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. Author manuscript; available in PMC: 2022 Oct 15.
Published in final edited form as: Acta Biomater. 2021 Jul 27;134:716–729. doi: 10.1016/j.actbio.2021.07.052

Translocator Protein-Targeted Photodynamic Therapy for Direct and Abscopal Immunogenic Cell Death in Colorectal Cancer

Qing Xie 1, Zhen Li 1,2, Yang Liu 1, Dawei Zhang 1,3, Meng Su 1, Hiroaki Niitsu 5, Robert J Coffey 4,5, Mingfeng Bai 1,6,7
PMCID: PMC8802307  NIHMSID: NIHMS1735075  PMID: 34329783

Abstract

Abscopal effect is an attractive cancer therapeutic effect referring to tumor regression at a location distant from the primary treatment site. Immunogenic cell death (ICD) offers a mechanistic link between the primary and remote therapeutic effects by activating favorable anti-tumor immune responses. In this study, we induced ICD in colorectal cancer (CRC) cell lines in vitro and in vivo by targeting the 18 kDa translocator protein (TSPO), a mitochondrial receptor overexpressed in CRC. Photodynamic therapy (PDT) using a TSPO-targeted photosensitizer, IR700DX-6T, caused effective apoptotic cell death in fourteen CRC cell lines. In a syngeneic immunocompetent CRC mouse model, the growth of tumors subjected to TSPO-PDT was greatly suppressed. Remarkably, untreated tumors in the opposing flank also showed marked growth suppression. Dendritic and CD8+ T cells were activated after TSPO-PDT treatment, accompanied by decreased Treg cells in both treated and non-treated tumors. In addition, a cancer vaccine developed from TSPO-PDT produced a significant tumor inhibition effect. These results indicate that TSPO-PDT could not only directly suppress tumor growth but also dramatically provoke host anti-tumor immunity, highlighting the potential of TSPO-PDT as a successful therapeutic for CRC that exhibits systemic effects.

Keywords: TSPO, photodynamic therapy, immunogenic cell death, colorectal cancer, anti-tumor immunity

Graphical abstract

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Introductions

Colorectal cancer (CRC) is the leading cause of death among digestive system cancers and ranks third in deadliness when comparing all types of cancer. By the close of 2020, as many as 104,610 new cases and 53,200 deaths in the United States are projected [1]. Despite advances in the treatment of CRC, the 5-year overall survival rate for CRC remains 58%−65% for all stages [1]. The median overall survival (OS) is only 2 years in patients with peritoneal or distant metastasis [2]. It is imperative to develop more effective treatments for CRC, while ensuring improved life quality of patients.

Photodynamic therapy (PDT) is an FDA-approved therapeutic modality, which combines a specific wavelength of light with a light-sensitive drug (photosensitizer) to kill cancer cells [3]. During the process of PDT, the photosensitizer (PS) is activated by light irradiation to produce reactive oxygen species (ROS), which consequently lead to cell death. PDT is minimally invasive, effective and highly controllable, and has become a popular alternative or addition to conventional cancer treatments, such as chemotherapy and surgery.

Recent studies suggest that PDT is an effective approach for preventing tumor recurrence or even treating localized metastasis, such as in the peritoneum, a main site of CRC metastasis. A phase II clinical trial involving PDT treatment of intraperitoneal tumors that originated chiefly from ovarian cancer, but included other sites, showed beneficial results in patients [4]. The first clinical trial involving PDT of CRC conducted by Herrera-Ornelas showed beneficial effects of PDT using hematoporphyrin derivative (HpD) on a cohort of 14 patients with unresectable CRC or recurrence after surgery. The results suggest that PDT may prevent pelvic perineal tumor recurrence from CRC [5]. Another type of phototherapy, photothermal therapy, has also been used to treat CRC. Yi Chen et al reported a nanoparticle, PCH-DI, which induced photothermal effect and released chemotherapeutic drug, doxorubicin, upon light irradiation. This combination therapy approach inhibited tumor growth in an HCT-116 tumor xenograft and AOM-induced murine orthotopic colorectal cancer model [6].

One major limitation of PDT in cancer treatment is that most of the currently used PSs can also cause phototoxicity to surrounding normal tissues due to a lack of tumor selectivity [7]. In this study, we chose the 18 kDa translocator protein (TSPO) as the target for PDT of CRC. Increased expression of TSPO is found in several types of cancers including colorectal [8], breast [9], prostate [10] and brain cancers [11]. Moreover, tumoral mitochondria are an attractive therapeutic target as excessive ROS production by PDT in the tumor mitochondria can trigger apoptosis [12]. In the present study, we examined the therapeutic potential of TSPO-PDT in the treatment of CRC using IR700DX-6T, a unique TSPO-targeted PS developed in our lab [13].

