Pancreatic ductal adenocarcinoma (PDAC) is an intractable cancer that is difficult to diagnose early, and there is no cure other than surgery. PDAC is classified as an adenocarcinoma that has limited effective anticancer drug and molecular-targeted therapies compared to adenocarcinoma found in other organs. A large number of cancer cell lines have been established from patients with PDAC that have different genetic abnormalities, including four driver genes; however, little is known about the differences in biological behaviors among these cell lines. Recent studies have shown that PDAC cell lines can be divided into epithelial and mesenchymal cell lines. In 3D cultures, morphological and functional differences between epithelial and mesenchymal PDAC cell lines were observed as well as the drug effects of different anticancer drugs. These effects included gemcitabine causing an increased growth inhibition of epithelial PDAC cells, while nab-paclitaxel caused greater mesenchymal PDAC cell inhibition. Thus, examining the characteristics of epithelial or mesenchymal PDAC cells with stromal cells using a 3D co-culture may lead to the development of new anticancer drugs. Pancreatic cancer is a malignant tumor disease with poor prognosis and a 5-year overall survival rate of approximately 11% ; therefoAmong all pancreatic malignancies, pancreatic ductal adenocarcinoma (PDAC) is the most common subtype, has high proliferative and metastatic potential in other organs, and accounts for approximately 90% of all pancreatic malignancies. Thus, pancreatic cancer and PDAC are often used interchangeably . AlthougBasic research on PDAC has mainly been conducted using cultured PDAC cell lines, experimental animals, and human surgical tissue specimens. Cultured PDAC cell lines were established from patients that had different gene mutations and expression profiles ,9. PDAC KRAS and the three tumor suppressor genes TP53, CDKN2A, and SMAD4, which have been thoroughly characterized in human tissue samples [Cell lines are ubiquitous experimental tools in cancer research. Cancer cell lines are developed from a patient’s cancer cells, and these lines are considered to meet the definition of tumors that grow autonomously and indefinitely. Based on these properties, cancer stem cells (CSCs) that have the capacity to self-renew are present in cultured cancer cell lines. Many cell lines have been used for pancreatic cancer research. The most frequently altered driver genes in PDAC include the oncogene  samples ,11,12,13 samples . Mutatio samples . Some PD samples . Since P samples . StudiesSeveral public cell banks supply cancer cell lines worldwide, such as the American Type Culture Collection (ATCC), European Collection of Cell Cultures (ECACC), Japanese Collection of Research Bioresources (JCRB) cell bank, and Riken Bioresource Center. Cellosaurus is a knowledge resource that aims to describe all cell lines used in biomedical research by providing information on immortalized cell lines, including cancer cell lines . There aAs of 20 April 2022, there are 417 pancreatic cancer cell lines, including 377 PDAC and 40 other pancreatic cancer cell types, in the Cellosaurus. In the PDAC cells, two types of cells mainly express epithelial or mesenchymal molecules. PANC-1 and MIA PaCa-2 have been classified into mesenchymal molecule-expressing PDAC cells and named quasi-mesenchymal or mesenchymal types in previous manuscripts ,16. The According to the 2006 symposium of the American Association of Cancer Research, a CSC is defined as “a cell within a tumor that possesses the capacity to self-renew and cause the heterogeneous lineages of cancer cells that comprise the tumor” . Three mCSC-specific markers are highly expressed in the cell membranes or cytoplasm of CSCs, and in human PDAC tissues, these markers can be detected using immunohistochemical and immunofluorescent staining. Pancreatic CSC markers including CD133, CD44, CD24, CXCR4, ATP-binding cassette sub-family G member 2 (ABCG2), ALDH1, nestin, and epithelial cell adhesion molecule  are detected in PDAC tissues at various levels (13.5–78.9%) ,24. In hSince CSCs more efficiently efflux anticancer drugs than non-CSCs, SP cells that rapidly efflux fluorescent dyes were selected as CSCs by flow cytometry. Zhou et al. showed that there were 2.1–8.7% of viable SP cells in PANC-1 cell populations . SP cellThe sphere formation assay indicates the self-renewal capacity of cancer cells that can form floating colonies called spheres when cultured in low-attachment dishes. PDAC cell lines can form spheres in low-attachment dishes, and sphere-forming cells possess stem cell abilities ,34,35. FAlthough genetic abnormalities are different in each PDAC cell line, differences in their biological behavior have not been well investigated. Therefore, some medical journals require that more than two cancer cell lines show similar experimental results, and reviewers often point out that authors should perform the same experiments using more than one PDAC cell line. When cultured in 2D, most PDAC cells showed similar pleomorphic morphology  mice after implantation of the cells into the pancreas [FGFR1, FGFR3, and CD44 [In 2D culture conditions, we examined the epithelial or mesenchymal features of PDAC cell lines. Among the eight PDAC cell lines , five epithelial cell lines written earlier had high E-cadherin mRNA expression and low vimentin mRNA expression, whereas three mesenchymal cell lines written later expressed low E-cadherin and high vimentin expression ,37 Tabl. There wpancreas . These Pand CD44 . It is sPathologically, pancreatic cancer cells proliferate sterically in both primary and metastatic lesions, forming masses in the human body. By culturing PDAC cells on a low-attachment plate using the same medium as the 2D culture, 3D spheres can be formed . The expBecause PDAC forms 3D tumors in vivo even in the early stages, 3D culture is considered to be an important in vitro experimental condition. However, studies using PDAC cell lines are primarily based on the results of 2D culture systems. Previous studies have shown that there are differences in the proliferative capacity of cells and the effects of anticancer drugs on PDAC cells, as well as mucus secretion that is characteristic of adenocarcinoma in cells lines cultured in 2D and 3D conditions ,17.Previous studies have reported that the proliferative capacity of cancer cells is higher in 2D cultures than in 3D cultures ,48. In m50) for gemcitabine and oxaliplatin in PDAC cells needs to be 200-fold higher in 3D culture spheres than in 2D culture [Almost all anticancer drugs are less effective in multicellular spheroid models than in 2D cell cultures ,49,50. I culture . When th culture . A previ culture . Further culture .It is unclear why the effects of anticancer drugs differ between epithelial and mesenchymal PDAC cell lines grown in 3D culture conditions, but these features may contribute to the efficacy of anticancer drugs. The spheres of PDAC cells with epithelial features are covered with surface lining cells, while the spheres of mesenchymal-featured PDAC are uncovered and remain separated from one another. This difference may assist in anticancer drug permeability into the spheres. In addition, the spheres of PDAC cell lines with mesenchymal features have more proliferative activity than the spheres of epithelial PDAC cell lines, with the proliferating cells confined to the surface cells of the spheres in PK-1 , which mThere have been several studies of the relationship between pancreatic cancer with low E-cadherin or vimentin expression and poor prognosis in human patients with PDAC ,53. TissThere is abundant desmoplastic stroma around PDAC cells in human PDAC tissues. PDAC stroma consists of stromal and inflammatory cells, and extracellular matrix proteins that all contribute to the aggressive biological behavior of the tumor . The tumAlthough their morphology is almost unchanged when cultured in 2D conditions, PDAC cell lines established from patients with PDAC are divided into those that express epithelial or mesenchymal proteins. However, in 3D culture, epithelial PDAC cells form small spheres with coated cells on the surface, while mesenchymal PDAC cells form spheres that are loosely bound together. Epithelial and mesenchymal PDAC spheres differ in cell proliferation and anticancer drug responses in 3D culture. More selective in vitro research on the characteristics of PDAC and effective anticancer agents will be advanced by 3D co-culture methods of epithelial or mesenchymal PDAC cells and stromal cells present around the tumor.


