The Capacity of Drug-Metabolising Enzymes in Modulating the Therapeutic Efficacy of Drugs to Treat Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a rare soft tissue sarcoma (STS) that predominantly affects children and teenagers. It is the most common STS in children (40%) and accounts for 5-8% of total childhood malignancies. Apart from surgery and radiotherapy in eligible patients, standard chemotherapy is the only therapeutic option clinically available for RMS patients. While survival rates for this childhood cancer have considerably improved over the last few decades for low-risk and intermediate-risk cases, the mortality rate remains exceptionally high in high-risk RMS patients with recurrent and/or metastatic disease. The intensification of chemotherapeutic protocols in advanced-stage RMS has historically induced aggravated toxicity with only very modest therapeutic gain. In this review, we critically analyse what has been achieved so far in RMS therapy and provide insight into how a diverse group of drug-metabolising enzymes (DMEs) possess the capacity to modify the clinical efficacy of chemotherapy. We provide suggestions for new therapeutic strategies that exploit the presence of DMEs for prodrug activation, targeted chemotherapy that does not rely on DMEs, and RMS-molecular-subtype-targeted therapies that have the potential to enter clinical evaluation.

Machine learning and bioinformatic analyses link the cell surface receptor transcript levels to the drug response of breast cancer cells and drug off-target effects.

Breast cancer responds variably to anticancer therapies, often leading to significant off-target effects. This study proposes that the variability in tumour responses and drug-induced adverse events is linked to the transcriptional profiles of cell surface receptors (CSRs) in breast tumours and normal tissues. We analysed multiple datasets to compare CSR expression in breast tumours with that in non-cancerous human tissues. Our findings correlate the drug responses of breast cancer cell lines with the expression levels of their targeted CSRs. Notably, we identified distinct differences in CSR expression between primary breast tumour subtypes and corresponding cell lines, which may influence drug response predictions. Additionally, we used clinical trial data to uncover associations between CSR gene expression in healthy tissues and the incidence of adverse drug reactions. This integrative approach facilitates the selection of optimal CSR targets for therapy, leveraging cell line dose-responses, CSR expression in normal tissues, and patient adverse event profiles.

AAV Immunotoxicity: Implications in Anti-HBV Gene Therapy.

Hepatitis B virus (HBV) has afflicted humankind for decades and there is still no treatment that can clear the infection. The development of recombinant adeno-associated virus (rAAV)-based gene therapy for HBV infection has become important in recent years and research has made exciting leaps. Initial studies, mainly using mouse models, showed that rAAVs are non-toxic and induce minimal immune responses. However, several later studies demonstrated rAAV toxicity, which is inextricably associated with immunogenicity. This is a major setback for the progression of rAAV-based therapies toward clinical application. Research aimed at understanding the mechanisms behind rAAV immunity and toxicity has contributed significantly to the inception of approaches to overcoming these challenges. The target tissue, the features of the vector, and the vector dose are some of the determinants of AAV toxicity, with the latter being associated with the most severe adverse events. This review discusses our current understanding of rAAV immunogenicity, toxicity, and approaches to overcoming these hurdles. How this information and current knowledge about HBV biology and immunity can be harnessed in the efforts to design safe and effective anti-HBV rAAVs is discussed.

Antitumor synergism between PAK4 silencing and immunogenic phototherapy of engineered extracellular vesicles.

Immunotherapy has revolutionized the landscape of cancer treatment. However, single immunotherapy only works well in a small subset of patients. Combined immunotherapy with antitumor synergism holds considerable potential to boost the therapeutic outcome. Nevertheless, the synergistic, additive or antagonistic antitumor effects of combined immunotherapies have been rarely explored. Herein, we established a novel combined cancer treatment modality by synergizing p21-activated kinase 4 (PAK4) silencing with immunogenic phototherapy in engineered extracellular vesicles (EVs) that were fabricated by coating M1 macrophage-derived EVs on the surface of the nano-complex cores assembled with siRNA against PAK4 and a photoactivatable polyethyleneimine. The engineered EVs induced potent PAK4 silencing and robust immunogenic phototherapy, thus contributing to effective antitumor effects in vitro and in vivo. Moreover, the antitumor synergism of the combined treatment was quantitatively determined by the CompuSyn method. The combination index (CI) and isobologram results confirmed that there was an antitumor synergism for the combined treatment. Furthermore, the dose reduction index (DRI) showed favorable dose reduction, revealing lower toxicity and higher biocompatibility of the engineered EVs. Collectively, the study presents a synergistically potentiated cancer treatment modality by combining PAK4 silencing with immunogenic phototherapy in engineered EVs, which is promising for boosting the therapeutic outcome of cancer immunotherapy.