Cancer cells are known to escape from anti-tumor immunity as they progress and metastasize. Therefore, restoring antitumor immunity is considered an effective approach for cancer therapy. Besides the direct effect of PDT on cancer cells, PDT was recently reported to activate antitumor immunity by inducing ICD [14]. ICD is a form of cell death that markedly increases the immunogenicity of dying cancer cells, resulting in regulated activation of the immune response [15]. Such antitumor immune responses lead to an abscopal effect, which refers to tumor regression at a location distant from the primary treatment site. ICD is thought to be highly stress-dependent as it requires induction of ROS and ER stress [14, 16]. During ICD, tumor cells express a large amount of damage-associated molecular patterns (DAMPs), including calreticulin (CRT), secreted ATP, heat shock protein 70/90 and HMGB1. These DAMP molecules are translocated from the cytoplasm to the cell surface or extracellular matrix and function as “eat me” signals [17]. As such, ICD makes cancer cells recognizable to dendritic cells (DCs), which phagocytose dying cancer cells and initiate strong antitumor response [18]. Results have suggested that therapeutic approaches that induce considerable ICD could be clinically beneficial [19, 20]. Here, we report that TSPO-PDT using IR700DX-6T caused direct ICD as well as an ICD abscopal effect.

Material and methods

Synthesis of Photosensitizer

The description of IR700DX-6T synthesis can be found in our previous paper [12].

Cells

Colorectal cell lines including MC38, LS 174T, SNU-C4, SNU-503, HCT 116, HT-29, DLD-1, RKO, SW620, and SW480 and a human breast cancer cell line, MCF7, were acquired from ATCC (American Type Culture Collection). KM12-SM, KM12-C, SC and CC are maintained in the Coffey lab. Detailed information of all were summarized in Supplementary Materials and Methods, and Table S1.

In vitro cell viability assay after PDT

Cells were seeded in 96-well plates and allowed to grow overnight. Cell medium was removed and replaced with fresh medium with IR700DX-6T or IR700DX. Cell were incubated for 16 hours, washed with fresh medium and then subjected to light irradiation. Cells were irradiated with LED light at a wavelength of 670–710 nm (peak at 690 nm) and an irradiation power indicated in the figures. After the PDT treatment, cells continued to grow for an additional 16 hours before cell viability was measured using cell counting kit-8 (Cat#: CK04–11, Dojindo Molecular Technology, Inc).

Immunoblotting

Procedure can be found in the Supplementary Materials and Methods.

Fluorescent imaging of CRC cells

Colocalization of IR700DX-6T: cells were seeded into Petri dishes and incubated overnight. IR700DX-6T (3 μM) was added for a 3 hours incubation. Two hundred nM Mitotracker Green (Cat#: M7514, Invitrogen)/LysoTracker Yellow (Cat#: L12491, Invitrogen)/ER-Tracker Green (Cat#: E34251, Invitrogen) and 100 nM Hoechst33342 (Cat#: 62249, Thermo Fisher) were added to stain the mitochondria and nuclei, respectively. After the cells were incubated for additional 30 mins, unbound probe was removed by washing with culture medium twice.

Blocking study: MC38 cells were seeded into 8-well chamber slides (Fisher Scientific) and incubated overnight. The cell culture medium was then replaced with serum-free DMEM. Cells were divided into 4 groups, and each group was treated with: (1) 3 μM IR700DX-6T for 4 hours; (2) 30 μM of DAA1106 (blocking agent) for 1 hour followed by 3 μM of IR700DX-6T for 4 hours. (3) 3 μM IR700DX for 4 hours; or (4) no treatment. Cells were then incubated with Mitotracker Green and Hoechst33342 with a concentration of 200 nM and 100 nM, respectively, for 30 mins. Unbound probes were removed by washing with 1X PBS twice.

A detailed fluorescent imaging procedure can be found in the Supplementary Materials and Methods.

Immunofluorescence and Immunohistochemistry (IHC)

Detailed information can be found in the Supplementary Materials and Methods.

In vivo PDT study

Animal studies have been approved by the Vanderbilt’s Institutional Animal Care and Use Committee (IACUC). Female C57BL/6 mice at 6–8 weeks old were purchased from Jackson Laboratory. MC38 cells (5×106) were subcutaneously injected into both sides of the flank of each mouse. Tumor-bearing mice were randomly separated into two groups (4 per group): (1) IR700DX-6T-PDT; and (2) a no treatment control. In vivo PDT was carried out at 7 days after cell injection. Tumor-bearing mice were i.v. injected with 10 nM of IR700DX-6T in 100 μL of 1X PBS or just 100 μL 1X PBS via the tail vein. Light irradiation (18 J/cm2) was applied 2 hours after the photosensitizer injection. The LED light was placed approximately 1.5 cm above the tumor area. The tumor sizes were measured daily by a caliper and the volume was calculated as (tumor length) × (tumor width)2/2.

For the vaccination study, DCs were isolated from the bone marrow of isogenic mice. Seven days later, DCs were stimulated by MC38 cells treated with TSPO-PDT (with 0.5 μM IR700DX-6T and 28 J/cm2 light dose) or 3 freeze/thaw (F/T) cycles at a ratio of 1 DC : 2 MC38 for 24 hours. DCs (5×106) were then intraperitoneally injected into each mouse. MC38 cancer cells (5×106) were inoculated into mice 7 days after vaccination, and tumor growth was monitored daily.

CD8+ T cell depletion

Mice received an i.p. injection of 150 μg of anti-CD8 antibody (Leinco, Product No. C380) 1 day before PDT treatment, followed by periodic depletion of T cells through injection of 50 μg of antibody every 4 days until the end of the experiment.

Flow cytometric analysis

The detailed information can be found in the Supplementary Materials and Methods.

Statistical analysis

All data presented are the mean ± SEM (standard error of the mean) of 4 independent measurements for in vitro PDT studies and 5 independent measurements in animal studies. Student’s t-test and two-way ANOVA were used to analyze in vitro and in vivo results, respectively. P values < 0.05 were considered statistically significant. The analyses were performed using Prism 7 (GraphPad Software, San Diego, CA92108).

