NFAT1 expression is regulated by GRK5 in a kinase independent manner, and loss of NFAT1 phenocopies GRK5 loss-of-function effects on the cell cycle alterations. Self-renewal of tumor propagating cells (TPCs) is thought to give rise to tumor relapse. We show that loss of GRK5 results in a significant reduction of RMS self-renewal capacity in part due to increased cell death. Treatment of human RMS xenografts in mice with CCG-215022, a GRK5-selective inhibitor, results in reduced tumor growth and self-renewal in both major subtypes of RMS. GRK5 represents a novel therapeutic target for the treatment of RMS.Rhabdomyosarcoma (RMS) is the most common soft-tissue pediatric sarcoma. Clinical outcomes for RMS patients with relapsed or metastatic disease remain poor. Treatment options remain limited, presenting an urgent need for novel therapeutic targets. Using a high-throughput siRNA screen against the human kinome, we identified GRK5, a G-protein receptor kinase, as a novel regulator of RMS tumor cell growth and self-renewal. Through functional assays  RAS pathway [Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue cancer. There are two major subtypes of RMS, each with distinct histologic features and genetic alterations. Embryonal rhabdomyosarcoma (ERMS) typically harbors mutations in the  pathway . Alveola pathway . Other R pathway , highlig pathway –7, and p pathway . In huma pathway . TargetiTherapeutic targeting of protein kinases has been demonstrated to be an effective treatment option for a variety of cancers . There eGRK5) belongs to a family of serine/threonine kinases [G-protein coupled receptor kinase 5 . Using the CRISPR/Cas9-based genetic editing strategy, we show that GRK5 loss-of-function reduces RMS self-renewal capacity turation , is a kein vitro assay for assessing the self-renewal capacity of tumor cells [FES, LTK, LYN, NME9, PIK3C2A, GRK5) showed differential effects on self-renewal compared to cell growth and were prioritized for further validation. We subsequently utilized a high-efficiency CRISPR/Cas9 gene targeting strategy [To identify potential candidate kinases that are essential for self-renewal of ERMS, we performed a siRNA library screen against the human kinome (714 kinases) in two ERMS cell lines (RD and 381T). Each cell line was transfected with a pool of 3 siRNAs against each kinase, along with control (scramble) siRNAs, in 384-well low attachment plates to induce sphere formation. The sphere assay was used as a surrogate or cells . RD and strategy  to validGRK5 mRNA expression levels were analyzed in 4 RMS cell lines  and compared against a primary myoblast line and an immortalized fibroblast line. In the 4 RMS cell lines, regardless of subtype, the expression level of GRK5 is at least 2-fold higher compared to normal cell types  and nuclear localization (NLS) functional domains of GRK5  in the sphere formation in 3  out of the 4 RMS cancer cell lines . Spheroids generated from cells harboring targeted disruption of GRK5 appeared to be both smaller and fewer in number  and ARMS (Rh5) xenografts in immunocompromised NOD-scid-IL2Rgammanull (NSG) mice. In both RMS subtypes, targeted disruption of GRK5 resulted in approximately 4 to 6-fold reduction in self-renewal frequency   and ERMS (381T and SMS-CTR) cell lines. Loss of GRK5 resulted in a significant reduction in cell viability in all 4 RMS cell lines ( < 0.05) . To asse of GRK5 . Even thK5  significantly reduced cell growth  and Rh30 with TP53(R237H) [TP53 mutations. Our findings indicate that GRK5 regulates RMS cell growth in a kinase- and TP53-independent manner.The studies characterizing the role of GRK5 in cancer pathogenesis to date have primarily investigated the functional requirement of its kinase activity , 34. TP53(R237H) , 36, redNFAT1 expression following targeted disruption of GRK5 in ERMS cell lines. We also show that GRK5 potentially interacts with NFAT1, and this interaction is abrogated with loss of GRK5. Our findings suggest a role for GRK5 acting in a kinase-independent manner to regulate the NFAT1 expression. CRISPR/Cas9 mediated-disruption of NFAT1 phenocopies the loss-of-function effects of GRK5 on cell cycle progression. NFAT likely functions as an important downstream mediator to promote oncogenic activity of GRK5. Further study is required to determine whether GRK5 directly or indirectly interacts with NFAT1 in ERMS in regulating ERMS tumor cell growth.Dysregulated activity of the NFAT family of transcription factors has been identified in cancer . While Nin vitro and in vivo. The stem-like RMS spheres harboring GRK5 knockout show increased cell death. Our findings indicate that GRK5 is a promising therapeutic target against RMS stem-like features. Further investigation is required to assess whether loss of GRK5 leads to reduced heterogeneity of RMS tumors and thereby reduces the potential for resistance against standard-of-care therapies.TPCs undergo self-renewal to recapitulate the complex heterogeneity of a given malignant tumor and are thought to be the major drivers of cancer relapse and metastasis in selected cancer types , 38. DisInhibitors selective to GRK5 are currently limited. Amlexanox, an FDA-approved anti-inflammatory drug, has been shown to inhibit GRK5 activity . Howeverin vitro and in vivo demonstrates that GRK5 regulates ERMS cell growth in a kinase-independent manner and is essential for RMS self-renewal capacity. A GRK5 inhibitor, CCG-215022, recapitulates the loss-of-function effects of GRK5. Thus, our findings demonstrate the promise of GRK5 as a therapeutic target against RMS disease progression and relapse.With treatment options against RMS remaining relatively unchanged over last 3 decades, there remains a need for more effective therapeutic targets. From a comprehensive siRNA library screen against the human kinome, we have identified GRK5 as a novel regulator of both RMS self-renewal and cell growth. Our functional characterization of GRK5 in vitro assay for assessing the self-renewal capacity of tumor cell. The ATP-based Cell Titer Glo assay  was performed on the siRNA-treated adherent cells, and high-content imaging was performed at the Quellos core facility at day 5 post-siRNA transfection.To identify potential candidate kinases that are important for the self-renewal of ERMS, we utilized the Quellos high-throughput screening core facility in the Institute of Stem Cell and Regenerative Medicine at the University of Washington to perform an siRNA library screen against the human kinome (714 kinases) in two different cell lines derived from ERMS (RD and 381T). Each cell line was transfected with a pool of 3 siRNAs against each kinase, along with control (scramble) siRNAs, in 384-well low attachment plates to induce sphere formation. The sphere assay was used as a surrogate Single gene knockout was accomplished using lentiviral transduction of RMS cells with Cas9 expressing and gene-specific double gRNA constructs. Lentiviral transduced cells were placed under antibiotic selection and plated for assays 7 days later. Cloning of Cas9 and gRNA expression constructs was performed as described previously . OverexpGRK5: gRNA1- GGACCTGGTCTCCCAGACGG; gRNA2- GGAGCAGCCCTTTCTTGGG; NFAT1: gRNA1- GACGGAGTGATCTCGATCCG; gRNA2- GATCCCACAAGGCGAGTCCG; FES: gRNA1-GGCCGAGCTTCGTCTACTGG; gRNA2- GAGCCTGCTCATCCGGGAA; LTK: gRNA1- GCTGGCTCCAAGATACTAGG; gRNA2- GACCAGCGTGGTGGTGACCG; LYN: gRNA1- GTAGCCTTGTACCCCTATGA; gRNA2- GGAATGGCATACATCGAG; NME9: gRNA1- GACCTCGATCCTCATCTTC; gRNA2- GATGTCCTCGAAAAGTACAG; PIK3C2A: gRNA1- GCACAGGTTTATAACAAGC; gRNA2 - GGGGCGCTTGCTAATATTTT.The following gRNAs were used for targeting genes in human RMS cell lines: RMS cell growth was assessed via cell counts or an ATP-based luminescent cell viability assay, Cell Titer Glo . Myogenic differentiation was performed following serum starvation of RMS cells in 2% horse serum/DMEM for 72 hours prior to fixation in 2% paraformaldehyde. Immunofluorescence against MF20 (myosin heavy chain) was then performed. Self-renewal was assessed by visual counts of the spheroids, induced in growth factors  enriched neurobasal medium as previous described . Apoptos6 RMS cells (RH5 or 381T) suspended in Matrigel. When the tumor size reached 20–75 mm3, CCG-215022 (10 mg/kg) or vehicle (DMSO) were given intraperitoneal every 3 days for up to 21 days or until tumors reached end point (750 mm3). Tumor measurements were made with calipers every 3–4 days at tumor onset until tumors reached end point or at the end of drug treatment, whichever came first. For limiting dilution experiments, 6 NSG mice were given RMS cell injections (Rh5 or 381T) of either control or GRK5 knock-out cells at either 2 × 103, 1 × 104, 5 × 104 dilutions in the same manner as previously described. Analysis of limiting dilution data was performed by the extreme limiting dilution (ELDA) software as previously described [All mouse experiments were approved by the University of Washington Subcommittee on Research Animal Care under IACUC protocol #4330–01. 6–7 immunocompromised NOD-SCID IL2rg−/− (NSG) mice were xenografted via subcutaneous injections into the flanks with approximately 1–2 × 10The RMS tissue microarray was obtained from Seattle Children’s Hospital. Immunohistochemistry was performed at the Histology and Imaging core facility at the University of Washington. Immunofluorescence was performed as previously described . DuolinkCell lysates from RMS cells were counted and lysed in RIPA buffer  with protease inhibitors plus 2× sample buffer . Equal amounts of protein lysates were electrophoresed on a 4–15% gradient SDS-polyacrylamide gel  and fast transferred to Immun-Blot PVDF membranes  using the Turbo-Blot Transfer system  We used the following antibodies: GRK5 (N145/20) , GAPDH (14C10) , Cleaved Caspase 3 (Asp175) . Goat anti-mouse or anti-rabbit HRP conjugated IgG secondary antibodies were obtained from Santa Cruz Biotechnology . Membranes were blocked in 5% milk in Tris Buffered Saline plus Tween . Quantitative analysis of Western blot images was performed on ImageJ.GRK5 FWD – GTCTGTCCACGAGTACCTGA; REV – CAGGCATACATTTTACCCGT; NFAT1 FWD - ACGAGCTTGACTTCTCCACC; REV - TGCATTCGGCTCTTCTTCGTRNA was collected from human cell lines lysates  using Qiagen RNeasy Plus Mini Kit . cDNA was then generated using High Capacity cDNA Reverse Transcription Kit from Applied Biosystems . RT-PCR reactions were then run with iTaq Universal SYBR Green mix on a CFX Connect Real Time System . RT-PCR primers used are listed below: Mann-Whitney statistical test was run on drug treat RMS tumor mouse experiments to assess statistical significance in differences between experimental and control. Two tailed Student’s t-test was applied when appropriate.


