The fusion of light and immunity: Advancements in photoimmunotherapy for melanoma

Abstract

Metastatic melanoma is an aggressive skin cancer with high mortality and recurrence rates. Despite the clinical success of recent immunotherapy approaches, prevailing resistance rates necessitate the continued development of novel therapeutic options. Photoimmunotherapy (PIT) is emerging as a promising immunotherapy strategy that uses photodynamic therapy (PDT) to unleash systemic immune responses against tumor sites while maintaining the superior tumor-specificity and minimally invasive nature of traditional PDT. In this review, we discuss recent advances in PIT and strategies for the management of melanoma using PIT. PIT can strongly induce immunogenic cell death, inviting the concomitant application of immune checkpoint blockade or adoptive cell therapies. PIT can also be leveraged to selectively remove the suppressive immune populations associated with immunotherapy resistance. The modular nature of PIT therapy design combined with the potential for patient-specific antigen selection or drug co-delivery makes PIT an alluring option for future personalized melanoma care.

Graphical Abstract

In this review, we discuss recent advances in photoimmunotherapy (PIT) based strategies for the management of melanoma. PIT has the potential to ignite systemic anti-tumor immunity, which may lead to enhanced immune checkpoint-blockade or adoptive cell therapy responses. PIT is highly customizable and can be tailored in a patient-specific manner.

Introduction

Melanoma, a potentially fatal cancer that often arises from cutaneous melanocytes, displays complex heterogeneity in its developmental pathways and presentation. Currently, 4 major types of melanomas have been delineated—superficial spreading, nodular, lentigo maligna, and acral lentiginous (1). Several risk factors are associated with melanoma, including environmental, genetic, or immune-related changes (2). Most notably, prolonged exposure to sunlight or artificial ultraviolet (UV) radiation through tanning beds results in significant DNA damage in skin cells, including melanocytes, which in turn leads to extensive mutational burdens, and ultimately melanoma formation (3). Certain phenotypical traits such as fair skin, red hair, and freckles, are also associated with UV light sensitivity and a high risk of developing melanoma (2).

The past two decades have seen significant advancements in melanoma treatment. Multiple targeted therapies have been approved for melanoma patients, which intervene in the MAPK signaling pathway by inhibiting BRAF or MEK. (4). Immunotherapies, namely immune checkpoint blockade (ICB) therapies, have demonstrated unprecedented clinical success in melanoma and other cancers. Approved ICB therapies target immune-regulatory checkpoint proteins such as programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte associated protein 4 (CTLA-4), and lymphocyte-activation gene 3 (LAG-3), to stimulate successful anti-tumor immune effector responses (5). However, ICB therapies are extremely expensive, and a majority of ICB patients still experience therapy resistance, ultimately leading to tumor relapse and fatality (6, 4). Thus, additional research is desperately needed to identify novel treatment options capable of overcoming or preventing resistance to ICB or other melanoma immunotherapy approaches.

One such emerging option relies on phototherapy, or photodynamic therapy (PDT). PDT consists of the administration of photosensitizing (or photoabsorbing) agents, which can accumulate in tumors and then be activated by agent-specific wavelengths of light. Upon activation, PDT destroys tumors through the production of reactive oxygen species (ROS) in cancer cells. PDT can effectively destroy tumors as a minimally invasive technique and continues to attain recognition for its promise in melanoma treatment (7). PDT is currently FDA approved for the dermatologic removal of actinic keratoses, precursors to nonmelanoma skin cancers (8). PDT with the photosensitizer porfimer sodium is also approved for select treatment applications in esophageal and endobronchial cancers, lung adenocarcinoma, and more (9).

A potentially effective use of PDT in melanoma may be through photoimmunotherapy (PIT). While PDT focuses on the direct achievement of tumor cytotoxicity, PIT aims to achieve tumor clearance by initiating or enhancing anti-tumor immune responses (10, 11). Traditionally, PDT has been restrained to primary tumor treatment due to the necessity for light penetration (12). PIT can stimulate systemic anti-tumor immunity, widening the feasibility of PDT well beyond skin lesions and into the realm of controlling advanced metastatic disease (13). The use of PIT in a primary tumor site to jumpstart systemic anti-tumor immunity as a precursor to ICB or other immunotherapy options appears to be very promising. Synergy with PIT may result in overcoming or preventing ICB resistance (14, 15), or improving responses from other immunotherapy options, such as adoptive cell therapies (ACTs) (16, 17). PIT can initiate the tumor-immune cycle by driving immunogenic cell death (ICD). PIT can also result in gene expression changes in tumors leading to the activation of immune-stimulating pathways or the removal of immune checkpoints (18, 19). Alternatively, PIT can selectively eliminate regulatory immune populations that accumulate in tumors and prevent successful immune or ICB responses (20). The conjugation of photosensitizing agents to a variety of delivery vectors, such as nanomaterial platforms, liposomes, or tumor-specific antibodies, has been shown to overcome previous limitations on PDT tumor specificity. Further, these diverse delivery mechanisms facilitate easy, tumor-directed co-application of other anti-cancer therapeutics, which can be leveraged to directly enhance PIT responses or improve anti-tumor immunity through a variety of other means. PIT is a minimally invasive option with extreme potential to synergize with current melanoma immunotherapies. In this review, we have focused on recent advancements in PIT for the management of melanoma.

