Osimertinib has revolutionized the treatment of EGFR-mutated tumors. Its current applications include the first-line setting, second-line setting, as well as the adjuvant setting. Although it represents a milestone in the context of targeted therapy, inevitably all tumors develop an acquired resistance, some mechanisms involve EGFR, others do so through alternative pathways leading to a bypass in osimertinib inhibition. It is key to understand these acquired mechanisms of resistance, both in the clinical setting, as well as in preclinical models, in order to develop and contribute to the identification of possible therapeutic strategies to overcome this acquired resistance.EGFR-mutated tumors represent a significant percentage of non-small cell lung cancer. Despite the increasing use of osimertinib, a treatment that has demonstrated an outstanding clinical benefit with a tolerable toxicity profile, EGFR tumors eventually acquire mechanisms of resistance. In the last years, multiple mechanisms of resistance have been identified; however, after progressing on osimertinib, treatment options remain bleak. In this review, we cover the most frequent alterations and potential therapeutic strategies to overcome them. EGFR mutations, ALK and ROS1 rearrangements, MET mutations and amplifications, BRAF and KRAS mutations, RET fusions, NTRK fusions, and HER2 mutations, among others [EGFR mutations were first described in 2004. The most frequent mutations are exon 21 point L858R mutation and in-frame deletions of exon 19, accounting for 40% and 50% of EGFR mutations, respectively, and conferring sensitivity to EGFR inhibitors. Other described mutations include G718X, S768I, and L861Q, as well as an in-frame insertion in exon 20 [EGFR mutations display a heterogeneous representation depending on ethnicity and region, accounting for 40–60% in South-East Asian patients, 9–67% in patients from the Americas, 22–27% in patients from the Indian subcontinent, around 12% in African Americans, and 10–20% in Caucasian patients [EGFR amplification concurrent with T790M, HER2 amplification, MET amplification, SCLC transformation, and others [EGFR [Non-small cell lung cancer (NSCLC) accounts for approximately 80–90% of lung cancers; adenocarcinoma represents the most frequent histologic subtype of NSCLC. Different molecular oncogene addiction drivers have been identified in NSCLC, particularly in adenocarcinomas, including  exon 20 ,4,5. In EGFR activating mutation [Osimertinib was designed to overcome the effects of the T790M EGFR-resistant mutation while targeting the initial mutation . It was mutation . Subsequmutation ,14.EGFR mutated tumors seem to benefit less, if anything, from immunotherapy compared to other patients with advanced NSCLC and no targetable alterations. Clinical trials combining osimertinib and immunotherapy showed intolerable toxicity, and, on the other hand, most clinical trials combining chemo- and immunotherapy lacked a significant number of patients with EGFR mutant NSCLC to draw definitive conclusions on efficacy [Despite all these achievements, significant challenges remain ahead. Patients with efficacy .EGFR mutant tumors. In this review, we present and analyze the most frequent alterations associated with resistance to osimertinib  for relevant studies. Our search criteria included the following terms: “osimertinib”, “resistance”, “non-small cell lung cancer”, “clinical trials”, “cell lines”, as well as keywords related to acquired mechanisms of resistance, including but not limited to “C797X”, “small-cell transformation”, “squamous cell transformation”, “MET amplification”, “HER2 amplification”, “HER2 mutation”, “RET fusions”, “BRAF mutations”, “BRAF amplification”, and “RAS mutations”.To identify published articles or abstracts that described acquired mechanisms of resistance against first- or second-line osimertinib in clinical practice, as well as in vivo and in vitro models, we systematically searched PubMed  were used and transduced with lentiviruses bearing genes encoding the del19/T790M/C797S and L858R/T790M/C797S mutations in order to study the growth inhibitor effects of the compound CH7233163 . AnotherThe incidence of C797X in the clinical setting differs depending on the treatment setting of osimertinib. In the first-line scenario, 6 of 91 patients (7%) acquired a C797X mutation in plasma samples from the FLAURA clinical trial . AnalysiSeveral therapeutic strategies have been proposed to tackle the emergence of these mutations; however, currently, chemotherapy remains the standard treatment after progression to osimertinib.Preclinical data suggest that, in the absence of T790M, cells remain sensitive to reversible binding EGFR TKIs , whose binding does not rely on C797 . HoweverIn the second-line setting, the position in which T790M is located, whether in the same (cis) or in a different (trans) position related to C797S, has a significant impact. In vitro studies have reported that trans-T790M/C797S cells were resistant to third-generation EGFR TKIs but sensitive to the combination of first- and third-generation TKIs, whereas cis-T790M/C797S cells were refractory to all EGFR TKIs tested, as well as their combinations .Case reports explored this clinical approach: a patient with a triple mutation tumor (del19/trans-T790M/C797S) received osimertinib and erlotinib after progression to osimertinib, reaching a PFS of 3 months . In anotIn the cis-T790M/C797S population, in vitro studies suggest that brigatinib (an ALK and ROS1 inhibitor with EGFR inhibition activity) could overcome this resistance mechanism with the combination of brigatinib plus an anti-EGFR antibody . A retroEGFR mutant NSCLC who had received osimertinib and had a median of four prior lines of therapy obtained an ORR of 39% (with complete responses in 2% of the patients) and an mPFS of 8.2 months. The efficacy of patritumab deruxtecan was maintained across various resistance mechanisms and different levels of HER3 expression. Interestingly, in the subgroup of patients with a known EGFR resistance mutation, ORR rose to 50%. This promising drug will be further evaluated in the HERTHENA-Lung01 trial [Preliminary data was presented recently about patritumab deruxtecan (HER3-DXd), an antibody–drug conjugate (ADC) against HER3 . Forty-f01 trial  and also01 trial .EGFR-mutated untreated NSCLC. One phase I/II study is exploring whether the combination of osimertinib and gefitinib could delay the emergence of acquired resistance mechanisms. To date, the combination has shown an acceptable toxicity profile [Preventive strategies are also being tested in patients with  profile .Less frequent mutations different to C797X have also been described. After progression to osimertinib in the FLAURA study , 2% of pThere is limited evidence regarding the functional effect of these less frequent mutations. G796X mutations occur close to C797 residue and thus, affect the interaction between osimertinib and its binding residue . L792X, EGFR L858R/L781Q tumor responded partially to a third-line of treatment with afatinib before progressing and identifying a KRAS mutation [A case report by Fang et al. described a patient whose mutation . To our EGFR-dependent mechanism of resistance is the amplification of EGFR, with an incidence of 33% and 31% in the first and later lines, respectively. In vitro studies have confirmed EGFR amplification as an independent resistance mechanism to osimertinib, even though, in most cases (up to 50%), it co-occurs with other EGFR resistance mutations [Another utations ,74,79. Outations .Given that the C797S mutation is the most frequently acquired resistance mechanism to osimertinib, fourth-generation EGFR TKIs are currently being developed. We describe some of the most relevant in the following section.EGFR co-occurring mutations [EAI045 is the first allosteric reversible, non-ATP competitive inhibitor targeting L858R/T790M/C979S utations . In vitrutations . EAI045 utations . No clinJBJ-04-125-02 is a reversible, non-ATP competitive allosteric inhibitor that was effective in preclinical models harboring the L858R/T790M/C979S mutations in combination with osimertinib, leading to increased apoptosis and an inhibition of cellular growth. JBJ-04-125-02 lacks binding affinity against exon19del; therefore, it is ineffective in this situation . At the EGFR del19/T790M/C797S, L858R/T790M/C797S, del19/T790M, L858R/T790M, del19, and L858R mutations [CH7233163 is a non-covalent ATP-competitive inhibitor, which has shown in vitro and in vivo activity overcoming the utations . HoweverBLU-945 is a potent, selective EGFR inhibitor with activity against del19/T790M/C797S, L858R/T790M/C797S, del19/T790M, and L858R/T790M in in vitro and in vivo assays ,27. The Early evidence is available for other inhibitors. TQB3804 is a drug that effectively inhibited del19/T790M/C797S, L858R/T790M/C797S, del19/T790M, and L858R/T790M in in vitro and in vivo assays . TRE-069EGFR mutant tumors to transform into small cell lung cancer (SCLC) are unknown; limited data due to the lack of tissue availability makes it difficult to understand this phenomenon [The underlying mechanisms that cause a number of enomenon . Its incenomenon ,85,86.Tissue biopsy at the time of progression to osimertinib remains of paramount importance in order to identify this transformation; however, in several cases, elevated serum levels of neuron-specific enolase can be found and suggest this phenomenon .EGFR mutation [EGFR expression levels drop, which explains why these tumors are not sensitive to EGFR-TKI therapy [TP53 (91%), Rb1 (58%), and PIK3CA (27%). Interestingly, in this study, these mutations were not described prior to histologic transformation [Consistently, most of the transformed tumors maintain the original activating mutation . However therapy . Common ormation .EGFR-mutated advanced NSCLC, a higher risk population for this transformation into SCLC has been defined by the presence of the concurrent loss of both TP53 and RB1. This population represents approximately 5% of all EGFR mutated tumors, and its incidence of transformation is 18%, in contrast to EGFR mutant tumors without mutation on RB1 and TP53, representing 3% of the cases. These patients have worse outcomes, with median OS in a retrospective cohort of 29.1 months compared to 56.4 months of patients with preserved RB1 and TP53 [Rb1 loss was significantly associated with SCLC transformation. The study also identified patients with worse outcomes as those with concurrent pretreatment TP53 and Rb1 loss [Among the population of and TP53 . A retroRb1 loss .A retrospective analysis examined a cohort of 67 patients diagnosed with transformed-SCLC, where up to 30% had received osimertinib. The most common treatment was platinum-etoposide, achieving an ORR of 54%, mPFS of 3.4 months, and mOS of 10.9 months since the transformation . InteresEGFR mutation. There was no clear molecular pattern after evolving into squamous tumors, with only one patient gaining a PIK3CA mutation.Schoenfeld et al. identified five cases of transformation into squamous cell carcinoma . The incCurrently, there are no clinical data regarding therapeutic strategies in these patients; however, a histology-based approach is recommended .MET  gene is located on chromosome 7q21-q31. It encodes for the MET polypeptide, which is processed into a glycoprotein and serves as a transmembrane receptor tyrosine kinase. MET becomes activated through the binding of the hepatocyte growth factor ligands, subsequently activating different pathways, such as RAS/ERK/MAP kinase, PI3K/AKT, Wnt/β-catenin, and STAT [The and STAT ,93.MET gene amplification has been identified as a resistance mechanism against osimertinib in HCC827 cells, which is a hypersensitive EGFR exon 19 mutant NSCLC cell line [MET amplification, as well as hyperactivation, were detected [ell line . Inductidetected  in the oMET amplifications are found between 9% and 24% of tumors that progress to osimertinib as second or later lines [MET amplification was found between 7% and 15%, representing the second most common resistance mechanism after C797X [In clinical practice, er lines ,67,79,85er C797X ,79.Multiple clinical trials are currently open to find effective strategies in this setting of unmet need, especially since osimertinib is now the current standard of treatment in the first line. Different combinations of both anti-EGFR and anti-MET drugs show promising results for these patients.EGFR-mutated and MET-amplified tumors across different clinical scenarios [The TATTON phase 1b study assessed the combination of osimertinib and savolitinib in patients with cenarios . The cohcenarios . One of cenarios .MET or EGFR/MET-based mechanism of resistance had a 50% ORR. Interestingly, the response rate rose to 90% in 10 patients with high expression by immunohistochemistry (defined as a combined EGFR and MET H-score > 400). However, of these high expressors, five of them had an unknown mechanism of resistance. The results of the general cohort showed an ORR of 36%, with an mDoR (median Duration of Response) of 9.6 months and an mPFS of 4.9 months. A cohort of the future CHRYSALIS-2 trial will try to validate these findings.The phase I clinical trial CHRYSALIS evaluated the combination of lazertinib (a third-generation EGFR TKI) and amivantamab (a bi-specific antibody that targets both MET and EGFR) in patients with osimertinib-relapsed and chemotherapy-naïve advanced NSCLC ,96. In tMET amplification [The INSIGHT 2 trial is evaluating tepotinib (a MET TKI) in monotherapy and its combination