The substrate promiscuity these CYP enzymes exhibit makes them a key factor for studying drug interactions. These proteins are equipped with a prosthetic group composed of iron protoporphyrin IX, bound by a cysteine thiolate ligand at their active site (in most proteins). This active site is anchored in the molecule’s center, posing a challenge for immediate interaction with target ligands (Schenkman and Jansson, 2006; Urban et al., 2018). Furthermore, the catalytic cycle of CYP enzymes requires a multicomponent system for the transfer of a pair of electrons, facilitated by redox partners such as NADPH-cytochrome P450 oxidoreductase (CPR) and, less commonly, cytochrome b5 (Waskell and Kim, 2015; Jeffreys et al., 2018). Although the structure is highly similar across all families, ligand access channels can influence the enzyme’s selectivity for the substrate (Urban et al., 2018). Residues within these channels confer affinity to certain types of molecules, prioritizing them for the catalysis of reactions such as hydroxylation, epoxidation, deamination, and monooxygenation, among others (Zhao et al., 2021). An example of this is the N-hydroxylation generated in dapsone, an anti-leprosy medication, catalyzed by CYP enzymes 2B6, 2D6, 3A4, 2C8, 2C19, 2E1, 2C18, 2C9, or the oxygenation generated by 3A4 in desogestrel, which is a contraceptive medication (Rendic and Guengerich, 2021).

In metabolism, drugs typically undergo three main phases. The first phase aims to increase the molecule’s polarity through reactions such as oxidation, reduction, or hydrolysis (Conan et al., 2021). In these processes, CYP enzymes participate in 96% of the reactions, while the remaining percentage is distributed among enzymes such as aldo-keto reductase, microsomal flavin monooxygenase, and monoamine oxidase (Rendic and Guengerich, 2015). The second phase involves further increasing the polarity of molecules through conjugation reactions mediated by transferase enzymes such as N-acetyltransferases (NAT) and sulfotransferases (SULT), among others (Conan et al., 2021). Finally, the last phase consists of the excretion and elimination of metabolized compounds through transporters like P-glycoprotein (Xu et al., 2005). It is important to note that, in the first phase, CYP enzymes also play a role in the bioactivation of prodrugs. These compounds exhibit a therapeutically inactive conformation and require transformation to generate the desired effect (Rendic and Guengerich, 2021).

The CYP enzymes are responsible for metabolizing ∼75% of marketed drugs (Guengerich, 2010). Families 1 to 3 of these enzymes account for over 80% of the involvement in drug-associated metabolic reactions (Zhao et al., 2021). Additionally, the isoforms 3A4, 2C9, 2C19, 2D6, and 1A2 are responsible for ∼95% of drug oxidations (Guengerich, 2010).

An example of metabolic activation is acetaminophen, a commonly used analgesic and antipyretic, which is metabolized by the P450 isoforms 3A4, 2E1, 2D6, 1A2, and 2A6 (Rendic and Guengerich, 2021). However, these CYP enzymes are also involved in the bioactivation of various chemotherapeutic prodrugs used in cancer therapy.

Cytochrome P450 enzymes are closely linked to cancer, playing various roles, from the chemical transformation of antineoplastic drugs to acting as metabolizers of carcinogens (Elfaki et al., 2018; Zhao et al., 2021). Although it has been observed that isoforms 1A1, 1A2, 1B1, 2A6, 2A13, 2E1, and 3A4 are significantly involved in the metabolism of various carcinogens, there is no information on the percentage of involvement of these CYP isoforms in the biotransformation of carcinogens (Guengerich, 2010).

Most anticancer drugs undergo metabolism through various CYP enzymes isoforms. For example, tamoxifen, an estrogen receptor modulator with anticancer properties, is metabolized through different isoforms, mainly 2D6 and 3A5 (Goetz et al., 2005; Serrano et al., 2011). Another case is ellipticine, an antineoplastic agent and topoisomerase II inhibitor, metabolized through various isoforms such as 1A2, 1A1, 2C19, 2E1, 2D6, 2C9, 3A4, 2B6, and 1B1. In addition to its anticancer function, raloxifene prevents osteoporosis and is metabolized by CYP3A4 and CYP2D6 (Rendic and Guengerich, 2021). Similarly, oxazaphosphorines like cyclophosphamide (CPA) and ifosfamide (IFA) are activated by CYP enzymes. CYP2B6 is primarily responsible for CPA metabolism, while CYP3A4 is responsible for IFA metabolism. However, other isoforms, such as 2A6, 3A5, 2C9, 2C18, and 2C19, are also involved in the bioactivation of these drugs. CPA and IFA are used as chemotherapeutic agents in various cancer types, including breast, prostate, some lymphomas, and leukemia (Roy et al., 1999; Jounaidi and Waxman, 2004).