Photodynamic therapy (PDT)

PDT relies on the combined application of light energy and photosensitizing agents to generate ROS in specific tissues or cell types. Upon photon-mediated excitation, photosensitizing agents can undergo intersystem crossing to enter a high energy, long-lived “triplet state.” In the triplet state, photosensitizing agents can react with surrounding substrates and release energy, enabling targeted ROS production. Type I PDTs generate type I ROS, such as superoxide, or other free radicals (O₂^{− •}, OH•, etc.), while type II PDTs generate type II ROS in the form of singlet oxygen (¹O₂). ROS, in turn, results in potent cytotoxicity (21–24). Type I and type II PDT responses are not necessarily mutually exclusive, and can occur simultaneously (22), but a majority of current PDT options rely on the type II pathway (23). PDT approaches for melanoma treatment are highly flexible and can include the direct targeting of melanoma cells (23), or indirectly mediate anti-cancer effects by targeting tumor vasculature (25) or tumor infiltrating cell types such as immune cells or fibroblasts (26–29). A recent randomized controlled clinical trial evaluating the safety and efficacy of PDT with the photosensitizing agent verteporfin in primary choroidal amelanotic melanoma (an ocular melanoma) achieved complete regression in 88% of patients, of which 56% did not experience recurrence after a mean follow-up time of 3.5 years. No patients experienced reduced visual acuity, highlighting the noninvasive nature and selectivity of this modality (30). Alteration of light application techniques and intensity, photosensitizing agents, and the optional inclusion of photosensitizing agent delivery vehicles provides PDT with high flexibility and multiple opportunities to improve specificity.

Despite these benefits, deployment of PDT in melanoma is still faced with numerous challenges. A major hurdle in this direction is achieving a sufficient degree of light penetration to tumor tissues, without which a biological response is not possible (31). While type I PDT can transfer electrons to protons, the more common type II PDT requires sufficient oxygen concentrations, diminishing feasibility in the often inevitable hypoxic melanoma microenvironment (23, 32, 33). Poor water solubility of photosensitizing agents (34), or insufficient cellular uptake can also confound successful targeting of cytotoxicity or can cause off-target damage if ROS is produced extracellularly (7). Cellular antioxidants are capable of neutralizing intracellular ROS produced by PDT (35, 33). Importantly to the case of melanoma, melanin, a pigment responsible for skin photoprotection against ultraviolet radiation (UV)-induced damages, can readily absorb visible light, preventing efficient photosensitizer activation and ROS generation in melanotic cases (36). In response to these problems, recent research and PDT approaches have aimed to overcome these challenges by combining photosensitizing agents with delivery mechanisms to enhance specificity and uptake. Most notably, photosensitizing agents are being combined with a wide range of nanoparticle systems and/or conjugated to antibodies for better tumor specificity (reviewed in (37, 31)). Next generation photosensitizing agents are also under constant development. These emerging photosensitizing agents can be activated well outside of the melanin absorption peak at 335 nm (38), and instead act in the near-infrared (NIR) range at ~700–860 nm, thus widening the applicability of PDT to melanoma regardless of pigmentation status while maintaining tissue-penetration (36, 39). Recently developed photosensitizers also display higher melanoma specificity compared to previous generation options and require less powerful light sources for their activation (40).

While the goal of traditional PDT has not focused on initiation of anti-tumor immunity, a toolkit of efficacious PDT delivery systems must be established for successfully initiating immunogenic cell death, shifting the targets of PDT to tumoral immune population constituents, or providing benefit to ICB responses. Therefore, overcoming the challenges to traditional PDT responses in melanoma is of paramount importance to facilitate successful PIT. Below, in Table 1, we briefly summarize recent examples of PDT approaches gaining attention for their ability to overcome these challenges in melanoma.

Table 1:

Recent photodynamic therapy (PDT) studies in melanoma that address previous PDT limitations.

