Theranostics 2020; 10(8):3397-3412. doi:10.7150/thno.42243

Research Paper

Novel calreticulin-nanoparticle in combination with focused ultrasound induces immunogenic cell death in melanoma to enhance antitumor immunity

Sri Nandhini Sethuraman1,#, Mohit Pratap Singh1,#, Girish Patil1, Shitao Li1, Steven Fiering2, P. Jack Hoopes2, Chandan Guha3, Jerry Malayer1, Ashish Ranjan1,✉

1. Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, Oklahoma 74074
2. Geisel School of Medicine, Dartmouth, Hanover, NH03755
3. Albert Einstein College of Medicine, Bronx, New York 10461
# Both authors contributed equally to the experimental study and drafting of the manuscript.

This is an open access article distributed under the terms of the Creative Commons Attribution License ( See for full terms and conditions.
Sethuraman SN, Singh MP, Patil G, Li S, Fiering S, Hoopes PJ, Guha C, Malayer J, Ranjan A. Novel calreticulin-nanoparticle in combination with focused ultrasound induces immunogenic cell death in melanoma to enhance antitumor immunity. Theranostics 2020; 10(8):3397-3412. doi:10.7150/thno.42243. Available from

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Rationale: Some studies have shown that the local activation of immunogenic cell death (ICD) by upregulating calreticulin (CRT) expression in solid tumors can improve antitumor effects. Although a promising approach, a key current challenge in ICD tumor therapy is the absence of a clinically translatable method for reproducibly inducing the CRT expression. Herein, we report a novel calreticulin-nanoparticle (CRT-NP) that enhances ICD and synergizes with focused ultrasound (FUS) to achieve local and systemic antitumor effects.

Methods: Full-length clone DNA of calreticulin was encapsulated in NPs made from DOTAP and cholesterol. Three CRT-NP intratumoral injections of 20 µg each were given 2 days apart, and FUS heating (42-45°C, ~15min) was applied sequentially 24h after each injection to induce ICD. To investigate ICD specific immune effect, the splenocytes of mice vaccinated with CRT-NP (± FUS) treated B16F10 cells were evaluated ex-vivo for TRP-2 antigen specific immunity. Additionally, the long-term protection was evaluated by re-challenging with the melanoma cells in the flank regions of tumor bearing mice.

Results: CRT-NP plus FUS (CFUS) upregulated CRT expression, expanded the population of melanoma TRP-2 specific functional CD4+ and CD8+ T cells and tumor-suppressing M1 phenotype, and increased PD-1 and PD-L1 marker expression in the T cells. Therapeutically, CFUS suppressed B16 melanoma growth by >85% vs. that seen in untreated controls, and >~50% vs. CRT-NP or FUS alone, and prevented tumor growth in distal untreated sites.

Conclusions: CRT-NP amplifies the FUS and ICD therapeutic outcomes against melanoma, suggesting that the proposed combinatorial methodology may be clinically translatable.

Keywords: Immunogenic cell death, calreticulin, nanoparticle, melanoma, focused ultrasound