CCND1-IGH fusion) when given in combination as compared to single agents. These data suggest that the combination of venetoclax and selinexor is effective and t may serve as a therapeutic marker for response and target for future clinical trials.Patients with multiple myeloma-bearing translocation t have recently been shown to benefit from the apoptosis-inducing drug venetoclax; however, the drug lacks FDA approval in multiple myeloma thus far due to a potential safety signal in the overall patient population. Selinexor is an inhibitor of nuclear export that is FDA-approved for patients with multiple myeloma refractory to multiple lines of therapy. Here, we report that in four patients with multiple myeloma with t, the concomitant administration of venetoclax and selinexor was safe and associated with disease response. Moreover, the combination was synergistic in t multiple myeloma cell lines and caused decreased levels of Cyclin D1 (which is overexpressed due to the  CCND1 at chromosome 11q13 is juxtaposed to the IGH gene at chromosome 14q32, resulting in overexpression of the protein Cyclin D14. Screening of multiple myeloma (MM) cell lines for sensitivity to venetoclax showed that high sensitivity was restricted to cell lines with CCND1 translocations6. Venetoclax induces apoptosis by acting as a BH3-mimetic to inhibit the anti-apoptotic factor BCL2 and is approved for use in certain B-cell malignancies and acute myeloid leukemia7. Venetoclax has been studied in combination with bortezomib and dexamethasone (versus placebo with bortezomib and dexamethasone) for relapsed/refractory myeloma; however, the benefit in progression-free survival (PFS) in the venetoclax arm was offset by increased mortality, thus hindering its path to approval8. A subset analysis showed that patients who possessed t and/or high BCL2 expression had clinical responses and longer PFS without increased mortality, leading to cautious off-label use of the medication in select patients. Selinexor is an inhibitor of nuclear export (SINE) that blocks the cargo-binding pocket of XPO1. XPO1 recognizes cargo proteins bearing a nuclear export signal and shuttles them out of the nucleus. These cargoes include tumor-suppressor proteins such as p53, as well as proteins binding to select mRNA including BCL29. Cyclin D1 is a known cargo protein of XPO111. Cyclin D1 is essential in the regulation of the cell cycle, and its overexpression can result in uncontrolled cell growth, contributing to cancer development and progression14. Selinexor is approved for patients with myeloma who have received at least four prior lines of therapy and are refractory to two proteasome inhibitors, two immunomodulating therapies, and a CD38-targeted monoclonal antibody16.In the t translocation, 17. All patients responded initially to venetoclax but ultimately developed resistance and progressive disease. The addition of selinexor recaptured responses and lead to clinical benefit, suggesting a synergistic effect of the combination. The combination of venetoclax and selinexor was further studied in MM cell lines with and without t translocations and showed enhanced synergy in those cell lines bearing the CCND1-IGH translocation.We report here, four patients with relapsed/refractory t MM who had progression of disease after multiple lines of treatment and were considered for venetoclax treatment based on previous data showing efficacy of venetoclax in t MM2 every two weeks and dexamethasone 12 mg weekly. The patient had 17 cycles of treatment before POD, at which point bortezomib was substituted for carfilzomib with continued POD. The patient was enrolled onto a clinical trial of a BCMAxCD3 bi-specific T-cell engager with POD. Given prior resistance to venetoclax-based therapy, he was given selinexor 60 mg weekly in combination with venetoclax 400 mg daily and dexamethasone 40 mg weekly with subsequent VGPR. His kappa light-chain levels correlated with response and are shown in Fig. A 55–60-year-old man with free kappa light-chain MM, R-ISS stage 1, and standard risk cytogenetics with t; Fig.  was treaAn additional three patients with t11;14) MM were treated with the combination of selinexor and venetoclax. The summary of their treatment history and responses to therapy are outlined in Fig. 1;14 MM wOverall, the combination of selinexor and venetoclax was well tolerated. Selinexor was prescribed at 60 mg or 80 mg weekly, and venetoclax was prescribed at either 400 mg or 800 mg daily based upon tolerance and efficacy. One patient had dose interruption of selinexor due to fatigue and dyspnea, however she was able to resume therapy without further issues. No other dose interruptions or reductions were required. Leukopenia, neutropenia, and thrombocytopenia were observed, but there was no incidence of bleeding or febrile neutropenia. Hyponatremia was also observed, however, did not require intervention and resolved.To further test our hypothesis of the preferential effects of selinexor and venetoclax in patients bearing the t11;14) translocation, we tested the synergy of the combination on human MM cell lines at various concentrations and then examined cell viability 72 h after treatment. MM cell lines with and without the t translocation are shown in Supplementary Table 1;14 tran18. As expected, with selinexor we saw a reduction in XPO1 protein levels that was further reduced with the combination in U266-B1 but no difference in selinexor and combination was seen in RPMI-8226. We then assessed the effects of treatment on cargo proteins that are known to be regulated by XPO1 and found an increase in tumor-suppressor p53 levels and a decrease in p65 levels with the treatment of selinexor and combination in both cell lines . There was a significant increase in overall survival of the mice in the combination treatment group compared to the other groups  we described above may have been due to either single drug alone; however, the in vitro synergy led us to hypothesize that the combination effect would be greater than the individual agents in vivo as well. We therefore utilized a xenograft multiple myeloma mouse model of the t myeloma cell line KMS12BM. After tumor engraftment, mice were randomized to receive vehicle, selinexor, venetoclax or the combination of selinexor and venetoclax. Overall, the treatments were well tolerated, and the mice received continuous treatment until the vehicle recipients were euthanized due to advanced tumor growth at 17 days. At this timepoint, the combination-treated group showed a significant decrease in tumor volume when compared to the other groups . To our knowledge, this is the first report of patients with MM treated with the combination of selinexor and venetoclax.In the past decade, the treatment landscape for MM has dramatically changed with proteasome inhibitors, immunomodulatory agents, and antibody therapies becoming the mainstays of therapy. Despite these innovations, cures for multiple myeloma remain elusive. However, successful management of the disease requires multiple lines of therapy and new drugs with novel mechanisms of action. Selinexor inhibits nuclear export with activity in penta-refractory MM. Venetoclax is approved in other hematologic malignancies and shows promise in a subset of MM patients with t. The combination of selinexor and venetoclax has shown preclinical synergy in other cancer typesCCND1-IGH fusion confirmed by FISH who were relapsed or refractory to prior therapies were able to achieve responses with the combination of selinexor and venetoclax, with two patients achieving a VGPR. Importantly, both patients progressed through prior venetoclax-based regimens, yet they still responded to a selinexor and venetoclax combination. Neither patient received single-agent selinexor, so we do not know whether the combination was required for the response. However, the doses of selinexor used were lower than that on the FDA label for use in refractory MM. Alongside the in vitro and in vivo synergy findings shown here, this suggests that there is an effect of the combination allowing for lower dosing of selinexor. Since adverse effects of selinexor have been shown to be dose-dependent, combining selinexor with venetoclax potentially allows for a more tolerable and therapeutic option for patients, such as the ones described, that are older, are heavily pre-treated, or not candidates for further intensive therapy. This may also serve as an option for patients who may not have access to clinical trials for relapsed and refractory disease or for certain reasons request a strictly oral regimen. It could also act as a bridge to therapies that require manufacturing time, such as CAR-T therapy.Four patients with t MM with 21. Positive correlation between Cyclin D1 and BCL2 and the role in oncogenesis has been described in other tumor types23. Our results did not show a difference in BCL2 levels after the combination therapy but other studies suggest the balance of BCL2 and other anti-apoptotic proteins, such as BCL-XL and MCL-1 against the pro-apoptotic proteins is more important than BCL2 expression levels alone27. Accordingly, we saw increases in MCL-1 levels with venetoclax monotherapy that was abrogated by the addition of selinexor. This effect was seen in both t and non-t myeloma cell lines but was more significantly decreased in the t. While MCL-1 has not been reported to be a direct cargo of XPO1, others have reported decreased MCL-1 levels by selinexor therapy potentially by disrupting protein and downregulated expression of mRNA29. Expression of MCL-1 is also regulated by NFκB signaling, and we observed decreases in NFκB p65 after combination therapy.The synergistic mechanism of the combination and selective preference in t multiple myeloma is an area of ongoing investigation. However, our preliminary studies suggest a role for Cyclin D1 itself as a target of selinexor and synergistic effect of the combination with venetoclax. Cyclin D1 is a cargo protein of XPO1, the direct target of selinexor, but the activity of venetoclax against Cyclin D1 expression is not well defined. Previous studies have correlated venetoclax sensitivity to the expression of BCL2, especially in cell lines or patients who possess tIn