Approaches	Outcomes	Sources
Direct hyaluronic-acid (HA) conjugation or HA-conjugated delivery system	Improved selectivity for CD44-expressing melanoma cells and intracellular intake	(33, 83–87, 32)
Various delivery methods employing catalase, MnO2 doping, perfluorocarbons and pyropheophorbide A polymers, Cu2O-coordinated carbon nitride compounds, hemoglobin liposomes	Sequester, increase, or maintain oxygen availability for ROS production in the local tumor microenvironment, resulting in enhanced melanoma cytotoxicity	(84, 33, 85, 86, 88, 59)
Nanoparticles containing the hypoxia-responsive prodrug tirapazamine	Utilize PDT-mediated hypoxia to activate pro-drug for anti-melanoma benefit	(89)
Various micelle or liposome delivery mechanisms	Enhanced specificity and uptake of photosensitizing agents to melanoma cells, enhanced photosensitizer stability, easy co-delivery of chemotherapies or other agents, enhanced ROS production and cell death	(90–100)
Monoclonal antibody conjugation or particle-coating	Improved specificity to melanoma cells and flexibility with targeting options	(95, 101, 102, 57)
Depigmentation agents, photobleaching, using light sources out of the melanin absorbance range	Enhanced PDT sensitivity and ROS production in melanotic melanoma	(103–105, 35)

Photoimmunotherapy (PIT)

While traditional PDT approaches have solely focused on maximizing the cytotoxic tumor burden, the goal of PIT is to maximize anti-cancer immune responses, making PIT a form of photodynamic immunotherapy. The most appreciated form of PIT aims to facilitate successful anti-tumor immunity by inducing ICD in tumors (41). The variety of PIT delivery vectors allows easy development and tumor-specific deployment of adjuvant therapies that can further enhance ICD, in turn, further strengthening anti-tumor immunity. NIR PIT is a recently developed method of antibody-conjugated PDT, specifically designed around ICD induction (42, 43). Alternatively, PIT approaches that do not result in complete tumor cytotoxicity can still enhance tumor immunogenicity and immune responses by activating immunostimulatory signaling pathways or preventing regulatory immune checkpoints from forming (18, 19). PIT can also be directed to the removal of regulatory immune populations, which can accumulate in tumors during melanoma progression and are associated with immunotherapy resistance (27, 28). The combination of PIT with ICB or ACT, or use of PIT as a primer for other immunotherapies, has incredible potential to initiate robust systemic anti-tumor immune responses (Figure 1).

Figure 1:

Photoimmunotherapy (PIT) for personalized melanoma treatment. PIT has the potential to ignite systemic anti-tumor immunity in metastatic melanoma patients, which may lead to enhanced immune checkpoint blockade or adoptive cell therapy responses. PIT is highly customizable and can be tailored to primary tumor makeups in a patient-specific manner, ultimately maximizing immune responses.

PIT is advantageous as it is administered to a single, localized tumor region, minimizing the chances of detrimental off-target toxicity, immune-related adverse events, or cutaneous adverse events, all of which are challenges faced by the systemic therapies used for melanoma treatment, such as chemotherapy, targeted therapy, or immunotherapy (44). Due to the significant adverse events of combining targeted therapy and ICB, a phase I clinical trial has faced early closure (45), and another phase II trial experienced grade 3–5 treatment-related adverse effects in 70% of the combined treatment arm patients (46). Further, chemotherapy is notorious for immunosuppression, making it a poor candidate to be combined with ICB (47) and, due to poor response rates, chemotherapy has not been the recommended standard for melanoma management since the development of targeted and immunotherapy options (48). Thus, the ability of PIT to induce a systemic immune response through only local application positions PIT as a promising therapeutic candidate to overcome the current barriers preventing combination strategies for melanoma treatment. PIT is emerging as a viable option to improve immunotherapy responses or prevent immunotherapy resistance for the successful treatment of metastatic melanoma and possibly for other cancers as well. Below we discuss recent advancements in this area of research.

PIT and Immunogenic Cell Death (ICD)

ICD is a form of cell death characterized by the exposure and release of various cellular constituents into the tumor microenvironment (TME). During PIT approaches attempting to induce ICD, cellular membranes are degraded, and proteins that are normally localized to the endoplasmic reticulum or nuclear space are released. Cellular contents normally only associated with their intracellular compartments can serve as potent damage-associated molecular patterns (DAMPs). De novo synthesis of other heat shock proteins (HSPs) and type I interferons (IFNs) can also occur during the ICD process and enhance immune responses (49, 50). The aggregate ICD cocktail of DAMPs and cytokines can activate local dendritic cells (DCs), encouraging their phagocytosis of tumor-associated antigens and/or tumor neoantigens that were also released during cell death. These stimulatory events enhance DC migratory capabilities, and the resulting mature DCs can effectively prime naïve T cells in draining lymph nodes. Activated tumor-specific CD8⁺ T cells expand and migrate back to the tumor to execute their effector functions. Thus, PIT application and ICD can subsequently initiate or potentiate the cancer immunity cycle. In the local TME, ICD-mediated DAMP and IFN expulsion may also stimulate tumor infiltrating T cell function, or enhance their recruitment (51, 52, 50, 53, 54). The ability of PIT-enabled ICD to establish or invigorate robust anti-tumor immunity serves as an obvious complement to immunotherapy approaches, and ICD has already been posited as a means to overcome immunotherapy resistance (55). Melanoma clinical trials recognizing this potential for synergy are already exploring combinations of ICB and ICD-inducing therapeutics (reviewed in (56)).