Bioprinting offers the opportunity to fabricate precise 3D tumor models to study tumor pathophysiology and progression. However, the choice of the bioink used is important. In this study, cell behavior was studied in three mechanically and biologically different hydrogels , and thiol-modified hyaluronan (HA-SH crosslinked with PEGDA)) with cells from breast cancer (MDA-MB-231 and MCF-7) and melanoma (Mel Im and MV3), by analyzing survival, growth, and the amount of metabolically active, living cells via WST-8 labeling. Material characteristics were analyzed by dynamic mechanical analysis. Cell lines revealed significantly increased cell numbers in low-percentage alginate and HA-SH from day 1 to 14, while only Mel Im also revealed an increase in ADA–GEL. MCF-7 showed a preference for 1% alginate. Melanoma cells tended to proliferate better in ADA–GEL and HA-SH than mammary carcinoma cells. In 1% alginate, breast cancer cells showed equally good proliferation compared to melanoma cell lines. A smaller area was colonized in high-percentage alginate-based hydrogels. Moreover, 3% alginate was the stiffest material, and 2.5% ADA–GEL was the softest material. The other hydrogels were in the same range in between. Therefore, cellular responses were not only stiffness-dependent. With 1% alginate and HA-SH, we identified matrices that enable proliferation of all tested tumor cell lines while maintaining expected tumor heterogeneity. By adapting hydrogels, differences could be accentuated. This opens up the possibility of understanding and analyzing tumor heterogeneity by biofabrication. Various types of cancers become an increasing problem in our aging society. Nowadays, one out of eight women in the USA develops breast cancer and approximately 3% of non-Hispanic white people develop invasive melanoma . InteresOver the years, many different natural and synthetic scaffold materials have been developed and used in 3D in vitro and in vivo models. HA is a naturally occurring glycosaminoglycan, consisting of glucuronic acid and N-acetylglucosamine, and it is one of the main components of the ECM with, for example, approximately 400–500 µg per gram skin . HA can 2+, it is easy to handle and frequently used for 3D models. However, mammalian cells cannot adhere to or degrade alginate. Material scientists modify the molecular structure by, for example, oxidizing alginate to alginate dialdehyde (ADA) or blending it with degradable proteins, like fibrin or gelatin, to optimize the material for 3D culture [Apart from HA-based tumor models that have to be highly defined for consistent results due to possible pro-carcinogenic effects, more inert hydrogels have been established for mammalian cells. Alginate is a polysaccharide of guluronic and mannuronic acid from cell walls of brown algae, providing them with stability and flexibility. Due to its ability to be easily crosslinked and form hydrogels after adding divalent ions like Ca culture ,22. Addi culture , leading culture . After p culture ,25.In this study, we exemplarily compared two mammary carcinoma cell lines (MDA-MB-231 and MCF-7) and two melanoma cell lines (Mel Im and MV3), to identify suitable hydrogels with different mechanical and biological characteristics  for 3D bioprinting. These cell lines are of different subtype and have different mutations to represent a range of diverse cancers with possibly varying requirements to the matrix. Material characteristics were analyzed via dynamic mechanical analysis (DMA). We focused on the effects of the hydrogels on cell survival, metabolic activity, and growth, comparing tumor cells of different origin and different subtypes.m/v and 3% m/v; ADA–GEL was used in 2.5% m/v and 4% m/v; HA-SH was used with 0.8% m/v Glycosil crosslinked with 0.5% m/v PEGDA and 1% m/v PEGDA.In this study, we compared the cell growth, survival, and metabolic activity of two mammary carcinoma cell lines (MDA-MB-231 and MCF-7) and two melanoma cell lines (Mel Im and MV3) in three different hydrogels, using two different hydrogel concentrations each. Alginate was used in 1% To demonstrate typical colony forming behavior of tumor cells in standardized assays, all cell lines  were cultivated both in agar and Matrigel . Except DMA measurements were performed to correlate the cellular behavior with the material properties . The stoOverall, 3% alginate was the stiffest material, followed by the other conditions. Moreover, 1% alginate, 4% ADA–GEL, and the two HA-SH (0.5% PEGDA and 1% PEGDA) gels are all in the same range, in terms of stiffness. The softest material was 2.5% ADA–GEL. The graphs of the storage modulus, E′ b are relThe metabolic activity of all cell lines was analyzed over 14 days with a WST-8 assay , at timeInterestingly, both melanoma cell lines showed a trend to higher metabolic activity in ADA–GEL and HA-SH than mammary carcinoma cells during long-term cultivation (>7 days). This effect was even stronger in higher concentrated or higher crosslinked hydrogels. Both melanoma cell lines had a significantly higher metabolic activity than both mammary carcinoma cell lines in HA-SH with 1% PEGDA. In contrast, in alginate, there was a tendency for a higher metabolic activity of breast cancer cells, as compared to melanoma cells. When increasing the concentration of alginate from 1% to 3%, however, this effect disappeared—all cell lines showed similar behavior in 3% alginate.Measured metabolic activity of all cell lines as a surrogate marker for cell proliferation increased significantly over 14 days, in 1% alginate and both concentrations of HA-SH, indicating a significant increase in cell number. Compared to 1% alginate, 3% alginate resulted in lower metabolic activity, in general. In contrast to alginate, both concentrations of ADA–GEL only led to a significant increase in metabolic activity in Mel Im. For MDA-MB-231 and MV3, there was a trend for an increase in cell metabolic activity over time in ADA–GEL, while MCF-7 even showed a decrease in metabolic activity over 14 days in this hydrogel. Similar to alginate, the low-concentrated 2.5% ADA–GEL resulted in stronger absorption values, as compared to the higher concentration of 4% ADA–GEL.−1 of each hydrogel with a Spearman correlation coefficient of ρ = −0.27. The individual cell lines showed distinct correlations. MDA-MB-231 had ρ = −0.6, MCF-7 had ρ = 0,34, Mel Im had ρ = −0.71, and MV3 had ρ = −0.26. No coefficients were significant.Overall, the mean WST-8 absorption of all cell lines on day 14 correlates b with thWith the help of Hoechst 33342, FITC-annexin V, and ethidium homodimer III, it is possible to distinguish between healthy (blue), apoptotic (green), and necrotic (red) cells by fluorescence microscopy. In all conditions, sporadic cell doublets of all cell lines were observed on day one, although they were plated from a single cell suspension. This is an indication for the ongoing mitotic activity within the gels. Cells were further stained and imaged at time points 7 and 14 days, to compare proliferation and survival over time. Both mammary and melanoma cell lines survived and proliferated over 14 days in alginate , ADA–GELIn 1% alginate, some channels full of cells a, aligneAll cell lines were able to escape the hydrogels and formed colonies on the bottom of the wells over 14 days. We observed this earlier in the thinner gels. An example of a colony of Mel Im bursting through the surface of a hydrogel is shown in Additionally, few MCF-7 started to differentiate and to form mammary-gland-like structures in the different hydrogels c. Those For analysis of survival after cell seeding in different hydrogels, cells were stained and evaluated at day one a. GeneraApoptosis rates on day one were relatively low in 1% alginate for all cell lines, with no significant difference between melanoma and mammary carcinoma cell lines. Necrosis rates tended to be a little higher, especially for MCF-7. This effect was pronounced when increasing the concentration to 3% alginate; MCF-7 had a significantly higher necrotic rate than the other cell lines. MDA-MB-231 also showed a significantly higher necrotic rate than both melanoma cell lines. MCF-7 had a trend for the highest apoptotic rates in 2.5% ADA–GEL, compared to the other cell lines. Here, MCF-7 had a significantly higher necrotic rate. In 4% ADA–GEL and in both HA-SH conditions, there were no significant differences between the cell lines.The measurement of the colonization on day 14 b revealeWith this study, we compared the functional characteristics, like growth and survival, from cell lines from breast cancer and melanoma in 3D hydrogels. According to the known tumor heterogeneity, we expected differences between cells of different origin, especially in 3D culture . It was the aim of this study to find a suitable material that enables cultivation and the subsequent use for bioprinting of reproducible 3D tumor models, while maintaining tumor heterogeneity.The cell lines of mammary carcinoma (MDA-MB-231 and MCF-7) and melanoma (Mel Im and MV3) are all of metastatic origin. The breast cancer cell lines are well-established models in many different laboratories. MDA-MB-231 is a highly invasive triple-negative breast cancer (TNBC) cell line, i.e., the cells are negative for hormone receptors (estrogen and progesterone) and HER2 making it more difficult to target them with therapies . MCF-7 iWe used Matrigel and agar as controls, to identify whether the cell lines are suitable for our model. Matrigel is a widely used hydrogel for various tumor models, since it can mimic, in part, the tumor microenvironment. However, its complex, varying, and ill-defined composition makes it difficult to use for many applications with specific questions. Matrigel is a basal membrane matrix composition from mouse sarcoma cells that is rich in growth factors, type IV collagen, laminin, entactin, and heparan sulfate proteoglycans. Therefore, they provide anchorage points that allow the cells to attach easily, while agar does not provide adhesion motifs for mammalian cells. All the cell lines were able to proliferate under both conditions . Hence, 2+ can be washed out over time, and the gels degrade, depending on the molar mass distribution of the alginate [Like agar, alginate also does not provide motifs for cell attachment. HA-SH (Glycosil) provides only binding for CD44 but no classic RGD sequences for integrin binding. CD44 binding can be reduced due to the thiol modification , dependialginate . The celOverall, apoptotic and necrotic rates were low in the different gels. Considering the different apoptotic and necrotic stainings, MCF-7 cells seem to be the ones with the worst overall survival. Further, we observed the highest necrosis rates in HA-SH gels. In these gels, a smaller pipette was used due to different crosslinking setups, which could have led to higher shear forces while pipetting. MCF-7 cells seem to be more susceptible to shear stress. A model for circulating tumor cells also reported a non-significant trend for this with fluid shear stress . Due to −1 with data of static Young’s moduli from literature, one can conclude a good agreement of both measurement techniques. The observed deviations can be caused by several factors, such as batch-to-batch variations, experimental variations, and different crosslinking techniques. The stiffest material in our experimental setup was 3% alginate with a mean storage modulus, E′, at 1 rad s−1 of 79.6 kPa, which decreased to a fifth for 1% alginate. Other groups reported a mean Young’s modulus, E, of 3.6 ± 0.5 kPa for 1% alginate, while stiffness increases 2.7- to 5.3-fold in 3% alginate up to 9.8 kPa to 19 kPa [All of our materials showed mainly elastic properties , as the o 19 kPa ,49. Howeo 19 kPa ) and theo 19 kPa . Huang e−1 was 16.3 kPa, it reduced to less than a third in 2.5% ADA–GEL. This was the softest material in our setup, although the polymer content was higher than in 1% alginate. The combination of the oxidation of alginate and the addition of gelatin weakens the crosslinking. Others reported a Young’s modulus of 16 kPa of 7.5% ADA–GEL at room temperature [While our mean storage modulus in 4% ADA–GEL at 1 rad sperature . In thisperature . Interes−1 a similar storage modulus to 1% alginate and 4% ADA–GEL. When considering the SD of the measurements, the PEGDA concentration had an insignificant effect on the stiffness of the material. It should be noted that PEGDA in the concentrations used does not crosslink all thiol groups of HA-SH—the remaining groups can crosslink via disulfide bonds between molecules.Our HA-SH gels in both PEGDA concentrations had with 15.1 kPa (1% PEGDA) and 21.9 kPa (0.5% PEGDA) at 1 rad sThe total correlation of the WST-8 on day 14 of all cell lines to the storage modulus has a small negative linear correlation. MCF-7 stood out with a small positive correlation, while the others had a larger negative correlation. None of those were significant. This is an indicator that not only material stiffness is key to distinct cellular behavior. The chemical and biological characteristics of the hydrogels play a major role, as well, again, depending on the cell line. Even