Recent advances in HCC treatment rely on physical therapies, such as trans-arterial radioembolization with ⁹⁰Ytrium, improved molecular targeted therapies such as multi-kinase inhibitors, and immune-modulation by anti-Programed Death Ligand 1 (anti-PD-L1) or anti-Cytotoxic T-Lymphocyte Antigen 4 (anti-CTLA4). In this context, PDT, as being a complex product of physics, chemistry, and biology, may provide a combined local and systemic approach to HCC treatment. However, the use of PDT for the treatment of liver tumors has been limited so far. Generally, the major issues with the first generation of PS were short wavelength of absorbance, poor in-vitro aqueous stability with short circulation half-life, lesser tumor selectivity, and skin phototoxicity ^[1][2][12,13]. This further aggravates when using PDT on the liver, where the high vasculature makes certain PS accumulate not only in the tumor but also in the healthy parenchyma. For instance, during 5-ALA PDT for HCC, we can observe higher accumulation in the healthy liver, since the liver is the center for heme synthesis, which is used to metabolize the pro-drug 5-ALA to the actual PS, protoporphyrin IX (PpIX) ^[5][41]. This heme biosynthesis pathway is further responsible for liver pigmentation thus altering the light penetration which decreases as the function of distance ^[6][42].

The latter issue may be addressed by using PS being more homogeneously distributed throughout the tumor at an optimal concentration and requiring a higher wavelength for their activation ^[5][41]. For instance, a study on rat liver reveals that meta-tetra(hydroxyphenyl)chlorin (mTHPC) requires less PS dosage than Photofrin^® and other hematoporphyrin-derived PS since it activates at a higher wavelength ^[5][6][7][41,42,43]. Moreover, this cytotoxic effect was further increased with near-infrared PS 5,10,15,20-tetrakis(m-hydroxyphenyl)bacteriochlorin (mTHPBC), a PS belonging to the same class of hydroporphyrins as mTHPC but with a higher wavelength of activation light ^[5][6][7][41,42,43].

Tumor selectivity and targeting could be facilitated by coupling PSs with nano-carriers. With the recent advances in nano-carrier technology, a lot of PSs are being modified and tested for enhanced efficacy. Wang et al. demonstrated that PDT mediated by IR780 and near-infrared (NIR) illumination could induce higher cell growth inhibition of HCC cell lines when delivered by a nanoparticle complex (Pullulan, Pluronic F68, and phospholipid) also encapsulating paclitaxel, with respect to IR780 or paclitaxel alone. In-Vivo studies further demonstrated reduced tumor growth and angiogenesis ^[8][44]. Zhang et al. further combined multiple approaches of hypoxia, PDT, and chemotherapy with an efficiently designed drug delivery system based on DNA aptamers and gold nanoparticles, to develop a targeted and effective HCC therapy ^[9][45].

Indocyanine green (ICG), a water-soluble tricarbocyanine dye, is a widely used agent in clinical practice for intraoperative HCC visualization ^[10][51] and liver function assessment ^[11][52]. Additionally, it has also been used for NIR PDT of several cancer models including HCC. Interestingly, when photoactivated, ICG also generates heat, which thereby contributes to a tumor-suppressive effect, known as PhotoThermal Therapy (PTT). Under PTT the PS is photo-excited, to generate vibrational energy in the form of heat, thereby inducing a cytotoxic effect ^[12][53]. An ICG-Lactosome nanoparticle complex has been developed showing higher accumulation in HCC, improved tumor visualization, and causing higher cell death when compared with ICG alone ^[13][46]. Therefore, ICG seems to have the potentiality to become the best candidate for PDT in HCC treatment. Nevertheless, the mixed impact of PDT and PTT has its equal drawbacks. As summed up by Giraudeau et al., ICG exhibits phototoxicity via PDT at a low power dose, and via PTT at a high dose; which are both relying on different molecular and cellular mechanisms ^[12][53].

Besides PS modification, PS-specific drug formulations can also be developed to enhance tumoral accumulation. For instance, 5-ALA can be administered with an iron-chelating agent which can eliminate the iron in the microenvironment, thereby inhibiting the metabolism of PpIX into heme and increasing PpIX accumulation ^[14][54]. In an in-vivo study, Chang et al. highlighted the use of iron chelator, 1,2-diethyl-3-hydroxypyridin-4-one (also known as CP94), caused double PpIX-based fluorescence than 5-ALA alone group and exhibit reduced skin photosensitization ^[15][55]. Vitamin D has also been proven to enhance PpIX levels in the cells ^[16][56]. Such drug formulations have not been studied for HCC but can significantly improve the efficacy of the therapy by augmenting the tumoral PS accumulation and decreasing non-tumoral cytotoxicity.