Nevertheless, enzyme-mediated bioactivation can lead to various possibilities. One possibility is that the prodrug may transform into its active form without changing, producing the desired effect. Another one is that the enzymes can cause an increase or loss of activity and even lead to toxicity for the individual (Guengerich, 2021). This last outcome is attributed to genetic polymorphisms (Gaedigk et al., 2018). In this context, individuals can exhibit different phenotypes based on the enzyme alleles they possess, and this allelic frequency varies among populations. Allelic variants of the enzyme have different affinities for the same substrate (Figure 1). The website Pharmacogene Variation Consortium (https://www.pharmvar.org/genes) gathers valuable information on the pharmacological interaction of various CYP polymorphisms.

Based on these polymorphisms, individuals have been classified as ultra-rapid metabolizer (UM), extensive metabolizer (EM), intermediate metabolizer (IM), and poor metabolizer (PM) (Mirabbasi et al., 2017; Caudle et al., 2020). This genetic diversity has a direct impact on how the body reacts to different medications, posing a challenge to healthcare as it can result in the lack of efficacy of treatments or the manifestation of adverse drug responses (Bosch et al., 2006). Due to the significant involvement of CYP enzymes in drug metabolism, it is crucial to identify their genetic peculiarities in an individual or population to optimize the efficacy and safety of pharmacological treatments (Goh et al., 2017). These CYP enzymes’ polymorphisms have been suggested to modulate the cancer risk of patients and contribute to individual susceptibility, particularly in the metabolism of tobacco-related compounds (Hernando-Rodriguez et al., 2012).

Among the CYP enzymes, 2D6, 2A6, and 2B6 show more polymorphisms in the Caucasian population and are closely related to drug metabolism (Preissner et al., 2013). For instance, the CYP2B6*6 allele, resulting from missense mutations (K262R, Q172H), leads to a decrease in its function due to its reduced expression in the liver (Ariyoshi et al., 2011; Preissner et al., 2013). However, the protein of the CYP2B6*6 allele shows a higher affinity for CPA, with a Km of 1.62 mM, compared to the normal allele, which has a Km of 2.68–4.03 mM (Ariyoshi et al., 2011; Lautier et al., 2016). In this case, the function depends more on the expression of the enzyme than on the affinity of a substrate. Conversely, the CYP1A2*1F allele with importance in processing antineoplastic drugs experiences an increase in its function; hence, individuals with the homozygous genotype (1*F/1*F) are classified as rapid metabolizers. Notably, the prevalence of this allele in European and American populations surpasses 60% (Neyshaburinezhad et al., 2021). An example where the activity is unaffected is the CYP2D6*2 allele, resulting from missense mutations (R296C, S486T), with more than a 30% allelic frequency in both the European and American populations (Neyshaburinezhad et al., 2021).

In vivo analysis of allelic variants provides relevant information for enzymatic engineering. Obtaining enzymes with improved characteristics opens the possibility of their use in gene therapy against cancer (i.e., GEDPT). This type of therapy offers particular benefits to individuals resistant to conventional cancer treatments, such as those with breast cancer carrying the CYP2C9*2 allele (R144C). This allele is associated with resistance to neoadjuvant chemotherapy, which is the initial step in treatment aimed primarily at halting tumor growth and reducing its size to facilitate surgical removal or improve the outcomes of radiotherapy in the future. The neoadjuvant chemotherapies utilize combinations of various drugs. For example, CMF includes CPA, methotrexate, and fluorouracil, while FAC comprises fluorouracil, doxorubicin, and CPA. The allelic frequency of CYP2C9*2 ranges between 6% and 13% in Europeans and Americans (Seredina et al., 2012; Neyshaburinezhad et al., 2021).

