Treatment of Conjunctival Melanoma Cell Lines With a Light-Activated Virus-Like Drug Conjugate Induces Immunogenic Cell Death

Abstract

Purpose: Conjunctival melanoma (CJM) is a rare malignant ocular surface tumor, which often leads to local recurrences and metastases. In murine models of subcutaneous tumors, treatment with a novel virus-like drug conjugate (VDC; Bel-sar) showed a dual mechanism of action with direct tumor cell killing as well as stimulation of an antitumoral immune response. Bel-sar is currently being evaluated for the treatment of primary uveal melanoma and indeterminate nevi in a phase III clinical trial. We determined whether Bel-sar also has direct antitumor efficiency and a potential immunostimulatory capacity in CJM cells.

Methods: Three human tumor-derived CJM lines were used. Bel-sar's subcellular and intracellular locations were determined with tracers. Following light activation of Bel-sar, cytotoxicity and exposure of damage-associated molecular patterns (DAMPs) were assessed. Treated tumor cells were co-cultured with THP-1 derived macrophages to assess tumor-cell phagocytosis.

Results: Bel-sar was bound and internalized by CJM cells and subsequently found in the cell membrane, lysosome, Golgi apparatus, and mitochondria. Bel-sar activation induced near complete cell death with half-maximal inhibitory concentration (IC₅₀) values between 30 pM and 60 pM. Finally, light-activated Bel-sar enhanced exposure of DAMPs, including calreticulin, heat shock protein 90, and stimulated phagocytosis by macrophages.

Conclusions: Treatment with a novel VDC (Bel-sar) induced pro-immunogenic cell death in all three CJM cell lines. The in vitro cytotoxicity was accompanied by exposure of DAMPs, suggesting Bel-sar is a potential treatment for CJM by a dual mechanism of action. This dual mechanism may provide a targeted and direct killing of tumor cells and induce an immune response which might decrease local recurrences and metastasis.

Conjunctival melanoma (CJM) is a rare and malignant ocular tumor, with an incidence of 0.15 to 0.49/million in adults and accounts for about 2% of ocular melanoma cases.^1,2 CJM arises from atypical melanocytes in the basal layer of the conjunctival epithelium or in precursor lesions, such as pigmented nevi. CJM is characterized by a high mutational burden and mainly harbors a BRAF or NRAS mutation, genetically resembling cutaneous melanoma.^3,4 The primary treatment of CJM is local excision combined with cryotherapy to the margins. Various adjuvant therapies, such as topical chemotherapy and radiotherapy, may follow surgical excision to reduce local recurrence rates.^2,5 There is currently no consensus on the preferred adjuvant therapy.⁶ In fact, first-line treatment followed by adjuvant therapy still shows local recurrence in 19% (median = 3.6 years) of cases (seen in a multicenter international data-sharing study).⁷ Up to 26% of patients may develop metastasis at 10 years and there are currently no approved drugs for metastasis.⁸ However, given that CJM is genetically similar to cutaneous melanoma and mucosal melanomas, immune therapy, using, for example, programmed cell death-1 (PD-1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA4) inhibitors, may be considered for patients with metastatic CJM. A limited number of cases with this approach has been reported.^9–12 

A stronger local and systemic immune response might reduce the recurrence and/or metastasis rate of CJM. This may be facilitated by photodynamic therapy (PDT). Photodynamic therapy is a clinically applied, minimally invasive tumor ablation method that involves the administration of a photosensitizer and subsequent activation by the appropriate laser wavelength. In the presence of oxygen, a photochemical reaction is initiated which then produces reactive oxygen species (ROS).¹³ The ROS can directly kill tumor cells or lead to vasculature damage, whereas it can also induce immunogenic cell death, characterized by an enhanced exposure or release of damage associated molecular patterns (DAMPs). These DAMPs, such as calreticulin (CRT) and heat-shock protein 90 (HSP90), have been shown to induce maturation of antigen-presenting cells (APCs) and stimulate an immune response.^14,15 Murine experiments have shown that PDT can serve as an adjuvant to induce a local as well as systemic immune response to reduce recurrence or metastasis in MC38 and CT26 subcutaneous tumors.^16,17 

