Chemokines exert crucial roles in inducing immune responses through ligation to their canonical receptors. Besides these receptors, there are other atypical chemokine receptors (ACKR1–4) that can bind to a wide range of chemokines and carry out various functions in the body. ACKR2, due to its ability to bind various CC chemokines, has attracted much attention during the past few years. ACKR2 has been shown to be expressed in different cells, including trophoblasts, myeloid cells, and especially lymphoid endothelial cells. In terms of molecular functions, ACKR2 scavenges various inflammatory chemokines and affects inflammatory microenvironments. In the period of pregnancy and fetal development, ACKR2 plays a pivotal role in maintaining the fetus from inflammatory reactions and inhibiting subsequent abortion. In adults, ACKR2 is thought to be a resolving agent in the body because it scavenges chemokines. This leads to the alleviation of inflammation in different situations, including cardiovascular diseases, autoimmune diseases, neurological disorders, and infections. In cancer, ACKR2 exerts conflicting roles, either tumor-promoting or tumor-suppressing. On the one hand, ACKR2 inhibits the recruitment of tumor-promoting cells and suppresses tumor-promoting inflammation to blockade inflammatory responses that are favorable for tumor growth. In contrast, scavenging chemokines in the tumor microenvironment might lead to disruption in NK cell recruitment to the tumor microenvironment. Other than its involvement in diseases, analyzing the expression of ACKR2 in body fluids and tissues can be used as a biomarker for diseases. In conclusion, this review study has tried to shed more light on the various effects of ACKR2 on different inflammatory conditions. Chemokines are small chemotactic cytokines produced by many cell types  that are primarily involved in regulating the movement of immune and non-immune cells during normal immune surveillance, inflammatory situations, and development . These cChemokine receptors, which are considered members of a family of seven transmembrane G-protein-coupled receptors (GPRs), mediate the particular actions of chemokines on their target cells . These rApart from the conventional chemokine receptors, a subfamily of atypical chemokine receptors (ACKRs) has been discovered . These rACKR2 cDNA showed 86% similarity and 71% identity to the murine Ackr2 gene, and regarding protein, both differ from other chemokine receptors as they replace the highly conserved DRYLAIVHA motif with DKYLEIVHA in human ACKR2 and mouse Ackr2, bringing an extra charged residue into the region (https://www.ncbi.nlm.nih.gov/gene/1238), the ACKR2 gene comprises three exons mapped to chromosome 3p22.1, a region of the genome that contains a cluster of genes encoding chemokine receptors. Moreover, according to the UniProt database (https://www.uniprot.org/uniprot/P21741), ACKR2 protein contains 384 amino acids, of which amino acid 19 is N-glycosylated, which is considered dispensable for ligand binding and high expression , in which a single amino acid change at position 41 in its binding site, i.e., the first three extracellular loops, sets V41A apart from wild-type ACKR2. It has been shown that this allele is structurally similar to the wild-type ACKR2, and there was no noticeable variation in the predicted folding patterns of proteins, and both have similar expression levels in transfected CHO-k1 cells. When compared to wild type, ACKR2-V41A is estimated to have minor hydrophilicity. In terms of binding affinity, the binding affinity for CCL2 and CCL4 is decreased in V41A, leading to a decreased scavenging efficiency of this allele. Regarding receptor recycling, it was also demonstrated that ACKR2-V41A has decreased receptor recycling and expression levels on the cell surface for a long period of time. Furthermore, Ca2+-dependent signaling is considerably lower in CCL2-activated cells that express the ACKR2-V41A allele than in the wild type, resulting in decreased signaling following CCL2 activation in this allele. These variations in binding, recycling, and signaling have shown different efficiency in various conditions, possibly leading to different responses in diseases , was first cloned by Nibbs et al. in 1997. The human e region . Accordipression , and thediseases . In sepadiseases . In addidiseases . Anotherdiseases . A possivia activating a β-arrestin-dependent pathway , some lymphocytes , myeloid progenitor-derived cells (such as mast cells and dendritic cells), blood endothelial cells, and lymphatic endothelial cells (LECs)—the major ACKR2-expressing cells in adults—have been shown to express ACKR2 –22. Acco pathway , 24. It  pathway . For theThis receptor is a highly versatile ACKR capable of binding to the majority of inflammatory CC chemokines, including CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL17, and CCL22 , 26. AltGATA1 gene induced ACKR2 expression in these cells, suggesting that the ACKR2 promoter possibly contains a binding site for GATA1 , cytokines (IL-6), leukocytes, the coagulation cascade, and the complement system. Regarding ACKR2, although it has been shown that placental ACKR2 is decreased at transcriptional and protein levels in preeclampsia in Cho et al.’s study, this downregulation has not been observed in Tersigni et al.’s investigation. Cho et al. have hypothesized that ACKR2 downregulation leads to higher inflammatory responses, resulting in excessive recruitment of macrophages to the maternal–fetal interface and an inappropriate response to systemic inflammation . In contnterface . Additionterface .+ macrophages into the mammary glands and subsequently controls the process of mammary gland branching morphogenesis during development. Ackr2-deficient mice have displayed premature development of mammary glands. This was linked to elevated infiltration of macrophages into the developing gland, resulting in an increased density of the ductal epithelial network. These findings show that ACKR2 is a key regulator of branching morphogenesis in a variety of biological situations and support the involvement of chemokines, playing critical roles in postnatal development processes , are developed at the epithelial ducts and promote network formation. A stromal component composed of fibroblasts, extracellular matrix (ECM), adipocytes, and immune cells leads to the achievement of this process. Mammary glands and surrounding TEBs contain macrophages, and their phagocytic and cytokine-producing characteristics are essential for several tissue remodeling events during developmental processes. In macrophage-deficient animals, mammary gland development is substantially disrupted, accompanied by abnormal TEB formation, ductal elongation throughout puberty, and lobuloalveolar development during pregnancy. In summary, these investigations demonstrate that macrophages play a critical role in the control of ductal branching in the developing mammary gland , 47. Macrocesses , 49. Furrocesses .The lymphatic vessels (lymphatics) and LECs play a vital role in the flow of fluid and leukocytes such as APCs from the peripheral tissues to the secondary lymphoid organs, especially lymph nodes. Human LECs but not vascular endothelial cells have been shown to express ACKR2 within non-inflamed tissues. Since this receptor has been found in a subset of lymphatic vessels, proposing that the heterogeneity in the expression of ACKR2 within the lymphatic vasculature might be a sign of function in this microenvironment. For example, only afferent lymphoid vessels in regions near the epidermis and deeper in the dermis express ACKR2 in the human skin sections. ACKR2 is also expressed in small and large intestines (in the villi), colon (lamina propria mucosa and muscular layer), appendix (in lamina muscularis externa), and other secondary lymphoid organs, including spleen (in red pulp) and tonsils (on sinuses like channels and parafollicular areas) . AccordiWhen the body’s tissues are exposed to harmful stimuli such as infections, damaged cells, or irritants, inflammation occurs. It is a defensive reaction involving immune cells, blood vessels, and molecular mediators, and it is a complicated biological response involving several factors. An inflammatory response’s primary goal is to remove the source of cell damage, which helps remove necrotic cells and tissues that have been destroyed by the initial harmful stimuli as well as the inflammatory response, leading to tissue healing . During Numerous investigations employing human samples or ACKR2-deficient animals have shown that ACKR2 expressed by lymphatic vessels plays a critical role in inflammatory diseases and malignancy. While the increasing consensus is that ACKR2 works as a negative regulator of inflammation, conflicting findings have been reported about its involvement in the modulation of adaptive immune responses and the development of autoimmune disease.The tumor microenvironment is the main interaction site between cancer cells and the host immune system. Through interactions between chemokines and chemokine receptors, diverse immune cell subsets are attracted to the tumor microenvironment, and these populations have unique impacts on tumor growth and treatment outcomes .de novo tumor development. CCL3 is highly elevated in tetradecanoyl phorbol acetate/dimethylbenz(a) anthracene -induced inflammation in B6/129 ACKR2-deficient mice, leading to recruitment and infiltration of polymorphonuclear cells (PMNs) and CD3+ T cells into papilloma. This hyperactive inflammatory response enhanced papilloma formation and made ACKR2-deficient mice more susceptible to the higher proliferation of keratinocytes  and elevated leukocyte infiltration  in the inflamed mucosa of the colon (The two most common types of inflammatory bowel illness are Crohn’s disease and ulcerative colitis (IBD). The production of pro-inflammatory chemokines is common in people with IBD. Additionally, the persistent mucosal inflammation seen in IBD has been linked to a higher incidence of colon cancer, also called colitis-associated cancer. Inflammatory cytokines have also been shown to play a role in the development of colonic inflammation into colon cancer. If pro-inflammatory chemokines are not properly controlled, it may lead to persistent intestinal inflammation and even cancer . In an ehe colon . These hhe colon . These dhe colon .–/– mice develop high levels of IL-1β, IL-6, IL-12, IL-17, TNF-α, CCL2, CCL3, CCL4, CCL5, CCL12, CCR1, CCR2, CCR5, FGF, and VEGF in oral SCC lesions, leading to higher infiltration of Ly6G+ neutrophils, mast cells, and F4/80+ macrophages in tumor tissues and proposing an unleashed inflammation in the tumor site. This inflammatory milieu results in the creation of highly proliferative tumor cells -treated ACKRor cells . The prior cells . Therefong VEGFA .