Single cell dissociation antibody staining and FACS sorting Cellular atlas DEGs Cellular interaction Ligand Recepto r Immunostaining Functional assays Correlation analysis Droplet-based scRNA-seq Data Cell 1 Cell 2 Cell x Gene 1 Gene 2 Gene y Highlights Single cell transcriptomic datasets are a valuable resource to dissect cellular diversity and intercellular crosstalk of human ICCs. Malignant cells displayed remarkable inter-tumor heterogeneity and Tregs revealed highly immunosuppressive characteristics. Six distinct fibroblast subsets were defined in ICCs and adjacent tissues. CD146 + vCAFs, comprising most of the fibroblasts, had tight interactions with malignant cells through IL-6/IL-6R axis. Tumor exosomal miR-9-5p elicited IL-6 expression in vCAFs, contributing to ICC progression via upregulation of EZH2.

Whole tumor cell lysates (TCL) have been implemented as tumor antigens for cancer vaccine development, although clinical outcomes of TCL-based antitumor immunotherapy remain unsatisfactory. In order to improve the efficacy of TCL-based vaccines, biomaterials have been employed to enhance antigen delivery and presentation. Here, we have developed chitosan nanoparticles (CTS NPs) with surface mannose (Man) moieties for specific dendritic cells (DCs) targeting (Man-CTS NPs). The Man-CTS NPs were then loaded with TCL generated from B16 melanoma cells (Man-CTS-TCL NPs) for in vitro and in vivo assessment. Potency of the Man-CTS-TCL NPs as cancer vaccine was also assessed in vivo by immunization of mice with Man-CTS-TCL NPs followed by re-challenge with B16 melanoma cell inoculation. We have shown here that Man-CTS-TCL NPs promote bone marrow-derived dendritic cells (BMDCs) maturation and antigen presentation in vitro. In vivo evaluation further demonstrated that the Man-CTS-TCL NPs were readily taken up by endogenous DCs within the draining lymph node (DLN) following subcutaneous administration accompanied by increasing in serum IFN-γ and IL-4 levels. Tumor growth was also significantly delayed in mice primed with Man-CTS-TCL NPs vaccine, attributable at least in part to cytotoxic T lymphocytes response. Moreover, Man-CTS-TCL NPs vaccine also exhibited therapeutic effects in mice with melanoma. Thus, we report here the Man-CTS-TCL NPs as effective anti-tumor vaccine for cancer immunotherapy.

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In this study, the photochemical internalization (PCI) technique was adopted in a nanoparticle-based antigen delivery system to enhance antigen-specific CD8 T cell immune response for cancer immunotherapy. Pheophorbide A, a hydrophobic photosensitizer, grafted with polyethylenimine (PheoA-PEI) with endosome escape activity and near-infrared imaging capability was prepared. A model antigen ovalbumin (OVA) was then complexed with PheoA-PEI to form PheoA-PEI/OVA nanoparticles (PheoA-PEI/OVA NPs) that are responsive to light. Flow cytometry analysis revealed increased endocytosis in a murine dendritic cell line (DC2.4) that was treated with PheoA-PEI/OVA NPs compared to free OVA. Generation of reactive oxygen species (ROS) in DC2.4 cells was also confirmed quantitatively and qualitatively using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Confocal laser scanning microscopy (CLSM) further demonstrated that the PheoA-PEI/OVA NPs enhanced cytosolic antigen release after light stimulation. Moreover, PheoA-PEI/OVA NP treated DC2.4 cells exhibited enhanced cross-presentation to B3Z T cell hybridoma in vitro after light irradiation, substantially increased compared to those treated with free OVA. Consistently, in vivo results revealed upregulation of CD3CD8T lymphocytes in tumors of mice treated with dendritic cells plus PheoA-PEI/OVA NPs and light irradiation. The activated T cell response is partly responsible for the inhibitory effect on E.G7 tumor growth in mice immunized with dendritic cells plus PheoA-PEI/OVA NPs and light irradiation. Our results demonstrate the feasibility to enhance antigen-specific CD8 T cell immune response by light-responsive nanoparticle-based vaccine delivery for cancer immunotherapy.