DLL3 might become a predictive immunohistochemical marker in small cell carcinoma of the lung (SCLC). We investigated the influence of pre-analytical handling of samples on the performance of DLL3 immunohistochemistry (IHC) using DLL3 SP347 ready to use assay (Ventana). DLL3 positive cell lines were subjected to different experimental conditions mimicking the pre-analytical variation in daily clinical practice. Formalin fixation of 24 h led to the most optimal results of DLL3 IHC. Longstanding fixation in Cytolyt, methanol-based fixative for cytology samples, but also decalcification using a mix of formic- and hydrochloracid resulted in decreased DLL3 staining. Postponed staining of blanc slides for 3 months also decreased DLL3 IHC. Postponed fixation of the SCLC cell lines did not influence the performance of DLL3 IHC, although this might be different in the tissues than in the cell lines. In conclusion, different pre-analytical variables decrease the performance of DLL3 IHC. These findings are essential for implementing novel predictive immunohistochemical biomarkers in daily pathology practice.The online version of this article (10.1007/s00428-020-02848-y) contains supplementary material, which is available to authorized users. DLL3 emerged as a potential target in small cell lung cancer (SCLC) from gene expression studies. DLL3 is a member of a Notch ligand receptor family and in normal cells inhibits the Notch pathway , 2 whichFrom the experience gained with PD-L1 as a widely used predictive IHC biomarker in pathology, it was recognized that preanalytical, analytical, and postanalytical variables can influence testing performance –8. Pre-aThe aim of this study is to examine the influence of different clinically relevant preanalytical variables on the performance of DLL3 IHC. We assessed the effect of different fixatives, fixation times, delay in fixation, and use of a decalcification procedure.Three cell lines were used: NCI-H69 [H69] (ATCC® HTB119™) human SCLC line reported to have a low DLL3 expression; NCI-H82 [H82] (ATCC® HTB175™) human SCLC line reported to have a medium DLL3 expression, comparable with that in patient SCLC samples and A549 (ATCC® CCL-185™); and human non-small cell cancer line previously reported to have virtually no DLL3 expression (negative control) .Three cell lines were separately grown under aseptic conditions in an incubator providing a humidified atmosphere of 5% CO2 in air. All cell lines were used between 3 and 9 passages after thawing to ensure complete revival and routinely tested for mycoplasma. All cell lines were cultured in RPMI base media supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, 1500 mg/L sodium bicarbonate, and 100 U/mL penicillin and 100 μg/mL streptomycin.After sufficient cells were cultured, H69 and H82 were centrifuged and subsequently objected to different conditions as described below. A549 was first treated with trypsin, and two times washed in PBS. All three cell lines were embedded in agar gel, processed in TissueTek Xpress X120 , and embedded in paraffin block.In Table In the following phase, postponed fixation was examined. The cells were centrifuged, washed, and left without bovine serum and without fixative for 3, 6, and 24 h on room temperature and 4 °C, respectively. Subsequently, cells were processed using the golden standard method which emerged from the previous step .Influence of decalcification was tested with two commonly used decalcification media:(1) 1:1 8 N-formic acid and 1-N sodium formate solution (pH 2.2), also known as Kristensen and (2) Sakura Reagent TDE30 decalcifier. In line with the clinical practice specimens were first fixated and subsequently decalcified. Different fixation times  in 10% buffered formalin were examined with subsequent longstanding decalcification for 24 or 48 h.Influence of postponed staining of blanc histological sections was examined, where the blancs were left on the room temperature or 4 °C for 1 week and 3 months, respectively.The whole series of experiments (including growing the cell cultures) was performed in duplo.From each block, 4-μm thick sections were cut and mounted on positively charged glass slides and stained using DLL3 ready-to-use assay , after pre-treatment with CC1 for 80 min, and using the OptiView detection kit (Ventana) on a Benchmark Ultra slide staining instrument (Ventana), following the manufacturer’s instructions.Staining intensity was scored using the H-score (range 0–300). In short, the percentage of cells at each staining intensity level  is scored, and H-score is calculated using the following formula: 1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+) .All slides were stained twice (in duplo staining) and scored by two investigators (in duplo reading).p value of < 0.05 was considered to be statistically significant.H-scores were summarized using means, medians, and standard deviations (SD). Statistical analysis was performed using R . Mixed-ep = 0.002) and the scores of two investigators . Since these differences were systematic and comparable in all experimental conditions  Fig. , most prp < 0.0001) with the interclass correlations between the H-scores within the conditions of 0.59 for H69 and 0.77 for H82.The differences between the 25 tested experimental conditions . Overall, DLL3 staining was lower after Cytolyt fixation compared with formalin in both cell lines in all conditions with the difference becoming more prominent after Cytolyt fixation of longer than 24 h .All three cell lines were subjected to fixation in 10% neutral buffered formalin and CytoLyt Fig.  for fourp = 0.0349 for H69; p = 0.59 for H82) but not to the level of the golden standard , although the differences were not statistically significant but showed a certain trend (p = 0.063). Interestingly, we noted a larger difference in the low-DLL3 expression cell line H69.The influence of postfixation in formalin was examined after the initial fixation in CytoLyt for three different times: 3 h CytoLyt followed by 24-h formalin  or the temperature .Cells were centrifuged, washed, and left without bovine serum and without fixative for 3, 6, and 24 h on room temperature and 4 °C followed by the golden standard fixation time of 24 h formalin . In contrast, decalcification in Sakura led to significant decrease in DLL3 intensity in all tested times . Moreover, we observed an artefactual positivity in the DLL3 negative cell line (A549) after Sakura decalcification of any duration  using the two decalcification methods (Kristensen and Sakura). We showed that decalcification in Kristensen did not lead to decrease in DLL3 staining intensity Fig.    decrease in staining intensity.Postponement of IHC of blanc slides left unstained of either 1 week or 3 months led to a decrease of the DLL3 staining intensity which was statistically non-significant (82) Fig. . The difThe influence of preanalytical variables on the staining intensity of DLL3 IHC revealed that formalin fixation of 24 h led to an optimal performance of DLL3 IHC staining and that longstanding fixation with Cytolyt as well as decalcification using Sakura Reagent TDE30 decalcifier resulted in essential reduction of DLL3 staining intensities.From what is reported in the literature, the diagnosis of SCLC is made on cytology samples in 15–31% patients as many patients have a broadly metastasized disease at the time of diagnosis . These pAntigen retrieval is an important step in the process of immunohistochemistry and is mainly based on heating induced protein modifications in order to release the antigen from methylene bridges formed between the proteins by formalin . Some anWe also demonstrated that type of decalcification can drastically decrease the performance of the IHC. Sakura is currently used in small biopsies for short decalcification (1–2 h) and contains a mix of formic acid and hydrochloric acid with pH of 0.1 . The KriIn our previous work, we demonstrated that postponed fixation of lung resection specimens resulted in a significant reduction of different IHC as cytokeratins, TTF1, but most importantly of PD-L1 . The proPostponed staining reduced the performance of the DLL3 staining irrespective of the temperature at which the blanc slides were kept. After the slides of 4 μm are cut and put on the glass, tissue is exposed to oxygen from the air. We hypothesize that oxidation of the proteins might lead to reduced staining, as described earlier . AlthougNotably, most differences between the conditions were observed in the H69 cell line, which has a low DLL3 epitope concentration. Samples with low epitope concentration  are considered optimal for validation of novel IHC tests for an equivalent outcome compared to the clinically validated test, because a slight variation in the test can lead to the altered outcome , 35. ThiIn conclusion, formalin fixation of 24 h led to the most optimal results of DLL3 IHC. Acceptable staining was reached already after 3 h of formalin fixation. Fixation in Cytolyt, longstanding decalcification using Sakura, and postponed staining of blanc slides led to decreased DLL3 IHC. It remains to be determined whether these differences will be clinically relevant when DLL3 is implemented in the clinical practice.Suppl Fig 1H-score variation between the experiments, plotted per cell line. Note the systematic differences between the two experiments. (PDF 5 kb)Suppl Figure 2Differences between the two raters (in duplo reading) were also systematic with a slight bigger differences in H69 (low DLL3 epitope concentration cell line). A, B. Plots of the mean H-scores of the two raters (each axis one rater). C,D. differences in H-score between two raters plotted against the mean H-scores between the raters. Note the systematic differences between the raters. (PDF 7 kb)Supplementary Figure 3When fixation was postponed for 3,6,24 hours (x-axis), there was no decrease of DLL3 staining found, irrespective of the temperature at which the cells were kept during this cold ischemia time. Red bars represent the DLL3 levels of both cell lines when golden standard of fixation is used . (PNG 120 kb)High resolution image (TIF 16905 kb)


GGT5 was highly expressed in cancer-associated fibroblasts (CAFs) in lung adenocarcinoma, predicting the unfavorable survival of patients with lung adenocarcinoma. Cell growth, foci formation and spheres formation analyses showed that cancer cell proliferation was attenuated under treatment with the conditioned media from GGT5-silenced CAFs. Moreover, high expression of GGT5 in CAFs enhanced the drug resistance of cancer cells by increasing intracellular glutathione and reducing the intracellular reactive oxygen species in cancer cells. In mouse xenograft model, we proved that targeting GGT5 with a small-molecule inhibitor GGsTop could inhibit tumor growth and increase the chemosensitivity of cancer cells. Taken together, our study illuminates that high level of GGT5 in CAFs contributes to cancer cell survival and drug resistance, indicating that GGT5 may be a promising therapeutic target in lung adenocarcinoma.Gamma-glutamyltransferase 5 (GGT5) is a member of the gamma-glutamyl transpeptidase gene family with the capacity of cleaving the gamma-glutamyl moiety of glutathione, but its role in cancer progression has never been revealed. In this study, we found that gene  Lung cancer is a highly aggressive neoplasm with an unfavorable survival . Non-smaGamma-glutamyltransferase (GGT) is a membrane-bound extracellular enzyme involved in the hydrolysis of gamma-glutamyl bonds of glutathione (GSH) and other gamma-glutamyl compounds. In animals, GGT is highly expressed in tissues with secretory or absorptive functions, such as kidney, intestine, liver, prostate and gallbladder . MoreoveAspergillus fumigatus induced asthma, GGT5 knockout mice have increased airway hyper-responsiveness [Gamma-glutamyltransferase 5 (GGT5), also known as GGT-like activity 1 (GGTLA1), is a member of the GGT protein family. Many tissue-local macrophages, such as Kupffer cells in the liver and alveolar macrophages, express GGT5 protein . GGT5 issiveness . These ein vitro and in vivo functional studies indicated that CAFs-derived GGT5 promoted cell proliferation and drug resistance by reducing the reactive oxygen species (ROS) level in LUAD cells, suggesting that GGT5 may be a promising therapeutic target for patients with LUAD.In the present study, we first reported that high expression of protein GGT5 in cancer-associated fibroblasts (CAFs) predicted the poorer survival of LUAD patients. Moreover, our GGT5 in human using Coexpedia indicated that GGT5 was closely associated with malignant cancer development, including lung cancer  suggested that both overall survival (P = 0.00054) and progression-free survival (P = 0.0099) of LUAD patients with high GGT5 expression  were significantly shorter than those with low levels of GGT5 expression   , 1F. TakGGT5 was high expressed in lung tissues from patients with idiopathic pulmonary fibrosis, a lethal disorder characterized by the aberrant accumulation of fibroblasts and myofibroblasts [GEO public cohort analysis (GSE2052) indicated that gene roblasts , than thGGT5 (siGGT5-M). First, the qPCR was used to confirm the knockdown of GGT5 by siRNA at mRNA level  was performed using Coexpedia. Analysis results showed that GGT5 was significantly related to cell differentiation and cell cycle regulation, hinting that GGT5 might mediate the crosstalk between CAFs and tumor cells . Hence, NA level . Resultsin vitro . Moreovein vitro  and sphein vitro  analysesin vitro. Results showed that down-regulation of GGT5 in CAFs impaired the chemoresistance of LUAD cells  markedly suppresses the tumor growth in nude mice xenograft model in A549 and LAC1 cells and does not affect the body weight of mice  and GGsTop  significantly inhibited the tumor growth . All samples used in this study were approved by the Committees for Ethical Review of research involving human subjects at the Sun Yat-sen University Cancer Center . LUAD cell line A549 was purchased from the American Type Culture Collection . The other LUAD cell line LAC1  were established in our laboratory , 37. AllIn brief, tissue slides with paraffin sections were deparaffinized by xylene, rehydrated using graded ethanol and rinsed with deionized water. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min at room temperature. For antigen retrieval, slides were high-pressure-treated and boiled in citrate buffer  for 15 min. Nonspecific binding was blocked with 5% normal goat serum for 30 min at room temperature. The slides were incubated with monoclonal antibodies against GGT5 , Ki67 , Fibronectin  or α-SMA  at 4°C overnight in a humidified chamber. For IHC staining, the slides were incubated with horseradish peroxidase-conjugated secondary antibody and stained with the DAB substrate. The representative figures of IHC staining were captured with light microscope . An immune-reactivity score system was applied as described previously . The perPrimary NFs or CAFs were established in our laboratory after tumor resection. In detail, fresh LUAD tissue and paired non-tumorous lung tissue were cut into as small pieces as possible by surgical scissors in sterile culture dish, respectively. And then the tissue homogenate was transferred into sterile tube supplemented with 1ml Liberase  for enzymolysis at 37°C for 30 min. The suspension was filtered through a sterile Cell Strainer  to collect a single-cell suspension. The filtrate was centrifuged at 1,500 rpm for 5 min at 4°C and washed twice with pure DMEM. The cell pellet was resuspended with 3 ml DMEM supplemented with 20% FBS and finally plated on 6-cm cell culture dishes. Based on the different adhesive capacity of fibroblasts and other cells, the unadherent cells (mainly tumor cells) were removed after culturing for 30 min at 37°C to obtain relative pure fibroblasts, because the adhesion time of fibroblasts is much shorter (~20-30 min) than tumor cells . Next, when the cell density reaches ~80% dish surface area, fibroblasts are further purified by short time digestion of trypsin (~30 s). The digested cells were cultured for further isolation of fibroblasts again. Finally, the pure fibroblasts are confirmed by immunostaining with antibody against Fibronectin .GGT5 in NFs or CAFs, full-length human GGT5 complementary DNA was amplified by reverse transcription-PCR and was cloned into pLenti6.3/V5 expression vector . For GGT5 interference assay, one small interfering RNA specifically targeting GGT5  and the scramble control siRNA was purchased from GeneCopoeia . GGT5 expression vector and control plasmids, siGGT5 and scramble control siRNA were transfected into fibroblasts using the HilyMax transfection reagent  according to the manufacturer’s instructions, respectively. The expression level of GGT5 was confirmed by quantitative real-time PCR (qPCR) using FastStart Universal SYBR Green Master (Roche) and a 96-well Real-Time PCR Detection System (Roche). ACTB was used as the internal control. The primers used were as follows: GGT5: 5'-GTCAGCCTAGTCCTGCTGG-3' (forward) and 5'-GGATGGCTCGTCCAATATCCG-3' (reverse); ACTB: 5'-CATGTACGTTGCTATCCAGGC-3'′ (forward) and 5'-CTCCTTAATGTCACGCACGAT-3' (reverse). The relative expression level (defined as fold change) of the target gene (2-ΔΔCt) was normalized to the endogenous ACTB reference (ΔCt).To study the function of 3 A549 cells were seeded in each well of a 6-well plate with fibroblasts-conditioned media. After a week of culture, surviving colonies (>50 cells per colony) were counted with crystal violet (Sigma) staining. Triplicate-independent experiments were performed.Briefly, 1×103 A549 or LAC1 cells were planted in each well of a 96-well plate, in which 10 μl CCK-8 was added to 90 μl of culture medium. And after 2 h incubation at 37 °C, the absorbance was measured at 450 nm. Triplicate independent experiments were performed.The Cell Counting Kit-8 (CCK-8) assay kit  was applied to measure the cell growth rate. A suspension of 1×10http://rsb.info.nih.gov/ij).Human paired NFs and CAFs were lysed with RIPA lysis buffer  supplemented with cocktail protease inhibitor (Roche). After boiling for 5 min, protein lysates were separated by 10% SDS-PAGE and then transferred to PVDF membranes . The membranes were blocked with 5% non-fat milk in TBS/Tween20  for 1 hour at room temperature, and incubated with primary antibodies against GGT5  and β-Tubulin  overnight at 4 °C. The membranes were washed three times with TBST buffer for 5 min per time, and then incubated with HRP-conjugated secondary antibodies for 2 hours at room temperature. The bands were visualized with the enhanced chemiluminescence , and the signals were quantified by ImageJ software  was used to determine the reduced GSH content in the different medium samples. γ-Glutamyltransferase Activity Fluorometric Assay Kit  was used to determine the GGT activity in fibroblasts or tumor cells. Experiments were performed with biological replicates.6 cells; LAC1: 4×106 cells) in 100μl phosphate buffered saline (PBS) were injected subcutaneously into nude mice. After tumor cells was injected, GGsTop  or Cisplatin  was administered intraperitoneally every two days for total five times. The length (L) and width (W) of tumor were measured every week by calipers, and tumor volumes were calculated as volume (mm3) = L × W2 × 0.5.All animal experiments were approved by Animal Ethics Committee at Sun Yat-sen University Cancer Center. Five-week-old female BALB/c nude mice were purchased from the Guangdong Medical Laboratory Animal Center (Guangzhou China). Different numbers of LUAD cells . Survival curves were obtained from Kaplan-Meier Plotter . Disease Ontology and Gene Ontology analyses were based on Coexpedia (http://www.coexpedia.org/). Results were considered statistically significant when P < 0.05.SPSS version 17.0  was used for all data analyses. An independent Student Supplementary Figures


The epidermal growth factor receptor (EGFR) is overexpressed by 80–90% of squamous cell carcinoma of head and neck (HNSCC). In addition to inhibiting EGFR signal transduction, cetuximab, a monoclonal antibody targeting EGFR can also bind to fragment crystallisable domain of immunoglobulins G1 present on natural killer (NK), causing antibody-dependent cellular cytotoxicity (ADCC). However, presence of cetuximab resistance limits effective clinical management of HNSCC.In this study, differences in induction of ADCC were investigated in a panel of ten HNSCC cell lines. Tumour cells were co-cultured with NK cells and monitored using the xCELLigence RTCA.While ADCC was not influenced by HPV status, hypoxia and cetuximab resistance did affect ADCC differentially. Intrinsic cetuximab-resistant cell lines showed an increased ADCC induction, whereas exposure to hypoxia reduced ADCC. Baseline EGFR expression was not correlated with ADCC. In contrast, EGFR internalisation following cetuximab treatment was positively correlated with ADCC.These findings support the possibility that resistance against cetuximab can be overcome by NK cell-based immune reactions. As such, it provides an incentive to combine cetuximab with immunotherapeutic approaches, thereby possibly enhancing the anti-tumoural immune responses and achieving greater clinical effectiveness of EGFR-targeting agents. Despite advances in conventional treatments, 5-year survival for metastatic HNSCC has remained only 40%.4 Therefore, innovative therapeutic strategies are necessary to increase survival outcomes and reduce substantial toxicities of conventional treatments.Head and neck squamous cell carcinoma (HNSCC) remains the sixth most common cancer type with incidence rates reaching nearly 750,000 cases/year.6 EGFR belongs to the human receptor tyrosine kinase family and signalling of EGFR regulates cellular processes involved in anti-apoptotic signalling, proliferation, differentiation, migration and angiogenesis.7 80–90% of HNSCC show overexpression of EGFR, making it a compelling drug target for inhibition.8 Two classes of EGFR inhibitors have been developed, namely the tyrosine kinase inhibitors (TKIs), including erlotinib and gefitinib, as well as monoclonal antibodies (mAbs) such as cetuximab and panitumumab.9 Nonetheless, although increased expression of EGFR is a negative prognostic factor and has been linked to poor prognosis in HNSCC patients,10 it has no clear predictive role for treatment with EGFR-targeted therapeutic agents.11Both targeted therapies and immunotherapies are now at the forefront of personalised cancer medicine. Aberrant signalling of the epidermal growth factor receptor (EGFR) plays an integral role in the tumorigenesis of multiple cancer types, including HNSCC.12 Clinical data of cetuximab showed improved overall survival (OS) in both locally advanced and recurrent/metastatic settings, when combined with radiation (RT) versus RT alone (median OS 49 vs. 29 months) or combined with chemotherapy (CT) versus CT alone (median OS 10.1 vs. 7.4 months), respectively.13 These results led to FDA approval of cetuximab for the treatment of locally advanced- as well as recurrent and metastatic- HNSCC. However, issues such as tumour heterogeneity and multiple resistance mechanisms have limited the observed response rates (RR) to only 13% for cetuximab monotherapy and 36% in combination with CT .14 Interestingly, besides its direct receptor blocking ability, both preclinical and clinical evidence have shown that cetuximab also induces immunologic anti-tumour effects.16 Cetuximab consists of an IgG1 backbone, through which it can bind CD16 fragment crystallisable (Fc) receptors located on natural killer (NK) cells, macrophages and granulocytes, of which NK cells have been proven to be the most potent effectors.17 Binding of the IgG1-Fc part of cetuximab to CD16 on NK cells triggers cytolytic activity called antibody-dependent cellular cytotoxicity (ADCC), which is predominantly mediated by perforin and granzymes.16 Furthermore, cetuximab has been shown to enhance cross-priming of cytotoxic T-lymphocytes via professional antigen-presenting cells, such as dendritic cells,18 mainly through induction of immunogenic cell death of tumour cells.19 These results confirmed the important immune-related mechanism of action of cetuximab, in addition to its receptor blocking effects.Cetuximab is a chimeric EGFR-blocking IgG1 mAb that binds the extracellular domain of EGFR, thereby preventing receptor activation and downstream signalling, as well as inducing internalisation.20 Furthermore, changes in oxygen level have been previously shown to affect NK cell activity as well.21 Together with the findings that hypoxia is frequently seen in most solid tumours,22 it is of crucial importance to take the hypoxic tumour microenvironment into account.Importantly, the tumour microenvironment (TME) plays a crucial role in the effectiveness of cancer therapies, e.g. through metabolic changes within the tumour cells. In this regard, we have previously shown that activation of hypoxia‐inducible factor (HIF) signalling is closely linked with EGFR-signalling, as HIFs are key survival factors under hypoxic conditions.23 we also determined whether ADCC was correlated with EGFR expression. Lastly, we investigated whether differences in ADCC could be associated with differences in EGFR internalisation  and/or HPV status in a broad panel of HNSCC cell lines. Furthermore, as we previously showed that EGFR expression within our panel of cell lines was highly cell line-dependent and was significantly altered under hypoxia,ion Fig. .Res) variants of the originally cetuximab-sensitive (CetSen) parental SCC22b and SC263 cell lines were generated as described previously (suffix R).24 As a control for vehicle exposure and an increased culture period, cells were also exposed to the vehicle control, i.e. PBS (suffix PBS). All other cell lines were previously shown to be intrinsically cetuximab-resistant (IntRes).23 To confirm permanent resistance, dose-response was re-assessed after culturing resistant cell lines for 6 weeks in absence of cetuximab.Ten human HNSCC cell lines with different sensitivities to cetuximab and HPV status were included. UM‐SCC‐104 and UM-SCC-090 were obtained from American Type Culture Collection  and Cal‐27 was obtained from Merck Millipore . SC263 and SQD9 were kindly provided by prof. Sandra Nuyts , and LICR‐HN1 and SCC22b were kindly provided by prof. Olivier De Wever . 