Membrane Targeting ICD-inducing PIT

Near-Infrared PIT (NIR-PIT) is an emerging PDT approach specifically focused on inducing ICD. Currently, NIR-PIT relies on the photosensitizer dye IRDye700DX (IR700) in conjugation with a monoclonal antibody, and activation with NIR light at 690 nm. Upon NIR exposure, IR700 switches from hydrophilic to hydrophobic, becoming membrane soluble to induce necroptosis (43, 57), a classic example of ICD (58). A variety of other PDT formulations that do not rely on IR700 can also serve as PIT by virtue of their ICD-inducing nature. So far, two NIR-PIT strategies are under investigation in melanoma.

NIR-PIT directed against CD29, a surface antigen often overexpressed in melanoma, has been shown to effectively kill melanoma cells in vitro and in established B16 melanoma tumors in mice, while sparing normal skin and vascular integrity (43). CD29 directed NIR-PIT also reduced melanoma tumor expression of ki67, a proliferation marker, potentially indicating a prolonged anti-proliferative effect even after therapy application. CD29 directed NIR-PIT was specific to melanoma cells, and did not diminish tumoral levels of immune populations important for tumor-immunity, such as DCs, T cells and natural killer (NK) cells. Combined with anti-CTLA4, an ICB therapy, CD29 directed NIR-PIT was found to further extend B16-bearing mouse survival, and enhance the expression of activation markers on tumor infiltrating CD8⁺ T cells, and DC maturation markers (43).

CD146 is another surface marker overexpressed in melanoma cell lines. In an A375 melanoma xenograft model, radiolabeled anti-CD146 preferentially accumulated in the tumor tissue compared to other sites. CD146-targeting NIR-PIT generated singlet oxygen in vitro and diminished tumor volume in A375 mouse xenograft models. The authors of this study speculated that CD146‐targeted NIR-PIT might lead to enhanced immune responses but did not assess immune-related impacts of their treatment (57). As NIR-PIT was only recently developed, further research to examine PIT-mediated immune-related benefits and to improve melanoma antigen-selection options is required to understand the best strategies or conditions needed for robust ICD induction. NIR-PIT is under investigation in multiple other cancer types, where it is also being applied alongside ICB to improve outcomes (reviewed in (10)).

While NIR-PIT focuses on necroptotic ICD via membrane rupture, endoplasmic reticulum (ER) disruption can also lead to ICD. Pardaxin, an endoplasmic reticulum targeting peptide, can adjust the ICD strategy to one based on ER targeting (59). Pardaxin was recently incorporated into liposomes, which contained the photosensitizing agent indocyanine green conjugated to gold nanospheres. Notably, hemoglobin (Hb) could also be contained within the liposomes to maintain oxygen levels during ROS production. In B16 cells, these liposomes successfully localized to the ER and induced ICD upon light activation. In mice bearing B16 melanoma tumors, liposome delivery and laser activation prevented tumor growth and induced ER-specific stress. Liposome treatment incorporating Hb, pardaxin, and the photosensitizing agent indocyanine green resulted the most robust anti-tumor response, which included production of in pro-inflammatory and cytotoxic cytokine production (interferon gamma (IFN-γ) and tumor-necrosis factor alpha (TNF-α) among others), as well as DC maturation and local and splenic CD8⁺ T cell expansion, suggesting the development of a systemic immune response (59). Regulatory T cells (Tregs), which are associated with impaired tumor immunity (60), were also reduced (59). Thus, targeting photosensitizing agents to specific cellular membranes is an emerging strategy to select for ICD over non-immunogenic cell death mechanisms, therefore enabling systemic anti-tumor immunity.

Alternative ICD-inducing PIT

With photosensitizing agents and nanoparticle delivery options experiencing continued innovation, certain recently discovered PIT options have been shown to induce ICD without help from membrane-targeting antibodies, peptides, or other external agents. A hydrazide (TPH)/Cu/Fe nanoplatform was recently combined with indocyanine green (acronymized as TCFI) (15). The TCFI nanoplatform displayed high uptake in B16F10 melanoma cells in vitro. Upon NIR-mediated activation, TCFI nanoparticles induced ferroptotic cell death and severe oxidative stress, resulting in the release of the hallmark ICD DAMPs, high mobility group box 1 protein (HMGB1) and calreticulin (CALR). In B16F10 mouse models, PIT with the TCFI nanoparticle enhanced DC mutation in tumor-draining lymph nodes and tumor T cell infiltration. TCFI PIT application to a single B16F10 melanoma tumor, in mice bearing tumors on both flanks, greatly enhanced responses to subsequent anti-PD-1 ICB administration. TCFI PIT treatment of primary tumors synergized with anti-PD-1 to greatly reduce tumor volume in distant sites, compared to either TCFI PIT or anti-PD-1 alone, suggesting systemic anti-tumor immunity potent enough to reduce metastatic disease (15).

