Tumor dissemination and recurrence is attributed to highly resistant cancer stem cells (CSCs) which may constitute a fraction of circulating tumor cells (CTCs). Small cell lung cancer (SCLC) constitutes a suitable model to investigate the relation of CTCs and CSCs due to rapid tumor spread and a high number of CTCs. Expansion of five SCLC CTC lines  in vitro at our institution allowed for the analysis of CSC markers and cytotoxicity of the CSC-selective drugs salinomycin and niclosamide against CTC single cell suspensions or CTC spheroids/ tumorospheres (TOS). Salinomycin exerted dose-dependent cytotoxicity against the SCLC lines but, with exception of BHGc7 TOS, there was no markedly enhanced activity against TOS. Similarly, niclosamide exhibits high activity against BHGc7 TOS and UHGc5 TOS but not against the other CTC spheroids. High expression of the CSC marker CD133 was restricted to three SCLC tumor lines and the BHGc10 CTC line. All SCLC CTCs are CD24-positive but lack expression of CD44 and ABCG2 in contrast to the SCLC tumor lines which show a phenotype more similar to that of CSCs. The stem cell marker SOX2 was found in all CTC lines and SCLC GLC14/16, whereas elevated expression of Oct-3/4 and Nanog was restricted to BHGc26 and UHGc5. In conclusion, the SCLC CTCs established from patients with relapsed disease lack a typical CSC phenotype in respect to chemosensitivity to CSC-selective drugs, surface markers, expression of pluripotent stem cell and transcription factors. Tumors seem to contain a small subpopulation of cancer stem cells (CSCs) which sustain primary tumors and metastases –4. CSCs liplatin . A few cliplatin , 27. In -catenin , 29. Add-catenin . The ant-catenin .Metastasis-initiating capabilities of CTCs based on stemness properties are difficult to detect since CTCs show extensive heterogeneity and only an extremely small fraction of these cells is able to establish secondary lesions , 33. In SCLC26A was established in our lab from a pleural effusion of a SCLC patient before treatment. GLC14 and GLC16 have been derived from the same patient before and after first cycles of chemotherapy , 42, DMSPictures of clusters of SCLC CTCs were acquired from tissue cultures and processed using Photoshop . Preparation of a TOS histological section was done as described previously .4 cells in form of single cells or tumorospheres (TOS) in 100 μl medium were distributed to wells of 96-well microtiter plates  and ten 2-fold dilutions of the test compounds were added from stock dilutions as described [50 values were determined from dose-response curves using Origin 9.1 software .1 × 10escribed . Assays Antibodies were obtained from Biolegend . Expression of CD133 (Clone 315-2C11), CD24 (ML5), CD44 (IM7) and ABCG2 (5D3) was determined in flow cytometry using corresponding secondary reagents (Sigma-Aldrich) and a Cytomics FC500 flow cytometer  in indirect immunofluorescence. Data analysis and histogram overlays were done employing the Kaluza flow analysis software (Beckman Coulter).Pluripotent stem cell markers were analyzed using the ARY010 Proteome Profiler Array  according to manufacturer’s instructions. Experiments were done in duplicate and the different tests were calibrated using the six reference spots included for each individual membrane. Arrays were evaluated using ImageJ and Origin 9.1 software .p < 0.05 regarded as significant difference using Origin software .Statistical significance was tested by t-tests and The cytotoxic activity of salinomycin against SCLC CTC and tumor cell lines was tested in MTT proliferation assays. An example of a dose-response curve for BHGc10 single cells and TOS is shown in Fig. 50 values for all SCLC cell lines are shown in Fig. 50 values in the 1 μM range. All other SCLC lines yield IC50 values for salinomycin in the 2–4 μM range, including BHGc7 single cells, BHGc10 and BHGc26TOS.The ICThe cytotoxic activity of niclosamide against CTC and tumor SCLC cell lines was tested in MTT proliferation assays Fig. . The celAnalysis of CSC marker expression was done using indirect immunofluorescence and flow cytometry. High expression of the CSC marker CD133 was detected in the BHGc10 CTC and DMS53 tumor cell lines, whereas expression was absent in the BHGc16 CTC cells Fig. .Fig. 4FlThese three SCLC cell lines showed expression of CD24 to a variable degree In addition to CD133-positive DMS53 and BHGc10, significant expression of this antigen in GLC14 and GLC16 SCLC lines and peak labeling of BHGc16 CTC and NCI-H69 cells was found , as well as unique mutations of p53 , 43. All+/CD24− CSC phenotype [50 values of salinomycin vary between 0.3 and 10 μM, depending on the cell type used and treatment period [With the single exception of the SCLC NCI-H69 cell line, salinomycin has more pronounced cytotoxic effects against the SCLC CTC cell lines including BHGc16, BHGc16 TOS, BHGc26 and UHGc5 TOS. TOS show higher resistance to chemotherapeutics such as cisplatin, topotecan and epirubicin compared to the corresponding single cell suspensions . Howeverhenotype . Cell dehenotype , 48–50. t period , 52. Howt period .Niclosamide showed high cytotoxic activity against most SCLC lines with exception of GLC14 and GLC16. Among the SCLC CTC lines, BHGc7 TOS and UHGc5 TOS were more resistant to niclosamide than the corresponding single cell fractions in contrast to BHGc10 TOS, BHGc16 TOS and BHGc26 TOS which exhibited the reversed effect. Again, this CSC-selective drug exhibits heterogenous activity against the SCLC CTC spheroids. Niclosamide has antitumor activity via inhibition of NF-κB, an increase in ROS and blocking of the Wnt/β-catenin, STAT3 and NOTCH signaling pathways but also targets mitochondria in cancer cells , 55. How+ CD90+ subpopulation expressing increased levels of Nanog and Oct-4. CD133 (Prominin 1) has been used to identify CSCs in lung cancer and was linked to high levels of ABCG2 and chemoresistance [−/CD87− cells showed the highest tumorigenic potential [SCLC is sensitive to the first-line therapy in most patients but invariably recurs as chemo- and radioresistant tumor within approximately one year . This nasistance . Severalsistance , 63. Thesistance . CD133 psistance , 66. Howotential . Signifiotential . In conchigh/CD24low cells, which have also been found in pleural metastases [+ CSCs were reported to be less proliferative and more resistant to chemotherapy compared to CD24− cells [+ SCLC cells and, in particular, from all SCLC CTC lines. No expression of the chemoresistance mediator ABCG2 could be detected in the SCLC lines studied here (data not shown). In conclusion, CD133 is not expressed in response to prior chemotherapy and a CD24+/CD44−/ABCG2− phenotype does not fit to the panel of markers generally expressed by typical CSCs of solid tumors, with the possible exception of BHGc10. Furthermore, SOX2 seems to represent a general marker of the neuroendocrine SCLC tumors which is not restricted to CSCs. Expression of Oct-3/4 and Nanog is limited to BHGc16 und BHGc26 which lack significant expression of CD133 and GLC14/16.In respect to CTCs, CD44 has been identified as CSC marker of gastric cancer and most of the CTC-positive patients showed CD44-positive CTCs . In breatastases . Recent tastases . Early dtastases . Our restastases , 42. CD24− cells . With ex−/CD24+/ABCG2− salinomycin-sensitive phenotype which lack CD133 expression. With exception of SOX2, pluripotent stem cell transcription factors Oct-3/4 and Nanog are expressed to a minor extent in the CTC cell lines studied. The SCLC CTC cell lines are chemosensitive to topotecan and epirubicin as single suspensions in good agreement with the lack of ABCG2 expression [The numerous studies reporting CSC traits in SCLC tumor cells point to a possible role of this phenotype in resident SCLC primaries and secondaries but our results preclude a role of CSC characteristics among SCLC CTCs , 74. It pression . Howeverpression , 76. CSC