CSPG4 as a target for the specific killing of triple-negative breast cancer cells by a recombinant SNAP-tag-based antibody-auristatin F drug conjugate.

PURPOSE: Triple-negative breast cancer (TNBC) is phenotypic of breast tumors lacking expression of the estrogen receptor (ER), the progesterone receptor (PgR), and the human epidermal growth factor receptor 2 (HER2). The paucity of well-defined molecular targets in TNBC, coupled with the increasing burden of breast cancer-related mortality, emphasizes the need to develop targeted diagnostics and therapeutics. While antibody-drug conjugates (ADCs) have emerged as revolutionary tools in the selective delivery of drugs to malignant cells, their widespread clinical use has been hampered by traditional strategies which often give rise to heterogeneous mixtures of ADC products. METHODS: Utilizing SNAP-tag technology as a cutting-edge site-specific conjugation method, a chondroitin sulfate proteoglycan 4 (CSPG4)-targeting ADC was engineered, encompassing a single-chain antibody fragment (scFv) conjugated to auristatin F (AURIF) via a click chemistry strategy. RESULTS: After showcasing the self-labeling potential of the SNAP-tag component, surface binding and internalization of the fluorescently labeled product were demonstrated on CSPG4-positive TNBC cell lines through confocal microscopy and flow cytometry. The cell-killing ability of the novel AURIF-based recombinant ADC was illustrated by the induction of a 50% reduction in cell viability at nanomolar to micromolar concentrations on target cell lines. CONCLUSION: This research underscores the applicability of SNAP-tag in the unambiguous generation of homogeneous and pharmaceutically relevant immunoconjugates that could potentially be instrumental in the management of a daunting disease like TNBC.

Technological advances in the use of viral and non-viral vectors for delivering genetic and non-genetic cargos for cancer therapy.

The burden of cancer is increasing globally. Several challenges facing its mainstream treatment approaches have formed the basis for the development of targeted delivery systems to carry and distribute anti-cancer payloads to their defined targets. This site-specific delivery of drug molecules and gene payloads to selectively target druggable biomarkers aimed at inducing cell death while sparing normal cells is the principal goal for cancer therapy. An important advantage of a delivery vector either viral or non-viral is the cumulative ability to penetrate the haphazardly arranged and immunosuppressive tumour microenvironment of solid tumours and or withstand antibody-mediated immune response. Biotechnological approaches incorporating rational protein engineering for the development of targeted delivery systems which may serve as vehicles for packaging and distribution of anti-cancer agents to selectively target and kill cancer cells are highly desired. Over the years, these chemically and genetically modified delivery systems have aimed at distribution and selective accumulation of drug molecules at receptor sites resulting in constant maintenance of high drug bioavailability for effective anti-tumour activity. In this review, we highlighted the state-of-the art viral and non-viral drug and gene delivery systems and those under developments focusing on cancer therapy.

Click Chemistry-Generated Auristatin F-Linker-Benzylguanine for a SNAP-Tag-Based Recombinant Antibody-Drug Conjugate Demonstrating Selective Cytotoxicity toward EGFR-Overexpressing Tumor Cells.

Antibody-drug conjugates (ADCs) are bifunctional molecules combining the targeting potential of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs. This simple yet intelligently designed system directly addresses the lack of specificity encountered with conventional anti-cancer treatment regimes. However, despite their initial success, the generation of clinically sustainable and effective ADCs has been plagued by poor tumor penetration, undefined chemical linkages, unpredictable pharmacokinetic profiles, and heterogeneous mixtures of products. To this end, we generated a SNAP-tag-based fusion protein targeting the epidermal growth factor receptor (EGFR)-a biomarker of aggressive and drug-resistant cancers. Here, we demonstrate the use of a novel click coupling strategy to engineer a benzylguanine (BG)-linker-auristatin F (AuriF) piece that can be covalently tethered to the EGFR-targeting SNAP-tag-based fusion protein in an irreversible 1:1 stoichiometric reaction to form a homogeneous product. Furthermore, using these recombinant ADCs to target EGFR-overexpressing tumor cells, we provide a proof-of-principle for generating biologically active antimitotic therapeutic proteins capable of inducing cell death in a dose-dependent manner, thus alleviating some of the challenges of early ADC development.