A novel virus-like particle drug conjugate (Bel-sar, previously named AU-011), is being used to treat small choroidal melanoma or indeterminate choroidal pigmented lesions (phase III trial, NCT06007690). The virus-like drug conjugate (VDC) is a combination of a virus-like particle (VLP) and the photosensitizer phthalocyanine. This VLP is derived from human papillomavirus and helps to deliver the photosensitizer to the tumor cells through multivalent binding to tumor-associated cell surface heparan sulfate proteoglycans.¹⁸ Irradiation with an infrared laser activates the conjugated phthalocyanine and a photochemical reaction is initiated. Previous work showed that Bel-sar treatment is effective in a rabbit orthotopic uveal melanoma model and induces immunogenic cell death in uveal melanoma cell lines.^19,20 We subsequently showed in mouse models that combining Bel-sar with immune checkpoint inhibitors, such as PD-L1, CTL-4, and LAG3, led to durable complete responses with an abscopal effect.¹⁷ These data suggest that Bel-sar may be used as an adjuvant to induce a stronger immune response and potentially reduce the recurrence or metastasis rate of CJM. 

As the photosensitizer plays a critical role in the effectiveness of PDT, its subcellular location in different cellular components may induce various pathways of cell death or exposure of DAMPs.²¹ The main effector, ROS, is highly reactive, with a lifetime in the order of 40 ns and a maximum action radius of about 20 nm.²² This short action radius together with the short duration means that the localization of the photosensitizer influences the site of action of PDT at the subcellular level. Targeting the lysosome, Golgi apparatus, and mitochondria could enhance the tumor's immunogenicity by inducing pyroptosis and preventing autophagy.^23–25 We therefore investigated the intracellular and subcellular location of Bel-sar in tumor cells at different time points and determined whether Bel-sar induced immunogenic cell death in three different human CJM cell lines. Our data show that after binding to the cell membrane, Bel-sar is found in several organelles, and light activation consistently leads to immunogenic cell death and increased phagocytosis across all cell lines. 

Materials and Methods

Cell Lines and Cell Culture

Conjunctival melanoma cell lines CRMM1 (primary, BRAF mutation), CRMM2 (primary, NRAS mutation), and CM2005.1 (recurrence, BRAF mutation) were cultured under 5% CO₂ at 37°C in F-12K and RPMI1640 medium (Life Technologies), supplemented with 10% or 20% heat-inactivated fetal calf serum (Greiner Bio-one, Alphen aan den Rijn).^26,27 

Binding and Uptake of Bel-sar

Cells were seeded in 96-well plates (Greiner) at 10,000 cells per well and allowed to attach for 24 hours. Bel-sar targets tumor cells by binding to HSPGs on the cell membrane and is then internalized into the cytoplasm. To investigate Bel-sar's binding ability to tumor cells, we performed the tests at 4°C, as this inhibits its uptake, allowing us to assess its binding efficiency to the tumor cell lines. They were then incubated with different concentrations (range from 3 pM to 300 pM) of Bel-sar during indicated time periods at 4°C (binding) or 37°C (uptake). After the indicated time period, the cells were washed with phosphate-buffered saline (PBS) and fixed with 4% formalin (J.T. Baker, Landsmeer, The Netherlands) for 30 minutes. The cells were washed 3 times and reconstituted in 100 uL flow cytometry buffer (PBS with 0.5% bovine serum albumin [BSA] and 0.02% sodium azide). The fluorescence of Bel-sar was then measured using an Aurora cytometer (Aurora, Cytek Biosciences). Bel-sar is provided by Aura Bioscience Inc. (Cambridge, MA, USA) and the production process of Bel-sar has been described in a previous publication.¹⁹ 

Fluorescence Microscopy

CRMM2 cells were seeded in 24-well glass plates (Greiner) at 25,000 cells per well and allowed to attach for 24 hours. For the subcellular location, the cells were first incubated with NucBlue live probes (Thermo Fisher) for 30 minutes at 37°C and then with PBS. After that, the cells were incubated with 3000 pM Bel-sar for 4 hours at 4°C or 37°C. The cells were then washed 3 times with PBS and incubated with a cell surface marker (CD44-FITC; Thermo Fisher) at 4°C for 30 minutes. After washing with PBS, the cells were imaged under an SP8-WLL fluorescence microscope (Leica). 

For the intracellular location, after incubation with NucBlue and washing, the cells were incubated with 3000 pM Bel-sar for 4, 8, and 24 hours at 37°C. Then, the cells were washed 3 times with PBS and incubated with 0.5 uM LysoTracker Green DND-26 for lysosomes, 5 uM BODIPY FL C5-Ceramide complexed to BSA for Golgi apparatus, 0.5 uM MitoTracker Green FM for mitochondria, 0.5 uM ER-Tracker Green for the endoplasmic reticulum (Thermo Fisher). Immediately, the cells were imaged under an SP8-WLL fluorescence microscope. 