Our results demonstrate that a short time interval between treatments caused more unrepaired DNA damages and more cell kill, especially at higher temperatures. Although hyperthermia before ionizing radiation may result in slightly more DNA damage, the sequence between hyperthermia and ionizing radiation yielded similar effects on cell survival. Cervical cancers are almost exclusively caused by an infection with the human papillomavirus (HPV). When patients suffering from cervical cancer have contraindications for chemoradiotherapy, radiotherapy combined with hyperthermia is a good treatment option. Radiation-induced DNA breaks can be repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Hyperthermia can temporarily inactivate homologous recombination. Therefore, combining radiotherapy with hyperthermia can result in the persistence of more fatal radiation-induced DNA breaks. However, there is no consensus on the optimal sequence of radiotherapy and hyperthermia and the optimal time interval between these modalities. Moreover, the temperature of hyperthermia and HPV-type may also be important in radiosensitization by hyperthermia. In this study we thoroughly investigated the impact of different temperatures (37–42 °C), and the sequence of and time interval (0 up to 4 h) between ionizing radiation and hyperthermia on HPV16 Most cases of cervical cancer are caused by infection with human papillomavirus (HPV). HPV types 16 and 18 are responsible for >70% of all cases of cervical cancer in the world ,3, incluHyperthermia has the ability to change the microenvironment and make cells more sensitive to radiotherapy. The precise mechanisms on how hyperthermia enhances tumor oxygenation are not fully understood. Hyperthermia can induce hypoxia-inducible factor-1 (HIF-1), a potent regulator of tumor vascularization and metabolism, which can push surviving cells toward glycolysis . HyperthMultiple mechanisms are responsible for the radiosensitization effect by hyperthermia, each operating at different temperatures and each with different optimal sequences and time intervals ,14. Hype+ (SiHa and Caski), HPV18+ (HeLa and C4I), and HPV negative (C33A and HT3) cell lines, which sequence and time interval (0 up to 4 h) between applying ionizing radiation and hyperthermia caused the most damage to the tumor cells at different temperatures (37 to 42 °C). Effects on cell cycle distribution, DNA damage level, and cell survival fraction were assessed. Finally, different mechanisms explaining these results will be discussed.In this study we investigated in in vitro models of HPV16+, HPV18+, and HPV-negative cervical carcinoma cell lines. A schematic overview of treatments is given in + SiHa (red) and Caski (orange) (+ HeLa (green) and C4I (yellow)  were tested and the effect of different temperatures was investigated for HPV16(orange) B; HPV18+(yellow) C; and HP(yellow) D. Per th+ SiHa (red) and Caski (orange) (+ HeLa (green) and C4I (yellow)  E; HPV18+(yellow) F; and HP(yellow) G cervica+ cell lines (+ cell lines HeLa (green), C4I (yellow), HPV-negative cell lines  between ionizing radiation and hyperthermia resulted in the lowest cell survival. Moreover, there is no significant difference between the different sequences in clonogenic capacity.Cell cycle distribution was studied by the incorporation of BrdU. A schematic overview of treatments is given in Apoptosis after different time intervals and for the different sequences of ionizing radiation (4 Gy) and hyperthermia (42 °C) was measured by the Nicoletti assay. A short time interval between ionizing radiation and hyperthermia resulted in the highest apoptotic levels measured by flow cytometry. All cell lines showed that approximately 30–35% of cells were apoptotic after a 0 h time interval between ionizing radiation and hyperthermia, compared to approximately 20–30% after a 4 h time interval between ionizing radiation and hyperthermia . All cel+ SiHa and Caski , was measured by nuclear γ-H2AX staining, which may be very important to understand the earlier described differences in cell survival. To prevent the counting of nonradiation induced DSBs, exclusion of S-phase cells was done using Edu-exclusion A. In Fignd Caski C, HPV18+ and C4I D, and HP and C4I E cell li+ cell lines  between the two treatment modalities, suggesting cells are halted and the damage is too severe to continue to the G2 phase. Since the number of cells in S phase was unchanged or larger after treatment, there is no indication that cells went into cell senescence. The outcome of ionizing radiation is affected by the cell cycle, mitotic cells are hypersensitive because they can inactivate DSB repair, so during interphase, cell survival is maximal when cells are irradiated during the early G1 and G2 phase of the cell cycle and is minimal during the S phase . PreviouHigher levels of DNA damage were observed after treatment with a shorter time interval between ionizing radiation and hyperthermia, indicating that there was more remaining DNA damage after a short time interval between the two therapies. Blocking DNA repair was clearly the dominant radiosensitizing effect of hyperthermia in our in vitro experiments, the question is: which pathways are involved? Currently, it is generally accepted that hyperthermia downregulates BRCA2 , a proteAll the in vitro data converge to the conclusion that a higher temperature 42 °C vs. 39 or 41 °C of HT and the shortest time interval (0 h) between ionizing radiation and hyperthermia results in the lowest cancer cell survival. In six different cervical cancer cell lines, all experiments were conducted independently and performed at least three times. Our results confirm the data reported in a study of Van Leeuwen et al. , in whicDecades ago, similar experiments were performed on mammalian cell lines, in which Chinese hamster ovary (HA-1) and mouse mammary sarcoma (EMT-6) cells were investigated regarding the interaction between hyperthermia and ionizing radiation. Li and Kal concluded that in HA-1 cells the radiosensitizing effect of hyperthermia was larger when applied before ionizing radiation , but in in vivo model, in which the tumor microenvironment is taken along as well as the relevant healthy tissues around the target region. In patients, a short time interval has been shown to result in lower in-field recurrence and a longer overall survival   data are expressed as the mean with standard deviation of at least three independent experiments. Statistical analyses for cell survival, γ-H2AX staining, and apoptosis levels were performed using two-way ANOVA analysis using GraphPad Prism version 8 .In summary, hyperthermia is an effective tumor sensitizer for ionizing radiation. However, optimizing therapies is important to find the best clinical outcome. Our in vitro results demonstrated that the time interval between hyperthermia and ionizing radiation is an important factor that determines the effectiveness of this combinational therapy. Moreover, our results may indicate that HPV-negative cell lines are already quite sensitive to ionizing radiation alone, while the HPV-positive cell lines require hyperthermia in order to reduce cell survival. The HPV16+ cell line seems to be more sensitive to the combination of hyperthermia and ionizing radiation, already at lower doses of ionizing radiation, compared to the HPV18+ cells. However, the survival fraction, cell cycle distribution, apoptotic levels, and DNA damage repair is more dependent on the time interval between the two treatments, rather than on the type of HPV. In conclusion: a shorter time interval between ionizing radiation and hyperthermia results in lower survival fractions than after a longer time interval, and the sequence between the two modalities did not demonstrate any significant differences on cervical cancer cell lines. However, studying the effects on normal tissue should be taken into account before the most optimal way of applying hyperthermia and radiotherapy can be determined.