Cancer gene therapy is a promising approach for treating cancer by introducing genetic material into cancer cells to fight the disease (Cross and Burmester, 2006). The key principle of cancer gene therapy involves delivering therapeutic genes into cancer cells or healthy tissue (e.g., immune cells) (Cesur-Ergün and Demir-Dora, 2023). Viral and non-viral vectors (e.g., liposomes and polymers) can carry the therapeutic genes and insert them into target cell DNA (Hwang et al., 2001; Sung and Kim, 2019; Manisha. B.; Shinde et al., 2020). Several approaches exist to achieve anti-cancer effects:

a. Gene therapy can replace mutated genes that cause or drive cancer growth and progression with normal functioning genes (gene correction, e.g., BRCA1 in breast cancer) (Obermiller et al., 2000).

This review further focuses on GDEPT, specifically the utilization of CYP enzymes therein, as a clever approach for cancer gene therapy. It introduces a group of well-studied enzymes and allows the use of already clinically approved drugs, aiming to improve their efficacy and specificity and reduce their side effects.

Gene-directed enzyme prodrug therapy (GDEPT) is a promising approach in cancer treatment, involving the delivery of a therapeutic gene encoding a foreign enzyme to tumor cells, where a systemically administered nontoxic chemotherapy prodrug can be converted into its active, cytotoxic metabolite upon expression of the enzyme, leading to cancer cell death (Figure 2) (Günther et al., 2006; Grohmann et al., 2009). Due to the selective genetic modification of tumor cells, this technique has shown potential to improve antitumor activity and selectivity for cancer cells of chemotherapy treatments (Mishra et al., 2018). GDEPT has been evaluated in various clinical trials using different enzyme/prodrug combinations, demonstrating its versatility and potential for clinical application (Wang et al., 2004; Hedley et al., 2007; Grohmann et al., 2009; Alekseenko et al., 2015). However, in terms of using GDEPT specifically for cancer treatment, sufficiently successful human clinical trials with the desired significant improvements are still lacking, despite promising results from preclinical studies as well as moderately successful early-stage clinical trials (Phases I and I/II, see Section 4.6) (Salmons et al., 2003; Braybrooke et al., 2005; Karjoo et al., 2016). Moreover, some studies have focused on optimizing GDEPT by incorporating novel enzymes and prodrugs, as well as improving the expression levels of prodrug-activating genes to enhance therapeutic efficacy (Chen and Waxman, 2002; Kratz et al., 2008; Gerth et al., 2019). Overall, GDEPT holds promise as a targeted and effective approach for cancer therapy, with ongoing research aiming to refine further and expand its applications.

The role of CYP enzymes in GDEPT is crucial for developing targeted cancer treatments. CYP enzymes, especially the CYP1, CYP2, and CYP3 families, metabolize endogenous and exogenous substances in the human body (Zanger and Schwab, 2013; Luo et al., 2021). They play a significant role in activating anticancer prodrugs within cancer cells, thereby converting non-cytotoxic prodrugs into cytotoxic drugs, which selectively target and kill cancer cells (Kumar, 2010). A wide range of clinically established anticancer drugs need these CYP enzymes to become activated to their cytotoxic form, with CYTOXAN (cyclophosphamide) and IFEX (ifosfamide) being thoroughly studied examples for use in GDEPT (Le Blanc and Waxman, 1989; Kivistö et al., 1995). Chen and Waxman (2002), Quiñones et al. (2008), provide a list of anti-cancer P450 prodrugs of interest for use in GDEPT. Furthermore, some commonly used chemotherapy drugs do not necessarily need the CYP enzymes to become active but still benefit from CYP metabolism by conversion into a more active metabolite. For example, CYP2B enzymes aid in the release of the cytotoxic aziridine moiety from Tepadina (Thiotepa), while CYP2D6 transforms the anti-estrogen breast cancer drug tamoxifen to the 100-fold more potent derivative 4-hydroxy-tamoxifen (Borgna and Rochefort, 1981; Waxman, 1993; Dehal and Kupfer, 1997).

However, for successful GDEPT in cancer patients, careful considerations must be considered. As mentioned before, multiallelic genetic polymorphisms in CYP enzymes influence the activity and function of these enzymes, leading to distinct pharmacogenetic phenotypes and, therefore, variations in the therapy responses of patients (Zhou et al., 2009; Zanger and Schwab, 2013). Furthermore, the modulation of CYP enzyme activity, e.g., by other drugs or dietary polyphenols, affects the pharmacokinetics and bioavailability of drugs, influencing the efficacy of chemotherapy, especially when combined with GDEPT (Korobkova, 2015).