+ NK cells, leading to enhanced tumor-homing efficiency of NK cells. ACKR2–/– mice have been demonstrated to have increased killing activity of NK cells through enhanced CCR2 expression and NK infiltration into the CCL2-expressing melanoma tumor microenvironment. As a result, ACKR2 expression in the tumor microenvironment decoys CCL2 and impairs the infiltration of NK cells  during the early stages of the metastasis process, losing cell-to-cell connections and epithelial properties in favor of mesenchymal ones that enable them to invade and metastasize to the surrounding or distant tissues. In this process, several transcription factors, including Twist, Slug, Snail, Gsc, and Zeb, are involved, leading to downregulation of epithelial markers (E-cadherin and cytokeratin) and upregulation of mesenchymal markers  . Cancer-As discussed, LECs are the main expressors of ACKR2 in the adult human body. Therefore, analyzing vascular tumors in terms of ACKR2 expression to find the origin of the tumor might be promising in detecting the type of cancer. In this regard, Nibbs et al. showed that ACKR2-immunoreactive tumor epithelial cells might be exclusively derived from LECs, suggesting that ACKR2 can be useful in detecting lymphatic tumor cells .Cervical cancer is the female reproductive system’s second most prevalent malignancy. Regional lymph node metastases are a critical prognostic factor in individuals with cervical cancer. Around half of the cervical cancer patients with pelvic lymph node metastases will have a disease recurrence, with the majority suffering from uncontrolled tumor growth. The reported 5-year survival rate for individuals with a recurrence is between 3% and 13%, and prognostic indicators for lymph node metastases in patients with cervical cancer are lacking . AnalyziAlthough there is no defined correlation of ACKR2 expression with other carcinogenic factors in cervical cancer, ACKR2 downregulation has been accompanied by dysregulation of metastatic factors such as MMP9 and VEGF in breast cancer. In this cancer, expression of ACKR2 was accompanied by ACKR1 and ACKR4 which were negatively correlated to axillary lymph node metastasis of breast tumor cells, suggesting favorable outcomes for co-expression of ACKR2, DARC, and CCX-CKR in breast cancer , 79. FurSince ACKR2 downregulation has been shown to contribute to the progression of colon adenocarcinoma, the correlation of ACKR2 expression in tumor tissues was analyzed to introduce its capability of being a biomarker in colon adenocarcinoma. Results showed that ACKR2 mRNA expression in colon adenocarcinoma tissues was decreased compared to unaffected mucosa. ACKR2 was also highly expressed in endothelial cells of the blood and lymphatic vessels of unaffected tissues compared with tumor tissues. Moreover, ACKR2 expression in tumor tissues was negatively correlated to tumor invasiveness, as its expression was lower in T3 and T4 grades than in T1 and T2 ones. All of the correlation was sex- and age-independent. In addition, ACKR2 expression was correlated to the expression of its counterpart ligand, i.e., CCL2, but not CCL5 and CCL8, suggesting that ACKR2 downregulation increases CCL2 in the tumor microenvironment to recruit more inflammatory cells and promote inflammation. CCL22, a Treg-recruiting chemokine, was another chemokine that was increased in the ACKR2-decreased tumor tissues, showing that ACKR2 downregulation also promotes tumor development by creating an immunosuppressive tumor microenvironment . TherefoIn another study, ACKR2 expression was tracked to analyze the trend of expression through the conversion of an adenoma into an adenocarcinoma sequence. Results have demonstrated that the expression ratio of ACKR2 was decreased through normal to tumor and increasing depth of tumor invasion. Moreover, the expression ratio of ACKR2 was decreased along with elevating tumor grade (adenoma to adenocarcinoma), whereas its expression was elevated through normal to pathological in adenoma tissues. Collectively, during the adenoma procedure, ACKR2 expression was insignificantly increased, and then its expression declined insignificantly in the process of malignancy. Moreover, the highest downregulation of ACKR2 was at the point that adenoma cells are capable of being malignant . Thus, tToday, it is well known that chemokines, their receptors, and other mediators are part of an interactive network. Therefore, analysis of a set or group of mediators would be helpful in predicting disease progression and prognosis. In a study to analyze the profile of expression of chemokine ligands and receptors in salivary adenoid cystic carcinoma (SACC), it has been shown that the expression of ACKR2 alongside CXR1, CXCR2, CMKLR1, and ACKR4 was higher in patients without recurrence, while inflammatory chemokines such as CXCL11, CCL5, and CCL20 were higher in metastatic cell lines compared with non-recurrent and less-invasive cells . AlthougACKR2 gene on cancer, it has been shown that rs2228468 is correlated with breast cancer metastasis. In SNP rs2228468, serine substitutes with tyrosine at location 373, and it has been found that this SNP shows limited differential effects on chemokine degradation ability. Analysis of this SNP in breast cancer has concluded that SNP rs2228468 has been associated with TNM and that rs2228468 serves as an indication of a changed metastatic propensity in Han Chinese patients  and Ly6Clow (after acute phase) subsets of monocytes in ischemic zones, and excessive infiltration of innate immune cells has been demonstrated to induce unfavorable remodeling of the left ventricle and heart rupture. The recruitment of inflammatory cells to the ischemic heart is mainly dependent on the CC chemokine members, namely, CCL2, CCL5, and CCL3 and their receptors, CCR2. ACKR2 was shown to be expressed in infarcted myocardium in humans and mice  include a wide range of conditions that affect the blood vessels and heart and may present themselves in life-threatening events such as myocardial infarction (MI), aneurysm, and stroke. Myocardial infarction (MI) is histologically characterized by the infiltration of leukocytes, namely, neutrophils (within the first 24 h) and Ly6Cand mice , 86. ACKfraction .Other than MI, recent research has found that pro-inflammatory molecules are overexpressed in the nucleus tractus solitarii (NTS) of spontaneously hypertensive rats (SHR) when compared to normotensive rats (Wistar–Kyoto rats: WKY), implying that the NTS of SHR may be inflamed abnormally. Therefore, the expression of inflammatory chemokines and cytokines might be involved in the formation of hypertensive phenotype in rats . RegardiAD is the most frequent neurodegenerative condition in the aged population and is the leading cause of dementia in this generation. AD is defined pathologically by the accumulation of amyloid-beta (Aβ) peptides in the gray matter, resulting in insoluble aggregated plaques that are considered to cause neurodegeneration and cognitive impairment. CCL2 is involved in the regulation of monocyte and dendritic cell migration across the blood–brain barrier, as well as the differentiation and migration of macrophages. CCL2 is mostly released by astrocytes and microglia in the brain, with only trace amounts secreted by endothelial cells. CCL2 is produced in the central nervous system (CNS) due to an immunological response to infection, damage, or inflammatory reaction in the CNS .In the animal model of AD, CCL2 is essential for causing chronic inflammation and activation of immune cells, as well as the release of other chemokines. The overexpression of CCL2 causes activated microglia cells to remain in close proximity to the inflamed location. This leads to enhanced activated microglia and elevation of key features of AD, such as amyloid plaques, plaque aggregation, and cognitive loss . In AD mCells of the microglia and astrocytes predominate in the CNS when it comes to ACKR2 expression , 94. Folvia the production of reactive oxygen species, nitric oxide, and glutamate from infiltrating immune cells and brain-resident microglia . Inflammation is a natural reaction to trauma and has neuroprotective and regenerative effects because it enables the clearance of cellular debris and the release of neurotrophic substances , 96. Theicroglia , 97. Furicroglia –100. Furicroglia , 100. Siicroglia , 94. Folfter TBI .+ mice compared to ACKR2-knockout mice decrease inflammation and demyelination of the spinal cord and have a lower incidence and severity of EAE episodes  peptide-induced ACKR2episodes . The admammation . In contammation . It has ammation . Finallyammation . Given ARegarding the human counterpart of EAE, i.e., MS, it has been shown that ACKR2 mRNA expression in PBMC derived from relapsing-remitting MS subjects was decreased. However, no link was found between this receptor and other cytokines implicated in the pathogenesis of MS, such as TNF-α and IL-33 . TherefoACKR2–/– mice under identical settings, but what was evoked at primary sites (probably not only IFN-γ) that increases ACKR2 expression at distant locations is still unknown , indicating the inflammation-regulatory role of ACKR2 at distant sites . This re unknown –107.ACKR2 gene and decrease ACKR2 mRNA and protein levels in HEK293 cells. ACKR2 expression is further downregulated in psoriasis patients after cell damage, which is a key trigger for the creation of new plaques . When cells are subjected to tensile stress, ACKR2 is rapidly downregulated, and miR-146b is simultaneously upregulated  in both ACKR2–/– and WT animals. After bleomycin exposure, Ifnb–/– mice showed increased pro-fibrotic indices. There is evidence to indicate that ACKR2 is one of the key players in preventing chronic inflammation-induced skin scarring caused by promoting IFN-β production . Moreover, downregulation of ACKR2 increased the recruitment and influx of CCR2+/CCR5+ Tγδ cells (anti-fibrotic and homeostatic T cells) in the lungs, which resulted from upregulation of CCL5, CCL12, and IFNγ. In conclusion, ACKR2 is activated during bleomycin-induced pulmonary fibrosis, and its deletion reduces mortality, lung damage, inflammation, and fibrosis. ACKR2 is a determinant for managing the recruitment of CCR2+/CCR5+ IFN-γ–producing Tγδ cells, which generate IFN-γ and regulate the development of the Th17 response and fibrosis intensity (Idiopathic pulmonary fibrosis (IPF) is a lethal disorder resulting from aberrant fibroblast and immune system activation, which leads to irreversible fibrotic changes in the lungs. This disorder like fibrosis in other tissues is caused by excessive immune responses and production of chemokines such as CCL2, CCL3, CCL11, and CXCL1–3 in the lungs, leading to initiating the inflammatory reactions and inappropriate activation of fibroblast. Although ACKR2 has been implicated as a chemokine scavenging receptor to reduce inflammatory responses in several tissues, it has been shown that ACKR2 plays a promoting role in IPF. In bleomycin-induced IPF in ntensity .Obesity is the outcome of a persistent anabolic state defined by increased caloric intake, decreased energy expenditure, or both . ExperimRheumatic disorders, often known as rheumatism, are a group of diseases that affect the connective tissues, especially the joints, causing chronic and often intermittent pain. Rheumatism is a general term that refers to a wide range of diseases, including arthritis and non-articular rheumatism, including spondyloarthropathies, lupus, Sjogren’s syndrome, systemic sclerosis, and sarcoidosis.ACKR2 mRNA and a lack of increase in serum levels of CCL2, CCL3 (weakly), and CCL4 in patients with SSC compared with healthy control, indicating that ACKR2 might be a response to alleviate the SSC pathogenesis , is an autoimmune rheumatic condition characterized by excessive collagen production and deposition in the skin and internal organs, as well as injury to small arteries, which is collectively called fibrosis . Therefoogenesis . In contogenesis . A possi–/– mice rejected their syngrafts 1 week after surgery. This is a surprising finding, given that