The Evolution of SNAP-Tag Labels.

The conjugation of proteins with synthetic molecules can be conducted in many different ways. In this Perspective, we focus on tag-based techniques and specifically on the SNAP-tag technology. The SNAP-tag technology makes use of a fusion protein between a protein of interest and an enzyme tag that enables the actual conjugation reaction. The SNAP-tag is based on the O6-alkylguanine-DNA alkyltransferase (AGT) enzyme and is optimized to react selectively with O6-benzylguanine (BG) substrates. BG-containing dye derivatives have frequently been used to introduce a fluorescent tag to a specific protein. We believe that the site-specific conjugation of polymers to proteins can significantly benefit from the SNAP-tag technology. Especially, polymers synthesized via reversible deactivation radical polymerization allow for the facile introduction of a BG end group to enable SNAP-tag conjugation.

TIM-3: a tumor-associated antigen beyond checkpoint inhibition?

Immune checkpoint inhibitors are one of the most remarkable immunomodulatory therapies of current times. Sabatolimab is a high-affinity, humanized anti-TIM-3 monoclonal antibody currently in development for patients with myeloproliferative disorders, including acute myeloid leukemia and myelodysplastic syndromes. By targeting TIM-3, a receptor expressed on various immune effector cells as well as myeloid cells, multiple mechanisms of action that are distinct from canonical immune checkpoint inhibitors are in play - (i) blockade of TIM-3 and its ligands PtdSer/galectin-9, (ii) modulation of leukemic cell self-renewal as well as (iii) antibody-dependent phagocytosis of TIM-3-expressing leukemic cells. Novel immunotherapies such as sabatolimab which enhance the antitumor immune response on converging fronts represent the promise of a continuously replenished armoury for the treatment of cancer.

The Potential of Antibody Technology and Silver Nanoparticles for Enhancing Photodynamic Therapy for Melanoma.

Melanoma is highly aggressive and is known to be efficient at resisting drug-induced apoptotic signals. Resection is currently the gold standard for melanoma management, but it only offers local control of the early stage of the disease. Metastatic melanoma is prone to recurrence, and has a poor prognosis and treatment response. Thus, the need for advanced theranostic alternatives is evident. Photodynamic therapy has been increasingly studied for melanoma treatment; however, it relies on passive drug accumulation, leading to off-target effects. Nanoparticles enhance drug biodistribution, uptake and intra-tumoural concentration and can be functionalised with monoclonal antibodies that offer selective biorecognition. Antibody-drug conjugates reduce passive drug accumulation and off-target effects. Nonetheless, one limitation of monoclonal antibodies and antibody-drug conjugates is their lack of versatility, given cancer's heterogeneity. Monoclonal antibodies suffer several additional limitations that make recombinant antibody fragments more desirable. SNAP-tag is a modified version of the human DNA-repair enzyme, O6-alkylguanine-DNA alkyltransferase. It reacts in an autocatalytic and covalent manner with benzylguanine-modified substrates, providing a simple protein labelling system. SNAP-tag can be genetically fused with antibody fragments, creating fusion proteins that can be easily labelled with benzylguanine-modified payloads for site-directed delivery. This review aims to highlight the benefits and limitations of the abovementioned approaches and to outline how their combination could enhance photodynamic therapy for melanoma.

Properties of Bulk In-Pt Intermetallic Compounds in Methanol Steam Reforming.

Heterogeneous catalysts are often complex materials containing different compounds. While this can lead to highly beneficial interfaces, it is difficult to identify the role of single components. In methanol steam reforming (MSR), the interplay between intermetallic compounds, supporting oxides and redox reactions leads to highly active and CO2 -selective materials. Herein, the intrinsic catalytic properties of unsupported In3 Pt2 , In2 Pt, and In7 Pt3 as model systems for Pt/In2 O3 -based catalytic materials in MSR are addressed. In2 Pt was identified as the essential compound responsible for the reported excellent CO2 -selectivity of 99.5 % at 300 °C in supported systems, showing a CO2 -selectivity above 99 % even at 400 °C. Additionally, the partial oxidation of In7 Pt3 revealed that too much In2 O3 is detrimental for the catalytic properties. The study highlights the crucial role of intermetallic In-Pt compounds in Pt/In2 O3 materials with excellent CO2 -selectivity.

Antibody-siRNA conjugates (ARCs) using multifunctional peptide as a tumor enzyme cleavable linker mediated effective intracellular delivery of siRNA.