93‐VU-147‐T was kindly provided by Dr. Josephine Dorsman . Acquired cetuximab-resistant . MEM was additionally supplemented with 1% non-essential amino acids . Cells were grown as monolayers and maintained in exponential growth in 5% CO2/95% air in a humidified incubator at 37 °C. All cell lines were confirmed free of mycoplasma infection through regular testing . Additionally, all cell line identities were validated through short tandem repeat profiling. Hypoxic conditions (1% O2) were achieved by the use of a Bactron IV anaerobic chamber (Shel Lab), as described previously.25 Hypoxic conditions were initiated immediately after addition of the treatment.All cell lines were HPV-negative, except for the HPV-positive 93‐VU-147‐T, UM‐SCC‐104 and UM-SCC-090 cell lines. All HNSCC cell lines were cultured in DMEM , except for the UM-SCC-090 cells, which were cultured in MEM . Base media were supplemented with 10% foetal bovine serum , 1% penicillin/streptomycin , and 2 mM +CD3− NK cells were obtained from PBMCs using a human NK cell isolation kit  according to the manufacturer's instructions. Purity of NK cell isolation was assessed on the Cytoflex flow cytometer (Beckman Coulter) using CD3-FITC  and CD56-PE  labelled mAbs. A cut-off of 80% CD56+CD3− NK cells was used to define an adequate isolation for further experiments. NK cells were resuspended in DMEM or MEM for further experiments.Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of healthy donors  using Lymphoprep density gradient centrifugation  and frozen in FBS containing 10% dimethyl sulfoxide  in liquid nitrogen (Nippon Gasses). For experiments, PBMC were thawed one day in advance in RPMI  supplemented with 10% FBS, [2 mM] L-glutamine, [1 mM] sodium pyruvate , 1% penicillin/streptomycin, and [1.5KU/ml] DNase I . CD56+ cells and these cells were subsequently sorted into CD16+ and CD16− populations using anti-human CD16 PE-conjugated mAb  on the FACSAria II (BD biosciences).Retrovirally transfected NK92 cells, expressing CD16, were kindly provided by Dr. Nadia Mensali  and maintained in X-vivo 10 medium supplemented with 5% human serum and [500U/ml] interleukin-2. The initial population of NK92 cells consisted of 86% CD1626 Cells were harvested using 0.05% Trypsin-EDTA  and counted using the TC20TM automated cell counter (Bio-Rad). Cells were seeded in a 96-well plate and left for overnight incubation. The following day, cetuximab [0–15 µg/ml] was added. After 48 h, cells were fixed with 10% trichloric acid and stained with 0.01% SRB followed by measurement of the optical density at 540 nm using the iMark microplate reader (Biorad).The growth inhibitory effects of cetuximab were assessed using the colorimetric sulforhodamine B (SRB) assay as described previously.27 Seeding density was optimised for each HNSCC cell line to ensure continuous growth until the end of the assay. Briefly, cells were harvested, counted and background impedance of the E-plate16 was measured before seeding of cells. After overnight incubation, cetuximab [2 µg/ml] or human immunoglobulin G1 kappa  , as an isotype control was added. Additionally, either cell-free medium or isolated NK cells were added to each well at an effector (NK cells) to target (tumour cells) (E:T) ratio of 5:1. The impedance was followed up by automated measurement every 15 min starting from cell seeding and ending 48 h after treatment with cetuximab both under normoxic and hypoxic conditions. Measurement of the impedance was expressed as Cell Index (CI). Each condition was performed in dublo.Cytotoxicity studies were performed using gold-coated 16-well plates (E-plate16) for the xCELLigence Real-Time Cell Analysis (RTCA) , as described previously.23 Briefly, cells were harvested, counted and stained with an EGFR PE‐conjugated antibody  or an isotype , as a negative control. Live/Dead Fixable Far‐Red Dead Cell Stain Kit (Thermo Fisher) was used to exclude dead cells. Samples were fixed in 4% formaldehyde for 10 min under normoxic and hypoxic conditions. Samples were measured on the FACSAccuri (BD Biosciences) and analysed through FLOWJO version 10.1 (TreeStar Inc.) The percentage positive cells (overton) and mean fluorescence intensities (ΔMFI) were calculated by subtracting the signal of the isotype from the signal of the sample.Previous experiments determined baseline EGFR expression in our panel of HNSCC cell lines through flow cytometry.2. Phase and red fluorescence channels were acquired with a 10× objective immediately after addition of Ab-Fab mix to cells and followed up for 24 h. Each condition was performed in triplo, with four images being captured and analysed per well, per time point. The total red object area (µm2/well) was quantified for each time point and normalised to the cell confluence (%) using the IncuCyte ZOOM software version 2018B.All HNSCC cell lines were seeded in 96-well plates and left overnight to adhere until a confluence of 40–50% was reached the next day. Cetuximab and an IgG1κ isotype were labelled with IncuCyte® Human FabFluor-pH Red Antibody Labeling Reagent  at a molar ratio of 1:3 (test Ab : labelling Fab), according to the manufacturer’s protocol. The intensity of the FabFluor reagent is pH-dependent. As such, a fluorogenic signal is observed as the Fab-Ab complex is internalised and processed via acidic lysosomes and endosomes. Ab-Fab mixes were added to cells at a concentration of [2 µg/mL] and plates were placed into the IncuCyte ZOOM Live-Cell Analysis System (Essen BioScience) inside an incubator at 37 °C and 5% COP-values < 0.05 were considered statistically significant.At least three independent experiments were performed for all experiments, with NK cells isolated from three different donors. Moreover, all conditions were plated at least in duplo. Results are presented as mean ± standard error of the mean (SEM). A linear mixed model was used to assess the influence of cetuximab sensitivity, HPV- and oxygenation-status on cetuximab-mediated ADCC and EGFR internalisation. A stepwise backward approach was applied, starting from a model with all fixed and random effects and their interactions. If the interaction term was not significant, a model with only the main effects was fitted. Significance between conditions was assessed using Tukey's multiple comparison. The possible correlation between EGFR expression, internalisation and cetuximab-mediated ADCC was evaluated through a pairwise Spearman’s rank correlation. All statistical analyses were conducted using SPSS statistics 25 (IBM) and JMP Pro 14 (SAS). 23 thereby classifying cell lines in CetSen, IntRes and AcqRes cell lines. To study cytotoxic effects of short-term exposure to cetuximab for 48 h (time point used in ADCC assays), we determined dose-response curves for the CetSen SCC22b-PBS, SC263-PBS cell lines and the AcqRes SCC22b-R, SC263-R cell lines  to growth inhibition by cetuximab compared to the corresponding isogenic CetSen cell lines. All other HNSCC cell lines were intrinsically resistant to cetuximab treatment (data not shown).We previously identified the sensitivity of our panel of ten HNSCC cell lines to cetuximab treatment for 168 h using the SRB assay,CD16-, NK92CD16+, respectively) (E:T = 5:1) to prove the attribution of ADCC in this setting   compared to cetuximab alone. Similar decreases were observed with addition of NK92CD16+ . However, addition of cetuximab and NK92CD16- cells to either SCC22b-PBS or SCC22b-R HNSCC cells did not result in a significant difference in cell survival compared to addition of NK92CD16- alone   and SCC22b-R  cell lines . These results confirm the minimal growth inhibitory effect of cetuximab after 48-h treatment and show increased NK-cell killing following cetuximab treatment can be attributed to interaction between cetuximab and CD16, inducing ADCC. Finally, these results also show that ADCC can potentially overcome cetuximab resistance.Next, using the xCELLigence RTCA system, we identified cetuximab-induced ADCC in two HNSCC cell lines (SCC22b-PBS and SCC22b-R) in co-culture with CD16-positive or CD16-negative NK92 cells  Fig. . Cetuxim81) Fig. . In contnes Fig. , compareSen cell lines SCC22b-PBS (82.91% ± 9.48) and SC263-PBS (62.23% ± 6.18) compared to isotype control (p ≤ 0.001). No decrease in survival was observed in the corresponding isogenic AcqRes clones (p > 0.201)  and UM-SCC-090  cell lines , except in the UM-SCC-104 . However, the addition of cetuximab plus NK cells from healthy donors to HNSCC cell lines induced a statistically significant decrease in cell survival in all ten HNSCC cell lines compared to NK cells alone   Fig. . Similarnes Fig. . In cont07) Fig. .Fig. 3HeSen cell lines with cetuximab [2 ug/ml] for 48 h showed a reduction in cell survival of 73.940 ± 8.40% and 69.59 ± 5.25% for SCC22b-PBS and SC263-PBS, respectively, compared to isotype control (p ≤ 0.004). Neither the IntRes nor AcqRes cell lines showed a significant decrease in survival following cetuximab treatment alone . Decrease in survival following co-culturing with NK cells under hypoxia was significant for the 93-VU-147-T and UM-SCC-104 cell lines  but not for the other HNSCC cell lines . Similar to normoxia, addition of cetuximab and healthy donor NK cells to HNSCC cell lines under hypoxia showed a significant induction of ADCC in all cell lines compared to isotype control   Fig. .p = 0.002), indicating a non-uniform effect of hypoxia on ADCC activity between the different groups of cell lines. Herein, hypoxia significantly reduced ADCC activity in the CetSen  and the AcqRes cell lines  but not in the IntRes cell lines   showed a significantly higher ADCC activity in the IntRes resistant cell lines compared to CetSen and AcqRes cell lines (p ≤ 0.007). Lastly, the influence of HPV status on hypoxia and cetuximab-mediated ADCC was assessed as well  and no significant differences in ADCC activity between HPV-positive and HPV-negative cell lines could be detected (p = 0.631).A multivariate linear mixed model was applied to define the role of cetuximab resistance, HPV status and/or hypoxia on ADCC activity. The fraction of ADCC was calculated through subtraction of the effect of cetuximab monotherapy Fig.  and used42) Fig. . Compariell Fig. . No signSen and AcqRes cell lines under hypoxia, while the IntRes cell lines maintain ADCC activity, compared to normoxia. Furthermore, we observed the highest induction of ADCC by healthy NK cells in the IntRes cell lines followed by the CetSen and AcqRes cell lines. Finally, HPV status had no significant influence on ADCC activity provoked by cetuximab treatment.Taken together, statistical analysis suggested that ADCC activity is reduced in the Cetp ≥ 0.143 and ΔMFI: p ≥ 0.170) and HPV status (overton: p ≥ 0.302 and ΔMFI: p ≥ 0.110).23 Under hypoxia, a significant increase in EGFR positivity  and ΔMFI  was observed for most cell lines.23 In this study, no correlation was found between EGFR expression and ADCC activity for each cell line  and under hypoxia . Thus, in our panel of HNSCC cell lines, baseline EGFR expression could not predict ADCC activity.Next, baseline EGFR expression levels were evaluated as a predictor of sensitivity to ADCC. Previously, a cell line-specific expression of EGFR positivity (16.59–80.54 ± 1.90–3.58) and ΔMFI (108.00–1150 ± 23.43–9.21) was observed. These data were shown to be independent of cetuximab sensitivity (overton: p < 0.001–0.050) compared to the isotype  compared to the AcqRes and IntRes cell lines (p = 0.069) . Interestingly, correlating EGFR internalisation with ADCC activity revealed a moderate correlation which showed a statistical trend   Fig. . In cont75) Fig. .Res cell lines, together with the positive correlation between both, our results indicate that a higher degree of internalisation might lead to a higher degree of cetuximab-induced ADCC.Based on these results, we suggest that ADCC activity is independent of EGFR baseline expression. Since both ADCC activity and internalisation were significantly higher in the in Int28 However, absence of durable responses (as a result of the development of resistance) is a limiting factor and a reason why long-term survival figures with this treatment are disappointingly low.29 In the present study, we show that cetuximab resistance induces a stronger ADCC response by healthy NK cells in a panel of HNSCC cell lines. Furthermore, we show that regulation of EGFR internalisation might possibly affect the degree of ADCC that can be induced. These findings may be promising for future combinations with immune-oncology drugs.The EXTREME trial showed that adding cetuximab to platinum-based CT not only improved response rate (36% vs 20%) but also median OS (10.1 vs 7.4 months) in patients with recurrent/metastatic HNSCC and led to its