ACKR2–/–mice are completely histocompatible with WT littermate mice and form such syngeneic graft rejection. However, the impact was attributed to an enhanced innate immune response in ACKR2–/– mice, indicating the participation of damage-associated molecular patterns (DAMPs) under the sterile circumstances of transplant surgery. Specifically, it was claimed that DC-expressed ACKR2 contributes to DC behavior modification by increasing maturation and allosensitization. Despite the reduced allospecific T-cell response shown in ACKR2–/– mice, no difference in allograft survival was observed between WT and ACKR2–/– mice, suggesting that any such impact was limited to syngeneic grafts (+ cells (LEC cells) in ACKR2–/– mice at the early post-graft stage, although lymphatic density was comparable to that of WT grafted mice. It was proposed that deletion of ACKR2 increases the proximity of pro-lymphangiogenic macrophages to LECs. Therefore, it was proposed that ACKR2, through modulating lymphangiogenesis, might be involved in the suppression of corneal allograft rejection  process and present corneal alloantigens to naive host T cells. The induction of an allospecific Th1 response then facilitates corneal transplant rejection. Syngeneic corneal transplants done in naive hosts, in contrast to allografts, are accepted forever. Regardless of the result, both corneal syngeneic and allogeneic transplants stimulate corneal hem- and lymphangiogenesis, which is thought to have a substantial impact on the graft’s outcome or the effectiveness of a subsequent graft , 133. ACc grafts . The altc grafts . Syngeneejection .high/Gr1+ myeloid cells, which express CCR2, exert immunomodulatory and immunoregulatory functions and recruit into tissues whereby CCL2 is produced  is the most serious clinical issue that may occur following allogeneic BM cell transplantation. GVHD’s target tissues are invaded by T cells, and it has been shown that local chemokine synthesis is considered a mediator of this invasion . It is nproduced . Therefoe marrow . In concIn vitro studies have shown that ACKR2 reduces the amount of leukocyte infiltration, inflammation, and fibrotic tissue remodeling that occurs following renal IRI, hence avoiding the development of chronic kidney disease , as well as greater levels of inflammatory CCL2  activation in the lungs and might promote COPD progression  is the formation of granulomas. Mycobacteria-reactive T cells, especially IFN-γ-secreting ones, play a critical role in this process  were protected against IAV, with a reduced viral load and dysfunction of the lungs -induced inflammation is a critical process for viral clearance, inducing adaptive immune responses, and getting the lung to a hemostatic status. However, an increased immune response characterized by excessive chemokine production might result in severe lung damage that contributes to death. Certain chemokines are linked to an increase in immunopathology after IAV infection, whereas others are necessary for the virus to be effectively controlled , 157. CChe lungs . It is t failure . CCR5-de process .In vitro, in vivo, and clinical studies have indicated that ACKR2 is dysregulated in different conditions in the body. Therefore, down- or upregulation of ACKR2 in these conditions prompted investigations to find underlying mechanisms concerning ACKR2’s involvement in diseases. In cancer, ACKR2 has been shown to act as a double-edged sword and play a paradoxical role. In some studies, ACKR2, through inhibiting inflammation, mediates the resolution of inflammation in various conditions such as infections, autoimmune diseases, and cancer. Therefore, using mechanisms that lead to upregulation of ACKR2 might be promising in the treatment of those diseases. For example, it has been shown that some miRNAs, such as miR-146b, can suppress ACKR2 expression. In this regard, targeted modulation of miR-146b through siRNA might be promising in the upregulation of ACKR2 to induce scavenging inflammatory chemokines , that leads to the formation of enormous and densely linked networks is a fascinating area of research. Protein interaction networks effectively develop biological and biomedical applications by abstracting core scientific concepts . Proteinin vitro and in vivo studies, which are almost animal models. As a result, it is proposed that future investigations can be more humanized to be translated into clinical trials. Finally, it is hoped that using ACKR2-based approaches in the treatment and diagnosis of diseases might broaden our knowledge of pathogenesis to overcome complications.Most studies regarding ACKR2 function and its mechanism of action in physiological and pathological situations are based on observations extracted from AS: Conceptualization, Investigation, and Writing—Original Draft. FJ-N: Investigation and Writing—Original Draft. HM: Investigation and Writing—Original Draft. FE: Investigation and Writing—Review and Editing. MO: Investigation and Writing—Original Draft. AJ: Investigation and Writing—Original Draft. JN: Conceptualization, Writing—Original Draft, Methodology, and Supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Calcium electroporation (CaEP) is an innovative anti-tumor treatment modality that induces cell death by introducing supraphysiological concentrations of calcium into cells with a limited effect on normal cells. The objective of the present study is to assess the effect of CaEP in uveal melanoma (UM) cell lines in comparison to electrochemotherapy (ECT) with bleomycin using 2D monolayer cell cultures as well as 3D tumor spheroid models in four different UM cell lines. The morphological changes of the spheroids, the cell viability, growth rate as well as the cytotoxic effect of electroporation (EP) with calcium chloride and bleomycin were evaluated with various drug concentrations. The results of CaEP and ECT both suggest a comparable dose-dependent reduction in cell viability and proliferation rate in all tested 2D cell lines and 3D tumor spheroids. These data point out that CaEP is an established anticancer treatment causing cell death by ATP depletion in in vitro and in vivo, representing an efficient alternative therapy with a lower cytotoxic potency for the local UM tumor control.Electrochemotherapy (ECT) is the combination of transient pore formation following electric pulse application with the administration of cytotoxic drugs, which enhances the cytotoxic effect of the applied agent due to membrane changes and permeabilization. Although EP represents an established therapeutic option for solid malignancies, recent advances shift to the investigation of non-cytotoxic agents, such as calcium, which can also induce cell death. The present study aims to evaluate the cytotoxic effect, the morphological changes in tumor spheroids, the effect on the cell viability, and the cell-specific growth rate following calcium electroporation (CaEP) in uveal melanoma (UM) 2D monolayer cell cultures as well as in 3D tumor spheroid models. The experiments were conducted in four cell lines, UM92.1, Mel270, and two primary UM cell lines, UPMD2 and UPMM3 (UPM). The 2D and 3D UM cell cultures were electroporated with eight rectangular pulses  of a 1000 V/cm pulse strength alone or in combination with 0.11 mg/mL, 0.28 mg/mL, 0.55 mg/mL or 1.11 mg/mL calcium chloride or 1.0 µg/mL or 2.5 µg/mL bleomycin. The application of calcium chloride alone induced an ATP reduction only in the UM92.1 2D cell cultures. Calcium alone had no significant effect on ATP levels in all four UM spheroids. A significant decrease in the intracellular adenosine triphosphate (ATP) level was documented in all four 2D and 3D cell cultures for both CaEP as well as ECT with bleomycin. The results suggest a dose-dependent ATP depletion with a wide range of sensitivity among the tested UM cell lines, control groups, and the applied settings in both 2D monolayer cell cultures and 3D tumor spheroid models. The colony formation capacity of the cell lines after two weeks reduced significantly after CaEP only with 0.5 mg/mL and 1.1 mg/mL, whereas the same effect could be achieved with both applied bleomycin concentrations, 1.0 µg/mL and 2.5 µg/mL, for the ECT group. The specific growth rate on day 7 following CaEP was significantly reduced in UM92.1 cell lines with 0.5 and 1.1 mg/mL calcium chloride, while Mel270 showed a similar effect only after administration of 1.1 mg/mL. UM92.1 and Mel270 spheroids exhibited lower adhesion and density after CaEP on day three in comparison to UPM spheroids showing detachment after day 7 following treatment. CaEP and bleomycin electroporation significantly reduce cell viability at similar applied voltage settings. CaEP may be a feasible and inexpensive therapeutic option for the local tumor control with fewer side effects, in comparison to other chemotherapeutic agents, for the treatment of uveal melanoma. The limited effect on normal cells and the surrounding tissue has already been investigated, but further research is necessary to clarify the effect on the surrounding tissue and to facilitate its application in a clinical setting for the eye. Uveal melanoma (UM) is a relatively rare disease with an incidence of approx. five cases per million in the USA and approx. 1–8 cases per million in Europe per year, however, constituting the most common primary intraocular tumor in adults . It arisFurthermore, the molecular pathogenesis of UM is characterized by chromosomal abnormalities and gene mutations. Most common are mutations in the guanine nucleotide-binding protein G(q) subunit alpha (GNAQ) and the guanine nucleotide-binding protein subunit alpha-11 (GNAQ/GNA11), which occur in a mutually exclusive manner in approx. 90% of posterior UMs . Other cTreatment options range from local therapy, including radiotherapy (brachytherapy or teletherapy), thermotherapy, and tumor resection  to enucleation . The proDespite advancements in the management of localized disease, adjuvant therapy of metastatic disease in the form of chemotherapy, adjuvant immunotherapy, and molecular targeted therapy may be necessary to prevent further progression, prolong life expectancy, and improve quality of life. The research and establishment of additional globe-preserving treatment strategies are, therefore, crucial for uveal melanoma patients .Electroporation-based techniques, such as irreversible electroporation, electrochemotherapy, and gene therapy, have already been established as effective alternative treatment modalities, especially for inoperable, recurring, and chemo-resistant malignancies in various tumor entities that do not adequately respond to the current treatment regimen. Electroporation (EP) is the induction of transient permeabilization of the cell membrane via short high voltage electric pulses, allowing passage of ions and molecules into and out of the cell. Electrochemotherapy is emerging (ECT) as an adjuvant treatment modality for local tumor control in various cancer entities. It combines the application of electric pulses to the tumor tissue with the administration of chemotherapeutic agents, rendering the cell membrane permeable to otherwise impermeant or poorly permeant anticancer drugs ,25,26,272+-ATPases to transport calcium out of the cells, and reduced production of ATP due to disruption in the mitochondrial membrane potential by the high intracellular calcium concentrations [Calcium electroporation (CaEP) is a prospective anticancer treatment where high concentrations