Cell Viability and Cytotoxicity

To detect the effect of Bel-sar on the viability of CJM cell lines with and without laser excitation, CJM cells were seeded onto the 96-well black plates (Thermo Fisher) and allowed to attach overnight. The next day, the tumor cells were incubated with Bel-sar (0.01–1000 pM) for 4 hours, washed 3 times in PBS, measured, and supplied with fresh medium. Immediately after, the cells were irradiated at a total light dose (fluence) of 25 J/cm2 and a light intensity (fluence rate) of 600 mW/cm2 using a 690 nm LED diode laser (CNI Laser, Changchun, China). Next, 20 µL MTS reagent (Cell Titer 96 Aqueous One, #G3581; Promega) was added to each well 24 hours post-irradiation and incubated for 3 hours at 37°C. The optical density (OD) value of each well was measured at 490 nm using a spectrum analyzer (Biolegend, Bio-Rad, iMark set). Cell viability is expressed in percentage (%) and calculated using the following formula: (OD 490 nm treated cells – OD 490 nm background) / (OD 490 nm untreated cells – OD 490 nm background) × 100. For cytotoxicity, 50,000 CJM cells were seeded in 24-well plates (Corning) in medium and allowed to attach overnight at 37°C and 5% CO₂ in an incubator. The cells were incubated with Bel-sar (3–300 pM). Then, the cells were washed 3 times with PBS and supplied with fresh medium. Immediately after, the cells were irradiated at a total light dose (fluence) of 25 J/cm2 and a light intensity (fluence rate) of 600 mW/cm2 using a 690 nm LED diode laser (CNI Laser, Changchun, China), unless indicated otherwise. At 24 hours after treatment, the tumor cells were stained with Annexin V-FITC (Thermo Fisher, Eugene, OR, USA) at 2.5 µL per sample and 0.25 mg/mL DAPI (Sigma) in Annexin V-binding buffer (Thermo Fisher, Eugene, OR, USA), followed by analysis using an Aurora cytometer (Aurora, Cytek Biosciences). 

Measurement of CRT and HSP90 at the Cell Surface

CJM cells were treated by Bel-sar PDT (after incubation with 30–300 pM Bel-sar, the cells were irradiated with a total light dose of 25 J/cm2 and a light intensity of 600 mW/cm2 using a 690 nm LED diode laser) or three cycles of freeze-thawing (F/T; freeze cells at –20°C for 1 hour and then thawed at 37°C for 1 hour, 3 cycles). After 24 hours of incubation, the cells were collected and washed with flow-cytometry buffer, and then resuspended in flow-cytometry buffer with Recombinant PE Anti-Calreticulin antibody (Abcam, Cambridge, UK), Recombinant Alexa 488-HSP90 antibody (Abcam, Cambridge, UK), and DAPI (Sigma-Aldrich, Zwijndrecht, The Netherlands) for 30 minutes at 4°C. Finally, the samples were analyzed by flow cytometry on an Aurora cytometer (Aurora, Cytek Biosciences). 

Phagocytosis Assay

To evaluate the immunostimulatory ability of Bel-sar treated CJM cells, we co-cultured the CJM cells with THP-1 derived macrophages (M0). Briefly, THP-1 cells were maintained in RPMI 1640 GlutaMAX medium supplemented with 10% heat-inactivated fetal calf serum (FCS) and a 2% mixture of penicillin and streptomycin (Thermo Fisher), and cultured under 5% CO₂ at 37°C. In order to obtain THP-1 derived Mφ, THP-1 cells were seeded at a density of 7 × 10⁶ cells in 25 mL in a T175 flask and stimulated with 50 ng/mL of Phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) for 48 hours, followed by further incubation in complete medium in the absence of PMA for 3 days. Tumor cells were first stained with CMFDA (1 uM, Chloromethyl fluorescein diacetate; Abcam) for 0.5 hour and then treated with Bel-sar and laser, as described above. Immediately thereafter, tumor cells were collected and co-cultured with THP-1 derived Mφ at a ratio of 1:1 or 1:3 for 2 hours. Cells were collected and stained with anti-CD11b-PE (clone ICRF44, Biolegend) or anti-CD68-BV785 (clone Y1/82A, Biolegend), and then analyzed by flow cytometry on an Aurora cytometer (Cytek Biosciences). The THP-1 derived Mφ that had phagocytosed tumor cells were CD1b+CMFDA+ positive. 