In a similar approach, gold nanocages were loaded with indocyanine green before envelopment in a monophosphoryl lipid A bilayer, termed MLI-AuNCs. B16F10 melanoma readily takes up MLI-AuNC, and activation (808 nm), results in potent cytotoxicity (61). In B16F10 xenograft models, MLI-AuNC treatment of established tumors diminished tumor growth, and acheived a complete, relapse-free, response in 2/5 mice. The anti-tumor effect was associated with increased in situ DC maturation, and T cells producing IFN-γ, suggestive of ICD-mediated immune activation. Accordingly, MLI-AuNC application to primary tumors was able to suppress growth at metastatic sites, reduce metastatic lesion count, and improve T cell infiltration at metastatic sites (61).

Ruthenium-containing complexes for PDT are being extensively investigated (62) and NIR-absorbing ruthenium (II) complexes capable of inducing ICD were recently described (63). In particular, the ruthenium (II) complex ML19B01-PDT induces cell death in B16F10 cells (630 nm and 730 nm activation range). ML19B01-PDT-induced B16F10 cell death was immunogenic, accompanied by release of the DAMPs ATP and HMGB1, and increased CALR cell surface expression. ML19B01-PDT treatment also enhanced DC phagocytosis of B16F10 cells, which resulted in the upregulation of DC activation markers. B16F10 cells were treated with ML19B01-PDT and irradiated, lysed, and used to vaccinate tumor-free mice before B16F10 challenge. Vaccination prolonged mouse survival and diminished tumor growth (63). ICD induction with PDT not only has potential for in vivo treatment, but also for the development and production of preventative or therapeutic cancer vaccines.

Co-delivery ICD-inducing PIT

The variety of mechanisms utilized for photosensitizer delivery allows the easy application of secondary therapeutic agents capable of acting on cancer cells, immune cells, or tumor features to enhance ICD outcomes as part of PIT. For example, ATP is an immunostimulatory molecule released during ICD, but tumors can degrade ATP with the ectoenzyme CD39 (64). To enhance PIT-mediated ICD, a recent study utilized microneedle delivery of lipid nanoparticles containing the photosensitizer IR780 and the CD39-inhibitor sodium polyoxotungstate. Microneedle delivery of these nanoparticles followed by an 808 nm light application reduced tumor volume in B16 melanoma bearing mice, which was accompanied by increased pro-inflammatory cytokines in the TME, such as TNF-α and the interleukins 6, 12, and 18. Treatment enhanced DC maturation and infiltration in situ and in draining lymph nodes, which was accompanied by increased CD4⁺ and CD8⁺ T cell tumor infiltration. While only primary tumors were treated, the observed lymph node changes are indicative of systemic anti-tumor immunity (64). The microneedle PIT delivery strategy is capable of delivering numerous other targeted therapies or chemotherapies, which may be a promising means to initiate systemic immunity from sufficient ICD at an easily accessible primary site (Figure 1).

Impaired tumor vasculature has been demonstrated to prevent successful tumor immune responses and decrease tumor T cell infiltration. Attempting to overcome this barrier, a NIR-activated nanoparticle containing pseudo‐semiconducting polymers as ROS-producing photosensitizing agents was paired with lenvatinib, a vascular endothelial growth factor receptor (VEGFR) inhibitor that can normalize tumor vasculature and restore T cell infiltration. Named “Combo-NP,” this nanoparticle combination displayed anti-melanoma cytotoxicity upon light activation in vitro (14). Combo-NP application and light activation in a co-culture of DCs with B16F10 melanoma enhanced DC activation, suggesting a significant immune response mediated by ICD as this model precludes any effects observed from lenvatinib-mediated vascular changes. In an OCM1-bearing uveal melanoma mouse model, Combo-NP treatment and photoactivation prevented tumor volume and weight growth without systemic toxicity, and Combo-NP accumulated preferentially in the tumor mass. Similar results were achieved in mice bearing subcutaneous B16F10 melanoma, where Combo-NP plus light activation alleviated hypoxia and facilitated ICD, as evidenced by HMGB1 release. These effects were accompanied by increased CD8⁺ T cell infiltration and DC maturation (14). Tregs, and myeloid derived suppressor cells (MDSCs), another suppressive, tumor-infiltrating immune population (65), were reduced (14). Combined with anti-PD-L1, an ICB therapy, Combo NP induced a systemic anti-tumor response. The combined effect was capable of reducing growth at metastatic tumor sites, and far exceeded the ability of anti-PD-L1 as a monotherapy. Distant tumors also experienced DC maturation, CD8⁺ T cell infiltration, and MDSC reductions (14).