The origin recognition complex (ORC) cooperates with CDC6, MCM2-7, and CDT1 to form pre-RC complexes at origins of DNA replication. Here, using tiling-sgRNA CRISPR screens, we report that each subunit of ORC and CDC6 is essential in human cells. Using an auxin-inducible degradation system, we created stable cell lines capable of ablating ORC2 rapidly, revealing multiple cell division cycle phenotypes. The primary defects in the absence of ORC2 were cells encountering difficulty in initiating DNA replication or progressing through the cell division cycle due to reduced MCM2-7 loading onto chromatin in G1 phase. The nuclei of ORC2-deficient cells were also large, with decompacted heterochromatin. Some ORC2-deficient cells that completed DNA replication entered into, but never exited mitosis. ORC1 knockout cells also demonstrated extremely slow cell proliferation and abnormal cell and nuclear morphology. Thus, ORC proteins and CDC6 are indispensable for normal cellular proliferation and contribute to nuclear organization. Saccharomyces cerevisiae is the best characterized system, from which individual proteins involved in DNA replication have been identified and studied extensively, including functional reconstitution of the entire pre-RC assembly and the regulated initiation of DNA replication from these pre-RCs with purified proteins , comprising Orc1-6 subunits, binding to each potential DNA replication origin  at origins of DNA replication throughout the genome . Among eproteins . In S. cn origin . Chromatn origin . The Mcmn origin . During n origin . The funn origin .S. cerevisiae and human cells, the ORC1-5 subunits contain a AAA+ or a AAA+-like domain and a winged-helix domain (WHD)  . In yeasin (WHD) . ORC binin (WHD) . On the in (WHD) . ORC subin (WHD) . One or  mitosis . In huma mitosis .S. cerevisiae, S. pombe, Xenopus, and Drosophila, since mutation or depletion of ORC prevents CDC6 binding and MCM loading onto DNA  cells in which expression of either ORC1 or ORC2 subunit was eliminated using CRISPR-Cas9-mediated gene ablation could still proliferate  strategies have shown that targeting regions within protein domains typically show significantly higher degree of negative selection phenotypes . This isll types . Other mproteins . Subsequferation .Streptococcus pyogenes Cas9) across each exon of ORC1-6 and CDC6 were designed and synthesized. Pooled CRISPR libraries also included control guide RNAs targeting either known core essential genes such as CDK1, PCNA etc. as positive controls, or those targeting non-essential gene loci or no loci at all as negative controls  since essential protein domains correlate with higher negative selection phenotype, we hypothesized that analyzing the effect of every possible guide RNA target site in the ORF might uncover new functional regions, and (b) a high-throughput screen of this nature would provide incontrovertible evidence about the essentiality of ORC and CDC6 proteins, at least in the cell lines tested. A recent study validated this approach by analyzing tiling-sgRNA data from Munoz et. al. and found that up to 17.7% of the regions that displayed a CRISPR knockout hyper-sensitive (CKHS) phenotype, did not overlap with previously annotated domain or known function . Thus, gcontrols . The totNeGeCKO’ , negativNeGeCKO’ , Rosa26,egatives  and 43 phttps://toolkit.tuebingen.mpg.de/frpred) server that calculates a conservation score based on amino acid variability as well as the probability of it being a functional ligand binding or catalytic site at each amino acid position of the input sequence (https://consurf.tau.ac.il/) server which analyses the probability of structural and functional conservation despite amino acid variability for any given position of input sequence (http://bioinf.cs.ucl.ac.uk/psipred/) that scores for intrinsically disordered regions (IDRs) that are usually not well conserved yet found to be functionally essential in many proteins  Consequence   dataset was compared to a combined RNAi dataset of cell lines, it indicated that using the CRISPR method, with a gene effect score of less than −0.5, ll lines . It is eed study . For ORChers not . Of note-1 cells . CompariORC4, which is entirely structured, and thus none of the sgRNAs target NAD regions. For all other genes, the fraction of NAD targeting sgRNAs that scored as essential, as well as their locations within these regions were comparable between cell lines rather than between proteins  – that is sgRNAs targeting AD regions showed significantly higher depletion compared to those targeting non-annotated domain (NAD) regions  and 7a. proteins . This suin vitro . In yeasin vitro . Anotherin vitro .p53 status of the cell, post-translational modifications, local structure of the gene locus can confound analyses from high-throughput tiling-sgRNA CRISPR screening  was constructed, and the ORC2 cDNA was edited to harbor multiple mismatches based on two of the four sgRNAs, ORC2-1 and ORC2-2  TIR1 (OsTIR1) gene that encodes a plant auxin‐binding receptor that interacts with the conserved E3 ubiquitin ligase SCF complex to degrade mAID-tagged proteins and the U2OS cell line. In the dropout CRISPR/Cas9 experiment, cells expressing a positive control RPA3 sgRNA and all four ORC2 sgRNAs, but not the negative control Neg15 sgRNA  or as rounded, mitotic-like cells (30%)  .gr proteins in ORC2_H-2, H-4, and H-5 cell lines were depleted following 4 hr of auxin treatment  and CHK1(S345) phosphorylation were detected in all three ORC2 mutant cell lines  (pH3S10) followed by flow cytometry analysis . During the second cell cycle, in the absence or presence of doxycycline and auxin, it took TO-HCT116 cells about 35–50 min to progress from early prophase to chromosome segregation , n = 10.ORC1 and ORC2 genes in p53-/- HCT116 cells (-/-ORC1 (B14 clone) and -/-ORC2 (P44 clone) cell lines described in this study as a gift from Dr. Anindya Dutta and performed several experiments on them. Using the ORC2 validated sgRNAs , we first tested if the -/-ORC2 cell line was sensitivity to the four ORC2 sgRNAs we used in our study  to determine the nature of the ORC2 transcripts in the -/-ORC2 cells . To determine the CNV status we performed SMASH . We also compared HCT116 -/-ORC1 (B14) and HCT116 -/-ORC2 (P44) cell lines with the parental lines, that is either +/+p53 and -/-p53 background by confocal microscopy .To further address this question of the role of ORC2, we performed a new G1 synchronization experiment with ORC2 depletion and the result is shown in new Figure 4—figure supplement 3. While arresting the cells in G1, we harvested two time points after auxin treatment. The nuclear volume remained unchanged. CENP-C staining was also unchanged (data not shown). We suggest that this abnormal heterochromatin phenotype is due to cells lacking ORC2 during S phase that affects sister chromatids cohesion loading , therefore, the centromeric region showed decompaction in late S/G2 phase. We have also provided new evidence of heterochromatin decondensation after 48 hr of ORC2 depletion in Figure 4—figure supplement 2.It is hard to tell whether aberrant mitosis is an indirect effect of incomplete DNA replication, checkpoint activation or due to a direct effect of ORC loss. Because most cells arrest at G1, late S, or G2 