with osimertinib in patients who progressed to first-line osimertinib with an acquired fication .Several case reports have used the combination of osimertinib and crizotinib in patients whose tumors had progressed to osimertinib as a second or later line and had acquired a MET amplification. Responses in the published cases differ, with benefits ranging from 2 to 7 months ,35,97.MET alterations in the pretreatment samples. Consequently, efficacy data in this setting is currently insufficient to draw any conclusion [Responses were also seen in the clinical trial that evaluated patritumab-deruxtecan; however, only 8% of the tumors had nclusion .HER2 is a tyrosine kinase receptor that belongs to the EGFR family. Among the different HER family proteins, HER2 has the strongest catalytic kinase activity ,99. Its In vitro studies have developed the HER2 exon 16 skipping (HER2D16) in HEK293 cells (human embryonic kidney cell line)  and H197HER2 aberrations have been identified as acquired mechanisms of resistance to osimertinib. In the first-line therapy, amplification was detected in 2% of the cases and HER2 mutations in 1% [HER2 amplifications (5%), HER2 amplifications co-occurring with EGFR L792X + C797X + PIK3CA amplification (1%), HER2 amplifications and EGFR G796S + MET amplification (1%), and HER2 amplifications and PIK3CA amplifications (1%) [HER2 deletion [HER2 amplification [In the clinical setting, ns in 1% . In secoons (1%) . The mosdeletion ,101,102.fication ,101, howHER2 alterations (mutations or amplifications) and benefited from the drug in terms of response [In the clinical trial evaluating patritumab-deruxtecan, some patients presented with response .EGFR mutations, including lapatinib, neratinib (HER2 and EGFR inhibitors) [Different targeted therapies against HER2 have been tested in NSCLC without co-occurring ibitors) ,37,103, RET proto-oncogene encodes a receptor tyrosine kinase (RTK). Its activation can develop as a consequence of gain of function amino acid substitutions and genomic rearrangements, leading to the formation of fusion proteins; RET fusions are usually generated by pericentric and paracentric inversions of chromosome 10 [RET alterations frequently coexist with other genomic alterations such as TP53, cell cycle-associated genes, the PI3K pathway, and mitogen-activated protein (MAP) kinase effectors [RET region of exon 11 to intron 11, which are sites of fragile DNA secondary structures [The osome 10 ,105. RETffectors . In the ffectors . Other Rffectors ,106,107.ructures . Liquid ructures .BRAF is a member of the RAF family of serine/threonine kinases. It is a part of the MAP kinase pathway, and its signaling follows downstream from BRAF to MEK 1 and 2 and ERK, which further phosphorylates multiple molecules .BRAF G469A as a resistance mechanism against osimertinib in PC9 cells (EGFR exon 19 delE746-A750). The cells were exposed to increasing concentrations of osimertinib (ranging from 10 to 500 nM) for nine months. When identifying possible resistance mechanisms, EGFR, HER2, or MET alterations were not identified, however, a BRAF G469 mutation was detected. The study observed that this mutation maintained the activity of the MAP kinases pathway. Combination treatment with osimertinib plus selumetinib or trametinib (both MEK 1/2 inhibitors) was effective in restoring the sensitivity of osimertinib to resistant BRAF G469A mutated cells [In vitro studies have identified mutations in ed cells .BRAF alterations were reported as a mechanism of resistance to osimertinib in 3% of the cases (BRAF V600E mutation). This mutation co-occurred with MET amplification and FGFR3-TACC3 fusion, with concurrent EGFR C797X mutation; other co-occurring alterations included MET amplification with BRAF V600E and CDK6 amplification [EGFR exon 19del/T790M, which had progressed to osimertinib, with acceptable tolerance to the treatment [In the clinical trial AURA3, fication ,109,110.fication . A combireatment . The usereatment .BRAF fusions represent approximately 2% of the cases of acquired resistance to osimertinib; described fusions include PJA2-BRAF, MKRN1-BRAF, and AGK-BRAF. The latter was shown to develop in the primary tumor but was absent in a metastatic lesion, highlighting the impact of clonal heterogeneity in resistance mechanisms [EGFR exon 19del and an AGK-BRAF fusion, observing a partial response to this treatment [chanisms ,113. Thereatment .KRAS is a member of the membrane-bound family proteins RAS. It possesses inherent GTPase activity. RAS can activate different effector molecules, such as RAF and the MAP kinase pathway, as well as PI3K, ultimately activating mTOR .NRAS E63K mutation. As well as NRAS G12V and G12R, KRAS copy number gains were also detected. PC9 cells were chronically treated with escalating concentrations of osimertinib and a single concentration of osimertinib; different models were used, exposing cells to a final concentration of osimertinib of 160 nM and 1500 nM in the different models. The treatment combination of osimertinib with either selumetinib or an Aurora kinase b inhibitor was effective in overcoming resistance to osimertinib [In vitro studies have detected RAS alterations as a resistance mechanism against first-line osimertinib in PC9 cells, developing an mertinib .KRAS mutations were reported as acquired resistance mechanisms in 1% (KRAS G12D) and 3%  in the AURA3 and FLAURA trials, respectively [KRAS mutations, including G12D, G12S, G61K, and Q61R, as well as an amplification of CDK4/KRAS/MDM2 [In the clinical setting, ectively ,66. OtheRAS/MDM2 ,118,119.RAS/MDM2  or adagrRAS/MDM2 , could bHER2 amplification, CCND2 and CCNE1 amplifications. PIK3CA E545K was the most frequent mutation detected [PI3K is activated through different upstream pathways involving tyrosine kinases, G coupled proteins, and RAS-related GTPases. Its activation can lead to diverging downstream pathways, such as Akt, TEC family tyrosine kinases, and mTOR . PIK3CA detected . In the detected . An in vdetected . A studydetected ,47.CCND, CCNE, and CDKN, among others [Cyclin D-dependent kinases (CDK4 and CDK6) are major oncogenic drivers; their sustained activation leads to cancer cells entering the cell cycle repeatedly by producing G1-S phase transitions and reducing the duration of the G1 phase. Genes associated with CDKs include g others .EGFR L858R and T790M mutations) through an increased expression of CDK4. Osimertinib resistance was achieved by exposing the cells to osimertinib concentrations from 5 nM to 1.5 µM for 22 weeks. Subsequently, H1975 OR (osimertinib resistant) cells were developed. No alterations in MET, KRAS, BRAF, MEK, or PI3K were detected, and neither C797X nor T790M loss was identified. H1975 OR cells had fewer G1 phase and more G2 phase cells than H1975 cells [Cell cycle alterations have been described in in vitro studies as a resistance mechanism to osimertinib in H1975 cells , including TYRO-3 and MER. AXL ligand is Gas6, which binds to the ectodomain of AXL; its activation leads to cellular growth, proliferation, motility, and invasion, involving different signaling pathways, such as MAP kinase and PI3K/Akt, among others. Furthermore, AXL has been implicated in the process of epithelial-mesenchymal transition ,126.MET amplification, as well as an AXL upregulation, as a mechanism of acquired resistance in EFGR TKI resistant NSCLC cell lines [In vitro studies have identified a concurrent ll lines ,128. Furll lines . A studyEGFR exon 19 delE746-A750), PC9 cells, H1975 cells, and HCC4006 cells  [MET amplification and AXL upregulation identified by Sanger sequencing [MET amplification and AXL expression were detected; however, T790M, C797S mutations of KRAS, NRAS, BRAF, and TP53 mutations were not detected [AXL activation has been described as an acquired resistance mechanism to first-line osimertinib in HCC827 cells  ,59,60. Hquencing . In anotdetected .AXL overexpression has been reported as an acquired mechanism of resistance to first- and second-generation EGFR TKIs, as well as to second-line osimertinib ,130. In Enapotamab vedotin and ADC specific against AXL has shown activity in an in vivo model of osimertinib resistant NSCLC  and is cThe IGF-1 receptor belongs to the insulin receptor family and has roles in cell growth and differentiation. It can be activated by IGF1, IGF2, and insulin .EGFR, MET, KRAS, MEK, BRAF, and PIK3CA, was performed. No alterations were detected; however, the IGF-1 receptor was detected to be overactivated in these cells [Activation of the IGF-1 receptor as an acquired resistance mechanism to osimertinib was described in in vitro studies using PC9 and H1975 cells. PC9 cells were exposed to gefitinib over 6 months, developing resistance through the T790M mutation. Subsequently, these cells were cultured with stepwise escalation to osimertinib in concentrations ranging from 150 nmol/L to 1 µmol/L over 6 months, thus developing osimertinib resistant cells. H1975 cells were exposed to osimertinib using the high-concentration method, culturing cells with 1µmol/L osimertinib for 3 months. Whole exome sequencing of several genes, including se cells .EGFR mutated osimertinib resistant and proposed as a mechanism of acquired resistance. Furthermore, treatment with linsitinib (an IGF-1 receptor inhibitor) and osimertinib restored sensitivity to osimertinib in in vitro studies [Activation of the IGF-1 receptor through IGF-2 overexpression has been identified both in clinical specimens and in cell lines of NSCLC  studies ,133. We EMT is a cellular process involved in different types of cancer, allowing cells to enhance invasive capacity, cancer stem-cell similar properties, as well as resistance to treatments. In this process, epithelial cells lose their cell polarity and cell-to-cell adhesion, including a downregulation of epithelial proteins, such as E-cadherin, and acquire mesenchymal characteristics, including increased migration and invasion properties, and an upregulation of proteins, such as N-cadherin and vimentin. EMT develops through the involvement of different proteins and pathways, including TGF-β, SMAD and MAP kinase pathways, induction of IGF-1 receptor, and Notch signaling, among others ,135,136.In vitro studies using H1975/AR cells (gefitinib resistant) were exposed to osimertinib through a stepwise escalation process, later detecting a decreased E-cadherin and increased vimentin expression, as well as the capability to form larger spheroids . In a diOther mechanisms of resistance to osimertinib have been described in in vitro studies, including the Src-AKT pathway and EGFR wild-type amplification in PC9 and H1975 cells. The cells were cultured with osimertinib at an initial concentration of 0.3 µmol/L and incrementally increased to 1 µmol/L. After several months of exposure, osimertinib resistant cells were developed .Other reported resistance mechanisms in the clinical setting to first and second-line osimertinib include fusions of SPTBN1-ALK, EML4-ALK, FGFR3-TACC3, NTRK1-TMP3, and GOPC-ROS1, among others ,138,139.EGFR mutations. Despite the long duration of response with osimertinib, eventually, all patients will progress. Up until now, chemotherapy remains the standard treatment in this setting. However, in the advent of new diagnostic genomic platforms, a myriad of diverse mechanisms of resistance to osimertinib have been identified. Different molecular mechanisms can coexist, as well as histologic transformation and molecular derangements. It is therefore of paramount importance to assess these genomic and phenotypic changes through a rebiopsy when feasible and alternatively using liquid biopsies. One of the main challenges when multiple alterations are found is to elucidate which mechanism is driving the resistance to efficiently target that mechanism and improve the chances of therapeutic success. In vitro studies can contribute to this. Another important challenge is that the mechanisms of resistance are diverse, thus, making it complex to design clinical trials encompassing all possible scenarios. Umbrella trials such as ORCHARD are good examples of maximizing the efficiency to address different mechanisms of resistance in one trial.Osimertinib has become the standard of care in the first-line setting for patients with advanced lung cancer and sensitizing Another strategy is designing drugs that can potentially overcome different mechanisms of resistance, such as patritumab-deruxtecan or even resistance mechanisms to different drivers, e.g., the phase 1 TROPION-PanTumor01 study. In this trial, datopotamab deruxtecan, an anti-TROP2 ADC, was used to treat patients with NSCLC and targetable genomic alterations (including EGFR mutation and ALK and RET fusions) who had progressed to prior treatment options (82% of patients had received ≥3 lines). The ORR was 35%, and the median DoR was 9.5 months, thus, positioning datopotamab deruxtecan as an attractive option in the future . The outUpfront combinations of osimertinib with chemotherapy or other targeted agents might also result in the delay of the emergence of resistance, but still, we need to pursue curative strategies in this scenario.To date, there are no available data on mechanisms of acquired resistance to osimertinib in the adjuvant setting; however, the increasing use of osimertinib in this early disease setting will probably lead to changes in the profile of resistance.The technological advances, along with the creative designs of clinical trials in these infrequent subpopulations, will be key to improving the outcomes of patients with EGFR mutant NSCLC.


