Malignant pleural mesothelioma (MPM) is a deadly thoracic malignancy with limited treatment options. Chemotherapy remains the most widely used first-line treatment for unresectable MPM but is hampered by drug resistance issues. Small molecule inhibitors and microRNA mimics have shown promising potential for the treatment of MPM in preclinical studies, but are yet to be successfully implemented in the clinical setting. Our study aims to provide an understanding of the molecular mechanism(s) that mediate drug response in MPM. The inhibitor of apoptosis family member, survivin, has been reported to be over-expressed in MPM and is associated with drug resistance. Therefore, we particularly focused on determining the cellular mechanism(s) that contribute to MPM cell response to a survivin small molecule inhibitor, YM155. Our study provides key information to facilitate a prediction of the potential utility of small molecule inhibitors and microRNA mimics as treatment options for MPM.ABCA6 and ABCA10, were upregulated in the MPM cell lines and correlated with poor sensitivity to YM155.Malignant pleural mesothelioma (MPM) is a deadly thoracic malignancy and existing treatment options are limited. Chemotherapy remains the most widely used first-line treatment regimen for patients with unresectable MPM, but is hampered by drug resistance issues. The current study demonstrated a modest enhancement of MPM cell sensitivity to chemotherapy drug treatment following microRNA (miRNA) transfection in MPM cell lines, albeit not for all tested miRNAs. This effect was more pronounced for FAK (PND-1186) small molecule inhibitor treatment; consistent with previously published data. We previously established that MPM response to survivin (YM155) small molecule inhibitor treatment is unrelated to basal survivin expression. Here, we showed that MPM response to YM155 treatment is enhanced following miRNA transfection of YM155-resistant MPM cells. We determined that YM155-resistant MPM cells secrete a higher level of exosomes in comparison to YM155-sensitive MPM cells. Despite this, an exosome inhibitor (GW4896) did not enhance MPM cell sensitivity to YM155. Additionally, our study showed no evidence of a correlation between the mRNA expression of inhibitor of apoptosis (IAP) gene family members and MPM cell sensitivity to YM155. However, two drug transporter genes,  Malignant pleural mesothelioma (MPM) is an aggressive tumour of the membrane lining of the lung which is highly related to asbestos exposure. There are limited effective treatment options available to MPM patients in the clinical setting which consequently attributes to a poor associated median survival of 12 to 18 months following first-line chemotherapy treatment with cisplatin and pemetrexed ,4. The aTumour resistance to chemotherapy drug treatment can partly be attributed to the tumour microenvironment (TME) protecting the tumour cells against treatment; known as extrinsic resistance. This extrinsic resistance arises from tumour cells interacting with their surrounding environment and can cause alterations in gene expression profiles via the exchange of small RNAs, such as microRNAs (miRNAs) ,7,8,9. ESurvivin is a key member of the inhibitor of apoptosis protein (IAP) family, which mediates the regulation of both apoptosis and cell division . The IAPMiRNAs are small non-coding RNAs that play a role in all essential biological pathways and consequently, their downregulation or dysfunction have been implicated in the development and/or progression of cancer, including MPM ,34. It hWe have previously shown that miRNAs that are associated with the FAK pathway are able to enhance MPM response to FAK small molecule inhibitor treatment with PND-1186, albeit with a broad spectrum of sensitivity across a range of MPM cell lines . AdditioPND-1186 and YM155 (Sepantronium Bromide) were purchased from Selleck Chemicals. TRIzol was purchased from Life Technologies . Cisplatin was purchased from McFarlane Medical & Scientific Pty Ltd.  Gemcitabine was purchased from Eli Lilly and Company 2.Five MPM cell lines  and the immortalised mesothelial cell line, MeT-5A, were obtained from American Type Culture Collection . The primary mesothelioma cell line, MM05, was generated at the University of Queensland Thoracic Research Centre , and Ren cells  were proABCA6 and ABCA10 genes were selected to investigate their involvement in MPM cell response to YM155 treatment  mRNA was determined by quantitative real time PCR (RT-qPCR) using KAPA SYBR® FAST reagents  and the Viia7 qPCR System . Probe Design software  was used for designing PCR primers  according to the manufacturer’s instructions. Reverse transcription (RT) reactions were performed using 200 ng of total RNA with an MMLV first strand cDNA kit  according to the manufacturer’s protocol. The expression of the IAP and drug transporter . RNU6B was used as an endogenous control. Post-transfection miRNA expression was calculated as fold change, relative to the miRNA expression of the untransfected control MSTO-211H and VMC23 cells, for each tested miRNA mimic. BIRC5 (survivin) and PTK2 (FAK) RT and RT-qPCR quantification was carried out on the miRNA-transfected MSTO-211H and VMC23 cells using the MMLV cDNA kit and KAPA SYBR® FAST methods, respectively, as described above. BIRC5 and PTK2 mRNA expression was determined as fold change, relative to the mRNA expression of the untransfected control cells, for each tested miRNA mimic.Following a 24 h incubation period after miRNA mimic transfection of the MSTO-211H and VMC23 cells, total RNA was extracted using Trizol reagent (Life Technologies) according to the manufacturer’s protocol. For miRNA quantification experiments, RT reactions were performed using 50 ng of total RNA with a TaqMan microRNA reverse transcription kit  in accordance with the manufacturer’s protocol. The miRNA expression was determined by RT-qPCR using KAPA PROBE FAST  and miRNA-specific TaqMan assay  reagents  using th® Elite ABC Kit, rabbit, 1:200) for 60 min at room temperature, followed