phase with checkpoint activation, only a very small percentage of cells enter mitosis to exhibit the abnormal phenotype. ORC2 localizes at the kinetochore during M phase, and we suggest that those mitotic cells lacking ORC2 cannot properly align and segregate sister chromatids during M phase. This could be due to direct outcome of ORC2 removal or that ORC2 is essential for recruiting other factors before M phase. In Figure 5 we’ve shown that cells can progress normally during first cell cycle upon release from double thymidine block, which indicates loss of ORC2 during mitosis doesn’t affect cell division. Nonetheless, ORC2 could still be part of an essential components for recruiting or loading important factors for chromosome congression, and this action can be traced back to G1 or S phase, which will then be hard to discriminate from its replication function.We have added new discussion about the loss of ORC1 in the cells and the possibilities of how they can have the limited proliferation. We have also added a discussion of a more recent paper by Shibata and Dutta, 2020, on the reported loss of ORC2 and ORC5 in human cell lines.


FGFR1 regulon to be involved in the characterization of the NB group, where neuroendocrine signature genes were significantly upregulated, and further β-catenin was shown to have significantly higher nuclear localization. LP groups were mainly driven by the regulons ERBB2, FOXA1, GATA3, and PPARG, and they showed upregulation of the genes involved in epithelial differentiation and urogenital development, while the activity of EGFR, FOXM1, STAT3, and HIF1A was implicated for the regulation of BS group. Collectively, our results and classifications may serve as an important guide for the selection and use of bladder cancer cell lines for experimental strategies, which aim to manipulate regulons critical for bladder cancer development.  Bladder cancer is one of the most frequent cancers and causes more than 150.000 deaths each year. During the last decade, several studies provided important aspects about genomic characterization, consensus subgroup definition, and transcriptional regulation of bladder cancer. Still, much more research needs to be done to characterize molecular signatures of this cancer in depth. At this point, the use of bladder cancer cell lines is quite useful for the identification and test of new signatures. In this study, we classified the bladder cancer cell lines according to the activities of regulons implicated in the regulation of primary bladder tumors. Our regulon gene expression-based classification revealed three groups, neuronal-basal (NB), luminal-papillary (LP), and basal-squamous (BS). These regulon gene expression-based classifications showed a quite good concordance with the consensus subgroups assigned by the primary bladder cancer classifier. Importantly, we identified  Bladder cancer is a heterogeneous group of tumors, where transitional cell carcinoma constitutes the great majority of the cases. Classically, bladder cancer is diagnosed in two histopathological classes as ‘muscle invasive bladder cancer (MIBC)’ and ‘non-muscle invasive bladder cancer (NMIBC)’ with different prognostic and molecular characteristics . In the last decade, there have been a number of studies characterizing the genomic landscape of both MIBC and NMIBC and defining the molecular subgroups . A more recent study aimed to define the consensus subgroups of MIBC using the gene expression data in combination with several studies , where the six consensus subgroups were referred to as ‘luminal papillary’, ‘luminal nonspecified’, ‘luminal unstable’, ‘stroma-rich’, ‘basal/squamous’, and ‘neuroendocrine-like’. In this study, the authors, in addition, associated these subgroups with distinct regulon activities, previously defined in . These regulons implicated in bladder carcinogenesis include transcription factors and growth factor receptors, determined according to their gene regulatory activity in bladder cancer . Bladder cancer cell lines have been extensively used for modeling the development, progression and molecular characteristics of bladder cancer. In addition to the focused characterization of cell lines, where only two/three of them are used , there are a few other studies, which provided details about the molecular and genomic characterization of bladder cancer cell lines collectively. In one study, a classification based on the subgroups defined by , ‘“Urobasal A”, “Urobasal B”, “Genomically Unstable”and “SCC-like” were established for 40 bladder cancer cell lines . Another study performed exome sequencing for 25 bladder cancer cell lines and identified the frequently mutated genes among analyzed cell lines . A more recent study provided a comprehensive review about molecular characteristics, origin, and tumorigenic properties of more than 150 murine and human bladder cancer cell lines . In addition, the Cancer Cell Line Encyclopedia of the Broad Institute (CCLE database) provides a unique source for the transcriptomic and genomic data produced in a variety of cancer cell lines including bladder cancer . Although regulon activities have been significantly associated with primary bladder cancer subgroups , there has not been yet a study, which characterized the bladder cancer cell lines according to regulon activities defined for the primary bladder cancers . In this study, we classified the bladder cancer cell lines into 3 groups according to their regulon activities and associated the upregulated genes in each cell line group with the targets of the regulons. Our results reveal previously unknown cooperative regulatory activities in bladder cancer cells and can serve as a guide for modeling bladder cancer according to different regulon activities. 2. The two bladder cancer cell lines 5637 and RT112 were obtained from DSMZ and J82 was kindly provided by Dr. S. Senturk . 5637 and RT112 were cultured in RPMI 1640 (Gibco BRL), J82 was cultured in DMEM (Dulbecco’s Modified Eagle Medium). All media were supplemented with %10 FBS and %1 Penicillin-Streptomycin. Cells were cultured at 37 °C and 5% COIn 24 well plates, J82 was plated 10000/well, RT112 was plated 20000/well, 5637 was plated 40000/well. Cells were incubated overnight on glass coverslips and rinsed with 1x PBS the following day. Cells were fixed with 4% formaldehyde for 15 min at RT, and 0.2% TritonX was used for permeabilization. Fixed cells were blocked with 2% Donkey serum for 45 min. Afterwards, cells were incubated with β-catenin antibody  diluted in 2% donkey serum overnight at 4°C. Next day, cells were rinsed 2 times with 1x PBS. Goat Anti-Rabbit Alexa Fluor 594 was used as a secondary antibody. DAPI was used for nucleus staining. Coverslips were mounted onto slides for imaging with Zeiss LSM880. Images were acquired as Z-stack using ZEN 2 software. Images with maximum intensity were used for further analysis. Quantification of the images were done with ImageJ program. Splitted DAPI channel images were used to determine region of interests for nuclear β-catenin signal intensities. A total of 17 cells per cell line were used for quantification. Integrated Density Values (IDV) were used for statistical analysis. CCLE RNAseq gene expression data for bladder cancer cell lines (RPKM) were downloaded