GDEPT can be utilized to introduce desired CYP enzymes to achieve cell-specific gene delivery and expression, controlled conversion of prodrugs to drugs in target cells, and expanded toxicity to the target cells’ neighbors through bystander effects (Zhang et al., 2015). The bystander effect is crucial for GDEPT success because it removes the need to transduce all target tumor cells with the therapeutic gene, which is currently unattainable with existing gene delivery methods. The introduced CYP enzymes activate bioreductive cytotoxins, thereby increasing the efficacy of targeted therapy for drug-resistant hypoxic tumors (Pidkovka et al., 2021). Moreover, genetic engineering allows to develop and engineer optimized CYP enzymes with desired metabolic function and drug selectivity for use in GDEPT (Li et al., 2020). Importantly, the tumor cell-specific activation of selective CYP enzyme function through GDEPT allows for the use of lower doses of already approved chemotherapy prodrugs due to the increased sensitivity of the modified cells, potentially reducing toxicity on healthy tissue while maintaining therapeutic efficacy, highlighting the importance of this approach in cancer therapy (Miura et al., 2015; Mishra et al., 2018).

Moreover, the overexpression of certain CYPs, such as CYP1B1, in some cancer cells has led to the development of CYP inhibitors for chemoprevention as well as chemotherapy prodrugs designed to be activated by CYPs specifically expressed in cancer cells for cell-specific cytotoxic effects, further demonstrating the potential for targeted cancer treatments utilizing CYP enzymes (Bruno and Njar, 2007).

In summary, the role of CYP enzymes in GDEPT is pivotal for developing targeted and selective cancer therapies. The ability of CYP enzymes to activate prodrugs within cancer cells, the impact of genetic polymorphisms on CYP enzyme activity, and the potential to reduce toxicity while maintaining therapeutic efficacy highlight the significance of CYP enzymes in GDEPT. Developing engineered CYP enzymes for use in GDEPT represents a promising approach to cancer gene therapy.

The design and optimization of CYP enzymes for cancer therapy requires a multidisciplinary approach, combining molecular biology, protein engineering, and pharmacology. As mentioned in Section 3, the genetic engineering of CYP enzymes has been explored for cancer therapy using the concept of GDEPT. In GDEPT, CYP enzymes activate prodrugs selectively within tumor cells, leading to localized cytotoxic effects. The ultimate goal is to develop CYP enzymes with improved expression, stability, catalytic activity, regioselectivity, and tumor-specific delivery to enhance the effectiveness of cancer treatment while minimizing side effects.

CYP enzymes can have multiple potential sites of metabolism on a given substrate molecule. One strategy is optimizing their regioselectivity to selectively target specific regions of a prodrug or anticancer agent to improve the efficacy and selectivity of cancer therapy (Sun et al., 2007). Importantly, the recognition of substrates, their access to the active site, and the binding of redox partners are not solely influenced by the residues within the active site but also by residues outside of it (Kumar et al., 2005; Sun et al., 2007), and should be considered during the engineering of these enzymes. Additionally, optimizing the catalytic activity and modifying the structure of prodrugs or anticancer agents could enhance substrate binding and catalysis and their specificity towards CYP enzymes (Chen et al., 2004; Jounaidi et al., 2006; Chen et al., 2007; Sun et al., 2007). A way to improve its catalytic activity is the co-expression of CYP reductases to allow proper electron transfer (Lengler et al., 2006; Touati et al., 2014; Sellner et al., 2021). Fusion of CYP enzymes with other proteins, such as reductases or targeting peptides, can enhance their stability, solubility, and specific delivery to tumor cells, improving the therapeutic potential for cancer therapy (Steffens et al., 2000; Kan et al., 2001; Jounaidi and Waxman, 2004; Tychopoulos et al., 2005; Jounaidi et al., 2006). Other recently explored alternatives are the addition of a detachable linker, which breaks down within the tumor microenvironment, and the addition of a functional carrier to the original anticancer drug that could help the release of a molecule that has enhanced pharmacokinetic and pharmacological properties at the tumor site, transforming traditional antineoplastic agents into prodrugs (Najjar and Karaman, 2019). Figure 3, summarizes some of the strategies employed and the expected outcomes.

