Restricting tumor lactic acid metabolism using dichloroacetate improves T cell functions.

BACKGROUND: Lactic acid produced by tumors has been shown to overcome immune surveillance, by suppressing the activation and function of T cells in the tumor microenvironment. The strategies employed to impair tumor cell glycolysis could improve immunosurveillance and tumor growth regulation. Dichloroacetate (DCA) limits the tumor-derived lactic acid by altering the cancer cell metabolism. In this study, the effects of lactic acid on the activation and function of T cells, were analyzed by assessing T cell proliferation, cytokine production and the cellular redox state of T cells. We examined the redox system in T cells by analyzing the intracellular level of reactive oxygen species (ROS), superoxide and glutathione and gene expression of some proteins that have a role in the redox system. Then we co-cultured DCA-treated tumor cells with T cells to examine the effect of reduced tumor-derived lactic acid on proliferative response, cytokine secretion and viability of T cells. RESULT: We found that lactic acid could dampen T cell function through suppression of T cell proliferation and cytokine production as well as restrain the redox system of T cells by decreasing the production of oxidant and antioxidant molecules. DCA decreased the concentration of tumor lactic acid by manipulating glucose metabolism in tumor cells. This led to increases in T cell proliferation and cytokine production and also rescued the T cells from apoptosis. CONCLUSION: Taken together, our results suggest accumulation of lactic acid in the tumor microenvironment restricts T cell responses and could prevent the success of T cell therapy. DCA supports anti-tumor responses of T cells by metabolic reprogramming of tumor cells.

A melanoma patient with macrophage-cancer cell hybrids in the primary tumor, a lymph node metastasis and a brain metastasis.

In 1911 it was proposed that cancer might result from fusion and hybridization between macrophages and cancer cells. Using immunohistochemistry it was determined that essentially all solid tumors expressed macrophage-like molecules on their cell surface. More recently we have used forensic (STR) genetics that allows one to detect DNA from more than one individual in the same sample. By studying biopsies from individuals receiving allogeneic stem cell transplants and later developed solid tumor metastases, we were able to detect both donor and patient DNA sequences suggesting that hybrids were present. Previously we found hybrids in biopsies of a renal cell carcinoma, a melanoma in a brain metastasis and a melanoma in a primary tumor with lymph node metastases. Here we have traced hybrids from a primary melanoma to an axillary lymph node to a brain metastasis. This is the first time that the entire metastatic process has been documented.

International Scientific Collaboration Is Needed to Bridge Science to Society: USERN2020 Consensus Statement.

Scientific collaboration has been a critical aspect of the development of all fields of science, particularly clinical medicine. It is well understood that myriads of benefits can be yielded by interdisciplinary and international collaboration. For instance, our rapidly growing knowledge on COVID-19 and vaccine development could not be attained without expanded collaborative activities. However, achieving fruitful results requires mastering specific tactics in collaborative efforts. These activities can enhance our knowledge, which ultimately benefits society. In addition to tackling the issue of the invisible border between different countries, institutes, and disciplines, the border between the scientific community and society needs to be addressed as well. International and transdisciplinary approaches can potentially be the best solution for bridging science and society. The Universal Scientific Education and Research Network (USERN) is a non-governmental, non-profit organization and network to promote professional, scientific research and education worldwide. The fifth annual congress of USERN was held in Tehran, Iran, in a hybrid manner on November 7-10, 2020, with key aims of bridging science to society and facilitating borderless science. Among speakers of the congress, a group of top scientists unanimously agreed on The USERN 2020 consensus, which is drafted with the goal of connecting society with scientific scholars and facilitating international and interdisciplinary scientific activities in all fields, including clinical medicine.

Chimeric antigen receptor T-cell therapy for melanoma.