from Cancer Cell Line Encyclopedia (CCLE) database  and were accessed at cbioportal . Regulon definitions were based on . Mutation data for bladder cancer cell lines were obtained using cbioportal . Neuroendocrine differentiation gene definitions are based on the information provided in Supplementary Table 3 from .Using the gene expression values for the regulon genes, we clustered 25 bladder cancer cell lines using kmeans option (k = 6), within pheatmap package (Kolde 2019). Only the regulons that have min 1 rpkm  expression value in at least one cell type analyzed were included in clustering. This resulted in 19 number of regulons which contributed to the clustering analysis. In order to determine the consensus classification of bladder cancer cell lines, we utilized the “Molecular Classification of Bladder Cancer” classifier developed by Kamoun et al.,  (134.157.229.105:3838/BLCAclassify). Gene expression matrix for the cell lines in rpkm ) was uploaded to the classifier and resulting consensus classifications are presented in Figure 1b and Supplementary Table S1.Differential gene expression analysis, where one cell line group was compared with the other groups, was performed using cbioportal . Basically, custom cell line groups were formed based on our classifications , and differentially expressed genes were identified using ‘Compare’ and ‘mRNA’ options. Upregulated genes were defined using q value threshold of 0.1 and log Ratio of 0.5. Gene ontology analysis for the upregulated gene sets was performed using the ConsensusPathDB (CPDB) database of Max Planck Institute . Overrepresentation function of the CPDB was used, and only Level 4 GO terms  were included for further analysis. “GOChord” function of “GOplot” R package was used for visualization . In chord graphs, maximum top 20 GO terms with adjusted p-value <0.05 were shown. For the limit parameter of the “GOChord” function, a minimum number of genes belonging to a specific GO term was determined as 5 if the number of the genes in upregulated gene set was >100, otherwise the number was set as 4 genes minimum. Genes, which are linked with at least 4 different GO terms, were displayed on the plots together with their logFC value representations. Regulon – target gene association table was downloaded from  (Table S2.25) . Genes, which are positively associated with the regulons , were referred to as the target of the respective regulons. Afterwards, upregulated genes for each cell line group were intersected with the targets of the regulons and the results were presented as percent intersection rate . Statistical analyzes were performed utilizing the R/Bioconductor packages (www.bioconductor.org). ANOVA was used to check the statistical difference among the groups for Figures 3a, 4a, 5a, and Supplementary Figure S2. Subsequently, Bonferroni post-hoc test was applied to the results of ANOVA test. Spearman correlation test was applied for Figures 3c, 3d, 4c, 4d, and 5b. Dunnett’s multiple comparisons test was used for statistical analysis of the immunostaining images . We determined the expression of the regulon genes in 25 bladder cancer cell lines and classified these cell lines according to the expression profile of the regulon genes. Our unsupervised clustering analysis using kmeans (k = 6) clustered the bladder cell lines into 3 groups . In order to find out to what extent our regulon-based classifications are legitimate, we additionally classified the cell lines using the consensus classifier algorithm provided in . This analysis identified 5 out of 9 cell lines in group 1 to be assigned to neuroendocrine-like subgroup; 6 out of 6 cell lines in group 2 were identified to belong to luminal papillary and 10 out of 10 cell lines in group 3 as basal-squamous . Among the group 1 cell lines, one cell line (J82) had almost equal annotation scores (0.383 vs 0.385) for neuroendocrine-like and basal squamous classes, and, for two of the cell lines (SW1710 and TCCSUP), annotation scores were rather close as well (Supplementary Table S1). Therefore, we named the group 1-3 as ‘neuronal-basal (NB)’, ‘luminal papillary (LP)’ and ‘basal squamous (BS)’, respectively.FGFR1, which is highly enriched in ‘stromal-rich’ subgroup in consensus classification of bladder cancer , mainly separates the NB group from the two other groups. The regulon cluster 4 driven by the expression of FGFR3, ERBB3, TP63, and FOXA1 was mainly enriched for LP class; regulon cluster 6 constituted by PPARG and GATA3 expression was enriched in LP class and partially in BS class. Regulon cluster 5, driven by luminal-papillary markers RARG, RXRA  and basal marker KLF4  was relatively enriched in LP class, with partial enrichments in NB and BS classes. Regulon cluster 3, dominated by the basal markers, EGFR, FOXM1, STAT3 ,and HIF1A  were similarly enriched in all cell line groups. Although luminal and basal terms are classically used for bladder cancer cell lines , our regulon expression-based analysis here brought additional features, characteristics of each group. Our analysis revealed that the expression status of For each of the 3 groups, we determined with the clustering analysis , we performed differential gene expression analysis contrasting one group with all other groups and determined the upregulated genes for each group. This analysis identified 327 and 570 upregulated genes in NB and LP classes, respectively. However, within the significance thresholds we used, we failed to detect upregulated genes for the BS class. The reason behind this can be attributed to the heterogeneous structure of this group, as it can be seen in the heatmap  and in PCA analysis  as well.FGFR1 targets (18.96%), followed by GATA6 (4.89%) and FOXM1 (4.89%) . FGFR1 was also significantly upregulated in the NB group . FGFR1 targets, which are upregulated in the NB class were mainly involved in neurogenesis, neuron differentiation, nervous system development . Further, expression of the genes VIM and ZEB1 implicated in epithelial to mesenchymal transition , highly correlated with the expression of FGFR1, emphasizing the role of this regulon in the transcriptomic constitution of the NB group .Having determined the upregulated genes in different cell line groups we defined, next, we tempted to relate those genes with the regulon targets. We identified the genes positively associated with the regulons using the information provided in . This analysis showed that cell line groups constituted according to regulon expression profiles were in concordance with the regulon activity. For the NB group, upregulated genes had the highest intersection rate with ERBB2, FOXA1, PPARG, ERBB3, FGFR3, RARG, and GATA3 targets . We identified that almost all these regulons were significantly upregulated in the LP class . Target genes of the regulons upregulated in LP class were involved in epithelial cell differentiation, cell junction organization, and urogenital system development . Remarkably, expressions of FOXA1 (ρ = 0.71) and GRHL3 (ρ = 0.60) significantly correlated with the expression of ERBB2 , indicating the luminal characteristics of the LP group. Upregulated genes in the LP class mainly intersected with FGFR1 targets upregulated in the NB group, prompted us to decipher this connection in more detail. As FGFR1 is the main player