Results

TSPO is overexpressed in CRC cells and tumors.

We first examined TSPO expression in 14 CRC cell lines by immunoblot analysis, including 13 human cell lines (see Supplementary Table 1) and MC38, which was derived from an azoxymethane (AOM)-induced adenoma in C57BL6/J mice. Despite the different nature and origin of the cell lines, TSPO expression was confirmed in all of these cell lines, while it is not expressed in normal colon tissue (Fig. 1A) or TSPO (−) MCF7 human breast cancer cells (Fig. 1D). TSPO expression was also examined in tissue from AOM/dextran sulfate sodium (DSS)-induced colon tumor mouse models, as well as tumors generated by subcutaneous transplantation of MC38 cells. TSPO expression in all the colon tumors was higher than case-matched adjacent normal colon (Fig. 1B and 1C). Overall, these CRC cell lines and mouse models of colonic neoplasia exhibited higher TSPO expression than the normal colon, supporting the strategy of employing TSPO-PDT for the treatment of CRC.

Fig. 1.

Fig. 1.

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TSPO expression in CRC cells and tumors. (A) Immunoblot of TSPO in 14 CRC cell lines, including 13 human CRC cells and a murine MC38 cell line. (B-C) Comparison of TSPO expression between normal colon and colonic tumors. B, Immunoblot; C, Immunohistochemistry. (D) Immunoblot of TSPO in MC38 cells compared to MCF7 cells. (CN: colonic neoplasia)

TSPO-PDT induces CRC cell death

We first confirmed the target-specific uptake of IR700DX-6T (structure shown in Fig. 2A). in CRC cells. As shown in Fig. 2B, uptake of IR700DX-6T was seen in TSPO-expressing MC38 cells, but not in TSPO (−) MCF7 cells. Consistent with the subcellular localization of TSPO in previous reports, fluorescence from IR700DX-6T mainly colocalized with mitochondria, but not ER or lysosome (Fig. 2B). When MC38 cells were treated with N-(2,5-Dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl) acetamide (DAA1106), a small molecule TSPO ligand with a high binding affinity [21, 22], prior to IR700DX-6T, the uptake of IR700DX-6T was markedly decreased as a result of competitive binding to TSPO (Fig. 2C). Furthermore, MC38 cells treated with non-targeted IR700DX showed little if any fluorescent signal. Taken together, these results indicated that IR700DX-6T specifically bound to TSPO.

Fig. 2.

Fig. 2.

Fig. 2.

Fig. 2.

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In vitro evaluation of TSPO-PDT with IR700DX-6T. (A) Structure of IR700DX-6T. (B-C) Fluorescent imaging of IR700DX-6T uptake (red). Mitochondria and nuclei were stained with Mitotracker Green (green) and Hoechst33342 (blue), respectively. Endoplasmic reticulum (ER) and lysosome were stained with green and yellow, respectively. Scale bar: 50 μm. (B) Comparison between TSPO (+) MC38 and TSPO (−) MCF7 cells. Co-localization of IR700DX-6T with mitotracker, ER-tracker and lysotracker. (C) Binding specificity of IR700DX-6T to TSPO. MC38 cells were divided into 4 groups and subjected to: (1) 3 μM of IR700DX-6T for 4 hours; (2) 30 μM of DAA1106 (blocking agent) for 1 hour, followed by 3 μM of IR700DX-6T for 4 hours; (3) 3 μM of IR700DX for 4 hours; or (4) a no treatment control. (D) The effects of TSPO-PDT in MC38 cells. MC38 cells were subjected to various concentrations of IR700DX-6T (0.5, 1.0, 2.5 and 5.0 μM) and light doses (3.6, 7.2, 10.8, 16.2, 21.6 and 27.0 J/cm2). (E) Effect of TPSO-PDT compared to non-targeted PDT in MC38 cells. MC38 cells were incubated with IR700DX-6T (1 μM) or IR700DX (1 μM) for 4 hours, followed by light irradiation at various doses (3.6, 7.2, 10.8, 16.2, 21.6 and 27.0 J/cm2). (F-G) Effects of PDT with IR700DX-6T in 14 CRC cell lines. Cells were incubated with IR700DX-6T (1 μM or 5 μM) for 16 hours, followed by light irradiation at 18 J/cm2. Percentage cell death was determined at 16 hours after PDT treatment. (H) Cell toxicity assay in 3 CRC cell lines. Cells were incubated with IR700DX-6T (from 0.625 μM to 10 μM) for 16 hours. Cell toxicity was determined by MTT assay. (I) Effects of PDT with IR700DX-6T on TSPO (−) MCF7 cells. Cells were incubated with IR700DX-6T (from 1 μM to 5 μM) for 16 hours, followed by light irradiation at 18 J/cm2. Percentage cell death was determined at 16 hours after the PDT treatment.

We next tested the effect of PDT with IR700DX-6T on MC38 cells. The therapeutic outcome presented a concentration- and light-dose dependent manner with 80% cell death when cells were subjected to 27.0 J/cm2 irradiation and over 2.5 μM IR700DX-6T (Fig. 2D). PDT using IR700DX (a free dye) showed significantly lower level of MC38 cell death than that using the same concentration of IR700DX-6T at all light doses (p < 0.01, 0.001, 0.001 and 0.0001 at 27, 10.8, 16.2, and 21.6 J/cm2, respectively), except for the lowest doses (3.6 and 7.2 J/cm2) where no obvious cell death was observed (Fig. 2E), suggesting a target-specific PDT effect of IR700DX-6T.