In-Vitro studies present a major setback for PDT research, the most significant being oxygen availability, since most of the cancers, including HCC, develop in a hypoxic background. The use of hypoxic chambers and organoids based 3-D cultures might prove beneficial for HCC modeling. Such systems, however, will not be cost-effective and require high skill sets. That is why a pre-clinical setup, using various humanized mouse models can help us understand the applications of PDT by giving a more detailed effect on the 3-D microenvironment. The most widely used models for this purpose are the subcutaneous tumors developed by either injecting human or murine HCC cell lines beneath the skin or transplanting small tumor pieces from one mouse to another. Many teams have developed orthotopic mouse models where the tumors are injected into the organs of origin, which gives a better model of cancer. Another approach can be the use of specific carcinogens to induce organ-specific cancers. However, these models can have major drawbacks as the light might not penetrate to its full efficiency thereby limiting the effectiveness of the therapy ^[17][47]. Our unpublished data have revealed that when excited with blue light, the fluorescence from PpIX could not be observed from the exterior but was successfully detected after the sacrifice and recuperation of the tumor core from the mice. Studies by two independent groups using 5-ALA PDT for different pre-clinical HCC models demonstrated a fluorescence-based selective accumulation of PpIX in the tumor, along with an anti-tumor effect ^[17][18][47,48]. The most interesting feature of these studies was PpIX accumulation and necrosis in the tumor core (up to 8 mm for mouse model). This reflects the penetrating capabilities of 5-ALA PDT, rather than a mere superficial effect. Hepatic resection has become a standard HCC treatment for early-stage patients, even in the presence of liver cirrhosis. However, long-term survival is often limited by intra-hepatic recurrences, which are not always prevented by anatomical resection or adequate surgical margins. Furthermore, small satellite nodules are hardly detected through visual inspection and intraoperative ultrasonography, especially in liver cirrhosis ^[17][47]. Hence, PDT can introduce itself as an intraoperative procedure to hepatic surgery, where it may be used both as a simple and rapid real-time fluorescence-based visual aid and as a complementary treatment targeting the tumor surrounding parenchyma. Fluorescence-guided hepatectomy using ICG has already become a standard, and one can take advantage of its potential as a PS to perform PDT during ICG-guided surgical resection. However, as discussed above, ICG might not be the ideal PS for intraoperative PDT. By contrast, 5-ALA PDT may be the best option with less photothermal-induced injury, especially to the biliary tract. In addition, to increase tumor selectivity, one could imagine the PS to be injected right into the tumor bed with the help of a trans-arterial catheter, a practice commonly used during trans-arterial chemo-embolization. At the end of the surgery, immediately after removal of the specimen, all conditions would be met to perform an efficient adjuvant PDT. The tumor bed would be exposed with clear access to the remaining peri-tumoral parenchyma, which might then adequately be submitted to illumination. Intraoperative PDT may not complicate the surgical procedure, which would be prolonged by no more than 20–30 min (based on our observations with adjuvant PDT performed during surgical resection of glioblastoma), and may also help the surgeon by providing fluorescence-guided imaging of the tumor at the beginning of the operation. Furthermore, even though ambient light in the operation room might induce photo-bleaching, this should not be a significant issue, given the lack of specificity and low energy of the ambient light which shall require a significantly longer illumination duration in order to induce significant cytotoxicity, compared to the red-light used during PDT ^[19][57]. We also believe that intraoperative adjuvant PDT may be of interest in terms of postoperative early recurrence, irrespective of the expected surgical margins. The 3-year and 5-year recurrence-free survival after R0-resection of HCC is still about 50% and 40% respectively, with no impact of surgical margin being superior to 10 mm ^[20][58] and indeed, many other studies showed that surgical margins do not influence the postoperative recurrence rates, overall survival, or recurrence pattern ^[21][22][23][59,60,61]. In addition, PDT will induce an anti-tumor immune response, which might further eliminate the possibility of tumor recurrence thereby giving long-lasting protection through the development of an immune memory, which will be discussed in the following section. However, current clinical data regarding the use of PDT in HCC patients are scarce, consisting only of small patient groups with short-term follow-up. Furthermore, there has been no study that correlates the efficacy of PDT with the cause of HCC. Particularly, PDT has not been tested yet on in-vivo HCC models arising in the context of non-alcoholic steatohepatitis (NASH). Given the increasing number of HCC in NASH cirrhosis, especially in western countries, this is certainly a field where clinicians and researchers should come together to design relevant clinical trials in the future. From a theoretical point of view, one might expect a difference in the efficiency of the PDT in NASH-HCC as compared with viral-HCC, as NASH-HCC more frequently arises before end-stage fibrosis ^[24][62] and, on the other hand, tumors are often larger than in other etiology. From a surgical point of view, in operable patients, the results of surgical resection tend to be slightly better in NASH-HCC as compared with viral-HCC ^[25][63] and thus, justify the idea of combining intraoperative PDT and liver resection in those patients to further optimize the results. It should be noteworthy that such procedures are applicable only to candidates eligible for hepatectomy i.e., BCLC stage 0 or A patients with early-stage solitary non-cirrhotic tumor, with good liver performance and no radiological evidence of vascular invasion.

34. Photodynamic Therapy May Induce an Anti-Tumor Immunity

Like any host response to an external stimulus, PDT-induced immune response will rely on an intricate network of inflammatory cytokines, chemokines, transcription factors and release of DAMPs by the PDT treated tumor. After treating colon cancer with pyropheophorbide-a methyl ester-based PDT, two waves of transcription factor NF-κB (Nuclear Factor κB) activation were observed ^[26][65]. NF-κB regulates the expression of a wide range of genes responsible for the activation of inflammation and immune response. Along with transcription factor AP-1, NF-κB induces the expression of cytokines such as IL-1α, IL-6, IL-8, TNFα ^[27][66]. Thus, PDT activates pro-inflammatory mediators thereby generating an acute inflammatory response. Studies involving various PS and cancer models have demonstrated the direct impact of PDT on immune components can be activating, suppressive, or lethal ^[28][67]. Garg et al. showed through an in-vitro set-up that when cancer cells are treated with reticulotropic PS, Hypericin based PDT, there is an exposure of calreticulin and HSP70, which then facilitate the tumor cell phagocytosis by Dendritic Cells (DCs), thereby highlighting the underlying mechanism of ICD by PDT ^[29][30][31][68,69,70]. Generally, PDT-induced tissue damage causes the infiltration of innate immune cells due to underlining oxidative stress, which leads to increased expression of Hypoxia Inducible Factor (HIF) ^[29][68]. Additionally, since this lowered level of oxygen typically resembles a site of wound or infection, HIF also causes secretion of other inflammatory cytokines and co-stimulatory factors to enhance the function of these infiltrating innate cells ^[29][68]. However, activation of adaptive immunity is the most important aspect, in order to impart a long-lasting tumor growth control. In light of that, Korbeliek et al. demonstrated an adoptive transfer of splenocytes from mice treated with Photofrin^® PDT against mammary sarcoma, which resulted in increased tumor regression post-PDT in the recipient SCID mice compared to the mice receiving just the PDT dose. This highlights that the presence of tumor-sensitized T cells in the spleen can have a significant impact on augmenting the impact of PDT ^[32][74].

45. PDT and Immune Response in HCC

Due to viral infection and cirrhosis, a majority of patients suffer from chronic inflammation in HCC. In the tumor microenvironment, there are a high prevalence of immuno-suppressive regulatory T cells (Tregs) and Myeloid-derived suppressor cells (MDSCs) (monocytes, macrophages, and DCs), along with an increased expression of immune checkpoint regulators ^[33][76]. Due to these immune suppressive populations, the tumor-infiltrating CD8+ T cell population gets exhausted and their capacity to present tumor-associated antigen is impaired, which further leads to tumor progression and poor prognosis ^[33][34][35][76,77,78]. All this develops a network of cytokines, chemokines, and other factors resulting in an intricate microenvironment. With the recent development in immune checkpoint inhibitor-based immunotherapy, the influence of the suppressive population in tumors has decreased. The two key targets are Programmed Death Ligand 1 (PD-L1) and Cytotoxic T Lymphocyte Antigen 4 (CTLA4). When these inhibitory signals bind to their receptors on T cells (CD8+ and/or CD4+), it reduces their proliferation. At the same time, they also reduce Treg apoptosis and contribute to their inhibitory function ^[33][76]. These signals are often overexpressed in the tumor microenvironment, thus contributing to the immune escape mechanism. Blockage of these signals, by using anti-PD-L1 and anti-CTLA4 antibodies, has given improved results in the clinic for a wide range of solid tumors, alone or in combination with existing chemo or radiotherapy. Regarding HCC management, the recent report on the combination of atezolizumab (a PDL-1 inhibitor) with bevacizumab (a VEGF inhibitor) ^[36][79], showing its superiority against sorafenib, has been a major step towards the use of immunotherapy as the first-line systemic treatment of advanced HCC. Thus, combining such immunotherapy strategies with PDT could be a relevant proposition. Immune checkpoint blockade-based immunotherapy along with PDT could increase infiltration of tumor-specific effector T cells and decrease secretion of TGFβ, an immunosuppressive cytokine secreted by Tregs which have an autocrine role ^[27][66], might lead to lower tumor recurrence and higher patient survival rate. However, a lot of research is still needed in both immunotherapy and PDT fields, especially for PDT dose and treatment standardization, along with guidelines from respective associations, before we could initiate a combinatorial approach for PDT and immunotherapy. The basic rationale for HCC treatment is the targeting of the primary tumor site along with the suppression of pro-tumor factors. The current treatment regimens only target one of the aspects of the rationale, while the persistence of the immune-suppressive microenvironment remains a hurdle. PDT causes tumor destruction, which results in a tissue injury and therefore release of tumor antigen. This initiates a host–tumor reaction, which results in infiltration of Tumor-Infiltrating Lymphocytes (TILs) to induce an anti-tumoral immune response and can be combined with immunotherapy to augment its impact.