The tissue-specific targeted delivery and efficient cellular uptake of siRNAs are the main obstacles to their clinical application. Antibody-siRNA-conjugates (ARCs) can deliver siRNA by exploiting the targeting property of antibodies like antibody-drug conjugates (ADCs). However, the effective conjugation of antibodies and siRNAs and the release of siRNAs specifically at target sites have posed challenges to the development of ARCs. In this study, the successful conjugation of antibodies and siRNAs was achieved using a multifunctional peptide as a linker, composed of a cell-penetrating peptide (CPP) and a substrate peptide (SP), which is highly expressed in solid tumors. The resulting antibody-multifunctional peptide (SP-CPP)-siRNA system delivered the siRNA to target tumor cells by the specific binding of the antibody. Once the enzymes on the tumor cell surface hydrolyzed the substrate peptide linker, siRNA-CPP was released from ARCs. The released siRNA-CPP entered the targeted cells via the cellular penetrating ability of CPP, resulting in improved siRNA-mediated gene silencing efficiency, verified both in vitro and in vivo. After intravenous administration, the designed ARCs achieved approximately 66.7% EGFP (Enhanced Green Fluorescent Protein) downregulation efficiency in nude mice xenografted with the HCT116-EGFP tumor model. The proposed system provides a prospective choice for ARC production and the safe and efficient delivery of siRNAs.

Isolation and light chain shuffling of a Plasmodium falciparum AMA1-specific human monoclonal antibody with growth inhibitory activity.

BACKGROUND: Plasmodium falciparum, the parasite causing malaria, affects populations in many endemic countries threatening mainly individuals with low malaria immunity, especially children. Despite the approval of the first malaria vaccine Mosquirix™ and very promising data using cryopreserved P. falciparum sporozoites (PfSPZ), further research is needed to elucidate the mechanisms of humoral immunity for the development of next-generation vaccines and alternative malaria therapies including antibody therapy. A high prevalence of antibodies against AMA1 in immune individuals has made this antigen one of the major blood-stage vaccine candidates. MATERIAL AND METHODS: Using antibody phage display, an AMA1-specific growth inhibitory human monoclonal antibody from a malaria-immune Fab library using a set of three AMA1 diversity covering variants (DiCo 1-3), which represents a wide range of AMA1 antigen sequences, was selected. The functionality of the selected clone was tested in vitro using a growth inhibition assay with P. falciparum strain 3D7. To potentially improve affinity and functional activity of the isolated antibody, a phage display mediated light chain shuffling was employed. The parental light chain was replaced with a light chain repertoire derived from the same population of human V genes, these selected antibodies were tested in binding tests and in functionality assays. RESULTS: The selected parental antibody achieved a 50% effective concentration (EC50) of 1.25 mg/mL. The subsequent light chain shuffling led to the generation of four derivatives of the parental clone with higher expression levels, similar or increased affinity and improved EC50 against 3D7 of 0.29 mg/mL. Pairwise epitope mapping gave evidence for binding to AMA1 domain II without competing with RON2. CONCLUSION: We have thus shown that a compact immune human phage display library is sufficient for the isolation of potent inhibitory monoclonal antibodies and that minor sequence mutations dramatically increase expression levels in Nicotiana benthamiana. Interestingly, the antibody blocks parasite inhibition independently of binding to RON2, thus having a yet undescribed mode of action.

Identification of the atypically modified autoantigen Ars2 as the target of B-cell receptors from activated B-cell-type diffuse large B-cell lymphoma.

It has been suggested that B-cell receptor (BCRs) stimulation by specific antigens plays a pathogenic role in diffuse large B-cell lymphoma (DLBCL). Here, it was the aim to screen for specific reactivities of DLBCL-BCRs in the spectrum of autoantigens and antigens of infectious origin. Arsenite resistance protein 2 (Ars2) was identified as the BCR target of 3/5 ABC-type DLBCL cell lines and 2/11 primary DLBCL cases. Compared to controls, Ars2 was hypo-phosphorylated exclusively in cases and cell lines with Ars2-specific BCRs. In a validation cohort, hypo-phosphorylated Ars2 was found in 8/31 ABC-type, but only 1/20 germinal center B cell (GBC)-like type DLBCL. Incubation with Ars2 induced BCR-pathway activation and increased proliferation, while an Ars2/ETA-toxin conjugate induced killing of cell lines with Ars2-reactive BCRs. Ars2 appears to play a role in a subgroup of ABC-type DLBCLs. Moreover, transformed DLBCL lines with Ars2-reactive BCRs still show growth advantage after incubation with Ars2. These results provide knowledge about the pathogenic role of a specific antigen stimulating the BCR pathway in DLCBL.

