Cancer chemotherapy and beyond: Current status, drug candidates, associated risks and progress in targeted therapeutics

Cancer is a heterogeneous and multifactorial disease in which a series of genomic/molecular alterations cause the uncontrolled growth and proliferation of the cells, causing a rapid increase in tissue mass in the affected parts of the body. Under normal conditions, a cell gets signals to die and to supplant the organism with a young and healthier cell. Cancer cells grow using the body's oxygen and supplements, depriving other cells of regular supplements and growth factors. These cells can turn the microenvironment in their favor, deceive the immune system of the body, and can exploit the physiology of other cells to accommodate their needs. 1 , 2 , 3 , 4 , 5 , 6 Some of the biomarkers which are currently been used to detect cancer including human epididymis protein 4 (HE4), carcinoembryonic antigen (CEA), legumain, mesothelin, osteopontin, and vitamin E-binding plasma protein. A plethora of anti-cancer drugs and natural medicinal compounds have been devised over the years, which can suppress tumor growth through diverse mechanisms. Some of the drugs/compounds work on crucial cellular enzymes, while others may alter cell metabolism. They have also shown the potential to interfere with some critical cellular processes, e.g., programmed cell death/apoptosis, drug resistance, DNA damage, DNA replication, or immune reactions. These drugs have distinct modes of action and selectivity for multiple cancer types 7 , 8 , 9 , 10 , 11 , 12 ; however, subtle alteration to their chemical structure could abolish their anti-cancer potency. 13 , 14 The idea of chemotherapy (utilizing toxic compounds and drugs to destroy cancerous cells) came into existence after the reports of mustard gas killing lymphatic tissues and bone marrow. The effects were later validated in mice using an effective derivative of the gas (nitrogen mustard) that showed effective regression of lymphoma tissues. 15 , 16 , 17 The first patient receiving this nitrogen mustard was forty-eight years old lymphosarcoma patient whose cancer softened and cleared initially. Unfortunately, later he died after relapsing, but this secret military trial at Yale University paved the way for chemicals in treating cancers and developing the field of cancer chemotherapy for treating a variety of cancers. 18 , 19 , 20 , 21 Chemotherapy primarily works by circumventing the cancer cells from further growth and division. Cancer cells usually divide and grow at a much-accelerated rate than normal cells and are physiologically possess very high endogenous stress. Therefore, the drugs can destroy them rapidly and more effectively in comparison to other surrounding cells. Some of the promising inhibitor therapies which have been used to treat solid cancers are polyadenosine diphosphate-ribose polymerase inhibitors, angiogenesis inhibitors, histone deacetylase (HDAC) inhibitors, mechanistic target of rapamycin (mTOR) inhibitors, poly (adenosine di-phosphate-ribose) polymerase (PARP) inhibitors, p53/mouse double minute 2 homolog (MDM2) inhibitors, hedgehog pathway blockers, tyrosine kinase inhibitors and proteasome inhibitors. 22 , 23 Chemotherapy may have different variations, which can affect the target cells in distinct manners. 24 Some of the treatments may directly alter the quality of cellular proteins, rendering them non-functional and affecting major cellular physiological pathways. Major chaperone repressors, autophagy suppressors, or proteasome inhibitors belong to these classes of molecules. 25 , 26 , 27 , 28 , 29 , 30 , 31 Other drugs may target some essential hormones and interfere with the body's overall metabolism. 32 , 33 The 2018 Nobel Prize attributed to immune checkpoint therapy research has highlighted the importance of immunotherapy, which underlies the possibilities of modulating our immune system in our favor to cope up with the cancer-like conditions and fight back against the disease. 24 , 34 The selection of chemo-preventive drugs or a combination of drugs is mostly based on the type and stage of cancer. The primary objective of these drugs is to neutralize the cancerous cells and ameliorate the stress caused by the tumor's growth. The dosage and timespan of the treatment are two important points of consideration. It has been observed that the medications are given at very high doses causing numerous side effects and harm to other healthy cells. 35 One major challenge is the relapse of the disease. During reoccurrence, it has been widely observed that the cancer cells attain drug resistance following longer exposure to the drugs. Despite enormous applications, most chemotherapies are given to prolong and ease the patient's survival, hence called palliative chemotherapy. 21 However, prolonged exposure to these drugs may have excessively adverse effects on a patient's physical and mental health, making it difficult to continue the ongoing treatment. Oncologists, for the most part, provide these medications with possible time intervals in between, which al