conclusion, though limited to four patients, we observed responses in four out of four patients the administration of selinexor and venetoclax combination was safe in t translocated relapsed and refractory MM patients. Preclinical studies in a xenograft mouse model of t multiple myeloma also showed combination efficacy and tolerability. Based on these results, we are planning a prospective clinical trial to test the effectiveness of venetoclax and selinexor combination in t multiple myeloma patients.30.Patients received treatment under regular clinical care but retrospectively provided written informed consent to be included in this study. The Sylvester Comprehensive Cancer Center (SCCC) institutional review board approved the study. Responses were assessed by IMWG criteriaThree-month-old female NSG-SGM3 mice were used for xenograft studies approved by the University of Miami Institutional Animal Care and Use Committee (IACUC) guidelines (Protocol #20-079).2 incubator and passaged every 2–3 days. Selinexor  and venetoclax  were obtained from Selleck Chemicals .RPMI-8226 and U266-B1 cell lines were purchased from ATCC . OPM2 and KMS12BM cell lines were a kind gift from Dr. Leif Bergsagel from Mayo Clinic. Cell lines were tested for mycoplasma with the MycoAlert mycoplasma detection kit . RPMI-8226 and OPM2 were cultured in RPMI-1640 with 10% FBS. U266-B1 and KMS12BM were cultured in RPMI-1640 with 15% FBS and 20% FBS, respectively. Cell lines were maintained at 37 °C in a CO4 cells/well in a 6 × 6 matrix of white, clear-bottom 96-well plates. Each cell line was exposed to increasing concentrations of each drug and a vehicle-only control (DMSO). After 72 h of incubation at 37 °C in a 5% CO2 incubator, the effects of cell viability were measured using CellTiter-Glo viability assay according to the manufacturer’s instructions . Synergy was analyzed via the Bliss independence model using Synergy Finder software (synergy.fimm.fi) as well as via the Chou–Talalay method by using CompuSyn software (combosyn.com). Synergy Finder model synergy score values above 10 indicates synergistic effects and values below −10 indicates antagonistic drug effects. CompuSyn combination index (CI) values CI < 1 are synergistic, CI = 1 is additive, and CI > 1 are antagonistic.Cell lines were seeded at 1 × 106 cells/well in a six-well plate. Whole-cell lysates were prepared with IP lysis buffer supplemented with protease and phosphatase inhibitors . Protein concentration was determined using BCA Protein Assay kit . Ten micrograms of total protein was separated by electrophoresis on a 4–12% bis-tris protein gel and transferred onto PVDF membranes and probed with antibodies against Cyclin D1 ; XPO1 and p53 ; and MCL-1 and p65 , β-actin  all were used at 1:1000 dilution in 1% BSA. Membranes were visualized by Clarity Western ECL substrate  following the manufacturer’s protocol. The following treatments were used: DMSO-only control, selinexor (200 nM), venetoclax (1 µM), and combination. β-actin was used as a loading control. Treatment of U266-B1 and RPMI-8226 was 24 h and treatment of KMS12BM and OPM2 was 16 h. Western blot quantification was performed using ImageJ and normalized against β-actin. All blots were derived from the same experiment and processed in parallel. Uncropped blot images are provided in Source Data File.Cells were plated at 1 × 103 or if weight loss was >20% for two consecutive days.The NSG-SGM3 mice were weighed, followed by anesthetized by exposure to 1–5% vaporized isoflurane in 100% oxygen. One million KMS12BM cells were injected into the mice subcutaneously. The following drugs were administered with a minimum of nine mice per experimental group: a vehicle on the same schedule as the treated groups, selinexor , venetoclax , and the combination on the same schedule. Mice were monitored daily, and tumor burden was measured using high-frequency ultrasound . Mice were sacrificed when tumor size reached >2 mmFurther information on research design is available in the Supplementary AppendixData Set 1REPORTING SUMMARY


In this review, we will summarize major approaches that prevent donor T cell migration into GVHD target tissues and approaches that augment tolerization of the infiltrating T cells in the GVHD target tissues while preserving strong GVL activity in the lympho-hematopoietic tissues.Allogeneic hematopoietic cell transplantation (Allo-HCT) is a curative therapy for hematological malignancies  due to the graft-versus-leukemia (GVL) activity mediated by alloreactive T cells that can eliminate residual malignant cells and prevent relapse. However, the same alloreactive T cells can cause a serious side effect, known as graft-versus-host disease (GVHD). GVHD and GVL occur in distinct organ and tissues, with GVHD occurring in target organs  and GVL in lympho-hematopoietic tissues where hematological cancer cells primarily reside. Currently used immunosuppressive drugs for the treatment of GVHD inhibit donor T cell activation and expansion, resulting in a decrease in both GVHD and GVL activity that is associated with cancer relapse. To prevent GVHD, it is important to allow full activation and expansion of alloreactive T cells in the lympho-hematopoietic tissues, as well as prevent donor T cells from migrating into the GVHD target tissues, and tolerize infiltrating T cells  Allogeneic hematopoietic cell transplantation (Allo-HCT) provides curative therapy for hematological malignancies such as lymphoma and leukemia by relying on the graft-versus-leukemia/lymphoma (GVL) effects mediated by alloreactive T cells –6. Howev+ Tregs can enhance GVL activity while suppressing GVHD (Acute GVHD (aGVHD) is a dysregulated and exaggerated alloimmune response whose pathogenesis has been elegantly described in previous and recent reviews –11. Althing GVHD –23. In t4β7integrins interacting with their ligands CXCL9, CXCL10, CXCL11, CCL25 or MAdCAM-1 migrate into gut  T cells  activity or even enhanced activity  in the draining lymph nodes . The S1PRs agonist FTY720 retains alloreactive T cells in lymphoid tissues and prevents T cell infiltration of GVHD target tissues, thereby preventing GVHD while preserving GVL effects in both MHC mismatched and MHC-haploidentical murine GVHD models –62. On tTissue inflammation caused by pretransplant conditioning regimens triggers migration of alloreactive T cell into GVHD target tissues . Delayedin vivo administration of anti-IL-6R mAb attenuates GVHD with significant expansion of Tregs and reduction of inflammatory Th1 and Th17 cells  which, in turn, up-regulate the chemokine receptors and their ligands that drive the migration of T cells to GVHD target tissues , 80, 81.17 cells , 88. Inhactivity . Anotheractivity . Neutralactivity , 91, 92.activity , while pactivity . In clinactivity . Similaractivity , 96.+ Tregs , adipose tissue (AT), and dental pulp –102. It + Tregs –118. Unlactivity –23. In cactivity . Co-injeactivity , and a cactivity .Exosomes are naturally occurring extracellular vesicles (EVs) that are released from many cell types and can be enriched from virtually all body fluids, including blood plasma, urine and saliva . Dependiin vitro. Progress will require improved understanding of the mechanisms of MSCs and the development of methods that define the optimal source, in vitro culture methods, measurement of potency, cell dose, and the timing and frequency of administration.Overall, the efficacy of MSCs treatment varies from study to study, possibly because MSCs are very heterogeneous, depending on their origin and the methods used to isolate and propagate them N) which have not yet encountered their corresponding antigens, and (2) antigen-experienced T cells, which include memory and effector T cells (TM) composed primarily of clonal expansions of T cells specific to their respective antigens (CM), effector memory T cells (TEM), and effector T cells (TE)  naïve T cells (Tantigens . Based olls (TE) . Most T CT model . In contraftment . This stN and TM subsets for their ability to cause GVHD using various murine GVHD models  from HLA-matched sibling donors after myeloablative conditioning (+ selected pan-T cell depleted (TCD) PBSCT recipients, although the incidence of moderate (grade II-III) aGVHD was not reduced. GVHD in these patients, however, was uniformly responsive to corticosteroids, with a very low incidence of grade III-IV GVHD. The incidence of cGVHD was strikingly reduced compared to BMT. More importantly, the presence of TM in the graft contributed to rapid recovery of T cells and the transfer of protective virus-specific immunity. No excessive rates of infection or relapse was observed   compared to historic T-cell depleted haplo HCT cohorts   –144. In rategies , 146.N-depleted allo-HCT are very encouraging, and the understanding obtained from various human studies generally correlate with the results of murine experiments. However, relapses still occur at a low incidence. The underlying mechanism by which TN and TM exert differential effects on alloreactivity remains unclear. It has been proposed that TN cause GVHD while TEM do not because they lack CD62L and CCR7, which are critical in directing TN toward to the sites of antigen presentation for GVHD initiation, such as lymph nodes (LN) and Peyer patches (PP). However, in murine GVHD model, Anderson et al. showed that CD62L and CCR7 were not required for TN-mediated GVHD, since CD62L-/- donor T cells still induced GVHD, and GVHD also developed in recipients that lacked LN and PP. Even when TEM constitutively express CD62L, they do not cause GVHD   , 160, whl models , 162. ILignaling . We founr1 cells . In lympactivity . Mainten tissues Figure 1 tissues  but has hiIFN-γ+IL-10+CD4+ T cell interactions with B cells. Our previous report showed that autoreactive IFN-γ+IL-10+CD4+ T cells can activate B cells to produce autoantibodies  nor ruxolitinib interfered with the GVL effect in vivo in MHC-mismatched murine models ; adm; adm+ T activity , potentiQS wrote the review manuscript; UN edited the manuscript; RN, PM, and DZ critically reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by National Institutes of Health Grant R01 CA228465 (to DZ) and National Natural Science Foundation of China (grant no. 82100226) to QS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