characterizing this group, we checked the enrichment of FGFR1 regulon activity in each consensus subgroup of primary bladder cancer . We discovered that although FGFR1 has the highest enrichment score in stromal-rich consensus subgroup , it was also moderately enriched in neuroendocrine-like subgroup  ). To strengthen this association further, we checked the expression of genes marker of neuroendocrine differentiation  in the cell line groups we determined. This analysis also revealed that genes involved in neuroendocrine differentiation were significantly higher expressed in NB group  . Additionally, expression of FGFR1 highly correlated with the expression of neuroendocrine markers . Collectively, these results highly argue for the neuronal characteristics of the NE group and involvement of FGFR1 in this signature. Our finding, which shows the enrichment of neurogenesis-related genes in the APC mutation and one had CTNNB1 mutation. On the contrary 2 had APC or CTNNB1 mutation in the two other cell line groups . Based on this information, we checked the β-catenin localization in one of the NB group cell lines we had in lab J82 and the other two cell lines, 5637 (BS group) and RT112 (LP group) as controls (no mutation in CTNNB1 or APC). The staining of β-catenin in 5637 and RT112 was concentrated at the cytoplasm and the membrane while in J82 it was concentrated at the nucleus of the cells. Our data showed that β-catenin showed significantly higher nuclear localization in J82 compared to the other two cell lines . This finding strengthens our conclusions about the involvement of WNT/β-catenin pathway in neuronal differentiation of bladder cancer cells. The information we provide for the potential involvement of FGFR1 in neuroendocrine features of bladder cancer , identification of significantly increased nuclear localization of β-catenin in a cell line belonging to NB group  collectively strengthens the neuronal/neuroendocrine characteristics of the cell lines present in NB group according to our classifications. We recently showed that the WNT/β-catenin pathway is associated with the active regulatory elements characterizing neuronal bladder cancer . Within this frame, to check any connection of the NB group with WNT/β-catenin pathway deregulation, we scanned the cell lines we used in this study for the mutation status of β-catenin and β-catenin destruction complex components. Among the NB group cell lines, 3 of them had Bladder cancer cell lines serve as important models for modeling bladder tumorigenesis, invasive characteristics and treatment responses . So far, several studies characterized the genomic and transcriptomic properties of bladder cancer cell lines . In this study, we aimed to characterize the bladder cancer lines in terms of their regulon activity, defined for the primary bladder cancers in literature . Our results showed that bladder cancer cell lines have differential regulon activities, reflecting their transcriptomic signatures and their consensus classifications . FGFR1 and were involved in neuronal differentiation. Accordingly, the expression of the genes marker of neuroendocrine differentiation  was significantly higher in the NB group compared to the two other cell line groups. In literature, FGFR1 has been shown be expressed at higher levels in bladder cancers showing mesenchymal features . Knock-down of FGFR1 in JMSU1 and UMUC3 cell lines, belonging to NB group in our results, resulted in a significant reduction in the anchorage-independent ability of these cells . Further FGFR1 expression was high in most small cell carcinoma of the bladder , which is a rare type of bladder cancer with neuroendocrine differentiation . These existing literature and our findings highly support the association of FGFR1 with NB characteristics and neuronal differentiation of bladder cancer. Genes significantly upregulated in cell lines belonging to the NB group were mainly intersected the targets of APC, a component of β-catenin destruction complex . It is known that the immune gene expression signature is relatively depleted from small cell neuroendocrine carcinoma of the bladder , and neuroendocrine-like bladder cancer show decreased levels of immune infiltrate . It was also identified that Wnt/β-catenin signaling can decrease the T-cell infiltration in melanoma mouse models. Thus, inhibition of Wnt signaling has been suggested to prevent immunotherapy resistance . In addition, inhibition of FGFR1 has been shown to enhance the immune checkpoint inhibitor response in breast cancer . Based on all these information, we checked the expression of CXCL16, T cell chemoattractant  in bladder cancer cell lines and identified a significant negative correlation with FGFR1 expression . Our data and existing literature together suggest a regulatory axis involving FGFR1, WNT/ β-catenin signaling, and tumor immune microenvironment in regulation of NB cell lines. Therefore, we suggest that combinatorial treatment strategies disrupting this regulatory axis can be applied on NB cell lines. We previously showed that WNT/β-catenin pathway is deregulated in neuronal subtype of bladder cancer . In this study, we identified significantly higher accumulation β-catenin in nucleus in J82 cell line belonging to NB group, which has a mutation in ERBB2 has been identified to be overexpressed in high-risk non-muscle invasive bladder cancer  and as one of the major prognostic factors for survival status of the patients . FOXA1 expression was adequate for separating non-basal subtype of bladder cancer from the basal subtype . Furthermore, GATA3, FOXA1, and PPARG have been shown to drive the luminal fate in a collaborative manner . Thus, within this frame, our regulon-based classifications confirm the luminal character of the LP class we defined. Regulons implicated in LP group cell lines are mainly known for early bladder cancer, mostly non-muscle invasive and luminal associations. EGFR, FOXM1 and STAT3 as the main regulons, driving the basal characterization of this group . EGFR has been previously shown to be enriched in basal-like bladder cancer, and some groups of muscle invasive bladder cancers have been determined to respond to EGFR inhibitors . In addition, expression of FOXM1 as a prognostic factor in the survival of muscle invasive bladder cancer patients , STAT3 expression, and phosphorylation was identified to be substantially higher in basal-like bladder cancer . Further, STAT3 activated transgenic mice directly developed invasive bladder cancer without going through the intermediate noninvasive stages . Our results here collectively emphasize the role of EGFR, FOXM1, and STAT3 in basal characteristics of BS cell lines. Our differential gene expression analysis did not identify significantly upregulated genes in the BS class, largely because of the heterogeneity of this group . However, we determined To conclude, our regulon-based classification of bladder cancer cell lines may serve as an important guideline for studying the different regulons implicated in bladder cancer and trial of drug candidates relevant for targeting regulons. Aleyna Eray: Design of the study, computational and experimental analysis, writing of the manuscript. Serap Erkek-Ozhan: Design, supervision of the study, writing of the manuscript.