The cancer cell metabolic mechanisms functionally overlap with the host cells, so cancer treatment is very challenging. 76 , 77 Designing drugs or therapeutics mainly focuses on selectivity, which can specifically kill the cancerous cells without affecting the non-cancerous cells ( Fig. 2 ). In a way, the anti-cancer therapy may resemble antimicrobials, but the cancerous cells and the bacterial cells have differences in terms of the metabolic and physiological characters. 78 , 79 , 80 For our immune cells, bacterial cell recognition is straightforward, while our transformed cells can hide cell surface receptors to evade the immune system. 81 Early-stage detection of cancers increases the chances of treatment and survival. Therefore, recognizing and diagnosing those transformed cells remains one of the most challenging tasks in oncological research. At early stages, most known biomarkers remain undetectable, and cancer cells' ability to hide from immune cells freezes our immune responses. 82 , 83 In a tumor subpopulation, transformed cells differ drastically from the other cells in terms of physiology, metabolism, proliferation rate, etc. 84 , 85 , 86 Besides that, the genetic diversity may pose additional challenges to target all kinds of cells in a particular tissue mass owing to the tumor heterogeneity. 87 , 88 A combination of drug therapy is applied to address this challenge, and the regime is devised according to the cancer phase and type ( Fig. 2 ). 89 , 90 In the subsequent section, we provide a comprehensive overview of various chemotherapeutic agents which have served the purpose for several decades in different cancer types. These drugs could be classified in different ways based on their chemical nature/source, molecular target, mechanism/mode of action, or effectiveness in model systems against various malignancies/cancer types ( Table 1 ). Table 1 Table listing chemotherapeutic drugs/agents used in current clinical practice along with details of their molecular targets, mode of action, and investigated model systems across diverse malignancies. Table 1 Drugs/Compound Molecular targets Mechanism of action Studied model system Reference/Source Antineoplastic alkylating agents Altretamine DNA polymerase alpha catalytic subunit Ribonucleoside-diphosphate reductase large subunit DNA Inhibitor Inhibitor Other/unknow B-cell chronic lymphocytic, leukemia/prolymphocytic leukemia/small lymphocytic lymphoma refractory, and refractory non-Hodgkin's lymphoma 278 Bendamustine DNA Intra- and inter-strand crosslinking Chronic lymphocytic leukaemia (CLL), follicular non-Hodgkin's lymphoma refractory, refractory Hodgkin lymphoma, refractory Mantle cell lymphoma, Waldenström's macroglobulinemia (WM), recurrent multiple myeloma, and refractory indolent B cell non-Hodgkin lymphoma 279 Busulfan DNA Cross-linking/alkylation Chronic myelogenous leukemia 279 Carboplatin DNA Cross-linking/alkylation Advanced cervical cancer, advanced Endometrial Cancer, advanced esophageal cancers, advanced head and neck Cancer, advanced melanoma, advanced non-small cell lung cancer, advanced ovarian carcinoma, advanced Sarcoma, metastatic breast cancer, neuroendocrine carcinoma of the skin, pleural mesothelioma, refractory Hodgkin lymphoma, retinoblastoma, advanced bladder cancer, advanced small cell lung cancer, advanced testicular cancer, advanced thymoma, advanced thymic carcinoma, and refractory non-Hodgkin's lymphoma 279 Carmustine DNA Glutathione reductase, mitochondrial RNA Other/unknown inhibitor Other/unknown Glioblastoma, brainstem glioma, medulloblastoma, astrocytoma, ependymoma and metastatic brain tumors, multiple myeloma, Hodgkin's disease, non-Hodgkin's lymphomas, and may be used on the skin (topically) for cutaneous T-cell lymphoma 280 Chlorambucil DNA Cross-linking/alkylation Chronic lymphocytic leukemia, lymphomas, and Hodgkin's disease 279 Cisplatin DNA DNA-3-methyladenine glycosylase Alpha-2-macroglobulin Serotransferrin Copper transport protein ATOX1 Cross-linking/alkylation Not Available Not Available Not Available Not Available Testicular cancer, ovarian cancer, bladder cancer, head and neck cancer, esophagus cancer, small cell and non-small cell lung cancer, non-Hodgkin's lymphoma and trophoblastic neoplasms 279 Cyclophosphamide DNA Nuclear receptor subfamily 1 group I member 2 Cross-linking/alkylation Not Available Lymphoma, multiple myeloma, leukemia, ovarian cancer, breast cancer, small cell lung cancer, neuroblastoma, and sarcoma. 279 Dacarbazine DNA DNA polymerase alpha subunit B 6-phosphogluconate dehydrogenase, decarboxylating Cross-linking/alkylation Other/unknown Inhibitor Melanoma skin cancer, soft tissue sarcoma, Hodgkin lymphoma 279 Daunorubicin DNA DNA topoisomerase 2-alpha DNA topoisomerase 2-beta Intercalation Inhibitor Inhibitor Acute nonlymphocytic leukemia in adults and acute lymphocytic leukemia in adults and children 279 Decitabine DNA DNA (cytosine-5)-methyltransferase 1 DNA (cytosine-5)-met