Statistical Analysis

Statistical analyses were performed in GraphPad Prism version 9.0 for Windows (GraphPad Software, La Jolla, CA, USA). A Student's unpaired two-tailed t-test was performed to compare two experimental groups for the analysis of DAMPs. The mRNA expression of DAMPs was compared using the Mann-Whitney U test. half-maximal effective concentration (IC₅₀) was calculated using a nonlinear regression analysis. Statistical differences were considered significant at * = P < 0.05, ** = P < 0.01, and *** = P < 0.001. 

Results

Subcellular and Intracellular Location of Bel-sar in Conjunctival Melanoma Cells

We determined whether Bel-sar bound to and was taken up by the three CJM cell lines by assessing its binding (at 4°C) and uptake (37°C) in vitro. Fluorescence microscopy showed that at 4°C, Bel-sar only bound to the membrane (Fig. 1A), whereas at 37°C it was partly located in the cytoplasm (Fig. 1B). We then quantified Bel-sar binding and uptake using flow cytometry analysis. In all cell lines, Bel-sar binding and uptake increased over time and with a higher concentration. At 37°C (uptake), the geometric mean (gMFI) was significantly higher than at 4°C (binding; see Fig. 1B). 

Figure 1.

 

Intracellular location, binding and uptake of Bel-sar by CJM cell lines. (A) Intracellular location of Bel-sar in CRMM2 cells after staining with CD44-FITC and DAPI imaged by fluorescence microscopy after 4 hours of incubation with 300 pM Bel-sar at 4°C or 37°C. Scale bar = 40 µm. (B) Binding (4°C) and uptake (37°C) at indicated concentrations in three CJM cell lines. The graphs show the average gMFI ± SEM of cells from three independent experiments.

After uptake into the cytoplasm, photosensitizers can target different organelles. It is known that the subcellular location of a photosensitizer is associated with the capacity to induce immunogenic cell death.²¹ Therefore, we analyzed the subcellular location of Bel-sar in CRMM2 cells at different time points. Bel-sar was first located in lysosomes 4 hours after incubation and not yet in the Golgi apparatus or mitochondria (Fig. 2A); after 24 hours, Bel-sar was mainly found in the lysosomes, whereas some accumulation was seen in the Golgi apparatus and mitochondria (Fig. 2B, Supplementary Fig. S1). This sequence and localization (see the Table) resembles the behavior of virus particles during the human papillomavirus infection process.²⁸ 

Figure 2.

 

Subcellular location of Bel-sar in the CRMM2 cell line at different time points: after 4 hours incubation (A) or 24 hours incubation (B) with 3000 pM Bel-sar at 37°C. The organelle tracers for lysosomes, Golgi apparatus, mitochondria, or ER are labeled green. (A) After 4 hours, Bel-sar only appears in the lysosomes. (B) After 24 hours, Bel-sar is mainly located in lysosomes and partly in the Golgi apparatus and the mitochondria. The ER tracker induces morphological changes in the CRMM2 cells, as well as in the other two cell types, which complicates the imaging of live cells. Consequently, we were unable to obtain clear results on ER uptake and have decided not to include comments on ER. Scale bar = 40 µm.

Table.

 

Kinetics of Transmembrane Localization of Bel-sar in the CRMM2 CJM Cell Line as Determined by Fluorescence Microscopy

 

Cell Viability and Light-Activated Bel-sar Induced Cytotoxicity In Vitro

We analyzed the effect of Bel-sar on the viability of CJM cells in the presence or absence of light. When the CJM cell lines were incubated with Bel-sar (concentration from 0.01 pM to 1000 pM) in the dark, no significant toxicity was seen (Fig. 3A). However, when exposed to light with the appropriate wavelength (690 nm), the MTS cytotoxicity assay showed that Bel-sar treatment led to significant killing with a half maximal inhibitory concentration (IC₅₀) of 30 to 60 pM (Fig. 3B). The IC₅₀ in CRMM1, CRMM2, and CM2005.1 was 40 pM, 31 pM, and 60 pM, respectively. Subsequently, PDT-treated cells were analyzed for apoptosis and necrosis, which showed agreement with the MTS assay results. The cytotoxicity of Bel-sar was enhanced with increasing concentrations (Fig. 3C) and increasing fluence (Fig. 3D). 

Figure 3.