gels with similar mechanical properties showed completely different cellular behavior, especially for MCF-7.The physiological in vivo Young’s modulus is approximately 1.8 kPa in normal breast tissue and can increase to 12 kPa in breast cancer . In diffApparently, MV3’s metabolic activity was positively influenced by the presence of gelatin or, less likely, ADA. The initial metabolic activity in the group “MV3 with 2.5% ADA–GEL” on day one of the assay tended to be higher than of the other cell lines. Compared to the microscopic analysis on day one, cell numbers were not noticeably higher in this group, although a few cell doublets had formed. Due to this high initial value, we could not detect a significant increase (only approximately 30% increase) in metabolic activity over 14 days. Nonetheless, metabolic throughput for MV3 tended to be still much higher in ADA–GEL, compared to breast cancer cell lines. Likewise, Mel Im showed the highest metabolic throughput in ADA–GEL, compared to the other cell lines. This effect increased in higher concentrated hydrogels. Similarly, in HA-SH, for both melanoma cell lines, a higher metabolic labeling was observed, indicating a higher cell number, compared to breast cancer cell lines, while this tendency became significant when increasing the PEGDA concentration up to 1%. By comparing these results to the in vivo situation, we can observe a surprising degree of similarity. In vivo, nodular melanoma is known for its aggressiveness and metastatic activity throughout the body . In the HAS2 mRNA and also HYAL2 [The presence of HA-SH itself within the hydrogels can have different effects on tumor cells. In our study, we could show that increasing the crosslinking of HA-SH by increasing the PEGDA concentration intensified the difference in tumor cell behavior. Previously, it has been shown that highly invasive cancer cell lines like the MDA-MB-231 have a high expression of so HYAL2 , resultiso HYAL2 . Furtherso HYAL2 ,61. It wso HYAL2 . TherefoNotably, some MCF-7 cells started to spontaneously form mammary-gland-like structures in all different hydrogels c. The ce3 and D-Glucono-δ-lactone can significantly improve the printing results without increasing the polymer density [In summary, the biggest advantages of the model presented in this study are the following. In contrast to other hydrogels, like Matrigel, the gels of this study are well-defined and tailorable, e.g., in terms of stiffness and growth factors. Therefore, they can be used to answer questions that are more sophisticated, also while accentuating tumor heterogeneity. Using extrusion-based 3D printing, we can further improve the models, e.g., with the implementation of stiffness gradients, the addition of other cells or stimulating factors in a defined spatial organization. In addition, a macroporous structure could ensure homogeneous crosslinking, which could be difficult for alginate-based bioinks otherwise. Thicker constructs may have low diffusion rates. Variations of the different hydrogels in this study have already been used for extrusion-based 3D printing. Although alginate itself has poor printing properties due to low shape fidelity, a pre-crosslinking step with CaCO density . Due to  density . Bioprin density . Here, tThis study confirms that we could successfully simulate in vivo conditions in which the different cell lines showed the expected heterogeneity, with respect to origin and subtype. We even could pronounce this effect by increasing the hydrogel concentration or crosslinking density. This makes it possible to study tumor heterogeneity further. Generally, all cells proliferated better in hydrogels of lower concentrations. In this in vitro model, various other cancer cell lines could be tested by using these hydrogels. Subsequently, it should be noted that establishing a 3D tumor model makes it necessary to carefully choose hydrogels for each cell line individually.−1, 0.1 mg mL−1, Sigma-Aldrich), and the incubator was set to 5% CO2, at 37 °C.All tumor cell lines in this study were of metastatic origin. MDA-MB-231 and MCF-7  were cultivated in DMEM high glucose , 2 mM L-glutamine , 10% FCS Superior , and non-essential amino acids . Mel Im was cultivated in DMEM low-glucose (Sigma-Aldrich) with 2 mM L-glutamine, MV3 in DMEM high-glucose (Sigma-Aldrich) with 2 mM L-glutamine, with the addition of 10% FCS each. MV3 were obtained from Peter Friedl . The media were supplemented with penicillin/streptomycin  in DMEM supplemented with 20% FCS (Sigma-Aldrich), 1.925 mg mL−1 sodium bicarbonate, and 1× non-essential amino acids. Then, 5 × 103 cells were seeded in triplicates, into 1 mL 0.3% m/v top agar (diluted from base agar composition with cell suspension in DMEM) per well. The plates were incubated for 14 days, and the colonies were observed under an inverted microscope .For the evaluation of attachment-independent growth, cells were seeded into soft agar. Six-wells were coated with 2 mL of a 0.5% base agar, which was prepared as a dilution of 2% 4 cells were suspended per mL Matrigel, and 100 µL was seeded per 96-well, in triplicates, and incubated at 37 °C, for 30 min. Afterward, 100 µL of medium was added. Pictures were taken at day 7, using an inverted microscope.The capability to form colonies from single cells was also evaluated by using Matrigel . Then, 2 × 10m/v respectively 3% m/v VIVAPHARM® Alginate PH 176  was dissolved in PBS without Ca2+ and Mg2+ (Sigma-Aldrich). A positive displacement pipette was used to resuspend the cells (1 × 106 mL−1) and form hydrogel beads by pipetting droplets of approximately 100–150 µL (3–4 mm in diameter) of the cell suspension into a CaCl2 bath . After 10 min of crosslinking, the beads were washed by using 100 mM HEPES (Sigma-Aldrich), for 10 min, and then transferred into cell culture medium and incubated at 37 °C and 5% CO2, for 14 days. Medium was changed three times per week.For alginate hydrogels 1% ® Alginate PH 163 S2 (JRS PHARMA GmbH & Co. KG), based on previous experiments [4 solution (Sigma-Aldrich) was added slowly to the stirring dispersion, in the dark. After 6 h, 10 mL of ethylene glycol was added to quench the reaction. After 30 min, most of the supernatant was removed, and the residual ADA filled into dialysis tubes  and dialyzed for 3–4 days. Afterward, the solution was lyophilized, using an Alpha 2-4 LSCplus .ADA was synthesized by using VIVAPHARMeriments . Brieflym/v respectively 8% m/v ADA was dissolved in PBS, without Ca2+ and Mg2+, and 5% m/v respectively 8% m/v solutions of porcine gelatin (Sigma-Aldrich) were prepared. The two solutions of the same concentration were mixed and crosslinked to ADA–GEL through stirring at 37 °C for 10 min. ADA–GEL beads were produced as described for alginate beads.Then, 5% m/v and PEGDA in a concentration of 0.5% m/v or 1% m/v. Cells were mixed with the components (1 × 106 mL−1) and 40 µL pipetted into glass cloning cylinders with an outer diameter of 6 mm and an inner diameter of 4 mm. After 30 min incubation in the incubator at 37 °C for the gelation process that utilizes the Michael addition, a piston was used to transfer the hydrogels into cell culture medium. Hydrogels were cultivated at 37 °C, at 5% CO2, for 14 days. Medium was changed three times per week.Hyaluronic-acid-based hydrogels were produced by using the HA-SH Glycosil, and polyethylene glycol diacrylate  . Glycosil was used in a final concentration of 0.8% The apoptotic/necrotic/healthy staining  was performed on days 1, 7, and 14. In short, hydrogels were washed with the kit’s binding buffer and incubated for 15 min in the staining solution containing FITC-annexin V (for apoptosis), ethidium homodimer III (for necrosis), and Hoechst 33342 (for DNA). Next, hydrogels were washed once again and evaluated, using an inverted fluorescence microscope. Cells that emit solely blue fluorescence were considered healthy, red fluorescence necrotic, and green fluorescence apoptotic.For the evaluation of the cell survival on day 1, the mean color intensities of the individual channels were measured after applying a background subtraction, using the rolling ball algorithm of Fiji Is Just ImageJ (Fiji) 1.52p , an exteTo evaluate the rate of colonization, quantification of phase-contrast images was performed on day 14, using Fiji. Pictures were taken close to the bottom of the constructs. The area of the colonies in focus within the gel was manually measured and set in relation to the visible gel area. Blurred colonies were excluded from quantification. Each time, one picture per individual experiment—in total, three representative images—were analyzed.WST-8 assays (PromoCell GmbH) were performed to test for the amount of metabolically active cells on days 1, 7, and 14. The hydrogel constructs were transferred into wells of a 96-well plate, in triplicates. New constructs were used each time. Then, 100 µL of medium and 10 µL of the tetrazolium salt WST-8 were added and incubated for 2 h, at 37 °C. Afterward, 100 µL was pipetted into new wells and measured by using a microplate reader (Thermo Fisher Scientific), at 450 nm, with a reference of 600 nm.2. ADA–GEL was crosslinked for 40 min. HA-SH gels were cast into 12 mm Teflon rings (226 µL per ring) and crosslinked for 30 min. The different gels were transferred into complete cell culture medium, put into the incubator at 37 °C, and measured for their mechanical characteristics, on day 1.For characterizing the viscoelastic properties of the samples, DMA tests were performed. The different gels were prepared in the same concentrations as described above. For alginate and ADA–GEL, films of 1 mL were prepared in a 12-well plate, resulting in circular samples with a diameter of 18 mm and a height of 2 mm. Alginate gels were crosslinked for 10 min in 100 mM CaCl−1 for determining suitable amplitudes for the following frequency sweeps. The amplitude was chosen as a trade-off between measurement noise at very low amplitudes and non-linearities, e.g., gel destruction or wall-slip, at larger amplitudes. The time and frequency dependent viscoelastic properties were determined by frequency sweeps. A range of 0.1 to 10 rad s−1 (0.02 to 1.5 Hz) is covered for all materials. This regime is relevant for revealing relations of mechanical properties and cell studies, since those are performed in a quasi-stationary state. Average values and standard deviations were calculated from at least 3 measurements of technical replicates. All measurements were additionally rated by the following criteria and only evaluated if all were met:The axial force recorded by the instrument did not drastically alter during the measurement.The oscillation force was significantly bigger than the lower limit given by the manufacturer.Raw data signals showed a sinusoidal function for recorded force and displacement.Both moduli  were positive and recorded by the instrument.A DHR-3 rheometer , equipped with a 20 mm plate–plate geometry for alginate and ADA–GEL samples and a 12 mm plate-plate geometry for HA-SH samples in oscillating compression mode, was used for all tests. An axial pre-force of 0.1 N was applied to all samples, except for the 2.5% ADA–GEL samples, since a drastic deformation was already observable under 0.1 N axial load and was therefore reduced to 0.05 N. All tests were performed with a constant temperature of 37 °C, controlled by the attached Peltier element. An equilibration time of 3 min prior to each measurement was proven as sufficient to ensure a homogeneous temperature distribution in preliminary tests. Amplitude sweeps were performed at a frequency of 1 rad sThe complex modulus |E*| was calculated as follows:n = 3). Statistical analysis was performed by using IBM SPSS statistics software V24 . Differences between groups were analyzed, using the Kruskal–Wallis H test, followed by a Mann–Whitney U test for post hoc analysis; the asymptotic significance was used. Significant p-value was set to ≤ 0.05. Figures show the mean ± standard deviation and were created with GraphPad Prism 8.1.2 . Depicted microscopic images were arranged and edited with CorelDRAW X6 and Corel PHOTO-PAINT X6 , to compensate differences in brightness and contrast to match the observed results.All experiments, except for DMA, were performed three times , with regard to tumor heterogeneity. It could be demonstrated that hydrogels have vastly differing mechanical properties and have to be carefully chosen according to the respective demands of each cell line or the experimental question to optimally mimic the in vivo condition of different tumor (sub)types. Generally, melanoma cells showed a tendency for a higher metabolic activity in ADA–GEL and HA-SH, compared to breast cancer cells, while this effect got stronger with increasing concentration of the hydrogels. In contrast, mammary carcinoma cells showed equally good proliferation in 1% alginate, as compared to melanoma cells. These different cell lines showed different characteristics in vitro that could also be observed in vivo, with respect to origin and subtype. We conclude that biofabrication of these hydrogels may offer a suitable model to study and understand tumor heterogeneity.