We then expanded the study to 13 human CRC cell lines. All of the lines displayed a concentration-dependent susceptibility to TSPO-PDT (Fig. 2F–2G). This result suggests that PDT coupled with IR700DX-6T is uniformly effective in TSPO (+) CRC cell lines. Furthermore, the cell toxicity was determined using 3 selected CRC cell lines. IR700DX-6T showed insignificant toxicity to these cell lines when light irradiation was not applied (Fig. 2H). To further verify the target specificity of TSPO-PDT, a TSPO (−) cell line, MCF7, was selected to conduct the PDT experiment. Our data showed only mild cell death after TSPO-PDT treatment, even at high concentrations of IR700DX-6T (Fig. 2I).

TSPO-PDT causes ICD in CRC cells

Apoptosis is a common type of cell death associated with ICD (previously named as immunogenic apoptosis) [15]. Since TSPO-PDT is expected to introduce excessive ROS to the mitochondria and subsequently cause mitochondrial damage and apoptosis, we set out to monitor morphological changes in the mitochondria associated with apoptosis after the TSPO-PDT treatment using live cell imaging with a fluorescent microscope (Fig. 3A and Supplementary videos). Before the PDT treatment, the mitochondria of MC38 cells showed threadlike or dotted structures, which turned into swollen circle-like structures (indicated by red arrows) after IR700DX-6T-PDT treatment (Fig. 3A). The morphological change of mitochondria was accompanied by evidence of apoptosis. A mass of membrane blebbing was discovered at 40 minutes after PDT treatment (Fig. 3A). Cell shrinkage (Fig. 3A) also started to appear at 40 minutes after TSPO-PDT and became more obvious at 2 hours. At later time points (3 and 5 hours), most of the MC38 cells showed distorted morphologies consistent with apoptosis (Fig. 3A). The generation of ROS was detected after the PDT treatment as well. As showing in Fig. 3E, remarkable ROS was found in the TSPO-PDT-treated group as compared with the untreated control.

Fig. 3.

Fig. 3.

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Apoptosis and ICD of MC38 cells induced by TSPO-PDT. (A) MC38 cells were incubated with 3 μM IR700DX-6T for 2 hours, followed by light irradiation at a power density of 30 J/cm2. Mitochondria was labeled by Mitotracker Green. Morphological change to cells and mitochondrial damage over time was monitored and recorded using fluorescent microscopy. Blue, yellow and green arrows indicate cell blebbing, shrinkage, and distorsion, respectively. Pink arrows show the normal structure of the mitochondria, and red arrows indicate damaged mitochondria after PDT treatment. Scale bars: 10 μm. (B-D) MC38 cells were treated with PDT using 0.5 μM of IR700DX-6T and 18 J/cm2 of light irradiation at 690 nm. At 3 hours (or indicated time points) after PDT treatment, cells were subjected to immunofluorescent imaging (red) of cleaved caspase-3 (B), calreticulin (C) and heat shock protein 70 (D). Nuclei were stained with DAPI (blue). Scale bars: 50 μm. (E) Change of ROS in MC38 cells compared to the control group after TSPO-PDT treatment. The data were shown as mean ± SD (n = 5); ***: P < 0.001. (F) Upregulation of ATF4 and Bip proteins after TSPO-PDT treatment. (G) TSPO-PDT-treated MC38 cells stimulated the maturation of dendritic cells in vitro. Bone marrow-derived DCs (BMDCs) were co-cultured with TSPO-PDT-treated MC38 cells, freeze/thaw-treated MC38 cells, or LPS (1 mg/L, positive control). CD11c was used to gate DCs, and cell surface markers including CD86, CD80 and MHC II were plotted as a histogram for comparison among the groups.

Caspase-3 staining further verified apoptotic cell death of TSPO-PDT-treated MC38 cells, and cleaved caspase-3 appeared at 2 hours after TSPO-PDT. At 3 hours post TSPO-PDT, a large number of apoptotic cells were visualized (Fig. 3B), and a few cells showed relatively low fluorescent signal, presumably indicating onset of the apoptotic process (Fig. 3B). Strong caspase-3 staining was also observed at later time points (5 and 9 hours after IR700DX-6T-PDT), providing additional evidence of increased apoptosis in cells by TSPO-PDT (Fig. 3B).

We next studied the association of the apoptosis of MC38 cells with ICD. ICD is triggered by releasing DAMPs from damaged cells [15]. Therefore, we evaluated two key DAMP molecules, calreticulin (CRT) and heat shock protein 70 (HSP70), using immunofluorescence. As shown in Fig. 3C and 3D, at 3 hours after PDT treatment, high levels of CRT and HSP70 were released compared to no treatment controls [23]. Thus, TSPO-PDT resulted in the characteristic features of ICD. Due to the close association of apoptosis with ER stress [24], we also investigated the effect of TSPO-PDT on ER stress. We observed upregulation of two ER stress markers, activating transcription factor 4 (ATF4) and binding immunoglobulin protein (Bip), at 0.5 and 1.5 hours after the PDT treatment, respectively (Fig. 3F), indicating that TSPO-PDT induced ER stress in CRC cells.