approval in that setting.23 had a limited effect on cell survival even at high concentrations. Interestingly, cetuximab treatment of the intrinsically cetuximab-resistant UM-SCC-104 cell line showed a significant decrease in cell survival after 48 h. However, this cell line was still classified as intrinsically resistant based on our previous published work. Herein, we showed that cetuximab treatment of the UM-SCC-104 cell line for 168 or 48 h had a similar decrease in survival.23 As opposed to this, 168-h treatment of the cetuximab-sensitive SCC22b-PBS and SC263-PBS cell lines showed a reduction of their mean survival well below 50%. This indicates that after the initial decrease, longer cetuximab treatment of the UM-SCC-104 does not decrease cell survival further and can be considered cetuximab resistant. A possible explanation for the time-dependent cytotoxic effect of cetuximab could be, as suggested by Narvi et al., that initial/short-term treatment with cetuximab mostly promotes cell cycle arrest, while a sustained inhibition of EFGR signalling initiates apoptotic signalling pathways.30The present study focused on short-term cetuximab treatment on HNSCC cell lines. Cetuximab treatment of cell lines previously identified as cetuximab sensitive,17 Interestingly, in comparison to other immune infiltrated tumour types, HNSCC tumours are marked by the highest levels of NK cell infiltration,31 showing the importance of NK cells in further investigations. Previous studies have demonstrated the ability of cetuximab to induce ADCC in various tumour types.33 Furthermore, inhibition of anti-inflammatory cytokines, such as TGF-β, or addition of pro-inflammatory cytokines, such as IL-2 and IL-15, enhanced cetuximab-mediated ADCC.34 However, knowledge regarding the effect of cetuximab sensitivity and/or HPV status on ADCC activity remains scarce. Therefore, in the present study we focused on determining ADCC activity of cetuximab in a panel of HNSCC cell lines with different cetuximab sensitivities and HPV status under both normoxic and hypoxic conditions. Addition of cetuximab to co-cultures of HNSCC cells with NK cells clearly induced ADCC in all cell lines. However, the extent of ADCC induced differed significantly among cell lines. In this regard, our study is the first to show that cell lines with acquired resistance against cetuximab are equally susceptible to cetuximab-mediated ADCC as their corresponding isogenic cetuximab sensitive cell lines. Surprisingly, intrinsically cetuximab-resistant cell lines were more susceptible to ADCC compared to sensitive and acquired resistant cell lines in our assay.Besides inhibition of EGFR signalling, cetuximab binds Fc receptors on NK cells.35 However, research investigating the possible effect of cetuximab sensitivity on ADCC induction has been limited. One mechanism described is the presence of an EGFR-K521 polymorphism, shown by Braig et al., which reduced the effectiveness of cetuximab binding to EGFR and thus reduced EGFR signalling and ADCC.36 To the best of our knowledge, the study of Braig et al. is the first to emphasise the unique immune-related effect of cetuximab resistance mechanisms.Considerable research has tried to elucidate cetuximab resistance mechanisms.37 others describe a correlation between EGFR expression and cetuximab-mediated ADCC.38 Clinical reports show discordant results as well40 and therefore EGFR expression is currently not a recognised predictive biomarker for cetuximab treatment in HNSCC. Similar results were observed for panitumumab, an IgG2 mAb against EGFR which functions exclusively through receptor inhibition.41 Together with our results, this suggests that (i) even low EGFR expression levels may be sufficient to induce maximal ADCC by NK cells, and (ii) the involvement of other biological mechanisms might be the cause for the discrepancy in literature and the difference in ADCC among our cell lines.We investigated two additional potential mechanisms, to unravel the complex interplay between ADCC and cetuximab sensitivity. First, we hypothesised that expression levels of EGFR might determine ADCC activity. However, we found no significant correlation between cetuximab-mediated ADCC and EGFR expression, both under normoxia and hypoxia. Other in vitro studies report varying results. While some studies do not observe a correlation,42 Interestingly, in contrast to ligand-bound EGFR internalisation, cetuximab-bound EGFR has an increased stability under endosomal pH levels and therefore is targeted for lysosomal degradation rather than recycling or trafficking to other cell organelles.43 Thus, binding of cetuximab leads to downregulation of EGFR, reducing the amount of available surface EGFR for ADCC induction. However, based on our data and those of Kol et al.,33 internalisation per se was not sufficient to impair ADCC induction. Therefore, we postulate that internalisation of EGFR is able to reduce—but not abolish—surface expression of EGFR. This has also been shown in colorectal cancer,44 and suggests that a minimal threshold for induction of ADCC exists and that this threshold is maintained following treatment with cetuximab. Moreover, our results showed that internalisation even increased ADCC effects. Further research investigating the mechanisms of internalisation within cell lines with different cetuximab sensitivities is necessary to improve therapeutic approaches considering NK cell-mediated ADCC through cetuximab.Second, to further investigate this matter, we determined internalisation of EGFR following treatment with cetuximab. Herein, one would expect a reduction in surface expression of EGFR to decrease the possibility of recognition by cetuximab and subsequently induction of ADCC as well. In our results, we showed that levels of EGFR internalisation varied between HNSCC cell lines and found a positive correlation between EGFR internalisation and ADCC. While receptor internalisation is known as a signal attenuation process, the idea that internalisation is required for activation of specific downstream transcription molecules is gaining interest.45 Others showed that levels of CD16 expression do not correlate with the intensity of ADCC,46 suggesting that hypoxia could abolish the natural cytotoxicity of NK cells but not ADCC. Our results are in line with these reports, showing that ADCC is maintained under hypoxia. However, while hypoxia did not affect ADCC sensitivity of acquired and intrinsic resistant cell lines, cetuximab-sensitive cell lines did become more resistant to NK cell-mediated ADCC. In this regard, hypoxia is known to modulate both proteomic and genomic changes within tumour cells.47 Our results suggest that modulation of these processes is different between cell lines with different cetuximab sensitivities and thereby differently influences susceptibility for ADCC. However, further research is warranted to elucidate the mechanisms behind the reduced ADCC within our cetuximab-sensitive cell lines compared to the acquired resistant and intrinsic resistant cell lines under hypoxia.Besides cetuximab resistance, we also investigated the role of hypoxia on ADCC. Previous studies have shown that hypoxia affects the functionality of NK cells. Both expression of the NKG2D and, albeit to a lesser extent, CD16 receptors as well as intracellular perforin and granzyme B were decreased under hypoxia.48 ADCC activity in HPV-positive cell lines was comparable to HPV-negative cell lines in our setting, showing only a statistical trend. Future studies with isogenic HPV-positive and -negative cell lines could provide more insight into the role of HPV. However, similarly to our results, a recent study in cetuximab-treated recurrent or metastatic HNSCC patients did not observe a relationship between HPV status and ADCC inducibility despite observing a better OS.15 In this regard, the prognostic benefit of HPV is suggested to be a result of a pre-existing immune response against HPV-positive cells.49 Studies have shown that HPV-positive HNSCC were associated with a higher number of intratumoural NK cells,50 which may partly explain the increased OS. However, as shown by Taylor et al., there was no difference in ADCC between HPV groups, taken into account the number of NK cells.15 Therefore, these results suggest that NK cells play a major role against HNSCC and that, in contrast to an increased number of NK cells, HPV status does not affect their activity. However, the mutational profile and molecular characteristics between HPV-positive and -negative HNSCC have been shown to differ as well, adding to the heterogeneous complexity of HNSCC and disparities in therapeutic responses.51Finally, although HPV is a known positive prognostic factor in HNSCC,In conclusion, our study presents novel insight into the impact of cetuximab resistance, HPV status and hypoxia on the efficiency of cetuximab-based tumour cell killing. We found that mechanisms of cetuximab resistance may enhance effector cell-based killing. Further research into these mechanisms could help overcome cetuximab resistance by combining immunotherapies with targeted therapies. Furthermore, we show that induction of ADCC is maintained, independent of surface EGFR expression levels and that internalisation of EGFR is closely linked with resistance to cetuximab. Further research into unravelling this mechanism will provide a better understanding of cetuximab-based therapies in the future.Supplemental material


A Trop‐2‐positive EC xenograft model was used to determine the in vivo activity of SG. Moderate‐to‐strong staining was detected in 84% (120/143) of EC samples, whereas 43% (3/7) of the primary EC cell lines tested overexpressed Trop‐2. EC cell lines overexpressing Trop‐2 were significantly more sensitive to SG compared to control ADC (P = 0.014 and P = 0.005). Both SG and the unconjugated parental antibody hRS7 mediated high ADCC against Trop‐2‐positive cell lines. Moreover, SG induced significant bystander killing of Trop‐2‐negative tumors cocultured with Trop‐2‐positive tumors. In the xenograft model, intravenous administration of SG twice weekly for three weeks was well tolerated and demonstrated impressive tumor growth inhibition against poorly differentiated, chemotherapy‐resistant EC xenografts (P = 0.011). In summary, SG is a novel ADC with remarkable preclinical activity against poorly differentiated EC cell lines overexpressing Trop‐2. These findings warrant future clinical trials.Endometrial cancer is the most common gynecologic malignancy in developed countries. The antibody–drug conjugate (ADC) sacituzumab govitecan (SG) targets trophoblast cell‐surface antigen‐2 (Trop‐2) – a cell‐surface glycoprotein highly expressed in many epithelial tumors – and delivers the active metabolite of irinotecan SN‐38 to Trop‐2‐positive tumor cells. We evaluated Trop‐2 expression in endometrial endometrioid carcinoma (EC) tissues and the activity of SG against primary poorly differentiated EC cell lines and xenografts. Trop‐2 expression was assessed in 143 formalin‐fixed–paraffin‐embedded tumors and seven primary tumor cell lines by immunohistochemistry and flow cytometry, respectively. Cell viability of primary tumor cell lines was assessed following exposure to SG, or control antibodies. Antibody‐dependent cell cytotoxicity (ADCC) against Trop‐2‐positive and Trop‐2‐negative EC cell lines was measured  TROP‐1 is overexpressed in the majority of endometrial cancers. Sacituzumab Gavitecan (SG) is highly effective, both in vitro as well as in vivo, against primary endometrial cancer cell lines and xenografts overexpressing TROP‐2. Clinical studies with SG in patients with recurrent, chemotherapy‐resistant endometrial cancer overexpressing TROP‐2 are warranted. ADCantibody–drug conjugateADCCantibody‐dependent cellular cytotoxicitycontrol ADCnontargeting control antibody–drug conjugateDARdrug‐to‐antibody ratioECendometrial endometrioid carcinomahRS7unconjugated monoclonal antibodyPBLsperipheral blood lymphocytesSGsacituzumab govitecanSCIDsevere combined immunodeficiencyTMAtissue microarrayTrop‐2trophoblast cell‐surface antigen‐21et al., Endometrial endometrioid carcinoma (EC) is the most prevalent gynecologic malignancy in the United States, with approximately 61 880 newly diagnosed cases and 12 160 deaths in 2019. Unlike ovarian and cervical cancer, the incidence and mortality rates from EC are globally increasing. If current trends continue, the incidence of EC in the United States will double by 2030  precisely target tumor cells as they are composed of a monoclonal antibody specific to surface antigens present on particular tumor cells and then kill these tumor cells with a highly potent chemical linker. ADCs may therefore optimize tumor targeting TACSTD2 gene on chromosome 1p32, which is differentially expressed in a variety of epithelial tumors  is a 45 kDa transmembrane glycoprotein encoded by the et al., et al., et al., et al., et al., et al., et al., et al., et al., Sacituzumab govitecan or IMMU‐132 (SG) is a novel ADC combining the humanized RS7 antibody targeting