Photothermal therapy (PTT) is an effective method for tumor eradication and has been successfully combined with immunotherapy. However, besides its cytotoxic effects, little is known about the effect of the PTT thermal dose on the immunogenicity of treated tumor cells. Therefore, we administered a range of thermal doses using Prussian blue nanoparticle-based photothermal therapy (PBNP-PTT) and assessed their effects on tumor cell death and concomitant immunogenicity correlates in two human neuroblastoma cell lines: SH-SY5Y (MYCN-non-amplified) and LAN-1 (MYCN-amplified). PBNP-PTT generated thermal dose-dependent tumor cell killing and immunogenic cell death (ICD) in both tumor lines in vitro. However, the effect of the thermal dose on ICD and the expression of costimulatory molecules, immune checkpoint molecules, major histocompatibility complexes, an NK cell-activating ligand, and a neuroblastoma-associated antigen were significantly more pronounced in SH-SY5Y cells compared with LAN-1 cells, consistent with the high-risk phenotype of LAN-1 cells. In functional co-culture studies in vitro, T cells exhibited significantly higher cytotoxicity toward SH-SY5Y cells relative to LAN-1 cells at equivalent thermal doses. This preliminary report suggests the importance of moving past the traditional focus of using PTT solely for tumor eradication to one that considers the immunogenic effects of PTT thermal dose to facilitate its success in cancer immunotherapy.

Adoptive T cell therapy (ATCT) has been successful in treating hematological malignancies and is currently under investigation for solid-tumor therapy. In contrast to existing chimeric antigen receptor (CAR) T cell and/or antigen-specific T cell approaches, which require known targets, and responsive to the need for targeting a broad repertoire of antigens in solid tumors, we describe the first use of immunostimulatory photothermal nanoparticles to generate tumor-specific T cells. Specifically, we subject whole tumor cells to Prussian blue nanoparticle-based photothermal therapy (PBNP-PTT) before culturing with dendritic cells (DCs), and subsequent stimulation of T cells. This strategy differs from previous approaches using tumor cell lysates because we use nanoparticles to mediate thermal and immunogenic cell death in tumor cells, rendering them enhanced antigen sources. In proof-of-concept studies using two glioblastoma (GBM) tumor cell lines, we first demonstrated that when PBNP-PTT was administered at a "thermal dose" targeted to induce the immunogenicity of U87 GBM cells, we effectively expanded U87-specific T cells. Further, we found that DCs cultured ex vivo with PBNP-PTT-treated U87 cells enabled 9- to 30-fold expansion of CD4+ and CD8+ T cells. Upon co-culture with target U87 cells, these T cells secreted interferon-ɣ in a tumor-specific and dose-dependent manner (up to 647-fold over controls). Furthermore, T cells manufactured using PBNP-PTT ex vivo expansion elicited specific cytolytic activity against target U87 cells (donor-dependent 32-93% killing at an effector to target cell (E:T) ratio of 20:1) while sparing normal human astrocytes and peripheral blood mononuclear cells from the same donors. In contrast, T cells generated using U87 cell lysates expanded only 6- to 24-fold and killed 2- to 3-fold less U87 target cells at matched E:T ratios compared with T cell products expanded using the PBNP-PTT approach. These results were reproducible even when a different GBM cell line (SNB19) was used, wherein the PBNP-PTT-mediated approach resulted in a 7- to 39-fold expansion of T cells, which elicited 25-66% killing of the SNB19 cells at an E:T ratio of 20:1, depending on the donor. These findings provide proof-of-concept data supporting the use of PBNP-PTT to stimulate and expand tumor-specific T cells ex vivo for potential use as an adoptive T cell therapy approach for the treatment of patients with solid tumors.