These results are of high significance for cell engineering and especially for exosome engineering.Five antibiotic resistance (AR) genes have been used to select for transgenic eukaryotic cell lines, with the BleoR, PuroR, HygR, NeoR, and BsdR cassettes conferring resistance to zeocin, puromycin, hygromycin, geneticin/G418, and blasticidin, respectively. We recently demonstrated that each AR gene establishes a distinct threshold of transgene expression below which no cell can survive, with BleoR selecting for the highest level of transgene expression, nearly ∼10-fold higher than in cells selected using the NeoR or BsdR markers. Here, we tested the hypothesis that there may be an inverse proportionality between AR protein function and the expression of linked, transgene-encoded, recombinant proteins. Specifically, we fused each AR protein to proteasome-targeting degron tags, used these to select for antibiotic-resistant cell lines, and then measured the expression of the linked, recombinant protein, mCherry, as a proxy marker of transgene expression. In each case, degron-tagged AR proteins selected for higher mCherry expression than their cognate WT AR proteins. ER50BleoR selected for the highest level of mCherry expression, greater than twofold higher than BleoR or any other AR gene. Interestingly, use of ER50BleoR as the selectable marker translated to an even higher, 3.5-fold increase in the exosomal loading of the exosomal cargo protein, CD63/Y235A. Although a putative CD63-binding peptide, CP05, has been used to decorate exosome membranes in a technology known as “exosome painting,” we show here that CP05 binds equally well to CD63 In the 4 decades since the invention of mammalian cell transgenesis , biomediWe recently reported that the choice of antibiotic resistance (AR) gene can also have a major impact on transgene expression . More spExosomes are small secreted organelles (diameter of ∼30–200 nm) that have the same topology as the cell, are highly enriched in a subset of proteins, lipids, and RNAs, and can transmit signals and molecules through an intercellular vesicle trafficking pathway . As a reEscherichia coli dihydrofolate reductase (ecDHFR) are particularly intriguing, as they can be stabilized in a dose-dependent manner by small molecules To determine whether destabilization domains could impact the levels of transgene expression, we generated pITRSB-based Sleeping Beauty transposons , 19 thatThe 14 polyclonal cell lines that emerged from this process were expanded for 2 additional weeks in selective media and then interrogated by flow cytometry to measure the mean, median, and individual mCherry expression levels across ∼20,000 cells from each cell line. Consistent with our operating hypothesis, degron-tagged forms of the BsdR, BleoR, PuroR, HygR, and NeoR proteins were selected for higher levels of expression of the linked recombinant protein mCherry . HoweverOf the five antibiotics commonly used in mammalian cell transgenesis experiments , blasticidin kills fastest of all, as addition of blasticidin killed 100% of 293F cells within 48 h. Curiously, when 293F cells were transfected with the transposon carrying the CMV-mCherry-2a-BsdR transgene, we observed relatively little cell death and robust growth of nearly all cells in the population. These results indicate that most of the cells (i) had been transfected, (ii) continued to express the BsdR protein, and (iii) expressed enough blasticidin deaminase enzyme activity to confer blasticidin resistance on the cells, regardless of the level of transgene expression.A). In fact, many of the blasticidin-resistant cells in this polyclonal cell line displayed levels of mCherry fluorescence that were no higher than the background fluorescence of 293F control cells. In contrast to the BsdR-selected cell line, the ER50BsdR-selected cell line displayed mean mCherry expression levels that were, on average, approximately fivefold higher than the BsdR-selected cell line, whereas the ecDHFRBsdR-selected cell line displayed average mCherry expression approximately sixfold higher than the BsdR-selected cell line (B and C). The pronounced effect of BsdR degron tagging on transgene expression is evident from a number of perspectives, which include (i) 122-fold higher and 168-fold higher median fluorescence levels, (ii) the threefold lower CV values (∼365), and (iii) the drop in mean/median ratio from ∼45 to ∼2.Consistent with this notion, we found that the blasticidin-resistant cell line resulting from transfection with the BsdR-based vector displayed the lowest level of mCherry expression and the greatest cell-to-cell heterogeneity in mCherry of any cell line tested A. In facell line , B and CA and B).We previously established that the BleoR gene selects for higher and more homogeneous transgene expression than the BsdR, PuroR, HygR, or NeoR selectable marker genes . We obseAs predicted from our operating hypothesis, this 2.3-fold increase in mean transgene expression led to a further reduction in the cell-to-cell heterogeneity of mCherry expression, shown here by the drop in the CV from 84 to 48. It should, however, be noted that >99% of the cells in both the BleoR and ER50BleoR-selected cell lines expressed mCherry fluorescence that was >1000-fold above background, evidence that both forms of the BleoR gene are quite good at generating cell lines in which nearly every surviving cell expresses a high level of transgene expression. That being said, the superior performance characteristics of the ER50BleoR marker can be seen throughout our data, even in the mean/median ratio that fell to 1.1, just shy of the theoretical optimum of 1. Taken together, these results demonstrate that the ER50BleoR AR gene can be used to rapidly select for polyclonal cell lines that express the very highest levels of linked transgene expression, without the need for isolating and screening large numbers of SCCs. However, if SCCs are needed, selection with the ER50BleoR marker is likely to eliminate all but the highest-expressing clones, reducing the number of clones that need to be screened to identify the highest-expressing cell lines.versus 6807 arbitrary units [a.u.], respectively), a level that was ∼40% the level selected by the BleoR marker , and higher than the levels of transgene expression observed in cell lines selected via the NeoR (4498 a.u.) or BsdR (1308 a.u.) markers . While the amplitude of this effect was less than we observed for the BsdR and BleoR AR genes, it did lead to reduced heterogeneity of expression, shown here by drop on CV from 107 to ∼80 and a fall in mean/median ratio, from 1.4 to 1.2.As for the degron-tagged forms of PuroR, they selected for ∼70% higher mean mCherry expression , A–C. WhA–C). This was, of course, unexpected, and raised questions about why degron tagging could have a strong effect on some AR proteins but little if any effect on others.In contrast to BsdR, BleoR, and PuroR, HygR was at most slightly sensitive to degron tagging, if at all. While both degron-tagged forms of the HygR marker resulted in slight increases in the homogeneity of expression, the increase in mean mCherry expression was either undetectable (in the case of ER50HygR-selected cells) or only 24% (in the case of ecDHFRHygR-selected cells) , A–C. ThA–C). Consistent with our previous report . These vectors were transfected into 293F cells, which were then incubated in nonselective media for 2 days, followed by addition of zeocin to select for transgenic cell lines. Two weeks later, all zeocin-resistant clones from each transfection were pooled, creating the cell lines F/YA22 (BleoR based) and F/YA24 (ER50BleoR based). These cell lines were expanded under selection for an additional 2 weeks and then examined for the expression and vesicular secretion of CD63.The primary question arising from these studies is whether use of the ER50BleoR marker dives improved cell engineering, and more specifically, an improvement in exosome engineering , 37, 38.Fig. 7B–D). As for the extent of this increase, immunoblot analysis of cell and exosome fractions collected from all three cell lines (E–G) revealed that F/YA22 cells contained ∼15-fold more CD63 , whereas F/YA24 cells contained ∼30-fold more CD63, relative to 293F cells  (E and G). The 2.1-fold higher level of CD63 expression seen for the ER50BleoR-selected cell line  is consonant with the 2.3-fold increase observed for mCherry expression, indicating that the ER50BleoR drives higher recombinant protein expression, even for proteins as dissimilar as the soluble cytoplasmic mCherry and the polytopic membrane protein CD63/Y235A.Immunofluorescence microscopy confirmed that the F/YA22 and F/YA24 cell lines did indeed express far more CD63 than the parental 293F cell line , B–D. Asll lines , E–G rev0.00044) , E and Gp = 0.00038) but that F/YA24-derived exosomes contained 22-fold more CD63 than 293F cells . This 3.5-fold increase in the loading of an exosomal cargo protein  raises the intriguing possibility that exosome cargo protein loading operates preferentially at the very highest levels of exosome cargo protein expression.In addition to its utility for cell engineering, our data indicate that this marker may be of even greater utility for exosome engineering. Specifically, we observed that F/YA22-derived exosomes contained sixfold more CD63 than 293F-derived exosomes , binds specifically to the extracellular domain of CD63, and moreover, that this peptide can be used to dock a diverse array of other peptides and oligonucleotides on the surface of exosomes, imbuing them with specific biological activities and tissue tropisms , 40, 41.