Trop‐2 coupled to a hydrolyzable linker that allows for a time‐dependent release of the payload, SN‐38, the active metabolite of irinotecan (7‐ethyl‐10‐hydroxycamptothecin) to the tumor tissue  approved this study, and patient consent was obtained per institutional guidelines prior to tissue collection. The EC cell lines were established from fresh tumor biopsy samples and de‐identified as described previously from our group (El‐Sahwi 2.2n = 143). Representative areas from primary EC were selected in hematoxylin/eosin‐stained preparations by a trained gynecologic surgical pathologist, and 0.6 mm cores were obtained and arrayed in a recipient block. In order to increase representation and capture possible marker heterogeneity, two cores were obtained from different areas of each tumor were included in the TMA samples. Sections of the resultant TMA samples were cut at 5 μm and then transferred to glass slides for further histologic staining and processing. The tissue samples were collected with either specific consent or a waiver of consent under an approved Yale Human Investigation Committee protocol. Next, purified goat polyclonal antibody against the recombinant human Trop‐2 extracellular domain  was applied for 1 h. After that, a secondary biotinylated anti‐goat antibody  and the streptavidin–biotin complex  were applied. Subsequently, 303‐diaminobenzidine (Dako) was used as a chromogen and the sections were counterstained by hematoxylin (Dako). For all of the above, appropriate positive and negative controls were used. The percentage of tumor cells with membranous Trop‐2 immunoreactivity was estimated, and the staining intensity was measured semiquantitatively using the following scale: 0 for no staining; 1+ for weak; 2+ for moderate; and 3+ for strong staining. In order to obtain the final immunoreactivity score, staining intensity  was multiplied by the percentage of positive tumor cells. The final immunoreactivity score was then classified into four ordinal categories: 0–9 negative (score 0), 10–99 weak (score 1), 100–199 moderate (score 2), and 200–300 strong (score 3).A retrospective cohort of stage I‐IV endometrioid EC tissue microarray (TMA) format samples were used in this study 2 immunoglobulin (FITC) that was used as a secondary reagent . The data were acquired using cell quest software . Mean fluorescence intensity (MFI) was evaluated using cell quest and prism 8. Cell lines with MFI greater than 100 were determined to have 3+ expression of Trop‐2 and with MFI of 51–100 2+, 21–50 1+, and 20 or less were considered negative for Trop‐2 expression.Flow cytometry to determine Trop‐2 expression was performed on primary poorly differentiated EC cell lines that were cultured 2.4m stock solution for the in vitro experiments. Drug‐to‐antibody ratio (DAR) of SG and control ADC was 6.78 and 6.84, respectively. For the in vitro experiments, the dosage of the drug was adjusted according to the DAR, in order to expose cells treated with SG and control ADC to equivalent quantities of SN‐38. For in vivo experiments, SG and the control ADC were reconstituted in sterile 0.9% sodium chloride as a 5 mg·mL−1 solution. hRS7 IgG (molecular weight: 150 kDa) was obtained in liquid form from Immunomedics, Inc., as a 10 mg·mL−1 solution.Sacituzumab govitecan (hRS7‐CL2A‐SN‐38), a nontargeting control ADC (h679‐CL2A‐SN‐38), and unconjugated monoclonal antibody hRS7 IgG were obtained from Immunomedics, Inc. . Lyophilized SG and control ADC were dissolved in sterile 0.9% sodium chloride as a 2 μ2.551Cr) release assays were performed in order to measure the cytotoxic reactivity of Ficoll–Hypaque‐separated peripheral blood lymphocytes (PBLs), in combination with the drug SG, the control ADC, and the hRS7 IgG against the EC cell lines at an effector to target ratio (E : T) of 5 : 1 and 10 : 1. The release of 51Cr from target cells was measured as evidence of tumor cell lysis after exposure of the tumor cells to a concentration of 2.5 µg·mL−1 of SG, the control ADC, or hRS7 IgG. Negative controls for this experiment included tumor cells incubated with PBLs and no ADCs. The positive control condition included 1% SDS to achieve complete lysis of target cells. Chimeric anti‐CD20 mAb rituxan (2.5 μg·mL−1) was used in all bioassays as a negative control for hRS7 IgG. The release of 51Cr by cytolysis from the target cells was counted in a gamma radiation counter . The percentage cytotoxicity of SG, the control ADC, or hRS7 IgG was calculated by the following formula: % cytotoxicity = 100 × (E − S)/(T − S), where E is the experimental release, S is the spontaneous release by target cells, and T is the maximum release by target cells lysed with 0.1% SDS. The results were reported as mean ± SEM.Standard 4‐h chromium . The viable cells were then quantified using a flow cytometry‐based assay that has been previously characterized  to identify the percentage of live versus dead cells that were present in each well. The bystander effect was assessed by comparing the percentage of live Trop‐2‐negative cells (GFP‐ARK4) when they were cocultured with Trop‐2‐positive cells (ARK2) and treated with either SG versus the control ADC.ARK4, a Trop‐2‐negative uterine serous cell line stably transfected with a green fluorescence protein (GFP) plasmid  was plated (80 000 cells/well) alone in a 6‐well plate (2 mL per well). A 1 : 1 ratio of this ARK4 cell line stably transfected with GFP was admixed with END(K)265, a 3+ Trop‐2 expressing EC cell line, and then plated in a 6‐well plate (2 mL per well). After overnight incubation, cells were treated with either SG or the control ADC at a concentration of 1 n2.8in vivo antitumor activity of SG, the control ADC, and hRS7 IgG was tested in xenograft models using the Trop‐2 + END(K)265 cell line, a grade 3 EC with mixed endometrioid and clear cell histology. Each mouse ; ENVIGO, Indianapolis, IN, USA) received a subcutaneous injection of 14 million END(K)265 cells suspended in 300 µL of a 1 : 1 solution of sterile PBS containing cells and Matrigel® (BD Biosciences). When a tumor volume of 0.3 cm3 was obtained, the mice were then randomized into four groups (six mice/group): saline control, SG, control ADC, and hRS7 IgG. SG, the control ADC, and hRS7 IgG were given at a dose of 500 μg in 100 μL IV twice per week for three weeks by retro‐orbital and/or tail vein injection. Tumor volume was measured twice weekly using Vernier calipers. Determination of tumor volume was obtained using the formula (A2 × B)/2, where B represented the largest tumor diameter size and A was the smallest perpendicular tumor diameter. The mice were subsequently euthanized when the group's average tumor volume reached 1.5 cm3. All animal care and euthanasia were carried out according to the regulations and rules established by the Institutional Animal Care and Use Committee (IACUC), which are in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals.The 2.950 extrapolations were performed using graphpad prism 8 . Five drug concentration points were used for the calculation of IC50 in agreement with the previous literature  was performed using a TMA containing 143 EC patient samples. Moderate‐to‐strong (score 2–3) Trop‐2 expression was found in 84% (120/143) of these tumors  (2 μg·mL−1) at E:T ratios of 5 : 1 and 10 : 1, they were resistant to PBL‐mediated cytotoxicity  of hRS7 IgG, SG, and the control ADC revealed that hRS7 IgG and SG were highly effective in inducing ADCC against primary EC cell lines expressing Trop‐2 at high levels 265 cell line) but the control ADC did not. As shown in Fig. E:T ratios of 5 : 1 and 10 : 1, a mean cytotoxicity ± SEM = 12.3 ± 3.1% for SG and 19.7 ± 5.4% for hRS7‐IgG was determined, respectively, while low/negligible killing  was induced by control ADC 265) and Trop‐2‐negative cell line 361) were both tested for their potential sensitivity to PBL‐mediated cytotoxicity using standard 4‐h 3.4P = 0.014 and P = 0.005, respectively)  .Subsequently, three primary EC cell lines (two were Trop‐2‐positive and one was Trop‐2‐negative) were treated with scalar concentrations of SG, the control ADC, and hRS7 IgG. There was a significant increase in cell cytotoxicity induced against Trop‐2‐positive EC cell lines treated with SG 265 and END(K)254, respectively) than that of those treated by the control ADC (3.5P = 0.001).In order to evaluate the potential of SG to mediate a bystander killing effect toward EC cell lines with heterogeneous Trop‐2 expression, we admixed cells with high Trop‐2 expression 265 cells) and those with low or negligible Trop‐2 expression  for 72 h. As shown in Fig. 3.6in vivo experiments comparing the potential antitumor activity of SG, the control ADC, hRS7 IgG, and saline. Within 4 days of therapy initiation , SG demonstrated a significant antitumor effect in these chemotherapy‐resistant tumors compared to all controls, including nonspecific ADC . This SG‐mediated inhibition of tumor growth was evident at all subsequent time points up to day 15 when the control ADC animals were euthanized secondary to progression of disease, which was defined as a tumor volume greater than 1.5 cm3 (P < 0.01). This antitumor effect translated into a significant survival benefit for SG‐treated animals in comparison with all the controls . Median survival for the SG group was 40 days versus 15 days for the control ADC, and 11 days for hRS7 IgG and saline 265 tumor cells in SCID mice, these xenograft models were chosen for the 4et al., et al., et al., et al., et al., High expression of Trop‐2 has been discovered in a variety of human epithelial tumors including cervical, uterine, and ovarian cancers and confers tumor cells with an increased ability to proliferate and migrate  of the EC samples evaluated by IHC and 43% of the primary EC cell lines assessed by flow cytometry. Importantly, we have shown Trop‐2‐positive EC cell lines to be highly sensitive to treatment with SG when compared to treatment with the control ADC and hRS7 IgG. In contrast, no differences were found in Trop‐2‐negative cell lines. These results demonstrate the need for Trop‐2 receptor expression on tumor cells for the induction of SG cytotoxic activity against EC.et al., et al., et al., Furthermore, we demonstrated that the mechanism by which SG induces tumor cell killing is not only due to the internalization of the ADC and the consequent intracellular release of the toxic payload SN‐38 but it may also be potentially mediated by immune system cells . Indeed, both SG and hRS7 IgG demonstrated significant induction of ADCC toward Trop‐2‐positive EC cell lines in the presence of PBL, while the control ADC generated only a low level of cytotoxicity. The killing activity was Trop‐2‐specific as demonstrated by the negligible ADCC induced by SG against Trop‐2‐negative tumors. These findings support previous results from our group evaluating the activity of the unconjugated humanized anti‐Trop‐2 monoclonal antibody hRS7 in ovarian cancer  demonstrated that a few injections of SG were highly effective in inducing regression of EC xenografts. Indeed, twice‐weekly administration of SG demonstrated a statistically significant difference in tumor growth inhibition of END(K)265 when compared to administration of the control ADC. It was considered that a possible limitation in the use of SG in the clinical setting is its potential toxicity. Thus, it is important to note that there was no evidence of acute or chronic toxicity detected in the animals treated with SG for the entire duration of the study. These results are the first to demonstrate the efficacy and safety profile of the in vivo activity of SG against biologically aggressive and poorly differentiated EC. These findings warrant the support of the use of SG in clinic trials.Nonetheless, the in vitro and in vivo results in biologically aggressive endometrial cancer, results from a phase I/II clinical study reported acceptable toxicity and encouraging therapeutic activity of SG against multiple recurrent human epithelial cancers including urothelial cancers and lung cancers  but more uniquely SG demonstrates a significant bystander killing effect, which may aid in the treatment of tumors with heterogeneous antigen expression. Nonetheless, EC xenografts overexpressing Trop‐2 were found to be highly sensitive to treatment with SG. Our results combined with recent phase II data demonstrating significant clinical responses in tumors resistant to standard chemotherapy regimens support the implementation of clinical trials for patients with recurrent and poorly differentiated EC that harbor Trop‐2‐positive tumor expression.Trop‐2 overexpression is found in over 80% of EC, and our All authors fulfill the conditions required for authorship. ADS declares research funding support to Institution from ImmunoMedics. AM declares that she receives research funding support from Spanish Medical Oncology Society.ADS designed research; EP, SL, BZ, SB, AB, LZ, AM, EB, PM, NB, GA, CH, GM, ER, D‐AS, MA, JTR performed research; PES, GS contributed new reagents/analytic tools, ADS and EP wrote the paper.