by Avidin-Biotin Complex  for 30 min. Visualization of cells with bound primary antibody was performed by exposing them to diaminobenzidine chromogenic (DAB) substrate for 1 min, followed by counterstaining with 10% hematoxylin for 3 min. All slides were mounted with coverslip and air dried overnight before imaging with a ZEISS Axio.M2 microscope with the 20× objective lens.Following a 24 h incubation period after miRNA mimic transfection of the MSTO-211H and VMC23 cells, the cells were fixed in 10% formalin and processed into paraffin-embedded cell blocks. The cell blocks were prepared as 5 µm sections and deparaffinized in three changes of xylene (3 min each), followed by three changes of 100% ethanol (3 min each) and three changes of 70% ethanol (3 min each). The sections were incubated with a citrate buffer  for 1 h at 86 °C and then stained with either anti-FAK  or anti-survivin  antibodies overnight at 4 °C. The sections were then subjected to 150 µL of secondary antibody . Experiments involving human cell lines were performed 3 times with 3 replicates each time.MPM cell response to drug treatment was assessed by the Alamar Blue cell death assay in 96-well plates with various concentrations of chemotherapeutic drugs and small molecule inhibitors. Briefly, following a 24 h incubation period after miRNA or siRNA transfection, cells were treated with a medium containing various drug concentrations of either cisplatin, gemcitabine, PND-1186 or YM155. In addition to the chemotherapy drugs and small molecule inhibitors, the exosome inhibitor, GW4869, was added to study the MPM drug response in relation to exosome inhibition. Following drug treatment, the cells were incubated for 72 h. Alamar Blue was prepared as previously described ; filter-6 MPM cells were seeded in a T75 flask with 10 mL growth media (containing 10% exosome depleted FCS). At 48 h post cell seeding, 10 mL of conditioned media was collected from each MPM cell line. Exosomes were isolated according to the manufacturer’s instruction using a total exosome isolation kit . Exosome protein analysis was performed using the Bradford assay.2 × 10y, was:To assess the statistical significance between the miRNA mimic-treated MPM cell proliferation and the inactive miRNA control mimic-treated MPM cell proliferation, the following method was applied as previously described . MPM cel50) of the miRNA control mimic-treated MPM cells (one component model), 50) for the miRNA mimic-treated MPM cells (two component model), p-value < 0.05), using the following formulae for chi-squared value and degrees of freedom:In the above formula,  package  in R [4950 was calculated as the sigmoidal transition point resulting from the two component model having the best fit . Fold change was calculated as the control’s IC50 divided by the drug’s IC50. The one component model  and the IC50 for each MPM cell line, as shown in detail in the R code text file (ICext file . To deteext file .BIRC5) and survivin protein expression in the MSTO-211H cells, with respect to the untransfected control cells, following miRNA transfection (PTK2) expression (with the exception of miR-17-5p), with respect to the untransfected control cells; albeit a decrease in FAK protein expression was less apparent (PTK2) and FAK protein expression, with respect to the untransfected control cells, following miRNA transfection  following miRNA transfection . Our ressfection A  to 300 nM (MM05).Mechanisms of MPM response to chemotherapy drug and FAK (PND-1186) small molecule inhibitor treatment were studied in our previous papers ,40. In tp-value = 1).We assessed whether exosome production potentially contributes to this variable MPM cell response to survivin small molecule inhibitor treatment by collecting conditioned media from a range of cultured MPM cell lines and performing a subsequent exosome prep and quantification analysis. Our results show that all tested MPM cell lines secreted a higher level of exosomes in comparison to the non-malignant Met-5A mesothelial cell control and that there was a statistically significant reduction in exosome secretion following treatment with the GW4896 exosome inhibitor for all tested MPM cell lines, with the exception of H28 and MSTO-211H B. AdditiBIRC5, XIAP, IAP1 and IAP2 members of the IAP gene family; the drug-transporting ABC transporters, MDR1 (ABCB1), MRP1 (ABCC1) and BCRP (ABCG2); the uptake transporters, OCT1 (SLC22A1); and the survivin regulator, ILF3 (ILF3). Upon conducting qPCR analyses to examine the mRNA levels of these genes in MPM cell lines, our data indicated that MDR1 (ABCB1) and IAP2 (genes known to mediate drug transport and anti-apoptosis [IAP2 and MDR1 (ABCB1) mRNA levels were found to be approximately 30- to 503-fold and 14- to 11,897-fold higher than the MeT-5A control, respectively, . In contrast, OCT1 (SLC22A1), BCRP (ABCG2), and BIRC5 mRNA levels were found to be less than 1-fold for the majority of tested MPM cell lines with respect to the MeT-5A control . There was no evident correlation between the up- or downregulated expression of these genes and MPM cell response to YM155 treatment .Upon establishing that there was no evident link between exosome secretion and MPM sensitivity to YM155 small molecule inhibitor treatment, we alternatively suspected that survivin-related gene expression may play a role. To determine whether survivin-related genetic alterations play a role in small molecule inhibitor drug response in MPM, we examined the gene expression levels of the IAP family members, including transporters involved in the cellular uptake and efflux of drug molecules, and correlated their expression with MPM cell response to YM155 small molecule inhibitor treatment. In particular, we assessed the expression of poptosis ,50, respctively, ; albeit  control , attribuABCA6 and ABCA10, in the MPM cells. The qPCR data revealed that ABCA6 and ABCA10 are upregulated in the majority of tested MPM cell lines with respect to the non-malignant mesothelial MeT-5A cell control  to small molecule inhibitor treatment