One notable characteristic of ICD is induction of dendritic cell (DC) maturation. We therefore carried out DC stimulation experiments using TSPO-PDT-treated MC38 cells. As shown in Fig. 3G, TSPO-PDT-treated MC38 cell lysates accelerated the maturation of DCs, while the freeze/thaw (F/T)-treated MC38 cell lysate showed a much reduced effect. This result provides additional confirmation that ICD in CRC cells is caused by TSPO-PDT.

TSPO-PDT inhibits tumor growth in animal models

In our previous study, we have shown target-specific therapeutic effect of IR700DX-6T-PDT in a breast cancer mouse model and we evaluated the direct PDT effect [13]. In this study, we mainly focus on investigating the abscopal effect induced by IR700DX-6T-PDT using a syngeneic immunocompetent CRC mouse model. We developed the CRC tumor mouse model using immunocompetent C57BL/6J mice in which MC38 cells were subcutaneously injected into both flanks of syngeneic mice. TSPO-PDT was applied only to the tumor on the left side (primary tumor) of each mouse. The tumors on the right side were considered remote tumors, enabling examination of a possible abscopal effect. Tumor and major organs were collected after IR700DX-6T injection to evaluate the biodistribution of the compound. As expected, IR700DX-6T showed high uptake in the tumor. The only organ that showed comparably high uptake is kidney, suggesting high tumor-targeting effect of IR700DX-6T (Fig. 4F).

Fig. 4.

Fig. 4.

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In vivo PDT effect of IR700DX-6T. (A) Experimental scheme. MC38 cells were subcutaneously injected into both flanks of each mouse. The tumor-bearing mice were divided into two groups, including (1) untreated; and (2) TSPO-PDT. PDT was applied to tumors on the left side of each mouse in the PDT group. (B) Primary tumor growth curves. (C) Gross appearance of primary tumors harvested on day 14. (D) Remote tumor growth curves. (E) Gross appearance of remote tumors harvested on day 14. Scale bars: 10 mm. (F) Biodistribution of IR700DX-6T. Quantified data from Ex vivo fluorescence imaging of organs removed from MC-38 tumor-bearing mice i.v. injected with IR700DX-6T at 2, 12, 24 and 48 hours post-injection.

As shown in Fig. 4, TSPO-PDT greatly inhibited the growth of both primary and remote tumors. At the end of the observation period (14 days), the primary tumors subjected to TSPO-PDT were significantly smaller than the non-treated control (164 ± 19 vs 999 ± 75 mm3, p < 0.001) (Fig. 4B and 4C). Remarkably, the growth of the remote tumors in the TSPO-PDT-treated mice was also significantly suppressed (p < 0.0001) in the treatment group (319 ± 39 mm3) versus the no treatment group (1007 ± 119 mm3) (Fig. 4D and 4E). Taken together, TSPO-PDT not only exhibited a direct inhibition effect on the CRC tumor growth, but also induced an abscopal effect.

We also closely monitored potential side effects in animals during the TSPO-PDT treatment. At 24 to 72 hours after PDT, mild swelling in and around the tumor area was noted but this disappeared promptly without any intervention. We observed a similar effect in our previous studies [13], likely due to an acute inflammatory response caused by PDT treatment. At the time of sacrifice, major organs and tissues surrounding the tumor were collected and histologically evaluated. No inflammation or morphological damage in animals subjected to PDT treatment was observed. In addition, tissues toxicity assay was conducted to further evaluate the safety of IR700DX-6T to animals. After a high dose (30 nM) of the PS was systemically delivered into C57BL/6J animals through tail vein injection, major organs were collected at 2 hours, 48 hours and 7 days post injection. Histological results showed no obvious morphological change in any organs at each time point (Supplementary Fig. S4), indicating low toxicity of this PS to the animals.

TSPO expression in harvested tumor samples was characterized by immunofluorescence (Supplementary Fig. S1). As expected, a strong fluorescent signal was observed in most cells in the tumor tissues, suggesting high TSPO expression in these tumors.

TSPO-PDT provokes host anti-tumor immune response

We further investigated the abscopal effect by studying the mechanism behind the anti-tumor immune response triggered by TSPO-PDT. We first studied DCs and Tregs isolated from the spleens of tumor-bearing mice. The percentage of mature DCs (CD11c+/CD80+, CD11c+/CD86+ or CD11c+/MHCII+) significantly increased in the TSPO-PDT group in comparison to the untreated group at all time points (Fig. 5B). Specifically, a significant increase of MHC II+ DCs was seen at 1 day after TSPO-PDT treatment (14% ± 2% in the PDT group vs. 10% ± 2% in the control group, p < 0.05). The increase persisted throughout the experimental period, although the difference was not statistically significant at the last time point (day 14, p = 0.054) (Fig. 5B–5D). The percentage of DCs expressing costimulatory molecules, CD80 and CD86, also increased in the TSPO-PDT group compared to the untreated group (18% to 80% increase). Statistical significance was discovered at 1 day (p < 0.05) after TSPO-PDT for CD80, and at 5 and 14 days after TSPO-PDT for CD86 (p < 0.05 and p < 0.01, respectively) (Fig. 5B–5D). These data suggest maturation of DCs, likely by antigen phagocytosis in TSPO-PDT-treated tumors. In addition, at 14 days after the TSPO-PDT treatment, the Treg population was 35% lower in the treatment than the control group, indicating more immune-active tumor microenvironment (Fig. 5E). We next examined tumor-infiltrating T lymphocytes (TILs). As shown in Fig. 5F–H, the numbers of TILs increased after TSPO-PDT treatment in primary and remote tumors. At 1 day after TSPO-PDT, the TILs in the primary and remote tumors were 2.2 (p < 0.05) and 2.7 (p < 0.01)-fold more than those in the untreated control, respectively (Fig. 5G). At 14 days, CD3+/CD8+ TILs still showed higher numbers in the PDT-treated primary (2.7-fold, p < 0.01) and remote (2.5-fold, p < 0.001) tumors than the control group (Fig. 5H). Taken together, these data indicate that TSPO-PDT remarkably provoked a host anti-tumor response through DC maturation and TIL production, while suppressing Treg cells.