Case Presentation. Case 1: A 60-year-old Chinese woman presented with a recent onset of worsening of blood pressure control and clinical features of Cushing's syndrome. Investigations confirmed ACTH-independent endogenous hypercortisolism, and a CT abdomen showed a 6 cm right adrenal mass. Twenty-four-hour urine steroid profiling revealed co-secretion of adrenal androgens and atypical steroid metabolites. Laparoscopic right adrenalectomy was performed, and pathology of the tumor was classified as an ACA by the Weiss scoring system. Four years later, the patient presented with an abrupt onset of severe hypercortisolism and was found to have a metastatic recurrence in the liver and peritoneum. The patient received a combination of mitotane, systemic chemotherapy, and palliative debulking surgery and succumbed 8.5 years after the initial presentation due to respiratory failure with extensive pulmonary metastases. Case 2: A 68-year-old Chinese woman presented with acute bilateral pulmonary embolism and was found to have a 3 cm left adrenal mass. Hormonal workup confirmed ACTH-independent endogenous hypercortisolism, and laparoscopic left adrenalectomy revealed an ACA according to the Weiss scoring system. Five years later, she presented with recurrent hypercortisolism due to hepatic and peritoneal metastases. The patient had progressive disease despite mitotane therapy and succumbed 7 years after initial presentation.  Adrenocortical carcinoma (ACC) is a rare endocrine malignancy. An accurate diagnosis of ACC is of paramount importance as it greatly impacts the management and prognosis of a patient. However, the differentiation between early stage, low-grade ACC and adrenocortical adenoma (ACA) may not always be straightforward. The recommended classification system, namely, the Weiss scoring system, is not without flaws. We herein report two cases of ACC which were initially diagnosed as ACA according to the Weiss scoring system but developed distant metastases in subsequent years.  Although the Weiss scoring system is recommended as the reference pathological classification system to diagnose adrenocortical carcinoma, there remain tumors of borderline malignant potential which may escape accurate classification. Various alternative classification systems and algorithms exist but none are proven to be perfect. Clinicians should recognize the potential limitation of these histological criteria and scoring systems and incorporate other clinical parameters, such as the pattern of hormonal secretion, urinary steroid profiling, and radiographic features, to improve the prognostication and surveillance strategy of these tumors. Adrenocortical carcinoma (ACC) is a rare, highly aggressive malignancy with a reported annual incidence of 0.7–2 per one million people, while adrenocortical adenoma (ACA) is one of the commonest adrenal tumors and is benign , 2. An aPatient A, a 60-year-old Chinese lady, who had a history of gastroesophageal reflux disease, hypertension, and hyperlipidemia, presented with insidious onset of right loin pain for 3 months in June 2012. She also had worsening of blood pressure control over the past few months and occasional palpitations. Physical examination was notable for moon face and buffalo hump suggesting Cushing's syndrome. Computed tomography (CT) of the abdomen performed for suspected urinary tract pathology showed a 6 cm right adrenal mass . SubsequThe patient underwent laparoscopic right adrenalectomy in August 2012. The pathology of the resected tumor was reported to be ACA. Postoperatively, hypertension was cured and antihypertensives were stopped. A follow-up CT at 9 months after the operation did not show any evidence of recurrence, and urine steroid profiling (USP) at 1.5 years postoperatively showed normalization of the previously abnormal steroid metabolites together with normal free cortisol and androgens. She also developed adrenal insufficiency and was maintained on hydrocortisone replacement postoperatively until 2015.18F-fluorodeoxyglucose positron emission tomography/computed tomography (18FDG-PET/CT) scan showed a large solid mass at the right lobe of the liver , a small nodule around the previous right adrenalectomy bed (SUVmax 2.1), and multiple foci of hypermetabolic peritoneal lesions in both sides of the abdominal cavity (highest SUVmax 4.6)  and biochemical hyperandrogenism . 18F-flumax 4.6) . A laparmax 4.6) . A pathoWe retrospectively reviewed the histology of the right adrenal tumor which was removed in August 2012 and compared it with that of the peritoneal nodule. The morphological appearance of the previous adrenal tumor shared a focal resemblance with that of the peritoneal nodule, suggesting that the current tumor might be arising from the original one, which would have been a well-differentiated ACC and had presently recurred and metastasized. The focal myxoid change was observed, and mitosis was up to 4/50 high power field (HPF). Atypical mitosis, necrosis, broad fibrous bands, vascular invasion, or capsular invasion was not identified. Although frankly malignant features were not present in the original adrenal tumor, the presence of a diffuse growth pattern and <25% clear tumor cells , which wMitotane was initiated once the diagnosis was made in July 2016 with the dose titrated to maintain a target mitotane level of 14–20 micrograms/ml. A follow-up CT abdomen 4 months later showed progressive disease with enlarging liver metastasis (up to ∼10 cm) and peritoneal nodules. Chemotherapy with etoposide (EP) and cisplatin was added in January 2017, and follow-up CT 2 months later showed a reduction of peritoneal metastases and stable liver metastasis. EP was continued for five cycles until May 2017, but in July 2017, progressive disease was noted again on interval imaging with enlarging liver metastasis. At the same time, the patient became highly symptomatic with marked hypercortisolism which was uncontrolled despite high-dose mitotane (up to 4.5 gm/day) and metyrapone (up to 6 g/day).AKT1, AKT3, ALK, ARAF, AURKA, BAP1, BRAF, BRCA1, BRCA2, CBFB, CCND1, CCND2, CCND3, CCNE1, CD79A, CD79B, CDH1, CDK4, CDK6, CDKN2A, CSF1R, CTNNB1, DDR2, EGFR, ERBB2, ERBB3, ERBB4, ESR1, EZH2, FGFR1, FGFR2, FGFR3, FLT1, FLT3, FLT4, GNA11, GNAQ, HGF, HIF1A, HRAS, IDH1, IDH2, IGF1R, JAK1, JAK2, JAK3, KDM6A, KDR, KIT, KRAS, LRRK2, MAP2K1, MAP2K2, MAP2K4, MAP3K1, MAP3K9, MAPK1, MAPK3, MET, MPL, MST1R, MTOR, MYD88, NF1, NOTCH1, NOTCH2, NOTCH3, NRAS, NRG1, NTRK1, NTRK2, NTRK3, PAK6, PDGFRA, PIK3CA, PIK3R1, PTCH1, PTEN, PTK2, PTK2B, RAF1, RET, RHOA, ROS1, SMO, SRC, STK11, SYK, TOP1, TSC1, and TSC2. The postoperative course was complicated with bilateral subphrenic collection, left pleural effusion, pulmonary embolism, and steroid withdrawal syndrome which necessitated several drainage procedures, anticoagulation, and prolonged hospitalization for 3 months. After extended rehabilitation, the patient gradually regained independent self-care and functional ability.A decision on palliative right hepatectomy and peritoneal nodule resection was made after multidisciplinary discussion and the operation was performed in September 2017. Further immunohistochemical studies showed intact immunoreactivity of mismatch repair proteins  and less than 1% PD-L1 expression. Targeted next-generation sequencing on extracted tumor cell DNA did not reveal any variants or small insertion/deletion in the following genes: In April 2018 , Patient A was found to have bilateral lung metastases. Chemotherapy was resumed with EP and subsequently the addition of doxorubicin  in view of progression in October 2018. She completed 6 cycles of EDP in May 2019 and enjoyed stable disease until August 2019, when she had disease progression with increasing pulmonary and peritoneal metastases. Gemcitabine and carboplatin were initiated as second-line palliative chemotherapy in September 2019. However, disease progression was again noted after 3 cycles, and chemotherapy was stopped. Patient A was then offered palliative care and she subsequently passed away due to respiratory failure with extensive pulmonary metastases in January 2021, almost 5 years after the relapse of the disease and 9 years after her initial presentation.Patient B, a 68-year-old Chinese lady, whose past medical history was remarkable for hypertension, presented to us in August 2011 for acute shortness of breath, chest discomfort, and transient loss of consciousness. She was later found to have extensive bilateral acute pulmonary embolism and was treated with intravenous thrombolysis followed by anticoagulation . After sLaparoscopic left adrenalectomy was performed in May 2012. Intraoperatively, a 5 cm left adrenal tumor was found and removed. Histologically, the lesion consisted of nests and trabeculae of medium-sized tumor cells and a rich capillary network. The tumor cells were polygonal, with pale to amphophilic cytoplasm, as well as round, deeply stained nuclei with nuclear pleomorphism . The maxHowever, in November 2016, the patient was noted to have the reappearance of Cushing's syndrome with central obesity and buffalo hump. Repeat hormonal testing showed relapse of endogenous ACTH-independent Cushing's syndrome as well as the presence of markedly elevated 17-hydroxyprogesterone (17-OHP) metabolites that were not seen in the previous urinary steroid profiling. An abdominal CT showed local recurrence of a 2 cm nodule at the left adrenal bed, a 4 cm liver mass, multiple peritoneal nodules, and recurrent pulmonary embolism . Apixaba18FDG-PET/CT 8 months later (in October 2017) showed a mild interval reduction of liver metastasis  and risk of open surgery, while percutaneous radiofrequency ablation was deemed not feasible anatomically. Mitotane was thus started as monotherapy in February 2017 and titrated gradually to 3 g daily, which was the maximally tolerated dose to achieve a mitotane level of approximately 11 micrograms/ml. Follow-up tastasis  togethertastasis . SystemiHistopathology remains the gold standard for diagnosing ACC and should be obtained in all patients . DespiteThe imperfection of the Weiss scoring system has instigated the development of a more unequivocal pathological classification system over the past three decades. In 2002, Aubert et al. modified the Weiss scoring system to include only five of the original nine items with a maximum score of 7. Being easier to use in clinical practice and more reproducible, the modified Weiss score has a reported specificity of 96.9%  , 4, 14. The diagnostic difficulty of ACC is well exemplified in the present two cases where the features of overt malignancy were not evident pathologically. The presence of only a diffuse growth pattern and <25% clear cells of the tumor volume in Patient A, and the presence of borderline mitosis of 5/50 HPF and regressive changes (fibrosis and hemorrhage) in Patient B would not be sufficient to classify either tumor as ACC under the Weiss scoring system or other systems Tables  and 5. OApart from the above histopathological clues that speak for the potential malignant behavior of an adrenocortical tumor, several clinical, radiological, and biochemical features in our present cases should have been recognized as incongruent with a simple benign ACA in retrospect. These included the large size of the tumor on presentation, an imaging phenotype inconsistent with adenoma, and co-secretion of adrenal cortisol and androgens and their precursors. The presence of both having a diameter greater than 4 cm and a CT tumor attenuation >10 HU are shown to be highly sensitive to adrenal malignancy –28. A moThe pattern of steroid secretion may also indicate if an adrenal lesion is an ACC as disorganized steroidogenesis is almost always observed in ACC. Co-secretion of androgens and cortisol, secretion of immature steroid precursors, or secretion of estradiol in males are highly suspicious for ACC . In receα, 16α, 20α-pregnenetriol and 3β, 16α, 20α-pregnenetriol to be highly specific for ACCs in addition to THS and these steroid metabolites were also observed in Patient A. USP can identify residual or recurrent ACCs even before the disease becomes radiologically apparent and is useful in the surveillance and monitoring of recurrent disease  and chromatin remodeling ), alterations in methylome and transcriptome, miRNAome, and chromosomal aberrations are emerging tools for further prognostication of ACC but yet to be incorporated into clinical practice due to the resource-intensive nature of pan-genomic bioinformatic analysis [In terms of treatment, complete surgical removal is the only curative therapy for localized ACC. In patients with unresectable tumors or incomplete tumor resection, the prognosis is significantly worse , 9, 40. analysis , 40–44. analysis . Despiteanalysis .p < 0.001) and no effect on overall survival as compared to streptozocin plus mitotane  [s disease control rate of 23% were observed in 39 patients over a median follow-up of 17.8 months [For patients who develop recurrent disease at least 12 months beyond the initial surgery, complete surgical removal of the recurrence is still recommended if it is achievable , 45. At  p=0.07) . EP comb p=0.07) . This re p=0.07) . Other s p=0.07) , 49, 50.8 months . Other p8 months .Adrenocortical carcinoma is an aggressive endocrine cancer in which therapeutic options are often limited and prognosis becomes gloomy once metastatic disease is manifested. It is also a heterogeneous disease with varying clinical behavior, where a small subset of patients harboring low-grade well-differentiated tumors may demonstrate a more indolent clinical course . It shou