N-acetylglucosamine, pyruvate and acetylcarnitine. Furthermore, the metabolism of XTC.UC1 was skewed towards the de novo synthesis of aspartate, an effect that persisted even in glucose-free media, pointing to reductive carboxylation. Our data suggests that metabolic reprogramming and a subtle balance between ROS generation and scavenging/conversion of intermediates may be involved in ROS-induced w-CIN in HCC and possibly also in rare cases of follicular thyroid cancer showing a NHG.Hürthle cell carcinoma (HCC) is a recurrent subtype of non-medullary thyroid cancer. HCC is characterized by profound whole-chromosome instability (w-CIN), resulting in a near-homozygous genome (NHG), a phenomenon recently attributed to reactive oxygen species (ROS) generated during mitosis by malfunctioning mitochondria. We studied shared metabolic traits during standard and glucose-depleted cell culture in thyroid cancer cell lines (TCCLs), with or without a NHG, using quantitative analysis of extra and intracellular metabolites and ROS production following inhibition of complex III with antimycin A. We found that the XTC.UC1 and FTC-236 cell lines (both NHG) are functionally impaired in complex I and produce significantly more superoxide radicals than SW579 and BHP 2–7 (non-NHG) after challenge with antimycin A. FTC-236 showed the lowest levels of glutathione and SOD2. XTC.UC1 and FTC-236 both exhibited reduced glycolytic activity and utilization of alternative sources to meet energy demands. Both cell lines also shared low levels of α-ketoglutarate and high levels of creatine, phosphocreatine, uridine diphosphate- Differentiated NMTC can be grossly subdivided in follicular and papillary variants. About 90–95% of differentiated NMTC can be cured by a combination of surgery and radioactive iodide treatment. Recurrence of differentiated NMTC is mainly seen in a subset of BRAF-mutated papillary thyroid cancers (PTC) and in Hürthle cell carcinomas (HCC). Although HCC explains only 3–10% of consecutive cases of differentiated NMTC, HCC accounts for up to 40% of recurrent cancers due to abrogation of sodium iodide (NaI) transport over the cellular membrane that impairs radioactive iodide therapy. HCC is now considered a distinct subtype of thyroid cancer2.Non-medullary thyroid cancer (NMTC) is the most common endocrine malignancy3. Analysis of the mitochondrial DNA (mtDNA) in oncocytic lesions from different origins revealed frequent inactivating/disrupting variants, concentrating in the genes encoding subunits of complex I (CI) of the electron transport chain (ETC), thus suggesting an impact on metabolism4. However, other studies also identified numerous mtDNA variations in other types of NMTC6. Furthermore, a substantial fraction of HCCs entirely lack mtDNA variants9. Thus, mtDNA mutations cannot solely explain the oncocytic phenotype and might be a secondary effect due to mitochondrial hyperplasia caused by a coupling defect11. It has even been suggested that the oncocytic phenotype may be caused by mutations in oncogenes and/or tumour suppressor genes, with mtDNA variations accumulating as a late effect12.The characteristic oncocytic metaplasia found in HCC is caused by abnormal proliferation of inadequately functioning mitochondria14. Near-haploidy can be easily detected by multiparameter DNA content analysis15 and is frequently followed by endoreduplication or genome doubling of the entire near-haploid genome13, a common trait of solid tumours18. Endoreduplication of an entire near-haploid genome is sometimes referred to as ‘uniparental disomy’20. Thus, many HCCs show a ‘near-homozygous genome’ (NHG), a term that covers both a near-haploid genome status and the status after endoreduplication. We recently suggested that NHGs in HCC are the net result of a continuous process of whole-chromosome instability (w-CIN). w-CIN may be driven by factors such as increased intracellular production of reactive oxygen species (ROS) as a by-product of metabolic changes21 following the loss of CI functionality22. Interestingly, a recent study of renal oncocytomas found that recurrent CI loss was the earliest detected genetic alteration accompanied by altered metabolism and affected the production of the ROS scavenger glutathione23.In addition to an oncocytic phenotype, HCCs frequently show near-haploidy due to loss of complete chromosomes, but with retention of chromosomes 5, 7 and 12 in a heterozygous state24 in two TCCLs with NHG  and two TCCLs without a NHG (SW579 and BHP 2–7). Furthermore, we performed spectrophotometric activity measurements of OXPHOS complexes in TCCLs. In addition we quantified superoxide and hydroxyl radical generation under normal culture conditions and after complex III (CIII) inhibition.The aim of this study was to elucidate the metabolic features of thyroid cancer cell lines (TCCLs), focusing on ROS-driven w-CIN. To do this, we used metabolomics analysis25, FTC-236, (derived from an FTC metastasis)26, SW579 (derived from a poorly differentiated thyroid carcinoma and deposited by A. Leibovitz) and XTC.UC1, the only model for HCC27. Cell lines were authenticated by a multiplex PCR , cultured for 20 passages and tested for mycoplasma at regular intervals (in-house PCR method). For detailed information, see Supplementary Table 2. Standard RPMI-1640 media contains 3.3 µM reduced glutathione (GSH).The following cell lines were used: BHP 2–7 (derived from a primary papillary thyroid cancer)For metabolite analysis, cells were cultured for two weeks in DMEM/F-12 or RPMI-1640. Subsequently, ten replicates of each cell line were grown for 72 hours. Six and 24 hours before cell extraction, culture media were refreshed (controls) or replaced by glucose-free DMEM/F-12 or RPMI-1640 supplemented with 5 mM galactose (as a glucose substitute). In parallel, cell-free DMEM/F-12 or RPMI-1640 were also incubated for the same time periods and used as controls to calculate the consumption and production of metabolites.The levels of ATP, ADP and AMP, as quantified from NMR spectra (see section “NMR spectroscopy and quantification of metabolites” below), were used to assess the adenylate energy charge (AEC) for each cell line. AEC values were 0.89 ± 0.02 for XTC.UC1, 0.90 ± 0.02 for FTC-236, 0.90 ± 0.01 for SW579 and 0.89 ± 0.01 for BHP 2–7.3:H2O, 8.1:0.9:1 (v/v/v)). Mixtures were stored at −80 °C for at least 30 min, allowing protein precipitation. The mixtures were then centrifuged at 16000 × g for 10 min at 4 °C. Supernatants were dried under nitrogen flow and samples were stored at -80 °C until further processing.Extracellular metabolites: Culture medium (100 μL) was collected prior to harvesting and immediately mixed with two volumes of a pre-chilled (−28 °C) solution of methanol/chloroform/water (MeOH:CHCl2 was discarded and intracellular metabolites were extracted at 4 °C with 1.5 mL pre-chilled (−28 °C) methanol/chloroform/water  and detached and lysed with the aid of a cell scraper. The solutions were transferred to 2 mL Eppendorf tubes and centrifuged at 16000 × g for 20 min at 4 °C. Supernatants were dried under nitrogen flow.Intracellular metabolites were extracted as follows. Cells were quickly washed with 5 mL MilliQ water at 37°C. Metabolic activity was immediately quenched by adding 15 mL liquid nitrogen to the petri dish and samples were stored on dry ice until further processing. The liquid N2HPO4/KH2PO4 buffer (pH = 7.4) prepared with 99.9% deuterated water (D2O) containing 0.2 mM NaN3 and 0.4 mM trimethylsilylpropionic-d4 acid sodium salt (TSP-d4). From each sample, 190 µL was transferred to 3 mm NMR tubes using a Gilson 215 robot. Remaining volumes from each sample were pooled according to each condition and used for 2D NMR experiments.Extracts were reconstituted in 250 μL of 0.15 M KCells were harvested using trypsin/EDTA and washed with HBSS. Cell counting was performed with a Muse cell analyser (Merck-Millipore) using the count and viability kit (product # MCH100102) according to the manufacturer’s instructions, after first calibrating the system with the Muse system check kit (product # MCH10010).24. Briefly, NMR data were recorded using a 14.1 T NMR spectrometer  under standardized instrumental settings for all samples. Per sample, a 1D 1H spectrum was collected using the noesygppr1d pulse sequence, which is implemented in the standard library of the NMR manufacturer. The spectral data were phase and baseline corrected and referenced to TSP-d4 methyl protons at δ 0.00 ppm and subsequently imported into Chenomx NMR suit 8 , allowing metabolite quantification. Metabolite assignment was based on the Bruker Bbiorefcode and Chenomx databases. The structures of all annotated metabolites were then confirmed in 2D NMR experiments of the pooled samples. The quantification of each metabolite was performed by integration of its proton peaks in the NMR spectrum using the deconvolution fitting algorithm of Chenomx NMR Suit. The calculated integrals were subsequently transformed to concentrations (mM), based on the known concentration of the internal standard TSP-d4 in each sample (0.05 mM). Finally, all concentrations were normalised to the cell population of each cell line. The quantities of ATP, ADP and AMP calculated from the NMR data were used to calculate the adenylate energy charge (AEC) of each sample. Uptake and release rates of metabolites by the cells, from and to the medium, respectively, were calculated using the following formula: r = (Cspent − Cfresh) × V × 1000/A, where Cspent (mM) is the concentration of each metabolite as quantified in the culture media after 72 h of incubation and Cfresh (mM) is the concentration of the same metabolite in a parallel incubation of cell-free medium; V in litres is the volume of the culture medium  and A is the area under the growth curve. The latter was calculated using the formula A = [(cells × d)/ln2] × (1-2−t/d), where cells represent the population of cells at 72 h, d is the doubling time and t is the time of incubation (i.e. 72 h). A positive value of r indicates release of a metabolite from the cell and a negative value indicates uptake in units of μmol/h/106 cells. To represent uptake and release in the figures, the r values of each metabolite were divided by the mean r of that metabolite and reported as a relative change in the figure legend.A detailed protocol for NMR spectroscopy and quantification of metabolites has been described previouslySuperoxide and hydroxyl radicals were analysed semi-quantitatively by flow cytometry. Cells  were treated and labelled according to the following scheme  after daily calibration. Fluorescence was collected using a 450/50 nm bandpass filter for detector B , and a 610/20 bandpass filter (MitoSOX RED fluorescence) and a 530/30 nm bandpass filter (CellROX Green) for detectors D and F (blue 488 nm laser line), respectively. Cyto-Cal beads FC3MV  were analysed allowing comparison of different measurements over time and expressing data as median equivalent