Photothermal Therapy Promotes Tumor Infiltration and Antitumor Activity of CAR T Cells

Abstract

Chimeric antigen receptor (CAR)-redirected T lymphocytes (CAR T cells) show modest therapeutic efficacy in solid tumors. The desmoplastic structure of the tumor and the immunosuppressive tumor microenvironment usually account for the reduced efficacy of CAR T cells in solid tumors. Mild hyperthermia of the tumor reduces its compact structure and interstitial fluid pressure, increases blood perfusion, releases antigens, and promotes the recruitment of endogenous immune cells. Therefore, the combination of mild hyperthermia with the adoptive transfer of CAR T cells can potentially increase the therapeutic index of these cells in solid tumors. It is found that the chondroitin sulfate proteoglycan-4 (CSPG4)-specific CAR T cells infused in Nod scid gamma mice engrafted with the human melanoma WM115 cell line have superior antitumor activity after photothermal ablation of the tumor. The findings suggest that photothermal therapy facilitates the accumulation and effector function of CAR T cells within solid tumors.

Keywords: CAR T cells; cell therapy; drug delivery; immunotherapy; photothermal therapy.

Figure 1.

Schematic illustration showing the effects of the mild heating of the tumor that causes enhanced infiltration and activation of adoptive transfer CAR.CSPG4⁺ T cells.

Figure 2.. Photothermal therapy of the tumor promotes CAR T cell proliferation and cytokine release.

(a) Hydrodynamic diameter of PLGA-ICG nanoparticles measured by dynamic light scattering. Insert is the TEM image of PLGA-ICG (Scale bar, 200 nm). (b) The UV–vis–NIR spectrum of PLGA-ICG, exhibiting high absorption in the near infrared region. (c & d) IR thermal images (c) and temperature curves (d) of PBS and PLGA-ICG under the 808-nm light irradiation for 5 min at the power density of 0.5 W/cm². Data are presented as mean ± s.e.m. (n=3) (e) Representative flow cytometry analysis of CAR.CSPG4⁺ T cells labeled with CFSE three days after the indicated treatments. (f) Mean fluorescence intensity of CFSE, indicating T cell proliferation. Data are presented as mean ± s.e.m. (n=4). (g & h) Detection of IL-2 (g) and IFN-γ (h) in the supernatant of CAR.CSPG4⁺ T cells, three days after the indicated treatments. Data are presented as mean ± s.e.m. (n=4). Statistical significance was calculated via one-way ANOVA with a Tukey post-hoc test. P value: *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3.. Photothermal therapy of the tumor modifies the tumor microenvironment.

(a) IR thermal images of WM115-tumor-bearing mice injected with PLGA-ICG or PBS with the 808 nm laser irradiation (0.3 W/cm², 20 min). (b) Changes of the tumor temperature measured by the IR thermal imaging. (c) Immunofluorescence imaging of tumors collected from mice 24 hours after photothermal therapy. Scale bar, 50 μm. (d) Ultrasound imaging illustrating the blood perfusion of the WM115 tumors. Microbubbles injected intravenously were used as the ultrasound contrast agent. (e) Representative hypoxia and HIF1-α immunofluorescence staining of the tumors after photothermal therapy (Scale bar, 50 μm). (f) Representative flow cytometry plots and quantification of murine CD45⁺ cells infiltrating the tumor after photothermal therapy. Data are presented as mean ± s.e.m. (n=10). (g & h) Representative flow cytometry plot and quantification of murine CD11c⁺ (g) and CD11b⁺ (h) cells gating on CD45⁺ cells. Data are presented as mean ± s.e.m. (n=10). (i) Quantification of chemokines in the tumor (n=10). Statistical significance was calculated via two-tailed Student’s t-test. P value: *P < 0.05; **P< 0.01; ***P < 0.001.

Figure 4.. Photothermal ablation of the tumor increases the infiltration of adoptively transferred CAR T cells.

(a) In vivo bioluminescence imaging of the CAR.CSPG4⁺ T cells. (b) Quantification of CAR.CSPG4⁺ T cells detected in the tumor with or without photothermal ablation. Data are presented as mean ± s.e.m. (n=3). (c) Representative flow cytometry plots of CAR.CSPG4⁺ T cells infiltrating the tumor. (d-e) Absolute frequency of CD3⁺ (d), CD4⁺ (e) and CD8⁺ T cells (f) within tumors. Data are presented as mean ± s.e.m. (n=4). (g) Representative immunofluorescence of tumors showing CD4⁺ and CD8⁺ CAR T cells infiltrating the tumor. Scale bar 50 μm. Statistical significance was calculated via two-tailed Student’s t-test. P value: *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5.. Combined photothermal ablation and adoptive transfer of CAR T cells inhibit the growth of the human melanoma WM115 in vivo.

(a) Representative bioluminescence of the WM115 tumors (n = 4). (b & c) Individual (b) and average (c) tumor growth kinetics in different groups. Day 0 indicate the day in which treatment was initiated. Data are presented as mean ± s.e.m. (n = 6). (d) Murine IL-6 levels detected in the tumors 7 days after the indicated treatments. Data are presented as mean ± s.e.m. (n = 8). (e & f) Human IL-2 and IFN-γ levels detected in the tumor 7 days after the indicated treatments. Data are presented as mean ± s.e.m. (n = 8). Statistical significance was calculated via one-way ANOVA with a Tukey post-hoc test. P value: *P < 0.05; **P < 0.01; ***P < 0.001.