−/− cell line that could be used as a negative control for CP05-based engineering studies. Toward this end, we transfected 293F cells with recombinant Cas9 protein mixed with a CD63-specific guide RNA (gRNA) that targets the 3′ end of fifth common exon of the CD63 gene  (A). The transfected cells were grown for several days  and then processed for fluorescence-activated cell sorting (FACS) using a fluorescently tagged anti-CD63 antibody, with individual CD63-deficient cells sorted into distinct wells of a 96-well plate. After 2 weeks of culture, numerous SCCs were interrogated by quantitative RT–PCR to identify cell lines that had a significant reduction in CD63 mRNA abundance, as many of the most severe mutations in human genes  result in mRNA destruction by the nonsense-mediated RNA decay machinery . These mutations preclude the proper splicing of the CD63 gene and appear to have induced nonsense-mediated RNA decay turnover of their cognate mRNAs, as seen in the approximately sevenfold reduction in total CD63 RNA abundance observed for this cell line (C). Our data cannot, however, exclude the possibility that a small amount of C-terminally truncated CD63 proteins might be expressed in this cell line, though these CD63 proteins would lack its C-terminal 54 amino acids, including the last 18 amino acids of the second extracellular loop and the entirety of the fourth transmembrane domain and cytoplasmic tail.In anticipation of using CP05 peptides to engineer exosomes, we sought to create a CD63in bold) A. The trachinery . Candidaintron 5 B. These ell line C. Our da−/− cell line, the parental 293F cell line, and the F/YA24 cell line by flow cytometry, using a commercially available fluorescently tagged monoclonal anti-CD63 antibody. The average staining intensity for the 293F/CD63−/− cell line  was approximately fivefold lower than we observed for the WT 293F cell line  and ∼200-fold lower than we observed for the F/YA24 cell line, which expresses high levels of CD63/Y235A . Thus, while we cannot exclude the possibility that CD63−/− cells express some small amount of a truncated CD63 protein, it clearly displayed less cell surface CD63 than these other cell lines, even though we failed to include the preferred control of a matched isotype staining in these experiments.To determine whether these mutations led to a drop in the cell surface expression of CD63, we interrogated the 293F/CD63D). Furthermore, when we repeated these experiments using a 10-fold higher concentration of CP05, we observed a 10-fold increase in the cell surface labeling of all three cell lines (E). Taken together, these results are consistent with prior observations showing that the CP05 peptide binds biological membranes but provide additional clarity to those results by showing that CP05 binds to cell membranes in a CD63-independent manner.With these three cell lines in hand, we next tested the foundation of our planned CP05-based exosome engineering platform and asked whether a fluorescently tagged CP05 peptide would bind the surface of these cell lines in a CD63-dependent manner. Specifically, we synthesized the peptide NH2-CRHSQMTVTSRL(K/Cy5)-amide, resuspended it in cell-labeling buffer, incubated it with all three cell lines, washed the cells, and then interrogated each by flow cytometry. Much to our surprise, we found that this peptide displayed strong binding to the surface of all three cell lines, evident here in the strong Cy5 fluorescence detected for all three cell populations D. Furthell lines E. Taken We previously established that choice of AR gene is a major factor in determining the level of transgene expression in antibiotic-resistant cell lines . The dati.e., zeocin) by chelating them in complexes with a 1:1 stoichiometry , which in just 12 amino acids displays three positively charged side chains , four hydroxylated side chains , and two polar side chains (C1 and Q5). In short, the CP05 peptide is a highly polar polycationic peptide nearly ideally suited for nonspecific binding to biological membranes, which are known to have a nearly unsaturable capacity for binding polar polycationic polymers. In conclusion, the CP05 peptide may be a great way to “paint” the exosome surface with CP05-coupled molecules, but we are skeptical about whether this ability is due to specific CD63–CP05 binding.In light of these considerations, our observation that the CP05 peptide bound equally well to F/CD63E. coli cells grown in ampicillin-containing Luria broth media.Plasmids used in this study were based on the Sleeping Beauty transposon–carrying vector pITRSB , 19. Eac2. For stable clone selection, 293F cells were transfected with plasmid DNAs using Lipofectamine 3000 (Thermo Fisher Scientific), incubated for 48 h in CM, and then split into selective media . Antibiotic-resistant clones were fed every 3 to 4 days in selective media until distinct drug-resistant clones were large enough to be seen by eye, typically 2 weeks. The thousands of antibiotic-resistant SCCs that arose from each transfection were then pooled to create a single polyclonal cell line from each transfection, which were then expanded for an additional 2 weeks under antibiotic selection. Cells were then processed for flow cytometry, fluorescence microscopy, exosome collection, and immunoblot.293F cells  were grown in complete media  at 37 °C and 5% CO7 cells per ml in HBSS containing 0.1% fetal bovine serum at 4 °C. Cell suspensions were maintained on ice, diluted to a concentration of 1 × 106 cells per ml, and examined for mCherry fluorescence by flow cytometry on a Guava easyCyte flow cytometer (Luminex) set to the appropriate excitation and detection wavelengths. The relative brightness was determined for thousands of individual cells in each cell line using InCyte software (Luminex) and replotted with FlowJo (Beckton Dickinson), version 10, as scatter plots, average relative brightness, and CV.For measurement of mCherry fluorescence, cells were washed in Hank’s buffered saline solution , released from tissue culture plates using trypsin/EDTA solution, and resuspended at a concentration of ∼1 × 107 cells per ml in HBSS. Cell suspensions were maintained on ice, diluted to a concentration of 1 × 106 cells per ml, incubated with either (i) fluorescently labeled anti-CD63 antibody  or (ii) CY5-labeled CP05 peptide (NH2-CRHSQMTVTSRL(K/Cy5)-amide; Vivitide; molecular weight = 11,910). Following the binding reaction, cells were washed five times in PBS and then examined by flow cytometry on a Beckman CytoFlex flow cytometer set at the appropriate excitation and emission wavelengths. The relative brightness was determined for thousands of individual cells in each cell line using Beckman CytExpert software (version 2.3.1.22) to calculate average brightness and CV as well as to generate scatter plots and histograms.For measurement of surface CD63 abundance and Cy5-CP05 peptide binding, cells were washed in HBSS, released from tissue culture plates using trypsin/EDTA solution, and resuspended at a concentration of ∼1 × 10l-lysine–coated cover glasses. Cells were then washed in PBS and fixed in 3.7% formaldehyde in PBS for 30 min. Cover glasses were then placed cells side down on 0.050 ml of FITC-labeled anti-CD63 antibody, diluted in PBS, for 30 min , washed five times in PBS, and then fixed to glass slides using 0.007 ml of Fluoromount-G (Electron Microscopy Sciences).Cells were grown overnight on poly-6 cells/ml in shaker flasks in a volume of approximately one-fourth the flask volume and grown for 3 days at a shaking speed of 110 rpm. Cells and large cell debris were removed by centrifugation at 5000g for 15 min. The supernatant was passed through an ∼200 nm pore size diameter sterile vacuum filtration unit  to yield a clarified tissue culture supernatant. The clarified tissue culture supernatant was then subjected to two 30 min-long spins at 10,000g to remove any contaminating microvesicles, followed by collection of exosomes by spinning the samples at 100,000g for 2 h, discarding the supernatant, and resuspending the exosomes in PBS.293F cells and derivative clones were seeded into FreeStyle 293 Expression Medium  at a density of 1.5 × 10Cells and exosomes were lysed in SDS-PAGE sample buffer, separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and blocked for 1 h in blocking solution . The blocked membranes were then incubated overnight with primary antibody diluted into blocking solution at 4 °C, with gentle rocking. Following extensive washes in TBST, the polyvinylidene difluoride membranes were incubated with horseradish peroxidase (HRP)–conjugated secondary antibodies, again in blocking solution. The membranes were again washed extensively in TBST, followed by chemiluminescent detection of HRP using ECL Plus detection reagents . Antibodies were obtained from commercial sources .293F cells were transfected with purified Cas9 protein  and a gRNA (Invitrogen) targeting the sequence 5′-AACGAGAAGGCGATCCATA[AGG]-3′ in exon 5 of the CD63 gene. Following 3 days of incubation, CD63-deficient cells were isolated by FACS using an FITC-conjugated anti-CD63 antibody  on a Sony SH800S FACS machine. Single cells were seeded into a 96-well plate and expanded until the media began to turn acidic. To identify the CD63 mutations in different SCCs, genomic DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen), amplified by PCR using locus-specific primers, subcloned into a plasmid vector, expanded as single clones in bacteria, and then six individual clones were sequenced from plasmids derived from each genomic DNA PCR. To measure CD63 mRNA abundance, RNA was extracted by Quick-RNA Microprep Kit (Zymo Research), followed by reverse transcription with High-Capacity RNA-to-cDNA Kit (Applied Biosystems) and quantitative real-time PCR using SYBR Green qPCR Master Mix (Bio-Rad), gene-specific primers for CD63 mRNA (5′-CAGTGGTCATCATCGCAGTG-3′ and 5′-ATCGAAGCAGTGTGGTTGTTT-3′) and 18S rRNA (5′-CGGCGACGACCCATTCGAAC-3′ and 5′-GAATCGAACCCTGATTCCCCGTC-3′), and the CFX96 Real-Time PCR detection system (Bio-Rad). The expression of CD63 mRNA relative to 18S ribosomal RNA was calculated using the ΔΔCT method.Statistical analysis was performed using GraphPad Prism 8 software for Windows/Mac  or Excel (Microsoft). Flow data are reported as mean, median, and CV, whereas immunoblot data are reported as mean ± standard error, with significance determined by one-way analysis of variance.All data are contained within this article.S. J. G. is a paid consultant for Capricor, holds equity in Capricor, and is coinventor of intellectual property licensed by Capricor. S. J. T. and C. G. are coinventors of intellectual property licensed by Capricor. Y.I. declares that he has no conflicts of interest with the contents of this article.