with YM155 . There was an even stronger correlation between the fold-change in expression of ABCA10 and MPM cell response (IC50) to small molecule inhibitor treatment with YM155 . The greater the increased expression of ABCA6 or ABCA10, the greater the MPM cell line’s resistance to YM155 treatment. Furthermore, we showed that siRNA treatment of the drug-resistant MSTO-211H cell line induced a reduction in the expression of the ABCA6 and ABCA10 genes , respectively , FAK over-expression has been linked to aggressive tumour behaviour, tumour survival and progression ,58,59,60Survivin is an attractive potential target for cancer therapies because it is rarely expressed in normal cells but remarkably upregulated in several types of cancers. YM155 is a selective small molecule inhibitor of survivin transcription that exhibits potent antitumor activity by inducing apoptosis and autophagy in various types of cancer, as well as enhancing the anti-tumour and anti-angiogenic effects of chemotherapy drugs such as cisplatin ,64,65. Id miRNA combinational treatment approach on MPM tumour-bearing mice is greatly warranted.Exosomes have been widely reported to play a key role in chemotherapy drug resistance for a range of cancer types  and theiABCA6 and ABCA10. Both of these genes are members of the ABC transporter gene family, which are known to contribute to chemotherapy drug resistance in cancer [ABCA6 and ABCA10 is associated with poor MPM cell response to YM155 small molecule inhibitor treatment and silencing of these genes re-sensitises MPM cells to YM155 small molecule inhibitor treatment  and survivin-related drug uptake/transporter genes that are known to mediate drug resistance in other cancer types ,75,76. On cancer ,78,79. Oreatment C,D, thusCollectively, the results from our study reinforce our understanding that chemotherapy and small molecule inhibitor drug response in MPM is complex and multi-faceted. There is an evident correlation between survivin expression and MPM drug response, however exosome production in MPM does not appear to play a direct role in survivin-mediated drug resistance. Rather, our study indicates that there is a link between the ABC drug transporter family and MPM response to survivin small molecule inhibitor treatment, which warrants further exploration in future preclinical studies. Furthermore, our study shows that restoration of tumour suppressor miRNA is able to enhance MPM cell response to chemotherapy drug and small molecule inhibitor treatment. Hence, prospective investigations aiming to explore combinational miRNA replacement with chemotherapy drug and/or small molecule inhibitors as a potential treatment option for MPM is greatly warranted.ABCA6 and ABCA10, and reduced MPM sensitivity to YM155.This study demonstrated that the combination of small molecule inhibitor targeting agents of survivin and FAK with tumour-suppressor miRNA mimics, constitute promising efficacy as potential combinational treatment options for MPM that warrants further investigation. Upon exploring the potential mechanism responsible for variable MPM cell sensitivity to the survivin small molecule inhibitor, YM155, it was established that exosome involvement does not appear to be a contributing factor despite a high level of exosome secretion for most of the tested MPM cell lines. Furthermore, there was no evident correlation between survivin-related gene expression and MPM response to YM155. Rather, we determined that there is a potential link between the over-expression of the drug transporter genes,


The importance of circulating tumor cells (CTC) is well recognized. However, the biological characteristics of CTC in the bloodstream have not yet been examined in detail, due to the limited number of CTC cell lines currently available. Thirty-nine CTC cell lines were reported by 2021. For successful cell culturing, these CTC cell lines were reviewed. Previous studies on short-term cultures of CTC also analyzed approaches for establishing the long-term culture of CTC. Negative selection, hypoxic conditions, three-dimensional conditions, and careful management are preferable for the long-term culture of CTC. However, the establishment of CTC cell lines is dependent on the specific characteristics of each cell type. Therefore, a method to establish CTC cell lines has not yet been developed. Further efforts are needed to resolve this issue.  The importance of circulating tumor cells (CTC) has recently been recognized. Although the cultivation of CTC is very promising as a preclinical model for human cancer, difficulties are associated with establishing CTC cell lines due to the low number of CTC in blood and the majority of these cells not being viable. Only a limited number of permanent cell lines of CTC are currently available and their management is more challenging than that of standard cell lines.CTC cell lines are important for examining the biology of CTC, but not for providing quick feedback to cancer patients. Furthermore, the translational relevance of cell lines is often questioned because prolonged cultures and multiple passages lead to phenotypes that are no longer representative of the original tumor in terms of cell epigenetics and gene expression profiles . TherefoWe previously established cancer cell lines from several tissue specimens, including esophageal and pancreatic cancer ,3. We idThe systematic detection of CTC was initially reported by other research groups. Racila et al.  used an The majority of conventional cancer cell lines generally proliferate on a two-dimensional (2D) environment, in contrast to the 3D environment of most CTC cell lines, such as spheroids or organoids. We established a small cell esophageal cancer cell line (TYUC-1) that actively grew with sphere formation in a standard culture flask from a tissue specimen . This exConcerning the approaches to long-term culture, we also examined the literature for short-term cultures. Our aim was to obtain a more detailed understanding of the characteristics of CTC and several methods that