of calcium are introduced into the cytosol following the application of electric pulses. The high intracellular concentrations of calcium ions can lead to an adenosine triphosphate (ATP) deficit ,29,30,31trations ,33,34,35Preclinical investigations suggest that this acute and severe ATP depletion leads to necrotic, apoptotic, or autophagic cell death ,29,30,31To reproduce the heterogenic characteristics of uveal melanoma, four UM cell lines with different genetic profiles were analyzed in the present study: two UM cell lines, UM92.1 and Mel270, and two uveal primary melanoma (UPM) cell lines, UPMD2 and UPMM3. The genetic and morphologic characteristics are summarized in All cell lines were authenticated by short tandem repeat profiling according to published data ,42,43,442).The UM cell lines UM92.1 and Mel270 were maintained in RPMI 1640 medium . The UPM cell lines UPMD2 and UPMM3 were cultivated in Ham/F12 medium . The cell medium was supplemented with 10% fetal calf serum and 1% penicillin-streptomycin (5000 U/mL) and was exchanged two times per week. The cell cultures were incubated in a humidified incubator  in 100 µL of the respective cell culture medium. The spheroid cultures were incubated in a humidified chamber  for one week until electroporation.The 3D spheroids were generated by seeding 5 × 105 cells were resuspended in 400 µL Hepes buffer containing 0, 0.11 mg/mL, 0.28 mg/mL, 0.55 mg/mL or 1.11 mg/mL calcium chloride or 1.0 µg/mL or 2.5 µg/mL bleomycin.The UM cells were trypsinized and transferred into Ingenio cuvettes  for the formation of monolayer cell cultures. A total of 5 × 10Seven days after generating the UM spheroids, the medium was gently removed and replaced with 100 µL Hepes buffer containing 0, 0.55 mg/mL, 1.11 mg/mL, 2.78 mg/mL, 5.55 mg/mL, and 8.33 mg/mL calcium chloride or 1.0 µg/mL or 2.5 µg/mL bleomycin. The spheroids were treated at higher concentrations of calcium chloride because preliminary tests showed no effect after treatment at the concentrations used on the single-cell model (data not shown).The monolayer cell cultures and the spheroids were then electroporated using a Genedrive voltage pulse generator  with an electroporator adapter for cuvettes and a two parallel aluminum electrode (4 mm apart) respectively. In this study, cells and spheroids were electroporated with eight rectangular pulses  of a 1000 V/cm pulse strength according to the European Standard Operating Procedure on Electrochemotherapy (ESOPE) alone or in combination with the aforementioned calcium and bleomycin concentrations.4 cells were transferred to white opaque-walled multi-well plates  in 100 µL medium (RPMI 1640) or Ham/F12 medium, supplemented with 10% fetal calf serum and 1% penicillin-streptomycin (5000 U/mL) per well. Changes in cellular ATP concentrations  were analyzed using the CellTiter-Glo 2.0 assay  according to the manufacturer’s instructions. Equal portions of CellTiter-Glo 2.0 and cell suspension were mixed and placed in the centrifuge (750 rpm) for two minutes. After a further 15 min of incubation in a dark chamber at room temperature, the luminescence was recorded using the plate reader ClarioStar Plus . The ATP luminescence was given in RLU (relative light units).Following electroporation of monolayer cell cultures, the cell suspensions, approx. 10n = 5–8 each condition) was assessed using the CellTiter-Glo 3D (Promega) according to the manufacturer’s instructions. Equal amounts of CellTiter-Glo 3D and spheroid cultures were mixed by pipetting up and down for 30 s to ensure complete lysis of the cells and the release of ATP. The mixture was transferred to white opaque-walled multi-well plates. After five minutes of incubation in the centrifuge (750 rpm) and a further 25 min in a dark chamber at room temperature, the luminescence was measured using a reader ClarioStar Plus. The ATP luminescence was given in RLU.The cellular ATP level in UM and UPM spheroids  were seeded on flat bottomed 96-well plates  with 104 cells in 100 µL medium per well. To determine cell viability and growth, the cells were fixated 24 h, 3 days, and 7 days after electroporation with 50 µL 10% trichloroacetic acid  for one hour at 4 °C and then stained with 50 µL 0.4% Sulforhodamine B solution . After approximately three washing steps with 1% acetic acid, the minoxanthene dye was then dissolved with 150 µL 10 mM Tris buffer (pH 10.5). Finally, the absorbance was recorded at 560 nm using the reader ClarioStar Plus . Based on these data, the cell line-specific growth rate (SGR) in % per day was calculated by using the following Equation (1).After the electroporation procedure, the UM and UPM cells , on day 3 (3 d), as well as on day 7 (7 d) after treatment using a Zeiss Primovert bright-field microscope at 4× magnification. The image and morphological analysis of the spheroids was carried out with a Zeiss Axiocam 105 and ZENcore software . The optic density  and the cross-sectional area were measured to evaluate the mass density of the spheroids. Due to frequent detachment of cells from the main spheroid core due to low adhesion and density following treatment, the Feret’s diameter was calculated. This value represents the mean diameter of the not dissociated spheroid as well as the mean distance between the most distant spheroid compartments. All measurements were performed using the open-source software Fiji ImageJ 1.53c .n = 3–6 each condition) were diluted in medium (RPMI or Ham’s F12) supplemented with 10% fetal calf serum and 1% penicillin-streptomycin (5000 U/mL) and seeded on Cellstar 6-well plates  with 2 × 103 cells in 3 mL medium per well.UM and UPM cell suspensions of the monolayer cell cultures  with 2 × 103 cells in 3 mL medium per well.One week after the electroporation procedure, spheroids treated the same were pooled and seeded on 6-well plates  with 2 × 102), the medium was removed, and the cells were stained with 4% crystal violet solution. To assess the survival fraction (SF) of proliferating cancer cells, colonies containing 50 or more cells were counted using a Leica S6D Greenough stereo microscope . The plating efficiency ) and, therefore, the survival fraction ) were calculated in percentage and statistically analyzed.After a two-week for monolayer and a three-week incubation phase for spheroids in a humidified chamber . A value of To assess the cytotoxicity of CaEP compared to ECT with bleomycin in UM and UPM cells and spheroids, an ATP assay was performed 24 h after treatment . The anaThe ATP levels of UM and UPM spheroids were only marginally affected following calcium chloride and bleomycin administration without EP A–D. Elecp < 0.005). The reduction in cell viability and the cytotoxic effect of the tested conditions were not statistically significant in the UPMM3 cells (The sulforhodamine B (SRB) assay was used for cell density determination and, therefore, as a cytotoxicity screening of the tested agents. The tumor growth was quantified by calculating the specific growth rate (SGR). The cytotoxic effect of 2.5 µg/mL bleomycin and EP alone in the UM92.1 A and MelM3 cells G. NevertOne week following CaEP, a significant reduction in the cell viability with a mean specific growth rate B of 17.7The image documentation was conducted before and on days 3 and 7 following treatment . The masThe survival fraction in the monolayer cell lines was significantly decreased in all tested cell lines for all tested conditions . A statiAn overall reduction in the survival fraction and, therefore, the colony formation capacity of the tested spheroids was documented for all cell lines following CaEP and ECT A–D. The 2+) plays an important role as a second messenger by maintaining cytoplasmic Ca2+ concentration low at rest and by mobilizing Ca2+ in response to stimulus, which in turn activates the cellular reaction [2+ level in concentrations about 20 to 40 nM [ECT is a well-established electroporation-based treatment modality with high response rates in various tumor entities ,37,38,46reaction . In addito 40 nM .Despite positive results in vitro and in vivo, the application of CaEP is mainly restricted to research purposes ,52,53,54UM92.1 cells show a more significant response than Mel270 in regard to the ATP reduction in 2D cell cultures, whereas the ATP levels in the spheroids are comparable for both cell lines. The difference between the two tested models could be accredited to a more accurate simulation of morphological and biochemical features of the original tissue cells in the spheroids since 3D cell cultures offer investigative conditions that closely resemble the in vivo situation. In respect of the cell viability, a significant reduction was detected with concentrations of calcium chloride higher than 0.28 mg/mL. For the UM92.1 and Mel270 cell lines, a similarly significant effect regarding cell viability and specific growth rate reduction could be achieved in monolayer cell cultures after ECT with both bleomycin concentrations and following CaEP only after application of 1.1 mg/mL calcium chloride. Analyzing the image documentation of the spheroids, a more evident size reduction and detachment manifest in the UM92.1 and Mel270 cell lines in comparison to the UPM spheroids after both treatment modalities and independently from the administered concentrations. The detachment is more prominent after CaEP in comparison to ECT for both UM92.1 and Mel270, whereas ECT has a lower effect in concern to the spheroid detachment and a higher impact on the spheroid size, especially in the UM92.1 cell line. The low cohesion and peripheral cell distribution seen in the spheroid imaging explains the increase in the mean gray value following ECT in both cell lines. The greater response and the robust growth development of the UM92.1 and Mel270 spheroids may be associated with the origin of the cell lines. Mel270 cells arose from a large recurrent tumor after prior irradiation . The UM9Calcium electroporation is postulated to have a limited impact on normal cells compared to malignant cells, as shown in 3D spheroid models and in vivo ,52,53,54Furthermore, the temperature and the time of administration prior to or after electroporation have been discussed as possible factors with an impact on the efficacy of CaEP . In thisThe present study aims to evaluate the cytotoxicity, the morphological changes of tumor spheroids, the effect on the cell viability, the specific growth rate, and the survival fraction following CaEP compared to ECT in 2D monolayer cell cultures as well as in 3D tumor spheroids in four UM cell lines. A dose-dependent reduction in ATP levels, cell viability, and specific growth rate with varying sensitivity among the tested cell lines was documented. The limitation of our study is the short lifespan of spheroids, which affects the size of the treated tumor as well as the central cell necrosis in the tumor organoids. CaEP is a novel treatment modality, and little knowledge has been generated up to now regarding its pathomechanism, and the optimal therapeutic settings and further dependent variables remain to be explored. The use of 3D spheroid models imitates an in vivo setting with higher precision, delivering more representative results for optimization of CaEP settings for targeted treatment. Our in vitro results indicate that CaEP may constitute an efficient and inexpensive therapeutic option for the local tumor control of UM and the prolongation of life expectancy in patients with progressive disease.