Antibody-Based Targeted Interventions for the Diagnosis and Treatment of Skin Cancers.

BACKGROUND: Cutaneous malignancies most commonly arise from skin epidermal cells. These cancers may rapidly progress from benign to a metastatic phase. Surgical resection represents the gold standard therapeutic treatment of non-metastatic skin cancer while chemo- and/or radiotherapy are often used against metastatic tumors. However, these therapeutic treatments are limited by the development of resistance and toxic side effects, resulting from the passive accumulation of cytotoxic drugs within healthy cells. OBJECTIVE: This review aims to elucidate how the use of monoclonal Antibodies (mAbs) targeting specific Tumor Associated Antigens (TAAs) is paving the way to improved treatment. These mAbs are used as therapeutic or diagnostic carriers that can specifically deliver cytotoxic molecules, fluorophores or radiolabels to cancer cells that overexpress specific target antigens. RESULTS: mAbs raised against TAAs are widely in use for e.g. differential diagnosis, prognosis and therapy of skin cancers. Antibody-Drug Conjugates (ADCs) particularly show remarkable potential. The safest ADCs reported to date use non-toxic photo-activatable Photosensitizers (PSs), allowing targeted Photodynamic Therapy (PDT) resulting in targeted delivery of PS into cancer cells and selective killing after light activation without harming the normal cell population. The use of near-infrared-emitting PSs enables both diagnostic and therapeutic applications upon light activation at the specific wavelengths. CONCLUSION: Antibody-based approaches are presenting an array of opportunities to complement and improve current methods employed for skin cancer diagnosis and treatment.

Recent advances in immunotherapies against infectious diseases.

Immunotherapies are disease management strategies that target or manipulate components of the immune system. Infectious diseases pose a significant threat to human health as evidenced by countries continuing to grapple with several emerging and re-emerging diseases, the most recent global health threat being the SARS-CoV2 pandemic. As such, various immunotherapeutic approaches are increasingly being investigated as alternative therapies for infectious diseases, resulting in significant advances towards the uncovering of pathogen-host immunity interactions. Novel and innovative therapeutic strategies are necessary to overcome the challenges typically faced by existing infectious disease prevention and control methods such as lack of adequate efficacy, drug toxicity, and the emergence of drug resistance. As evidenced by recent developments and success of pharmaceuticals such as monoclonal antibodies (mAbs), immunotherapies already show abundant promise to overcome such limitations while also advancing the frontiers of medicine. In this review, we summarize some of the most notable inroads made to combat infectious disease, over mainly the last 5 years, through the use of immunotherapies such as vaccines, mAb-based therapies, T-cell-based therapies, manipulation of cytokine levels, and checkpoint inhibition. While its most general applications are founded in cancer treatment, advances made towards the curative treatment of human immunodeficiency virus, tuberculosis, malaria, zika virus and, most recently COVID-19, reinforce the role of immunotherapeutic strategies in the broader field of disease control. Ultimately, the comprehensive specificity, safety, and cost of immunotherapeutics will impact its widespread implementation.

Using the SNAP-Tag technology to easily measure and demonstrate apoptotic changes in cancer and blood cells with different dyes.

In vitro and ex vivo development of novel therapeutic agents requires reliable and accurate analyses of the cell conditions they were preclinical tested for, such as apoptosis. The detection of apoptotic cells by annexin V (AV) coupled to fluorophores has often shown limitations in the choice of the dye due to interference with other fluorescent-labeled cell markers. The SNAP-tag technology is an easy, rapid and versatile method for functionalization of proteins and was therefore used for labeling AV with various fluorophores. We generated the fusion protein AV-SNAP and analyzed its capacity for the specific display of apoptotic cells in various assays with therapeutic agents. AV-SNAP showed an efficient coupling reaction with five different fluorescent dyes. Two selected fluorophores were tested with suspension, adherent and peripheral blood cells, treated by heat-shock or apoptosis-inducing therapeutic agents. Flow cytometry analysis of apoptotic cells revealed a strong visualization using AV-SNAP coupled to these two fluorophores exemplary, which was comparable to a commercial AV-Assay-kit. The combination of the apoptosis-specific binding protein AV with the SNAP-tag provides a novel solid method to facilitate protein labeling using several, easy to change, fluorescent dyes at once. It avoids high costs and allows an ordinary exchange of dyes and easier use of other fluorescent-labeled cell markers, which is of high interest for the preclinical testing of therapeutic agents in e.g. cancer research.