Instead of vasculature, remodeling the extracellular matrix (ECM) is another method to improve immune infiltration (66). The dense ECM can also inhibit oxygen flow, preventing the successful ROS generation PDT relies on (67). To execute such ECM remodeling, microneedle patches were recently formulated with hyaluronidases (HAases) to degrade hyaluronic acid (HA) in the TME/ECM. In B16-bearing mice, HAase-assisted microneedle delivery enhanced penetration and retention of a formulation containing mesoporous prussian blue nanoparticles, (which can provide additional oxygen), and the photosensitzer chlorin e6. In a DC and B16 melanoma co-culture, activation of the conjugated chlorin e6 resulted in ATP and HMGB1 release and DC maturation, indicating ICD induction. The delivery and activation of this formulation could synergize with anti-PD-1 therapy, diminishing tumor volume at the primary treatment site and in distant tumors. Distant tumors also experienced enhanced CD8⁺ T cell infiltration, IFN-γ/TNF-α enrichment, and decreased tumor-associated macrophage levels. Systemic immunity was further evidenced by CD8⁺ T cell and DC splenic accumulation (67).

Certain chemotherapeutics can also induce ICD, which can be advantageous for PIT combination development. Indocyanine green was recently loaded into albumin nanoparticles alongside doxorubicin, an ICD-inducing chemotherapy. B16F10 cells readily internalized treatment-carrying albumin nanoparticles, and irradiation resulted in severe cytotoxicity via ICD, as evidenced by surface CALR expression and production of HSPs HSP70 and HSP90 (68). As we are beginning to recognize the immunostimulatory potential certain chemotherapeutics exhibit (69), clear candidates for similar PIT co-delivery strategies may emerge in the near future and display promising pre-clinical effects.

ICD-focused PIT has already demonstrated durable responses in pre-clinical melanoma models, synergized with current ICB approaches, and provides extreme flexibility for future co-delivery options capable of further improving outcomes. The current evidence suggests PIT applied to a primary tumor site may be a viable means of inducing ICD and systemic anti-tumor immunity, priming patients for better ICB outcomes.

PIT and Immunostimulatory Signaling

In cases where PIT does not result in immediate ICD, it can still serve to improve anti-tumor immunity by stimulating pro-immune pathways within cancer cells or at the tumor-cancer interface. Methyl-aminolevulinic (Me-ALA) acid is a pro-drug that is metabolized into its active form, the photosensitzer protoporphyrin IX. Me-ALA application to B16 melanoma followed by visible light irradiation strongly induced type I IFN signaling, as evidenced by expression of the type I IFNs (IFN-α and IFN-β) and phosphorylation of transcription factors activated during type I IFN responses (IRF3 and STAT1) (18). The generation of type I IFN responses to overcome immunotherapy resistance in melanoma is an active area of investigation (70) and can enhance ICD (18). Accordingly, transwell co-culture of Me-ALA treated B16 melanoma cells with DCs enhanced DC maturation and chemotaxis, which occurred in an IFN-α receptor-dependent manner. Me-ALA also increased expression of cytokines associated with the type I IFN response, such as CXCL10 (18), which is also associated with positive ICB responses (71). Activating the type I IFN response, or other similar pathways, with PIT may be an alluring method to sensitize resistant tumors to ICB or initiate anti-tumor immunity.

PIT with the photosensitizer redaporfin was shown to stimulate CD80 expression in B16F10 melanoma, which can bind, in a same-cell cis interaction, to programmed death-ligand 1 PD-L1 (19). This interaction reduced opportunities to stimulate the regulatory T cell PD-1 and CTLA-4 checkpoints (whose ligands are PD-L1 and CD80, respectively) thereby preventing immune exhaustion and stalled anti-tumor immunity (19, 72). Redaporfin-PIT combined with anti-CTLA-4 was found to extend survival of B16F10-bearing mice (19). PIT has potential to stimulate anti-melanoma immunity by activating tumor-intrinsic signaling cascades that generate pro-inflammatory cytokines, or by modulating availability of immune-signaling ligands on cancer cell surfaces. Additional future research is needed to show what photosensitizing agents can provide immune benefit by altering expression or availability in immune related pathways. While this strategy is so far specific to the tumor, “non-lethal PIT” that does not result in any cytotoxicity, may eventually be developed and applied to immune cells as a means to stimulate expression of pro-inflammatory pathways in the local TME.