P. lanceolata inflorescences, namely, phenylethanoid glucosides, acteoside, plantamajoside, and a flavonoid, isorhamnetin-3-O-rutinoside-4′-O-glucoside, possessed cytotoxic activity against the selected cancer cell lines. The potential antitumor effects of two phenylethanoid glycosides and one flavonoid were evaluated via MTT -2,5-diphenyltetrazolium bromide) assay on seven human carcinoma cell lines  and one nontumorigenic mammary epithelial cell line (MCF-12A). For the first time, acteoside was studied in ovarian cancer cell line OVCAR-3, and plantamajoside in all cell lines except breast adenocarcinoma MDA-MB-281 and hepatocarcinoma HepG2. The phenylethanoid glycosides showed stronger cytotoxic activity than that of the glycoside flavonoid. Acteoside and plantamajoside, at concentrations of 200 and 300 μM, respectively, had a highly toxic effect on the selected two cancer cell lines of breast adenocarcinoma MDA-MB-231 and MCF-7, ovarian cancer cell line OVCAR-3, glioblastoma cell line U138-MG, and hepatocarcinoma cell line HepG2. Both glycosides were significantly less cytotoxic towards nontumorigenic cell line MCF-12A; the effect appeared at a concentration of 400 μM. For the first time, the activity of acteoside and plantamajoside was compared in one parallel investigation. The results are discussed against a broad background of existing knowledge on biological effects, their mechanisms, and structure–activity relationships. Phenylethanoids may be potential compounds with cytotoxic activity against the selected cancer types. The aim of the study is to investigate whether the bioactive compounds isolated from  Plantago lanceolata L. (ribwort plantain), belonging to the Plantaginaceae family, is a perennial species with a global distribution, including the flora of Europe .Acteoside, when packed in liposomic nanoparticles, was less cytotoxic in normal human lung cells MRC-5  , melanocThere are several biological effects underlying the anticancer activity of acteoside, and numerous investigations have disclosed the molecular mechanisms responsible for those effects. The main effects are oxidative stress, apoptosis, and antiproliferative, antiangiogenesis, antimetastasis, anti-invasion, antitumor, and anti-inflammatory effects, and synergistic effects with anticancer drugs ,27. 50 of 85.0 and 44.0 µM to IC50 2.9 and 4.0 µM in glioblastoma cell lines T98G and U-138 MG, respectively, through an increase in apoptosis-associated proteins p35 and caspase-3 [Acteoside activity can be enhanced with a specific formulation. Acteoside in 190 nm liposomal nanoparticles, able to penetrate the membrane in the process of macropinocytosis, showed significantly enhanced activity from ICaspase-3 . Acteosiaspase-3 . Anisomeles indica (L.) Kuntze (Lamiaceae) [C-glucosylflavone, orientin, with a free 3′,4′-dihydroxy system showed weaker activity in glioblastoma cell lines T98G and U-138MG, both free (IC50 > 100 µM) and nanoformulated in liposomic substance (IC50 12.0 and 19.5 µM), than that of acteoside  (85) [Less frequent reports on the anticancer activity of plantamajoside have indicated similar effects to those of acteoside. In human breast cancer cell line MDA-MB-231, plantamajoside inhibited cell proliferation, migration, and invasion by decreasing the activity of matrix metalloproteinase-9 and -2 . In the miaceae) . A C-gluµM) (85) . Plantago lanceolata has been analysed a few times, but no study has related it to the presence of phenylethanoid glycosides. The methanol extract of P. lanceolata showed 50% growth-inhibitory values (GI50%) of >250, 47.17 and 50.58 µg/mL in renal adenocarcinoma (TK-10), breast carcinoma (MCF-7) and melanoma (UACC-62) cell lines, respectively. The activity was assigned to the flavonoids luteolin 7-O-glucoside and luteolin, and the mechanism of their action was topoisomerase I-mediated DNA damage [50 values of 172.3, 142.8, 405.5, and 551.7 µg/mL, respectively [50 23.7 µg/mL); the inhibition of the viability of normal embryonic fibroblast cells (MEFs) was much weaker (IC50 430 µg/mL) [The anticancer activity of Plantago genus with a high content of acteoside and plantamajoside, e.g., P. lanceolata-stabilized callus line with 28.4 mg/g of plantamajoside [P. media with a 93.0 mg/g acteoside content, the roots of this micropropagated species with a plantamajoside content of 44.1 mg/g [P. ovata agitated in liquid artificial media producing 33.02 mg/g of acteoside [Plantago species are a promising system for the biosynthesis of bioactive phenylethanoid glycosides.These studies confirmed the significant pharmacological effect of phenylethanoid glycosides in important disease entities. This group of compounds is widespread in about 20 families of higher plants and is widely studied for pharmaceutical, medical, and industrial applications ,14,58,59majoside , shoots 4.1 mg/g , and thecteoside . In termPhenylethanoids are potential compounds with cytotoxic activity against the selected cancer types. For the first time, the activities of acteoside and plantamajoside were compared in one parallel investigation. The two compounds acted on the same cancer cell lines, but acteoside was generally more potent. The difference in one sugar in the chemical structure of these two phenylethanoids influences their cytotoxic effect. Despite the numerous anticancer effects and mechanisms of action found especially for acteoside, the compounds under study appeared to be poorly effective in glioblastoma cell line U251-MG.


L promising targets. This study evaluates the effects of highly specific inhibitors for Bcl-xL (WEHI-539), Bcl-2 (ABT-199) and Mcl-1 (S63845) as radiosensitizers. Covering a broad spectrum of solid tumors, Non-Small-Cell Lung Cancer (NSCLC), Head and Neck Squamous Cell Carcinoma (HNSCC) and synovial sarcoma cell lines were exposed to fractionated radiation as standard therapy with or without Bcl-2 protein inhibition. Protein expression was detected by Western blot and cell death was assessed by flow cytometry measuring apoptosis. In contrast to NSCLC, a high level of Bcl-xL and its upregulation during radiotherapy indicated radioresistance in HNSCC and synovial sarcoma. Radioresistant cell lines across all entities benefited synergistically from combined therapy with Bcl-xL inhibition and fractionated radiation. In NSCLC cell lines, Mcl-1 inhibition significantly augmented radiotherapy independent of the expression level. Our data suggest that among antiapoptotic Bcl-2 proteins, targeting Bcl-xL may break resistance to radiation in HNSCC, synovial sarcoma and NSCLC in vitro. In NSCLC, Mcl-1 might be a promising target that needs further investigation. Avoidance of therapy-induced apoptosis is a hallmark of acquired resistance towards radiotherapy. Thus, breaking resistance still challenges modern cancer therapy. The Bcl-2 protein family is known for its regulatory role in apoptosis signaling, making Bcl-2, Mcl-1 and Bcl-x L and Mcl-1 are the most prominent representatives of the antiapoptotic branch of the family. They inhibit apoptosis by sequestering their proapoptotic relatives—a mechanism that is widely exploited by malignant cells to survive. Thus, resisting cell death is a hallmark of cancer harboring therapeutic implications [The Bcl-2 protein family regulates a cell’s fate by keeping a balance between pro- and anti-apoptotic proteins. The proapoptotic family members  react to apoptosis inducing signals by permeabilization of the outer mitochondrial membrane followed by subsequent cell death execution. BH3-only proteins  operate proapoptosis by binding antiapoptotic proteins ,2. Bcl-2L  and Mcl-1  have been evaluated in preclinical studies and are currently entering early clinical investigation. Promising results for solid tumors in terms of safety and efficiency from early clinical data will lead to further trials [Following this deliberation, inhibitors of antiapoptotic Bcl-2 family members (BH3 mimetics) were developed. The selective Bcl-2 inhibitor Venetoclax ABT-199) was the first compound approved for treatment of chronic lymphocytic leukemia (CLL) and certain types of acute myeloid leukemia (AML) ,5,6. Sper trials ,8,9.9 was theRadiotherapy is one of the cornerstones of many cancer therapies. Approximately 60% of patients with solid tumors receive an either curative or palliative irradiation within their treatment . By doseThe effect of ionizing radiation has been described in many studies. Amongst various forms of cell death, radiation-induced apoptosis is the most important effect ,13. IoniAs for many other tumor types, modern radiotherapy is a fundamental part of  treatment strategies in non-small-cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC) and soft tissue sarcomas such as synovial sarcoma. These tumor types may represent the variety of tumor histologies targeted by radiotherapy ,19,20. TCell death was assessed by flow cytometry via DNA fragmentation after using dose-fractionation schemes ranging from 4 × 0 to 4 × 5 Gy on NSCLC, HNSCC and synovial sarcoma cells . RadiatiWe then validated our findings by Western blotting for phosphorylated H2A histone family member X (pH2AX) and cleaved Poly-ADP-ribose polymerases (cleaved PARP) at 4 × 0 Gy and 4 × 2 Gy; cells were harvested 24 h after the last radiation session. pH2AX was used to measure additional double-strand DNA damage induced by radiation. Excluding SW982  and A549 (NSCLC), all other cell lines showed a detectable increase in pH2AX due to radiation. In line with the shown FACS data, there were no or barely detectable cleaved PARP in those cell lines described as being resistant above confirming very low apoptosis rates B.L were most resistant to radiation. The data also indicate that for these cell lines, upregulation could play a role in escaping radiation-mediated cell death, though densitometric analysis showed no significance in upregulation. Bcl-2 and Mcl-1 appear to play a minor role with expression levels being low, and apart from a slight upregulation of Bcl-2 among HNSCC cell lines, no detectable up- or down-regulation  B. Interegulation B.L seemed to play a role in radiation resistance among HNSCC and synovial sarcoma cell lines. Thus, a Western blot including all six cell lines was performed to adequately compare basal expression levels. Relative Bcl-xL expression was measured by densitometric analysis as described above and plotted against delta cell death at 4 × 4 Gy. The coefficient of determination  was calculated to measure the closeness of the data to the fitted linear regression (n = 6), a correlation of R2 = −0.78 , ABT-199 (Bcl-2) and S63845 (Mcl-1) were used to inhibit the prosurvival Bcl-2 protein family. Dose titration results were depicted graphically as a heat