The two (11- and 14-color) antibody combinations allowed for robust and sensitive detection of 19 and 23 IMC populations, respectively. Highly reproducible identification and enumeration of IMC populations was achieved, independently of anticoagulant, type of FCM instrument and center, particularly when database/software-guided automated  gating was used. Whereas no significant changes were observed in identification of IMC populations for up to 24h delayed sample processing, a significant impact was observed in their absolute counts after >12h delay. Therefore, accurate identification and quantitation of IMC populations requires sample processing on the same day. Significantly different counts were observed in PB for multiple IMC populations according to age and sex. Consequently, PB samples from 116 healthy donors (8-69 years) were used for collecting age and sex related reference values for all IMC populations. In summary, the two antibody combinations and FCM approach allow for rapid, standardized, automated and reproducible identification of 19 and 23 IMC populations in PB, suited for monitoring of innate immune responses in clinical and translational research settings.Innate myeloid cell (IMC) populations form an essential part of innate immunity. Flow cytometric (FCM) monitoring of IMCs in peripheral blood (PB) has great clinical potential for disease monitoring due to their role in maintenance of tissue homeostasis and ability to sense micro-environmental changes, such as inflammatory processes and tissue damage. However, the lack of standardized and validated approaches has hampered broad clinical implementation. For accurate identification and separation of IMC populations, 62 antibodies against 44 different proteins were evaluated. In multiple rounds of EuroFlow-based design-testing-evaluation-redesign, finally 16 antibodies were selected for their non-redundancy and separation power. Accordingly, two antibody combinations were designed for fast, sensitive, and reproducible FCM monitoring of IMC populations in PB in clinical settings  and translational research . Performance of pre-analytical and analytical variables among different instruments, together with optimized post-analytical data analysis and reference values were assessed. Overall, 265 blood samples were used for design and validation of the antibody combinations and   Whilenditions –12. Thisnditions –24.+ myeloid dendritic cells (myDCs) are now known to include different functional subsets, that can be identified based on CD14 expression (CD14- non-inflammatory and a CD14lo pro-inflammatory CD1c+ myDC population)  has been described recently, which was previously included in the plasmacytoid dendritic cell (pDC) population and exhibits mixed gene expression and functional profiles between pDCs and myDCs   and CD5:onocytes , 42. In nd myDCs . In para1+ myDCs , 43.In recent years, different 8-12 to 38 color panels have been designed and proposed for monitoring monocytic and DC populations in PB by flow cytometry (FCM) and mass cytometry, respectively , 44–46. Here we designed and validated two (11- and 14-color) antibody panels for standardized, automated, and reproducible identification of 19 to 23 IMC populations in human blood by FCM, and provide age and sex-matched reference values for more objective interpretation of altered IMC profiles in multicentric clinical settings. Ultimately, the antibody panels developed will allow to set a new benchmark for IMC in both clinical and translational research settings.For this study, 261 PB samples (195 ethylenediaminetetraacetic acid -EDTA- and 66 sodium heparin-anticoagulated) obtained from 205 healthy donors (HD) were evaluated. From them, 242 samples from 197 donors were used for antibody panel development and evaluation . For assessment of the technical performance of the antibody panels, construction & validation of the reference database for automated gating  57 sampl7 cells, employing the antibodies (www.EuroFlow.org ).Samples were processed within 4 hours (h) after collection, according to the EuroFlow bulk lysis and sample preparation and staining standard operating procedures (SOP) , 53 for Stained cells were stored at 4°C and measured within 1h by FCM. Absolute counts were assessed employing a double platform method based on quantitation of nucleated cells obtained in the Sysmex XP-300 automated hematological analyzer .in vitro activation assays were performed using sodium heparin anti-coagulated PB, as described elsewhere (2 in the presence of 100 ng/ml of lipopolysaccharide (LPS) (Sigma-Aldrich). For those experiments in which intracellular detection of cytokines was performed, 10 μg/ml of Brefeldin A (Sigma-Aldrich) was added to block cytokine secretion. For each condition, an unstimulated aliquot of the same sample was processed in parallel in an identical way. Stimulated PB samples were then stained with a panel of monoclonal antibodies (MoAb) (www.EuroFlow.org) as previously described (Short-term lsewhere . Brieflyescribed .6 cells were measured using LSR Fortessa (Becton Dickinson Biosciences (BD), San José, CA) instruments equipped with 4 lasers  or a 3-laser  Aurora  instrument. BD Fortessa flow cytometers were set-up at each center according to the EuroFlow guidelines (www.EuroFlow.org) and calibrated daily by use of BD™ Setup and Tracking (CS&T) beads (BD Biosciences), their performance being checked daily by acquisition of SPHERO™ Rainbow calibration particles . Calibration and daily quality control of the Aurora flow cytometer was performed according to the SOP recommended by the manufacturer. For data analysis, the Infinicyt™ software  was used. Gates were defined based on internal negative and fluorescence-minus-one (FMO) controls, for general population identification and immunophenotypic characterization, respectively.For each sample evaluated, 2.5 to 5 x 10PRP – MFI NRP)/2 x rSDNRP; where MFI, median fluorescence intensity; PRP, positive reference population; NRP, negative reference population; rSD, robust standard deviation], as previously described  .In a subsequent step, selection of the minimum set of the most informative markers for identification of additional subsets of IMC was performed per cell population, e.g., pDCs, myDCs, Axl (MDSCs) Table 1, (MDSCs) . For Axl+ CD14- myDCs; ii) CD34 for identification of hematopoietic precursor cells (HPCs) and CD100+ CD34int pre-DCs; and iii) CD192 for identification of M-MDSCs. Additionally, the fluorochrome conjugated to CD62L was changed to minimize its spread on the FcεRI channel, as required for clear cut subsetting of cMos  (assessed in 7 paired PB samples stained with versions 2 (n=3) and 3 (n=4) of the IMC tube; vs. delayed   and fresh vs. frozen  staining of (EDTA anti-coagulated) PB samples. To compare the performance of the EuroFlow IMC tube in different instruments , PB samples from 5 donors were stained with version 4 of the tube  for different anti-coagulants  was determined in duplicates of 5 EDTA-anticoagulated PB samples, processed in parallel  sample files from 6 adult HD (stained with version 3 of the EuroFlow IMC tube) were independently analyzed in parallel by an experienced (expert 1 – E1) and a novice (expert 2 – E2) flow cytometrist. Intra-operator variability was assessed for E1, who analyzed the files twice within a time lapse of ≥2 months.http://periscope-project.eu/): LUMC (n=5), USAL (n=8), RIVM (n=2), and UTU (n=3). Flow cytometry data files from those 18 samples that fulfilled all the selection criteria (described in detail in Supplementary Methods) were then merged into a single data file used as database tool, implemented in Infinicyt™ (Cytognos) , a second set of PB samples from HD (n=6), processed and acquired at LUMC (n=3) and USAL (n=3), was prospectively used. For these samples, analysis was repeated at two different timepoints set ≥2 months apart from each other.For construction of the database for automated analysis of the 11-color version of the EuroFlow IMC tube  . For val+/CD14-/CD16- cell compartment (vs. CD123 (26.3%) and CD304 (14.6%)      , 60–62. profile) . Likewisint CD100hi immunophenotype  and functional characteristics of myDCs   and pDCs (p<0.003 for IL1β and IL8)   . Therefovs. mature neutrophils, PCA-based evaluation of the performance of the IMC tube extended backbone  vs. the extended backbone plus CD11b, CD15 and CD66b, for identification of different polymorphonuclear (PMN) cells, including immature neutrophils, was performed. Of note, combined usage of cell size (forward scatter – FSC-) and internal complexity (side scatter – SSC-) plus CD45 allowed for clear separation of granulocyte and lymphocyte populations. Likewise, the SSChi CD16- CD33lo CD62Lhi, SSClo CD16- CD33hi CD62Lhi and SSChi CD16hi CD33lo CD62Lhi phenotypic profiles allowed clear discrimination among eosinophils, basophils, and mature neutrophils, respectively, as well as their distinction from SSCint CD16-/lo CD33+ CD62L-/lo immature neutrophils, with no clear added value of the other myeloid markers evaluated  neutrophils. Interestingly, neutrophils could be further subclassified based on expression of CD16 and CD62L (hi CD62L+), a phenotype previously reported to be associated with segmented neutrophils , myelocytes (CD11b+) and metamyelocytes (CD16lo/+)  . As expeD16lo/+) , signifi+ CD11b+ (or CD33+) CD15− and HLA-DR−/lo cells  up-regulation of CD16 and down-regulation of CD123 and CD192 on CD14+/HLA-DR-/lo cells vs. cMos -certified instruments , and an extended 14-color version , 9 populations of monocytes  and 5 populations of DC  , no significant differences were observed in the stain index of individual markers between samples collected with these two anticoagulants (data not shown). However, despite the lower CD300e expression on heparin-anticoagulated samples, multivariate PCA analyses revealed no significant impact on the overall discrimination of the distinct populations of IMCs (+ CD14lo myDC observed in heparin samples (p=0.02)  is required, which might affect the staining patterns and quantification of IMC populations , staininvs. delayed sample preparation and staining for 