INTRODUCTION: In recent years, chimeric antigen receptor (CAR) T cell therapy has emerged as a cancer treatment. After initial therapeutic success for hematologic malignancies, this approach has been extended for the treatment of solid tumors including melanoma. AREAS COVERED: T cells need to be reprogramed to recognize specific antigens expressed only in tumor cells, a difficult problem since cancer cells are simply transformed normal cells. Tumor antigens, namely, CSPG4, CD70, and GD2 have been targeted by CAR-T cells for melanoma. Moreover, different co-stimulatory signaling domains need to be selected to direct T cell fate. In this review, various approaches for the treatment of melanoma and their effectiveness are comprehensively reviewed and the current status, challenges, and future perspective of CAR-T cell therapy for melanoma are discussed. Literature search was accomplished in three databases (PubMed, Google scholar, and Clinicaltrials.gov). Published papers and clinical trials were screened and relevant documents were included by checking pre-defined eligibility criteria. EXPERT OPINION: Despite obstacles and the risk of adverse events, CAR T cell therapy could be used for patients with treatment-resistant cancer. Clinical trials are underway to determine the efficacy of this approach for the treatment of melanoma.

A metabolic switch to memory CAR T cells: Implications for cancer treatment.

Therapeutic efficacy of chimeric antigen receptor (CAR) T cells is associated with their expansion, persistence and effector function. Although CAR T cell therapy has shown remarkable therapeutic effects in hematological malignancies, its therapeutic efficacy has been limited in some types of cancers - in particular, solid tumors - partially due to the cells' inability to persist and the acquisition of T cell dysfunction within a harsh immunosuppressive tumor microenvironment. Therefore, it would be expected that generation of CAR T cells with intrinsic properties for functional longevity, such as the cells with early-memory phenotypes, could beneficially enhance antitumor immunity. Furthermore, because the metabolic pathways of CAR T cells help determine cellular differentiation and lifespan, therapies targeting such pathways like glycolysis and oxidative phosphorylation, can alter CAR T cell fate and durability within tumors. Here we discuss how reprogramming of CAR T cell metabolism and metabolic switch to memory CAR T cells influences their antitumor activity. We also offer potential strategies for targeting these metabolic circuits in the setting of adoptive CAR T cell therapy, aiming to better unleash the potential of adoptive CAR T cell therapy in the clinic.

Recent Advances in Studies of Skin Color and Skin Cancer.

The relationship between skin color and skin cancer is well established: the less melanin in one's skin the greater the risk for developing skin cancer. This review is in two parts. First, we summarize the current understanding of the cutaneous pigmentary system and trace melanin from its synthesis in the pigment cell melanosomes through its transfer to keratinocytes. We also present new methods for reducing melanin content in hyper-pigmented areas of skin such as solar lentigenes, melasma, and post-inflammatory hyperpigmentation. Second, we present evidence that at least one mechanism for the development of metastatic melanoma and other solid tumors is fusion and hybridization of leucocytes such as macrophages with primary tumor cells. In this scenario, hybrid cells express both the chemotactic motility of the leucocyte and the de-regulated cell division of the tumor cell, causing the cells to migrate a deadly journey to lymph nodes, distant organs, and tissues.

Roles of cell fusion, hybridization and polyploid cell formation in cancer metastasis.

Cell-cell fusion is a normal biological process playing essential roles in organ formation and tissue differentiation, repair and regeneration. Through cell fusion somatic cells undergo rapid nuclear reprogramming and epigenetic modifications to form hybrid cells with new genetic and phenotypic properties at a rate exceeding that achievable by random mutations. Factors that stimulate cell fusion are inflammation and hypoxia. Fusion of cancer cells with non-neoplastic cells facilitates several malignancy-related cell phenotypes, e.g., reprogramming of somatic cell into induced pluripotent stem cells and epithelial to mesenchymal transition. There is now considerable in vitro, in vivo and clinical evidence that fusion of cancer cells with motile leucocytes such as macrophages plays a major role in cancer metastasis. Of the many changes in cancer cells after hybridizing with leucocytes, it is notable that hybrids acquire resistance to chemo- and radiation therapy. One phenomenon that has been largely overlooked yet plays a role in these processes is polyploidization. Regardless of the mechanism of polyploid cell formation, it happens in response to genotoxic stresses and enhances a cancer cell's ability to survive. Here we summarize the recent progress in research of cell fusion and with a focus on an important role for polyploid cells in cancer metastasis. In addition, we discuss the clinical evidence and the importance of cell fusion and polyploidization in solid tumors.