fluorochrome (MEF). The experiment was repeated three times.Data were analysed using WinList 8.0 . Ten thousand single live cell events were selected after doublet and dead cell discrimination using SSC (side light scatter)-W (pulse width) vs. SSC-H (pulse height), FSC (forward light scatter)-W vs. FSC-H, and FSC-A (pulse area) vs. DAPI-A dot plots, respectively. Median relative fluorescence intensities (MFI) were converted to MEF values. An ANOVA test was used to study statistical significance.29.Isolated mitochondria were used for enzymatic measurements. Enzyme activities of the OXPHOS complexes were measured as previously describedPrimary antibodies: anti-α-Tubulin Mouse, 1:50,000 ; anti-SOD2 Rabbit mAb, 1:1000 . Secondary antibodies: Goat anti-Rabbit IRDye 800CW, 1:10000 ; Goat anti-mouse IRDye 680LT, 1:10000 .30. Cells were washed with ice cold PBS and lysed with Hot-SDS buffer containing PhosSTOP  and cOmplete .Western blotting was performed as described previouslyProtein concentrations were determined with the Bio-Rad DC Protein Assay . Ten µg lysates were mixed with 4x Laemmli Sample Buffer  containing βME, heated for 5 minutes at 100 °C and loaded onto a 1.5 mm 10% acrylamide gel . Electrophoresis was performed at 50 V. For blotting, the Bio-Rad semi-dry Trans-Blot Turbo Transfer System was used . Blots were then washed in Tris-buffered saline (TBS), blocked in Odyssey Blocking Buffer  at a 1:1 ratio with TBS for 1 hour, washed in TBS and incubated overnight at 4 °C with primary antibodies in TBS, 0.1% Tween-20, 5% BSA .The next day, blots were washed in TBS and incubated for one hour with the secondary antibodies in TBS/0.1% Tween-20/5% BSA protected from light. After washing, blots were air dried and imaged at high-resolution with the Odyssey Infrared Imaging System  using intensities 1.5 and 3.0 for the fluorescent colour channels red (700) and green (800), respectively. Images were analysed using Image Studio Lite Ver 5.2 software package .http://www.R-project.org/) using the packages pheatmap, dplyr, ggplot, ggsci and RColorbrewer, as well as GraphPad Prism version 8.0 . Pairwise comparisons of metabolite levels between the cell lines were performed using the Mann Whitney U test and p-values were corrected using the False Discovery Rate method of Benjamini-Hochberg (BH). Cluster analysis of cell lines and metabolites was performed in R using the factoextra package, with Spearman correlation analysis.Analysis and visualization of metabolomics data was performed in R statistical software  leads to increases in oxidized glutathione (GSSG), so higher levels of intracellular GSSG generally indicate increased ROS production. We used quantitative NMR to measure the intracellular levels of GSH and GSSG. Our analysis showed only minimal differences in the ratio of GSH/GSSG in the four cell lines, with XTC.UC1 and BHP2–7 showing a positive ratio (more GSH) while FTC-236 and SW579 had more GSSG than GSH Fig. . It shouWe also measured GSH/GSSG after challenging the cells by culturing in glucose-depleted medium for 6 and 24 h, as lack of glucose shifts dependency to OXPHOS in terms of energy needs. We found that both GSH and GSSG were only slightly decreased Fig. , and GSH2O2 scavenger, differ between the cell lines we then asked whether ROS production under normal culture conditions correlates with glutathione levels. We used quantitative flow cytometry to measure superoxide anion (O2−) under basal conditions and during inhibition of oxidation of the ETC component ubiquinol (CIII) using 100 µM antimycin A (AMA) for 2 hours . AMA treatment also induced a strong increase of CellROX Green fluorescence at this concentration, a ROS indicator mainly responsive to OH−, an increase that could be significantly inhibited in all cell lines when simultaneously treated with N-acetyl cysteine (NAC), an H2O2 and OH. scavenger. The signal amplitude of MitoSOX Red, which mainly reacts with the O2− radical, was only slightly reduced by NAC.Basal levels of Ones Fig. . However2− to H2O2 and O2. BHP2-7 and XTC.UC1 showed relatively higher expression of SOD2 compared to SW579 and FTC-236, the latter showing the lowest level , creatine, phosphocreatine (PCr) and acetylcarnitine were similar in XTC.UC1 and FTC-236 and significantly (p < 0.05) higher compared to the non-NHG SW579 and BHP 2-7 cell lines   Fig. . XTC.UC1ine Fig. , while cine Fig. . In additle Fig. . Finallytle Fig. .Figure 56 cells and 25.61 +/− 5.59 µM/106 cells, respectively), and these ratios were significantly higher than in XTC.UC1 (9.28 +/− 0.76 µM/106 cells) and FTC-236 (9.25 +/− 0.59 µM/106 cells). Collectively, these data indicate that XTC.UC1 is able to utilize multiple sources to produce high amounts of aspartate 21. The loss of entire chromosomes has been attributed to aberrant interactions with reactive oxygen species (ROS)21. Interestingly, recent findings showed that ROS vary with the cell cycle phase and reach their highest levels during mitosis33. Thus, in cells with malfunctioning mitochondria, e.g. oncocytic cells such as XTC.UC1, mitosis might lead to greater cellular stress, increased levels of ROS and subsequent ROS-induced aneuploidy by chromosomal missegregation35. This mechanism may be regulated via enhanced CHK2 phosphorylation, as seen in mitotic cells after AMA treatment21, which could in turn contribute to lagging chromosomes and missegregation36.Metabolic adaptation plays an important role in the development of cancers characterized by abundant malfunctioning mitochondria (oncocytic phenotype), including thyroid Hürthle cell carcinoma (HCC)2−) and hydroxyl radical (OH−) production in thyroid cancer cell lines with (XTC.UC1 and FTC-236) and without an NHG (SW579 and BHP 2–7), and studied the effects of inhibiting CIII using antimycin A. We also studied OXPHOS complexes in TCCLs by spectrophotometric activity measurements and used standard and glucose-depleted cultures to evaluate glycolytic activity, GSH synthesis and additional metabolic alterations.In this study we investigated the basal levels of superoxide  in the four TCCLs tested, despite differences in mtDNA variants21. This suggests that under standard culture conditions mitochondrial H2O2 is rapidly catabolized by enzymes such as catalase, glutathione peroxidase and peroxiredoxin 338, and only a fraction is converted to OH−, as shown by the limited effect of NAC. Interestingly, efficient detoxification of ROS is a suggested mechanism in HCC39. When we stressed the cell lines with 100 µm AMA, the NHG cell lines XTC.UC1 and FTC-236 generated significantly more O2− than BHP 2–7 and SW579. Simultaneously a strong increase of CellROX fluorescence was observed, which could be reduced significantly by NAC. This shows that AMA treatment drives redox homeostasis towards H2O2, which can be neutralized by NAC. By contrast, MitoSOX fluorescence (which reacts mainly with O2−) was left relatively unaffected by NAC, indicating that SOD2-mediated dismutation to H2O2 is likely favoured above scavenging by NAC in the presence of active SOD240.Similar to earlier studies of cybrid models carrying homoplasmy and various degrees of heteroplasmy41 and levels increase significantly during AMA treatment42. Our data indicate that the ETC in TCCLs is  functional even in the presence of a disruptive and/or damaging mtDNA variants21 and CI impairment in XTC.UC1 and FTC-236. Furthermore, overexpression of CIII and CIV, as described by Baris et al., suggests a compensatory mechanism for the relative loss of electron flow39. A functional ETC in the presence of CI mtDNA disruptive/damaging alterations might also be explained by the formation of respiratory supercomplexes that partially compensate for an impaired CI activity, as found in XTC.UC1 and in FTC-236 and as demonstrated in Ndufs4−/− mice43. This process might also play a role in HCC44 and in renal oncocytomas showing loss of CI activity45. The latter studies showed that, like XTC.UC1, renal oncocytomas produce high levels of glutathione as a defence against partial impairment of CI of the ETC. Furthermore, Gopal et al.23 found that cysteine and glycine, precursors of glutathione, were reduced in renal oncocytomas, suggesting that their utilization may be increased due to flux towards glutathione synthesis. We observed no difference in the uptake of cysteine between XTC.UC1 and FTC-236 46 and de novo synthesis of aspartate47, together with its precursors pyruvate, fumarate, malate and citrate, and comparable to what was found in XTC.UC1 cells. This might reflect cytosolic GOT1 activity in the context of an impaired CI48, which partly counterbalances loss of mitochondrial aspartate synthesis49. The use of α-KG might indicate direct scavenging and the utilization of reductive carboxylation of α-KG by XTC.UC1 and FTC-236 to support growth compromised by defective mitochondria51. In addition, all TCCLs exhibited increased aspartate synthesis  and the NRF1 and TFAM transcription factors are upregulated in HCC56, highly comparable to the observations in acetylcarnitine-supplemented aging rats. The downstream effects of PRC were confirmed in XTC.UC111. Thus, the high levels of acetylcarnitine in XTC.UC1 and FTC-236 might be a response to oxidative stress57, and the poor condition of the mitochondria in XTC.UC1 and FTC-236 might reflect accelerated aging55. Further support is found in the significantly lower levels of GSH, glutamate and glycine in FTC-236 (derived from a follicular thyroid cancer) compared to XTC.UC1 (HCC). This could indicate that the ROS scavenging capacity of FTC-236 is greatly reduced in comparison to XTC.UC1, BHP 2–7 and SW579, or that the GSH to GSSG turnover in FTC-236 is higher than in the other three cell lines. Yet another factor is the level of SOD2 expression, which is about two-fold lower in FTC-236 compared to XTC.UC1. Thus, in FTC-236 the conversion of O2− to H2O2 might be rate-limiting due to lower SOD2 expression compared to XTC.UC1. Interestingly α-KG, likewise identified as an H2O2 scavenger59, was significantly lower in XTC.UC1 and FTC-236 compared to SW579 and BHP 2–7, which might indicate an active role in H2O2 scavenging in the first two cell lines. Despite the lack of excess O2− and OH− under standard culture conditions, in XTC.UC1 we observed not only higher GSH but also more GSH compared to its oxidized form GSSG, suggesting lower consumption due to a low proliferative activity and a balanced flux of GSH synthesis, as long as there is a constant supply of nutrients.Both XTC.UC1 and FTC-236 showed high levels of acetylcarnitine, which plays a key role during β-oxidation of fatty acids to generate ATP61. We showed that XTC.UC1 and FTC-236 are less glycolytic and less OXPHOS competent compared to the BHP2-7 and SW579 cell lines. At first glance, FTC-236 appears to show classic Warburg-like characteristics due to low levels of TCA intermediates and low oxidative respiration. However, and similarly to XTC.UC1, FTC-236 showed lower levels of