In this study, we report a differential response of mitogen-activated protein kinase–kinase (MEK) inhibitor trametinib in 20 head and neck squamous cell carcinoma (HNSCC) patients’ tumor-derived cell cultures. Relatively sensitive and resistant cases to trametinib were identified using high throughput metabolic assays and validated in extended dose response studies in vitro. High throughput metabolic assays exploring combination therapies with trametinib were subjected to synergy models and maximal synergistic dose analyses. These yielded several candidates, including axtinib, GDC-0032, GSK-690693, and SGX-523. The combination regimen of trametinib and AXL/MET/VEGFR inhibitor glesatinib showed initial efficacy both in vitro and in vivo (92% reduction in tumor volume). Sensitivity was validated in vivo in a patient-derived xenograft (PDX) model in which trametinib as a single agent effected reduction in tumor volume up to 72%. Reverse Phase Protein Arrays (RPPA) demonstrated differentially expressed proteins and phosphoproteins upon trametinib treatment. Furthermore, resistant cell lines showed a compensatory mechanism via increases in MAPK and non-MAPK pathway proteins that may represent targets for future combination regimens. Intrinsic-targeted options have potential to address paucity of medical treatment options for HNSCC cancer patients, enhance response to extrinsic targeted agents, and/or reduce morbidity as neoadjuvant to surgical treatments. The 8-year survival of HNSCC is approximately 30.2% for HPV negative disease and approximately 70.9% for HPV positive disease.5 In this study, we focus on HPV negative disease, which is more aggressive. The standard treatment for HNSCC largely depends on tumor location, TNM staging, and medical comorbidities. Treatment incorporates surgery, chemotherapy, and radiotherapy singly and in combination. Chemoradiotherapy is the standard of care in unresectable, locally advanced disease.4 Thus far, two PD1 targeted immunotherapies, pembrolizumab and nivolumab, and one tumor intrinsic EGFR targeted therapy, cetuximab, are FDA approved for treatment of HNSCC.6Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide, resulting in 450,000 deaths annually, with 10,030 deaths in the United States.8 Additionally, cetuximab, the only tumor-intrinsic targeted therapy, was approved for use in combination with radiotherapy.9 However, cetuximab has declined in favorability as radiotherapy alone is no longer the standard of care.5 When cetuximab is used in combination with chemotherapy in the recurrent/metastatic setting, it extends progression-free survival (PFS) and overall survival for less than 3 months.9 Unfortunately, cetuximab also carries multiple side effects, resulting in an increase in adverse events.5 These limited therapeutic options represent a paucity in targeted therapeutics available for the treatment of advanced HNSCC, underscoring a need for new approaches.Although the introduction of new immunotherapies has greatly expanded HNSCC treatment options, only 13–18% of recurrent or metastatic cases are responsive, with all but 5% ultimately progressing to death.BRAF V600E or V600K mutation is present. In melanoma, trametinib single agent increased PFS by 4–7 months in BRAF V600E mutant tumors.10 Interestingly, there was a partial response rate of 10% in BRAF wild-type tumors, demonstrating that a subset of non-BRAF mutant tumors may benefit from MAPK inhibition. In HNSCC, Phase II randomized clinical trials with single-agent trametinib have shown modest results, with >25% reduction in tumor volume in 11/17 patients treated prior to tumor resection.11While estimates vary, the RAS-MAPK pathway has been found to be mutated in as high as 18% of HNSCC.6 Our underlying hypothesis is that patient-specific tumor intrinsic targeting has a role in improving therapeutic options and treatment efficacy. In this study, we focus on the more aggressive HPV negative HNSCC, examining trametinib as a therapeutic agent across HNSCC patient-derived primary and early secondary cell cultures; demonstrate patient-specific efficacy in vivo; and identify potential combination therapies that may synergize with trametinib treatment.MEK inhibitors targeting the MAPK pathway are used in combination with BRAF inhibitors in melanoma, metastatic lung cancer, and metastatic anaplastic thyroid cancer when a Primary tumor cell cultures derived from 20 HNSCC patients were subjected to a drug screen of 200+ inhibitors, natural products, and combinations . Trametinib was identified as showing selective sensitivity and resistance in the HNSCC cohort, demonstrating a non-uniform metabolic rate profile across cases . For the12 Additionally, trametinib, but not TAK-773 (a MEK1 inhibitor), showed reductions in metabolic rate, demonstrating that both MEK1 and MEK2 inhibition are necessary for cellular response at clinically relevant concentrations . Notably, sensitive case 10292 carries an activating HRAS mutation. HRAS mutations in non-HNSCC cancer cell lines have previously shown sensitization to MAPK inhibitors.13 In cell line 10336, many inhibitors, including trametinib, resulted in increases in metabolic rate, highlighting the importance of stratifying patient populations by drug sensitivity.To confirm selective sensitivity, relatively sensitive cell lines 10004 and 10292 and resistant/insensitive cell lines 10336, 10326, and 20003 were further characterized using an extended dose range in triplicate experiments, confirming case-specific dose-dependent inhibition . Cell li14 Bliss beta coefficients were calculated using SynScreen high throughput combination software.15 Chou-Talalay method for analyzing combination indices yielded similar results to the Bliss beta calculations, with a − .65 Spearman correlation16 . Furthermore, the Combination index (CI) methodology allowed for the identification of regimen dosing that yielded the minimum CI value across the drug dose ranges.16 This allowed us to identify both the synergistic drug targets and the optimal synergistic drug dosing regimens (Supplementary Table S1).After a preliminary screen of trametinib in binary combinations with 150+ other drugs and natural products, we identified and selected synergistic and antagonist drug combinations to be further validated (data not shown). The combination panel was further expanded using drugs with more specific drug interactions to characterize drug effects and pathways. To analyze the combination synergies, we used two different models for analyzing combination synergies, the Bliss independence model and the Loewe additivity model.The Bliss