Antimetabolites are the next class of substances that may compete, replace, or inhibit some specific metabolites inside the cells and thus meddle with the cellular metabolism ( Fig. 2 ). 93 These molecules mostly possess a structure similar to a cellular metabolite or enzyme–substrate, usually identified and processed by an enzyme to meet the cellular needs. 94 Tetrahydrofolates are formed from unreduced dietary folates by the enzyme dihydrofolate reductase. For example, various medications, such as aminopterin, methotrexate (amethopterin), pyrimethamine, trimethoprim, and triamterene, act directly on the production of these metabolites, hindering the enzyme from producing folate insufficiency. 95 Methotrexate, an antifolate drug, is one of the exceptionally successful anti-cancer medications that represses dihydrofolate reductase (DHFR) and obstructs the transformation of dihydrofolic acid (DHFA) into tetrahydrofolic acid (THFA). 96 This coenzyme activity is indispensable to the cells as they are essential for the nucleotide synthesis pathways. Methotrexate is an anti-inflammatory molecule and is hence used as a medication for many other inflammatory diseases, like rheumatoid arthritis and extreme psoriasis, apart from various cancer types. A detailed description of common antimetabolites is comprehensively provided in Table 1 .

Besides these three established classes, several other antineoplastic agents are also enrolled in the clinical practice of cancer chemotherapy ( Table 1 ). Given their clinical promises, in the subsequent section, we described the biological activities, targets, and mechanisms of key chemotherapeutic drugs that may belong to one or multiple drug classes and can affect more than one cellular pathway in the cancer cells and induce apoptosis by different mechanisms ( Fig. 2 ). A succinct review of well-known anti-cancer drugs that are in routine clinical practice is provided as follows.

Etoposide is a semi-synthetic derivative of a plant glycoside called podophyllotoxin. 117 , 118 It causes DNA damage by altering DNA topoisomerase-II function, and posing G2 cell cycle arrest. 119 Etoposide creates a ternary complex with DNA and the topoisomerase–II complex, preventing the latter's binding to the DNA strands, while in doing so, it causes damage to DNA strands. 120 Cancer cells depend on this enzyme more than healthy cells, as they divide more quickly; therefore, it causes defects in DNA synthesis that promotes apoptosis of the cancer cells. 121 Etoposide is effective against several aggressive malignancies including Kaposi's sarcoma, Ewing's sarcoma, testicular disease, lung disease, lymphoma, non-lymphocytic leukemia, and glioblastoma multiforme ( Table 1 ). 122

A transcription inhibitor chromopeptide molecule actinomycin D (also called dactinomycin) binds to DNA while initiating transcription at initiation complex and inhibits RNA elongation steps. 133 It can intercalate, preferably between guanidine and cytosine, or bind the DNA strand's minor grooves to form a highly stable drug-DNA complex, preventing DNA unwinding and thus stalling the progress of the RNA chain. 134 Dactinomycin was initially obtained from Streptomyces sp., identified as an antibiotic agent, but later its anti-tumor potential was recognized, and thus it was inducted in the chemotherapy as an anticancer antibiotic to treat Ewing's sarcoma, Wilms' tumor, rhabdomyosarcoma, trophoblastic neoplasm, testicular and ovarian cancers, etc ( Table 1 ). 135

Daunorubicin causes a break in DNA strands by actuating topoisomerase-II and producing quinone-type free radicals. Daunorubicin, otherwise called daunomycin, is a chemotherapy medicine used to treat cancer growth in acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), and Kaposi's sarcoma, etc. 141 , 142 It is applied by infusion into a vein, and following its intercalation with DNA, it hinders macromolecular biosynthesis. This predominantly obstructs the movement of the enzyme topoisomerase-II, which loosens up the DNA supercoiling. 143 Daunorubicin impedes the topoisomerase–II complex after it unties the DNA chain for replication and thereby it represses the DNA double helix from resealing and therefore interferes with the replication to proceed ( Table 1 ). 144 , 145