Cytidine decreases melanin content in a reconstituted three-dimensional human epidermal model.

The process of melanin biosynthesis and its distribution throughout the skin is regulated by complex processes involving several enzymes in melanocytes. Recently, Diwakar et al. demonstrated that cytidine-a sialyltransferase inhibitor, 6'-sialyllactose (6'-SL) and 3'-sialyllactose (3'-SL) inhibited melanogenesis and melanosome transfer process. In this study, we have furthered this research, considering cytidine as a commercially viable and safe option over 6'-SL and 3'-SL. The efficacy of 2% w/v cytidine was studied in MelanoDerm™ skin equivalents in comparison with the positive control 1% w/v kojic acid and the vehicle control. Both the positive control and cytidine demonstrated a significant reduction in melanin content relative to the vehicle control. These experiments conclude that cytidine can effectively reduce melanin content in a skin equivalence assay and suggests that cytidine may be a good candidate for a skin lightening agent for human skin.

Leukocyte⁻Cancer Cell Fusion-Genesis of a Deadly Journey.

According to estimates from the International Agency for Research on Cancer, by the year 2030 there will be 22 million new cancer cases and 13 million deaths per year. The main cause of cancer mortality is not the primary tumor itself but metastasis to distant organs and tissues, yet the mechanisms of this process remain poorly understood. Leukocyte⁻cancer cell fusion and hybrid formation as an initiator of metastasis was proposed more than a century ago by the German pathologist Prof. Otto Aichel. This proposal has since been confirmed in more than 50 animal models and more recently in one patient with renal cell carcinoma and two patients with malignant melanoma. Leukocyte⁻tumor cell fusion provides a unifying explanation for metastasis. While primary tumors arise in a wide variety of tissues representing not a single disease but many different diseases, metastatic cancer may be only one disease arising from a common, nonmutational event: Fusion of primary tumor cells with leukocytes. From the findings to date, it would appear that such hybrid formation is a major pathway for metastasis. Studies on the mechanisms involved could uncover new targets for therapeutic intervention.

Clinical evaluation of the lightening effect of cytidine on hyperpigmented skin.

BACKGROUND: Melanocytes, which reside in the basal layer of the epidermis, produce the pigment melanin in cytoplasmic organelles known as melanosomes. Melanosomes are transferred to keratinocytes which provide the color in our skin. Recently, Diwakar et  al reported the crucial roles of protein glycosylation in both melanogenesis and melanosome transfer to keratinocytes, and each was inhibited by the nucleotide cytidine. OBJECTIVE: The main objective of this study was to determine the clinical effects of topical application of cytidine to the hyperpigmented regions of the face in a group of human volunteers. METHODS: A randomized, vehicle-controlled study was conducted for 12 weeks on healthy Korean female subjects. Cytidine was formulated into the lotion at concentrations of 2%, 3%, and 4% (w/w) and compared to the vehicle control formulation. The clinical outcomes were evaluated by performing visual assessment grading, measuring melanin index, skin brightness, and skin color parameters. In vitro skin penetration studies were conducted using Franz cell chambers for the 2% cytidine test formulation. RESULTS: The test group showed significant improvements in the visual assessment scores, melanin index, skin brightness, and skin color compared to the control group. Although significant dose-dependent improvements were seen in the clinical study, the in vitro Franz cell studies indicated that the clinical efficacy and potency of cytidine might be further enhanced by formulating a better topical delivery system, which will be the goal of our future studies. CONCLUSIONS: This randomized, double-blind, 12-week clinical study successfully demonstrated the efficacy of cytidine on skin depigmentation in a dose-dependent manner.

