Inhalable CAR-T cell-derived exosomes as paclitaxel carriers for treating lung cancer

Abstract

Background: Non-small cell lung cancer (NSCLC) is a worldwide health threat with high annual morbidity and mortality. Chemotherapeutic drugs such as paclitaxel (PTX) have been widely applied clinically. However, systemic toxicity due to the non-specific circulation of PTX often leads to multi-organ damage, including to the liver and kidney. Thus, it is necessary to develop a novel strategy to enhance the targeted antitumor effects of PTX.

Methods: Here, we engineered exosomes derived from T cells expressing the chimeric antigen receptor (CAR-Exos), which targeted mesothelin (MSLN)-expressing Lewis lung cancer (MSLN-LLC) through the anti-MSLN single-chain variable fragment (scFv) of CAR-Exos. PTX was encapsulated into CAR-Exos (PTX@CAR-Exos) and administered via inhalation to an orthotopic lung cancer mouse model.

Results: Inhaled PTX@CAR-Exos accumulated within the tumor area, reduced tumor size, and prolonged survival with little toxicity. In addition, PTX@CAR-Exos reprogrammed the tumor microenvironment and reversed the immunosuppression, which was attributed to infiltrating CD8⁺ T cells and elevated IFN-γ and TNF-α levels.

Conclusions: Our study provides a nanovesicle-based delivery platform to promote the efficacy of chemotherapeutic drugs with fewer side effects. This novel strategy may ameliorate the present obstacles to the clinical treatment of lung cancer.

Keywords: CAR-T; Exosomes; Inhalation; Lung cancer; Paclitaxel; Targeted delivery.

Scheme 1.

Schematic illustration of inhalable CAR exosomes derived from CAR-T cells as PTX carriers for treating lung cancer. A Preparation process of PTX@CAR-Exo. B Antitumor effect of inhalable PTX@CAR-Exo against lung cancer. CAR-T cell-derived exosomes (CAR-Exos) inherited the targeted cytolytic effects from parental cells by releasing granzyme B and perforins. The loaded PTX was delivered into the tumor area to stimulate immune responses. In contrast to conventional CAR-T cell therapy and intravenous administration of PTX, the inhalable PTX@CAR-Exos used in an orthotopic lung cancer model exhibited superior antitumor effects and improved the bioavailability of both CAR-Exos and PTX, with decreased systemic toxicity

Fig. 1

Preparation and characterization of CAR-T cells. A Molecular design of anti-MSLN mCAR. B The transfection efficiency of lentiviral in T cells was assessed by GFP expression using fluorescence microscopy. Scale bar: 20 μm. C Anti-MSLN CAR expression was assessed by Protein L staining and flow cytometry. Representative Protein L staining results are shown for mock transduced T cells and MSLN CAR-T cells. D Fluorescence images of LLC cells stably expressing GFP-MSLN after puromycin selection. Scale bar: 20 μm. E Representative flow cytometry histogram of the expression level of MSLN on LLC cells. An anti-MSLN antibody was used to confirm MSLN expression. F Cytolysis of CAR-T cells against MSLN-LLC cells. The cytotoxic activity of CAR-T and control T cells against cancer cell lines was assessed by an LDH-release assay at the indicated effector-to-target (E:T) ratios. G The level of IFN-γ evaluated by ELISA after co-culture of CAR-T cells with MSLN-LLC cells for 24 h. Data are presented as the mean ± SD of three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant

Fig. 2

Preparation and characterization of CAR-Exos. A Representative TEM image of T-Exo and CAR-Exo. Scale bar: 100 nm. B Size distribution and zeta potential of T-Exos and CAR-Exos measured by NTA and DLS. Data are presented as the mean ± SD of three biological replicates. C The expression of CAR protein and exosomal markers in CAR-Exos or whole-cell lysates of CAR-T cells were analyzed by Western blotting. D Flow cytometric analyses of CAR-Exos conjugated to latex beads or CAR-T cells. The histograms shown isotype controls (black) and positive expression (red). E Representative Confocal laser scanning microscopy (CLSM) images of CAR-Exos bound to the surface of MSLN-LLC cells (green). T-Exos and CAR-Exos were labeled with DiI (red) and DAPI (blue) labeled nuclei of MSLN-LLC cells. CAR-Exos showed a higher binding affinity to the MSLN protein than T-Exos. Scale bar: 10 μm

Fig. 3

The enhanced antitumor effect of PTX@CAR-Exos in vitro. A Flow cytometric analyses of granzyme B and perforin expression of CAR exosomes conjugated to latex beads or CAR-T cells. The histograms shown isotype controls (black) and positive expression (red). B The expression of granzyme B and perforin in CAR-Exos or whole-cell lysates of CAR-T cells were analyzed by western blotting. C The cytotoxic activity of different concentrations of CAR exosomes against MSLN-LLC cells. D In vitro drug release profile of paclitaxel-loaded CAR exosomes was evaluated in PBS at pH 7.4 and different temperatures. The percentage of drug release (%) = OD value of the PTX released from the CAR exosomes /OD value of the total PTX in the CAR Exosomes × 100%. E Cytotoxicity and Calcein-AM/PI staining analysis in vivo. Fluorescent microscopic imaging of MSLN-LLC cells after treatment with PBS, T-Exos, CAR-Exos, PTX, PTX@T-Exos, or PTX@CAR-Exos for 24 h, cells were stained with Calcein-AM/PI (original magnification, 10 ×). Scale bar: 100 μm. F Quantitative analysis of cell death rate analyzed by Calcein-AM/PI staining. Data are presented as the mean ± SD of three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant

Fig. 4

The biodistribution of DiR-labeled CAR-Exos via inhalation in vivo. A The biodistribution of CAR-Exos in vivo via intravenous injection or inhalation. B The quantification of DiR intensity from A. C The intensity of inhaled CAR-Exos at different time points in vivo. D The quantification of DiR intensity at different time points. E The intensity of inhaled CAR-Exos at different concentrations of exosomes in vivo. F The quantification of DiR intensity at different doses. Data are presented as the mean ± SD of three biological replicates

Fig. 5

The therapeutic efficacy of PTX@CAR-Exos in orthotopic lung tumor-bearing mice. A The designed diagram for evaluating the efficacy of PBS, T-Exos, CAR-Exos, or PTX@CAR-Exos treated orthotopic lung tumor-bearing mice. Mice were given inhaled therapy via nebulizer for 2 weeks, starting on day 7 after establishment of the orthotopic model; IVIS imaging was performed on days 14 and 21 post implantation. B Respective bioluminescence images of MSLN-LLC cells in mice on days 7, 14, and 21 post implantation. C Statistical analysis of the signal intensity from B. D Survival analysis of tumor bearing mice after different treatments. E Representative images of tumor nodules in different groups and H&E-stained slice images of the lungs. Scale bar: 100 μm. Data are presented as the mean ± SD of three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant

Fig. 6

Improvement in the tumor microenvironment following PTX@CAR-Exos treatment in vivo. A, C Representative flow cytometry plots and statistical analysis of CD4⁺ T cells and CD8⁺ T cells infiltration at the tumor site. B, D Representative flow cytometry plots and statistical analysis of infiltrated CD8⁺GzmB⁺ T cells at the tumor site. Cytokine concentrations E TNF-α and F IFN-γ in tumor tissues as quantified by ELISA assay. Data are presented the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant