Translational issues with phototherapy of cancer

Highlights

• •The field would benefit from a general review of the use of photo-based therapy methods concerning the micro-environmental stimulating factors of the tumor such as hypoxia, which are essential for maintaining the stemness properties of CSCs, the change of microenvironmental niche to induce dormancy and metastasis, and the direct effect on angiogenesis following the induction of environmental stress.
• •The different levels of autophagy induction in response to PDT and PTT can play important roles.
• •The direct impact of the consequences of various perturbations by the phototherapy-based therapeutic on stressor-induced signaling pathways that directly and indirectly affect the induction of dormancy and ultimately the induction of tumor recurrence is very important.
• •The conclusion of this review paper demonstrates that all the parameters in PDT and PTT therapy like pharmacokinetics of nanoparticles shape and concentration, power of laser, wavelength of laser, curation time, induced temperature, ROS production, etc., must be tuned before entering in the clinical trials, for avoiding undesired sophisticated responses including enhanced recurrence and/or invasiveness of tumor cells.

Abstract

Cancer is one of the most common and deadly diseases which has challenged human knowledge since its discovery. There are a variety of therapies, such as radiotherapy, chemotherapy, palliative surgery, medicine, and gene therapy, used for patients suffering from cancer, all of which represent, to some extent, a kind of failure. However, a number of novel strategies based on nanotechnology (including, photodynamic therapy [PDT] and photothermal therapy [PTT]) have been introduced in recent years, which appear to have the ability to be used as an auxiliary treatment for chemotherapy and radiotherapy in the future. Interestingly, cancer cells, in response to various therapeutic factors, can utilize certain cellular mechanisms and processes for their survival and growth. For example, macro autophagy/autophagy, can alter the cancer cell fate, from autophagic cell death to dormancy (as a key factor in tumor recurrence). Nonetheless, there have been conflicting conclusions regarding the interactions of phototherapy methods with cells as well as their wanted or unwanted impacts on these cells. In addition, there are complicated and controversial associations among the most common cell death mechanisms such as apoptosis, autophagy, and necrosis as well as tumor treatment and recurrence. The present review aims to describe various responses of cancer cells to photo-based therapy, which can control cell death or dormancy. Moreover, we discuss whether the incorrect application of various phototherapy parameters not only fails to treat and eradicate cancerous tumors, but also mediates the entry of tumor cells into the dormancy phase or even promotes a cancer flare up.

Graphical abstract

Introduction

Cancer is one of the most common and deadly diseases that has challenged human knowledge since its discovery. Unfortunately, despite a tremendous amount of research on this topic, relatively little progress has been made with regard to the effective treatment of cancer. Cancer is a non-contagious disease and the second leading cause of death worldwide. In 2024, approximately 1.9 million new cases of cancer are expected to be diagnosed in the United States, and over 600,000 deaths will be recorded due to cancer in the same year [1]. Overall, almost all of therapeutic strategies after a certain interval (from several months to several years), lead to recurrence of most cancers from either the same starting point or another location. In fact, due to the complicated and adaptable nature of cancers currently used treatments change the therapeutic equations in favor of the tumor. For example, migrated and reprogrammed macrophages to the M2 phenotype (producing anti-inflammatory cytokines) into the tumor microenvironment (TME), in response to platinum-based drugs as well-established chemotherapeutic agents, resulted in tumor progression and aggressiveness [2,3]. Cancer recurrency is highly sophisticated processes along with the inability of the immune system to accurately identify cancer cells because of the lack of completely specific markers, remains one of the most important challenges in cancer treatment [4]. Furthermore, studies over the past century have indicated that cancer cells resist therapeutic strategies through a variety of mechanisms, including reduced expression of specific receptors, specific transporters, and altered intracellular signaling [5], as well as through the use of certain inherent cellular mechanisms such as autophagy and dormancy [6]. Surprisingly, the mentioned mechanisms strictly correlated with the type of programmed cell death PCD including (1) apoptosis (previously referred to as type I PCD), (2) autophagy (type II PCD), and (3) programmed necrosis or necroptosis (type III PCD) [7]. More importantly, PCD dysfunction can result in a variety of diseases, including degenerative diseases and cancers. The ability of cancer cells to resist apoptosis and activate autophagy can be considered as a crucial factor for cancer development [8]. It is important to note that an identical stimulus can often simultaneously initiate apoptosis, autophagy, and necrosis. Nevertheless, there are complicated and controversial associations among apoptosis, autophagy, and necrosis; for example, autophagy has the ability to promote, delay, or inhibit the occurrence of apoptosis [9,10] (Fig. 1). One of the most famous examples related to crosstalk between the type of stress and the corresponding cellular response, is activating autophagy in response to doxorubicin (Dox) administration [11]. Occurring autophagy could be coupled with highly sophisticated intracellular mechanisms that finally results in dormancy of cancer cells until environmental clues lead to the recurrence of the cancer cells, which are typically even more aggressive than before the treatment [12,13]. Therefore, the type, quantity and quality of the cellular death completely could change the outcomes of chosen therapeutic strategy as external stress to cancer cells.

Fig. 1.

A heterogeneous tumor can enter into one of three main cell death mechanisms in response to different internal or external stimuli. Apoptotic cells are vascularized (pink) which will be removed by immune cells; necrotic cells burst (orange), which releases all growth factors and pro-inflammatory cytokines into the tumor microenvironment; and the effect of autophagy (blue), which is based on the autophagy magnitude and crosstalk with other molecules can end in autophagic cell death, apoptosis or survival.

In the case of photo-based therapies (PDT and PTT), as relatively newborn treatments mostly built on nanotechnology, undesirable side effects which could be happened according to exerted parameters has been neglected in the past two decades. For instance, we recently demonstrated that mild hyperthermia induced by gold nanorods acts as a dual‑edged blade in the fate of SH‑SY5Y cells via autophagy. Importantly, our findings also suggested that significant changes occur in genes related to the crosstalk between autophagy, dormancy, and metastatic activity of the treated cells. Also, we illustrated that PTT (which recently entered into human clinical trials) [14], enhances the aggressiveness of cancer cells at 43 °C, in contrast to 48 °C dependent on the regulation of autophagy [15]. In the parallel study, our team have shown than intracellularly heating the cancer stem like cells (CSC) increase the invasiveness of the cells through autophagy activated manner. Therefore, it seems the impact of stress which induced to cancer cells would be changed the fate of cancer cells. Given the above, the present review tries to describe and summarize a variety of cancer cell responses to photo-based therapy, as well as explains the key players controlling cell death or dormancy.

Impact of crosstalk between autophagy, apoptosis, and necrosis on tumor growth

Introduction

Despite their considerably distinct morphological characteristics and physiological processes, apoptosis, autophagy, and programmed necrosis still exhibit complicated interrelationships. Apoptosis and autophagy show synergetic effects under some conditions, while otherwise autophagy can be triggered only following apoptosis suppression [16,17]. In this section, we will discuss the crosstalk between three classical forms of cell death, including apoptosis, autophagy, and necrosis.

Crosstalk between apoptosis, autophagy, and necroptosis

CFLAR/cFLIP (CASP8 and FADD like apoptosis regulator) proteins possess CASP8-like structures but lack proteolytic activity. CFLAR possesses three main isoforms consisting of one long protein, CFLAR-L/cFLIPL, and two short proteins, CFLAR-S/cFLIPS and CFLAR-R/cFLIPR, in humans [18]. Nonetheless, these three isoforms take advantage of two death receptor domains at the N-termini, which mediate their interaction with the adaptor protein FADD to form the DISC complex. CFLAR has the ability to regulate both the death receptor-mediated extrinsic apoptosis pathway and death receptor-independent apoptosis pathways. In complex IIb/ripoptosome, homodimeric CASP8 induces apoptosis through cleaving RIPK1 and disassembling complex IIb/ripoptosome. Once pro-CASP8 forms a heterodimer with CFLAR-L/cFLIPL, not only does the cleavage of RIPK1 inhibit necroptosis but also the lack of activated CASP8 formation blocks apoptosis [19]. Nevertheless, the formation of a heterodimer by pro-CASP8 with CFLAR-S/cFLIPS-CFLAR-R/cFLIPR induces necroptosis due to the lack of proteolytic cleavage of RIPK1 [20] and simultaneously cannot trigger CASP8-dependent apoptosis. As a result, the presence of CFLAR isoforms in the ripoptosome specifies whether cells will execute RIPK1-dependent necroptosis or CASP8-dependent apoptosis. CFLAR, in addition to the regulation of apoptosis and necroptosis, is a negative regulator of autophagy. When autophagosome is being formed, ATG3 binds to MAP1LC3/LC3 (microtubule associated protein 1 light chain 3), a homolog of yeast Atg8 that is involved in autophagosome maturation. Importantly, the interaction of ATG3 and LC3 is inhibited by CFLAR due to competitive binding of ATG3, which consequently inhibits autophagy [21]. Although the process in which CFLAR regulates apoptosis and necroptosis via ripoptosome formation takes place in the plasma membrane, autophagy is prevented by CFLAR at the autophagosome formation sites (Fig. 2). Therefore, various subcellular localizations of CFLAR may play a critical role in its actions [21,22].

Fig. 2.

The effect of the nature of cell death on the fate of tumor cells. As discussed in the text, cytokines released to the microenvironment of the tumor following necrosis can stimulate cell proliferation, invasion, metastasis, or immune system paralysis. The magnitude of autophagy can lead to EMT and it associated pathways and can be combined with the above-mentioned consequences, which are shown the in figure as a reciprocal relationship between two purple boxes. The reciprocal relationship is indicated with up and down arrows.

The good, the bad, and the ugly

Based on cancer biology and treatment, it seems that apoptosis can be the best option for the elimination of cancer cells in comparison with other types of cell death (especially necrosis) (Fig. 2). As mentioned above, this is due to the ability of necrosis to release cancer cell contents to the tumor environment. Consequently, the release of pro-inflammatory cytokines, such as growth factors, IL4, TGFB1, and CSF1 (colony stimulating factor 1) stimulates the growth and survival of cancer cells through the activation of transcription factors, such as NFKB (nuclear factor kappa B), STAT3 (signal transducer and activator of transcription 3), and AP-1 [23]. In addition, these chemokines lead to more invasive stimulation through invoking innate immune cells specially tumor-associated macrophages (TAMs) and secretion of enzymes digesting extracellular matrixes, resulting in tumor cells becoming more invasive [24]. It should be said that this mechanism is also true in inflammation after the tumor growth, so that inflammation results in increased survival and growth of tumor cells; this growth then leads to secretion of inflammatory factors and other agents intensifying inflammation as a cycle. It is therefore important for scientists to realize that necrosis is designed and genetically controlled by the tumor cells for the development and survival of the entire tumor [25]. Therefore, STAT3, one of the cellular survival signaling intermediates, is activated in response to growth factors and pro-inflammatory factors, including IL6, HGF (hepatocyte growth factor), IL22, IL11 and EGF (epidermal growth factor), released from cancer cells, resulting in their growth and survival [26]. As mentioned above, these factors are originated from the major intermediates of inflammation caused by tumors, which have an undeniable impact on tumor recurrence and growth [27]. Moreover, the importance of cytokines has been shown through the effect of danger/damaged-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs) on tumor growth. Autophagy, as compared with the two methods inducing cell death as mentioned above, can be used for both cell death induction and survival; in addition, based on the above, an important question is whether or not tumor therapeutic protocols leading to increased stress induction to the tumor can promote tumor growth in the long term. Results from extensive studies on drug delivery to the tumor have indicated that various agents, such as high hydrostatic pressure, hypoxic environment, and, in some cases, the lack of angiogenesis, do not allow the entry of therapeutic agents to deep parts of the tumor [28]. Consequently, most common treatments remove only some parts of the tumor through different mechanisms. Thus, we can conclude that different types of therapy will stimulate different types of cell death. In fact, we face three options of the good (apoptosis), the bad (autophagy), and the ugly (necrosis). Nevertheless, it is also important to note that apoptosis has been inhibited in most cancer cells, and gene therapy (like TNFRSF10A/TRAIL [TNF receptor superfamily member 10a] delivery to cells combined with chemotherapy drugs) associated with other therapies seems to be a suitable solution to circumvent this problem. In the following, we discuss which type of cell death is further activated by photo-based therapy.

Dormancy and recurrence of cancer

Tumor recurrence and metastasis, leading to a high percentage of cancer-related death, are phenomena occurring long after the tumor treatment. These outcomes emerge in such a way that the patient has no clinical symptoms after treatment and before the recurrence of the tumor due to dormancy of the tumor cells. Dormancy of tumors was first shown by Willis [29], in which the dormant cells stop growing at the G₀-G₁ stage of the cell cycle [30]. In fact, under certain conditions (especially environmental stress) such as hypoxia, food deprivation, and stimulation by various drugs and phototherapy-based methods, the dormant cells experience a balance between apoptosis and cell division. There are a variety of factors playing roles in the induction of this state, including formation of cell attachment to the extracellular matrix, various pro-inflammatory and growth factors in the tumor microenvironment, interaction between various cell types in this environment, especially immune system cells with dormant cells, ROS and thermal stress, as well as intracellular signaling of dormant cells (Fig. 3 and Table 1) [31]. In general, this state is a highly effective defense mechanism in tumor cells, resulting in the escape of cancer cells from the factors damaging the cells [32]. However, what is important about dormancy is that these cells have the ability to pass this state and re-grow, a phenomenon called tumor recurrence. Another important issue is that dormancy can occur both at the tumor level and in isolated tumor cells, in particular cancer stem cells (CSCs). Furthermore, in recent years, clinical findings have reported cancer cell dormancy in response to different types of stresses like chemotherapeutics in various cancers [33] (Table 1).

Fig. 3.

Tumor recurrence and metastasis, leading to a high percentage of death from cancer, are phenomena occurring long after the treatment of the tumor. Dormant cells stop growing in the G₀-G₁ stage of the cell cycle. Different stimulators are shown as a blue ellipsoid and inhibitors of dormancy are shown as a green ellipsoid in the figure. “+” and “-” indicate stimulatory and inhibitory effects and down arrows are used to depict a decrease of receptors.

Table 1.

Dormancy inductive and reactivation factors.

Extracellular inductive factors for dormancy	Intracellular inductive factors for dormancy	Reactivation factors	Ref.
TGFB2-TGFBR3, BMP4, BMP7-BMPR2, AXL -GAS6, TBK1, Mir222/223, Mir23b, LIFR-STAT3-SOCS3, BHLHE41	MAPK14 CDKN2A/p16, CDKN1A/p21, PSMD9/p27, TRP53, MAP2K4, NR2F1-RARB-SOX9	MAPK1, PI3K-AKT1, TSC22D3, FOXO3, TGFB3, MERTK, POSTN, SFK, FOSL1, DAND5	[34]

AXL, AXL receptor tyrosine kinase; BHLHE41, basic helix-loop-helix family member e41; BMP4, bone morphogenetic protein 4; DAND5, DAN domain BMP antagonist family member 5; FOSL1, FOS like 1, AP1 transcription factor subunit; FOXO3, forkhead box O3; GAS6, growth arrest specific 6; LIFR, LIF receptor subunit alpha; MAP2K4, mitogen-activated protein kinase 4; MERTK, MER proto-oncogene tyrosine kinase; NR2F1, nuclear receptor subfamily 2 group F member 1; POSTN, periostin; PSMD9, proteasome 26S subunit, non-ATPase 9; RARB, retinoic acid receptor beta; SFK, Src family tyrosine kinases; SOCS3, suppressor of cytokine signaling 3; SOX9, SRY-box transcription factor 9; TBK1, TANK binding kinase 1; TGFBR3/TGFB1-RIII, transforming growth factor beta receptor 3; TSC22D3, TSC22 domain family member 3.

It is important to note that, among different types of PCD, only autophagy shows crosstalk with cancer dormancy. The first study on the role of autophagy and its crosstalk with dormancy effectors was presented by Gupta et al. using a PDGFR (platelet derived growth factor receptor) inhibitor; they indicated that a group of gastrointestinal tumor cells experiences dormancy through autophagy [35]. In addition, Lu et al. demonstrated the role of a RAS homolog inhibitor (DIRAS3/ARHI [DIRAS family GTPase 3]) in the induction of reversible dormancy induced by autophagy [36]. Such studies, showing a close relationship between autophagy (and especially its level) with dormancy and metastasis, led to the testing of autoimmune inhibitors in clinical and preclinical trials in order to enhance the therapeutic benefits of common methods and treatments [37]. Furthermore, it should be noted that these findings may be a promising platform in the future for the recognition of mechanisms involved in the above-mentioned processes (which, in most cases, differ from one type cancer to another); in this light, it seems that gene-based therapies and inhibitor drugs might be designed for any type of cancer with its own specific characteristics [38,39].

Why and how photo-based therapy induces apoptosis, necrosis, and autophagy

Photo-based therapy definition

Photo-based cancer therapy has emerged as a novel therapeutic strategy to tackle a wide variety of cancers. Cancer phototherapy strategies have received increased attention in recent years because of their unique characteristics, including minimal systemic toxicity, non-invasiveness, tumor specificity, and localized cancer therapy [14,40]. In general, cancer phototherapies induced by ultraviolet (UV), visible (Vis), and near-infrared (NIR) lights have the ability to penetrate into body tissues, which typically depends on the phototherapeutic agents [41]. There are a variety of phototherapies with different mechanisms, including low-level laser therapy (LLLT), photodynamic therapy, photothermal therapy, photo-induced chemotherapy [42], and photo-immunotherapy (PIT) [43]. Various studies have revealed the impact of light on biological processes, known as photo-biomodulation (PBM) [44,45]; this phenomenon refers to the use of special light spectra that can interact with biological materials and cells and alter their behaviors (Fig. 4). NIR is a light spectrum which is important in PBM. The therapeutic effect of NIR light was demonstrated on healing wounds, repairing damaged cells as well as stimulating physiological processes. This phenomenon was first indicated clinically in the form of a low-level laser therapy (LLLT) in 1960.

Fig. 4.

Various studies have revealed the impact of light on biological processes known as photobiomodulation (PBM). This phenomenon is based on the use of special light spectra that can interact with biological materials and cells and alter their behaviors.

Translationally into the clinics, the substantial question about the use of lasers for various purposes, such as rejuvenation of skin or improvement of muscle performance in athletes [46], is what impact will these radiation exposures have on the stimulation of tumors in the case of undetected tumors in individual people [47]? Another question, posed in previous sections and repeated here again, is whether or not phototherapy is able to change the genetic expression of the treated cells and the signaling pathways related to autophagy and dormancy, and what are the consequences? Of course, as mentioned above, this feature completely depends on different parameters such as wavelength, laser intensity, radiation duration, and the cancer type [48] (Fig. 5). Importantly, our recent work on highly heat-resistant human neuroblastoma cells revealed that acute and intracellular induction of mild hyperthermia (43–48 °C) leads to increased aggressiveness of remnant cells via the stimulation of autophagy at the genomic level (86 genes involved in autophagy process) and based on a tumor spheroid formation assay [49,50]. Our findings, alongside the entrance of PTT into the human clinical trial phase, raises alarms for all cancer researchers. It seems that PTT may lead to a temporary reduction in the size of the cancerous tumor; however, will the remaining cells survive with the same phenotype and even the previous genotype? Or rather will they become more aggressive due to the biological changes discussed below. Our study is the first to show the relationship among acute intracellular temperature stress, signaling pathways and cell death and the consequences on the aggressive behavior of cancer cells. Therefore, it seems that more detailed studies are needed for different treatments to select the best treatment option. In the following, we discuss the impacts of different laser intensities on processes and signaling pathways, as well as their effects on various cell organelles.

Fig. 5.

Different parameters of the indicated lasers are indicated.

Low level laser therapy

Introduction

Generally, low-level laser therapy, a minimally-invasive modality for cancer therapy, is used as a monochromatic coherent light at low energy levels, typically radiation from 1 to 1000 mW/cm² [44,51]. LLLT is able to regulate different biological possesses, such as differentiation [52], proliferation [53], cell viability [54], and cell death [55]. The molecular mechanism by which laser radiation exerts its effect has not been completely understood. The photo-biological effects of LLLT occur when the light energy is absorbed by intracellular chromophores, such as NADH, FADH₂, cytochrome C oxidase, mitochondria, and cAMP which can be induced according to PDT parameters (Fig. 6), eventually leading to the generation of a photo-signal [56,57]. The photo-signal leads to increased cellular ATP levels [58], transiently increased intracellular calcium concentration [59], the phosphorylation of MAPK [60], production of ROS [61], and upregulation of certain proliferation and cell death genes [62] (Fig. 6).

Fig. 6.

LLLT interactions with important cellular chromophores, including NADH, FADH₂, cytochrome c oxidase, mitochondria, and cAMP are depicted, along with the photo-signal in the cells. Arrows show pathways inhibited or stimulated by photo-signals.

Furthermore, ROS produced under laser irradiation have the ability to activate a number of intracellular and extracellular biological pathways, including apoptosis, autophagy and necrosis (Fig. 7) [63]. More recently, a number of studies have investigated the effect of ROS on the mechanism of cell death [64]. It was found that ROS play a crucial role in the induction of autophagy and apoptosis through various mechanisms, such as inactivating the de-lipidation activity of ATG4 [65], activating AMPK [66] altering the expression of BECN1, inhibiting PI3K-AKT1 (AKT serine/threonine kinase 1)-MTOR-RPS6KB1 (ribosomal protein S6 kinase B1), activating MAPK8/JNK1 signaling pathways [67], inactivating AKT1-GSK3B (glycogen synthase kinase 3 beta) [68], and activating NFKB [69], caspase family proteins and PARP1 [70]. In the following section, we will explain the association between phototherapy and these intermediaries in greater depth (some examples are included in Fig. 7 and Table 2). It is worth mentioning that the biological and cell death capability of LLLT may depend on the cell types and laser parameters such as wavelength [71], exposure time, applied energy, focal spot size, energy density, and power density [72]. LLLT containing different irradiation energies and powers demonstrates a biphasic dose response curve, so that its low dose seems to have useful therapeutic effects (photo-biomodulation), whereas higher doses are detrimental (phototoxic) [72].

Fig. 7.

ROS produced by PDT have different effects on cellular functions.

Table 2.

The effects of low-level laser therapy on cell signaling pathway.

Laser Wavelength	Cell exposed	Effects	Signal pathway activation	Refs.
830 nm	PMN cell	Balance alteration between expression of pro-apoptotic (BAX and TRP53) and anti-apoptotic (BCL2) proteins	Apoptotic cell death
A continuous wave 810 nm laser	OECM-1 cell Ca9–22 cell	Enhanced CASP3-CASP7 activity ROS-mediated RELA activation Increased expression of BECN1 and LC3-II	Apoptotic cell death Autophagy protective role	[73]
Nd-YVO4 (532 nm laser)	U251 glioma cell	Increased ROS-mediated BECN1 and LC3-II	Autophagy protective role	[74]
1064 nm 980 nm 810 nm 660 nm	Rat model of mucositis	Expression of Atg5, Becn1, and Ulk1 increased by 1064-nm laser wavelength, significant decrease in the expression of Becn1and Ulk1 by 980-nm laser wavelength, decreased expression of Atg5, Becn1, and Ulk1 by 810-nm laser wavelength, decreased expression of Atg5 and Becn1 by the 660-nm laser wavelength Ulk1 is increased at all wavelengths except 660 nm	Stimulate autophagy on all wavelengths, except 660-nm laser wavelength	[75]

RELA, RELA proto-oncogene, NF-kB subunit.

Low-level laser therapy and autophagy

LLLT can activate autophagy in cancer cells in which autophagy may not only serve as a cell survival mechanism (a protective role) but may also induce cancer cell death [76,77]. It seems that activated autophagy, in response to LLLT, may be highly dependent on the cell type, tissue type and gene expression profile that regulates autophagy [78]. A study by Bostanciklioglu et al. investigated the effects of various laser wavelengths (660, 810, 980, and 1064 nm) on the expression of autophagy genes (Atg5, Atg7, Becn1, and Ulk1) [79], showing that gene expression profiles of Ulk1 can be promoted at all wavelengths, except for 660 nm [79]. Moreover, the expression profile of Atg5, Atg7 and Becn1 genes increases in laser radiation with 1064 nm but decreases in other wavelengths (Table 2). Interestingly, in a low-level of laser therapy, autophagy plays a protective role by repairing the damage in ER and mitochondria; however, both apoptosis and autophagy may induce cell death with an increased level of phototherapy [80]. Frigo et al. indicated that the low-dose (150 J/cm²) 660-nm laser treatment regimen may reduce the size of the superficial melanoma tumors, but a high dose (1050 J/cm²) greatly increases the tumor size [81]. In this study, the authors did not indicate the exact mechanisms involved in this cellular behavior. Based on the above-mentioned examples, however, it can be concluded that treatment of cancer cells with low-power lasers may unintentionally result in the increased survival of the cancer tumor and stimulate its growth and stability, which can greatly influence the treatment results. It should also be considered that, due to the sharp decline in laser radiated to the target tissue (after absorption) by various biological entities [82], the intensity of the laser radiated to the tumor site alters and, ultimately, a laser dose reaching the cells unintentionally leads to further growth due to secretion of different types of pro-inflammatory cytokines or tumor dormancy through using the mechanisms mentioned above. Consequently, one of our suggestions in this review is to conduct research in this field to determine a suitable dose of laser treatment to minimize this possibility. However, given the cases already described in this review, the type of cell death (such as necrosis) occurring in some cells may have played a role in this phenomenon. For example, the release of inflammatory and pro-inflammatory factors or changes in different cellular signaling states might be a fundamental reason for the results obtained. This claim is somewhat supported by a study carried out by Hao et al. [83]. In another study conducted on a nude mouse model of thyroid carcinoma, Rhee et al. showed that HIF1A (hypoxia inducible factor 1 subunit alpha) and phosphorylated AKT1 signaling increase in response to a laser (650 nm, 100 mW/cm²) and, consequently, tumor growth is stimulated [84].

From translational points of view, these conflicting outcomes in the cell behavior become more complex in response to photo-based therapy methods when a laser is used to stimulate other molecules. Photodynamic and photothermal therapies are modern therapies, in which the stimulant molecules are used to produce ROS and heat. Consequently, the impact of the sum of laser and photo-synthesizer molecules or light-to-heat converting molecules on the behavior and fate of cells becomes much more complicated in terms of the type of cell death or autophagy induction and dormancy. Given the above, it can be concluded that the type of optical stress applied to the cell can have a direct impact on the cell fate. As mentioned, because of the increased survival of cancer cells due to low-intensity laser radiation, this treatment seems to pose a serious danger to researchers for designing effective therapeutic procedures. This means that treatments may not only lead to tumor improvement, but also unwittingly encourage cancer cells to survive and resist treatment.

Photodynamic therapy

Introduction

PDT is a promising therapeutic approach used for a variety of cancer therapies [85]. PDT has been approved as a treatment option by the United States Food and Drug Administration/FDA for endo-bronchial and endo-esophageal cancer [86], as well as for premalignant and early malignant lesions of skin (actinic keratosis), bladder, breast, stomach and oral cavities [87]. PDT is based on the application of a photosensitizer (PS) with irradiation of low energy (non-thermal) light [88]. Cells containing photosensitizer agents are exposed to low-energy light, which activates PS molecules that then react with oxygen molecules and finally produce different cellular ROS inducing singlet oxygen (1O²), hydroxyl radical (OH°), hydrogen peroxide (H₂O₂), and superoxide (O²−), resulting in photo-oxidative damage in biological molecules [89] Fig. 8.

Fig. 8.

Photosensitizer type, charge, hydrophobicity and their tendency to accumulate within a specific organelle are illustrated. All PS molecules in different organelles invoke the same pathways; however, this occurs through different mechanisms and may have alternate outcomes.

As mentioned above, the exact mechanism taking place in PDT includes alteration of an inert PS through electron transfer from the light photon to the PS. In addition, the active PS can also lose energy by development of a type I photochemical reaction. This Fenton reaction makes highly destructive free radicals. Nevertheless, generation of a type II photochemical reaction, also known as PDR, is considered to be the most significant pathway for clinical PDT. Here, the PS interacts with oxygen for the generation of singlet oxygen, a process considered as a basis of PDT for tumor and vascular ablation. The half-life of singlet oxygen is found to be around 40 nanoseconds, allowing for destruction of a radius of 20 nanometers [90]. PDT-induced tumor destruction can happen through programmed (apoptotic) or non-programmed (necrosis) pathways [91,92]. In general, the application of high light intensity leads to rapid elimination of tumor cells through necrosis [93]. The cellular and subcellular membrane is rapidly destroyed in this manner.

In PDT, ROS produced by synthetic PS are effective only in the subcellular organelles where the PS is localized. Thus, the subcellular organelles targeted by PS are considered as a main parameter to elucidate the mechanism of PS-induced cytotoxicity against cancer cells [94]. Various PSs used can be localized in different subcellular organelles mainly in the endoplasmic reticulum, mitochondria, lysosomes, plasma membrane, and nucleus (Table 3). Finally, PDT is able to cause cancer cell death through induction of apoptosis, necrosis, necroptosis, and autophagy [95]. Nonetheless, the exact mechanisms and relationships are complicated and depend on the photosensitizer type, cell density [96], cellular genotype (for example, MCF-7 is deficient in ATG7) [97], treatment doses, and subcellular localization of PS [97,98]. These effects will be discussed separately in the next subsections.

Table 3.

The effects of photodynamic therapy on cell signaling pathways.

Photosensitizer type	Laser wavelength	Cell exposed	Subcellular target	Effect	Signal pathway activation	Refs.
UCNP-GQD/TRITC nanoparticles	980 nm	4T1 cells	Mitochondria	CASP3 activation	Apoptotic cell death	[99]
MPPa	LED light (635 nm)	A549 cells	Mitochondria	CASP3-CASP9 activation BCL2 expression decreased BAX expression increased	Light and drug dose-dependent apoptotic cell death	[100]
5-ALA	LED light	PC12 and CL1–0 cells	Mitochondria	AMPK activation	Autophagic cell death	[101]
MPPa	LED light	MG- 63 cells	Mitochondria	Increasing expression levels of cleaved CASP3, CYCS/cytochrome c release, increased BAX expression, decreased BCL2 expression Increasing phosphorylated MAPK8, LC3-II and BECN1	Apoptotic cell death Pro-death autophagy	[102]
phthalocyanine	> 650nm	MCF-7c3	Lysosomes	CASP3 activation production of tBID from BID cleavage of PARP1 loss of BCL2	Apoptotic cell death	[103]
NPe6	LED light	ASTC-a-1	Lysosomes	BAX activation CYCS/cytochrome c release CASP3-CASP9 activation	Apoptotic cell death	[104]
NPe6	664 nm	Glioblastoma T98G cells	Mitochondrial	RIPK1 and RIPK3 pathways	Concentration-dependent necroptosis	[105]
PpIX	633 nm	PROM1/CD133+ cells	Not reported	Increased expression of LC3-II, ATG5, ATG12	Autophagy protective role	[106]

GQD, GQDs, graphene quantum dots; MPPa, methyl pyropheophorbide-a; NPe6, mono-L-aspartyl chlorine e6 (photosensitizer); PROM1, prominin 1; UCNP, upconverting nanoparticles.

Photodynamic therapy and autophagy

Autophagy, as a mode of cell death following PDT, may be a dominant factor in the absence of apoptosis [107,108]. Autophagy can serve as a factor in PDT at two different points in the sequence of events. The first includes the capability of autophagy to repair photo-damaged cellular components, such as organelles [109]. In the context of PDT, the nature of most photosensitizing agents tends to cause their migration to relatively hydrophobic intracellular sites, including membranes; reaction with important proteins plays a role in apoptosis and autophagy, including BECN1 [110] and ITPR3/IP3 [111] in the ER (ROS-mediated damage to the sarco/endoplasmic calcium-ATPase ATP2A2/SERCA2 [ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2] pump, followed by ER-calcium depletion) [112] and anti-apoptotic proteins of the BCL2 family, BAX and BAK1 (BCL2 antagonist/killer 1) located in the mitochondria [113,114]. The optical toxicity caused by the treatment with this light spectrum is evoked by stimulation, while ER stress is induced by ATF4 (activating transcription factor 4) and autophagy induction. For example, in a study conducted in 2017, it was indicated that PBM can intensify the autophagy- and apoptosis-based cell death induced by radiotherapy [115].

Second, and more interestingly, the double-edged nature of autophagy means that it may act as a mechanism involved in stimulating cell death or cell survival [116]. Autophagy can block apoptotic pathways, protecting cancer cells from treatment [117], and PDT may induce autophagy, based on the ROS type and oxidative injury degree [118] (Table 4).

Table 4.

ROS interaction with intracellular compartments.

Types of ROS	Interacting Cell Component	Effect	Refs.
[O2]•− and H2O2	SHC/p66shc	Apoptosis Growth signaling	[119,120]
H2O2 or HOCl	React with thiol side chains of cysteine in a nonspecific manner	Two-electron mediated oxidation and releases of H2O or Cl− and finally formation of sulfenic acid	[121,122]
H2O2	PTPN1/PTP1B (protein tyrosine phosphate non-receptor type 1)	Redox system imperfection	[123,124]
Sulfenic acids (highly reactive intermediates of ROS)	Internal thiols	Intramolecular disulfide species formation	[125,126]
H2O2	PTEN, PTPN1	Reversibly deactivation of these phosphatase	[127,128]
H2O2	YAP1, FOXO4	Activation of redox regulator genes	[129,130]
H2O2	MMP7 (matrix metallopeptidase 7)	Activation of the pro-enzyme	[131]
H2O2	Histone deacetylase, HDAC4 control by activation of TXN	Cardiac hypertrophy	[132]
Oxidative stress	Redox-cycling metal ions such as Fe2+ and Cu2+, Conversion of the side chain amines to carbonyls	Lysine, arginine, proline and histidine residues oxidation	[133]
HOCl	Tyrosine residues	Impaired protein function in atherosclerosis, diminished airway function in children with cystic fibrosis	[134,135]
H2O2	PerR (TF)	Regulates redox defense genes	[136]
[O2]•−	SoxR (TF)	Oxidation of an iron-sulfur cluster	[137]
ROS	NADPH/NOX	Inhibit tumor apoptosis and stimulate tumor proliferation	[138]
ROS	KRAS	Increase expression and activity of NOX enzymes: promote ROS/RNS generation	[139]
H2O2	PI3K-AKT	Stimulate cancer cell proliferation	[140]
Increased detoxification of ROS	Metabolic reprogramming	Increase of endogenous antioxidant machinery	[141]
H2O2	Signal transduction	Cell proliferation by STAT3 activation.	[142]
(•OH) (ONOO-)	H2 gases as trace element in some signaling pathway	Antioxidant, anti-inflammatory, anti-apoptotic, anti-tumor functions Pancreatitis, systemic inflammatory syndrome, sepsis, and neurodegenerative diseases.	[143]
ROS	H2 gas	SOD and CAT activation Regulation of oxidative stress damage Activation of MAPK14/p38, MAPK1-MAPK3, (NFKB), MAPK/JNK, and NF2EL2 MPO, malondialdehyde, 8-isoprostaglandin F2a, and thiobarbituric acid reduction	[144]
ROS	H2 gas	Downregulation of IL1B, IL6, (TNF), CCL2, intercellular cell adhesion molecules, (HMGB1), and prostaglandin E2. Restoration of regulatory T cells	[145,146]

CAT, catalase; HMGB1, high mobility group box 1; MPO, myeloperoxidase; PTEN, phosphatase and tensin homolog; PTPN1/PTP1B, protein tyrosine phosphate non-receptor type 1; SHC/p66shc, SHC adaptor protein; SOD, superoxide dismutase; TXN, thioredoxin.

In addition, autophagy plays a protective role against PDT in CSCs, demonstrating that targeting autophagy can be tailored, as a treatment strategy, for the increased sensitivity of CSCs to PDT [147]. However, generally, the associations between changes in the autophagic and dormancy pathways with the ROS levels have not been well defined, and different results have been indicated with different cancer cells. The cellular defense mechanisms leading to cellular survival, which are activated in response to PDT, can fall into two categories. The first one includes intrinsic cellular defense systems such as induction of intracellular antioxidant enzymes [148]. The second one consists of direct effects of PDT on apoptotic pathways, the expression profile of certain cell receptors, changes in the expression of the multidrug-resistant protein family, and, ultimately, increased expression of intracellular chaperone proteins [149,150]. Another established mechanism involved in cancer cell survival is the production of a stress response by PDT-generated ROS, which, as part of a cell survival mechanism, helps cancer cells get rid of the PDT-induced oxidative stress and cell damage. The above-mentioned survival pathways are mediated by transcription factors activator protein 1 (AP-1), NFE2L2 (NFE2 like bZIP transcription factor 2), HIF1A, and NFKB, as well as those that facilitate proteotoxic stress responses. The survival pathways are thought to render some types of cancer recalcitrant to PDT and change the tumor microenvironment in favor of tumor survival [151].

Furthermore, it was documented that the autophagy response plays different roles in apoptosis-deficient and apoptosis-competent cells. In the case of PDT, the induction of autophagy plays an undeniable role in resistance to PDT treatment and serves as a survival response in cells capable of developing an apoptotic response [152,153]. In an exciting study, Ji et al. demonstrated that PC12 and CL1–0 cell deaths induced by 5-aminolevulinic acid-PDT can be impeded by an autophagy inhibitor, rather than a CASP inhibitor, showing that the cell death is autophagic in nature [154]. Their results are in agreement with those of Wang et al. who reported that the inhibition of autophagy decreases induced apoptosis in prostate cancer cells, suggesting that the apoptosis is autophagy dependent [155].

Moreover, it was demonstrated that apoptosis is one of the leading mechanisms mediating PDT-induced tumor cell death [156]. Recently, Ouyang et al. have reported that autophagy and apoptosis are induced by protoporphyrin IX (PpIX)-PDT in HCT116 cells, and the inhibition of autophagy using chloroquine and ATG7 gene silencing strengthens the pro-apoptotic effects of PpIX-PDT [157]. However, it has been reported that autophagy works as a programmed cell death pathway if apoptotic pathways are impaired or when cells are overexposed to photodynamic therapy [[158], [159], [160]]. In addition, it has been described that mitochondrial photo-damage can lead to the activation of the autophagy pathway. According to the above, it could be concluded that the application of mitochondrial-targeting PS molecules may lead to increased PDT benefits. In the following, we discuss the effects of PDT targeting on lysosomes, a highly important organelle in autophagy. Lysosomes are involved in the late steps in autophagy; this may explain why lysosomes may be an especially appropriate target for the initiation of lethal photo-damage [76]. The efficacy of lysosomal photo-damage appears to be partly from both promotion of autophagic stress and suppression of autophagic pro-survival functions [228]. In one study, Kessel et al. showed that the autophagy process is suppressed in cells with photo-damaged lysosomes due to the lack of functional lysosomes [24]. Such a blockage might suppress the potential pro-survival effects of autophagy and/or actually contribute to the cytotoxicity of the photosensitizer [24]. Consistent with these reports, Tsubone et al. showed that apoptosis is not the main cell death mechanism after lysosomal photo-damage [210]. TPPS2a (a porphyrin-based photosensitizer)-triggered HeLa cell death can be related to the loss of autophagic capability rather than the classical LMP [210].

Based on what we have described for lysosomes and their undeniable role in autophagy, this process seems to be activated to induce apoptosis-dependent or -independent cell death in a manner involving lysosomal targeting. Although autophagosomes can be developed in the absence of lysosomes, autophagy cannot be completed because of the lack of this organelle. Last, it seems that autophagy and apoptosis rates are dependent upon the cancer cell type, photosensitizer, and light dosage.

Photothermal therapy

Photothermal therapy definition

Hyperthermia is a method in which cells cultured on a plate and in a hot water bath are kept for different periods at different temperatures. Indeed, physically, heat is slowly transmitted to the molecules of the cell culture medium and then to the intracellular fluid. The results of such therapeutic procedure include improved oxygenation inside tumors due to temperature-induced increased blood flow, increased sensitivity of tumors to anticancer drugs and irradiation [161], and finally increased migration of immune cells to targets, allowing for a better control of tumor burden [162]. Perhaps the fundamental difference in photothermal therapies (PTT) induced by hyperthermia is that the pyrogenic agent is inside the cell, and, suddenly and usually, it takes a few minutes to transmit the temperature into the cell [163] Fig. 9. Based on this difference, information on the induction of thermal shock using these methods is more related to hyperthermia. Using modern therapeutic methods may allow the elimination of a cancer tumor at a particular point, even though one cannot completely immerse the entire body of the patient into hot water! Consequently, PTT can introduce the benefits of not having side effects to physicians more effectively than traditional hyperthermia.

Fig. 9.

The left section of the figure depicts the tremendous effects of PTT on cell compartments, including genetic defects, membrane defects, and protein aggregation. The right section of the figure illustrates cellular defense mechanisms.

Photothermal therapy and autophagy

Among other advantages of nanotechnology on PTT, we can refer to the ability to load chemotherapy agents to NPs and induce chemo-photothermal therapy. For example, recently, Nam et al. have managed to destroy 85% of the cancer cells injected into mice through using this method [164]. Of course, it should be kept in mind that different types of NPs alone can also induce dispersity-dependent autophagy in different ways [165]. Autophagy inhibition can increase the cell killing ability of PTT through the apoptosis pathway, suggesting that combination therapy holds potential promise for future research and potential application of PTT assisted with autophagy modulators in cancer therapy [166]. Heat shock proteins (HSPs) and autophagy play a critical role in thermo-resistance. A variety of studies show that heat-induced apoptosis can be resisted by a class of proteins known as HSPs [167]. In PTT, heat converted from photothermal effects leads to damage of cytoplasmic components, resulting in the activation of autophagy. There are some studies showing that autophagy reduces cytotoxic effects of hyperthermia [168,169]. Xie et al. have shown that autophagosomes accumulate after heat stress as a result of increased autophagic flux in non-small cell lung cancer cells, as mediated by ER stress. Mocan et al. for the first time have revealed that both autophagy and apoptosis simultaneously occur in the same cell populations treated with near ablative temperatures, and that necroptosis may also be associated with this response [170,171]. However, there are studies showing autophagy or necroptotic cell death mechanisms after PTT [172]. Autophagy is proposed as an alternative cell death mechanism to apoptosis in response to PTT [173] (Table 5). Conversely, researchers have further demonstrated that heat-induced autophagy serves as a survival mechanism against apoptosis under in vitro and in vivo conditions [174]. Interestingly, studies have revealed that hyperthermia can result in increased ROS. In a recently-documented study, it has been shown that hyperthermia itself induces more sensitivity to radiotherapy through the production of ROS in colorectal cancer cells by autophagy-induced cell death [175]. This study is consistent with a study showing that the simultaneous use of oxidative stress with hyperthermia could lead to HT-29 cell death by autophagy [176].

Table 5.

Photothermal effects on cell signaling pathways.

Plasmonic NP	Laser Wavelength	Energy	Cell exposed	Subcellular target	Effect	Signal pathway activation	Refs.
Gold nanoprisms/NPRs	1064 nm	30 W/cm2 2 min	Vero cells	Cytosol	Cell membrane rupture	Necrotic cell death	[177]
Gold nanoprisms	(1064 nm	5W/cm2 2 min	MEF cells	Lysosome	BID activation	Intrinsic apoptosis pathway	[178]
Gold nanorod (GNR)	808 nm	3W/cm2 5 min	HeLa cells	Lysosome	apoptosis	Autophagy (pro-survival role)	[179]
Anti-EGFR-GNPs	820 nm	1.5 W/cm2 for 3 min	MDA-MB-231	Mitochondria Lysosome	Inhibiting the AKT1-MTOR signaling pathway	Autophagic cell death	[180]
GNR	808 nm	0.956 W 1.275 W 1.593 W 15 min	BL6-B16 melanoma cells	Cytosolic signaling	RIPK1 activation	Temperature-dependent necroptotic cell death	[181]
GNR	808 nm	20–50 W/cm2 5 min	HCT-116 ATCC	Perinuclear region Lysosomes	Cell membrane and nucleus membrane rupture	Apoptosis	[182]
GNR	808 nm	5.8 W/cm2 Different time course	HSC-3	Nuclear region	Increased phenylalanine	Apoptotic cell death	[183]

Photo-based therapy and tumor recurrence mediators

Autophagy crosstalk by signaling pathway

Studies have shown the role of autophagy in the emergence of tumor dormancy and recurrence by different mechanisms (Fig. 10 and Table 6) [184,185]. Lu et al. have shown that transient expression of this kinase can result in reversible autophagy in ovarian cancer cells, while its chronic expression leads to irreversible autophagy [186]. Furthermore, their studies suggested that the autophagy induced by this kinase (DIRAS3/ARHI) can make the cells enter a hibernation phase and then lead to tumor recurrence. More recently, studies have demonstrated that the suppression of KRAS causes increased autophagy in KRAS^-/- pancreatic ductal adenocarcinoma (PDAC). Similar findings were also documented with the suppression of the KRAS effector, MAPK1 (mitogen-activated protein kinase 1), which might result in promising pharmacological combination strategies for the effective treatment of PDAC [187]. In other words, inhibition of KRAS-RAF-MAP2K/MEK-MAPK1/ERK signaling leads to autophagy, which is capable of protecting PDA cells from the cytotoxic effects of KRAS pathway inhibition. Mechanistically, MAP2K1/MEK1-MAP2K2/MEK2 inhibition results in activation of the STK11/LKB1 (serine/threonine kinase 11)-AMPK-ULK1 signaling axis, a central regulator of autophagy. In addition, combinational inhibition of MAP2K1-MAP2K2 plus autophagy exhibits synergistic anti-proliferative effects against PDA cell lines in vitro and increases regression of xenografted patient-derived PDA tumors in mice. In addition, results from that study indicate that growth factors and pre-inflammatory factors in the tissue can propel the autophagy of this kinase from autoimmune death to survival and tumor recurrence. It is noteworthy that cell dormancy is one of the vital characteristics for CSCs [188].

Fig. 10.

All interactions between photo-based unwanted stresses and important processes which end in tumor metastasis, invasion, post transcriptional changes, angiogenesis, and tumor recurrence are depicted.

Table 6.

Autophagy-regulating factors and tumor fate.

Effectors	Autophagy Level	Signaling Pathway	Tumor Fate	Refs.
ATG4 ATG5 ATG7	High induction	PI3K-AKT1-MTOR and MAPK	Tumor dormancy and recurrence	[184,185]
DIRAS3/ARHI transient expression	Reversible induction	PI3K-AKT1-MTOR and MAPK	Tumor survival	[189,190]
DIRAS3 chronic expression	Irreversible induction	PI3K-AKT1-MTOR and MAPK inhibition	Tumor growth inhibition	[190]
Pro-inflammatory and growth factors	Induction	DIRAS3-dependent survival	Tumor dormancy and recurrence	[188]
Sh3glb1/Bif-1, Uvrag, Sqstm1, Wipi	Induction	DIRAS3-dependent survival	tumor dormancy and recurrence	[191,192]
Bafilomycin A1	Low Induction	ROS generation	Tumor growth inhibition	[193]
Inhibiting the integrin-PTK2-SRC pathway	Unknown	Dormancy	Tumor recurrence	[194,195]
Dormancy	Inhibition	MTOR inhibition	Tumor recurrence	[196]

In the previous section, it was pointed out that the pre-inflammatory factors induced by the explosion of some naturally necrotic cells or following a response to a therapeutic treatment are likely to use the above crosstalk and lead to the survival and recurrence of tumors. In addition to this phenomenon, it seems that some other genes related to autophagy, such as Sh3glb1/Bif-1 (SH3 domain containing GRB2 like, endophilin B1), UVRAG (UV radiation resistance associated), Sqstm1 [192], and Wipi (WD repeat domain, phosphoinositide interacting) [191], activate the same mechanisms. Moreover, in a recent study performed by Ramirez et al. on tumors of the mouse and human models of breast cancer, it has been shown that the Atg7/ATG7 gene plays a critical role in autophagy induction. They, once again, proved that inhibition of autophagy flux considerably eliminates metastatic cells using ROS and mitochondrial damage [197]. This means that the control of autophagy, as one of the best therapeutic strategies, in combination with other therapeutic methods in the clinical and preclinical phases is being advanced; in this regard, we recommend a useful review provided by Rebecca and Amaravadi [198]. It is noteworthy that, in addition to self-induced autophagy in response to the environmental stresses, the type of autophagy inhibitors also affects the therapeutic benefit through apoptosis induction. For example, Komata et al. have revealed that bafilomycin A₁ has a more fatal effect on glioma cells when compared with 3-MA [199]. As described previously, it has been confirmed that the effect of inhibiting the integrin-PTK2/FAK (protein tyrosine kinase 2)-SRC (SRC proto-oncogene, non-receptor tyrosine kinase) in breast cancer cells and increasing the strength (hardness) of the extracellular matrix in lung cancer for metastatic growth is one of the signaling and communication pathways leading to dormancy [199]. In fact, in these studies the inhibitory role of signaling pathways has been shown to be the cellular connections via integrins and the effect of the tumor micro-environment on the metastatic growth of tumors (Table 7). Whether photo-based therapy, as an environmental stressor, activates or deactivates these pathways is a very important question that must be further addressed. In general, some studies have indicated that genetic changes in cancer cells, especially CSCs, have occurred so that they can experience transient growth in response to environmental stresses, even with the activation of oncogenes [200,201]. Nowadays, a variety of results have been obtained about the genetic damages induced by physical and chemical treatments such as radiotherapy, chemotherapy, photodynamic therapy, and photothermal therapy that lead to autophagy induction [202,203]. As pointed out previously, autophagy can have cytoprotective effects in cancer cells [203,204]. Nevertheless, very interestingly, Lu et al. indicated that this dual role of autophagy is fully dependent on the degree of autophagy that occurs in the cell in response to the imposed stress [205]. In fact, based on the results obtained from their valuable study, the group concluded that there might be an autophagy threshold in the cells to propel them to death or survival. They also recognized that the magnitude and rate of autophagy are tied to some cellular survival signaling pathways, in particular involving PI3K. The concept of cellular survival is very important in cancer cells, especially detached tumor cells [206]. That remain in another part of the body in a dormant phase, and it seems to be one of the main reasons for the recurrence of tumors [207,208].

Table 7.

Autophagic dormancy and metastasis mediators and their effects on tumor recurrence and invasion.

Process	Signaling Pathway	Controllers	Effectors	Results	Tumor Fate	Refs.
Autophagic dormancy	Different types of stress	ECM physicochemical characteristic	Cellular stress	Quiescence	Tumor recurrence	[[209], [210], [211]]
		Niche hypoxia miRNA	PDT stress	Quiescence
		HIF1A	UPR	MTOR inhibition
		BECN1, ATG14	NOTCH	Recurrence of dormant cells
		ROS	HIF1A, MAPK, AKT1(PDT stress)	Senescence phase
		ROS	PDT stress (separation of cells from the ECM)	Anoikis
		Drugs (DOX)	autophagy	Quiescence
Autophagic metastasis	EMT	SMAD4, TWIST, SQSTM1, ULK2	TGFB1	Cell phenotype alteration	Tumor invasion	[212,213]
		ATF4 ATG4 IKK	Extracellular dissociation	Cell growth and differentiation
		RHOA	RHO	Cell invasion
		Genetic control	PTK2 activity and recycling	Cell motility
		Genetic control	IL6	Increased MMPs

IKK/IKB kinase, I kappa B kinases; RHO, ras homolog family; RHOA, ras homolog family member A; SMAD4, SMAD family member 4.

The role of the unfolded protein response in metastasis induced by photo-based therapy mediators

The role of UPR proteins, especially HSPA5/GRP78 (heat shock protein family A (Hsp70) member 5) and HSP90B1/GRP94 (heat shock protein 90 beta family member 1), has been confirmed in cancer patients with asymptomatic recurrent tumors and poor prognosis [214]. The study of the signaling pathways involved in autophagy and dormancy illustrates that the ER unfolded protein response and autophagy response are in line with each other. Studies on patient and animal models have indicated that the expression of UPR-regulating genes (HSPA5, HSP90B1, PDI, SERPINH1/HSP47 (serpin family H member 1), and PPIB/cyclophilin B) increases in dormant cells [215,216] (Table 8). The ERN1-XBP1 (X-box binding protein 1) pathway, is the most conserved branch of the UPR [217]. This molecule is an ultimate effector in the advancement and survival of breast tumor. In addition, the study on UPRs revealed that MAPK14/p38 increases the expression of EIF2AK3 and chaperone HSPA5 in response to the toxicity of drugs, consequently leading to drug resistance of HEp3 cells [218]. In the same regard, studies have revealed that the UPRs of the cancer cells are translocated to the cell surface followed by different types of stresses [219]. Table 8 indicates some of the relevant experiments in relation to the interaction of the UPR and EMT.

Table 8.

Evoked UPR signaling effects on metastasis.

Type of UPR induced by photo-based therapies	Intracellular Effects	Tumor Fate	Refs.
HSPA5, HSP90B1, PDI, SERPINH1/HSP47 and PPIB	Autophagic dormancy	Tumorigenicity and tumor recurrence	[216,220]
ATF6, ERN1	Cell cycle stoppage		[221]
XBP1	Dormancy, survival		[222]
EIF2AK3	Survival by increased CCND1, CCND3, and CCNA2, HSPA5		[223]
EIF2AK3-EIF2S1	EMT (FN1, SERPINE1 secretion)		[224]
ATF4	ATG5, ATG7, and ULK1 upregulation	Metastasis	[225]
EIF2AK3 (ATF4-dependent)	LAMP3 (autophagy activation)	Metastasis	[224]
ERN1	XBP1	Metastasis	[226]

EIF2S1, eukaryotic translation initiation factor 2 subunit alpha; FN1, fibronectin 1; SERPINE1/PAI1, serpin family E member 1.

As shown in the next sections, tumor hypoxia is one of the features of tumors always controlled developmentally by genetic and epigenetic factors. Another important branch involved in the UPR pathway is ERN1-XBP1, which is significant in the induction of metastasis. Studies have indicated that XBP1 expression increases in CSCs [227], which is associated with angiogenesis, recurrence and metastasis (Fig. 10 and Table 8). Ultimately, the result of this discussion is that the application of phototherapy techniques, which can considerably activate the ER, may play a major role in metastasis and dormancy induction of tumor cells. Unfortunately, in light of what has been discussed in the previous sections, this pathway is one of the main targets involved in photo-based therapy methods. It is recommended that future studies attempt to select the photo-based therapy parameters in such a way that they make the least interaction with this system. These strategies can even concentrate on the development and increased pharmacological inhibition of the ER or its effector pathways.

The autophagic dormancy and post-transcriptional changes following photo-based therapy and metastasis

As discussed previously, metastatic dormancy emerges as a process in which cells separated from the tumor or in the tumor (particularly in CSCs) can stop growing (dormancy) under certain conditions and environmental stress and will grow again and produce a new tumor sometime later under favorable conditions. The exact mechanisms involved in this process have not been fully determined; however, the role of some physiological cellular processes, such as autophagy, which inherently play a protective role in the cell, has been proven. In addition to the role of autophagy in the induction of dormancy, there are studies demonstrating that autophagy results in an increased and activated metastasis process. Metastasis is a process in which the invasion and motility of cancer cells are facilitated by the epithelial-to-mesenchymal transition process. In this process, cancer cells via increased expression of transcription factors such as SNAI2 (snail family transcriptional repressor 2), TWIST1 (twist family bHLH transcription factor 1) and ZEBRA1 change the nature of cells and enable invasion, extracellular remodeling, escape from the immune system and resistance to apoptosis [228]. These behavioral characteristics originated from the EMT pathway cause the cancer cells to exit the tumor and enter the bloodstream or lymphatic system, resulting in invasion to the extravasation or intravasation organs [229]. It is noteworthy that dormancy is a process in which its occurrence does not depend on environmental stress but rather can happen after separation of the cells from the tumor site [230]. Nevertheless, as discussed above, it has definitely been shown that the induction of dormancy depends on the balance between stimulatory factors or cellular dormancy inhibitors [231,232]. Physicochemical characteristics of the extracellular matrix and epigenetic regulators, such as retinoic acid and hypoxia, can be referred to as some of the most important factors in tumor recurrence and the exit of dormant cells from quiescence [33,233]. To confirm the impact of environmental stress on the development of proper conditions for the exit of dormant cells from quiescence, it was indicated in a study that PDT-induced stress can increase cell metastasis via increased miRNAs involved in hypoxia [234]. Induction in the tumor niche and dormancy. In addition to the direct role of hypoxia in the characteristics of the stemness of the stem cells in the tumor [235], the hypoxic cells stimulate the cell survival pathways by inhibition of MTOR signaling (through activation of transcription factors induced by HIF1A) [236] and stimulation of the compatible responses with the intermediation of the UPR [237]. Following the application of these processes, the EMT pathway is activated, and consequently, by altering the cancer cell enzymes profile, increased invasion of the extracellular matrix and resistance to apoptosis occur.

Beside the various mediators involved in dormancy and autophagy-induced metastasis, the NOTCH (notch receptor) pathway also plays a key role in cancer metastasis. Different studies have indicated that this signaling pathway controls a variety of intracellular biological processes and pathways [238,239]. One of these processes is the activation of biological signaling cascades of regulatory T cells. The role of various cells of the immune system in the growth and survival of cancer cells was discussed above. Reports indicate that NOTCH intracellular domain (NIC) activity in these cells has been demonstrated by MTOR signaling, along with other proteins such as RPTOR (regulatory associated protein of MTOR complex 1) that is used to identify the stressor conditions (Fig. 10). However, the exact mechanisms of this interaction are not well defined although NIC activity is related to BECN1 and ATG14 (important components of the PtdIns3K complex). Scientists have concluded that the relationship between autophagy and NOTCH signaling is another critical factor playing a crucial role in determining the fate of cancer cells [239]. Different clinical studies have revealed that increased expression of NOTCH1 in patients with breast cancer is associated with poor prognosis and a very high percentage of tumor recurrence [240]. In 2015, a study conducted by Abravanel et al. showed that pharmacological inhibition of NOTCH signaling significantly inhibits the recurrence of dormant cells in breast cancer tumors. The results indicate that dormancy is a very significant, rate-limiting step in tumor progression and development, which is affected by NOTCH signaling; this can be considered as a therapeutic target [241]. Another issue is the role of ROS in metastatic and dormancy signaling pathways. In addition to their role in genome instability, ROS can regulate important tumorigenic pathways (such as HIF1A, MAPK, and AKT1) and enter the cell into the senescence phase when their levels increase [242]. The role of ROS has also been demonstrated in metastases, especially in CSCs. In addition, activation of ROS-dependent anoikis (cell separation from the extracellular matrix) has been shown [243]. It seems that the ability of cancer cells to maintain the balance between oxidative stress and ROS levels, is directly related to the activation of signaling pathways associated with cell survival, which control cell clusters and communication [244]. In fact, an important question is whether it is possible to make the cancer cells protect themselves by the formation of cell clusters and, ultimately, create tumors after increased intracellular ROS production using photo-based therapy [245].

For clinical translational purposes, metastasis and dormancy can have two important therapeutic aspects: First, each of the pathways involved in this process can be considered as a clinical therapeutic goal, and second, the parameters involved in therapeutic modalities must be carefully studied to specify the best therapeutic strategy for their unwanted activation. It appears that these approaches can be considered to be similar to the personalized medicine discussion. For example, as mentioned above, the types of cancers (with different pathways of mediators involved in autophagic dormancy induction) should be considered separately. In other words, different parameters of the same treatment procedures should not be considered adequate for all types of cancers. Furthermore, because the level of autophagy plays a critical role in determining the fate of the cancer cells in terms of survival and death, stresses applied to the tumors should be optimized through various therapeutic methods (phototherapy, radiotherapy, and chemotherapy) to have the least chance of metastasis induction. For example, the wavelength, intensity, and duration of radiation should be selected in phototherapy in such a way that they do not lead unwittingly to autophagy-induced dormancy. Our suggestion for such an optimization is to conduct extensive studies on the likelihood of the impact of various parameters on the expression or activation of the pathways involved in these processes. To the best of our knowledge, there are very few studies to date in this area and, in most papers, the researchers have focused only on the inhibition of autophagy and its impact on the benefit of therapeutic strategies. It seems that the lack of studies investigating the relationship between how different temperatures induce different pathways involved in the induction of dormancy, and metastasis is significant.

In clinical set up usage, the stress applied to cells in phototherapy (PDT, PTT) procedures can drive the invasiveness, metastasis, or dormancy of the cancer cells. To better answer this question, we should also consider other questions, including whether the parameters involved in photo-based therapy, such as light intensity, radiated light wavelength, ROS produced following PDT, heat produced in photothermal therapy, and even the thermal shock applied in the phototherapy process (Δt or temperature change per time unit), be considered as factors affecting the onset of the above trends. As discussed, according to our studies, there are very few reports that have examined the altered expression of the above-mentioned genes in response to phototherapy, which will be addressed separately.

In addition to the genetic and physical effects of the cell in response to phototherapy, studies have indicated that this therapeutic strategy also affects post-transcriptional pathways of cells through influencing the expression and inhibition of miRNAs (Fig. 10 and Table 9). Different studies have revealed that the laser type, PS type, laser dose, and radiation duration differently affect the expression of miRNAs in various types of cells. In this section, we will investigate the impacts of phototherapy on the expression of cell cycle, apoptosis and angiogenesis regulator miRNAs derived from factors facilitating tumorigenesis and recurrence as well as cancer metastasis; Table 10 summarizes some of the important studies. For example, MIR335 (microRNA 335) that regulates the proliferation and migration of stem cells increases with LLLT [254]. Furthermore, studies have shown that LLLT also changes the expression of miRNAs involved in cell differentiation [255,256]. In addition to the impact of phototherapy on stem cells, alterations in miRNA expression are associated with tumorigenicity, malignancy phase, differentiation, and growth of various types of tumors [[256], [257], [258], [259]]. According to recent theories it has been determined that hypoxia is essential in the cancer cell niche to maintain the characteristics of CSC stemness [260]. Therefore, increased expression of MIR210, which is expressed under hypoxic conditions, appears to be a practical response, as a defensive mechanism applied by tumors, for the induction of hypoxia in response to PDT. Similarly, a study carried out by Bach et al. showed that the profile of MIR8 that regulates the growth and differentiation of cancer cells significantly increases from its baseline state after 5 h of PDT [261]. However, the same group showed that 1223 miRNAs in humans show no expression change after PDT, based on molecular arrays. Of course, as mentioned in the previous sections, it seems that the reason for this behavioral difference is related to the type of cells as well as the various PDT parameters. For example, in further studies, the same group has shown that some miRNAs (MIR1260B, MIR1260, MIR1280, and MIR1246) exhibit a slight increase in response to a type of photosensitizer [262]. This is while the same profile only has a transient increase in response to treatment with laser light. These findings clarify the importance of further study on photo-based therapy more than ever, as they show how much these treatments affect the various biological pathways. As noted, it seems that such studies have been used more to examine the impact of temperature on tumors. This is a semi-invasive technique in which the patient should be floated in hot water for the therapeutic results to be investigated. As rightly noted, the fundamental difference with photothermal therapies seems to be that the pyrogenic agent is inside the cell; it usually takes a few minutes to transmit the external temperature to the cell. Thus, these approaches may be very different from the previous traditional method in terms of how the thermal shock is applied and the time the cell takes to respond to stress.

Table 9.

MicroRNA profile and their effects on tumor fate in response to photo-based therapy.

Type of photo-based stress	Type of induced MicroRNA	Cellular effects	Tumor fate	Refs.
LLLT	MIR193	Stem cell proliferation	Tumor growth	[246,247]
	MIR193	Genes involved in the cell cycle (CDK2 and ING5)
	miRNAs	cell migration, tumorigenesity, malignancy phase
PDT	Apoptosis-specific miRNAs	Apoptosis induction	Tumor growth inhibition	[248,249]
	MIR210	Hypoxic augmentation of tumor microenvironment	Stemness characteristic of CSCs, tumor growth
	MIR296	Angiogenesis	Tumor growth and metastasis
	MIR634, MIR1246, MIR1290 and MIR487B	Regulation of cell stress, apoptosis, cell adherence and proliferation	Tumor suppression
PTT	miRNA control by HSPs	AGO2 expression	Tumor survival	[250,251]
	MIR10B, MIR15B and MIR139, MIR21	Oncogenic effects	Tumor invasion and recurrence
Cell stress	MIR21, let-7 family	Invasion to target cells	Metastasis invasion	[252,253]

AGO2, argonaut RISC catalytic component 2; ING5, inhibitor of growth family member 5.

Table 10.

Different stressors induced by photo-based therapy affecting angiogenesis.

Different types of stressors inducing angiogenesis	Mediators	Signaling pathway	Refs.
Hypoxia	VEGF increase	HIF1A-XBP1- or ATF4-dependent manner	[111]
ER stress after PDT	VEGF increase	HIF1A, non-AMPK-dependent manner	[271,272]
UPR	HSPA5	VEGF-dependent manner	[273,274]

Nonetheless, different studies have also shown the impact of hyperthermia on the expression of different miRNAs. The expression of miRNAs in different cancers depends on the cancer type and tissue and plays multiple roles such as altering the expression of various growth proteins, growth inhibition, and invasion to target cells [263,264]. In addition, the mentioned roles of miRNAs in cancer cell behavior are also very diverse and vary from tumorigenicity and differentiation to playing a role in EMT [265,266]. One of the most important roles of these molecules discussed in this review is the regulation of metastatic cascades, cellular migration, cellular invasion, binding and alteration of extracellular matrix of the cells nested in different tissues and EMT [267]. MIR21 and the let-7 family are two very important examples playing a vital role in tumorigenesis and metastasis [268]. Different studies have shown that, in normal conditions and in response to environmental stresses, cancer cells secrete these mentioned important miRNAs (in metastasis) to the blood in various packages, such as exosomes, and they communicate with other cells in the form of cell-to-cell communications that affect metastasis pathways [269]. To verify the effect of hyperthermia on miRNA secretion from breast cancer cells, Erbes et al. suggested that breast cancer cells release MIR10B, MIR15B and MIR139 in response to a 42-degree hyperthermia [251]. Given that the secretion of various miRNAs to the blood has been one of the most important characteristics of cancer cells and is now employed as a marker for diagnosis of cancer and metastasis, once again the following question is posed: Can phototherapy activate these pathways more and lead to more metastatic induction and tumor invasion to other healthy areas of the body? Moreover, the more important question is: Have we carefully investigated the parameters affecting this process?

The effect of photo-based therapy on angiogenesis

Angiogenesis, which serves to provide oxygen and nutrients, and therefore energy, in tumors, plays a highly significant role in metastasis [207]. This significance becomes more specified when we know that angiogenesis occurs from the cell dysplasia stage. Important mediators having crucial roles in the cell fate in response to photo-based therapy were discussed above, and Hassanpour et al. have recently reviewed the distinct role of autophagy in angiogenesis [270] (Table 10).

The effect of photo-based therapy on heat shock proteins

In recent years, a wide variety of studies have been conducted on the protective effects of HSPs and their mechanisms in response to various stresses, such as the effects of ROS produced by photo-based therapy, especially in response to the increased cellular temperature. Scientists have also discovered other important cellular protective roles of HSPs such as anti-apoptotic effects, intracellular signaling pathways, regulation of inflammatory factors and the immune system, and effects on autophagy [275]. The signaling pathways in which HSPs participate and their relationship to photo-based therapy are summarized in Fig. 10 and Table 11.

Table 11.

Roles of HSPs in cancer cell fate in response to different types of stresses.

Types of Heat shock proteins	Stressor	Intracellular effects	Cells fate	Refs.
HSP	PDT	Chaperone-mediated autophagy	Survival	[276]
HSPB8-HSPA4	PDT	Prevent protein accumulation	Survival by autophagy induction	[277]
HSPB1	Cisplatin	Autophagy	Apoptosis	[98,278]
HSPA1A	Autophagy	MAPK8 signaling pathway activation	Inhibition of apoptosis	[279,280]
HSP90AA1	Heat shock	CDC37-HSP90AA1 ULK1 phosphorylation and ATG13 release	Autophagy induction	[281]
HSP90AA1	Heat shock	HSF1	ATG7, autophagy induction	[282]
HSPB1	PDT	Inhibiting the release of CYCS/cytochrome c	Inhibition of apoptosis	[283]
HSPD1	Heat shock	Inhibiting CASP3	Anti-apoptotic	[284]
HSPD1-HSP90AA1	Cellular stress	TRAP1	Mitochondrial membrane stabilization	[285]
HSPA4	Heat shock	BAX-HSPA4	Inhibition of pro-CASP9	[286]
HSPA4-HSF1	Stress	Autophagosome formation	Apoptosis inhibition	[287]
HSPA4	Drugs (chemotherapy)	LC3 and ATG7 HSF1 knockdown	Resistance to chemotherapy	[282]
HSP90AA1	PDT	AKT1 activation	Cell survival	[288]

CDC37, cell division cycle 37, HSP90 cochaperone; HSPA1A/HSP72, heat shock protein family A (Hsp70) member 1A; HSPB8/HSP22, heat shock protein family B (small) member 8; TRAP1, TNF receptor associated protein 1.

The effect of photo-based therapy on immunotherapy

As discussed at the beginning of this review, it seems that inducing apoptotic cell death pathways is the best therapeutic intervention for tumors, which has the least possible negative consequences due to decreased release of tumor-growth factors. One of the most recent methods developed using phototherapy relies on the immune system. In recent years, the use of various stressors to stimulate cancer cells for expressing cell death antigens has been presented as a new therapeutic strategy [289]. (Fig. 10). In 2016, Malladi et al. indicated that in the dormant and quiescence phases, metastatic cells express fewer immune system receptors on their cell membrane through the inhibition of the WNT signaling pathway [290]; consequently, they remain less sensitive to a response from the immune system. In this method, cancer cells are driven by certain stresses to express molecular danger/damage-related patterns (DAMPs) in order to be identified and absorbed by the immune cells having receptors that can recognize these antigens [291]. Concerning this issue, some studies have been performed on methods of killing cancer cells via inducing the expression of the above-mentioned receptors following PDT [292,293]. Some of the work in this field is summarized in Table 12.

Table 12.

Relationship between photo-based therapy and immunotherapy.

Immunomodulatory agent	Cellular Inductive factors	Inflammatory response	Cell fate	Refs.
PDT	Increased expression of specific DAMPs	Identified by macrophages	Killing by immune cells	[291,293,294]
	ATP, HMGB1, CALR, and HSP expression increased in cell surface	Identified by macrophages
	HSPA4 and HSP90AA1 transmitted to the cell membrane	Identified by macrophage toll like receptors
PTT	Autophagy induced by ROS	CD4+ CD8+ are activated	Tumor elimination by immune cells	[[295], [296], [297]]
	Expression of immune-associated proteins such as IL6	Tumor microenvironment change

CALR, calreticulin.

Conclusions and perspectives

Considering all of the studies discussed above, we can summarize with the following:

• (1)As emphasized in this review about the importance of cancer cell death mechanisms on the outcome of treatment, we strongly suggest researchers who work in this field to develop and modulate different parameters of photo-based therapy for inducing apoptosis and autophagy-mediated cell death instead of necrotic cell death.
• (2)The different levels of autophagy induction in response to different environmental stresses (in the case of this review, HSPs and PDT-induced ROS) can play important roles. Thus, if we know which levels of cellular stress will bring about which level of autophagy, we will likely have better therapeutic outcomes.
• (3)The direct impact of the consequences of various perturbations on stressor-induced signaling pathways that directly and indirectly affect the induction of dormancy and ultimately the induction of tumor recurrence is very important; perhaps it will be possible to increase the phototherapy-based therapeutic benefit through the application of a simple pharmacological agent.
• (4)The field would benefit from a general review of the use of photo-based therapy methods concerning the micro-environmental stimulating factors of the tumor such as hypoxia, which are essential for maintaining the stemness properties of CSCs, the change of microenvironmental niche to induce dormancy and metastasis, and the direct effect on angiogenesis following the induction of environmental stress.
• (5)One recommendation that has been frequently posed throughout this review is that in future studies researchers should focus strongly on the investigation of the impacts of all parameters involved in phototherapy on the fate of cancer cells such as photo-synthesizers, radiated light wavelength, ROS levels, different levels of autophagy induction, wavelength intensity, and appliance of temperature change (sudden or stable).
• (6)It is important to carry out an assessment of changes in vital organelles during autophagy induction, such as microtubules and lysosomes, following the appliance of phototherapy as well as their stimulation or inhibition in order to change the therapeutic benefits of the above methods.
• (7)Various mediators such as secretion of miRNAs involved in invasion, growth, and differentiation of cancer cells must be studied more deeply in order for us to increase our knowledge of what patients are actually facing in response to photo-based therapies.
• (8)Overall, PTT and PDT have been shown to have great advantages compared to commonly used chemotherapy agents that are listed in Table 13. However, some critical issues related to all possible undesired outcomes following PTT and PDT remain uncovered and require urgent consideration in the field of translational oncology.

Table 13.

Comparison the advantages and disadvantages of PTT, PDT and chemotherapy.

Treatment Strategy	Tumoricidal mechanism(s)	Advantages	Disadvantages	Cellular Fate	Refs.
PTT and PDT	-Localized immunosurveillance following immune cell death -Activation anti-tumor cellular reactions -TME remodeling	-Targeting capability -Physical eradication of cancer cells -Saturation of the cellular defensive mechanisms -Combination therapy with other treatments -Induction of UPR inside the cells -High target/off-target ratio -Fast damage process	-Poor pharmacokinetic features of NDDS -Complicated development of nanostructures -Fine tuning of the generated heat and ROS -Possible evoking of the undesired biochemical process (Resistance autophagy, dormancy, invasiveness of the remnant cells)	-Apoptosis -ICD -Necroptosis -Autophagic death	[291,293,294]
Traditional Chemotherapy	-Inhibition of the cell's proliferation processes (DNA breaking, DNA methylation, cell structural defects such as microtubule formation inhibition)	-Relative killing of cancer cells (mostly non-stem like cancer cells) -Identified pharmacokinetic and pharmacodynamic features	-Uncontrolled biodistribution -Systemic toxicity -Non-targetability -Induction of resistive mechanisms against cell death mechanisms -Low targeting/off-target ratio -Relatively long-term medication therapy (increase the cancer cells adjustment with induced stress)	-Apoptosis -Necrosis -Ferroptosis	[[295], [296], [297]]

Financial and competing interest

The authors declare no conflicts of interest and no competing financial interest in this work.

CRediT authorship contribution statement

Maryam Ghafarkhani: Writing – original draft, Investigation, Data curation. Meghdad Abdollahpour-Alitappeh: Writing – review & editing, Writing – original draft. Zahra Alizadeh: Investigation, Data curation. Solmaz Tabibi Azar: Investigation, Data curation. Mahdieh Nemati: Investigation, Data curation. Amir Zarebkohan: Writing – review & editing, Supervision, Conceptualization. Daniel J. Klionsky: Writing – review & editing, Validation, Supervision.

Declaration of competing interest

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.