support the establishment of long-term cultures from short-term cultures. Based on previous findings and our own research, we herein discussed critical issues facing the development of CTC culture methodologies and proposed a rational strategy for the long-term culture of CTC.In this review, a long-term culture (CTC cell line) is defined as a culture period of longer than 1 year or continuous passages for more than 6 months. A short-term culture is defined as a culture period of longer than 1 week. We briefly discussed culture-related factors, such as methods for enriching CTC, the characteristics of CTC in the bloodstream, and stem cell biology. We excluded methods for ultra-short term (less than 1 week) cultures and expression assays because the focus of these methods is clinical evaluation, not long-term cultures. We mainly concentrated on the development of a reproducible protocol for establishing CTC cell lines. We also excluded details on the CTC capture system, clinical applications, genetic analyses, CTC-derived xenografts, and cultures of bone marrow disseminated tumor cells.Thirty-nine CTC cell lines were reported by 2021: 11 from colon cancer, 13 from breast cancer, 2 from prostate cancer, 2 from gastroesophageal cancer, 5 from small cell lung cancer, 2 from non-small cell lung cancer, and 4 from malignant melanoma. Establishment rates ranged between 1 and 17%, with most studies achieving a rate <10%  cells should survive in the blood because they negligibly express EpCAM. Therefore, EpCAM is not a standard marker for the establishment of cell lines. However, some cells express EpCAM and maintain epithelial characteristics. Additionally, many cells express stem cell markers, such as CD133, CD44, CD24, and ALDH1. These findings indicate that CTC have a mixed epithelial phenotype, an intermediate epithelial/mesenchymal phenotype, and stem cell-like properties.Stem cell concepts have been employed for long-term CTC cultures. CTC as non-adherent spheres may reflect the intrinsic properties of cancer stem cells that remain viable in the bloodstream after the loss of their attachment to the basement membrane . HoweverThe majority of CTC cell lines have been established using 6 to 18 mL of blood. A blood volume >10 mL is a suitable source for CTC cultures. Furthermore, since the establishment of cell lines was found to be slightly easier from patients with a high CTC count (>30/mL), a CTC count >300 is preferable for the success of cultures. However, a high CTC count only does not guarantee the establishment of a permanent cell line. Furthermore, a conventional CTC count is generally assessed using EpCAM(+) cells. Since EpCAM(−) CTC have been overlooked, CTC counts are not accurate. The most recent study on CTC used a 96 mL sample obtained from leukapheresis for a cell culture . Leukaph®). Although antibody-based approaches were employed in three studies, capture with antibodies against surface antigens is harmful, precluding its use for CTC cultures [An enrichment step for CTC is indispensable before cells are cultured. Viable CTC isolation may be divided into three types: antibody-based, physical property-based, and function-based approaches. The most successful enrichment method for long-term cultures is the negative selection method  has been used as the start medium. The culture medium is then changed to ordinal standard medium. The culture medium is important during the initial 2 weeks. The proliferation of most cells peaks after approximately 21 days. CTC with proliferative potential may then grow without special growth supplement, except for FCS .Epidermal growth factor (EGF) and basic fibroblast growth factor (FGF) are often listed in recipes for stem cell cultures. Recipes for serum-free CTC culture media mostly include proprietary B27 and N2 supplements, together with insulin, transferrin, and selenium. Y27632, a cell-permeable inhibitor of Rho-associated coiled-coil containing protein kinase, is also used. However, the optimal conditions for ex vivo CTC cultures have yet to be established.To appropriately culture CTC, it is important to understand the progression of CTC growth in each step. In this section, we focus on the growth progression of selected CTC cell lines.1.CTC-1, CTC2, and CTC3Zhang et al.  previous2.CTC-MCC-41In the case of CTC-MCC-41 cells, viable CTC were still observed after 4 days of culture in serum-reduced medium under hypoxic conditions. After 10 days, CTC started to proliferate and formed spheres. After a few weeks, the cell culture was switched to normoxic conditions. Single colonies from the CD45(−) population that appeared during the first week of the cell culture were transferred into a 24-well plate for further growth .3.BHGc7 and BHGc10BHGc cells initially grew as typical small spheroids that eventually showed the outgrowth of adherent tumor cells and shedding of apoptotic cell fragments . Prolife4.CTC-TJH-01During an ex vivo culture of CTC-TJH-01 cells, contaminating blood cells and a small number of CTC underwent rapid cell death. The majority of surviving CTC adhered to the plate, but remained dormant throughout the initial two weeks, suggesting the low in vitro clonogenic potential of CTC. CTC started to proliferate after two weeks, and the culture was switched back to normoxic conditions. CTC slowly proliferated, formed clusters, and then exhibited sustained proliferation ,20.5.CTC-ITB-01CTC-ITB-01 cells grew in parallel as adherent and non-adherent cell fractions with various cell sizes. Non-adherent cells grew from adherent cells. CTC showed an interchangeable adherent and non-adherent cell population similar to cancer stem cells .6.UWG01CTC and UWG02CTCIn the case of UWG01CTC and UWG02CTC cells, viable, relatively pure cultures were observed within 3 weeks and rapidly expanded. UWG01CTC is adherent and requires trypsinization for passaging; however, a loose adherent spheroid phenotype was induced in a hypoxic environment and serum-free media. In contrast, UWG02CTC grew in long mucinous, loosely aggregated, and weakly