The detection of volatile organic compounds (VOCs) exhaled by human body fluids is a recent and promising method to reveal tumor formations. In this feasibility study, a patented device, based on nanostructured chemoresistive gas sensors, was employed to explore the gaseous exhalations of tumoral, immortalized, and healthy cell lines, with the aim of distinguishing their VOC patterns. The analysis of the device output to the cell VOCs, emanated at different incubation times and initial plating concentrations, was performed to evaluate the device suitability to identify the cell types and to monitor their growth. The sensors ST25 (based on tin and titanium oxides), STN , and TiTaV  used here, gave progressively increasing responses upon the cell density increase and incubation time; the sensor W11 (based on tungsten oxide) gave instead unreliable responses to all cell lines. All sensors (except for W11) gave large and consistent responses to RKO and HEK293 cells, while they were less responsive to CHO, A549, and CACO-2 ones. The encouraging results presented here, although preliminary, foresee the development of sensor arrays capable of identifying tumor presence and its type. Tumor detection and monitoring are fundamental topics in medicine worldwide. According to American Cancer Society statistics, one out of three people will be diagnosed with cancer in their lifetime ,2. For tTumor gaseous biomarkers are volatile organic compounds (VOCs) produced by cancer cells mainly as a result of cell metabolic process alterations  and cell2 molecules that are quickly released into the atmosphere , entirely designed, produced, and assembled in the “Laboratorio Sensori” of the University of Ferrara, Italy. The working principle of these sensors is based on the chemoresistivity, i.e., the capability of varying the film conductivity as a consequence of the chemical reactions occurring between the surface of the grains and the gaseous molecules of the surrounding atmosphere. The chemoresistivity process requires the thermal activation of the sensor that consists in maximizing the number of electrons (in conduction band) able to overcome the potential barrier  in water or alcohol solution , during which several changes in the gel structure and properties occur as polycondensation, lead to an increase in thickness of the colloidal particles and to the reduction of their porosity.The last step is the drying and calcination: the GEL is dried first (at about 100 °C), and then calcinated at higher temperatures (at about 400–800 °C) to completely remove the residuals of solvents and of other chemical additives. In this process, the temperature and the relative humidity greatly influence the quality of the final MOX powder.To optimize the performances of a chemoresistive MOX gas sensor, it is primarily necessary to increase the surface–volume ratio of its sensitive porous material as much as possible, to maximize its interaction with the surrounding gases. With this aim, the sol-gel chemical process was employed to synthesize networks of MOX nanometric spherical grains, precisely interconnected ,43,44,45The MOX nanopowder, synthesized through the sol–gel technique ,42,43, wST25, based on tin oxides and titanium (25%) and gold nanoparticles (1%);W11, based on tungsten oxide;STN, based on tin, titanium, and niobium oxides;TiTaV, based on titanium, tantalum and vanadium oxides.The four MOX sensors hosted in SCENT B1, listed below, were selected on the basis of previous laboratory tests carried out on pure standard gases, on VOCs exhaled by blood, feces, biopsies, and preliminary tests on cultured cells ,32,33,34All sensors were thermally treated with a firing temperature of 650 °C and were heated to the working temperature of 450 °C by the platinum coil ,32,33,34uctivity ,32. The In order to assess the reliability of the sensor responses on the cell samples presented in the following, four different STN sensors were tested, as an example, in both wet , but the human skin fibroblasts were:A549, explant culture of lung carcinomatous tissue;CACO-2, human colorectal adenocarcinoma cell line;CHO, epithelial cell line derived from the ovary of the Chinese hamster;HEK293, specific immortalized cell line originally derived from human embryonic kidney;RKO, human colorectal cancer cells.The cell types employed here, purchased from the American Type Culture Collection  taken out from the incubator were quickly cooled to room temperature on a copper plate. Room temperature was monitored and kept approximately constant at 22.0 ± 1.5 °C.4, 2.5 × 105, 5 × 105, 1 × 106 cells per dish (3.5 cm of diameter); all dishes were kept in the same incubator at 37 °C in a humidified atmosphere containing 5% of CO2.To maximize the sample exhaling surface, in order to improve the signal-to-noise ratio, three petri dishes were simultaneously measured by placing them in the sample chamber stacked in an holding tray . Each roSince the cells were kept and grown in media rich in organic compounds, it was necessary to measure 2O at 37 °C  upon the cell density increase and withdrawal time from incubator .However, all sensors poorly discriminated among the 80, 250, and 500 K cells/dish plated at 24 h, except for the 1 M one. To check that the responses to the first three cell concentrations were small, they were averaged  and are shown in Sensor responses to CHO cells, an epithelial line derived from the ovary of the Chinese hamster, and A549 cells, an explant culture of lung carcinomatous tissue, were negligible after 24 h of plating, regardless of the plating concentration. In particular, the A549 responses were smaller than one, i.e., they were smaller than the sensor response to culture medium only. Both these cell types grew quite fast, and at a concentration of 500 K, cells/dish were already confluent after 48 h of plating , insert;Lastly, the cell lines CACO-2 and human skin fibroblasts gave negligible responses . This waIn summary, it is possible that the VOCs emitted by CHO, A549, and CACO-2 cell lines, although they are expected to be quite sustained since these cells are fast growing and therefore have a high metabolism, are poorly detected by the sensors. If this is the case, it may become advantageous, since the sensors here employed, giving different response patterns with different cancer cells, could be used in the future not only to detect them (hopefully to an early stage), but also to identify their type. To further explore this idea, as shown in The same sensor gave different response amplitudes to different cell lines a, or difThe sensors ST25, STN, and TiTaV used in this work gave progressively increasing responses upon increasing the cell number, no matter if it was attained by the initial seeding concentration or by the cell growth. The sensor W11 gave instead unreliable responses to all the cell lines here employed, and therefore, they were discarded in the final analysis. The sensors gave large responses to the RKO and HEK-293 cell lines, while they were progressively less responsive to the CHO, A549, and CACO-2 cell lines. This suggests that a particular cell type could be identified by properly combining the response to its VOCs via a different sensor, allowing for the future development of sensor sets that are able to identify the presence of a tumor and even its type. The novelty of this approach, in respect to the current methods in development for tumor identification, is the employment of a portable, compact, and fast (the outcome is attained within 10–15 min) device, not requiring expensive consumables or complex and expensive analysis of the samples (that may require days), and that can be operated by untrained people.Malagù, C.; Gherardi, S.; Zonta, G.; Landini, N.; Giberti, A.; Fabbri, B.; Gaiardo, A.; Anania, G.; Rispoli, G.; Scagliarini, L. Combinazione di materiali semiconduttori nanoparticolati per uso nel distinguere cellule normali da cellule tumorali (2015), National #:102015000057717 .


Understanding the impact of spatial heterogeneities that are known to occur in large‐scale cell culture bioreactors remains a significant challenge. This work presents a novel methodology for mimicking the effects of pH and dissolved oxygen heterogeneities on Chinese hamster ovary (CHO) cell culture performance and antibody quality characteristics, using an automated miniature bioreactor system. Cultures of 4 different cell lines, expressing 3 IgG molecules and one fusion protein, were exposed to repeated pH and dissolved oxygen tension (DOT) fluctuations between pH 7.0–7.5 and DOT 10%–30%, respectively, for durations of 15, 30, and 60 min. Fluctuations in pH had a minimal impact on growth, productivity, and product quality although some changes in lactate metabolism were observed. DOT fluctuations were found to have a more significant impact; a 35% decrease in cell growth and product titre was observed in the fastest growing cell line tested, while all cell lines exhibited a significant increase in lactate accumulation. Product quality analysis yielded varied results; two cell lines showed an increase in the G0F glycan and decrease in G1F, G2F, and Man5; however, another line showed the opposite trend. The study suggests that the response of CHO cells to the effects of fluctuating culture conditions is cell line specific and that higher growing cell lines are most impacted. The miniature bioreactor system described in this work therefore provides a platform for use during early stage cell culture process development to identify cell lines that may be adversely impacted by the pH and DOT heterogeneities encountered on scale‐up. This experimental data can be combined with computational modeling approaches to predict overall cell culture performance in large‐scale bioreactors. Small‐scale bioreactor models allow for time and cost‐efficient development and optimization of cell culture processes. During initial cell line selection, miniature bioreactors within the microliter and milliliter scales are used for high‐throughput experimentation to reduce development time and costs.3 vessel and Lara et al3 stirred tank reactor (STR). Others have shown that in vessels up to 8000 L poor mixing can lead to high pH excursions after base addition.In the case of microbial fermentations, a number of publications have described the effects of heterogeneous conditions caused by poor mixing.Despite the few examples of large‐scale bioreactor studies in literature, detailed and reliable data is still missing.A number of scale‐down approaches have been developed to study the effects of heterogeneities on mammalian cell culture experimentally. In cell culture the addition of base, typically sodium hydroxide or sodium bicarbonate, is used for pH control and these additions can give rise to regions of high pH around the “addition zone.”Saccharomyces cerevisiae fermentation in a 300 m3 STR. Others suggest this to be higher, around 1%Several groups have developed scale‐down compartment models to investigate heterogeneity effects.Traditional compartment models using STRs and PFRs can be complex to set up and are not amenable to modern high throughput experimentation. This study presents a new approach to creation of a single‐compartment model using the ambr®15 automated miniature bioreactor system. The aim is to mimic the conditions within a small zone of a large‐scale vessel where the cells would be exposed to heterogeneities. The utility of this approach is that it will enable rapid identification of cell lines which might be vulnerable to fluctuations in pH and/or DOT. This study describes the development of a fluctuation methodology in ambr®15 bioreactors and initial evaluation of a number of cell lines with different growth characteristics. The impact of pH and DOT fluctuations on growth and productivity as well as product quality was assessed. The data obtained from these scaled‐down studies could be fed into computational population balance models which could then predict overall behavior of the cell lines at large‐scale as has been demonstrated with microbial fermentations.22.11 MAbs. Prior to bioreactor inoculation a vial of the cell line stock was thawed and cultured aseptically in a 250 ml shake flask in a shaken incubator  at 140 rpm, 5% vol/vol CO2, and 36.5°C. The culture was passaged every 3–4 days until used for bioreactor inoculation.Four recombinant GS‐CHO cell lines were used in this study: AZCL_1, AZCL_3, AZCL_4, and AZCL_5 from AstraZeneca. All cell lines were developed in proprietary media and produce monoclonal antibodies (MAbs). AZCL_1 produces a bispecific fab fragment fusion antibody and the other cell lines produce different IgG2.26 cells ml−1 in an initial volume of 13.5 ml. The temperature of each vessel was controlled at 35.5 ± 0.5°C. The pH was controlled to a set point of 7.0 ± 0.1 using CO2 (max flowrate 1.23 ml min−1) and 1 M sodium bicarbonate. DOT was controlled at 50% of air saturation using oxygen. These set points were used for all experiments unless otherwise stated, that is, when