6h, 12h and 24h with the EuroFlow IMC tube, similar stain index values were observed for all markers evaluated (data not shown), except for CD16  and Slan , also confirmed by PCA analyses, as all populations evaluated for all timepoints tested clustered within one standard deviation of the 0h staining pattern (vs. 0h >10% for 62.5% (15/24) of the populations evaluated  when sample preparation was delayed for >12h  DC populations (Regarding immediate vs. frozen PB mononuclear cells (PBMCs) revealed that, despite freezing induced a significant (>15%) reduction in the MFI of PRP for Slan (-24.8% ± 5.7%), CD192 (-28.4% ± 3.9%), CD5 (-33.0% ± 5.0%) and CD62L (-52.2% ± 10.1%), with the exception of immature neutrophils, which could not be detected in frozen samples, all IMC populations could be identified in both conditions  were stained and measured in the same instrument and analyzed manually by an expert cytometrist. Overall, an average intra-assay %CV of 5.0% ± 4.5% was observed across the 26 populations evaluated, with 80.8% (21/26) of the populations displaying an intra-assay %CV <10% and only CD36vs. spectral, and 3- vs. 4-laser flow cytometers) was evaluated. Overall, a significant correlation  was observed for virtually all  IMC populations identified, with no significant differences and a limited bias  being detected between instruments. The only exceptions were CD62L+ FcεRI+ cMos and CD36+ Slan+ ncMos which were overestimated (bias: +17.5%) and underestimated (bias: -32%) in the data files generated in the Aurora vs. Fortessa X20 instruments, respectively .To further evaluate the feasibility of using the EuroFlow IMC tube in multicentric settings, 21 samples were locally collected, processed, and measured at 4 distinct facilities  using 5 distinct instruments. PCA revealed fully comparable and reproducible results for all centers/instruments  with a limited bias - absolute mean normalized bias (MNB) <15% - in population counts was observed between the experts  (+ DCs). To establish the intra-operator variability, expert E1 repeated the analysis of the files with a ≥2-month interval. Of note, even though the overall degree of correlation increased compared to expert E1 vs. E2  and agreement , the same patterns for populations with lower degree of agreement , were observed  and novice (E2) analysts in 6 ,FCS files, stained with version 3 Table 1 vs. database-guided automated gating showed a better degree of correlation  and agreement , compared to intra- and inter-operator manual analysis  performed at two different timepoints displayed a 100% correlation and degree of agreement for the 28 (IMC and non-IMC) populations tested, which clearly improves reproducibility compared to both intra- and inter-operator manual analysis.Comparison of manual expert-based analysis Table 3,+ Slan- ncMo, CD141+ myDCs, HPC, M-MDSC and preDCs  showed reduced numbers until the age of 10-17y with a rise again in young adults (18-39y) and stable numbers thereafter (+ Slan+ ncMos (- FcεRI- cMos (+ FcεRI- cMos peaked in young adults (18-39y), while they were reduced in older individuals (>55y) (+ CD14- myDCs showed a decrease until 18-39y and remained stable thereafter (- and CD62L+) displayed similar kinetics, peaking in middle aged adults (40-55y) and declining in older adults (>55y) , whereas CD62L- immature neutrophils (p=0.004) and CD62L- cMo populations  were significantly increased in men vs. women.When evaluating the potential impact of sex on the distribution of different IMC populations in PB, significantly lower mature neutrophil counts were observed in younger adults (18-39y) populations of monocytes and DC , 44–46.+ DCs and preDCs: an 11-color tube (with 13 antibodies), compatible with CE-IVD certified FCM instruments which identifies 19 IMC populations, and an extended 14-color variant (with 16 antibodies), allowing identification of 4 additional, less frequently reported, IMC populations. For fast translation to diagnostic laboratories, we evaluated the impact on both IMC population phenotypes and counts in PB, of different anticoagulants, immediate vs. delayed sample preparation and the usage of distinct types  of FCM instruments in single vs. multicenter settings. Finally, we developed a database-guided automated analysis approach for standardized data analysis and provided normal age- and sex-matched reference values as a basis for future immune-monitoring in patient care.Here, we employed for the first time a stepwise unbiased approach for the development of two alternative antibody combinations for monitoring of up to 23 different IMC populations in normal PB, including recently described populations, such as Axl+ and CD62L-), iMo and four populations of ncMos (defined based on CD36 and Slan expression). Of note, previous reports suggested that CD9 instead of CD36 might also be used for ncMo subsetting within the Slan+ compartment populations , 39, 67,partment . However(+ cells  and CD36lo expression (data not shown).In a second step, markers classically employed for identification of pDCs  , 31, 41 + myDCs (or cDC2) are comprised of functionally distinct subsets that can be discriminated based on CD14 expression (CD14lo inflammatory myDCs vs. CD14- myDCs)  CD1c+ CD14lo myDCs, ii) CD1c+ CD14- CD5 myDCs - and iii) CD1c+ CD14- CD5+ myDCs. Since both the CD14lo and CD14- CD5- subsets of CD1c+ myDCs have been recently shown to display gene expression patterns closer to monocytes  . Likewisonocytes , 42, fur+ DCs . This CD303lo Axl+ DC population also showed unique functional features both at steady-state and in response to LPS. As described by Villani et al , another Axl-expressing DC population has been reported in the literature (CD11c+ CD123lo Axl+ DCs), which exhibits an immunophenotypic profile (CD11c+ CD14- CD5+)  , 40 simiferation , suggesthi CD34int phenotype, ability to proliferate and differentiate into CD1c+ myDCs and CD141+ myDCs has been reported   gradient centrifugation step   overlapping IMC populations. Further functional, immunophenotypic, biochemical and molecular studies   , 64, but CD66b+) , as prev CD66b+) –76. In fion step , previouion step . In lineHLA-DR-) Figure 4HLA-DR-) , an incr VEGFR1) , 79, 80 vs. M-MDSCs has been previously performed. In line with earlier reports , aimed for the clinical setting, in which available IVD-certified instruments frequently have the ability to detect fewer parameters, and an extended 14-color tube (16 antibodies), that further allows identification of less frequent and/or more recently discovered IMC populations , mostly aimed at the discovery/research settings, in which instruments allowing simultaneous detection of >12 colors are more commonly available.vs. sodium heparin anticoagulated samples, although slightly lower counts of CD1c+ CD14lo myDCs might be detected in heparin samples. Similarly, no significant impact on the overall staining patterns and individual marker resolution was observed for samples stored at RT for up to 24h prior to staining, except for lower CD16 and Slan levels, according to previously reported findings for CD16 (vs. 0h) cell counts, in line with previous studies , leading to an overestimation of their counts, which might be due to the lower viability of more frequent populations, as supported by an increased percentage of cell debris, particularly at 24h. Conversely, underestimation of populations of (particularly CD36-) ncMos was observed after 12h, probably because ncMos have been reported to be more prone to spontaneous apoptosis  instruments are used in a research setting. Noteworthy, as the frequency of some of the IMC population can be as low as 0.1 cells/μL in healthy donors, to reliably and reproducibility identify and quantify these populations also in situations in which a significantly decreased frequency is observed, staining of 107 cells is recommended. While the EuroFlow IMC combination can be employed for processing of lower numbers of cells, in case of limited sample availability, the limits of detection (LOD) and quantitation (LOQ)  should be taken into account for data analysis and reporting.Further evaluation of the EuroFlow IMC tube showed a very good reproducibility both in single center, multi-instrument, and multi-center settings. Of note, the highly comparable results obtained in conventional vs. expert-based manual analysis was observed for population identification and quantification, in line with previous reports  and less labor-intensive features of AGI, further support the potential of database-guided automated analysis to reduce operator-related variability and allow for more efficient and reproducible data analysis. These features become particularly relevant in the diagnostic clinical setting and in cases where a high number of parameters and/or IMC populations are investigated , most likely associated with the physiological changes observed in puberty . In turn, other IMC populations showed modulation in older adults , potentially as a result of a skewing of hematopoiesis towards myeloid vs. lymphoid lineages, decrease in the function of neutrophil, monocytes and DCs and possibly also low-grade inflammation also known as “inflamm-aging” , similarly to what has been previously reported for neutrophils and lymphocytes , immature neutrophils and CD62Lphocytes , 91.In summary, we developed two standardized, and highly reproducible versions of the EuroFlow IMC tube, which are suitable for clinical and research/discovery studies, even in multi-instrument and multi-center settings, allowing for robust and accurate identification and quantitation of 19 to 23 IMC populations in blood. By addressing distinct  variables that might impact the reproducibility of laboratory testing, and providing normal age- and sex-related reference ranges, our study sets the basis for standardized immune-monitoring of IMC in distinct disease and treatment conditions, in the context of clinical trials and/or patient care such as in inflammatory diseases, various forms of tissue damage as well as for monitoring immune responses to infectious diseases, vaccination or immunotherapy.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Medical Research Ethics Committees United , Medisch Ethische Toetsingscommissie (METC) – LUMC, Comité de Ética de la Investigación con medicamentos del Área de Salud de Salamanca. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.in vitro activation assays. GF, provided the cord blood samples. DD, AH-D, JA, JVG, A-MB, QH, AO, and CT were involved in the multicentric validation. KP, CT, IL, BN, PD, AJ, A-MD, MB, BM, PV, GB and RG contributed to the collection, processing and/or analysis of samples of healthy donors for calculation of the reference values. AS-G, FB, A-MD, MB, BM, and RG performed the technical evaluation of the tube. AH-D, DD, and CT constructed and validated the database for automated analysis. CT, KP, and IK made the figures and performed the statistical analyses. KP, CT, and JD wrote the manuscript. All authors contributed to manuscript revision and approved the submitted version.CT, AO, and JD designed the study. KP, SB-V, DD, WB, IL, BN, PD, SK, AL, and AJ performed the flow cytometry experiments. KP and IL performed the The presented work was funded by the European Research Council under the European Union’s Horizon 2020 Research and Innovation Programme with an ERC Advanced Grant  and the IMI2 PERISCOPE project, financed by the Innovative Medicines Initiative 2 Joint Undertaking (grant number 115910). This joint undertaking receives support from the European Union’s Horizon 2020 Research and Innovation Programme, the European Federation of Pharmaceutical Industries and Associations (EFPIA), and The Bill and Melinda Gates Foundation (BMGF). This manuscript only reflects the author’s views. The Joint Undertaking is not responsible for any use that may be made of the information this manuscript contains. The coordination and innovation processes of this study were supported by the EuroFlow Consortium. The EuroFlow Consortium received support from the FP6-2004-LIFESCIHEALTH-5 program of the European Commission (grant LSHB-CT-2006-018708) as Specific Targeted Research Project (STREP).https://www.lumc.nl/research/facilities/fcf), coordinated by M. Hameetman, run by the FCF operators, D.M. Lowie, S. van de Pas, G.IJ. Reyneveld, and former FCF coordinator dr. K Schepers and former operators J.P. Jansen, E.F.E de Haas, G.M. De Roo, R.J. Mclaughlin and S.A.J. Veld  for their technical support.We would like to acknowledge and thank the LUMC Vrijwillige Donoren Service – Direct Gebruik  and donors for supplying the peripheral blood samples (request code L18.001) and the Flow cytometry Core Facility (FCF) of the Leiden University Medical Center (LUMC), The Netherlands  and a Scientific Advisor Agreement with Cytognos; all related fees and honoraria are for the involved university departments at Leiden University Medical Center and University of Salamanca. AH-D is an employee of Cytognos . Lastly, JD, CT, AO, JA, WB, KP, MB, AD, DD, and AH-D, are listed as (co)inventors on the patent “Means and methods for multiparameter cytometry-based leukocyte subsetting” , owned by the EuroFlow scientific consortium, which describes the flow cytometry panels developed in this study.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


