced effects seen with the combination of αRPT and chemotherapy, from cellular, molecular-pathway, and/or transport viewpoints.

High-energy, short-range (40-80µm in tissue) α-particles unequivocally kill cancer cells by causing double-strand DNA-breaks. αRPT is impervious to resistance and ideal for treating BCBM, because irradiation of the peripheral brain is minimal. However, cancer cells not being hit by α-particles will not be killed. Therefore, we developed a delivery strategy to spread the radioactivity within brain tumors; αRPT is attached on systemically injected ultrasmall-nanoparticles, dendrimers, that selectively accumulate in brain tumors and associate with tumor-promoting tumor associated macrophages (TAMs). TAMs are known to extensively infiltrate tumors, therefore, they spread αRPT within tumors while carrying the radiolabeled nanoparticles. We discovered that when alpha-particle radiotherapy acts on same cancer cells as the low-dose chemotherapy, they form an even more powerful cocktail. Therefore, since there is no perfect delivery strategy for truly uniform tumor infiltration and irradiation by αRPT to address the delivery heterogeneities, we hypothesize that the combination of αRPT with low doses of chemotherapy creates a new, potent cocktail to address the tumor cell intrinsic heterogeneities especially in sub tumoral regions locally receiving lower-than-lethal αRPT doses.

Previous studies [3-9] have explored the synergistic effect of combining radiotherapy and chemotherapy; however, these studies were in the context of ionizing radiation of much lower LET such as X-rays and/or employing significantly greater doses of chemotherapy.

Methods:

In vitro studies: Clonogenic assays and/or 3D spheroid models were established using 4T1 cells, a murine TNBC cell line, and were treated with Actinium-225 labeled dendrimer nanoparticles with and without low doses of cisplatin [2]. Viability was assessed by quantifying colony formation and/or percentage of spheroid regrowth compared to untreated controls, respectively.
Cell cycle analysis using flow cytometry was utilized to analyze DNA content and quantify the distribution of 4T1 cells in different phases of the cell cycle (G0/G1, S, and G2/M) following different treatments.

In vivo studies: BALB/c mice were divided into four cohorts and intracranially injected with 500 4T1 cells. Subsequently, they were treated with either 700nCi radiolabeled dendrimer alone (=50% MTD), 700nCi radiolabeled dendrimer combined with 5mg/kg cisplatin, or 5mg/kg cisplatin alone, with a control non-treated group included. Survival outcomes were assessed and plotted using Kaplan-Meier curves.

Results and Discussion: Our preliminary experiments reveal that the addition of low-dose chemotherapy enhances the effectiveness of αRPT both in vitro on monolayers and 3D models, as well as in vivo by enabling longer survival relative to each of the modalities alone. Initial investigations into the underlying mechanisms suggest that this enhancement is partly attributed to (1) enabling improved intratumoral spreading of the dendrimer-delivered radiotherapy, as deciphered by studies in 3D spheroids, and/or (2) generating a highly potent DNA-attacking drug combination. Crucially, the co-localization of these two modalities occurs exclusively within the tumor, minimizing off-target toxicities.

Conclusions and Future Directions: Encouraged by our current results we aim to assess the impact of this combination therapy on cellular properties, such as migration using scratch assay and transwell migration assays. At the molecular level, we will investigate the upregulation of signaling pathways involved in cell survival, proliferation, repair, and regulation, including PI3K-Akt, MAPK/ERK, and p53 pathways, which can be affected by radiotherapy and chemotherapy using phosphokinase assays, western blots. Furthermore, to elucidate the immune response elicited by our treatment regimens, we propose conducting immunohistochemistry on mouse brain tissues obtained from mice subjected to radioactivity alone, the combination therapy, and cisplatin alone. This analysis aims to discern variations in immune cell infiltration across different treatment conditions. The successful completion of these experiments will provide insights into the underlying biological and transport mechanisms, facilitating the development of combination treatment regimens involving α-particle therapy and chemotherapy to enhance therapeutic outcomes for patients with BCBM.

References:

1. American Cancer Society. Cancer Facts & Figures. 2024. , 2024.

2. A. Howe, O. Bhatavdekar, D. Salerno, A. Josefsson, J. Pacheco-Torres, Z.M. Bhujwalla, K.L. Gabrielson, G. Sgouros, S. Sofou, Combination of carriers, with complementary intratumoral microdistributions of delivered α-particles, may realize the promise for Actinium-225 in large solid tumors, Journal of Nuclear Medicine 63 (2022) 1223-1230.

3. Zhang, Z., et al., Radiotherapy combined with immunotherapy: the dawn of cancer treatment. Signal transduction and targeted therapy, 2022. 7(1): p. 258.

4. Li, M., et al., Targeted Alpha-Particle Radiotherapy and Immune Checkpoint Inhibitors Induces Cooperative Inhibition on Tumor Growth of Malignant Melanoma. Cancers, 2021. 13(15): p. 3676. doi:10.3390/cancers13153676.

5. Ku, A., et al., Auger electrons for cancer therapy - a review. EJNMMI radiopharmacy and chemistry, 2019. 4(1): p. 27-2.

6. Zhang, L., et al., A Multimodal Therapeutic Nanoplatform Overcoming Tumor Hypoxia Heterogeneity for Improved Tumor Chemoradiotherapy. Advanced functional materials, 2022. 32(34): p. 2204629-n/a.

7. Cytryniak, A., et al., Cubosomal Lipid Formulation for Combination Cancer Treatment: Delivery of a Chemotherapeutic Agent and Complexed α‑Particle Emitter 213Bi. Molecular pharmaceutics, 2022.19(8): p. 2818-2831.

8. Shrivastava, S., et al., Cisplatin Chemoradiotherapy vs Radiotherapy in FIGO Stage IIIB Squamous Cell Carcinoma of the Uterine Cervix: A Randomized Clinical Trial. JAMA oncology, 2018. 4(4): p. 506-513.

9. Nishri Y, Vatarescu M, Luz I, Epstein L, Dumančić M, Del Mare S, Shai A, Schmidt M, Deutsch L, Den RB, Kelson I, Keisari Y, Arazi L, Cooks T, Domankevich V. Diffusing alpha-emitters radiation therapy in combination with temozolomide or bevacizumab in human glioblastoma multiforme xenografts. Front Oncol. 2022 Sep 27;12:888100. doi: 10.3389/fonc.2022.888100. PMID: 36237307; PMCID: PMC9552201.