The speed at which a cell fate decision in nematode worms evolves is due to the number of genes that control the decision, rather than to a high mutation rate. Related research article Besnard F, Picao-Osorio J, Dubois C, Félix MA. 2020. A broad mutational target explains a fast rate of phenotypic evolution. eLife9:e54928. doi: 10.7554/eLife.54928Evolution is a two-step process: first, variation is generated, for example through random mutations; and then events such as natural selection determine whether specific traits become more or less common in a population. Some traits evolve faster than others, and different explanations for this emphasize the relative importance of one or the other of the two steps. For example, a fast-evolving trait could be under sustained and intense selection pressure, so that when a favorable new variant arises, it quickly spreads throughout the population. Alternatively, mutations might affect certain traits more than others, leading to more variation in these traits. Since variation is the fuel of evolution, this could make the affected traits change faster.At the molecular level, two mechanisms can bias which traits are more likely to be affected by mutations. On one hand, the genes that affect a particular trait could be located in genomic areas with high mutation rates, known as ‘mutational hotspots’ ; on the Caenorhabditis elegans and C. briggsae worms have two sexes – males, which are very rare, and hermaphrodites. In hermaphrodites, the cell divisions and fate decisions that occur during development are nearly identical for individuals within a species. However, a cell called P3.p, which is involved in the development of the vulva, sometimes divides and sometimes does not, even among worms with the same genome (e genome . A decade genome . Now, ine genome .C. elegans or the closely related C. briggsae. These lines started from the offspring of a single individual and were bred in parallel. All of the lines were inbred: in each line a single selfing hermaphrodite was the parent of the next generation. This meant that if the parent had a new mutation, there would be a good chance that its offspring would inherit it. In this environment, the effects of natural selection were minimized because the only requirement for a line to endure was that a single hermaphrodite from that line survived and reproduced. After many generations, each line accumulated its own constellation of new mutations. Since the lines all started out genetically identical and selection was minimal, any differences in traits between the lines were be due to the different mutations.To show this, Besnard et al. used mutation accumulation lines of either The mutation accumulation lines used in these experiments were evolved for 250 generations , after wThis broad mutational target is consistent with many genome-wide association studies and genetic mapping studies . These eCaenorhabditis species in this work reinforces the power of this model system in research on developmental and evolutionary biology. Because so much is known about worm cell fates, Besnard et al. were able to go beyond identifying genes and pose testable hypotheses for why this particular cell was more variable than other vulval precursors. For example, they point out that the P3.p cell is at the far end of a gradient of cell fate-inducing molecules that are secreted from the tail end of the animal. The Félix lab previously showed that P3.p cell division is much more sensitive to variations in the dose of these molecules than cells closer to the source of the gradient (The use of gradient . This suDevelopmental processes are responsible for generating the effects of many mutations. Therefore, to fully understand how mutations influence evolution, we must first understand the developmental context in which they occur.


The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production attributes and is currently used for manufacturing of several therapeutic proteins and viral vectors. Despite the increased popularity of this strain we still have limited knowledge on the genetic composition of its derivatives. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes , a pattern validated in 63 human adherent or suspension cell lines of other origin. Even though the Chinese hamster ovary (CHO) cell line is a successfully used mammalian platform for the production of advanced recombinant proteins with the need for proper protein folding and post translational modifications, there is an increasing demand for improved and more efficient bioproduction platforms. With an increasing number of difficult-to-express proteins entering clinical development, including bispecific antibodies and antibody–drug conjugates, alternative or engineered expression hosts are being explored. Extensive omics profiling of CHO cells has been carried out during recent years12, which has paved the way for cell line engineering efforts aiming to improve bioproduction efficiency and product quality15. Moreover, human production cell lines, such as HEK293, have served as convenient expression hosts for proteins with specific requirement for human post-translational modifications17.The production of protein therapeutics is a fast-growing field as it allows for the generation of sophisticated molecules with high specificity and activity in humans19. The expression of E1A and E1B enable continuous culturing of HEK293 cells by inhibiting apoptosis and interfering with transcription and cell cycle control pathways20. In addition, E1A and E1B are essential helper factors for adeno associated virus (AAV) production, which makes HEK293 cells attractive production hosts for recombinant AAV particles21. HEK293 cell lines have been reported to have a pseudotriploid genome with the adenoviral DNA inserted on chromosome 1923. The organization of the HEK293 genome is continuously evolving through the events of chromosomal translocations and copy number alterations, suggesting that long-term cultivation and subcloning of cells result in karyotypic drift24. Such abnormalities and genomic instability is, however, characteristic for immortalized cells and have also been reported for CHO cells28.The human cell line HEK293 is the most commonly utilized human cell line for expression of recombinant proteins for a multitude of research applications. This cell line originate from the kidney of an aborted human female embryo and was originally immortalized in 1973 by the integration of a 4 kbp adenoviral 5 (Ad5) genome fragment including the E1A and E1B genes, at chromosome 1917. Two examples are 293T29 and 293E31 cell lines, constitutively expressing the temperature sensitive allele of the large T antigen of Simian virus 4029, or the Epstein-Barr virus nuclear antigen EBNA1, respectively31. In addition, several HEK293 cell lines have been adapted to high-density suspension growth in serum-free medium34, enabling large-scale cultivation and bioproduction in bioreactors24. Two industrially relevant suspension cell lines are 293-F and 293-H , which both enable fast growth and high transfectivity in serum-free medium. In addition, the 293-H cell line, which was originally derived from a more adherent HEK293 cell clone, shows strong adherence during plaque assays. Despite extensive usage of CHO and HEK in both suspension and adherent mode and several empirical protocols for adaptation in either direction, molecular knowledge of the key genes involved in the transition between the two growth states are limited. While adherent cells have traditionally been widely used for the production of viruses, e.g. AAV and lenti virus for clinical research, suspension growth is the platform of choice for bioproduction of therapeutic proteins. Whereas certain experimental steps are more efficient in adherent mode, e.g. chemical transfection and viral infection, the ability to increase the volumetric cell density by growth in suspension without cell clump formation, which results in oxygen limitations, is a key step from a manufacturing perspective.Several HEK293 cell lineages have been established from the parental HEK293 lineage with the objective to improve recombinant protein production and are used for the production of therapeutic proteinsEven though different HEK293 strains have all been derived from the same original cell line, significant genomic and transcriptomic changes between parental and progenitor cell lines can be expected due to the genomic instability of HEK293 as discussed above. Here, we present a genomic and transcriptomic analysis of the HEK293 parental cell line along with five widely used HEK293 derivatives. An overall analysis of the differences in genomic landscape and transcriptomic profiles was performed in order to provide novel molecular insights into the differences between cell lines that have occurred during the process of clonal isolation and expansion. Furthermore, we focus on transcriptomic differences between adherent and suspension HEK293 cells and the impact of the differentially expressed genes on metabolic pathways and the phenotype of the cells from a bioprocess perspective.In this study, six industrially relevant HEK293 cell lines Fig. a were su18, which includes the E1A and E1B genes. In this study, overall high mRNA levels of E1A and E1B were observed in all HEK293 cell lines  by geneticin (G418) selection. The pRSVneo plasmid contains a truncated version of the Large T gene (according to the AddGene vector Database), which aligns perfectly with the truncated Large T sequence found in the 293E genome  gene, had a log2-fold copy ratio of < -1 in 293E, 293-F and Freestyle 293-F cell lines compared to the parental HEK293  and suspension (293-H and 293-F) progeny cell lines showed significant expression differences of similar gene sets as in the comparison between progeny cell lines and the parental HEK293 Figs. . For ins35, was used to generate a set of metabolic genes and their assigned pathways to find metabolic pathways with altered expression between adherent and suspension HEK293 progeny cell lines. As shown in Fig. In order to evaluate what metabolic impact the differentially expressed genes may have on cells in suspension compared to the adherent state, a generic human metabolic model, HMR236, predicted the cholesterol biosynthetic process pathway as the cellular pathway most affected by this expression variation  of all the six cell lines  have previously been shown to have a role in the epithelial to mesenchymal transition (EMT) pathway38 were analyzed. Principal component analysis of these cell lines resulted in clustering of suspension cell lines in a distinct group separated from adherent cell lines  had significant different expression levels between adherent and suspension cell lines with p-values < 0.01  were found to only maintain half the number of FH gene copies compared