Several methods and techniques for engineering CYP enzymes have been employed together or separately over the past decades, yielding enzymes with improved metabolic efficiency toward specific anticancer drugs (Table 1, Supplementary Table S1). Nevertheless, other approaches are becoming new alternatives to produce robust, more stable, and novel substrate affinity CYP enzymes. The next paragraphs describe these alternatives:

Some challenges of CYP enzyme’s genetic engineering include achieving successful and efficient expression, as these enzymes are often membrane-bound proteins that require specific conditions and co-expression of other proteins, such as redox partners, to be active (Lengler et al., 2006). Efficient electron transfer from redox partners could require the construction of linkers between them to facilitate their interactions (Touati et al., 2014).

An alternative under exploration is introducing non-human redox partners for P450 enzymes in gene therapy. CYP enzymes can be reconstituted with surrogate redox partners, enabling electron transfer during catalysis. For instance, a study by Liu X. et al. (2022) compared three pairs of frequently-used surrogate redox partners: Fdx1499/FdR0978, Adx/AdR, and Pdx/PdR, and found that Fdx1499/FdR0978 showed the most promise in terms of electron transfer properties (Liu X. et al., 2022). Moreover, the biological diversity of P450 redox partner systems is vast, and several new types of P450 redox partner systems have been characterized (Sadeghi and Gilardi, 2013; McLean et al., 2015). This suggests that there is potential for using alternative redox partners to modulate the catalytic activity of P450s, which could be beneficial for gene therapy applications. However, non-human redox partners must be compatible with human cells and not elicit an immune response. Moreover, producing non-human redox partners in human cells can be difficult, and ensuring their stability within the cellular environment adds another layer of complexity. It is important to carefully evaluate the potential benefits of combining cytochrome P450 and redox partner in vivo models that are relevant to the specific tissue being treated, as the effect of redox partner on the activity of CYP enzymes seems to differ depending on the type of cell being used (Lengler et al., 2006). Alternatively, “Molecular Lego” has been used to engineer catalytically self-sufficient CYP enzymes (Dodhia et al., 2006). This modular assembly of different protein domains could create chimeric proteins with desired properties. For instance, the human P450 domain has been fused with a non-human redox partner (Bacillus megaterium reductase-BMR), creating a single polypeptide chain that functions effectively within human cells (Gilardi et al., 2002; Fairhead et al., 2005; Dodhia et al., 2006; Catucci et al., 2022).

Moreover, several studies described the development of fusion proteins that combine CYP enzymes with other enzymes, such as monomeric sarcosine oxidase (CYP152B1-polyG-MSOX fusion protein) (Giuriato et al., 2022) or to promote CYP enzymes’ peroxygenase catalysis (CYP102A1, CYP152B1) (Paul et al., 2014; Shoji et al., 2016; Hardiyanti Oktavia et al., 2023) to enable P450-catalyzed reactions without the need for an external electron donor. These recent developments in P450 fusion proteins represent significant progress toward creating efficient and versatile biocatalysts for various applications, including gene therapy. Nevertheless, it is important to overcome the limitations of using an in situ H₂O₂ generation approach, such as potential cytotoxicity due to reactive oxygen species, lack of specificity in targeting cancer cells, and challenges in controlling H₂O₂ levels effectively.

Another challenge is finding appropriate enzyme-specific promoters, which will only drive expression in the target tissue, which is also a limitation in other GDEPTs (Robson and Hirst, 2003). The “Molecular Lego” approach could also aid designing expression vectors that allow for the modular assembly of genetic elements, such as promoters, enhancers, coding sequences for P450 enzymes, and targeting sequences. This modular design could enable the precise control of CYP enzyme expression levels, tissue specificity, and subcellular localization, optimizing therapeutic efficacy and minimizing off-target effects (Dodhia et al., 2006).

CYP enzymes exhibit a wide range of substrate specificities, and this specificity is not only given by the interaction between residues in the catalytic site but also by residues outside it (Kumar et al., 2005). Therefore, it can be challenging to engineer them to accept new substrates or alter their selectivity towards specific compounds. The successful modification will usually require a combination of engineering methods. Additionally, since they are promiscuous enzymes, they could show unwanted interactions with other drugs, affecting the therapy efficacy or creating unpredicted side effects (Ekroos and Sjögren, 2006; Nath et al., 2010).

These challenges (Figure 4) highlight the complexity of engineering CYP enzymes and the need for a multidisciplinary approach combining molecular biology, biochemistry, and computational methods.