PIT and Immunosuppression Removal

During melanoma progression, a variety of immunosuppressive cell types can infiltrate tumors and suppress effector immune responses. Tregs secrete an abundance of immunoregulatory cytokines and metabolites and can directly signal to inhibitory receptors on other immune cells, diminishing their effector functions or preventing their activation. Therefore, Treg-targeting or Treg depleting therapies are under investigation as a means to overcome ICB resistance and potentiate melanoma immune responses (73, 74). While PDT has traditionally relied on the accumulation of photosensitizing agents in cancer cells and cancer cell targeting, repurposing specificity-yielding strategies allows PIT to be leveraged against suppressive immune populations. PIT was recently used to deplete Tregs in melanoma models using a conjugate of anti-CD25 and chlorin e6 (20). CD25 is a Treg marker and has previously been examined for its Treg depletion potential (74, 20). In B16F10 tumor bearing mice, the anti-CD25-chlorin e6 conjugate displayed specificity to Tregs and could deplete them from tumors upon light irradiation. Anti-CD25-chlorin e6 activation reduced tumor volume well beyond the effect of anti-CD25 alone, increased tumor infiltration of CD8⁺ T cells, and increased T cell activation, as monitored by IFN-γ (20). These outcomes suggest PIT directed to Tregs is a viable strategy to stimulate immune activation and potentially reverse the Treg-mediated suppression associated with ICB resistance.

Apart from Tregs, other immunosuppressive cell populations can be targeted using PIT, such as MDSCs and tumor-associated macrophages (TAMs), which are known to accumulate in melanoma tumors, confound effective anti-tumor immunity, and prevent ICB responses (75). Cancer-associated fibroblasts (CAFs) are yet another population capable of promoting melanoma progression and impairing immunity through the secretion of regulatory cytokines (76). Immune-targeting PIT approaches aiming to deplete MDSCs have achieved promising results for colon cancer treatment (27). Immune-targeting PIT has also already shown promise for overcoming therapy resistance; anti-macrophage PIT has demonstrated effectiveness in models of therapy-resistant breast cancer (77), and CAF-directed PIT can overcome therapy resistance in esophageal cancer (78, 11). Given the importance of these immunosuppressive populations in preventing melanoma immunotherapy responses, and the outcome of PIT-mediated Treg depletion as a melanoma monotherapy, future research may assess the ability of PIT to target these populations as a means to overcome ICB resistance in melanoma.

PIT and Adoptive Cell Therapy (ACT)

ACTs rely on the isolation of immune cells followed by ex vivo stimulation, expansion, and sometimes modification, before patient infusion. Often, ACT is performed autologously, as a personalized treatment (79). For example, tumor-infiltrating lymphocyte (TIL) ACTs, such as lifileucel, rely on ex vivo expansion and stimulation of autologous tumor infiltrating T cells and have gained attention for the treatment of ICB resistant melanoma (80, 79). T cells can also be genetically engineered with chimeric antigen receptors (CARs), which are composed of an antigen-targeting region fused to signaling domains from the T cell receptor and co-stimulatory molecules, the basis of CAR-T therapy. Adoptive transfer of NK cells or natural killer T cells (NKT) are T-cell alternatives that have also garnered attention for promising pre-clinical efficacy in a range of cancers (81, 17). PIT is emerging as a promising strategy to prime tumors for subsequent ACT, destroying physical barriers to infiltration while simultaneously igniting a pro-inflammatory TME favoring sustained TIL, CAR-T cell, or NK cell activation and effector functions upon infusion.

CAR-T cell therapy has demonstrated robust effects in the treatment of hematological malignancies, but translation into solid tumors, such as melanoma, remains challenging, partially due to poor CAR-T infiltration and the extensive prevalence of immunosuppressive populations residing in the tumor before treatment (16). CAR-T cells targeting the melanoma antigen chondroitin sulfate proteoglycan-4 (CSPG4) could be strongly activated by a PIT-experienced CSPG4-expressing WM115 melanoma cells, using poly(lactic-co-glycolic) (PLGA) loaded with indocyanine green (PLGA-ICG) as the photosensitizing agent. In mice bearing subcutaneous WM115 tumors, PIT with PLGA-ICG inflamed the TME and dilated tumor vasculature, which was accompanied by an influx of DCs, monocytes, and the expression of multiple pro-inflammatory chemokines. Mice administered PLGA-ICG and irradiated before CAR-T cell delivery experienced the most CAR-T tumor trafficking and infiltration, and more effective CAR-T-mediated tumor clearance, inhibiting the growth of WM115 xenografts (16).

PIT with poly(ethylene glycol)-PLGA conjugated with PBIBDF-BT (PBT), making NP-PBT, is another approach recently examined for ACT priming. NP-PBT irradiation in mice resulted in extensive expression of chemokines associated with NKT chemotaxis and increased vascular permeability. Mice bearing B16F10 melanoma tumors and subjected to NP-PBT plus tumor irradiation experienced increased DC infiltration and DC maturation, increased NKT infiltration, and increased NKT activation. Accordingly, NP-BPT PIT priming of B16F10 tumors before adoptive, spleen-isolated, ex vivo expanded NKT therapy, also improved NKT tumor infiltration, and NKT activation. NKT delivery in PIT-primed tumors also enhanced CD8⁺ T cell infiltration and IFN-γ production. In a mouse model containing a primary and distant tumor site, NP-PBT PIT directed to the primary tumor, followed by NKT transfer, resulted in complete primary tumor clearance in 4/8 mice, and complete distant tumor clearance in 2/7 mice, suggesting extensive systemic anti-tumor immunity. Previously treated B16F10-bearing mice were able to reject B16F10 challenge, further suggesting long-term immunological memory development (17). TIL therapy and other ACTs continue to gain attention for melanoma treatment. The application of PIT as a primer for ACT is a nascent field of study, but current results using this combination already demonstrate complete tumor clearance in pre-clinical models. Further research in additional melanoma models using PIT as a precursor to ACT and ICB are desperately required, as resistance to these therapies remains challenging.