map . H838, a more resistant cell line, underwent a radiation schedule of 4 ×3 Gy, while A549, the most resistant NSCLC cell line, was irradiated with 4 × 5 Gy. Interestingly, H838 expressed the highest levels of Mcl-1 , Bcl-2 (ABT-199) and Mcl-1 (S63845) in the context of fractionated photon irradiation in exemplary solid tumors. We investigated a soft-tissue sarcoma subtype  as well as two different epithelial tumors (NSCLC and HNSCC), tumor types in which irradiation is an indispensable part of therapeutic concepts. Our HNSCC cell lines showed Human Papilloma Virus (HPV) negativity, and thus would correlate, in a clinical context, with a worse prognosis due to elevated resistance to radiotherapy and chemotherapy [L as a mechanism to escape cell death via tyrosine kinase inhibitors and sensitized them via BH3 mimetics [The objective of this study was to evaluate the effects of specific inhibitors for Bcl-xotherapy . This ismimetics . Interesmimetics .L in cancer survival and even demonstrate that cancer cells with focal amplifications of MCL1 and BCL2L1, the gene encoding Bcl-xL, especially depend on Mcl-1 and Bcl-xL for growth and survival, making both proteins ideal targets of molecular inhibition [Even though radiotherapy is a mainstay in treatment for different stages of synovial sarcoma, HNSCC and NSCLC, repair mechanisms and resistances still compromise the outcome. To this day, radiotherapy protocols are complemented by conventional cytotoxic agents such as Cisplatin in HNSCC and NSCLC to overcome the above-mentioned obstacles ,25. The hibition .L expression levels to be more resistant to irradiation and to increase Bcl-xL expression under radiotherapy. Those results file into existing discoveries from other studies. HNSCC cell lines with high Bcl-xL expression demonstrated an inferior response to chemotherapy or ionizing radiation and even turned out to be associated with poor clinical outcomes [L expression as a major factor for treatment response, while resistance to radiation may be associated with high Mcl-1 levels [Our findings revealed HNSCC and synovial sarcoma cell lines with high Bcl-xoutcomes ,28. Ow e1 levels . Even th1 levels . Coincid1 levels . Bcl-2 i1 levels . TherefoL level, meaning that a high Mcl-1-to-Bcl-xL mRNA ratio determines whether the cells entrust their survival to Mcl-1. All NSCLC cell lines we used showed a relatively identical high Mcl-1 expression. In a comparison of adenocarcinomas of the lung, Mcl-1 turned out to secure its survival and to be critical for its development [The effect of Mcl-1 inhibition in NSCLC with concomitant radiotherapy warrants further attention. Other studies have shown that Mcl-1 is found to be a major regulator in avoiding apoptosis in NSCLC . More prelopment . MunkhbaL high SW982. In combination with radiotherapy, Bcl-xL inhibition induces massive cell death presumably due to insufficient DNA repair mechanisms. For synovial sarcoma, Barrott et al. already suggested the in vitro therapeutic potential of Bcl-xL inhibition, underlining our findings [Sole treatment with WEHI-539 does not strongly affect the viability of Bcl-xfindings .L inhibitor ABT-263 showed in clinical trials dose-limiting side effects with thrombocytopenia, attributed to inhibition of Bcl-xL, and leukocytopenia, due to Bcl-2 inhibition [L inhibition with radiotherapy, thereby reducing the probability of adverse effects while still managing to obtain a notable increase in cell death. Our in vitro approach might be exploited in vivo, ultimately leading to clinical translation.Toxicity is a limiting factor for Bcl-2 inhibitor application. The combined Bcl-2/Bcl-xhibition ,40,41. Ahibition . We wereL inhibition needs further attention and may harbor great advantages for possible treatment strategies in the future in combination with radiotherapy.In conclusion, this study further unveils the capabilities of BH3 mimetics in solid tumors. The antiapoptotic Bcl-2 protein family seems to play an important role in ensuring survival in a tumor-type-specific pattern. Exclusively for NSCLC, Mcl-1 inhibition should be considered as radiosensitizer in follow-up studies. Applicable to all tumor types investigated here, Bcl-x2 in a humid atmosphere and cultured in Gibco’s RPMI 1640 Medium  supplemented with 10% fetal calf serum  and 1% Penicillin/Streptomycin (PAA). Solely A549 was grown and maintained in supplemented Gibco’s F-12K (Kaighn’s) Medium . Selective BH3 mimetics ABT-199  was provided by Abbvie, S63845 (Mcl-1-inhibitor) was purchased from Selleckchem  and WEHI-539 (Bcl-xL-inhibitor) from Hycultec .All human NSCLC and synovial sarcoma cell lines were purchased via ATCC , and HNSCC cell lines via CLS . The following cell lines were used: H838, A549, H1650 and H1975 (NSCLC). SW982, Fuji and SYO-1 . Detroit-562, FaDu and Cal-27 (HNSCC). All NSCLC cell lines are adenocarcinomas. H1975 has mutant Tumor protein 53 (TP53) (R273H) and the Epidermal growth factor receptor (EGFR) has L858R/T790M double mutation while H1650 has wild-type p53 and EGFR exon 19 deletion del E746-A750) . Cell li-A750 . CL , Mcl-1 , Bcl-2 , Cleaved PARP , pH2AX , Tubulin , and Actin . Secondary antibodies  were detected using PerkinElmer’s  Enhanced Chemiluminescence Substrate (# NEL103001EA), and signal intensity was measured with ImageJ® . Densitometric analysis was then performed by norming the expression band to their respective loading control. The value of 1.0 was assigned to a cell line of reference and densitometric values were plotted against the amount of induced cell death at 4 × 4 Gy.Cells were seeded as indicated for protein analysis. Protein isolation was performed and lysates separated using a 12% acrylamide concentration for the separating gel. After separation and solubilization via Sodium dodecyl sulfate–Polyacrylamide gel electrophoresis (SDS-PAGE), the samples were blotted onto a nitrocellulose membrane using a wet blot transfer apparatus  following standard procedures. Immunodetection was performed with following primary antibodies: Bcl-xw/v) sodium citrate and 0.1% (v/v) Triton X-100. After a 24 h incubation period at 4 °C in the dark, cell death was measured following the protocol of Nicoletti et al. using a CANTO II flow cytometer  [Cells were treated according to protocol before the supernatant and cells were transferred to the corresponding FACS tubes. Accutase™  was used for cell detachment prior to flow cytometry to reduce potential proteolytic effects of trypsinization. After centrifugation, the supernatant was discarded and the cell pellet resuspended in buffer consisting of 50 µg/mL propidium iodide , 0.1%  . Cell cyIn vitro experiments with ionizing radiation were performed with the X-Rad 320 x-Ray system cabinet from Precision X-Ray , parameters were set to 1 Gy/min at 320 kV and 12.5 mA for all experiments. A focus-to-skin distance (FSD) of 50 cm was chosen with the adjustable sample shelf and photon beam filtration was achieved using a filter consisting of 1.5 mm aluminum, 0.25 mm copper and 0.75 mm tin. Radiation dosage was applied with a fractionated schedule of 4 consecutive days of radiation with 1 to 5 Gy in each session to mimic clinical partitioning of the overall dose to smaller fractions. Cells were seeded 24 h prior to radiation to reach a confluency of 70–80% and split after the second radiation session to circumvent overgrowth. The harvesting point was chosen 24 h after the last session. Figures depicting the experimental setup were created using BioRender.com (accessed on 14 July 2022).p < 0.05; ** p < 0.01; *** p < 0.001.Statistical analysis was performed using GraphPad PRISM 9.0 . In vitro experiments were executed in biological triplicates and summarized as mean values with standard deviation. Normally distributed data were analyzed using an unpaired t-test, and for comparisons of more than two variables a two-way analysis of variance (two-way ANOVA) matching both factors was conducted. Correlations were verified using linear regression models. Statistical significance is indicated as *


Environmental exposure to fibers of respirable size has been identified as a risk for public health. Experimental evidence has revealed that a variety of fibers, including fluoro-edenite, can develop chronic respiratory diseases and elicit carcinogenic effects in humans. Fluoro-edenite (FE) is a silicate mineral first found in Biancavilla  in 1997. Environmental exposure to its fibers has been correlated with a cluster of malignant pleural mesotheliomas. This neoplasm represents a public health problem due to its long latency and to its aggression not alerted by specific symptoms. Having several biomarkers providing us with data on the health state of those exposed to FE fibers or allowing an early diagnosis on malignant pleural mesothelioma, still asymptomatic patients, would be a remarkable goal. To these purposes, we reported the miRNA transcriptome in human normal mesothelial cell line (MeT-5A) and in the human malignant mesothelioma cell line (JU77) exposed and not exposed to FE fibers. The results showed a difference in the number of deregulated miRNAs between tumor and nontumor samples both exposed and not exposed to FE fibers. As a matter of fact, the effect of exposure to FE fibers is more evident in the expression of miRNA in the tumor samples than in the nontumor samples. In the present paper, several pathways involved in the pathogenesis of malignant pleural mesothelioma have been analyzed. We especially noticed the involvement of pathways that have important functions in inflammatory processes, angiogenesis, apoptosis, and necrosis. Besides this amount of data, further studies will be designed for the selection of the most significant miRNAs to test and validate their diagnostic potential, alone or in combination with other protein biomarkers, in high-risk individuals’ liquid biopsy to have a noninvasive tool of diagnosis for this neoplasm. Malignant pleural mesothelioma (MPM) is an aggressive neoplasm of the pleural surface causally related to exposure to asbestos fibers . In factEnvironmental exposure to these carcinogen fibers represents a public health problem due to the aggression of MPM not alerted by specific symptoms and to its long latency. It is clear that the first step to prevent carcinogen fibers exposure-related diseases is to reduce the presence of such airborne fibers through confinement, encapsulation, and reclamation of the artifacts that contain them . These fAs is widely known, miRNAs are evolutionarily conserved long 18–25 nucleotide (nt) single-stranded RNAs that negatively modulate the expression of their target mRNAs. miRNAs are important molecules in gene expression regulation at the post-transcriptional