Photo-activated chemo-immunotherapy for metastatic cancer using a synergistic graphene nanosystem
Introduction
Triple-negative breast cancer (TNBC) accounts for 10–20% of diagnosed breast cancers, with limited options and poor prognosis [[1], [2], [3]]. More importantly, compared to hormone-positive breast cancer, TNBC is common in women younger than 40 years [4]. The cure rate of cancer has gradually increased in the past decades with the development of new cancer therapies, including target molecules, immune checkpoint inhibitors, vaccines, and CAR-T. However, the cure for TNBC is still elusive [[5], [6], [7], [8]]. For the unsatisfactory outcomes in treating TNBC, the major reason is the poor tumor immunogenicity and the strong immunosuppressive tumor microenvironment [9,10].
Chemotherapy has been one of the primary clinical modalities. However, the immunosuppressive effects of chemotherapeutic agents have been well known. As a few exceptions, doxorubicin (Dox) and mitoxantrone (MTX) induce topoisomerase II associated DNA damage in the cell nucleus, causing immunogenic cell death (ICD) that could trigger a tumor-specific immunity by releasing damage-associated molecular patterns (DAMPs) and tumor antigens [[11], [12], [13], [14]]. However, drug resistance is still the big challenge in chemotherapy, resulting in recurrence and drug-resistant metastases in the treated TNBC patients [15].
TGF-β is a pleiotropic cytokine that maintains an immunosuppressive environment to help established tumor escape from the host immune system [16]. Many tumors overexpression of TGF-β correlate with metastasis and poor prognosis. Therefore, TGF-β was considered as an ideal target and TGF-β inhibitors were developed for the treatment of cancer [17,18]. One of the TGF-β inhibitors, SB-431542, was found to decrease lung metastasis, however, there was no effect on the growth of the primary tumor in 4T1 xenograft tumor model [17]. LY2157299 prevented reestablishment of tumors after paclitaxel treatment of 4T1 xenograft [18]. Either chemotherapy agents or TGF-β inhibitors face severe limitations in treating TNBC due to delivery challenges and toxicity profiles.
Nano-graphene oxide (nGO) has been widely used as a drug carrier and photo-agent for cancer theranostics, due to its principal characteristics of 2D film structure and near-infrared absorption spectrum. Especially, PEG-nGO was found to activate macrophages with a potential to enhance the antitumor immunity [19]. We hypothesized that a combination of an in situ tumor vaccine with improved tumor microenvironment, should lead to an effective antitumor immunity. In addition, local application of chemotherapeutic and immunotherapeutic agents could be effective against tumors while reducing the systemic side effect.
Herein, we developed a graphene-based nano-system by conjugating rGO with MTX and SB for photo-activated, synergistic chemo-immunotherapy to treat metastatic TNBC using 4T1 mouse breast tumor model Fig. 1. The therapeutic efficacy of photo-activated chemo-immunotherapy was investigated. The host immunity, including induced ICD, antitumor vaccination, tumor-specific T cells, and the abscopal effect, were also investigated.
Section snippets
Synthesis and characterization of rGO/MTX/SB
The base of the nanoplatform, rGO, was conjugated with mPEG2000-NH2 following a well-established EDC/NHS protocol and purified by a 100 kDa filter through centrifugation. MTX and SB were then loaded onto rGO-PEG via π-π stacking interaction. The detailed protocol and analysis were shown in Supplemental Methods.
Cell culture and cellular treatment
4T1 murine breast cancer cell line and CT26 colon carcinoma cell line were both purchased from ATCC (Manassas, VA, USA), bone marrow macrophages and DCs were harvested from BALB/c mice,
Characterization of rGO/MTX/SB
The biocompatibility of rGO was improved by conjugating with mPEG2000-NH2, followed by MTX and SB loading via π-π stacking (Figs. S1-5). A UV–vis absorption spectrum of rGO/MTX/SB showed the characteristic absorption peaks of MTX and SB, indicating successful loading of MTX and SB, with a final size of 100–200 nm (Fig. 2A and B). The temperature increase of rGO/MTX/SB solutions detected under laser irradiation with an 805-nm laser indicated that rGO/MTX/SB kept the photothermal transfer effects
Discussion
The potent therapeutic strategies for metastatic cancer require efficient antitumor responses by eradicating primary tumors and controlling distal metastases. To this end, chemo-immunotherapy was developed as a combination therapeutic strategy by synergizing the tumor killing effect and antitumor immunomodulatory effect to control primary tumors and metastases with reduced drug doses. However, drug resistance and toxicity are still the big challenges due to the systemic delivery of
Credit author statement
Feifan Zhou, Designed and directed the project, Performed experiments, Analyzed data, Wrote the paper. Meng Wang, Designed and performed experiments, Analyzed data. Teng Luo, Performed experiments. Junle Qu, Designed and directed the project, Provided scientific input, Analyzed data, Wrote the paper. Wei R. Chen, Designed and directed the project, Provided scientific input, Analyzed data, Wrote the paper.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This study was supported in part by grants from the US National Institutes of Health R01 CA205348 (W.R.C.), the Oklahoma Center for Advancement of Science and Technology HR16-085 (W.R.C.), the National Natural Science Foundation of China (61722508/61525503/61620106016/61835009/81727804) (J.Q.), Guangdong Province Key Area R&D Program 2019B110233004 and Hainan University R&D Program (KYQD(ZR)20074) (F.Z.), Shenzhen Fundamental Research Program JCYJ20190808114609361 (M.W.).
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Both authors contributed equally to this manuscript.