 

Cytotoxic effect of Bel-sar on three CJM cell lines, in the dark (A) or with light activation (B), determined by MTS assay. The effect of (C) concentration and (D) fluence on in vitro cytotoxicity of Bel-sar on CRMM1, CRMM2, and CM2005.1 cells, measured by flow cytometry after staining with Annexin V and DAPI. The DAPI indicates late apoptosis cells while AV (Annexin V) staining indicates early apoptotic cells.

Membrane Exposure of Damage-Associated Molecular Patterns and Immunostimulatory Effect of Bel-sar Treatment

Apart from direct cell death, we analyzed whether Bel-sar treatment may be able to induce immunogenic cell death, which is characterized by the exposure or release of DAMPs, such as CRT and HSP90. These DAMPs together with apoptosis and necrosis can initiate pro-inflammatory responses in the tumor area, and then induce an antitumor immune response through interaction with APCs. The exposure of CRT and HSP90 was measured by flow cytometry. The data show that CRT (Fig. 4A) and HSP90 (Fig. 4B) exposure were enhanced by Bel-sar treatment in all three conjunctival melanoma cell lines in a concentration-dependent manner. Freeze/thaw was used as the positive control for CRT and HSP90 exposure. We conclude that Bel-sar treatment consistently increased DAMP exposure in the three CJM cell lines, regardless of the type of mutation. 

Figure 4.

 

Light-activation of Bel-sar on three CJM cell lines led to enhanced exposure of DAMPs and increased phagocytosis of tumor cells by THP1-derived macrophage. Increased levels of CRT (A) and HSP90 (B) on the cell surface as determined by flow cytometry on DAPI-negative cells of the CRMM1, CRMM2, and CM2005.1 cell lines. Tumor cells were incubated with Bel-sar at the indicated concentrations followed by light activation or left untreated and analyzed 24 hours later. As a positive control, cells were frozen (−20°C) and thawed (37°C; F/T) three times. (C) After Bel-sar treatment, 3 conjunctival melanoma cells were co-cultured at 2 different ratios (1:1 and 1:3) with THP-1 derived macrophage for 2 hours. Cell phagocytosis by THP-1 derived macrophages was scored as the percentage of CD11b+ CMFDA+ cells (mean values ± SEM). Data are shown as the mean ± SEM of three independent experiments and were statistically analyzed by an unpaired two-tailed Student’s t-test.

In order to test whether cell death or DAMP exposure may stimulate APCs and then initiate a specific immune response against the tumor, we evaluated the immunostimulatory capacity of Bel-sar treatment by co-culturing macrophages with tumor cells. Mφ were obtained by polarizing THP-1 cells with TMA, with THP-1 derived Mφ expressing high CD11b and CD68 (data not show). THP-1 derived Mφ engulfed more of the Bel-sar treated tumor cells than non-treated tumor cells (Fig. 4C). 

Taken together, the data show that after binding to the membrane, Bel-sar was distributed to lysosomes, the Golgi apparatus, and mitochondria. Following laser activation, Bel-sar induced effective cell death in all three CJM cell lines, with the characteristics of immunogenic cell death (enhanced exposure of DAMPs and increased engulfment by THP-1 derived Mφ). 

Discussion

The subcellular location of a photosensitizer is the most important factor that may influence the efficacy of treatment with PDT as it determines where and how photosensitizers may affect their cytotoxicity within the tumor or tumor tissue.^29–31 Photosensitizers can localize in the plasma membrane, lysosomes, endoplasmic reticulum, mitochondria, or the Golgi apparatus. This subcellular location in turn is determined by the chemical nature of the photosensitizer, the concentration, incubation time, and the cell type.³² Turubanova et al. compared the subcellular location between photosens and phenothiazine and the difference in subcellular location induced different types of cell death and affected the antitumor efficiency in vitro and vivo.³³ When a photosensitizer mainly accumulates in the cytoplasmic membrane, the reaction of singlet oxygen or ROS with the membrane lipids will result in lipid peroxidation and can lead to disruption of cellular membranes and direct cell death (apoptosis).^32,34 Our results show that Bel-sar was first observed at the cell membrane and was then engulfed into the cytoplasm. Lysosomes which contain a number of enzyme and hydrogen ions, play a crucial role in degrading intracellular material and cellular survival. Targeting lysosomes may furthermore enhance the induction of pyroptosis and ferroptosis which are considered examples of immunogenic cell death.^21,35 Dysfunction of lysosomes may block autophagic degradation and also induce pyroptosis via activating the NLRP3/GSDMD and caspase-3/GSDME pathways.²³ After engulfment into the cytoplasm, Bel-sar was first observed in the lysosome and was then transferred to the Golgi apparatus and mitochondria. Similar to the situation with regard to lysosomes, targeting mitochondria and the Golgi apparatus enhanced the tumor immunogenicity by pyroptosis.^24,25,36 Targeting multiple organelles may help contribute to an effective cytotoxicity and immunostimulatory ability in PDT. 