intracellular (Fig. 62. This is in line with the increased levels of pyruvate in XTC.UC1 and FTC-236 (see Fig. 63. Furthermore, lactate has also been identified as a TCA cycle carbon source in non-small-cell lung cancers. This was most apparent in patients with an aggressive tumour showing a high 18fluorodeoxyglucose uptake64, as can be observed in HCC65. In this context it is noteworthy that both XTC.UC1 and FTC-236 has been derived from metastasis. Low levels of lactate might also contribute to the relative low proliferative activity of XTC.UC1 compared to the other TCCLs (Supplementary Table Many earlier studies reported an increased dependency of tumours on glycolysis. More recently, the importance of other metabolic pathways in tumorigenesis, tumour progression and survival has become clearlar Fig.  lactate lar Fig.  and 4d. see Fig.  compared69, in line with a relatively quiescent glycolytic state70. Apparently, glucose is used to fuel other glycolytic pathways, such as the hexosamine pathway which supports UDP-GlcNAc synthesis (Supplementary Fig. The high levels of creatine and phosphocreatine might point to an alternative route to ATP generation in XTC.UC1 and FTC-236 and direct ROS scavenging via creatine71 and associates with formate and alanine excretion, found at high levels in all four TCCLs. This underscores this pathway as an interesting target for inhibition when combined with decreased glycolytic ATP production.The important role of glutamine in the metabolism of XTC.UC1 is reflected in an approximately four-fold increase after 6 h in a glucose-depleted medium see Fig. . This ma22, and which might correlate with a NHG in these tumours. Furthermore, our data points to a subtle balance between ROS generation and scavenging, directly via creatine and/or α-KG and/or conversion of ROS intermediates72. This might explain ROS-induced w-CIN in HCC and perhaps other rare cases of follicular thyroid cancer showing w-CIN73. Moreover, our data suggests phosphocreatine cycling and β-oxidation as alternative routes for generating ATP in XTC.UC1 and FTC-236, otherwise relatively quiescent glycolytic cell lines. Besides the pivotal role of acetylcarnitine in β-oxidation, the high levels found in the NHG cell lines XTC.UC1 and FTC-236 might provide protection against aged mitochondria. Importantly, we also found a profound metabolic switch of the HCC cell line XTC.UC1 towards the synthesis of aspartate. Bearing in mind the recent findings regarding the role of aspartate in cancer metabolism47, to the best of our knowledge our data shows, for the first time, that novo aspartate synthesis also plays a key role in HCC cells and might be considered a treatment target in this type of thyroid cancer.To define common metabolic characteristics associated with NHGs in TC, we studied four distinct TCCLs with and without a NHG. We demonstrated that XTC.UC1 and FTC-236 are CI impaired, a phenomenon found earlier in HCCSupplementary information.Supplementary Tables.


In a grouped analysis based on AR status, there were 2,127 down-regulated circRNAs in androgen independent cell lines compared to 2,236 in androgen dependent cell lines, thus identifying a potential circRNA signature reflective of androgen dependency. Through a bioinformatics approach, the parental genes associated with the top 10 differentially expressed circRNAs were identified such as hsa_circ_0064644, whose predicted parental gene target is RBMS3, and hsa_circ_0060539, whose predicted gene target is SDC4. Furthermore, we identified three circRNAs associated with the parental gene Caprin1 . Other studies have shown the importance of Caprin1 in PCa cell survival and drug resistance. Given the modified circRNA expression signatures identified here, these hypothesis generating results suggest that circRNAs may serve as potential putative diagnostic and predictive markers in PCa. However, further validation studies are required to assess the true potential of these markers in the clinical setting.Circular RNAs (circRNAs), a recently discovered non-coding RNA, have a number of functions including the regulation of miRNA expression. They have been detected in a number of malignancies including prostate cancer (PCa). The differential expression pattern of circRNAs associated with PCa and androgen receptor (AR) status was investigated in this study. circRNA profiling was performed using a high throughout microarray assay on a panel of prostate cell lines, which consisted of normal, benign, and malignant cells ( Non-coding RNAs (ncRNAs), which include microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) , play anThe aim of this study was to profile circRNAs in a panel of prostate cell lines and identify potential signatures associated with malignancy and androgen dependency. We propose that circRNAs have the potential to serve as useful biomarkers to improve diagnostic screening for PCa and/or identify men who are at risk of developing castration-resistant disease.A panel of malignant and benign prostate cell lines were used in this project, all of which were purchased from the ATCC . The benign prostate cell lines  were cultured in Keratinocyte Serum Free Media , containing 0.05 mg/mL bovine pituitary extract (BPE) and 5 ng/mL Epidermal growth factor (EGF) (Thermo Fisher Scientific) and 1% Penicillin Streptomycin (P/S) . The benign prostatic hyperplasia (BPH-1) and PCa cell lines  were cultured in RPMI-1640 (Merck KGaA) supplemented with 10% Fetal Bovine Serum (FBS) (Merck KGaA) and 1% P/S. PC-3 was cultured in ATCC-formulated F-12K Medium (Thermo Fisher Scientific) supplemented with 10% FBS and 1% P/S.® Reagent (Thermo Fisher Scientific). For phase separation, 200 μL chloroform (Merck KGaA) was added to the cell lysate. The sample were vortexed vigorously for 15 s, incubated at RT for 5 min and centrifuged at 12,000 × g for 15 min at 4°C. The upper aqueous phase was transferred into a fresh tube and the RNA precipitated from the aqueous phase, by the addition of 400 μL isopropyl alcohol (Merck KGaA). The sample was mixed, incubated for 5 min at RT and centrifuged at 12,000 × g for 15 min at 4°C. One milliliter 75% EtOH (Merck KGaA) was added to pellet, and centrifuged at 12,000 × g for 10 min at 4°C. The EtOH was removed and a repeat pulse centrifuge was performed. Surplus EtOH was removed and the pellet air-dried. The sample was re-suspended in 30 μL molecular grade H2O before DNase treatment with Ambion® TURBOTM DNase (Thermo Fisher Scientific), and an additional RNA clean-up using standard EtOH precipitation.Total RNA was prepared from cell lines  according to manufacturer’s instructions. Briefly, cells were lysed directly with 2 mL TRIzolThe panel of cell lines  were profiled using the Arraystar Human circRNA Array version 2.0 . The sample preparation and microarray hybridization were performed according to manufacturer’s instructions. Briefly, total RNA was digested with RNAse R  to remove linear RNAs and enrich for circRNAs. The enriched circRNAs were amplified and transcribed into fluorescent cRNA using a random priming method with Arraystar Super RNA Labeling Kit (Arraystar). The labeled cRNAs were hybridized onto the Arraystar Human circRNA Array V2 (8 × 15 K). The array slides were washed and scanned on the Agilent Scanner G2505C. Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images.t-test was used to identify significantly altered circRNAs. The false discovery rate (FDR) was applied to determine the threshold of p-value. An FDR of < 0.05 was used. Changes in expression were identified using unsupervised clustering analysis (euclidean distance measure and the “average” agglomeration method) and associated heat-maps generated.Quantile normalization and subsequent data processing were executed using the R software package . circRNAThe human circular RNA microarray (Arraystar) version 2.0 covers 13,617 previously discovered human circRNAs. Our cell line panel consisted of malignant , benign (BPH-1), and normal  cell lines. AR expression in 22Rv1, LNCaP, and VCaP was confirmed using qPCR . In totap-value  according to FC and p-value . We firsp = 0.001), located on chromosome 3  and NFIX (hsa_circ_0049657), both known to be involved in the regulation of the AR  in PCa  has been shown to be involved in the inhibition of PCa stem cell invasion, migration and proliferation, as has KRT7 (hsa_circ_0026358), which has been identified along with FAM129A, MME and SOD2 as part of a four-gene androgen regulated panel  in PCa  via tran) in PCa . hsa_cire in PCa . Similared panel .p < 0.001) is an exonic circRNA located on chromosome 11 and is associated with the gene SIAE . Caprin1 plays an important role in PCa cell survival and its upregulation has been shown to be associated with drug resistance, and is increased in SPOP mutated cell lines  and androgen independent/castration resistant) cell lines DU145, PC-3). Response to androgen ablation was demonstrated by confirming the presence or absence of AR-FL across cell lines see . CircRNA5, PC-3. ene SIAE . mRNA exh cancer . We idenll lines .p < 0.0001) located on chromosome 18 and is associated with the gene MTCL1 (FLNA (hsa_circ_0091934 and hsa_circ_0091894) located on the X chromosome, which plays a significant role in PCa development and progression, and has been highlighted as a possible biomarker for disease screening and detection  and SDC4 (hsa_circ_0060539). Interestingly, we identified three circRNAs associated with the parental gene Caprin1  and two circRNAs associated with FLNA (hsa_circ_0091934 and hsa_circ_0091894). Many of these genes are currently under active investigation as biomarkers in PCa, and identifying their associated circRNAs may contribute to ongoing research in this field (Parental genes of interest were is field .There is evidence to suggest that circRNAs regulate miRNA function by competing for the pool of miRNA binding sites to influence the activities of miRNAs in regulating gene expression . Interesin vitro study which identified multiple potential genes and ongoing validation is required in cell lines and in vivo to confirm our findings here.This study had several limitations. Firstly, we used a circRNA microarray platform to detect circRNAs compared to RNA-seq methods which may have higher sensitivity and accuracy to detect circRNAs. Secondly, this was a preliminary In conclusion, circRNAs were modified between benign and malignant cell lines, as well as androgen dependent and independent cell lines, which suggests a role for circRNAs in PCa initiation and progression. This study highlights the importance of circRNAs in PCa and their potential role in identifying the disease and monitoring response to treatment. The dysregulated signature identified here may prove useful for the development of a blood-based assay with both diagnostic and predictive value in this disease. However, this would require validation in larger patient cohorts.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.