Fig. 5.

Fig. 5.

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Anti-tumor immunity induced by TSPO-PDT. (A) Experimental scheme. MC38 cells were injected into both flanks of isogenic C57BL/6J mice. After 7 days, TSPO-PDT was applied to the tumors on the left side of each mouse in the PDT group. Spleens and tumors were harvested and studied for immune cell populations. (B-D) Percentages of CD80+, CD86, and MHC II+ cells in CD11c gated cells were compared at 1, 5 and 14 days after the PDT treatment (B-D). (E) Percentages of splenic CD25+ FOXP3+ cells were compared at 14 days after PDT treatment. (F) Representative immunofluorescent images showing tumor-infiltrated CD8+ T cells in the tumor tissue at 1 day and 14 days after TSPO-PDT. CD3 (green), CD8 (red) and DAPI (blue) are shown. Scale bars: 50 μm. (G) and (H) Numbers of CD3+ CD8+ cells in the tumors at 1 day (G) or 14 days (H) after treatment in the untreated control (UT), primary tumor (PT) and remote tumor (RT), calculated from Fig. F.

TSPO-PDT-DC vaccine suppresses tumor progression

To determine the potential of prophylactic vaccines against tumor progression, MC38 cells were treated with TSPO-PDT and the cell lysates were used as tumor antigens to stimulate DCs (TSPO-PDT-DC vaccine). The results were compared with a DC vaccine developed from F/T-treated MC-38 cells (F/T-DC vaccine). After i.p. injection of the vaccine, tumor growth inhibition was observed in both vaccinated groups, with a more pronounced effect in the TSPO-PDT DC vaccine group (Fig. 6B).

Fig. 6.

Fig. 6.

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Inhibition of tumor progression by TSPO-PDT-DC vaccine. (A) Experimental scheme. DC vaccines were injected intraperitoneally into each mouse 1 week prior to the inoculation of MC38 tumors. Single cells were prepared from the spleen and tumor-draining lymph nodes at 5 and 14 days, respectively. (B) Tumor growth curves in three different groups, including TSPO-PDT-DC vaccine, F/T-DC vaccine and no treatment groups. (C), (D), (H) and (I), The number of DCs was analyzed by counting CD11c+ cells (C: 5 days; H: 14 days). DC maturation levels were evaluated by analyzing the percentage of CD80+/CD86+ double-positive cells in CD11c+ populations (D: 5 days; I: 14 days). (E), (F), (J) and (K), The CD8+ T cell population was determined by flow cytometry. Representative plots are shown in E (5 days) and J (14 days); and the percentages of CD8+ T cell within the CD3+ T cell population are shown in F (5 days) and K (14 days). (G) and (L), Regulatory T cells was determined by the percentage of Foxp3+ cells in the CD3+/CD4+ cell population. (PDT-Vaccine: DC vaccine stimulated by TSPO-PDT-treated MC38 cell lysates; F/T-Vaccine: DC vaccine stimulated by freeze/thaw-treated MC38 cell lysates. * P<0.05, ** P<0.01, *** P<0.001).

DCs, CD8+ T cells and Tregs in the spleen and lymph nodes were analyzed to determine the immunological status of the vaccinated animals. In the spleen and lymph nodes, both vaccinated groups showed higher number of DCs (CD11c+) than the untreated group, and the TSPO-PDT-DC vaccinated group showed a significantly higher mature DC level (% of CD86+/CD80+ in CD11c+ cells) than the F/T-DC vaccine and untreated group at 5 days after vaccination (Fig. 6C, 6D, S2A, and S2B). In addition, in both the spleen and lymph nodes, The percentage of CD8+ T cells within the gated CD3+ T cell population was significantly higher in the TSPO-PDT-DC vaccinated groups than the F/T-DC and untreated group (Fig. 6E, F), which was accompanied by a significant decrease of the Treg cell percentage (% of Foxp3+ in CD3+/CD4+ cells) in the TSPO-PDT-DC vaccinated group compared to the F/T DC and untreated group at 5 days after vaccination (Fig. 6G and Supplementary Fig. S2C). At 14 days after vaccination, DC number and maturation levels, as well as the Treg cell percentage, returned to similar levels to the untreated group (Fig. 6H, I, L and Supplementary Fig. S2D, E, F), whereas the level of cytotoxic CD8+ T cells in the lymph nodes of the TSPO-PDT-DC vaccinated group remained significantly higher than the other groups (Fig. 6K). The combined evidences indicate that the TSPO-PDT-DC vaccine induced highly active anti-tumor immunity.