to the original HEK293 , which has previously been associated with different cancers44, was observed in all progeny cell lines except 293-H compared to HEK293  gene copies. The loss of FH copies was previously observed for HEK293 by Lin and coworkers and was suggested to play an important part in the transformed phenotype of the cell line45. Notably, DSG2 and DSC2 are located in the region on chromosome 18 that had gained genomic copies in all progeny cell lines except 293E compared to the parental strain 48. As suspension cell lines are cultivated under serum free conditions, the increased expression of genes associated with for instance cholesterol in suspension cell lines may be a result of a lower cholesterol content in the medium. However, as cholesterol is a major component of the cell membrane and has an important function for membrane structure and cell signaling49, the differential expression of genes associated with cholesterol synthesis and metabolism may also be of importance for the different morphologies between adherent and suspension HEK293 cells. Indeed, previous studies have shown that cholesterol plays a critical role in regulating the formation of cell-to-cell interactions in endothelial cells50 and that depletion of cholesterol reduces cell adhesion and increases endothelial cell stiffness52. Increased cell surface stiffness has been reported for HEK293 cells in suspension compared to adherent state as a result of up-regulation and re-organization of the actin cytoskeleton53. This may partly be a result of altered cholesterol levels in the cell membrane since cholesterol is a regulator of the actin cytoskeleton and cholesterol depletion has been shown to induce actin polymerization54. Interestingly, the Insulin-induced gene 1 protein (INSIG1), which was up-regulated in suspension compared to adherent HEK293, is a negative regulator of cholesterol synthesis and important for cholesterol homeostasis48 and knockout of INSIG1 has previously been shown to result in cholesterol accumulation55. Notably, a lower cholesterol biosynthesis in suspension cell lines compared to the original HEK293 strain was indeed predicted using IPA  that were consistently differentially expressed between adherent and suspension HEK293 have previously been annotated to play a role in epithelial to mesenchymal transition (EMT)38 , an enzyme responsible for the covalent cross-linking between elastin and collagen in the extracellular matrix, has been shown to be important for cell–matrix adhesion formation, supporting the adherent phenotype of adherent cells but is also associated with cell invasion and induction of EMT63. Besides the EMT-related genes, the additional three genes , consistently up-regulated in adherent cells compared to suspension cell lines, have previously been associated with increased cell adhesion through the retinoic acid signaling pathway67. In line with this, retinol metabolism was found to be down-regulated in suspension cells in the metabolic gene set analysis , with differential expression between all adherent and suspension comparisons of HEK293 Fig. a, were sOur study has outlined the genomic and transcriptomic variations between six industrially relevant HEK293 cell lines, in an attempt to improve the understanding of their respective differences in phenotype. We report a selective pressure to develop certain expression profiles during the evolution and continuous cultivation, evidenced by the numerous genes and pathways detailed here. The key common changes between HEK293 and its progeny cell lines involve in particular cell membrane proteins and processes related to cell adhesion, motility and the organization of various cellular components such as the cytoskeleton and extracellular matrix. In addition, changes associated with differences between adherent and suspension cell growth in particularly involve changes in cell adhesion protein expression, cholesterol metabolism and a set of six key genes  with potentially key roles in the differentiation between the two groups. These results could be of importance when pursuing further cell line engineering or bioprocess optimization of these and other human cell lines.2 in air. Suspension cell lines 293-F, 293-H and Freestyle 293-F (Gibco) were obtained from Thermo Fisher Scientific and cultivated in 293 SFM II medium (Gibco) supplemented with Glutamax at a final concentration of 4 mM (Gibco). Suspension cells were cultivated in 125-ml Erlenmeyer shake flasks (Corning) at 37 °C and 120 rpm in a humidified incubator with 8% CO2 in air. All cells were propagated from frozen stocks for no longer than 20 passages.The adherent cell lines HEK293 (ATCC-CRL-1573), HEK293T (ATCC-CRL-3216) and 293E (ATCC-CRL-10852) were obtained from ATCC and propagated in DMEM (D6429) supplemented with 10% FBS at 37 °C in a humidified incubator with 5% COAdherent cells were detached by trypsinization and both adherent and suspension cells were harvested by centrifugation. Genomic DNA was extracted using the Blood and Cell Culture DNA Mini Kit (Qiagen) according to the manufacturer’s guidelines and concentrations were determined by using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). Genome sequencing was performed at the National Genomics Infrastructure  using the Illumina HiSeq X platform. For RNA extraction, cells grown in log phase from three biological replicates were collected (derived from successive propagations). Cell pellets were resuspended in RNAlater Stabilization Solution (Invitrogen) according to the manufacturer’s recommendations until RNA extraction. Total RNA was extracted from three replicates of each cell line using Qiagen’s RNeasy plus Mini Kit according to the manufacturer’s instructions. Concentrations were determined with a NanoDrop ND-1000 spectrophotometer and RNA quality was assessed on a 2100 Bioanalyzer (Agilent Technologies) using RNA 6000 Nano chips (Agilent Technologies). All samples had an RNA integrity number of at least 9.9. RNA sequencing was performed at GATC  using the Inview Transcriptome Advance service and an Illumina HiSeq instrument.68. The raw alignments were then deduplicated, recalibrated and cleaned using GATK 69. Quality control information was gathered using Qualimap (v2.2)70. SNVs and indels have been called using the GATK HaplotypeCaller71. These variants were then functionally annotated using snpEff (4.1) and snpEff reference GRCh37.7572. The Piper pipeline from the National Genomics Infrastructure was used73. The correlation between BAM files was assessed using multibamsummary and its plotCorrelation function from deepTools274. Spearman was used to calculate correlation coefficients between samples, and the clusters are joined with the nearest neighbor. The R package seqCAT75 was used to compare SNVs between samples, its compare_profiles function mode parameter was set to the default value “intersection”. The heatmap in Supplementary Fig. 76. To compare the Large T antigen sequences of 293T and 293E, unmapped reads were extracted to new bam files using SAMtools77, converted to fastq with BEDTools78, and de novo assembled with MEGAHIT79. NCBI BLAST was used to identify the Large T antigen in the assembled contigs. To evaluate and visualize copy number variations, CNVkit80 was used with its whole-genome sequencing method, cbs segmentation82 and the HEK293 alignment as reference. GO enrichment analysis of genes with high or moderate impact SNPs was performed using PANTHER classification system83.Genome sequencing reads were aligned to the reference (human_g1k_v37.fasta) using bwa (0.7.12)84 was used to quantify transcripts by pseudo-alignment based on human genome assembly version GrCh37. Log transformed normalized data by DESeq2 was used for cell line clustering and calculation of Euclidean distances of samples. The expression comparison of the viral elements was based on normalized counts from DESeq2. Significant testing of differential mRNA expression of E1A/B elements was done by Welch two sample t-test85. For differential expression analysis, raw count data from Kallisto was imported using the tximport package86 and analyzed with DESeq287. Wald tests were used to calculate p-values, and the BH method was used for multiple testing correction. Throughout the article a gene is considered differentially expressed if log2- fold change >  ± 1 and FDR < 0.05. In the differential expression analysis between adherent and suspension cells, all suspension cell-lines were compared to all adherent cell-lines, and additionally, all pairwise combinations between suspension and adherent cell-lines were evaluated. For evaluating differential expression of 38 common differentially expressed genes between adherent and suspension HEK293 cell lines in a set of 63 human cell lines, RNA-seq data from each cell line deposited in the HPA database was used. Based on the growth characteristics, cells were divided into two groups of adherent and suspension cells. A Mann–Whitney U-test was used to compare normalized counts based on library size between the two groups for each of the 38 differentially expressed genes40.Kallisto88. The adjusted p-values and fold changes from the differential expression was used in combination with a gene set collection based on “goslim_generic Biological Process”. The heatmap for the progeny cells lines vs. HEK293 was based on the consensus score calculated based on GO term rank aggregation in Piano for each directionality from all pairwise gene set statistics calculations with Wilcoxon rank-sum test. The heatmap for suspension cells  vs. adherent cells  was based on the consensus score from gene set statistics calculations with mean, median, sum, Stouffer and tailStrength tests and was calculated with Piano’s consensusHeatmap function. To produce the network plot, gene sets were exported from HMR289. For finding differentially expressed pathways of genes between adherent and suspension cell lines, we used the Wilcoxon statistical test and filtered gene sets with adjusted p-value lower than 0.05 as significantly changed. In addition, for gene set analysis of 38 common DE genes between adherent and suspension cell lines we used EnrichR and GO biological process as gene set collection36.To discover significant alterations of gene sets and metabolic pathways between HEK293 cell lines, the Piano package in R was usedIn order to predict the pathway changes between cell lines based on differentially expressed genes from pairwise comparisons, ingenuity pathway analysis  was performed. To consider a gene as differentially expressed we used log2 fold change > 1 or < 1 and adjusted p-value < 0.05. For filtering results of gene set analysis by IPA we used Benjamini–Hochberg multiple testing corrected p-values lower than 0.05 to find gene sets with a different expression pattern.Supplementary Tables.Supplementary Figures.