adherent strands that only required gentle mechanical dissociation for passaging .7.CTC-3CTC-3 cells began to proliferate and form clusters after 14 days of culture . HoweverSeveral conditions were used for the expansion of CTC. However, suitable conditions depended on the characteristics of each cancer cell. Furthermore, the optimal timing and culture conditions for CTC expansion have not yet been established. Representative cases are described below.1.CTC-1, CTC-2, and CTC-3Adherent plates were used during the first week with growth factor-enriched stem cell culture medium. The medium was changed to EpiCult-C medium between 8 and 21 days. Standard medium with 10% FCS was then used for maintenance .2.Mel-167, PEM-22, Mel-182, and PEM-78CTC were cultured in 24-well ultra-low attachment plates for between 4 and 8 weeks. A 3D fibrin Matrigel culture was then used, and this was followed by a switch to an anchorage-independent culture [3.CTC-MCC-41CTC were cultured in 24-well non-adherent plates for the 1st week. Cells were then transferred to new 24-well non-adherent plates in the 2nd week and maintained in a T25 flask .4.CTC-3A Matrigel-coated plate was used in the first 2 weeks, and cells were then transferred to a normal 6-well plate .To establish approaches to long-term culture, short-term culture was reviewed. Information that will contribute to the establishment of long-term cultures of CTC was obtained .We successfully performed a short-term culture of CTC using a magnetic cell sorting system (MACS) with anti-Human Epithelial Antigen (HEA) 125 in 1999 . We dete®). Few studies employed RBC cell lysis only. Under optimal conditions, the introduction of an RBC lysis step does not markedly affect the viability of the nucleated cell fraction [® (filtration using a porous polycarbonate membrane) selection [Similar to long-term cultures, most enrichment methods for cultures involve the negative selection method  [® detects cancer cells not only on the separating membrane, but also on the plastic bottom of the 6-well plate [The MetaCellof 8 μm) ,45,47,51ll plate . Membran2.On-chip cultureZhang et al.  describeKapeleris et al.  suggesteRegarding CD45, a significant population of “double positive” cells with hematopoietic and epithelial markers (CK+/CD45+) were detected in many patient samples . FurtherThus, most of the double positive cells may be the heterogeneous CTCs . From thKhoo et al.  maintainThe viability and morphology of expanded CTC were examined by LIVE/DEAD staining .Few studies have employed low attachment plates and hypoxic conditions. Therefore, short-term cultures are generally aimed at clinical use, not long-term cultures. Therefore, they are routinely conducted under adherent and normoxic conditions. Furthermore, most studies used simple standard culture medium with FCS.Tapered microwells are preferred over conventional cylindrical microwells for cluster formation. Cultures are sheltered within microwells with minimal disturbance during medium changes, which reduces shear stress and cell loss to allow for subsequent expansion . This meCarmona-Ule et al.  suggeste® was 65%. Despite the leukapheresis approach, CTC from only a few patients have been successfully cultured. A success rate of 100% (12/12 samples) was achieved by Ficoll-Paque selection [The success rate of cultures was previously reported to be more than 50%. The total success rate of MetaCellelection .The typical cell culture period is <14 days and few cells may be cultured for more than 3 months. Typical spheres were shown to increase in size over time from 7–21 days . Khoo etCarmona-Ule et al.  investigAlthough proliferation was not observed due to the challenging ex vivo growth environment, the maintenance of CTC viability was guaranteed for at least two months . EliasovKhoo et al.  showed tKhoo et al. ,63 demonThe morphological spheroid construct typically consists of three different presentable types: large-sized, cohesive round-shaped spheroids; small-sized cohesive irregular or round spheroids; and discohesive “grape-like” spheroids . Some sa® system, CTC grew through the membrane on the bottom of the well after 14 days in an in vitro culture exhibiting two different phenotypes, epithelial-like and stem cell-like. Stem cell-like cells formed visible cell clumps. Cells growing on the bottom showed a very plastic morphology [In the MetaCellrphology . CTC incrphology .Non-adherent blood cells provide a cushion layer that keeps CTC in suspension . CTC havAfter 1 week of a cell culture under hypoxic conditions, cells are generally switched to standard cell culture conditions. When 85% confluence is reached, growing cells are transferred to an ultra-low attachment 24-well plate . HoweverRegarding the establishment of CTC cell lines, the majority of cultured CTC lack the ability to proliferate. Therefore, 2 months is a sufficient time period to assess whether CTC exhibit the ability to continuously proliferate. Future studies that compare CTC lines with CTC that stop proliferating after a short-term culture will facilitate the development of optimal molecular conditions for the permanent survival and growth of CTC .Cell growth depends on the proliferative potential of each cell. Among 21 conventional esophageal cancer cell lines, the time after the initiation of the primary culture to the second culture varied from 2.5 weeks to 48 weeks, depending on cell growth patterns, such as the adhesion-dependent type, spheroid type, and mixed type. Adaptability to a new environment is also important [We previously reported the maintenance of several cancer cells in protein-free medium without other supplements for 2 years . These cBased on our experience and previous findings, we preliminarily performed a culture of CTC collected from patients with prostate cancer and renal cell carcinoma. We recently developed a CTC capture system . We firs®-pot (porous polycarbonate membrane selection) with Lymphoprep or negative selection using RosetteSep® CD45, CD36, or CD56. A short-term culture of CTC was successful for 