Purpose: Triple-negative breast cancer (TNBC) is one type of breast cancer with invasive and metastatic properties (Carey et al., 2010, Garrido-Castro et al., 2019). The development of therapeutics to kill cancer cells through immunogenic cell death (ICD) is a promising strategy to elicit antitumor immunity and improve the efficacy of immunotherapy for TNBC (Feng et al., 2020).DSF is an FDA-approved anti-alcoholism drug that has copper-dependent anticancer effects. Diethyldithiocarbamate (DDC, a metabolite of DSF) and copper ions (Cu2+) form active anticancer copper diethyldithiocarbamate complex [Cu(DDC)2] to kill cancer cells. Previously, we prepared the Cu(DDC)2 NP formulation with a stabilized metal ion ligand complex (SMILE) technology developed in our laboratory. The Cu(DDC)2 NP showed excellent anticancer efficacies against breast cancer and prostate cancer in the preclinical setting (Chang et al., 2020, Chen et al., 2018, Huang et al., 2020). However, the amount of Cu(DDC)2 NP in non-tumor sites remain a concern. In the current work, we propose to use a novel CDL combination therapy to form Cu(DDC)2 NP in situ and explore the anti-cancer mechanism Methods: CuS NP was synthesized with a reported biomineralization method using albumin as the stabilizer. For most of the studies, 4T1 breast cancer cells were treated with CuS, 0.1mM; DQ, 2 μM; Laser, 2.54 W/cm2 for 5 minutes or other control groups. Cell viabilities were determined with MTT assay and Calcein-AM/PI staining in 2D and 3D tumor model. Intracellular ROS were determined with DCFH-DA dye. The percentage of percentage of early-stage apoptotic cells (annexin V+/PI-) and late-stage apoptotic cells (annexin V+/PI+) with flow cytometry. ICD markers were determined by immune florescence and ELISA. Statistical significance was performed with the unpaired two-tailed Student’s t-test. Data are presented as the mean ± SD, n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001 compared with the negative control group; # P < 0.05, # # P < 0.01, # # # P < 0.001, compared with CDL treatment group). Results: In this study, we developed a novel combination therapy for cancer treatment. We select Triple negative breast cancer (TNBC) as a model to perform proof-of-concept studies to evaluate CDL-mediated anticancer efficacy and understand molecular mechanisms. Fig. 2 A&B verified that CDL combination therapy could induce the highest ROS level. Fig2C indicated that CDL combination therapy shown most potent efficacy in inducing cell apoptosis. In addition, Cells undergoing ICD were often characterized by three biomarkers: (1) Cell-surface translocation of CRT; (2) ATP release; and (3) HMGB1 release. we determined the ICD biomarkers in 4T1 cells treated with CDL therapy (Fig. 3). The CDL therapy group showed the highest level of cell surface CRT, it also resulted in the most significant ATP and HMGB1release, suggesting CDL can effectively induce ICD due to their effects on ER stress and ROS generation. Conclusion: In this study, we developed a CDL combination therapy as a novel therapeutic approach for treating TNBC. The CDL combination therapy can enhance anticancer efficacy due to the combination of multiple anticancer mechanisms, including Cu(DDC)2 chemotherapy, the amplified oxidative stress.. This therapy will have minimal toxic side effects due to tumor-specific NIR light-activated “nontoxic-to-toxic” transition. Because of its unique mechanism of action, the CDL combination therapy can be used as a broad-spectrum anticancer therapy for different cancers. Cancer cells with elevated ROS levels will be more sensitive to CDL therapy. In addition, the CDL therapy will also have the potential to enhance the anticancer efficacy through eliciting antitumor immunity. References: 1. Carey L, Winer E, Viale G, Cameron D, Gianni L. Triple-negative breast cancer: disease entity or title of convenience?. Nature reviews Clinical oncology. 2010 Dec;7(12):683-92. 2. Garrido-Castro AC, Lin NU, Polyak K. Insights into molecular classifications of triple-negative breast cancer: improving patient selection for treatment. Cancer discovery. 2019 Feb 1;9(2):176-98. 3. Feng B, Niu Z, Hou B, Zhou L, Li Y, Yu H. Enhancing triple negative breast cancer immunotherapy by ICG‐templated self‐assembly of paclitaxel nanoparticles. Advanced Functional Materials. 2020 Feb;30(6):1906605. 4. Chang Y, Jiang J, Chen W, Yang W, Chen L, Chen P, Shen J, Qian S, Zhou T, Wu L, Hong L. Biomimetic metal-organic nanoparticles prepared with a 3D-printed microfluidic device as a novel formulation for disulfiram-based therapy against breast cancer. Applied materials today. 2020 Mar 1;18:100492. 5. Chen W, Yang W, Chen P, Huang Y, Li F. Disulfiram copper nanoparticles prepared with a stabilized metal ion ligand complex method for treating drug-resistant prostate cancers. ACS applied materials & interfaces. 2018 Nov 16;10(48):41118-28. 6. Fujiwara T, Liu XM, Ohya Y, Wang Y, editors. Polymers in Therapeutic Delivery. American Chemical Society; 2020.
Acknowledgements: This work was financially supported by the following resources: Launch Innovation Award (F, Li), Auburn University start-up fund (F. Li), NIH MIRA R35GM133795 (P. Chen). We thank Dr. Melissa Boersma at Auburn University Mass Spectrometry Center for LC/MS analysis.
Tumor specific activation of prodrug combination therapy can directly kill primary tumor, induce immunogenic cell death, and activate antitumor immunity to suppress metastatic tumor (Fig 1).
Intracellular ROS levels of 4T1 cells receiving different treatments were determined with DCFH-DA dye based method. (A) Fluorescence image and (B) fluorescence intensity determined with fluorescence spectrometer. (C) Annexin V/PI apoptosis assay.
Biomarkers of ICD. (A) Cell surface CRT determined with flow cytometry. (B) ATP release was determined with ATP bioluminescence detection kit. (C) HMGB1 release was determined with ELISA. (CuS, 0.1 mM; DQ, 2 μM; Laser, 2.54 W/cm2 for 5 min, OXP 5 μM). Data are presented as the mean ± SD, n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001, compared with the negative control group; # P < 0.05, # # P < 0.01, # # # P < 0.001, compared with CDL treatment group).
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