It is of an interest to document the molecular docking analysis of fluoroquinolones and other natural and synthetic compounds with the HCV NS3 helicase. Data shows that three fluoroquinolones interacted with the NS3 helicase in the catalytic region,targeting some of the amino acids known to play a crucial role in NS3 helicase activity. Similarly, binding energy shows that the fluoroquinolones were comparable to the thiazolpiperazinyl derivatives, while superior to several of the synthetic and naturalderivatives. The results show three fluoroquinolones to be potent helicase inhibitors that can be repurposed as supplemental therapy against HCV especially in cases non-responsive to DAAAs. Hepatitis C virus (HCV) is still a major public health issue in the 20th century, responsible for about 71 million infections worldwide .The N-tThe HCVNS3 helicase crystal structures (genotype 1a: PDB ID:1A1V and genotype 1b: PDB ID: 1CU1) wereretrieved from PDB database  in .pdb We compared the efficacy of 20 previously published synthetic and natural NS3 helicase inhibitors, namely, Ring-expanded (fat) nucleoside analogs , DRBT 1, manoaliFor the analysis of drug-protein interactions, molecular docking simulations were performed. Molecular docking studies and conformational analysis were performed using a web-based version of Autodock Vina, Webina 1.0.3 ,and intThe molecular docking analysis revealed that, in the case of NS3 helicase from genotype 1a, the fluoroquinolones; of loxacin, moxifloxacin, and sparfloxacin, as well as other NS3 inhibitors, formed interactions with the NS3 helicase in the catalytic groove. On the contrary, in the case of NS3 helicase from genotype 1b, the ligands formed interactions with NS3 helicase in three different regions, where sparfloxacin, thiazolpiperazinyl derivatives 3 and 5, and acridone derivative bound in the catalytic domain of the helicase, while ofloxacin, TBBT, fatsol, and thiazolpiperazinyl derivatives 1, 2 and 4  bound to the allosteric site of the helicase . Analysis of the docking energies revealed astrong binding affinity between NS3 helicase and the acridone derivatives (-11.4KJ/mol), compound 17 -10.3KJ/mol), and thiazolpiperazinyl derivatives 1-5  .The fluoroquinolones namely, moxifloxacin (-9.1KJ/mol) and sparfloxacin (-9.2KJ/mol) also exhibited a high binding affinity with the NS3 helicase, comparable to thiazolpiperazinyl derivatives 1-5, and superior to several of the synthetic and natural derivatives. In the case of genotype 1b (1CU1), analysis of docking energies revealed a strong binding affinity between NS3 helicase and the acridone derivatives (-11.9KJ/mol), compound 17 (-10.8KJ/mol), and thiazolpiperazinyl derivatives 1-5. Besides this, fluoroquinolones namely, ofloxacin (-9.2KJ/mol) and sparfloxacin (-9.2KJ/mol) also showed a higher affinity towards the catalytic region, which was superior to several ofthe synthetic and natural derivatives . Based on the binding energy values, the order of efficacy of interaction in between the fluoroquinolones and NS3 enzyme is in order, ofloxacin > moxifloxacin > sparfloxacin forgenotype 1a. For genotype 1a, analysis of the drug-protein interactions revealed that amino acids, Glutamine-493, Proline-230, Aspartate-296, Threonine-295, Glutamate-460, and Glutamic acid-496 in the groove of the active site, were involved in the binding withall the three fluoroquinolones, whereas other synthetic and natural derivatives made strong hydrogen bonds with Valine-372, Lysine-373, and Tyrosine-350.These amino acids located within these catalytic regions are reported to be crucial for the enzyme activity andthe HCV lifecycle, where Threonine-295 and Proline-230 mediate ATPase dependent binding to the active site, and aids in the maintenance of the electrostatic environment needed for ssDNA and RNA binding, thereby controlling the helicase activity. Targeti.3KJ/mol,Data shows fluoroquinolones that because of their established safety profile can be repurposed as a supplemental therapy against HCV, especially against the infections that arenon-responsive to DAAAs.


IL2RB is implicated in regulation of T cell-mediated immune response. However, the role of IL2RB in pan-cancer is unknown. The present premise sought to explore the role of IL2RB in tumorigenesis, tumor metabolism, and tumor immunity in pan-cancer.  Interleukin-2 receptor  Data were retrieved from multiple data resources including GTEx data resource, CCLE data resource, TCGA data resource, UCSC data resource, and TISIDB web server. These data were adopted to assess the expression, prognosis value, relationship between IL2RB and immune microenvironment, microsatellite instability, immune new antigen, gene mutation, immune modulatory factors immune checkpoint and TMB, and immune or molecular subtypes of IL2RB in various tumors. Estimate analysis and GSEA were conducted to assess the role of IL2RB in pan-cancer.  Differential analysis illustrated that L2RB was remarkably elevated in pan-cancer, notably in solid tumors compared with normal tissues. Survival analysis indicated that IL2RB was linked to pan-cancer prognosis, and elevated IL2RB contents were remarkably linked to dismal prognosis patients in diverse kinds of cancers. The findings illustrated that IL2RB contents were remarkably linked to tumor immune invasion, tumor microenvironment, TMB, MSI, DNA repair genes, methyl transferases, immune modulatory factors, and immune or molecular subtypes in pan-cancer. IL2RB gene mutation was evident in numerous cancers. The data illustrated that IL2RB contents were remarkably enriched in multiple signaling cascades which modulate tumorigenesis, tumor metabolism along with immunity.  The findings of the present premise illustrate that IL2RB plays an indispensible role in tumorigenesis, tumor metabolism, and immunity. Therefore, it is a prospective target gene in tumor-target therapy and tumor immune therapy. IL2RB is also a valuable predictive biomarker in most solid tumors. According to the most recent report from the International Agency for Research on Cancer (IARC), about 19.3 million new cases of cancer along with 10.0 million cancer-linked deaths were recorded globally in 2020 [α (coded by IL2RA), IL-2Rβ (coded by IL2RB), and IL-2Rγ (coded by IL2RG) [Interleukin-2 (IL-2) is a primary immunoregulatory cytokine produced from activated T cells. IL-2 exerts its effects by combining with IL-2R (interleukin-2 receptor) expressed on T cells along with natural killer (NK) cells . IL-2R cy IL2RG) , 5. IL2Ry IL2RG) –8. Howevhttps://gtexportal.org/), UCSC data resource (https://xenabrowser.net/datapages/), and TCGA data resource . Data for 31 cancer samples were obtained from GTEx datasets; data for 5 cancer samples were retrieved from UCSC data resource and 27 cancer samples were obtained from TCGA datasets for analysis in the present study. The CCLE data resource  was utilized to abstract expression data for every tumor cell line. Besides, TIMER data resource (https://cistrome.shinyapps.io/timer/) was employed to abstract pan-cancer immune invasion cell score data. Tumor name acronyms and the match meanings include LUSC (lung squamous cell carcinoma), ACC , WT (high-risk Wilms' tumor), STAD (Stomach adenocarcinoma), BLCA , CESC , CHOL (cholangiocarcinoma), NB (neuroblastoma), COAD (colon adenocarcinoma), LGG (brain lower grade glioma), COADREAD , READ , SARC (sarcoma), BRCA (breast invasive carcinoma), DLBC (lymphoid neoplasm diffuse large B-cell lymphoma), PCPG (pheochromocytoma and paraganglioma), READ (rectum adenocarcinoma), GBM (glioblastoma multiforme), GBMLGG (glioma), MESO (mesothelioma), HNSC (head and neck squamous cell carcinoma), KICH (kidney chromophobe), KIRC , LAML (acute myeloid leukemia), LIHC (liver hepatocellular carcinoma), LUAD (lung adenocarcinoma), OV (ovarian serous cystadenocarcinoma), ESCA , PAAD (pancreatic adenocarcinoma), PRAD (prostate adenocarcinoma), SKCM (skin cutaneous melanoma), ALL (acute lymphoblastic leukemia), STES , TGCT (testicular germ cell tumors), THCA (thyroid carcinoma), THYM (thymoma), UCEC , UVM , OS (osteosarcoma), and KIRC .Gene expression matrix along with clinical data in tumor and nontumorous samples were retrieved from the GTEx data resource  is a measurable marker for quantifying mutations present in a tumor cell. We employed Spearman's rank correlation to assess the TMB of each cancer sample. When comparing tumor tissue to nonmalignant tissue, microsatellite instability (MSI) involves the presence of a novel microsatellite allele. It constitutes any change in the length of a microsatellite produced by the insertion or deletion of a repeat unit. We utilized the Spearman's rank correlation to explore the relationship of IL2RB contents with MSI.P < 0.05 along with R > 0.20.Mismatch repair is a process for repairing mismatches inside cells. When critical genes lose their function as a result of this process, DNA replication mistakes occur that cannot be corrected. These mistakes result in a significant number of somatic mutations. Using expression profile data from the TCGA data resource, the association of five MMR genes  with IL2RB contents was investigated. DNA methylation is a chemical alteration of DNA that affects epigenetic inheritance and modulates gene expression without changing the DNA sequence. The link between IL2RB contents and the expression of four methyltransferases was explored. The ggplot R tool was utilized to visualize the data. The associations were remarkably positive at P < .05, and FDR 0.25. Cascades that satisfied these criteria were deemed to be remarkably enriched [Gene set enrichment analysis (GSEA) compares genes to predetermined gene sets in order to assess their expression status within a functional gene set. It is also utilized to assess if the expression level is linked to a biological process, a molecular function, or a cellular component . The Kyoenriched .il2rb gene was evaluated using TISIDB web server. Statistical significance was defined at P < .05.The TISIDB website comprises 998 immune-linked anticancer genes originated from 4176 records in 2530 studies. The web server is used for analysis of target genes in the tumor immune interplay via analyses of high-throughput data or literature abstraction . Herein,The contents of IL2RB in numerous tissues were determined using GTEx data resources, and the expression trend of IL2RB in 31 tissues was described . The expThe relationships of the IL2RB