16 out of 27 samples; however, expansion was very challenging to overcome. One reason is that we did not obtain a high number of viable CTC from patients because most had already received several courses of chemotherapy. The most important issue is that we have not yet established a specific protocol for individual carcinoma cells.We also used VIVANT-CELLRegarding double positive (CK+/CD45+) cells, we also detected these cells in the short-term culture of CTC. Some of these cells were large, polymorphic in shape and polynuclear . These mIn several trials, we developed three cell lines with the spontaneous immortalization of lymphocytes from different patients under conditions involving several growth supplements, such as Y-27632 and Z-VAD-fmk. We previously reported the incidental establishment of B-cell cell lines (ITSM), derived from EB virus spontaneous activation during an ordinary cell culture from pseudomyxoma peritonei . The lonCTC cell lines have unique intermediate characteristics between primary tumors and distant metastasis. CTC are not simple travelers in the bloodstream. Although most CTC are apoptotic and sensitive to stress, a few viable CTC survived.The CTC count of samples is the most important factor for the successful establishment of CTC cell lines. A CTC count of >100 cells/mL blood is favorable. Leukapheresis may be the most powerful method for enriching CTC; however, its success rate is low.There is currently no evidence to support the contribution of CD45+ cells to the success of long-term culture. However, a reconsideration of the parameters for CTC isolation is warranted .It is important to note that because optimal CTC culture conditions have yet to be defined, current research relies on stem cell culture methods that ensure maximal CTC expansion. Each protocol is designed based on a researcher’s own expertise and empirical results, and not by a rationale based on the biological features of CTC, which are presently not defined .Moreover, activated lymphocyte proliferation is transient, ending with programmed activation-induced cell death. Macrophages are the only cell type among peripheral blood mononuclear cells that are capable of surviving for weeks in culture. However, they are also a terminally differentiated cell type that is incapable of ex vivo proliferation .Other key technical limitations include the maintenance of the CTC phenotype and the composition of a stable population in culture. Previous studies suggested that normal human mesenchymal stem cells are prone to genomic changes and subsequent malignant transformation in long-term cultures .Therefore, the further optimization of CTC culture conditions and a more detailed understanding of the differences between non-adherent CTC cell lines and adherent cell lines as well as CTC cultures and patient samples is required .As described above, the recommended protocol for CTC cell lines is as follows . With caA careful review of previous studies suggested that CTC in long- and short-term cultures have different characteristics. Not only the culture method, but also the characteristics of CTC themselves affect the success of establishing CTC cell lines.In the circulation, CTC from any type of cancer encounter the same environment, which differs from their solid tissue location. Only cells with specific characteristics, such as stem cell-like cells, may survive in the bloodstream. CTC without these characteristics are protected by surrounding cells, such as leukocytes and platelets. However, it currently remains unclear whether only CTC with stem cell features are suitable long-term culture.Low attachment plates and hypoxic conditions may be the ideal conditions for the initial culture steps. A successful culture medium contains several growth factors and anti-apoptotic supplements. However, as described above, cancer cells have already acquired growth and survival autonomy. Therefore, CTC cultured in the presence of growth factors and small molecule inhibitors may not be ideal for modeling cancer and stromal interactions. The most important factors are the timing of CTC transfer to the next step and the careful management of medium changes.Short-term culture is the initial approach to long-term culture; however, prolonged protocols sometimes deviate from the main expansive route for establishing CTC cell lines. The success of short-term cultures is not always affected by a high number of CTC; however, long-term cultures generally require high numbers of CTC. Therefore, the culture method may only prolong cell survival; it does not affect permanent proliferative activity. The most important issue is obtaining CTC that are suitable for long-term culture; however, a method to identify these CTC has not yet been established.There is currently no gold standard method. The selection of an optimal method for each sample may be achieved through careful management and continued efforts. Extensive trials are needed to establish long-term cultures of CTC. Without these efforts, CTC that are suitable for expansion will not be identified.


Protein essentiality is usually accepted to be a conditional trait and strongly affected by cellular environments. However, existing computational methods often do not take such characteristics into account, preferring to incorporate all available data and train a general model for all cell lines. In addition, the lack of model interpretability limits further exploration and analysis of essential protein predictions.In this study, we proposed DeepCellEss, a sequence-based interpretable deep learning framework for cell line-specific essential protein predictions. DeepCellEss utilizes a convolutional neural network and bidirectional long short-term memory to learn short- and long-range latent information from protein sequences. Further, a multi-head self-attention mechanism is used to provide residue-level model interpretability. For model construction, we collected extremely large-scale benchmark datasets across 323 cell lines. Extensive computational experiments