conducting cultures with fluctuating pH or DOT conditions as discussed later. Pure nitrogen was sparged at a constant rate of 0.15 or 1.00 ml min−1. A solution of 0.5% vol/vol Antifoam C  was added to each vessel at an average rate of 20 μl per day. Proprietary media and feeds were used. Nutrient feeds were added periodically and glucose was maintained with daily additions of 500 g L−1 glucose stock solution.The ambr®15 system  is widely used in industry as a tool for high‐throughput process development and is described in detail elsewhere.2.32 and pCO2 measurements were also taken every 2 days using an ABL90 Flex Plus  blood gas analyzer. Beginning on day 2, samples were also taken for antibody titre measurements. Supernatant was analyzed using the Octet  which uses a protein A‐based quantification method.Daily measurements of metabolite concentrations (glucose and lactate) were made using a YSI 2900 analyzer . All other measurements were made every 2 days. Viable cell density and viability measurements were made using a ViCell XR  which uses the trypan blue dye exclusion method. Offline pH, pO2.4Harvested cell culture broth (cultures end on day 14) was centrifuged and then filtered through a 0.22 μm SteriFlip vacuum filter  before being purified using a TECAN automated liquid handling system  equipped with RoboColumns containing MabSelect SuRe Protein A resin (Sigma‐Aldrich). Antibody purity and aggregation was determined using a standard HPLC‐SEC method using an Agilent 1200/1100 HPLC system  with a TSKgel column (Sigma‐Aldrich). Protein fragmentation was determined using a standard capillary electrophoresis (CE‐SDS) method with a Beckman PA800 Plus instrument (Beckman Coulter). Glycan profiles were obtained using a semi‐quantitative method of reversed phase HPLC (RP‐HPLC) combined with quadruple time‐of‐flight (QTOF) mass spectrometry (MS). Samples were reduced with dithiothreitol (DTT) and glycans released using N‐Glycosidase F . Samples were injected onto an Acquity UPLC BEH300 column , from which the eluate was directly fed into the SYNAPT mass spectrometer (Waters UK) with electrospray ionization (ESI). The mass to charge raw data was deconvoluted to a relative mass; the largest peak in each chromatogram is always the G0F glycoform and all other glycoforms are inferred based on their mass relative to the G0F peak.2.5Gluc), lactate production (qLac) and protein production (qp) rates were determined by linear regression of cumulative production (or consumption) against the cumulative integral of viable cell density (cIVC), using the following equations:IVCt is the cumulative integral of viable density at time t (cells‐day ml−1), VCD is the viable cell density (cells ml−1), and t is time (days). The maximum specific growth rate was determined from the highest specific growth rate of the culture calculated between measured time points using the equation below:Average cell‐specific glucose consumption . Conditions yielding 33.1Two‐compartment scale‐down models used to study large‐scale bioreactor heterogeneities have complex setups and required liter quantities of media making them time consuming and expensive to implement. The current work addresses this problem by using an automated miniature bioreactor platform to mimic just a portion of a larger scale vessel, representing the heterogeneous environment that a small volume of cells is exposed to. The simulation of heterogeneity is achieved by introducing programmed pH and DOT fluctuations in a well‐mixed miniature bioreactor. The pH and DOT set points are changed at fixed regular intervals and durations, similar to the approach taken by Osman et al.Initial work focused on establishing a robust experimental methodology for creating fluctuations in the ambr®15. Figure 2 or O2 stripping and therefore drop pH or DOT more rapidly. Fluctuations were mainly driven by manipulating the controller settings; increasing both proportional (P) and integral (I) components to induce a more aggressive response from the controller, hence creating the fluctuations. Due to the nature of PI control in the ambr®15, the pH and DOT signals exhibited over and under‐shoot as can be seen in Figure The method for achieving fluctuations in the ambr®15 was transferred from preliminary studies using another miniature bioreactor system; the micro24®  (data not shown).Figure 2 flow into the bioreactor, allowing the nitrogen to strip the CO2 and adding bicarbonate base to raise the pH. Longer durations give the system more time to raise the pH. This difference between the programmed and actual range is likely due to the nature of PI controls of the ambr®15, the buffering capacity of the cell culture media and lactate accumulation within the medium.The amplitude of the fluctuations achieved at the three frequencies were lower than programmed and many cultures struggled to reach pH 7.5. Table 2 was being sparged into the system. This effect is more dramatic in AZCL_3 cultures and increases with fluctuations of longer duration  the pH fluctuations tapered off and the pH remained at the lower  set point for several hours or days before the fluctuations restarted. During this period no COn Figure . This is2 flow rate; the oxygen consumption rate of the cells caused the DOT to drop even quicker. As a result, the response is much quicker than with pH and hence better fluctuations are achieved. AZCL_3 cultures grew to higher densities and therefore exhibited more noise in their DOT profiles and the fluctuation amplitudes were higher due to PI controller overshoot. In general, the pH and DOT fluctuations achieved were stable. Specific control of DOT was easier to achieve than pH. This initial work demonstrates that it is possible to programme and maintain fluctuations in the ambr®15 bioreactors throughout the course of a CHO fed‐batch culture.In contrast, stable DOT fluctuations were achieved with all cell lines across all frequencies. This is because the DOT control system does not rely on addition of liquid and is not limited by the buffering capacity of the media, which inherently makes it more difficult to maintain a stable pH set point. The DOT fluctuations were driven by the manipulation of the O3.26 cells ml−1) and the other with a “high” peak VCD (~60 x 106 cells ml−1). This section describes the investigation into the “low” growing cell line (AZCL_1).Preliminary studies using the micro24® bioreactor indicated that the effects of fluctuations are likely to be cell line dependent.−1) as encountered in the cited works. For this reason, the pH fluctuations reported here were induced by changing controller set points and PI settings, reducing excessive base addition. The ambr®15 automatically added base if the pH fell below 6.9. In the case of AZCL_1 lactate build‐up during the culture did not exceed the buffering capacity of the medium hence no additional base was used. However, without base addition, the amplitude of the pH fluctuations was limited.Previous studies investigating the effects of pH heterogeneity have typically introduced fluctuations by adding base either on a fixed daily basis2 purge gas flow rate and PI settings. Controls 1 and 2 have the “new” set up; the former using all the new conditions, that is, higher N2 flowrate and PI values and the latter keeps the original PI values but has a higher N2 flow rate. All vessels were cultured with the standard (lower) PI and N2 flowrates until day 2, at which point fluctuations were initiated.The pH and DOT fluctuations achieved experimentally are shown in the left column of Figure 6 cells ml−1. Control 3 appears to grow 22% less than the other two controls with higher gas flowrate, peaking at only 15 x 106 cells ml−1. This is also reflected in the lower lactate accumulation, suggesting that the lower N2 flowrate may be the cause of lower growth and lactate production (qlac) in AZCL_1 cultures. Interestingly however, the titre was not affected in the same way, meaning the qp of Control 3 was higher than the two other controls  below 2 gl−1 compared to the pH and DOT fluctuated vessels that peaked around 2.3 gl−1. However, this difference was found not to be statistically significant. All cultures experience a metabolic shift into lactate consumption around day 10. A slight difference in viable cell density between the 15, 30, and 60 min pH fluctuated cultures was observed; the cultures with longer fluctuation frequencies (60 min) had lower growth, however this difference was also not significant (p > 0.05). This difference is not seen in cultures with DOT fluctuation.Comparing across the rows in Figure gluc) was observed. Fluctuated vessels did, however, have higher initial lactate production (qlac), particularly in pH‐fluctuated vessels which had lactate production rates twice as high as controls between day 0 and 6. Fluctuations appeared to also slightly increase the growth rate (μmax), as seen in Table Looking at the derived culture parameters in Table 3.3Higher growing cell lines require better bioreactor control as the high‐cell densities achieved can lead to high‐oxygen uptake rates and increased lactate accumulation. For this reason, high‐cell density cultures performed at large scale could be more susceptible to spatial gradients and heterogeneities.6 cells ml−1, which again match the growth profiles seen in previous 5 L cultures. The 60 min pH fluctuations did appeared to have slightly lower growth and product titre on average, compared to the other fluctuation frequencies. However, the 15 and 30 min pH‐fluctuated vessels had higher peak product titre (5.5 gL−1) compared to the control vessels (4.9 gL−1). This was a slightly surprising result although these differences were not statistically significant.As with the low‐growing cell line (AZCL_1), pH fluctuations are seen to have little effect on growth, productivity or lactate levels in AZCL_3 cultures. The control and pH‐fluctuated vessels had very similar growth profiles peaking around 62 × 10−1 on day 6 when the metabolic shift occurred and lactate consumption began. Lactate increased toward the end of the culture as the cells died. Control 2 had a slightly different profile to the other controls, particularly with regard to lactate. This control was set at 30% DOT to test whether the effects of fluctuating DOT between 10% and 30% were not simply due to the overall lower DOT (compared to 50% DOT). Control 2 had a higher initial lactate production, qlac, and the culture switched to consumption on day 5; but had a much lower consumption rate than the other controls . The specific productivity, qp, was not affected however (Table lac [day 0−6]) than controls, as seen in Table gluc, was also affected by DOT fluctuations; all DO‐fluctuated vessels exhibited higher qgluc than controls, as seen in Table In contrast, cultures with fluctuating DOT conditions demonstrated significant differences to control cultures in the case of the “high” growing AZCL_3 cell line. DOT‐fluctuated cultures showed a 35% decrease in growth compared to controls. This decrease impacts the productivity by the same degree; DOT‐fluctuated cultures had peak antibody titres between 2.5 and 3.1 gler Table . The DOTA previous study has found that fluctuating DOT in a hybridoma cell line caused higher lactate yield.3.4All cultures shown in Figures 3.4.1p = 0.004). There was no fragmentation observed in AZCL_3 cultures. The AZCL_1 cell line produces a bispecific fusion product, so upon reduction fragmentation is expected, although there were no significant differences between fluctuated vessels and controls. However, in both cell lines, an increase in LMW species before reduction was observed in DOT‐fluctuated vessels compared to controls. Upon reduction a similar increase was observed in the levels of the light chain (LC) fragment, suggesting that DOT‐fluctuated cultures over‐produced free‐LC. As well as the increased level of LC these cultures also exhibited a decreased level of heavy chain (HC) species.As discussed above, pH fluctuations had little impact on the growth or productivity of both cell lines and this was also seen in product quality data. With regard to product aggregation and fragmentation, there appeared to be more variation between triplicate vessels with fluctuated conditions however pH fluctuations did not show significant differences in either AZCL_1 or AZCL_3 cultures.3.4.2Glycan analysis was focused on AZCL_3 cultures due to the observed impact of fluctuations on growth and productivity. The glycan profiles of all the cell lines used in this study are dominated by the bi‐antennary glycan with no terminal galactose (G0F), which makes up >68% of the overall profile. The next largest peaks are typically the galactosylated glycans G1F and G2F, which have been reported grouped and make up between 15% and 30% of the profile. High‐mannose structures (Man5) are another key glycan that is typically monitored and the cell lines had <5% of these species present.Figure While DOT fluctuations had a pronounced impact on cell growth Figure , they ha3.52 flowrate