level, indeed, a single miRNA can control several mRNAs’ expression, and a single mRNA may be targeted by more than one miRNA, thus creating a network effect of cooperative regulation . Many stAs mentioned above, in this study, we reported a small RNA-Seq transcriptome profiling of human normal mesothelial cell line (MeT-5A) and in human malignant mesothelioma cell line (JU77) exposed and not exposed to FE fibers. Both these cell lines have been processed with and without FE fibers exposure, as reported in the experimental workflow . Subsequ2. The MeT-5A and JU77 cell lines were split 1:3 and 1:6, respectively, twice a week.A normal human mesothelial cell line (MeT-5A) and a malignant human mesothelioma cell line (JU77) were obtained from the American Type Culture Collection . Both cell lines have been cultured in Roswell Park Memorial Institute 1640 (RPMI 1640) medium supplemented with 10% fetal bovine serum, 1% L-glutamine , 1% non-essential amino acids solution , and 1% penicillin/streptomycin . The culture conditions were 37 °C in a humidified atmosphere with 5% COCells in confluent condition were separated from the culture flask  using 0.25% trypsin in 2.21 mM EDTA solution  and counted using Bürker chamber by Trypan Blue Stain 0.4% . The cells used for the experiments were between II/III passages.FE fibers were obtained from the Biancavilla quarry . These were sampled using magnifiers, needles and tweezers. Subsequently the fibers were weighed, sterilized under UV light for 10 min, suspended in a known volume of RPMI 1640 medium, and sonicated through Omni-Ruptor 4000 Ultrasonic Homogenizer  for 10 min. The stock solution was then diluted appropriately to obtain the different concentrations for in vitro treatments.3 cells/50 μL, while JU77 were plated at the density of 4 × 103 cells/50 μL. After 24 h of incubation, 50 μL of FE fibers solutions were added to the cell cultures in amounts corresponding to final concentrations from 200 to 0.78 μg/mL. Both cell lines grown in FE-free medium were used as controls. At each time point (from 6 to 72 h of FE exposure) in cell culture, 10% MTT in Dulbecco’s Phosphate-Buffered Saline (DPBS)  has been added to each well. After 4 h of incubation, the lysis solution  has been added to each well. The optical density was measured with an absorbance microplate reader  at λ = 620 nm. For each sample, three replicates were performed. Cell viability was calculated as the percentage of viable cells treated with FE fibers vs. untreated control cells as follows: Cell viability (%) = [OD (Treatment) − OD (Blank)]/[OD (Control) − OD (Blank)] ×100 [p < 0.05 was considered statistically significant.The in vitro functional experiments were preceded by the determination of the dose-response curves for both cell lines. MeT-5A were plated onto 96-well plates  for the dose-response curve at the density of 6 × 106 cells while JU77 was plated at the density of 8.5 × 105 cells onto 100 × 20 mm Petri Dishes  for these functional experiments. After 24 h-incubation, the medium of both cell lines has been replaced with FE fibers solutions to final concentrations of 50 and 10 μg/mL [MeT-5A was plated at the density of 1 × 1010 μg/mL . MeT-5A g for 5 min at 4 °C and suspended in 1 mL cold DPBS. The cell solution was transferred to Eppendorf tubes and cells were centrifuged at 0.8× g for 5 min at 4 °C [After removing the supernatant, cells were harvested on ice by scraping in cold DPBS. Cells are then centrifuged at 0.2×  at 4 °C . After rTotal RNA containing small non-coding RNA was extracted from the cell lines using miRNeasy Mini Kit  according to the manufacturer’s recommended protocols (miRNeasy Mini Handbook 11/2020). The RNA was quantified by the absorbance ratio at λ = 260/280 nm through NanoDrop (ND 1000) UV–Vis spectrophotometer . All samThe QIAseq miRNA library kit  was used for small RNA-Seq library preparation following the manufacturer’s instructions. RNA samples were quantified and quality tested by Agilent 2100 Bioanalyzer RNA assay . Libraries were then checked with both Qubit 2.0 Fluorometer  and Agilent Bioanalyzer DNA assay or Caliper . Finally, libraries were prepared for sequencing and sequenced on single-end 150 bp mode on NovaSeq 6000  .p-value (Benjamini–Hochberg correction) <0.05 were considered differentially expressed. Finally, the impact of differentially expressed miRNAs on biological pathways was evaluated by using MITHrIL [p-values without a Log2FC cutoff in order to evaluate also the impact of slightly differentially expressed miRNAs on biological pathways. All the above-mentioned steps of the analysis were performed using RNAdetector [Low-quality reads and adapters were trimmed using Trim Galore, which is a wrapper of FASTQC  and Cuta MITHrIL . In thisdetector  softwareThe in vitro functional experiments were preceded by the determination of the dose-response curves for both cell lines. The results showed a significant difference between 6 h treatments and all other time points  at all tested concentrations exposure to FE fibers. Finally, an exposure time to the fibers equal to 48 h was chosen because longer exposure times did not cause statistically significant differences in cell viability .In order to identify dysregulation in miRNAs induced by FE fibers, we performed an RNA-Seq transcriptome profiling of unexposed and exposed normal mesothelial (MeT5A) and malignant mesothelioma (JU77) cell lines. The differentially expressed miRNAs were: 333 untreated JU77 vs. MeT5A (145 up-regulated and 188 down-regulated), 323 in JU77 vs. MeT5A exposed to 10 μg/mL FE fibers (101 up-regulated and 222 down-regulated), and 325 in JU77 vs. MeT5A exposed to 50 μg/mL FE fibers (124 up-regulated and 201 down-regulated) . Among tHowever, when we compared miRNAs expression between unexposed vs. exposed MeT-5A, the results showed very few differentially expressed molecules. In particular, there were 40 differentially expressed miRNAs in MeT5A untreated vs. exposed to 10 μg/mL FE fibers (22 up-regulated and 18 down-regulated) and 35 differentially expressed miRNAs in MeT5A untreated vs. exposed to 50 μg/mL FE fibers (8 up-regulated and 27 down-regulated). On the other hand, the comparison between untreated vs. FE-treated JU77 showed several differentially expressed miRNAs. In particular, miRNAs differentially expressed in JU77 unexposed vs. exposed to 10 and 50 μg/mL FE fibers were 113 (24 up-regulated and 89 down-regulated) and 125 (22 up-regulated and 103 down-regulated), respectively . The miROnce the differentially expressed miRNAs for each comparison were identified, we investigated the impact of their dysregulation in metabolic and signaling pathways by using MITHrIL . MITHrILThe results demonstrated clear patterns of positive and negative perturbation scores involving 96 different pathways between the JU77 and MeT5A untreated and exposed to 10 and 50 μg/mL FE fibers. Of these, 28 showed mildly positive perturbation scores, while 68 showed mildly strong negative perturbation scores. Among these latter pathways, strong, negative patterns were observed in 25 pathways, including apoptosis; regulation of lipolysis in adipocytes; endocytosis; ovarian steroidogenesis; focal adhesion; progesterone-mediated oocyte maturation; axon guidance; adherence junction; longevity regulating pathway; regulation of actin cytoskeleton; sphingolipid signaling pathway; chemokine signaling pathway; leukocyte trans-endothelial migration; signaling pathways regulating pluripotency of stem cells; and specific signaling pathways of Rap 1, cGMP-PKG, AMPK, Hippo, PI3K-Akt, TGF-beta, ErbB, cAMP, Ras, and FoxO .The pathway analysis performed between untreated vs. FE fibers treated JU77 and MeT-5A and the main impacted pathways showed clear patterns of positive and negative correlations involving 121 different pathways. Of these, 66 showed mildly strong, positive correlations and, among these 25, showed strong, positive correlations including signaling pathways of FoxO, PI3K-Akt, Rap1, VEGF, p53, and Ras; the regulation of actin cytoskeleton; signaling pathways of chemokine, prolactin, sphingolipid, estrogen, insulin, thyroid hormone, and oxytocin; long-term depression; progesterone-mediated oocyte maturation; natural killer cell-mediated cytotoxicity; Gap junction; serotonergic and cholinergic synapse; axon guidance; signaling pathways regulating the pluripotency of stem cells; longevity regulating pathways; and focal adhesion. Mildly strong, negative correlations were observed for 55 patterns, and among these, 14 showed strong, negative correlations, including metabolic pathways of arginine and proline, alanine, aspartate, and glutamate; glycolysis/gluconeogenesis; signaling pathways of adipocytokine; NF-kappa B; Toll-like receptor; glucagon; HIF-1; Jak-STAT; osteoclast differentiation; glycosaminoglycan degradation; cytokine–cytokine receptor interaction; and the adherence junction. The correlations between these pathways were quite obvious for the JU77 cell line untreated vs. treated with FE fibers .Small RNA-Seq transcriptome profiling of healthy mesothelium and MPM in vitro has been evaluated to highlight the deregulated miRNAs and the various pathways involved in an aggressive cancer such as MPM.Certainly, the results showed a big difference in the number of deregulated miRNAs between tumor and nontumor samples both exposed and not exposed to FE fibers. As a matter of fact, the effect of exposure to FE fibers is more evident in the expression of miRNAs in tumor samples than in nontumor samples.In the present paper, we analyzed several pathways that are involved in the pathogenesis of MPM. It is very interesting to point out the strong perturbation scores involving the above-mentioned pathways in the MPM vs. healthy mesothelial cell line. Among these, certain pathways were involved that play important roles in inflammatory processes and angiogenesis. Inflammation has a central role, since mesothelioma is a multicentric neoplasm that originates from inflammatory foci. Inflammation has been correlated with cancer, enhancing the development of malignancies . In partSpecific signaling pathways have been found to be involved in malignant mesothelioma. Among these, Ras and p53, the commonly mutated genes associated with cancer, are rarely targeted in malignant mesothelioma . Ras hasOur goal was the validation of the most promising results in a subset of patients chronically exposed to FE fibers, using the liquid biopsy, to provide a minimally invasive screening tool for the secondary prevention of MPM. Early detection of circulating tumor biomarkers represents one of the most promising strategies to enhance the survival of cancer patients by increasing treatment efficiency . Besides