demonstrate that DeepCellEss yields effective prediction performance for different cell lines and outperforms existing sequence-based methods as well as network-based centrality measures. Finally, we conducted some case studies to illustrate the necessity of considering specific cell lines and the superiority of DeepCellEss. We believe that DeepCellEss can serve as a useful tool for predicting essential proteins across different cell lines.http://csuligroup.com:8000/DeepCellEss. The source code and data underlying this study can be obtained from https://github.com/CSUBioGroup/DeepCellEss.The DeepCellEss web server is available at Bioinformatics online. Essential genes are indispensable for the survival of a single-celled organism, a cell line or a multicellular organism  to capture long-range dependencies between residues. Finally, a fully connected layer with a sigmoid function performs the classification task.We conducted extensive experiments to evaluate the performance of DeepCellEss. In comparison, DeepCellEss shows greater effectiveness in predicting essential proteins than existing sequence-based methods. Compared to network-based centrality measures under cell line-specific networks, the results demonstrate that DeepCellEss effectively compensates for the limitations of network-based centrality measures. Furthermore, we performed some case studies which show the advantages of taking cell line-specificity into consideration. In addition, we carried out ablation studies to demonstrate the benefits of our proposed network architecture. Finally, we built a user-friendly webserver to expand our tool's accessibility.To construct a practical cell line-specific prediction model, we collected protein essentiality data in extremely large-scale cell lines. We downloaded the essentiality data generated by the Wellcome Sanger Institutes (Release 1) from the Project Score database (We collected sequence information from the Consensus CoDing Sequence (CCDS) database (Release 22)  Xeon(R) Gold 5220 CPU @ 2.20 GHz, 256GB memory and a Nvidia GeForce RTX 2080 Ti GPU. The hyper-parameter settings were determined by grid search techniques.We evaluated our models on the independent test sets of different cell lines. The model performance was assessed by the area under the receiver-operating characteristic curve (AUROC) and the area under precision-recall curve (AUPRC), which can measure the ranking ability for prediction models. It should be noted that AUPRC is more sensitive to the positive samples i.e. essential proteins and thus can provide more comprehensive evaluation.To evaluate the performance of DeepCellEss, we trained and tested DeepCellEss on a large collection of benchmark datasets across different cell lines. Specifically, DeepCellEss was trained independently on 323 cell line benchmark datasets using the same model optimization settings. After all training processes are completed, we carried out the tests on the corresponding independent test set of each cell line model. The detailed performance results of all cell line models are listed in In this section, we carried out comparison experiments to investigate the effectiveness of DeepCellEss for essential protein prediction. We compared the performance of DeepCellEss with five sequence-based baseline methods (described in Section 3.4) on the independent test set of HCT-116 cell line. The comparison results are shown in In addition, we can see that Pheg gets AUROC and AUPRC scores of 0.427 and 0.450, respectively, which are lower than the other methods. This can be explained by the fact that Pheg web server only provides a general human gene essentiality predictor, and it ignores specific differences in the essentiality of genes and the encoded products across cell lines, resulting in poor prediction performance on cell line-specific test datasets. Such results indicate the difficulty of identifying cell line-specific essential genes and proteins using a general model trained on common essential samples in cell lines. Thus, training cell line-specific models is necessary for discovering specific essential genes and proteins in different cell lines.Moreover, we observed that the performance of DeepCellEss is better than DeepCellEss-nc, which means that protein sequence features are more effective than nucleotide sequence features for DeepCellEss model. The results may be thanks to that: (i) protein sequences are composed of 21 types of amino acids while nucleotide sequences are made up of four different types of nucleotides, resulting in protein sequences has a more diverse sequence information; (ii) the encoded protein sequence is much shorter than the nucleotide sequence for a gene, which can reduce computational consumption and processing complexity; and (iii) protein sequence features are more informative for essentiality prediction.Over the past two decades, many studies have reported that the essentiality of proteins is highly related to the topological properties of PPI networks. Extensive network-based centrality measures were developed for discovering new essential proteins. These methods can efficiently mine latent information from network topology and rank essentiality for proteins in PPI networks. However, the network-based methods suffer from several major drawbacks: (i) these methods cannot be directly used for proteins that are not in the PPI networks; (ii) their prediction ability for essential proteins with low degrees is greatly limited. As a sequence-based method, DeepCellEss is able to compensate for the shortcomings of network-based centrality measures.In order to investigate whether DeepCellEss can achieve promising prediction performance without interaction information, we designed the following experiments: We first downloaded protein interaction data of HCT-116 cell line from the BioPlex 3.0 database  of essential and non-essential proteins. We ranked the result scores predicted by the six network-based methods and DeepCellEss from highest to lowest and compared the cumulative counts of essential proteins