A Melanoma Lymph Node Metastasis with a Donor-Patient Hybrid Genome following Bone Marrow Transplantation: A Second Case of Leucocyte-Tumor Cell Hybridization in Cancer Metastasis.

BACKGROUND: Metastatic disease is the principal cause of mortality in cancer, yet the underlying mechanisms are not fully understood. Macrophage-cancer cell fusion as a cause of metastasis was proposed more than a century ago by German pathologist Prof. Otto Aichel. Since then this theory has been confirmed in numerous animal studies and recently in a patient with metastatic melanoma. METHODS: Here we analyzed tumor DNA from a 51-year-old man who, 8 years following an allogeneic BMT from his brother for treatment of chronic myelogenous leukemia (CML), developed a nodular malignant melanoma on the upper back with spread to an axillary sentinal lymph node. We used laser microdissection to isolate FFPE tumor cells free of leucocytes. They were genotyped using forensic short tandem repeat (STR) length-polymorphisms to distinguish donor and patient genomes. Tumor and pre-transplant blood lymphocyte DNAs were analyzed for donor and patient alleles at 15 autosomal STR loci and the sex chromosomes. RESULTS: DNA analysis of the primary melanoma and the nodal metastasis exhibit alleles at each STR locus that are consistent with both the patient and donor. The doses vary between these samples indicative of the relative amounts of genomic DNA derived from the patient and donor. CONCLUSION: The evidence supports fusion and hybridization between donor and patient cells as the initiator of metastasis in this patient. That this phenomenon has now been seen in a second case suggests that fusion is likely to play a significant role for melanoma and other solid tumor metastasis, perhaps leading to new avenues of treatment for this most problematic disease.

Curcumin: A new candidate for melanoma therapy?

Melanoma remains among the most lethal cancers and, in spite of great attempts that have been made to increase the life span of patients with metastatic disease, durable and complete remissions are rare. Plants and plant extracts have long been used to treat a variety of human conditions; however, in many cases, effective doses of herbal remedies are associated with serious adverse effects. Curcumin is a natural polyphenol that shows a variety of pharmacological activities including anti-cancer effects, and only minimal adverse effects have been reported for this phytochemical. The anti-cancer effects of curcumin are the result of its anti-angiogenic, pro-apoptotic and immunomodulatory properties. At the molecular and cellular level, curcumin can blunt epithelial-to-mesenchymal transition and affect many targets that are involved in melanoma initiation and progression (e.g., BCl2, MAPKS, p21 and some microRNAs). However, curcumin has a low oral bioavailability that may limit its maximal benefits. The emergence of tailored formulations of curcumin and new delivery systems such as nanoparticles, liposomes, micelles and phospholipid complexes has led to the enhancement of curcumin bioavailability. Although in vitro and in vivo studies have demonstrated that curcumin and its analogues can be used as novel therapeutic agents in melanoma, curcumin has not yet been tested against melanoma in clinical practice. In this review, we summarized reported anti-melanoma effects of curcumin as well as studies on new curcumin formulations and delivery systems that show increased bioavailability. Such tailored delivery systems could pave the way for enhancement of the anti-melanoma effects of curcumin.

Isolation and Analysis of Suppressor Mutations in Tumor-Targeted msbB Salmonella.

Tumor-targeted Salmonella offers a promising approach to the delivery of therapeutics for the treatment of cancer. The Salmonella strains used, however, must be stably attenuated in order to provide sufficient safety for administration. Approaches to the generation of attenuated Salmonella strains have included deletion of the msbB gene that is responsible for addition of the terminal myristol group to lipid A. In the absence of myristoylation, lipid A is no longer capable of inducing septic shock, resulting in a significant enhancement in safety. However, msbB Salmonella strains also exhibit an unusual set of additional physiological characteristics, including sensitivities to NaCl, EGTA, deoxycholate, polymyxin, and CO2. Suppressor mutations that compensate for these sensitivities include somA, Suwwan, pmrA (C), and zwf. We describe here methods for isolation of strains with compensatory mutations that suppress these types of sensitivities and techniques for determining their underlying genetic changes and analysis of their effects in murine tumor models.