Aggressive tumors are primarily marked with uncontrolled and extensive cell division that may compromise the normal physiology of other non-transformed cells in the vicinity of affected organs/tissues. In many ways, cancer cells achieve super-specialized metabolic and survival potential with the capacity to tolerate extreme stresses like low oxygen and nutrient supplies. Cancers might start from mutation of one type or the other, but as the disease progresses, 149 , 150 multiple mutations and genetic abnormalities accumulate that lead to increased tumor heterogeneity. 87 The heterogeneous nature of tumor mass plays a vital role in developing resistance against most of the drugs that we have described here. This leads to a challenge of its kind, leaving doctors with the only option of changing the medication after certain intervals as one type of drug stops acting against tumors. In many cases, the combination therapy that includes more than one drug at a time could be an effective strategy against some form/type of cancers, such as cancers affecting bone marrow (leukemia) and lymph nodes (lymphoma). Clinical trials help a lot in understanding the effects of new combinations and thus have rapidly improved the cancer chemotherapy paradigm. The sustainability of chemotherapy relies upon its continuous effect on cell division and tumor growth. As discussed above, the majority of the treatments work by compromising the stability or integrity of nucleic acids that tend to stall the cell cycle or activate programmed cell death pathways. Other drugs may act by generating extreme proteotoxic stress inside the cells by interfering with the protein folding or degradation machinery, including chaperone, proteasome, and autophagy. 151 , 152 , 153 , 154 The idea is to generate cellular stresses so that the cancer cells that are already under compromised physiology may die while other cells can survive. However, it is evident from the above descriptions that most of these drugs act by interfering with one or multiple cellular pathways of extreme importance. 155 Therefore, chemotherapeutic drugs are mostly referred to as cytotoxic agents, causing a plethora of side effects. In most cases, long-term treatments may lead to enormous toxicity causing severe damage or failure of multiple organs. This may lead to enormous physiological and psychological stress to the patients under chemotherapeutic medication. Table 2 summarizes the adverse effects of various chemotherapeutic agents and the current stage of their clinical progress. The side effects of these drugs may depend on many factors and variables, such as type, stage, and location of the tumor, along with age, health, and other clinical aspects of the patients. The most common and readily visible side effects originate from the gut that may lead to nausea, vomiting, constipation, and diarrhea. 156 , 157 Most of the anticancer drugs can directly interrupt the cell division pathways; therefore, the first line of affected organs may also include those which are formed with continuously dividing cells, such as skin, hair, and intestinal linings. 158 , 148 Skin rashes, alopecia, and mucositis are the most visible side effects that one can observe in patients undergoing chemotherapy. 157 , 159 Radiation also contributes significantly to these debilitating changes. A general problem observed in many patients is fatigue that can occur due to multiple factors, including depression, anemia, high doses of radiation therapy, etc. 160 Chemotherapeutic drugs may also directly injure the bone marrow and cause a steep decline in blood cell count, exposing patients to several other types of possible infections. 161 Thrombocytopenia, a condition of excessive bleeding because of lower platelet numbers, is observed more frequently in patients taking alkylating and/or antimetabolites agents. 162 Alkylating agents may further trigger conditions, such as hemorrhagic cystitis leading to microscopic haematuria, nocturia, dysuria, etc. along with mild or intense suprapubic pain. 163 Whereas, antimetabolites can generate enormous toxicity in vital organs, like the liver and kidneys. 164 , 165 The most devastating of all is their direct impact on cardiovascular health. Different classes of chemotherapeutic drugs have varying impacts on cardiac cells, leading to multiple types of systemic conditions adversely affecting patients' survival. 166 Loss of libido and sexual dysfunctions are other conditions caused due to long-term exposure to these drugs, which affects not only physical well-being but also the patient's psychological health ( Table 2 ). 167 , 168 These drugs and radiation therapies may have several direct or indirect impacts on cognitive functioning leading to a condition, which is now commonly referred to as chemo brain. 169 , 170 This is a condition of the inability of someone undergoing chemo-drug therapies to do their routine cognitive functions, which may be related to their memory, c