mRNA contents with OS , DSS , DFS , and PFI  of 44 cancers were explored. Kaplan-Meier (KM) survival curve coupled with Cox proportional hazards models were utilized to evaluate the prognosis value of IL2RB. The results for Cox regression analyses and correlations between IL2RB with OS, DSS, DFI, and PFI in various cancers were presented as forest plots. The data illustrated that elevated IL2RB contents were not remarkably correlated with DFI . HoweverThe results of the KM curve analysis showed that high IL2RB mRNA contents were linked to worse OS in GBM, KIRP, LGG, THYM, and UVM, whereas high IL2RB mRNA levels were associated with better OS in HNSC, SKCM, and SARC . In addiR = 0.463, 0.585, and 0.684, P < 0.001), CD4 + T cells , CD8 + T cells , dendritic , macrophage , and neutrophil  in ACC, BRCA, and CHOL cancers. Moreover, the stromal score and immune score of individual cancer samples were evaluated via the R package Estimate to explore the roles of IL2RB in tumor immune microenvironment in the course of tumor development. The results showed that the top three tumors with the most remarkable correlation between IL2RB contents and stromal score among the 33 cancers were BLCA , CESC , and ESCA , CESC , and CHOL  were the top three cancers with the most remarkable correlation between IL2RB contents with immune score among 33 cancers , BLCA , and CESC  were the cancers with the most remarkable correlation between IL2RB contents and estimate immune score . BLCA , UCEC , CESC , THCA , and LGG   .TMB is a measurable marker for quantifying mutations present in malignant cells. TMB constitutes somatic mutation number in the coding sections of a tumor cell genome that occurs at an average of 1 M bases. The total number of nonsynonymous mutations can be easily expressed. Single nucleotide variations (SNVs) along with tiny insertions/deletions constitute the two kinds of mutations that are frequent in many kinds of mutations. Spearman's rank correlation was adopted to assess the relationship of IL2RB contents with TMB in each tumor type. In BLCA, BRCA, COAD, KIRC, LAML, LGG, SARC, THYM, and UCEC, IL2RB contents were positively linked with TMB, whereas in KRIP, PAAD, PRAD, and THCA were inversely linked to TMB .MSI involves the presence of a novel microsatellite allele in a tumor as compared to neighboring healthy cells as a result of any change in the length of a microsatellite produced by the insertion or deletion of a repeat unit. The relationship of MSI with IL2RB contents was assessed via Spearman's rank correlation. Only COAD was positively linked to IL2RB contents, whereas DLBC, SKCM, ESCA, HNSC, OV, KIRP, MESO, SARC, STAD, LUSC, and TGCT had an inverse relationship with MSI .As illustrated in To assess the influence of gene expression on cancers, human pan-cancer samples were stratified into two groups on the basis of the IL2RB contents: high and low. We utilized GSEA to determine the signaling cascades enriched in the KEGG and HALLMARK data resources in both high and low expression groups. Tables The relationship between IL2RB expression and abundance of tumor-infiltrating lymphocytes (TILs) was explored to determine the role of IL2RB in immune system and its implication in progression of cancers. The data illustrated that IL2RB contents were linked to relative abundance of TILs  in most human cancers . AssociaThe TISIDB website was adopted to examine the impact of IL2RB contents on immunological, as well as molecular subtypes in Pan-cancers. C1 , C4 (lymphocyte deficient), C2 (IFN-gamma dominant), C5 , C3 (inflammatory), and C6 (TGF-b dominant) are the six kinds of immune subtypes. The data exhibiting differential IL2RB expression was among the immune subtypes in various cancers. Expression level of IL2RB was remarkably different among immune subtypes in each cancer type. The top 4 cancer types with the most significant differential IL2RB expression are presented in Data were retrieved from Drug Bank to explore whether IL2RB is a potential target for antitumor drugs. The findings showed that IL2RB gene was a potential target gene for antitumor drugs such as Denileukin diftitox, Aldesleukin, Daclizumab, and Basiliximab . This inInterleukin-2 (IL-2) along with the IL-2 receptor (IL-2R) is the key factor in the regulation of immune system homeostasis and tolerance. IL-2 constitutes a T cell growth factor, which promotes proliferation along with differentiation of activated T cells. IL2RB is part of a receptor signaling complex with pleiotropic functions . IL2RB iThe findings of the present work exhibited higher contents of IL2RB in pan-cancers, especially in solid tumors in contrast with nontumorous tissues. Moreover, high expression of IL2RB was correlated with worse prognosis in the most tumor types. This indicates that IL2RB plays a key role in tumorigenesis, tumor proliferation, and tumor metastasis.Tumor invading lymphocytes in TME are independent predictors for cancer prognosis and immunotherapeutic efficacy , 18. StuIL2RB is receptor of IL-2, which regulates human immunity by binding IL-2. Studies report that mutations in IL2RB gene reduce surface expression and IL-2 binding, causing T and NK cell-driven immune dysregulation. In addition, high expression of wild-type IL2RB increases IL-2 responsiveness of human T lymphocytes in vitro , 36. SimGene set enrichment analysis was performed to explore the role of IL2RB in pan-cancer that was performed for high as well as low IL2RB expression groups. The data illustrated that IL2RB is implicated in several signaling pathways, such as compliment signaling, cytokine-cytokine receptor interaction signaling, PI3K-Akt-mTOR signaling, and allograft-rejection signaling. The highest enrichment scores of these signaling cascades were observed in the high-expression subgroup of IL2RB.This indicates that high IL2RB expression positively modulates these signaling pathways. Moreover, these data illustrate that IL2RB plays a vital role in promoting immune activation, modulating tumor cell viability, and promoting proliferation of tumor cell by upregulating the related signaling cascades.In summary, IL2RB expression level is higher in pan-caner tissues relative to the expression level in normal tissues. High IL2RB expression level is associated with worse clinical prognosis in pan-cancers. TMB, MSI, MMRs, and DNA methylation dysregulate IL2RB expression in pan-cancer. In addition, IL2RB is remarkably linked to tumor immune microenvironment and is a potential therapeutic target gene for tumor immunotherapy.


Oct4, Sox2, Klf4, and c-Myc) into Bactrian camel fetal fibroblasts (BCFFs) using retroviruses with a large host range to obtain Bactrian camel induced pluripotent stem cells (bciPSCs). They were comprehensively identified based on cell morphology, pluripotency gene and marker expression, chromosome number, transcriptome sequencing, and differentiation potential. The results showed the pluripotency of bciPSCs. However, unlike stem cells of other species, late formation of stem cell clones was observed; moreover, the immunofluorescence of SSEA1, SSEA3, and SSEA4 were positive, and teratoma formation took four months. These findings may be related to the extremely long gestation period and species specificity of Bactrian camels. By mining RNA sequence data, 85 potential unique pluripotent genes of Bactrian camels were predicted, which could be used as candidate genes for the production of bciPSC in the future. Among them, ASF1B, DTL, CDCA5, PROM1, CYTL1, NUP210, Epha3, and SYT13 are more attractive. In conclusion, we generated bciPSCs for the first time and obtained their transcriptome information, expanding the iPSC genetic information database and exploring the applicability of iPSCs in livestock. Our results can provide an experimental basis for Bactrian camel ESC establishment, developmental research, and genetic resource conservation.Induced pluripotent stem cells (iPSCs) can differentiate into all types of cells and can be used in livestock for research on biological development, genetic breeding, and in vitro genetic resource conservation. The Bactrian camel is a large domestic animal that inhabits extreme environments and holds value in the treatment of various diseases and the development of the local economy. Therefore, we transferred four mouse genes ( Oct4, Sox2, Klf4, and c-Myc) into mouse embryonic fibroblasts and obtained cells similar to ESCs, which were termed induced pluripotent stem cells (iPSCs). This technology provides a practical pathway to acquire pluripotent stem cells from large livestock. With the development of this technology, iPSCs have been successfully generated from humans, mice, sheep, goats, cattle, canines, rats, rabbits, pigs, horses, and monkeys [Myc, N-Myc, and Glis1, which can replace c-Myc [Klf1, Klf2, Klf5, Esrrb, and Bmp4 as replacements for Klf4 [Rcor2, GMNN, TH2A, TH2B, Obox1, Sox1, and Sox3 as replacements for Sox2 [Nr5a1, Nr5a2, E-cadherin, TCL-1A, Brn4, and Tet1 could efficiently replace Oct4 [The first embryonic stem cells (ESCs) were generated about 40 years ago by culturing mouse inner cell mass (ICM) on feeder layers . ESCs ar monkeys ,11,12,13ce c-Myc ,15; Klf1for Klf4 ,17,18; Rfor Sox2 ,21,22,23ace Oct4 ,26,27,28The Bactrian camel is endemic to desert and semi-desert areas. It possesses a distinctive reproductive physiological structure and can withstand high temperatures, severe cold, and salinity; however, its reproductive efficiency is low. Therefore, it has an important scientific research value. Being a multi-purpose livestock, the Bactrian camel also has important economic value owing to its meat, milk, and wool ,30. WithOct4, Sox2, Klf4, and c-Myc) into BCFFs to obtain bciPSCs, which were identified based on various aspects, including cell morphology, pluripotency markers, differentiation potential, and transcriptome analysis. Moreover, 85 potential pluripotency genes were predicted using the transcriptome analysis of the Bactrian camels. In this study, we aimed to provide an experimental basis for Bactrian camel ESC establishment, developmental research, genetic resource conservation, and basic research and genome data on iPSC enrichment.In this study, we used retroviruses to transfer four mouse genes  gene expression was significantly increased in all three bciPSC lines   Figure A.In the 11th-generation bciPSC-A3 cells, nine pluripotency-related proteins were selected for immunofluorescence detection, and all marker proteins were expressed B.FOXA2), mesoderm , and ectoderm  genes were expressed in the differentiated cells but not in undifferentiated cells  upregulated and downregulated genes from seven bciPSC lines to create a heatmap (We selected 7561 significantly (|log heatmap B. To cre heatmap B. A Venn heatmap C. The reThe top 20 GO term bubble diagrams of bciPSCs indicated that they mainly belong to cellular components . The enrSAP30 and CDKN1A (P21) were consistent with those of mouse and human stem cells , DNA methyl oxidases (TET1 and TET3), and histone demethylases  expressed in bciPSCs. The changes in their expression were consistent with reported iPSC formation and similar to those in human ESC   , 511 genBased on the analysis of the reported inducible genes, 43 inducible genes were identified , and 34 The venn diagram of the two data groups B revealeAlthough the production of and research on iPSCs is growing, it remains limited to human and model organisms. Establishing iPSC lines and genetic information databases for various non-model organisms is significant for studying iPSC mechanisms and the resource development of non-model organisms. Based on previous reports on the establishment of iPSCs in other species ,7,8,9,10TERT, a marker of ES cells in several species, was active [We verified the pluripotency of bciPSCs using various methods. bciPSCs have a high nucleocytoplasmic ratio, show epithelioid cell