Surface phenotype and NanoString gene expression profiling indicated the closest steady-state counterpart of these cells to be monocyte-derived macrophages. In GVHD, however, there was upregulation of monocyte antigens SIRPα and S100A8/9 transcripts associated with leukocyte trafficking, pattern recognition, antigen presentation, and costimulation. Isolated GVHD macrophages stimulated greater proliferation and activation of allogeneic T cells and secreted higher levels of inflammatory cytokines than their steady-state counterparts. In HLA-matched mixed leukocyte reactions, we also observed differentiation of activated macrophages with a similar phenotype. These exhibited cytopathicity to a keratinocyte cell line and mediated pathological damage to skin explants independently of T cells. Together, these results define the origin, functional properties, and potential pathogenic roles of human GVHD macrophages.Myelopoiesis is invariably present and contributes to pathology in animal models of graft-versus-host disease (GVHD). In humans, a rich inflammatory infiltrate bearing macrophage markers has also been described in histological studies. In order to determine the origin, functional properties, and role in pathogenesis of these cells, we isolated single-cell suspensions from acute cutaneous GVHD and subjected them to genotype, transcriptome, and in vitro functional analysis. A donor-derived population of CD11c GVHD cohorts . Despite cohorts .Animal models previously demonstrated that immunocompetent donor myeloid cells enhance GVHD, without specifying a particular cell type , 11. MacIn humans, a number of reports highlight an increase in myeloid cells bearing macrophage markers, showing that the level of infiltration correlates with clinical severity and outcome , 20, 21.The role of (recipient) myeloid cells in responding to danger signals is integral to most models of GVHD, but it is not known whether human GVHD infiltrates bearing macrophage markers are recipient or donor in origin and immunogenic or antiinflammatory in activity.Although donor myelopoiesis usually dominates the peripheral blood compartment during GVHD, recipient dermal macrophages have very slow kinetics of turnover in humans  and pote+CD14+ myeloid cells with the genotype, phenotype, and transcriptional profiles of donor monocyte–derived macrophages. These cells have potent immunological functions that are likely to contribute to the pathogenesis of GVHD and may offer opportunities for therapeutic intervention.Here, we employ direct methods of isolation and testing to show that acute GVHD lesions in human skin are dominated by CD11chttps://doi.org/10.1172/JCI133909DS1). BMT control patients without GVHD were biopsied on median day 83 after transplantation . GVHD skin biopsies were taken at the onset of an acute onset erythematous rash, before initiation of therapy. Classical acute GVHD, immunosuppression withdrawal acute GVHD, and acute GVHD following donor lymphocyte infusion were all included. A pathological diagnosis of acute-type GVHD was confirmed by standard histological criteria in all cases, and patients with clinical or histological features of chronic GVHD were excluded. The median day of biopsy was day 53 . In situ analysis showed an increase in CD3+ T cells and CD11c+ myeloid cells in a perivascular and epidermal interface distribution in GVHD  low lymphocytes and HLA-DRescribed , 25. Surescribed . A relatthy skin . Myeloidthy skin –24, 26. +CD14+ myeloid cells were expanded more than 10-fold compared with healthy control skin or BMT skin without GVHD  and cDC1 , but showed upregulation of monocyte-associated antigens , CD11c+CD14+ GVHD cells were segregated with steady-state monocyte-macrophages and resident dermal macrophages, away from DC populations , recruitment (CCL24) and stimulation of lymphocytes (CD82), stimulation of proinflammatory cytokines (SPP1), and leukocyte extravasation (SELPLG)  . Differe(SELPLG) .+ cDC2, as described in GVHD skin , FCGR3A/B (Fc receptor/CD16), GNLY (granulysin), and IFN-response genes IFITM1 and GBP1  chemokine receptor-ligand pairs . The prohe blood .MRC1, CCR5, and PPBP, upregulated in GVHD macrophages in vivo, were also highly upregulated in MLR macrophages. MLR-activated macrophages also expressed cytotoxic molecules perforin, granzyme A, granulolysin, and TRAIL, similarly to GVHD macrophages . The cytokine milieu of an HLA-matched MLR was similar to that observed when GVHD skin was cultured , and a prophages . Many ofonocytes .The expression of cytotoxic molecules prompted us to test the possibility that MLR-activated macrophages might mediate cytotoxicity to epidermal cells. We observed that MLR-activated macrophages were directly cytotoxic to a keratinocyte cell line in vitro in a dose-dependent manner . In ordeIn this study, we have defined the role of myeloid cells in human cutaneous acute GVHD by characterizing mononuclear infiltrates from primary tissue, isolating the dominant myeloid cell, and defining its origin, transcriptional profile, and functional properties. The data indicate that human GVHD lesions are highly infiltrated with donor monocyte–derived macrophages with enhanced allostimulatory activity and the potential to mediate epidermal pathology.Myeloid cells found in GVHD have previously been characterized as macrophages based on histopathology describing a small number of surface antigens. These studies lack further details of the biological characteristics or potential pathogenic role of macrophages in GVHD , 20–22. Numerically, macrophages show the greatest fold increase in GVHD of any mononuclear cell and constitute the most consistent “cellular signature” of acute GVHD relative to those of recipients without GVHD or healthy control skin. The macrophage/DC ratio is sensitive and specific relative to BMT control skin without GVHD, increasing more than 100-fold in the presence of GVHD. Previous studies of human GVHD have placed emphasis upon the potential existence of a pathognomonic subset of T cells in GVHD, although none has been consistently identified –8. Unlik+CD14+ cells based on the most direct means of distinguishing the infiltrate from autofluorescent CD11c– resident macrophages by flow cytometry. Several lines of evidence point to a monocyte origin; most notably, they are donor derived and therefore unlikely to arise by proliferation of resident-recipient macrophages. Additional staining demonstrated high expression of monocyte antigens S100A8/A9 and SIRPA (CD172), consistent with recent emigration of monocytes from the blood . Biopsies were performed at the onset of patient rashes clinically compatible with GVHD. An independent clinical pathologist provided diagnosis and histological grading of GVHD. BMT recipient controls with no evidence of any rash were biopsied when seen for routine assessments at day 28 or day 100 after transplant. Biopsies were transported in serum-free medium  and analyzed within 24 hours. An independent clinical pathologist provided diagnosis and histological grading of GVHD in controls and GVHD biopsies. Healthy control skin was obtained from patients undergoing mammoplasty or abdominoplasty, as previously described , and 10% heat-inactivated FBS (Sera Lab). MLR macrophages were generated from coculture of PBMCs from HLA-matched BMT donor and recipient pairs. Recipient PBMCs were irradiated (20 Gy) and used as stimulators for donor PBMCs at a 1:1 ratio. Cultures containing 1 to 5 × 107 donor PBMCs were maintained in RPMI 1640 with 10% human AB serum (MilliporeSigma) for 7 days. HaCaT cells were obtained from Accegen.Unless stated otherwise, all cells were cultured in RPMI 1640 (Lonza) with 100 IU/mL penicillin, 10 μg/mL streptomycin, 2 mM Skin shave biopsies were used whole or split into dermis and epidermis by treatment with dispase 0.5–1.0 mg/mL in RPMI for 60 to 90 minutes at 37°C  before digestion in medium with 1.6 mg/mL type IV collagenase (Worthington) for 12 to 16 hours at 37°C in RPMI with 10% heat-inactivated FCS. Gentle dissociation and passage through a 100 μm filter generated single-cell suspensions.For some experiments, an enzyme-free preparation of leukocytes was required. Skin was cultured as above, but without collagenase. After 48 hours, migratory leukocytes were harvested from the supernatant. Supernatants were stored at –80°C and later used for cytokine analysis by Luminex assay (see below)Formalin-fixed and paraffin-embedded skin shave biopsies from GVHD diagnostic material were used. Sections of 4 μm were made. Antigen retrieval and staining were performed using the BenchMark autostainer (Ventana). CD3, CD163, CD11c, factor XIII, and Ki67 primary antibodies and the ultraView Detection Kit were used (Roche).Antibodies used for microscopy are listed in Antibodies used for flow cytometry are listed in Sorted cells were spun onto slides, fixed in methanol and acetic acid, and prepared with dual-labeled XY FISH probes on a ThermoBrite System (Abbott) in accordance with the manufacturer’s instructions.ASIP, C19orf59, CCL17, CD1C, CD207, CLEC10A, CLEC9A, CLNK, COBLL1, CXCL5, DBN1, F13A1, FGD6, FLT3, GCSAM, GGT5, MKI67, LPAR2, LYVE1, MAFF, MERTK, NDRG2, PACSIN1, PPM1N, PRAM1, S100A12, SIRPA, TMEM14A, UPK3A and ZBTB46). From 3000 to 10,000 sorted cells were pelleted and lysed in 5 μl RLT buffer (QIAGEN) plus 1% β-mercaptoethanol, yielding 50 to 150 ng total RNA for analysis. Hybridization was performed according to the manufacturer’s instructions using a NanoString Prep Station and Digital Analyzer. nSolver Analysis software,version 3.0, was used for background correction and normalization.Gene expression was quantified by NanoString, using the Human Immunology, version 2, panel with a custom 30-gene add-on targeting monocytes, macrophages, and DC genes  and labeled with 1 μM CSFE (Invitrogen). T cells were cocultured with sorted macrophages or DCs at a 25:1 ratio for 7 days, incubated with antibodies to CD3, CD4, CD8, HLA-DR, and CD69 , and anaE. coli (MilliporeSigma) and harvested at 10 hours. Supernatants were cryopreserved at –80°C and batch analyzed by Luminex assay  using QIAGEN LiquiChip 200 running Luminex 100 integrated system software, version 2.3. Procartaplex Analyst, version 1.0, was used to define standard curves.Quantification was performed on medium from isolated macrophage populations stimulated ex vivo, and medium was conditioned by explanted GVHD skin and by donor-recipient MLR. For macrophage stimulation, FACS-sorted skin macrophages were stimulated with 100 ng/mL LPS from 7 donor PBMCs were maintained in RPMI with 10% human AB serum (MilliporeSigma) for 7 days. Macrophages and T cells were sorted from MLR outputs as HLA-DR+CD14+CD11c+ cells and SSCloCD3+ cells, respectively. Shave biopsies of recipient skin were taken before BMT conditioning. Biopsies were divided and incubated with medium alone (negative control), 2 × 105 MLR lymphocytes, or 2 × 105 MLR macrophages for 3 days in RPMI 1640 with 20% heat-inactivated autologous serum. Explants were fixed in 10% buffered formalin, sectioned, and stained with H&E. Severity of histological damage was graded using the Lerner scale by an experienced assessor blinded to experimental conditions.Blood from BMT donor and recipient pairs was prospectively collected into EDTA before transplantation. Recipient PBMCs were irradiated (20 Gy) and used as stimulators for donor PBMCs at a 1:1 ratio. Cultures containing 1 to 5 × 10t tests were performed in MultiExperiment Viewer software, version 4.8 ; or Newcastle Biobank, Newcastle and North Tyneside Research Ethics Committee 1 (reference 17/NE/0361).LJ, MC, and AJS conceived the project. LJ, MC, VB, and SP devised methodology. LJ, UC, MG, KG, GR, SP, MP, CAL, AKL, and VB performed experiments. LJ and XNW performed formal analysis. MC and LJ wrote the original draft of the manuscript. MC and LJ revised and edited the manuscript. LJ, MC, and AJS acquired funding. MC, EH, AP, SN, GHJ, VB, GR, and MH provided resources. MC and AJS supervised the project.