The major drawback of most chemotherapeutic treatment plans is their non-specificity or inability to ascertain and target cancerous cells directly. Therefore, this lack of specificity poses a major therapeutic limit reflected in the failure of delivering drugs locally to the cancer tissue mass. In the past few decades, many new approaches have been constituted and tested, and have given hopes for future drug delivery ( Fig. 3 A). Nanoparticle-based delivery methods, targeted antibodies, aptamer functionalization, and specific drugs like Herceptin (in breast cancer) offered the promise that can harness great success in the upcoming years. 179 , 180 , 181 Cancer cell-directed antibodies are at the forefront of targeted drug delivery methods and have accelerated the hunt for more effective or potentially improvised approaches. 182 , 183 Different types of nanocarriers and formulations of nano-drugs have also improved the delivery of drug compounds to the cancer cells. 184 , 185 , 186 The most significant advantage of targeted drug deliveries is that the enormous drug-mediated toxicities on other adjacent tissues and organs can be minimized. We can also avoid most of the collateral damage leading to stress and organ failures. However, the effectiveness of many of these delivery methods in patients is still under clinical evaluation. Further details regarding these delivery systems can be accessed in the previously published reports. 187 , 188

Advances in cancer therapy have prompted scientists to look for natural tumoricidal chemicals, such as antimicrobial peptides (AMPs), that could be used for cancer treatment. 202 , 203 , 204 In addition to antibacterial action, several AMPs have been shown to have intrinsic anticancer potential. 205 , 206 These molecules constitute an intriguing new resource for anticancer drugs, and they provide several distinct therapeutic advantages over other anticancer treatments since they do not bind directly to any particular receptor in cancer cells. 207 The polycationic and amphipathic character of AMPs has been postulated as a possible explanation for the preferential interaction of AMPs with cancer cells, as compared to normal cells. 208 , 209 Membrane disruption has been identified as a critical stage in the microbial killing mechanism of several AMPs. These anticancer effects of AMPs are unaffected by molecular heterogeneity inside a tumor or across tumors, nor are they counteracted by cancer cells' flexibility and alternate survival pathways, as is commonly found in cancer resistance ( Fig. 4 ). 204 , 209 , 210 Figure 4 Advantages of antimicrobial peptides as anti-tumor agents. Fig. 4 Cathelicidins are members of the antimicrobial peptide family found in granules of vertebrate neutrophils. 211 The only human cathelicidin is the cationic peptide LL-37. Cathelicidins are composed of a highly conserved N-terminal signal peptide including a cathelin domain and a C-terminal cationic antimicrobial peptide. Apart from their antimicrobial activity, cathelicidin-derived antimicrobial peptides exhibit a range of biological roles, including antiviral, antifungal, anticancer, and immunomodulatory properties. 212 , 213 LL-37 exhibits anticancer activity against oral cancer cells and has been identified as a molecular target for colon cancer. 214 Immunohistochemical staining of colon cancer tissue showed that LL-37 is abundantly expressed in normal colon mucosa but downregulated in colon cancer tissues due to DNA methylation in the LL-37 promoter. 215 In human oral squamous cell carcinoma SAS-H1 cells, the active domain of LL-37 induces mitochondrial depolarization, which results in caspase-independent apoptosis. Furthermore, mice lacking cathelicidin are more susceptible to azoxymethane-induced colon carcinogenesis. 216 Many important advancements have been achieved using antimicrobial peptides against colorectal cancer. 217 Using LL-37 on the surface of magnetic nanoparticles led to a significant reduction in cell viability of colon cancer cell lines, inducing apoptosis at a much higher rate than in the case of LL-37 peptide alone. 218 Fan et al created a biodegradable and injectable thermosensitive hydrogel containing docetaxel and LL-37 polymeric nanoparticles. Intraperitoneal injection of this hydrogel substantially inhibited the development of HCT116 carcinomatosis in a mouse model of colorectal carcinoma. 219 In comparison to existing chemotherapeutic agents, the flexible nature of peptide structures, along with non-receptor activity, might confer strategic benefits on AMPs for the treatment of colon cancer in the future. 220 New treatment approaches for patients with advanced-stage, unresectable melanomas continue to be of significant clinical interest, as these patients cannot be treated surgically and have only a modest or temporary response to chemotherapy and radiation. In phase 1 clinical trial NCT02225366 , intra-tumoral injections of LL-37 are being explored for melanoma patients with cutaneous metastases to determine the most effective dose of LL-37 that could be administered. Patients will receive weekly intra-tumoral injections of LL-37 (starting dose, 250 μg/tumor) every 7 days for up to 8 weeks. Researchers are also interested in understanding whether LL37 can activate the immune system, which could help in the control of the disease. 221 Although AMPs have the potential to develop into a more effective and safer alternative to conventional cancer therapy, future research and development of anticancer peptide therapeutics will most likely focus on the poor pharmacokinetics, the control of selectivity, toxicity, and routes of administration through the establishment of appropriate in vivo models. 222