Application of Mesenchymal Stem Cells in Melanoma: A Potential Therapeutic Strategy for Delivery of Targeted Agents.

Melanoma is a leading cause of mortality from skin cancer and has a poor prognosis. Despite rapid advances in the treatment of this tumor type, the efficacy of current chemo-/targeted-therapies is still limited owing to the lack of sufficient drug accumulation in the tumor tissue and development of chemo-resistance. Recently, the application of mesenchymal stem cells (MSCs) in cancer therapy has gained substantial attention, suggesting their potential roles as an intriguing vehicle in improving the delivery of targeted agents. MSCs are genetically modified with suicide tumor suppressor genes to inhibit cell signaling pathways associated with the progression and metastatic features of melanoma. Here we describe the clinical application of MSCs in melanoma with a particular emphasis on recent findings on the role of MSC expressing a distinct set of biologically functional chemokines and tumor suppressing agents. Accumulating data has shown the tumor- oriented homing capacity of MSCs and their applications as a vehicle (e.g., adipose derived mesenchymal stem cells expressing TRAIL, interferon-α/γ, pigment epithelium-derived factor and cytosine deaminase). Several questions regarding possible potential and intrinsic mechanisms that might induce tumorigenesis and drug resistance are yet to be addressed for tailoring MSC-nbased treatment of melanoma.

Evidence for glycosylation as a regulator of the pigmentary system: key roles of sialyl(α2-6)gal/GalNAc-terminated glycans in melanin synthesis and transfer.

UNLABELLED: The major regulators of melanogenesis are glycoproteins, however no role for glycosylation in the pathway has yet been described. We stained skin biopsies and melanocyte-keratinocyte co-cultures with a panel of 20 lectins as oligosaccharide markers. Notably, the Elderberry Bark Lectin (EBL/SNA) stained melanocytes in both systems. EBL binds the sequence Neu5Ac(α(2-6)Gal/GalNAc)- at the termini of some oligosaccharide antennae. We used inhibitors of synthesis and/or binding of this sequence to assess effects on pigmentation. METHODS: Cell culture, lectin histochemistry, siRNA transfection, and assays for dopa oxidase and melanin were carried out by standard techniques. RESULTS: 6'-sialyllactose, a short homolog of the sequence in question, anti-sialyltransferase 6 (ST6) siRNA, and cytidine, a sialyltransferase (ST) inhibitor, each inhibited EBL binding, melanogenesis and melanosome transfer. Unexpectedly, 3'-sialyllactose and siRNA for ST3, chosen as a negative controls, also inhibited these processes. Though strong inhibitors of melanization, none of the agents affected tyrosinase/dopa oxidase activity, indicating previously unrecognized post-tyrosinase regulation of melanization. CONCLUSIONS: We report for the first time that Neu5Ac (α(2-6)Gal/GalNAc)- and possibly Neu5Ac(α(2-3)Gal/GalNAc)-terminated oligosaccharides play multiple roles in melanin synthesis and transfer.

Fusion of bone marrow-derived cells with cancer cells: metastasis as a secondary disease in cancer.

This perspective article highlights the leukocyte-cancer cell hybrid theory as a mechanism for cancer metastasis. Beginning from the first proposal of the theory more than a century ago and continuing today with the first proof for this theory in a human cancer, the hybrid theory offers a unifying explanation for metastasis. In this scenario, leukocyte fusion with a cancer cell is a secondary disease superimposed upon the early tumor, giving birth to a new, malignant cell with a leukocyte-cancer cell hybrid epigenome.

Targeting ER stress-induced autophagy overcomes BRAF inhibitor resistance in melanoma.