was set to 1.00 ml min−1 in AZCL_5 cultures and 0.15 ml min−1 in AZCL_4 cultures to assess if DOT fluctuations could be achieved with standard flow rates. Stable DOT fluctuations were achieved with both N2 flowrates. AZCL_5 and AZCL_4 cultures peaked at around 35 × 106 cells ml−1 and 40 × 106 cells ml−1, respectively, so they are not as low growing as AZCL_1 but not as high as AZCL_3. In both additional cell lines DO‐fluctuated cultures had lower peak VCD values and higher lactate accumulation compared to controls, but these differences were less substantial than ones observed in AZCL_3 cultures. There was no discernible difference between the negative control and DOT‐fluctuated vessels, suggesting the effects may be due to the overall lower DOT. The results with these additional cell lines support the hypothesis that higher growing cell lines are more susceptible to DOT fluctuations (or shifts in DOT set points). The effects also appear to be highly cell line specific, which is in agreement with the mixed results seen in other published studies.Two additional cell lines (AZCL_4 and AZCL_5) were used to further explore the effects of DOT fluctuations. Similar experiments were set up with AZCL_5 and AZCL_4 where DOT was fluctuated between 10% and 30% in the same manner. A positive (30% DOT) and negative (10% DOT) control were used with the higher PI settings, as described in Table Figure 4A novel methodology using an automated miniature bioreactor system to investigate the effects of pH and DOT fluctuations on CHO cell culture performance is presented. This provides a high throughput platform for assessing the potential impact of large‐scale bioreactor heterogeneities on different cell lines and antibody products. In the two different cell lines used to exemplify the utility of the platform, fluctuations in pH had minimal impact on growth and productivity. An increase in lactate production was observed which matched observations from similar literature studies.Overall, this work suggests that the impact of pH and DOT fluctuations are cell line and product specific and are more likely to affect higher growing cell lines. The automated miniature bioreactor therefore provides a platform for rapid investigation of potential large‐scale culture heterogeneities on candidate cell lines and antibody products. Data from fluctuating and non‐fluctuating bioreactor studies is currently being used to try and predict overall culture performance at large scale using population balance approaches.Roman Zakrzewski: Conceptualization (lead); data curation (lead); formal analysis (lead); investigation (lead); methodology (lead); writing – original draft (lead); writing ‐ review and editing . Gary J. Lye: Conceptualization (supporting); supervision (supporting); writing – review and editing . Kenneth Lee: Conceptualization (supporting); data curation (supporting); supervision (supporting); writing – review and editing (supporting).https://publons.com/publon/10.1002/btpr.3264.The peer review history for this article is available at


Currently there is no effective treatment for UM. Multi-kinase inhibitors targeting dysregulated pro-tumorigenic signalling pathways have revolutionised anti-cancer treatment but, as yet, their efficacy in UM has not been established. Here, we identified the multi-kinase inhibitor afatinib as a highly effective agent that exerts anti-UM effects in in vitroWe assessed the anti-cancer effects of afatinib using cell viability, cell death and cell cycle assays in in vitro and ex vivo UM models. The signaling pathways involved in the anti-UM effects of afatinib were evaluated by Western blotting. The in vivo activity of afatinib was evaluated in UM xenograft models using tumour mass measurement, PET scan, immunohistochemical staining and TUNEL assays.We found that afatinib reduced cell viability and activated apoptosis and cell cycle arrest in multiple established UM cell lines and in patient tumour-derived primary cell lines. Afatinib impaired cell migration and enhanced reproductive death in these UM cell models. Afatinib-induced cell death was accompanied by activation of STAT1 expression and downregulation of Bcl-xL and cyclin D1 expression, which control cell survival and cell cycle progression. Afatinib attenuated HER2-AKT/ERK/PI3K signalling in UM cell lines. Consistent with these observations, we found that afatinib suppressed tumour growth in UM xenografted mice.Our data indicate that afatinib activates UM cell death and targets the HER2-mediated cascade, which modulates STAT1-Bcl-xL/cyclin D1 signalling. Thus, targeting HER2 with agents like afatinib may be a novel therapeutic strategy to treat UM and to prevent metastasis.The online version contains supplementary material available at 10.1007/s13402-022-00686-5. Uveal melanoma (UM) accounts for ~85% of ocular melanomas and 3% of all melanomas in humans. The National Organization for Rare Diseases estimates that the incidence of UM is ~5–7 per million in the general population , 2 AlthoCurrently, front-line treatment for UM includes radiotherapy, phototherapy and surgery, but vision impairment, blindness or eye removal are common clinical consequences following these treatments . BecauseMost UM tumours exhibit mutations in genes encoding the G protein-alpha subunits GNAQ or GNA11 that activate the mitogen-activated protein kinase (MAPK) and phosphoinositide-3 kinase (PI3K)/Akt signalling pathways . InitialThere are four isoforms in the ErbB lineage of proteins (ErbB1–4) that function as homo- and heterodimers . The dim, ex vivo and in vivo models. Thus, afatinib emerges as a new candidate for clinical evaluation in UM patients.HER2 is expressed in UM tumour cells . In the Dulbecco’s Modified Eagle Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI-1640), Fetal Bovine Serum (FBS), Insulin-Transferrin-Selenium (ITS), Penicillin-Streptomycin (P/S) and L-Glutamine were purchased from Thermo Scientific . Giant cell tumour (GCT) conditioned medium was obtained from United Biosciences . Dimethyl sulfoxide (DMSO), L-glutamic acid monosodium salt hydrate and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich . The MKIs afatinib, bosutinib, cediranib, foretinib, lapatinib, erlotinib, gefitinib, neratinib, pelitinib, vandetanib, crizotinib, sorafenib and sunitinib were obtained from Selleck Chemicals  ; all MKI2) at 37 °C and used within 20 passages after thawing.The human Mel202 UM cell line was kindly provided by Prof. B. Ksander . The 92.1 cell line was a gift from Prof. M.J. Jager  and the OMM-1 cell line was a gift from Prof. G.P. Luyten . The C918 cell line was purchased from BioScientific  and the BeNa Culture Collection . All cell lines were authenticated in-house or by the respective commercial suppliers and routinely checked for mycoplasma contamination every 6 months using a MycoAlert Mycoplasma Detection kit . They were always negative. C918, Mel202 and 92.1 cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS (v/v), 1% P/S and 1% L-Glutamine . OMM-1 cells were cultured in DMEM medium supplemented with 10% heat-inactivated FBS (v/v), 1% P/S and 1% L-Glutamine. All cell lines were maintained in a humidified incubator (5% CO4 cells/well) for 24 h. Subsequently, cells were treated with MKIs (10 μM in 0.1% DMSO) in RPMI-1640 or DMEM containing 1% FBS (v/v) for 24 h; 0.1% DMSO was used as the negative control. Following treatments, cells were incubated with MTT (0.5 mg/ml) in the dark for 3 h and then washed with phosphate-buffered saline . Next, the cells were treated with DMSO and the plate was shaken for 10 min at room temperature. Absorbance values were measured at 550 nm in a microplate reader   Ga-NOTA-PRGD2 tracer, fresh 68Ga activity was eluted from a 68Ge/68Ga generator with 0.05 M HCl; 1.5 ml fractions were collected. The radioactive fraction (~5.26 MBq) was added to 1 M sodium acetate buffer containing 50 μg NOTA-PRGD2. The mixture was then heated at 97 °C for 10 min and loaded onto a C18 column  using deionized water and then eluted with ethanol containing 10 mM HCl. On the day PET scans were conducted, ~3.7 MBq of 68Ga labelled tracer was administered to the nude mice under isoflurane anaesthesia via tail vein injection. PET scans were performed using an Inveon microPET scanner . Dynamic image acquisition continued for 60 min after administration. For each scan, regions of interest (ROIs) were determined using vendor software (ASI Pro 5.2.4.0) of decay-corrected whole-body coronal images. The radioactivity concentrations (accumulation) were obtained from mean pixel values within the multiple ROI volume and then converted to MBq/ml. Assuming a tissue density of 1 g/ml, these values were then divided by the administered activity to obtain an image-ROI-derived percent injected dose per gram.To prepare [Tumour tissues embedded in paraffin were sectioned (8 μm thickness) and then stained with hematoxylin and eosin . For immunohistochemical staining, the sections were incubated with an anti-Ki67 antibody  at 4 °C overnight and then incubated with HRP-conjugated secondary antibodies. The sections were visualized using a DAB substrate kit  and an Olympus light microscope .Apoptotic cells were assessed using a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay in paraffin-embedded tumour sections fixed on slides. The slides were stained using a TUNEL assay kit  following the manufacturer’s instructions. Nuclei were counter-stained with hematoxylin. Staining was visualized using a Magscanner KF-PRO-120 .p < 0.05. In vivo studies were randomized, and observers didn’t know the group allocation. Statistical analyses were performed using GraphPad Prism 9.0 software with one-way ANOVA followed by Dunnett’s post-hoc test when comparing multiple independent groups. An unpaired t-test was used to analyse differences between two groups. Two-way ANOVA was used to analyse data from treatment and control groups with or without serum stimulation.Data are reported throughout as mean ± standard deviation with significance defined as 50 values for afatinib in four cell lines that represent primary and subcutis metastatic UM were in the range 3.43–5.29 μM  to decrease cell viability was assessed in Mel202, 92.1, C918 and OMM-1 UM. The Mel202, 92.1 and C918 cell lines were derived from primary UM tumours, while OMM-1 is a well-established subcutis metastatic UM cell model. The concentration of 10 μM was selected in initial screening experiments because it exceeds the reported serum trough levels in patients who were treated with the MKIs tested in the present study. This was done to ensure effective cell killing across multiple UM cell lines. The most active agent across the four UM cell lines (< 20% viability remaining) was afatinib, while pelitinib was also active in Mel202 and OMM-1 cells; cediranib, foretinib, lapatinib and neratinib were most effective in OMM-1 cells  and Bcl-xL (anti-apoptotic) - that regulate apoptosis, was assessed in UM cells Fig. . We founSTAT1 acts as a tumour suppressor in a range of cancer types and is associated with the expression of Bcl-xL and cyclin D1 . We founAfatinib has been reported to act as a dual inhibitor of EGFR and HER2 in non-small cell lung cancer and other cancer cells . In thisp < 0.01; Fig. The activity of afatinib was further evaluated in an in vivo C918 UM cell xenograft mouse model. We found that afatinib treatment (15 mg/kg daily for 16 days) markedly decreased the final weights of C918-derived UM tumours in mice Fig.  and stroSubsequently, we performed immunohistochemical staining of the tumour tissues harvested from the xenografted mice Fig. . We founEGFR mutations, ALK fusions and ERBB2/HER2-amplification, respectively [UM has a poor prognosis and, currently, there are no effective treatment options. MKI drugs have revolutionised the treatment of many cancer types by targeting kinases that drive important tumorigenic mechanisms such as proliferation, survival, motility and angiogenesis. Gefitinib, crizotinib and afatinib are kinase inhibitors that are used to treat tumours that exhibit ectively –41. Otheectively . MKIs thectively , 15.The EGFR family encompasses four ErbB members (ErbB1–4) that form homo- and hetero-dimers . Apart fConsistent with findings in the present study, it has been reported that HER2 is expressed in UM cells . ForsberAfatinib-induced apoptosis is associated with decreased expression of the anti-apoptotic Bcl-xL protein, the cell cycle progression regulator cyclin D1, and a decrease in the Bcl-xL/BAX ratio in UM cell lines. Afatinib-driven cellular apoptosis was also accompanied by an elevated expression of STAT1. STAT1 plays an important role in cell survival, viability and responses to pathogens. STAT1 induces cell cycle arrest in response to interferon-γ by interacting with D-type cyclins and cyclin-dependent kinase-4 in fibrosarcoma cells . STAT1 aWe confirmed HER2 activation upon acute afatinib treatment in all the four UM cell lines tested, which suggests that HER2 is likely the molecular target of afatinib and, thus, may be a viable anti-cancer target in UM. Unlike the EGFR inhibitors erlotinib and gefitinib that act in a reversible fashion, the HER2 inhibitors afatinib, pelitinib and neratinib also include an irreversible component in their inhibitory mechanism . These aSeveral signalling cascades, including the AKT, PI3K and ERK pathways, have been shown to be activated by HER2 , 60. Themax of afatinib is ~0.16 μM after administration of multiple daily oral doses of 50 mg in patients [Afatinib is clinically approved to treat non-small cell lung carcinoma (NSCLC) and head and neck squamous carcinoma –64. A phpatients . Brain ppatients , 68. TheIn summary, we found that afatinib has potent anti-cancer properties in in vitro, ex vivo and in vivo UM models. HER2 signalling has emerged as a likely molecular target that activates apoptosis upon afatinib treatment in UM cells. Afatinib also has the advantage of preventing UM cell migration and enhancing reproductive cell death, which may contribute to suppressing UM metastasis. Together, our data indicate that afatinib may serve as a novel candidate drug with an improved therapeutic effect and selectivity in treating UM by targeting HER2.ESM 1(DOCX 721 kb)