Advances in epidermal growth factor receptor specific immunotherapy: lessons to be learned from armed antibodies.

The epidermal growth factor receptor (EGFR) has been recognized as an important therapeutic target in oncology. It is commonly overexpressed in a variety of solid tumors and is critically involved in cell survival, proliferation, metastasis, and angiogenesis. This multi-dimensional role of EGFR in the progression and aggressiveness of cancer, has evolved from conventional to more targeted therapeutic approaches. With the advent of hybridoma technology and phage display techniques, the first anti-EGFR monoclonal antibodies (mAbs) (Cetuximab and Panitumumab) were developed. Due to major limitations including host immune reactions and poor tumor penetration, these antibodies were modified and used as guiding mechanisms for the specific delivery of readily available chemotherapeutic agents or plants/bacterial toxins, giving rise to antibody-drug conjugates (ADCs) and immunotoxins (ITs), respectively. Continued refinement of ITs led to deimmunization strategies based on depletion of B and T-cell epitopes or substitution of non-human toxins leading to a growing repertoire of human enzymes capable of inducing cell death. Similarly, the modification of classical ADCs has resulted in the first, fully recombinant versions. In this review, we discuss significant advancements in EGFR-targeting immunoconjugates, including ITs and recombinant photoactivable ADCs, which serve as a blueprint for further developments in the evolving domain of cancer immunotherapy.

Antibody-Based Immunotherapy: Alternative Approaches for the Treatment of Metastatic Melanoma.

Melanoma is the least common form of skin cancer and is associated with the highest mortality. Where melanoma is mostly unresponsive to conventional therapies (e.g., chemotherapy), BRAF inhibitor treatment has shown improved therapeutic outcomes. Photodynamic therapy (PDT) relies on a light-activated compound to produce death-inducing amounts of reactive oxygen species (ROS). Their capacity to selectively accumulate in tumor cells has been confirmed in melanoma treatment with some encouraging results. However, this treatment approach has not reached clinical fruition for melanoma due to major limitations associated with the development of resistance and subsequent side effects. These adverse effects might be bypassed by immunotherapy in the form of antibody-drug conjugates (ADCs) relying on the ability of monoclonal antibodies (mAbs) to target specific tumor-associated antigens (TAAs) and to be used as carriers to specifically deliver cytotoxic warheads into corresponding tumor cells. Of late, the continued refinement of ADC therapeutic efficacy has given rise to photoimmunotherapy (PIT) (a light-sensitive compound conjugated to mAbs), which by virtue of requiring light activation only exerts its toxic effect on light-irradiated cells. As such, this review aims to highlight the potential clinical benefits of various armed antibody-based immunotherapies, including PDT, as alternative approaches for the treatment of metastatic melanoma.

Desensitization of metastatic melanoma cells to therapeutic treatment through repeated exposure to dacarbazine.

Aberrant anti-cancer drug efflux mediated by membrane protein ABC transporters (ABCB5 and ABCG2) is thought to characterize melanoma heterogeneous chemoresistant populations, presumed to have unlimited proliferative and self-renewal abilities. Therefore, this study primarily aimed to investigate whether continuous exposure of melanoma cells to dacarbazine (DTIC) chemotherapeutic drug enriches cultures with therapy resistant cells. Thereafter, we sought to determine whether combining the genotoxic activity of DTIC with the oxidative insults of hypericin activated photodynamic therapy (HYP-PDT) could synergized to kill heterogenous chemoresistant melanoma populations. This study revealed that DTIC resistant (UCT Mel-1DTICR2) melanoma cells were less sensitive to all therapies than parental melanoma cells (UCT Mel-1), yet combination therapy was the most efficient. At the exception of DTIC treatment, both HYP-PDT and the combination therapy were effective in significantly reducing the Hoechst non-effluxing dye melanoma main populations (MP) compared to their side population (SP) counterparts. Likewise, HYP-PDT and combination therapy significantly reduced self-renewal capacity, increased expression of ABCB5 and ABCG2 transporters and differentially induced cell cycle arrest and cell death (apoptosis or necrosis) depending on the melanoma MP cell type. Collectively, combination therapy could synergistically reduce melanoma proliferative and clonogenic potential. However, further research is needed to decipher the cellular mechanisms underlying this resistance which would enable combination therapy to reach therapeutic fruition.