Future Directions and Conclusions

Immunotherapy options have drastically enhanced treatment outcomes for metastatic melanoma patients. However, the continued prevalence of therapy resistance highlights the unmet and dire need for additional novel approaches, such as immune sensitization treatments or combination therapies, capable of expanding complete responses to the majority of patients. PDT, which involves the application of a photosensitizing agent and subsequent ROS-mediated tumor cytotoxicity upon light excitation, has dynamically evolved in the past 10 years. Flexible delivery mechanisms, including liposomes, nanomaterial platforms, and microneedles, enhance tumor specificity and accumulation while allowing the easy co-delivery of other agents that can overcome the traditional pitfalls of PDT or act in combination with PDT. Recent PIT studies have demonstrated robust effects in B16-F10 melanoma, which is highly pigmented and melanin rich (82), exemplifying the ability of next generation photosensitization agents and PIT approaches to retain efficacy regardless of melanin status in melanoma. Most importantly, PIT is an emerging photodynamic immunotherapy option that opens the realm of PDT to the effective treatment of metastatic disease. PIT utilizes primary tumors as launching platforms for the activation of systemic anti-tumor immune responses. Numerous PIT strategies promoting ICD in primary melanoma tumors have successfully initiated such responses with clear abscopal benefits. PIT has demonstrated robust synergy in pre-clinical melanoma models combining PIT with ICB therapy or ACT, suggesting priming patients by activating systemic tumor immune responses with primary-tumor-directed PIT may invite subsequent successful ICB or ACT responses at the level of metastatic disease. NIR-PIT has also been directed to remove suppressive immune populations in tumors, potentially providing a strategy for the re-sensitization of resistant tumors to current immunotherapy options. Together, PIT is emerging as a highly flexible and promising therapy that directs decades of traditional PDT innovations towards initiating anti-tumor immunity, providing an alluring opportunity for combinatorial synergy with current melanoma immunotherapy options. The modular design of PIT and the ease of personalization, especially through antibody conjugation or co-delivery of different therapeutics in a patient-specific manner, may push PIT to the frontier of personalized care as the cancer field continues to advance in this direction.

Biographies

Pranav Volety is a final-year neurobiology and bioengineering undergraduate student at the University of Wisconsin-Madison and pursuing his research under the mentorship of Dr. Nihal Ahmad at the Department of Dermatology, University of Wisconsin-Madison. His research interests lie in proto-oncogenes, photobiology, phototherapy, and cancer therapies. Post-graduation, he plans to pursue a PhD program in comparative biological studies, aiming to make meaningful contributions to both science and the community through translational medical therapies.

Carl A. Shirley is a senior undergraduate student trainee in the Dr. Nihal Ahmad’s Lab at the University of Wisconsin-Madison. Carl is passionate about cancer biology and immunology and aspires to pursue PhD studies before an eventual research career. His interests lie in understanding cancer immunotherapy resistance mechanisms and identifying their therapeutic vulnerabilities.

Gagan Chhabra received his PhD from Jawaharlal Nehru University, New Delhi, India. He completed postdoctoral training at the University of Illinois at Chicago and the University of Wisconsin-Madison. He is currently working as a Scientist at the Department of Dermatology, University of Wisconsin-Madison. His current research focuses on cancer immune signaling, melanoma biology, cutaneous UV responses, cancer chemoprevention and experimental therapeutics.

Nihal Ahmad received his PhD from the University of Lucknow in Lucknow, India. Following his postdoctoral training at Case Western Reserve University (CWRU) in Cleveland, Ohio, he joined the Department of Dermatology at CWRU as an Assistant Professor in 2000. In 2002, he moved to the University of Wisconsin-Madison as an Assistant Professor where he is currently Professor (with Tenure) and Vice Chair for Research in the Department of Dermatology. He also serves as the co-leader of the ‘Cancer Prevention and Control (CPC)’ Program of the Carbone Comprehensive Cancer Center. The research in his laboratory is focused on three major lines of investigation; i) mechanism of cancer development and identification of molecular targets for intervention, ii) mechanism of cutaneous UV responses including photocarcinogenesis, and iii) chemoprevention and experimental therapeutics of cancer.