Hsp104 is a hexameric AAA+ yeast disaggregase capable of solubilizing disordered aggregates and amyloid. Hsp104 couples ATP hydrolysis to polypeptide translocation through its central channel. Substrate binding by Hsp104 is mediated primarily by two conserved tyrosine residues in nucleotide binding domain (NBD) 1 and NBD2. Recent structural studies have revealed that an additional tyrosine residue (Y650) located in NBD2 appears to contact substrate and may play an important role in Hsp104 function. Here, we functionally analyze the properties of this proposed Hsp104 –substrate interaction. We find that Y650 is not essential for Hsp104 to confer thermotolerance. Supporting these findings, in a potentiated Hsp104 variant background, the Y650A mutation does not abolish potentiation. However, modulation of this site does have subtle effects on the activity of this potentiated Hsp104 variant. We therefore suggest that while Y650 is not essential for Hsp104 function, its modulation may be useful for fine-tuning Hsp104 properties. Protein disaggregases engage and dissolve proteins that aggregate and form amyloid. Protein aggregation is implicated in a range of diseases, and so it is important to understand the structure and mechanism of disaggregases. Hsp104, a member of the Hsp100 family, is a hexameric AAA+ protein disaggregase from yeast that is highly conserved in bacteria and eukaryotes, though it is absent in metazoa. Hsp104 assembles into a ring structure with a central channel through which substrate is threaded. ATP hydProtein misfolding underpins numerous disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), and diabetes, 11. BecC. elegans model of PD[To enhance Hsp104 activity, efforts have been made to tune Hsp104 to boost its activity and modulate its substrate-specificity–23. Thesdel of PD. A majordel of PD, 19, 20.H641A showed a loss of ~50% activity relative to ClpBWT[In order to apply rational protein design principles to further engineer Hsp104, it is important to comprehensively understand its structure and mechanism. Recently, a series of near-atomic level resolution cryo-electron microscopy (cryo-EM) structures of Hsp104 were reported. These sto ClpBWT. Howeverto ClpBWT. It is tHere, to investigate the functional role of this putative substrate binding region, we employed a series of yeast-based assays. Using a combination of thermotolerance, temperature sensitivity, and disease-substrate toxicity assays, we find that removal of the tyrosine 650 side chain from this pore loop does not ablate Hsp104 function as occurs when the canonical pore loop residues are mutated. However, the Y650A mutation does subtly modulate the function of a potentiated Hsp104 variant. Our results suggest that modulation of this site may prove useful for tuning Hsp104 activity.Fig 1)[Fig 1)[Substrate binding by Hsp104 is mediated primarily by Y257 and Y662, located in nucleotide binding domain 1 (NBD1) and NBD2, respectively. These 12 residues project into the central channel of Hsp104 to bind substrate and mediate its translocation via a ratchet-like mechanism , 28. An Y650A would display impaired thermotolerance. We constructed Hsp104Y650A, Hsp104Y662A, and Hsp104Y257A-Y662A in W303Δhsp104 yeast with Hsp104 expression under the control of the heat-shock element (HSE), which is present in the promoter region of Hsp104. This promoter allows low basal expression of Hsp104 that is significantly enhanced following stress. Following a brief pre-treatment at 37°C to induce the heat shock response and enhance Hsp104 expression, we exposed the cells to a severe heat shock at 50°C for 0 to 30 minutes. Following outgrowth, we noted that the strain expressing Hsp104Y650A displayed thermotolerance nearly identical to that of Hsp104WT. Strains expressing Hsp104Y662A and Hsp104Y257A-Y662A did not confer thermotolerance, and displayed growth similar to the vector strain, which does not express Hsp104 . We confirmed expression of each variant via immunoblotting, and observed that the expression pattern was consistent with Hsp104 expression being driven by the HSE promoter . Our findings demonstrate that residue Y650 plays little role in conferring thermotolerance, suggesting that if the Y650 residues contact substrate, this interaction is not essential for maintaining substrate remodeling activity.Upon exposure to environmental stress, Hsp104 expression is upregulated to broadly counter protein aggregation and promote cell survival, thereby conferring thermotolerance, 7. FollY257F-A503V-Y662F, in which the Y257F and Y662F pore loop mutations are introduced into the potentiated Hsp104A503V background, still rescues the toxicity of TDP-43, FUS, and α-syn[A503V. Hsp104Y257F-A503V-Y662F confers thermotolerance at levels similar to Hsp104WT, while Hsp104A503V is impaired in thermotolerance. Additionally, Hsp104Y257F-A503V-Y662F confers diminished temperature sensitivity relative to Hsp104A503V. Enhanced thermotolerance and diminished temperature sensitivity in yeast likely reflects decreased off-target effects and suggests that modulation of the Hsp104 pore loops is a viable way to tune disaggregase properties[Potentiated Hsp104 variants confer a therapeutic gain of function and robustly rescue the toxicity of TDP-43 and FUS (implicated in ALS) as well as α-synuclein (implicated in PD), 29. It and α-syn. Howeverroperties.A503V background: Hsp104A503V-Y650A, Hsp104A503V-Y662A, and Hsp104Y257A-A503V-Y662A. We chose to introduce alanine rather than phenylalanine mutations, anticipating that this strategy would more robustly demonstrate if these residues played a role in substrate binding. While a Y650F mutation would likely maintain a rescue regardless of the role of Y650 in substrate binding, ablating Y650 via mutation to alanine should impair the capacity of Hsp104A503V to confer a rescue if Y650 binds substrate. We employed the 416GAL vector to drive galactose-inducible expression of the genes, and we co-expressed these variants with TDP-43, FUS, and α-synuclein. Potentiated Hsp104A503V robustly rescued toxicity of all three substrates, while Hsp104A503V-Y662A and Hsp104Y257A-A503V-Y662A did not rescue the toxicity of any of these substrates. Hsp104A503V-Y650A still strongly rescued toxicity, indicating that the variant retained substrate binding, and supporting our results in the thermotolerance assays. However, we did note a modest decrease in rescue of TDP-43 toxicity when compared to Hsp104A503V. Here the rescue decreases from a score of 3 for Hsp104A503V to a score of 2 for Hsp104A503V-Y650A . However, Hsp104A503V-Y650A shows only a subtle decrease in rescue of FUS and α-synuclein toxicity as compared to Hsp104A503V, and the average score for this variant is equal to that of Hsp104A503V against FUS and α-synuclein . We confirmed consistent expression of each of the Hsp104 variants and disease substrates using immunoblotting . Our findings suggest that while the Y650 residue is not crucial for mediating substrate remodeling, modulation at this position could be a useful way to tune the properties of potentiated Hsp104 variants.We therefore designed experiments to both corroborate our findings that Y650 does not drive substrate remodeling, and also test if modulation of Y650 might be a way to tune Hsp104 substrate-specificity. We constructed a series of variants in the potentiated Hsp104A503V background subtly diminishes the toxicity rescue conferred by Hsp104A503V, we decided to perform a final functional test to assess the temperature sensitivity of these variants. While Hsp104A503V confers thermotolerance nearly similar to Hsp104WT  [A503V also confers a temperature sensitive phenotype at 37°C, displaying impaired growth when Hsp104A503V is expressed during a prolonged heat stress treatment. This temperature sensitive phenotype is diminished for Hsp104Y257F-A503V-Y662F [A503V, as do the verified substrate binding residues Y257 and Y662. However, we found that Hsp104A503V-Y650A displayed a similar temperature sensitive phenotype to Hsp104A503V, while Hsp104A503V-Y662A and Hsp104Y257A-A503V-Y662A displayed diminished temperature sensitivity . These results support the idea that Y650 does not play a crucial role in substrate remodeling in contrast to the essential roles played by residues Y257 and Y662.Because the introduction of Y650A into the potentiated Hsp104 , 30, Hsp3V-Y662F , 18. We Y662A and Hsp104V663G are defective in thermotolerance[A503V background to generate Hsp104A503V-E648A and Hsp104A503V-K649A. We tested these variants for suppression of TDP-43, FUS, and α-Syn toxicity and found that, like with Hsp104A503V-Y650A, ablating the side chains of E648 and K649 only subtly modulates the potentiation of Hsp104A503V . Our findings suggest that while modulation of this pore loop region might be a useful way to tune the properties of Hsp104A503V, this pore loop region does not play a major role in Hsp104 –substrate remodeling.It has been demonstrated that residues neighboring key substrate-binding residues Y257 and Y662 play an important role in Hsp104 substrate binding. For instolerance. To asseY650A is fully functional in thermotolerance, indicating that Hsp104 can still remodel substrate without the Y650 side chain. To investigate the role of this site further, we tested Hsp104A503V-Y650A in toxicity suppression assays. We find that the Y650A mutation does not abolish activity, supporting our findings in the thermotolerance assays, but that it does subtly decrease the rescue of TDP-43, FUS, and α-syn toxicity by potentiated Hsp104A503V. Our findings are in contrast to recent work with the bacterial homolog of Hsp104, ClpB. ClpBH631A, in which this third pore loop is ablated, shows impaired activity in a luciferase reactivation assay[Amyloid and aggregate dissolution by Hsp104 is driven by substrate binding and translocation through its central pore. Binding is mediated via residues Y257 and Y662 leading to translocation via a ratchet-like mechanism[ion assay. These dhsp104 [All yeast were WT W303aΔ, ade2-1). Yeast w, ade2-1). pRS416G600nm = 1.5, serially diluted, and spotted in duplicate onto synthetic dropout media containing glucose or galactose. Plates were analyzed after growth for 2–3 days at 30ºC. Each experiment was repeated with three independent transformations.Yeast were transformed according to standard protocols using polyethylene glycol and lithium acetate. Hsp104 hsp104 yeast were transformed with the indicated pHSE-Hsp104 plasmid. Yeast were grown to saturation overnight at 30°C in glucose dropout media. Cultures were normalized to A600nm = 0.3 and grown in glucose dropout media for an additional 4h. Cultures were then normalized to A600nm = 0.6 and incubated at 37°C for 30 min. Cultures were then heat shocked at 50ºC for 0-30min and then cooled for 2min on ice. Cultures were serially diluted and spotted on glucose dropout media, and the plates were incubated at 30ºC for 2–3 days. Experiments were repeated with three independent transformations.W303Δhsp104 yeast were transformed with the indicated pRS416GAL-Hsp104 plasmid. Yeast were diluted and grown in synthetic raffinose medium overnight. Cultures were grown in synthetic raffinose medium to A600nm = 2.0 and spotted onto SD-Ura or SGal-Ura media and incubated at 30ºC or 37°C. Plates were analyzed after 48-72h of growth. Experiments were repeated with three independent transformations.W303Δ600nm = 0.6, 3ml cells were harvested, treated in 0.1M NaOH for 5 min at room temperature, and cell pellets were then resuspended into 1x SDS sample buffer and boiled for 4min. Lysates were cleared by centrifugation at 14,000 rpm for 2min and then separated by SDS-PAGE , and transferred to a PVDF membrane. Membranes were blocked in Odyssey Blocking Buffer (LI-COR). Primary antibody incubations were performed at 4ºC overnight. Antibodies used: anti-GFP monoclonal (Roche Applied Science), anti-TDP-43 polyclonal (Proteintech), anti-FUS polyclonal (Bethyl Laboratories), anti-Hsp104 polyclonal (Enzo Life Sciences), and anti-PGK monoclonal (Invitrogen). Membranes were imaged using a LI-COR Odyssey FC Imaging system.Yeast were grown and induced in galactose containing medium for 5h from overnight cultures supplemented with raffinose. For plasmids under the control of the HSE promoter, cells were grown as for the thermotolerance assay and harvested after the incubation at 37ºC. Cultures were normalized to AS1 Supporting Information(DOCX)Click here for additional data file.