in the top 10%, top 20%, 30% and top 40% proteins. The results in We further explored the prediction performance of DeepCellEss on the proteins with low degree in PPI networks. According to the Centrality-Lethality Rule . DDX59 is a member of the DEAD box helicase family proteins, which involves in all aspects of RNA metabolism and plays an important role in many cellular activities. Last, we explored the performance of DeepCellEss on the proteins that have no protein interaction information in our HCT-116 benchmark dataset. Based on our statistics, there are 438 essential proteins that do not appear in the HCT-110 network, which means they are not able to be identified by network-based approaches. Then, we tested them using our re-trained HCT-116-specific model. The results show that 63.9% (280) of them are correctly predicted, indicating that our model has practical and effective prediction ability for essential protein prediction without PPI information.Overall, these experiment results and comparative analysis of DeepCellEss and network-based methods confirm that DeepCellEss can achieve promising performance for essential proteins with no PPI information or low degree in PPI networks. DeepCellEss effectively compensates for the limitations of network-based methods and offers a more practical approach to essential protein prediction.A major advantage of our proposed model is the capability to learn and predict protein essentialities across different cell lines. To demonstrate the effectiveness of our model for cell line-specific prediction, we used ‘G1/S-specific cyclin-D1’  as an example to compar DeepCellEss with other two available servers (Pheg and EP-GBDT). From pre-existing biological experiments, CCND1 performs obvious differences within various cellular environments in terms of essentiality. For instance, it is identified as essential in CL-11 cell line while non-essential in RPMI-B226 cell line. Moreover, DeepCellEss leverages the advantage of the attention mechanism to assign residue-level attention scores for query proteins, and provides the visual heatmap for interpretation. The JAB1/MPN/Mov34 metalloenzyme (JAMM) motif is highly conserved, typically consisting of a canonical sequence of ‘H-[NST]-H-x(7)-S-x(2)-D’. JAMM-containing proteins are metal-dependent proteases and responsible for providing the active site for isopeptidase activity . ResearcAdditionally, we analyzed the performance of DeepCellEss on intrinsically disordered proteins (IDPs), which are widely distributed in eukaryotes and closely associated with human diseases. From the cancer-related protein dataset of DisProt database, we found a conditional essential IDP with 100% disorder content, called ‘nuclear factor erythroid 2-related factor 2’ . Several studies have revealed that NFE2L2 is highly related to lung cancers  indicateTo measure the contributions of individual components to DeepCellEss structure, we conducted ablation studies by re-training and validating DeepCellEss without different components. Specifically, four main components, including skip connection, CNN module, multi-head self-attention module and bi-LSTM module, were separately removed, and we obtained four variants of DeepCellEss model. Then, we trained and validated these four models. The strategies for dataset splitting and model training remain unchanged as the raw DeepCellEss. http://csuligroup.com:8000/DeepCellEss. The DeepCellEss web server provides cell line-specific essential protein prediction and visualization for a large amount of cell lines. To facilitate the access to DeepCellEss, we developed a user-friendly web server, The identification of cancer cell line-specific essential proteins is particularly relevant for the discovery of novel precision cancer drug targets. However, existing computational methods have not taken into account the specificity of essential proteins in different cell lines, and lack practical and interpretable tools for human essential protein prediction. In this study, we proposed DeepCellEss, a cell line-specific interpretable deep learning prediction method based on the attention mechanism. The main contributions of DeepCellEss are summarized as follows:To the best of our knowledge, DeepCellEss is the first computational method that supports cell line-specific essential protein predictions, which makes it possible to predict protein essentialities in different cellular environments;DeepCellEss implements an interpretable deep-learning model through residue-level attention scores from multi-head self-attention mechanism. The attention scores enable to locate the most important sequence regions for different prediction results, and further make more comprehensive analysis and comparison for cell line-specific essential proteins;For real practical applications of our cell line-specific model, we constructed extremely large-scale datasets across 323 cell lines. Moreover, we provided a user-friendly web server of cell line-specific essential protein predictions. It is expected to help discover potential diagnostic biomarkers and therapeutic targets for precision cancer therapy.Although the extensive results show that DeepCellEss is an effective predictor for cell line-specific essential proteins and outperformers existing sequence-based methods, we would like to point out its limitations. The main limitation is that we do not consider the relations of different cell lines under the same tissue or cancer type. In our reported results, the models of different cell lines under the same cancer type show varying prediction performance. For example, in the cancer type of Colorectal Carcinoma, SNU-C1 model yields the best AUROC (0.825) and AUPRC (0.826), while MDST8 model gets the worst AUROC (0.728) and AUPRC (0.731). Therefore, future efforts could be devoted to improving the poor performance for some cell lines by introducing the relations between different cell lines. One potential solution is to use transfer learning techniques (btac779_Supplementary_DataClick here for additional data file.