Cytotoxic T lymphocytes (CTL) play a pivotal role in TSPO-PDT-induced anti-tumor immune response

The role of CTLs in TSPO-PDT-induced anti-tumor immunity was further confirmed by inhibition of CD8+ T cells. CD8 antibody was injected to MC38 tumor-bearing mice subjected to TSPO-PDT treatment (anti-CD8 TSPO-PDT group). For the primary tumors, although T-cell inhibition reduced the therapeutic effect of TSPO-PDT, the anti-CD8 TSPO-PDT group still showed a considerable tumor suppressive effect, presumably due to ROS produced from TSPO-PDT (Fig. 7B). Remarkably, the remote tumors in the anti-CD8 TSPO-PDT group grew even faster than those in the untreated group, with a 37.6% larger volume that reached statistical significance (p < 0.05) (Fig. 7C), suggesting that CD8+ T cells play pivotal roles in a TSPO-PDT-induced anti-tumor immune response.

Fig. 7.

Fig. 7.

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The pivotal role of CD8+ T cell in the TSPO-PDT-activated anti-tumor immunity. (A) Experimental scheme. MC38 cells were injected into both flanks of isogenic C57BL/6J mice. Animals were divided into three groups and subjected to: (1) no treatment; (2) TSPO-PDT; or (3) anti-CD8 antibody 1 day before TSPO-PDT. Light irradiation was applied to tumors on the left side of each mouse in the TSPO-PDT and anti-CD8 TSPO-PDT groups. Single cells were prepared from the spleens and lymph nodes to perform flow cytometric experiments. (B) and (C), Tumor growth curve among different groups. (D), (E), (I) and (J), DC percentages based on total cell counts were analyzed by counting CD11c+ cells (D: 5 days; I: 14 days). DC maturation levels were evaluated by analyzing the percentage of CD80+/CD86+ double-positive cells in the CD11c+ population (E: 5 days; J: 14 days). (F), (G), (K) and (L), The CD8+ T cell population was determined by using flow cytometry. Representative plots are shown in F (5 days) and K (14 days); and the percentages of CD8+ T cells in the CD3+ T cell population are shown in G (5 days) and L (14 days). (H) and (M), Regulatory T cells were determined by the percentage of Foxp3+ cells in the CD3+/CD4+ cell population. (* P<0.05, ** P<0.01, *** P<0.001).

Similar to the vaccination study, we compared DCs, CD8+ T cells and Tregs among the three groups (untreated, TSPO-PDT and anti-CD8 TSPO-PDT) at early (5 days) and late (14 days) stages. At the early stage, DCs were activated in both TSPO-PDT groups regardless of the presence of the CD8+ T cells, with increased CD11c+ cell percentages observed in both the spleen and lymph nodes (Fig. 7D, E and Supplementary Fig. S3A, B). Both TSPO-PDT and anti-CD8 TSPO-PDT groups showed upregulation of CD80 and CD86 expression as well. Interestingly, the DC maturation level in the anti-CD8 TSPO-PDT group is even higher than the TSPO-PDT group (Fig. 7E, Supplementary Fig. S3B). All these data indicate that the innate immune system was rapidly activated after TSPO-PDT treatment. The increase in CD8+ T cell levels and decrease in Treg levels in the TSPO-PDT group compared to the untreated group were reversed by the anti-CD8 treatment (Fig. 7F–H), which apparently modified the anti-tumor microenvironment to an immunosuppressive one. Similar results were collected at the late stage (Fig. 7I–M), suggesting that TSPO-PDT treatment provoked an efficient and long-lasting anti-tumor immune response, in which the CD8+ T cells played a pivotal role.

Discussion

Mitochondria have been studied as a therapeutic target against tumors for years, not only because they are an indispensable organelle that supplies energy to cancer cells and regulates apoptosis, but also because they participate in tumorigenesis and tumor growth [25]. For example, a recent study using a mitochondria-targeted PS achieved success in killing CRC cells [19]. However, most mitochondria-targeted PSs are not specific to cancer cells. Since all cells contain mitochondria, it is necessary to improve tumor specificity to avoid damage to normal cells. Therefore, tumor-associated TSPO was utilized as the binding target for our PS in this study. Our TSPO-targeted PS, IR700DX-6T, showed high tumor specificity as verified by the in vitro uptake and blocking experiments (Fig. 1), as well as our previous in vivo studies using a TSPO (+) breast cancer model [13]. IR700DX-6T also showed no significant dark toxicity to the major organs (Fig.S4). Further, PDT using IR700DX-6T displayed a remarkable killing effect, whereas treatment using the non-targeted IR700DX showed a negligible effect against MC38 cells. These results confirm that IR700DX-6T specifically binds to tumoral TSPO, highlighting an advantage over previously reported mitochondria-binding PSs that lack tumor specificity.