Here, we have investigated the differential expression of nAChR subunits in human breast cancer cell lines and breast epithelial cell lines at mRNA and protein levels and the effects of the αO-conotoxin GeXIVA, antagonist of α9α10 nAChR, on human breast cancer cells. Reverse transcription polymerase chain reaction (PCR) demonstrated that all nAChR subunits, except α6, were expressed in the 20 tested cell lines. Real time quantitative PCR (QRT-PCR) suggested that the mRNA of α5, α7, α9 and β4 nAChR subunits were overexpressed in all the breast cancer cell lines compared with the normal epithelial cell line HS578BST. α9 nAChR was highly expressed in almost all the breast cancer cell lines in comparison to normal cells. The different expression is prominent ( Cancer is one of the three principal diseases which lead to human mortality and was responsible for 9.6 million deaths worldwide in 2018 . Nicotinic acetylcholine receptors (nAChRs), the ligand-activated neurotransmitter receptors in mammals, are divided into muscle and neural nAChRs. The nAChRs are either homomeric or heteromeric pentamers and are comprised of α and non- subunits ,5,6,7,8.Conus generalis, which potently blocks α9α10 nAChR [As competitive antagonists of nAChRs, α-conotoxins (α-Ctxs), have been particularly useful in developing ligands that selectively target one nAChR subtype over another. Some of them have therapeutic potential ,23,24,2510 nAChR . In vivo10 nAChR ,28. Rece10 nAChR . αO-conoThe present study aimed to detect the differential expression of nAChRs in human breast cancer and human normal breast epithelial cells and the nAChR subunits that may be potential targets for breast cancer, to further evaluates the effects of αO-conotoxin GeXIVA on cellular proliferation.Previous studies demonstrated that nAChRs as ligand-gated ion channels were regulated by binding with acetylcholine (ACh) or nicotinic agonists, such as nicotine and nicotine-containing products . In addiThe LBD cDNAs for α3, α4, α5, α7, α9, α10, β2, β3 and β4 neuronal-type nAChR subunits were amplified with expected size respectively in all the 17 breast cancer cell lines and the three human normal mammary gland epithelial cell lines . It meanAs described above, all the tested cell lines were found to express neuronal nAChR subunits of α3, α4, α5, α7, α9, α10, β2, β3 and β4. Moreover, previous studies showed that nAChRs play a crucial role in the course of human cancer development ,12,32. IFor α5 nAChR subunit C, the hiCompared with normal cell line of HS578BST, α7 nAChR subunit had the highest expression level of mRNA in HCC1806 cancer cell line with ~600-fold higher than in the normal cell line D. The seFor α9 nAChR subunit E, the hiThe most significant upregulation for β4 nAChRs mRNA level was observed in the breast cancer cell line AU565, which had ~1500-fold more than the normal cell line HS578BST. There were three breast cancer cell lines, SK-BR-3, HCC1806 and MDA-MB-231 with 120~300-fold of β4 subunit mRNA higher than that of the HS578BST. Six breast cancer cell lines, MDA-MB-361, Bcap37, HCC1395, ZR-75-30, MCF-7 and HCC1937 showed 25~60-fold of β4 mRNA higher than that of the HS578BST. There was 2~10-fold increase in the amount of β4 nAChRs mRNA in MDA-MB-453, BT549, HS578T, BT483, BT20 and MDA-MB-157 cell lines compared to normal cell line HS578BST I.Compared with human normal mammary gland epithelial cell line HS578BST, there were 14 breast cancer cell lines expressing 2~20-fold higher levels of α3 nAChR subunit, including MDA-MB-453, BT549, HS578T, Bcap-37, HCC1395, BT483, SK-BR-3, BT20, ZR-75-30, HCC1806, AU565, HCC1937, MDA-MB-231 and MDA-MB-157. MDA-MB-361 and MCF-7 did not show significant changes in α3 nAChR expression from cancer cells compared to normal epithelial cells A. For α4 nAChR subunit B, the hiThe mRNA expression level of α10 nAChR subunit in five breast cancer cell lines was similar with the normal cell line respectively, which included MDA-MB-453, BT549, SK-BR-3, ZR-75-30 and HCC1937 F. The otCompared to normal cell line HS578BST, there were nine breast cancer cell lines including MDA-MB-453, Bcap37, HCC1395, BT483, BT20, ZR-75-30, HCC1806, MDA-MB-231 and MDA-MB-157 with expression of β2 nAChR subunit mRNA 4~25-fold higher than HS578BST G. There For β3 nAChR subunit mRNA expression H, there It is shown from quantitative PCR results that only four nAChR subunits that is, α5, α7, α9 and β4 nAChRs are highly expressed in all tested human breast cancer cell lines compared to normal mammary gland epithelial fibrocystic cells .p < 0.001) except MDA-MB-453 and HCC1395 cell lines ) and analyzed by flow cytometry (FCM). The specificity of these antibodies was confirmed by Western blotting in some cell lines, such as MCF-7, MAD-MB-157, MDA-MB-231, BT20, SK-BR-3, BT483, HCC1395, MDA-MB-157 α9 KO, HEK-293T, Hela, A549 . Take MDll lines .p < 0.01). There were three cancer cell lines, BT483, MDA-MB-231 and MDA-MB-157 cell lines that were statistically significant p < 0.05. In our study, the mRNA and protein expression differences for the α9 nAChR subunit in different human cancer cell lines have shown in the above results. Thus, further research is warranted to determine the expression differences of α9-containing nAChR protein in these cell lines by Western blot . Using aPrevious experiments have established that the over-expression of α9-nAChR at mRNA and protein level in almost all breast cancer compared to normal epithelial cells HS578BST  and αO-c50 values ranging from 35 to 130 μM. However, TxID had no effect on breast cancer cell line BT20. Co-treatment of BT20 cells with GeXIVA and TxID resulted in no significant difference of these cells to GeXIVA. Compared with normal cells treated with the same drug at the same concentration, the inhibition of cancer cells at any concentration of GeXIVA used was significantly higher than that of normal cells. The effects of different treatments on cells proliferation in breast cancer cells were determined by CCK-8 assay. In breast cancer cells, GeXIVA significantly inhibited the proliferation of cancers . AnalysiRecent gene expression profiling studies have categorized breast cancer into four groups including luminal subtypes A and B, HER2+/ER-, Basal-like ,33  belonged to HER2 over-expression subtype. Three cell lines  belonged to Luminal A subtype. Two cell lines (BT-474 and MDA-MB-361) fell within subtype of Luminal B. Twelve cell lines  fell within the Basal-like subtype. We designed the ten neuronal nAChR subunits gene specific primers and demonstrated for the first time that normal human mammary glands epithelial cells and mammary cancer cells express the α3, α4, α5, α6, α7, α9, α10, β2, β3, β4 neuronal nAChR subunits and examined the expression level of each subunit. All the existing neuronal nAChR subunits mRNA were analyzed by RT-PCR, which presented in almost all the cell lines except for α6 nAChR subunit . But, th2+ permeability [2+ influx into cancer cells and trigger membrane depolarization, which activates voltage-gated Ca2+ channels and subsequently activates the MAPK pathway, which may result in inhibition of apoptosis [p < 0.001) except MDA-MB-453 and HCC1395 cell lines  according to the manufacturer’s protocol. The concentration and purity of RNA were determined by NanoDrop 2000 spectrophotometry . Equal amounts of RNA (1 μg) from each sample were reverse-transcribed into first-strand cDNA with High Capacity cDNA Reverse Transcription Kit , then the first-strand cDNA subsequently amplified by RT-PCR using specific primers. Based on the reported mRNA sequence of ligand binding domain of human (homo sapiens) nAChR subunits, the specific primers for each subunit were designed , which w2O with total volume of 20 µL. The cycling conditions were 95 °C for 10 min  followed by 40 cycles of denaturation at 95 °C for 15 s, annealing at 58 °C for 40 s and extension at 60 °C for 60 s. To correct minor variations in mRNA extraction and reverse transcription, the gene expression values were normalized using the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Relative quantification of the mRNA level was computed by the comparative Ct (2−∆∆Ct) method [Quantitative real-time PCR (qRT-PCR) was performed using Analytikjena qTOWER3G Real-Time PCR system to amplify various nAChR subunits in each cell line, including α3, α4, α5, α7, α9, α10, β2, β3 and β4 nAChR subunits. Each qPCR reaction mix contained 5 µL of SYBR Green I Master , 2 µM of each primer, 1 µL cDNA and DNase and RNase-free HThe cultured cells were washed with cold phosphate buffer saline (PBS) for three times and harvested using a cell lysis buffer containing protease inhibitors PMSF . Equal amounts of protein from control and treated cell lysates were separated using a 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel under reducing conditions and transferred onto polyvinylidene fluoride (PVDF) membranes  that were subsequently probed with primary antibodies . In all Western blot experiments, membranes were additionally probed with an antibody for GAPDH  to ensure equal loading of protein among samples. Horseradish peroxidase-conjugated secondary antibodies  were used with enhanced chemoluminescence reagent  to visualize the protein bands. Images of the films are captured using the Alpha FluorChem E .A guava easyCyteTM flow cytometer with InCyte software  was used to validate the presence of the nAChRs. The cultured cells were harvested and washed with PBS for twice, fixed the cells with 4% PFA for 20 min, washed the cells twice by centrifugation at 1000 rpm for 5 min each time. Suspend the cells in 0.1% Triton X-100 in 1 × PBS buffer for 10 min and washed the cells twice with PBS by centrifugation at 1300 rpm for 5 min each time. The cells were blocked with 3% BSA in 1 × PBS for 30 min and then the cells were washed twice with PBS by centrifugation at 1300 rpm. Add primary antibody at an appropriate dilution and incubate for 2 h at room temperature, washed and incubated with secondary antibody (FITC-conjugated Goat anti-rabbit Ig (G + L)) for 1 h at room temperature. Finally, the cells were washed twice and re-suspended in 300 μL 1 × PBS and the expression of nAChRs was analyzed by flow cytometry. The changes of mean fluorescence intensities of each antibody were calculated and it represent the protein expression level of nAChRs in each cell line. The primary antibodies used were as follows—CHRNA3 Rabbit Polychonal antibody , anti-Nicotinic Acetylcholine Receptor α4 antibody , anti-CHRNA5 antibody , CHRNA6 Rabbit Polychonal antibody , anti-CHRNA7 antibody , CHRNA9 Rabbit Polychonal antibody , anti-CHRNA10 antibody , anti-nicotinic acetylcholine receptor β2 antibody , anti-CHRNB3 antibody  and CHRNB4 antibody . H-tetrazolium, monosodium salt (WST-8) assay kit . Exponentially growth cells were seeded onto 96-well plates (1 × 10 4 cells per well) and allowed to grow overnight. The cells were treated with five different concentration of αO-conotoxin GeXIVA . After 24 h, WST-8 was added into each well for 4 h before the measurement according to the manufacturer’s instruction. The absorbance 450 nm was measured by a microplate reader .The effect of αO-conotoxin GeXIVA on all tested breast cancer cell lines and normal cells HS578BST proliferation were examined by 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2t-test was used and a p-value < 0.05 was considered significant. Analysis was performed with the SPSS software package (Version 17.0).Three to five independent repeats were conducted for all experiments. Error bars represent these repeats. Statistical comparisons between groups were performed using ANOVA (Prism GraphPad Software and SPSS 17.0). A Student’s It is well known that the nAChRs are involved in processes of oncogenesis and inflammation. All the compounds mentioned above provided molecular evidence for the possible chemotherapeutic ability of them for smoking-mediated tumorigenesis through nAChRs pathway. Specifically, α-conotoxins as a common tool in the studies of nAChRs, which blocked various subtypes of nAChRs selectively and potently. In this article, expression of neuronal nAChR subunits in each cell line was delineated through different methods, which suggested α-conotoxins targeting various nAChR subtypes may display potent and additional therapeutic effects on related cancers. This study, the high expression of α9-nAChR in cancer cells was correlated with the pathogenesis. Our study has confirmed αO-conotoxin GeXIVA significantly inhibit growth of cancer cells compared with normal cells HS578BST in vitro. Due to its high potency, GeXIVA may possess great potential as anticancer agent for α9α10 nAChRs overexpressed breast cancer cell lines.