The incidence of primary liver tumors, hepatocellular carcinoma (HCC), intrahepatic cholangiocellular carcinoma (ICC), and combined HCC/ICC (cHCC/CC) is increasing. For ICC, targeted therapy exists only for a small subpopulation of patients, while for HCC, Sorafenib and Lenvatinib are in use. Diagnosis of cHCC/CC is a great challenge and its incidence is underestimated, bearing the risk of unintended non-treatment of ICC. Here, we investigated effects of targeted inhibitors on human ICC cell lines , in comparison to extrahepatic (E)CC lines , and HCC/hepatoblastoma cell lines . Cells were challenged with: AKT inhibitor MK-2206; multikinase inhibitors Sorafenib, Lenvatinib and Dasatinib; PI3-kinase inhibitors BKM-120, Wortmannin, LY294002, and CAL-101; and mTOR inhibitor Rapamycin. Dosage of the substances was based on the large number of published data of recent years. Proliferation was analyzed daily for four days. All cell lines were highly responsive to MK-2206. Thereby, MK-2206 reduced expression of phospho(p)-AKT in all ICC, ECC, and HCC lines, which mostly corresponded to reduction of p-mTOR, whereas p-ERK1/2 was upregulated in many cases. Lenvatinib showed inhibitory effects on the two HCC cell lines, but not on HEPG2, ICCs and ECCs. Sorafenib inhibited proliferation of all cells, except the ECC line CCC5. However, at reduced dosage, we observed increased cell numbers in some ICC experiments. Dasatinib was highly effective especially in ICC cell lines. Inhibitory effects were observed with all four PI3-kinase inhibitors. However, cell type-specific differences were also evident here. Rapamycin was most effective in the two HCC cell lines. Our studies show that the nine inhibitors differentially target ICC, ECC, and HCC/hepatoblastoma lines. Caution should be taken with Lenvatinib and Sorafenib administration in patients with cHCC/CC as the drugs may have no effects on, or might even stimulate, ICC.The online version contains supplementary material available at 10.1186/s12885-022-10247-6. Hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) are the most common primary hepatic malignancies, with 841,000 new cases worldwide in 2018, out of which 75%-85% accounted for HCC and 10%-15% for CCC [Fibroblast growth factor receptor-2 gene mutation or fusion, which only represent 10–15% of ICC patients [The prognosis for liver cancer patients is very poor. For example, in the United States of America, the 5-year survival rate for patients with HCC has remained at a low 18% . Approxipatients , 10. Indpatients , and thepatients , are apppatients , 12.A particular problem of primary liver cancer is the occurrence of combined HCC/ICC (cHCC/CC). Its incidence is reported to be highly variable. Due to great diagnostic challenge, its occurrence is likely to be massively underestimated. Of note, a single pass biopsy of a non-representative area will result in an incorrect diagnosis . BesidesTP53 is among the most frequently mutated genes [In both HCC and the small number of cHCC/CC tested, tumor protein ed genes , 16. Thied genes , 18. TP5ed genes . Oncogened genes . The PI3ed genes , compareed genes .There are a considerable number of studies and clinical trials on targeted drugs in HCC and CCC. If not mentioned otherwise, all chemicals and reagents were purchased from Sigma-Aldrich  and Merck . All inhibitors were purchased from Selleckchem . Inhibitors were: Sorafenib, Lenvatinib (multi-kinase inhibitor), Dasatinib (tyrosine-kinase inhibitor), BKM120, Wortmannin, LY294002, and CAL-101 (PI3K inhibitors), MK-2206 (AKT inhibitor), and Rapamycin (mTOR inhibitor). The inhibitors were dissolved in dimethyl sulfoxide (DMSO) . 2.The cells were grown in appropriate medium , ECC , HCC  and hepatoblastoma (HEPG2) cell lines over a course of 96 h using crystal violet proliferation assays. Nine potential inhibitors including Sorafenib and Lenvatinib, which are FDA approved drugs to treat HCC, were used. Based on literature and our previous studies [Proliferation assays were performed as described . Shortly500,000 cells per well were seeded into 6-well-culture plates, and allowed to adhere overnight at 37 °C. The cells were treated with 25 µM MK-2206 diluted in DMSO for 3, 8, and 24 h. Controls were performed with DMSO, in accordance to the highest concentration used in the test group. The wells were rinsed with washing buffer followed by lysis buffer. The wells were scraped with a cell scraper and the cell lysate was collected for Western blots.Protein extraction and Western blot were performed as described previously . Cells w*p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 with 95% level of confidence.The data were analyzed using Microsoft® Excel 2019 and Graph pad Prism version 4 software . All experimental errors were calculated as SEM. Statistical significance was calculated with one-way analysis of variance (ANOVA) between treatment and control groups at all time points. Statistical level of significance was accepted at Our results show that the ICC cell lines  incubated only in cell culture media and media containing DMSO increased in number in an almost linear fashion. Highly significant inhibition was observed with MK-2206, where we noticed a significant reduction in cell proliferation in a dose dependent manner. The cell numbers were reduced up to 95% compared to initial cell number Fig. A.Fig. 1PBy Western blot analysis, distinct down-regulation of p-AKT (Ser473) was noticed at 3—24 h in all of the studied ICC cell lines after MK-2206 treatment Fig. . SimilarIn parallel to ICC cell line, our studies also showed a highly significant effect of 10 µM MK-2206 on ECC cell lines EGI1 and TFK1. However, CCC5 was less susceptible . We noticed a reduction of about 90% at 96 h treatment with 25 µM MK-2206 in comparison to respective controls  and CCC5 (ECC cell line) Fig. A.Fig. 4PAt 1 µM and 5 µM, Lenvatinib significantly inhibited growth of HCC cell lines HEP3B and HUH7, but not hepatoblastoma cell line HEPG2. We did not observe any inhibitory effects on ICC and ECC cells Fig. B.Dasatinib showed maximum reduction in cell numbers on ICC cells where the dosage of 1 µM was already very effective. The results for ECC and HCC cell lines were less impressive, and only the higher dose of 10 µM induced consistent reduction of cell proliferation Fig. C.Since tumor suppressors of the PIK3 pathway such as PTEN (phosphatase and tensin homolog) were found to be mutated in combined HCC/ICC , we tested four different PI3K inhibitors, three of them inhibit predominantly PIK3CA  and one PIK3CD (CAL-101). The effects were relatively similar. PIK3CA inhibitors BKM-120 and Wortmannin showed significant inhibitory effects at higher concentrations on all cell lines, while LY294002 at higher dose had significant effect on all cell lines but RBE (ICC cell line), EGI1 (ECC cell line), and HEPG2 (hepatoblastoma cell line). The effects of CAL-101 were moderate Figs. A-D. The Finally, we studied the mTOR inhibitor Rapamycin. At a concentration of 10 nM, Rapamycin could significantly reduce the cell numbers of CCC5 (ECC cell line), HEP3B and HUH7 (HCC cell lines), while the proliferation was unaffected in other experiments Fig. .Fig. 6Pr. These preclinical rodent models can be used to investigate tumor specific and tumor type overlapping effects of therapeutics. Our preliminary data indicate overexpression of p-AKT in the murine TAA model (data not shown). Here, we searched for targeted drugs that inhibited in-vitro cell growth either in a cell type-specific or comprehensive manner.We previously developed a preclinical ICC model in mice by oral application of thioacetamide (TAA) in drinking water. Besides typical cirrhosis we observed development of ICC after 22 weeks of TAA . InteresWe observed differential effects of nine inhibitors targeting the PI3K/AKT/mTOR pathway in ICC, ECC, and HCC cell lines. The PI3K/AKT pathway plays an important role in proliferation, angiogenesis, apoptosis, cytoskeletal organization, and cell survival. This pathway is also known to interact with other signaling pathways such as the RAS-RAF pathway. Consequently, alterations in the PI3K/AKT pathway affect other signaling pathways, leading to uncontrolled cell proliferation and survival and eventually tumorigenesis by inducing transcription of the most commonly mutated genes such as AT-Rich Interaction Domain 1A (ARID1), BRCA1 Associated Protein 1 (BAP1), and Polybromo 1 (PBRM1), which are downstream of the PI3K/AKT pathway .The four PI3-kinase inhibitors tested  showed promising results by significantly reducing proliferation, with some variation depending on the tumor cell type. CAL-101 showed good effects in ICC cell lines, BKM120 and Wortmannin inhibited proliferation in all cell lines effectively. Thereby, Wortmannin showed better results in ICC and HCC than in ECC. The effects of LY294002 were somewhat intermediate. Therefore, PI3-kinase appears to be a promising target for the treatment of primary liver cancers.The AKT inhibitor MK-2206 potently blocked proliferation of all cell lines tested. This is in line with the marked down-regulation of p-AKT in the cells, whereas the expression of p-mTOR was only weakly or not at all changed in the cells. In fact, the mTOR inhibitor Rapamycin did not reveal any anti-proliferative effects in 7 out of 9 cell lines tested. In other cell types, it has been shown that AKT phosphorylates mTORC1 at Ser2448 leading to cell survival, proliferation, and metabolic changes . Our obsWhile MK-2206 has a comprehensive effect in ICC, ECC, and HCC, Dasatinib was most effective in ICC and inhibited cell growth effectively already at 1 µM. Dasatinib inhibits tyrosine-kinases including cKIT and EPH, and is approved for the treatment of chronic myeloid leukemia and acute lymphatic leukemia , 37. UprSorafenib, which is approved for HCC, was very effective in HCC/hepatoblastoma cell lines at 5 µM, but much less effective in two of three ECC cell lines. In ICC, proliferation was decreased greatly at 5 µM, but was even increased at some time points with 1 µM. Sorafenib has a very broad spectrum of activity , 38, andLenvatinib showed inhibitory effects on HCC proliferation with both concentrations, but not in the hepatoblastoma cell line HEPG2. In ICC and ECC, however, neither the lower nor the higher concentration demonstrated any anti-tumor activity. Therefore, caution should be taken with Lenvatinib administration in cHCC/CC.In sum, our studies show that primary liver tumor cell lines show distinct but overlapping patterns of reactivity to targeted molecular therapeutics. Thereby, as in case of Sorafenib, dosage is an important matter, as insufficient quantities might even induce pro-proliferative effects. For targeted therapy, it will be important to properly diagnose the incidence of combined HCC-ICC, as ICC may not or even inversely respond to Lenvatinib and Sorafenib. Customized drug therapy may be the next step to optimize therapy, tailored according to the nature of the tumor and the individual reactivity of patient-derived tumor cells.Additional file 1: Suppl. Figure 1. Uncropped Western-blot images of 25µM MK-2206-treated HUH28 and RBE and SSP25 with specific antibodies against total AKT and p-AKT, mTOR and p-mTOR, ERK1/2 and p-ERK1/2. α-Tubulin was used as loading control. Note: Images correspond to figure 1B. Suppl. Figure 2. Uncropped Western-blot images of 25µM MK-2206-treated EGI1 and CCC5 with specific antibodies against total AKT and p-AKT, mTOR and p-mTOR, ERK1/2 and p-ERK1/2. α-Tubulin was used as loading control. Note: Images correspond to figure 2B. Suppl. Figure 3. Uncropped Western-blot images of 25µM MK-2206-treated HEP3B and HUH7 with specific antibodies against total AKT and p-AKT, mTOR and p-mTOR, ERK1/2 and p-ERK1/2. α-Tubulin was used as loading control. Note: Images correspond to figure 3B.