In this study, the mitochondrion was selected as our target mainly due to the association of apoptosis with ICD. Different types of cell death have distinct functional perspectives as they relate to ICD. Necrosis has long been acknowledged to play a key role in inflammation and immune-related processes [26]. However, it was recently reported that accidental necrosis was not able to induce an effective anti-tumor immunity [27]. Increasingly, reports have shown that apoptosis has great potential in inducing ICD by releasing DAMPs, which subsequently activate DCs and provoke an antitumor immune response [28]. In a study focused on developing a prophylactic tumor vaccination, researchers found that apoptosis was more immunogenic than necrosis, and it triggered a strong CD8+ T cell response and inhibited tumor growth in a CT26 mouse colon cancer model [29]. Immunogenic apoptosis appears to be closely associated with ER stress, as persistent ER stress initiates apoptotic signaling pathways [30]. ER stress is a strong ICD inducer, which releases CRT from its membrane to translocate to cell surface. CRT then acts as an “eat me” signal to facilitate phagocytosis of dying cancer cells by DCs.

In our study, we noticed that CRC cells after TSPO-PDT showed morphological changes that are characteristic of apoptotic cell death, which was further supported by an increase of cleaved caspase-3. In addition, CRT were found to be translocated from the cytoplasm to the cell surface, accompanied by release of another key DAMP molecule, HSP70. Moreover, TSPO-PDT caused ER stress, as evidenced by upregulation ATF4 and Bip. Mitochondrial dysfunction and immunogenic apoptosis are reported to be directly linked to ER stress [31] [30]. It is likely that our TSPO-PDT treatment produced a large amount of ROS in the tumor mitochondria, leading to mitochondrial dysfunction and ER stress. The ER stress then mediated translocation of CRT, which is considered an ER resident protein [32]. Moreover, a recent report has shown that targeting mitochondria could induce a significant amount of ICD in cancer cells and solid tumors [19]. As such, we think mitochondria-targeted PDT may represent a promising strategy to activate ER stress and subsequent ICD.

The potential of PDT in inducing ICD and subsequently an anti-tumor immune response has been demonstrated in a few recent reports. Victoria and colleagues [14] reported that PDT using photosens and photodithazine effectively induced ICD. Photodithazine is amphiphilic and mainly distributes in the membranes of both the ER and Golgi apparatus, whereas photosens is a hydrophilic PS that enters cells by active endocytosis and mainly localizes to lysosomes. Hypericin-PDT has been reported to induce surface exposure of CRT and HSP70 [33], which is similar with our results. Hypericin is a PS that primarily localizes to mitochondria and lysosomes [34]. Unfortunately, these PSs all lack of tumor selectivity, which could cause significant side effects by damaging normal tissue surrounding the tumor. In contrast, our TSPO-PDT agent has a distinct advantage in inducing ICD with tumor specificity. In this study, no obvious side effects on normal tissues surrounding tumors were observed.

Utilizing an isogenic allograft model for CRC, we addressed the mechanism of TSPO-PDT on tumors in an immunocompetent host. The administration of a TSPO-PDT-DC vaccine showed a significant therapeutic effect, highlighting that TSPO-PDT-induced ICD has great potential in provoking a host antitumor immune response. We found greater numbers of mature DCs and CD8+ T cells in the TSPO-PDT group than the untreated group at an early stage after TSPO-PDT treatment, and the cell levels remained high till the end of the study (Fig. 6, 7). Moreover, CD8+ T cells were identified as the pivotal effectors in our TSPO-PDT approach, as evidenced by the reversed therapeutic effect in the remote tumors after administration of anti-CD8 antibody. These results suggest that DCs and CD8+ T cells were activated soon after TSPO-PDT, and the cytotoxic immune cells trafficked to the remote tumors, leading to a clear abscopal effect.

Conclusions

Taken together, we show that TSPO-PDT treatment reduces the growth of multiple TSPO-expressing CRC cell lines by inducing ICD. Notably, a direct and abscopal effect was observed in mouse tumor-derived MC38 cells when injected into syngeneic immunocompetent mice. If comparable effects could be achieved in humans, it would establish a novel paradigm for treating micro- and macro-metastasis. We also show the efficacy of a MC38-derived DC vaccine and that the immunogenic effects are mediated, at least in part, via CD8 T cells.

Supplementary Material

1

Statement of significance.

Abscopal effect is an attractive cancer therapeutic effect referring to tumor regression at a location distant from the primary treatment site. Immunogenic cell death (ICD) offers a mechanistic link between the primary and remote therapeutic effects by activating favorable anti-tumor immune responses. In this study, we report a new therapeutic approach that can reduce the growth of multiple CRC cell lines by inducing ICD. Notably, a direct and abscopal effect was observed in mouse tumor-derived MC38 cells when injected into syngeneic immunocompetent mice. If comparable effects could be achieved in humans, it would establish a novel paradigm for treating micro- and macro-metastasis.

Acknowledgments

We thank Sarah E. Glass for editorial assistance.

Funding:

RJC was supported by National Cancer Institute R35 CA197570 and the Vanderbilt-Ingram Cancer Center GI Special Programs of Research Excellence (VICC GI SPORE) P50CA236733. MB was supported by an American Cancer Society - CEOs Against Cancer - Pennsylvania Chapter Research Scholar Grant, (RSG-17-131-01-CDD), the VICC GI SPORE P50CA236733 and startup funding provided by Vanderbilt University Medical Center.

Footnotes

Declaration of interests

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.

Data availability

The raw data required to reproduce these findings are available to download from https://vumc.box.com/s/93u4tnxnfkhemk5z9lqslzelrgtqtwtr. The processed data required to reproduce these findings are available to download from https://vumc.box.com/s/93u4tnxnfkhemk5z9lqslzelrgtqtwtr.

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