Melanomas that result from mutations in the gene encoding BRAF often become resistant to BRAF inhibition (BRAFi), with multiple mechanisms contributing to resistance. While therapy-induced autophagy promotes resistance to a number of therapies, especially those that target PI3K/mTOR signaling, its role as an adaptive resistance mechanism to BRAFi is not well characterized. Using tumor biopsies from BRAF(V600E) melanoma patients treated either with BRAFi or with combined BRAF and MEK inhibition, we found that BRAFi-resistant tumors had increased levels of autophagy compared with baseline. Patients with higher levels of therapy-induced autophagy had drastically lower response rates to BRAFi and a shorter duration of progression-free survival. In BRAF(V600E) melanoma cell lines, BRAFi or BRAF/MEK inhibition induced cytoprotective autophagy, and autophagy inhibition enhanced BRAFi-induced cell death. Shortly after BRAF inhibitor treatment in melanoma cell lines, mutant BRAF bound the ER stress gatekeeper GRP78, which rapidly expanded the ER. Disassociation of GRP78 from the PKR-like ER-kinase (PERK) promoted a PERK-dependent ER stress response that subsequently activated cytoprotective autophagy. Combined BRAF and autophagy inhibition promoted tumor regression in BRAFi-resistant xenografts. These data identify a molecular pathway for drug resistance connecting BRAFi, the ER stress response, and autophagy and provide a rationale for combination approaches targeting this resistance pathway.

A Melanoma Brain Metastasis with a Donor-Patient Hybrid Genome following Bone Marrow Transplantation: First Evidence for Fusion in Human Cancer.

BACKGROUND: Tumor cell fusion with motile bone marrow-derived cells (BMDCs) has long been posited as a mechanism for cancer metastasis. While there is much support for this from cell culture and animal studies, it has yet to be confirmed in human cancer, as tumor and marrow-derived cells from the same patient cannot be easily distinguished genetically. METHODS: We carried out genotyping of a metastatic melanoma to the brain that arose following allogeneic bone-marrow transplantation (BMT), using forensic short tandem repeat (STR) length-polymorphisms to distinguish donor and patient genomes. Tumor cells were isolated free of leucocytes by laser microdissection, and tumor and pre-transplant blood lymphocyte DNAs were analyzed for donor and patient alleles at 14 autosomal STR loci and the sex chromosomes. RESULTS: All alleles in the donor and patient pre-BMT lymphocytes were found in tumor cells. The alleles showed disproportionate relative abundances in similar patterns throughout the tumor, indicating the tumor was initiated by a clonal fusion event. CONCLUSIONS: Our results strongly support fusion between a BMDC and a tumor cell playing a role in the origin of this metastasis. Depending on the frequency of such events, the findings could have important implications for understanding the generation of metastases, including the origins of tumor initiating cells and the cancer epigenome.

L-tyrosine and L-dihydroxyphenylalanine as hormone-like regulators of melanocyte functions.

There is evidence that L-tyrosine and L-dihydroxyphenylalanine (L-DOPA), besides serving as substrates and intermediates of melanogenesis, are also bioregulatory agents acting not only as inducers and positive regulators of melanogenesis but also as regulators of other cellular functions. These can be mediated through action on specific receptors or through non-receptor-mediated mechanisms. The substrate induced (L-tyrosine and/or L-DOPA) melanogenic pathway would autoregulate itself as well as regulate the melanocyte functions through the activity of its structural or regulatory proteins and through intermediates of melanogenesis and melanin itself. Dissection of regulatory and autoregulatory elements of this process may elucidate how substrate-induced autoregulatory pathways have evolved from prokaryotic or simple eukaryotic organisms to complex systems in vertebrates. This could substantiate an older theory proposing that receptors for amino acid-derived hormones arose from the receptors for those amino acids, and that nuclear receptors evolved from primitive intracellular receptors binding nutritional factors or metabolic intermediates.