The design and optimization of CYP enzymes for cancer therapy often involve strategies to minimize off-target effects and enhance tumor-specific activation of prodrugs.

GDEPT involves the delivery of CYP enzymes and their redox partners directly to tumor cells using gene therapy approaches. This reduces the side effects usually seen in the systemic delivery of prodrugs, which are activated by the CYP enzymes in the liver. Furthermore, the use of drug-metabolizing enzymes that are intrinsic to the tumor could mediate the local generation of cytotoxins, eliminating the need for complex delivery systems (e.g., CYP4B1 predominantly expressed in lung cells) (Travica et al., 2013). Another strategy is the coexpression of CYP enzymes with their redox partners in tumor cells to enhance tumor-specific activation of prodrugs (Tychopoulos et al., 2005; Mahato et al., 2011). CYP enzymes can be fused to targeting peptides to improve their tumor-specific delivery, as seen in other GDEPTs (Liu X. et al., 2022), and can also benefit from utilizing viral or non-viral vectors to deliver CYP enzymes selectively to tumor cells (Quester et al., 2017; Tapia-Moreno et al., 2017). Additionally, the “Molecular Lego” approach has the potential to enhance the accuracy of controlling the levels of CYP enzyme expression, as well as its specificity to certain tissues and its location within cells. By doing so, it can optimize the effectiveness of therapeutic treatments while reducing any unintended side effects (Dodhia et al., 2006).

CYP enzymes have gained attention in cancer therapy due to their potential role in targeted drug delivery and treatment. While this field of research is still evolving, there have been some promising results in their use for cancer therapy and their engineering for the same purpose. Table 1; Supplementary Table S1 summarize the genetic engineering efforts for obtaining CYP enzymes with desirable gene therapy features.

Preclinical studies have been conducted to evaluate GDEPT, including the involvement of cytochrome P450 enzymes, as demonstrated by a study conducted by McErlane and colleagues in 2005. In this study, the enzyme CYP2B6 was utilized in combination with radiation, along with the prodrugs banoxantrone (AQ4N) and CPA. DNA damage was observed in RIF-1 cells transfected with the enzyme and treated with AQ4N, supporting its ability to metabolize the drug. The antitumor capacity of the treatment was confirmed in a murine RIF-1 tumor model. Additionally, it was highlighted that the same gene can function effectively in oxygen-rich environments and under hypoxic conditions, as CPA requires oxygen for its toxic activity, unlike AQ4N, which can achieve the same goal in hypoxic conditions (McErlane et al., 2005).

Regarding clinical trials, promising results have been observed, as seen in the phase I-II study conducted by Löhr and colleagues in 2003. In this study, 14 patients with inoperable stage III-IV pancreatic adenocarcinoma were treated, and patients who had previously undergone chemotherapy were not accepted. Procedures involved the intra-arterial administration of microencapsulated cells with CYP2B1, followed by the application of ifosfamide. The results showed no toxicity greater than grade II in any 14 patients. Throughout the study, no increase in tumor size was observed in any participants (stable disease), and two exhibited a tumor volume reduction exceeding 50% (partial response). These findings indicate favorable treatment tolerance and suggest a positive impact on reducing tumor burden in certain individuals (Löhr et al., 2003).

Another phase I/II clinical trial employed a retroviral vector MetXia-P450 to induce the expression of CYP2B6. This approach aimed to transfect cancer cells, thereby generating enzyme expression and activating CPA to trigger its toxic effect in the affected cells. The study involved 9 patients with advanced breast cancer skin nodules and 3 patients with melanoma, who received two intratumoral injections of MetXia-P450. In some cases, the results revealed a partial response, with four patients maintaining a stable disease condition while the rest experienced disease progression. Although ten out of twelve participants showed positive transfection control, it is important to note that transduction affected less than 1% of tumor cells in this specific trial (Hunt, 2001; Braybrooke et al., 2005). Due to the very low transduction percentage, progress to other phases of clinical studies has not been made.

As of the writing date of this manuscript, no clinical trials beyond those mentioned in this article have been identified. This absence could be attributed to the focus of applying this therapy primarily to treating the primary tumor, with less attention given to metastasis. However, it is important to note that this therapeutic modality could be integrated with other anticancer treatments, thereby expanding its applicability and effectiveness in the comprehensive approach to cancer therapy.