The antitumor efficiency induced by PDT may not only depend on the photochemical properties and biological effects of the photosensitizer, its accumulation, and biodistribution, the total energy for irradiation, and, more importantly, but also on the genetic, epigenetic, phenotypic tumor profiles, and tumor microenvironment.^13,21–37 Tumors from the same origin and with the same subclassification may still differ, which may contribute to local discrepancies in photosensitizer uptake, cytotoxicity, and immunostimulatory effect triggered by PDT.²¹ Here, we used primary and recurrent CJM cell lines (two with BRAF and one with an NRAS mutation), and tried to investigate whether the mutation status affected the antitumor efficiency in vitro. The three cell lines showed an effective uptake of Bel-sar in IC₅₀ ranges as low as 30 to 60 pM. This is consistent with two previous studies which showed that Bel-sar was effective against different types of cancer and in a panel of uveal melanoma cell lines using picomolar ranges of IC₅₀.^19,20 This may be due to the unique structure of Bel-sar, which specifically targets tumor cells and effectively delivers a large amount of photosensitizer, contributing to its high accumulation in tumor cells regardless of mutations. 

PDT is particularly effective in generating rapidly abundant alarm/danger signals, known as DAMPs. Locally released DAMPs can be detected by the innate immunity alert elements, and then activate antigen-presenting cells, which then may initiate a systemic immune response.^14,15,38 Light-activated Bel-sar induced immunogenic cell death in three CJM cell lines, which was characterized by enhanced exposure of DAMPs, such as CRT and HSP90, and Bel-sar-treated tumor cells enhanced engulfment by macrophages. This indicates that Bel-sar treatment induced a strong local immune response. A local acute inflammatory reaction with DAMP exposure or release, an increase in vascular permeability, and release of cytokines recruits more immune cells, such as neutrophils, monocytes/ macrophages in order to remove debris or dying cells.^37,39 These infiltrating cells in turn modify an immunosuppressive tumor microenvironment to a more pro-inflammatory state. In our previous paper using a murine cutaneous melanoma model, Bel-sar treatment induced infiltration of especially M1 and less M2 macrophages.⁴⁰ The M2 macrophages are considered to contribute to an immuno-suppressive micro-environment and the polarization of M2 to M1 type macrophages helps to further enhance an antitumoral immune response. 

Frequent local tumor recurrences and lack of standard therapies for metastases are still main challenges in the management of patients with CJM. A combination strategy may be necessary to induce a strong local and systemic immune response to prevent recurrences or metastases. CJM is characterized by a very high mutation load, composed of approximately 500 somatic mutations in exonic regions.³ The genetic similarity to cutaneous melanoma and the high mutational burden suggest that patients with CJM could benefit from similar treatments that are being applied for cutaneous melanoma, such as immune checkpoint inhibitors, independent of the type of mutation.¹² A strong immune response could first be initiated by the Bel-sar treatment and further enhanced by the combination of immune checkpoint inhibitors, such as anti-PD-L1 and LAG-3. 

In our previous study, immune checkpoint inhibitors enhanced the efficacy of Bel-sar against established tumors in mice, leading to durable complete responses with an abscopal effect.¹⁷ The proposed synergistic effect of photodynamic therapy and immune checkpoint inhibitors could be the result of expansion of the numbers of tumor-infiltrating CD4+ and CD8+ T cells, further induced after immunogenic cell death.¹⁶ These data support combining light-activated Bel-sar with immune checkpoint inhibitors as a new strategy to treat primary CJM to reduce recurrences and metastasis formation. Further in vivo studies and clinical development may be supported by these preclinical data. 

Acknowledgments

Supported by a grant from Health Holland grant to M.J.J. and Aura Biosciences. 

The authors specially thank Annelies Boonzaier-Van der Laan from the light microscopy facility at the LUMC for her help in the live cell imaging microscopy. 

Disclosure: S. Ma, (F); R.V. Huis In't Veld, None; E. de los Pinos, (C); F.A. Ossendorp, None; M.J. Jager, None