Combination therapy may offer the advantages of the fast tumor regression of targeted therapies with the long-lasting responses induced by immunotherapy. 235 Immunotherapy may be able to consolidate the dramatic tumor responses achieved with targeted therapy into durable, long-lasting remissions, in which sustained host responses targeting multiple antigens may reduce the risk of developing potentially lethal drug-resistant clones. 236 Combining cancer vaccines with immune checkpoint inhibitors (ICIs) such as those targeting cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) is one approach to avoid T cell anergy. 237 , 238 Moreover, T-cell and NK-cell invasion may be facilitated by targeted drugs that modify the tumor endothelium. 239 Gemcitabine, for example, is a deoxycytidine analog that induces tumor cell death, which results in antigen cross-presentation and cross-priming. 240 , 241 Carboplatin, pemetrexed, and pembrolizumab are now commonly used in the treatment of non-small cell lung cancer as first-line agents. 242 For triple-negative breast cancer, nab-paclitaxel and atezolizumab (anti-PD-L1) were recently authorized based on an improvement in progression-free survival (PFS) over chemotherapy. 243 Adenosine receptor antagonists (ARs) are involved in regulating tumor progression. 244 The four ARs discovered so far, A 1 , A 2A , A 2B and A 3 , belong to the class A family of G protein-coupled receptors (GPCRs). 245 The A 2B R seems more promising as a target in chemotherapeutic interventions, 246 whereas the A 2A R is the main target in the immunotherapeutic approach. 247 , 248 The existence of dual compounds, i.e., molecules acting on the A 2A R and on the A 2B R, has prompted their use in both immunotherapeutic and chemotherapeutic interventions. 249 , 250 Epigenetic modulators may have a dual function in the tumor microenvironment, boosting both antigen expression and T cell effector activity. 251 , 252 ICI-induced reactivation of exhausted T cells is linked with considerable chromatin remodeling, indicating that epigenetic modification may relieve certain patients with exhausted T cell function. 253 , 254 While many strategies focus on modifying the native tumor microenvironment, an alternative strategy involves the direct infusion of engineered immune cells or cellular receptors into the patient via adoptive cellular therapy with the goal of increasing the number of effector cells that recognize tumor-expressed antigens. 255 Upon reinfusion, lymphocytes grown ex vivo may respond more potently to tumor antigen since they have not been continuously exposed to tolerogenic microenvironmental signals. 256 Tumor infiltrating lymphocytes (TIL) treatment, 257 chimeric antigen receptor (CAR) engineered T-cells, 258 , 259 and TCR-engineered cell therapy 260 , 261 are the three main forms of adoptive T cell therapy currently being investigated. 257 , 262 , 263 Injecting oncolytic viruses directly into the tumor microenvironment is an alternate technique for improving tumor antigen recognition and strengthening T cell responses. 264 Talimogene laherparepvec (T-VEC) is a modified herpes simplex virus that is injected intra-lesionally into the melanoma tumor in individuals who have failed to get their tumor removed. It induces immediate lysis of tumor cells and the production of granulocyte-macrophage colony-stimulating factor (GM-CSF), which serves as a cytokine to stimulate the recruitment, maturation, and activity of antigen presenting cells (APCs). 265 Several studies combining immunotherapy with molecularly targeted treatment, either concurrently or sequentially, have shown hepatotoxicity as a major concern. 266 Combinations of dabrafenib, trametinib, and anti-PD-1 treatment have been associated with a greater risk of grade 3/4 adverse events in melanoma than would be expected with targeted therapy alone. 256 Emerging drugs are beginning to take a more comprehensive perspective of the tumor microenvironment (TME) and are revealing complementary techniques to simultaneously restore and enhance immune activity and promote therapeutic synergy. 267 , 268 Future research of combined therapy should continue to concentrate on exploring the intricate interplay between targeted drugs and immunotherapy, as well as optimizing parameters such as administration time, dose, and sequencing that may enhance the therapeutic index. 240

This research was funded by "Agencia Canaria de Investigación, Innovación y Sociedad de la Información ( 10.13039/501100007757 ACIISI ) del Gobierno de Canarias” (No. ProID2020010134), and óCaja Canarias (Project No. 2019SP43).