morphology, express pluripotency genes, are AP-positive, and have normal diploid chromosomes 2n = 74) . Moreoves active ,58,59. T . Moreovs active . bciPSCss active ,60,61. TThe RNA-Seq results showed that bciPSCs and BCFFs had many distinct genes, and the overall modifications were similar to those of human ESC. We further analyzed the pluripotency-related signaling pathway and pluripotency-related genes and DNA methylases, and the results indicated that the bciPSCs are pluripotent. We also found significant differences among the stem cells of Bactrian camels, humans , pigs 66, mice , bovinesCYTL1, TTK, CDC45, ASF1B, SGO1, CDC6, CENPW, KIAA0101, AREG, DTL, HJURP, and SAPCD2) [MEOX2, CTSC, and CSRP2) [PROM1, FOXM1, CDCA5, CD74, MEST, FZD, and RAD54L) [CLSTN3, DLG2, and E2F8) [ASF1B [DTL [CDCA5 [PROM1 [CYTL1 [NUP210 [Epha3 [SYT13 [According to the screening of reported inducible genes, 34 were primarily distributed in intracellular membrane-bound organelles and regulated transcription from the RNA polymerase II promoter of cell processes in the GO database. The KEGG database had four signalaling pathways: signaling pathways regulating the privacy of stem cells, pathways in cancer, the Hippo signaling path, and the TGF-beta signaling path. Four hundred and one genes were obtained after screening. According to the direct analysis of RNA-Seq data, 511 genes were obtained. After a comparison of the two data sets, 46 core genes were obtained. We also selected 42 supplementary genes according to the order of differential gene expression levels. Comparing core and supplemental genes revealed very few shared genes. This shows that supplementary genes can also compensate for subjective screening. Through RNA-Seq data mining of bciPSCs, we predicted 85 potentially unique pluripotency genes in Bactrian camels, which can be used as potential marker genes for bciPSCs. They are mainly related to cell proliferation (such as  SAPCD2) ,72,73,74d CSRP2) ,76,77, p RAD54L) ,82,83,84nd E2F8) . Based o) [ASF1B , DTL [73L [CDCA5 , PROM1 [1 [CYTL1 , NUP210  [NUP210 , Epha3 [0 [Epha3 , and SYT3 [SYT13  are partEGFP as a template, we designed primers  supplemented with 10% fetal bovine serum (FBS) . The cells were subcultured once they reached 90% confluence. The first three generations of BCFFs were preserved in liquid nitrogen.BCFFs were derived from 3-month-old Bactrian camel fetuses and grown in a tissue block adherent culture after digestion and separation . The celMouse embryonic fibroblasts (MEFs) were derived from 13.5-day-old mouse embryos. The culture method and number of cryopreserved generations were the same as those of BCFFs. Mitomycin C -treated MEFs were coated on plates to increase the bciPSC number.Mycoplasma contamination using the Mycoplasma Staining Detection Kit .The two types of fibroblasts obtained in primary culture were identified as follows. The cells were fixed in 4% paraformaldehyde for 30 min at 25 °C. After washing twice with phosphate-buffered saline (PBS) , the cells were treated with PBS containing 0.2% Triton X-100 for 20 min. The cells were then incubated in PBS supplemented with 5% bovine serum albumin (BSA)  for 30 min. After three washes with PBS, the cells were incubated overnight at 4 °C in PBS containing the primary antibody anti-mouse Vim . The secondary antibody used was Alexa488-conjugated goat anti-mouse IgG . The cells were imaged after incubation with the secondary antibody diluted in PBS for 1 h at 37 °C in the dark. The cells used in the experiment were assessed for 2.The Lanzhou Veterinary Research Institute of the Chinese Academy of Sciences donated 293T cells. The cells were cultured in DMEM/high glucose  supplemented with 10% FBS and incubated at 37 °C under 5% COAll experimental procedures were approved by the Animal Care and Use Committee of the College of Veterinary Medicine at Gansu Agricultural University.Oct4, Sox2, Klf4, and c-Myc), and one contained a reporter gene (EGFP), and transduced them into laboratory-preserved BCFFs. The culture medium was replaced with an iPSC induction medium composed of KnockOut DMEM/F12 medium  supplemented with 20% KnockOut Serum Replacement , 2 mmol non-essential amino acids (NEAAs) , 0.1 mmol β-mercaptoethanol , and 1 mM valproic acid . The cells were subcultured after seven days. However, because of the lack of certainty in producing these cells, we evaluated six different subsequent culture protocols  and group B (supplemented with a missing base pair) according to the different pMXS-Sox2 plasmids in the induction combination.The detection primers for pluripotency genes were designed using the NCBI Primer-BLAST tool , and theSOX2 and nNANOG) (TERT)  according to the manufacturer’s protocol and stored at −80 °C. cDNA was prepared using the RevertAid First Strand cDNA Synthesis Kit  following the manufacturer’s protocol. Quanti Nova SYBR Green PCR Kit  was used for qRT-PCR assays. All qRT-PCR reactions were performed on a Veriti 96-well thermal cycler . This method was used to detect endogenous genes (such as n nNANOG) , the tel) (TERT) , and RNA) (TERT) . SpecifiAlkaline phosphatase (AP) staining was performed using the Alkaline Phosphatase Detection Kit .The cells were transferred to a T25 bottle. When their density reached approximately 70%, colchicine  was added to the final concentration of 0.25 µg/mL; the cells were digested and collected with trypsin after a 2.5 h treatment. The cells were then resuspended in 8 mL of potassium chloride solution , allowed to stand at 37 °C for 30 min, and 1 mL of freshly prepared fixative  was added to collect the cells. A few drops of the fixative were slowly added and mixed well, and another 8 mL was added to collect the cells. The cells were resuspended in 0.3 mL of the fixative, and 2–3 drops of the suspension were dropped onto the 4 °C glass slide. The slide was blown onto and baked several times with an alcohol lamp. The slide was then treated with 1% Giemsa solution  dye solution for 10–15 min; the seal film was washed off, observed, and micrographed with an oil lens. As there was no standard banding mode map for Bactrian camels, only a statistical analysis of the number of chromosomes was performed. The number of three bciPSCs for mitotic-phase spreads assessment was 22 (bciPSCs-A2), 25 (bciPSCs-A3), and 20 (bciPSCs-B13), respectively.Immunofluorescence was performed using the same steps as in fibroblast identification, outlined above. Primary antibodies included anti-mouseOCT4 , anti-mouseSOX2 , anti-mouseNANOG , anti-mouseTRA-1-60 , anti-mouseTRA-1-80 , anti-mouseSSEA1 , anti-mouseSSEA3 , anti-mouseSSEA4 , and anti-mouseREX1 . The secondary antibody used was Alexa647-conjugated goat anti-mouse IgG .Three bciPSC cell lines were harvested by treatment with trypsin  and transferred to 35 mm Petri dishes  in DMEM/F12 containing 10% FBS , 2 mM L-glutamine , 1% NEAA , and 0.1 mM β-mercaptoethanol  to induce embryonic body (EB) formation. After seven days of suspension culture, EBs were transferred onto gelatin-coated tissue culture plates and cultured in a medium supplemented with 10% FBS for another seven days. The three germ layer marker genes were detected using qRT-PCR, and protein levels were identified by immunofluorescence. Primary antibodies include anti-rabbit FOX2 , anti-rabbit DES , anti-mouse GFAP , and anti-mouse β-tubulin . The secondary antibodies were Alexa647-conjugated goat anti-mouse IgG  and Alexa647-conjugated goat anti-rabbit IgG .7 cells/site), which were then resuspended and transplanted into the dorsal flank of immune-deficient BALB/cNude mice. Three mice were used for each cell, for a total of twelve mice. Six- to eight-week-old nude mice were housed under pathogen-free conditions in a temperature-controlled room on a 12/12 h light/dark schedule with food and water ad libitum. After four months, the mice were sacrificed, and the tumors were dissected. Teratomas were then analyzed by hematoxylin and eosin (HE) staining , a reticular fiber staining kit , and collagen fiber staining . Simultaneously, the three-germ layer identification antibody was used for the immunohistochemical identification of teratomas.To evaluate pluripotency in vivo, we used matrix glue  to attach BCFFs and three bciPSC lines  were selected for transcriptome sequencing. The cell samples were sent to Genedenovo Biotechnology Co., Ltd.  on dry ice to establish a cDNA library and for subsequent analyses.https://www.ncbi.nlm.nih.gov/genome/?term=Camelus+bactrianus (accessed on 24 September 2021)) was used as the reference genome. RNA-seq and microarray data were analyzed using edgeR and GEO2R software, respectively.HISAT2 was used to align RNA sequencing reads, and the Camelus bactrianus Ca_bactrianus_MBC_1.0 genome assembly  analysis used to observe the main changes in the genetic information. We compared the differentially expressed genes in the RNA-Seq data and the GEO dataset. We also analyzed the enriched signaling pathways, pluripotency-related genes, and important genes in the reprogramming process in RNA-Seq data. The online software of the sequencing company was used to visualize the sequencing data.First, we used RNA-Seq sequencing data from bciPSCs to perform direct screening. We summarized upregulated genes involved in the signaling pathways identified during data visualization and the first 20 terms of the GO analysis in the seven bciPSCs. The upregulated genes were screened using counts ≤50 and ≥20 in BCFFs and bciPSCs, respectively, and labeled as group 1. Next, we performed screening based on known induced genes. Based on the relevant literature review, we compiled a comprehensive list of inducible genes across different species. Each bciPSC gene in the RNA-Seq data was annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database  and the GO databases . Subsequently, we selected the main subdatasets containing the summarized inducible genes at the six annotation levels and screened the upregulated genes. The upregulated genes in the new data set were screened using counts of ≤50 in BCFFs and ≥20 in bciPSCs and designated as group 2.http://string.embl.de/ (accessed on 28 November 2021)), to both predict gene interactions and select core genes. The Bactrian camels were not included in the reference species list. Accordingly, the most closely related Bos taurus was selected as the reference species.Ven map analysis for groups 1 and 2 determined whether to combine the analyses based on the degree of dissimilarity between the gene sets. After confirmation, the screened genes were imported to the Search Tool for the Retrieval of Interacting Genes database(STRING, To compensate for the disadvantages of selecting groups 1 and 2, we screened the upregulated genes without any screening treatment using counts of ≤50 in BCFFs and ≥20 in bciPSCs and designated the gene set as group 3. The genes of each of the seven bciPSCs in group 3 were sorted in descending order of copy number. The first 30 genes were summarized and used as supplementary genes. Finally, the core genes and complementary genes were combined to form the final candidate gene set.This is the first study to obtain bciPSCs with stable cell morphology, expression of pluripotency genes and markers, and the ability to differentiate in vitro and in vivo. Furthermore, we predicted 85 candidate genes that can be used for reprogramming Bactrian camel cells. Our efforts in transcriptome data mining and basic research support the development of the iPSC genetic information database and the use of iPSC in livestock by providing biological materials and data for establishing the Bactrian camel ESC, developmental research, genetic breeding, and the conservation of genetic resources. Considering the development of research on the disease treatment of Bactrian camels, this research is expected to contribute to the transformation of regenerative medicine in the future.