beta values show a normal distribution around the Bliss additivity value of 1, with sensitive cell lines accounting for the majority of the synergistic combinations . Stratifying by cell line, 10336 is identified as being particularly resistant to multiple inhibitors, skewing the distribution of the resistant cell lines . GDC-0032, a PIK3CA inhibitor, was synergistic in 5/6 cell lines, reduced metabolic rate by up to 91.5%, and sensitized 2/3 of the resistant cell lines to trametinib treatment .Figure Trametinib induced a dose-dependent decrease in the phosphorylation levels of ERK regardless of the sensitivities of the cells . This suggests that while trametinib is effective at blocking MAPK pathway inhibition, only a subset of cells will experience a decrease in metabolism. In the sensitive line, 10004, and the resistant line, 10336, trametinib treatment induced an increase in Phosho-AKT levels . This could partially explain trametinib synergies between PIK3CAi, AKTi, VEGFRi, METi, and AXLi, all of which either directly target or target an upstream regulator of Phospho-AKT.In3 to 82 mm3) (3 to 29 mm3), compared to trametinib alone . Although glesatinib alone showed little effect as a single agent in vitro, suggesting that the combination regimen and not glesatinib as a single agent is responsible for the tumor response, the absence of a glesatinib single-agent control in vivo is limitation of our in vivo study would need to be addressed in follow-up studies .To evaluate the relationship of trametinib sensitivity in vitro to efficacy in vivo, we established a patient-derived xenograft (PDX) model using patient-specific primary tumor cells. Trametinib significantly decreased tumor volume in 10004-PDX models  . While nib alone . Remissiib alone . The inh18 The relevance of the PDX model in our study is based upon several lines of evidence. The epithelial tumor cell morphology was indistinguishable in the patient-derived primary cultures and the mouse tumor derived primary culture of that patient after reestablishment of the PDX tumors in culture. Additionally, vimentin-keratin staining of the cultured cells showed the same epithelial to mesenchymal transition (EMT) characteristics in both the PDX and original tumor: 100% epithelial with 4% mesenchymal characteristics. Similarly, tumor pathology of the original case and the PDX tumor from that case were similar based upon H&E staining of tissue sections from each tumor . Finally, the cells from the mouse and primary tumor exhibited similar drug sensitivities .PDX models have been shown to be reliable in terms of replication of original tumor and predicting drug sensitivities .20 Basal proteomic levels of cell lysate showed no significant correlations with drug response.Reverse Phase Protein Array (RPPA) was conducted in order to determine the molecularly and functionally relevant phosphoproteomic changes in response to MEK inhibition. Lysate collected from cells treated with trametinib or vehicle for 24 hours revealed that maximum ERK inhibition occurred at 100 nM regardless of the functional response to trametinib, consistent with other studies .To test the functional relevance of MEK inhibition, lysate was collected from cells treated with trametinib or vehicle at the IC50 value for 3 days. The resistant cell lines were treated at maximum dose of 312 nM. Prior to RPPA analyses, immunoblot and metabolic rate assays confirmed the expected functional and molecular responses, ensuring the integrity of the cell lines and analyses . Both 24-hour and 3-day treatment samples were collected and sent to MD Anderson for RPPA analyses.All trametinib-treated cell lines showed a significant decrease in MAPK phosphorylation at T202 and Y204 . Although the MAPK phosphorylation antibody used for these results was designated as “use with caution” and therefore not used in the primary analysis, the decreases in MAPK phosphorylation indicates that trametinib was effective in inhibiting its primary target.Only MD Anderson-validated antibodies for differential affinity analyses between 24-hour and 72-hour groups . The 72-22 Together, these proteomic changes, specific to the resistant group, are suggestive of a MAPK reactivation mechanism available to trametinib-resistant cell lines. Phosphorylated heat shock protein was differentially increased in both resistant and sensitive groups, suggesting cellular stress in both resistant and sensitive cell lines. CDC25C, DNMT1, and RB1 phosphorylation were decreased across both resistant and sensitive cell lines at 72-hours. AURKB, CDC6, CCNB1, and FOXM1 showed differential decreases in resistant cell lines at both the 24-hours and 72-hour time points but failed to reach significance in the sensitive cell lines.When comparing between resistant and sensitive cell lines, the resistant groups showed a greater number of protein antigens differentially expressed at both the 24- and 48-hour time points, compared to the sensitive groups . Eight g11 These mixed results likely relate to the complexity and heterogeneity of HNSCC. HNSCC is a particularly diverse cancer in terms of molecular classification, biological characteristics, genomics, and HPV status, as well as in anatomical sites.23 Our exploration into the landscape of combination therapies offers the opportunity of increasing treatment efficacy amongst this heterogenous population of patients.24 Rational optimization of trametinib therapy for HNSCC will be enhanced by further evaluation of the differential targets in HNSCC, pathways underlying sensitivity or resistance, dose selection, and identification of promising synergistic combinations. This approach to new targets and pathway synergies is relevant to other cancers.In this study, we show that within a cohort of patients’ HNSCC derived primary tumor cell cultures, trametinib showed selective sensitivity in reducing tumor metabolic rate in a subset of HNSCC. Recently, trametinib as a treatment for HNSCC has been supported by clinical data as the result of the Phase II randomized clinical trial of trametinib in HNSCC showing mixed results, with observed reduction in tumor volume in 65% of patients.26 We also observe a statistically significant increase in AKT phosphorylation at 72 hours. This is concordant with dose response immunoblotting on the patient-derived cell lines . The increase in AKT phosphorylation is a plausible mechanism behind the synergies observed in the PIK3CA and AKT inhibitors in combination with trametinib.In order to further enhance the response of trametinib, we evaluated the synergistic response of both trametinib-sensitive and trametinib-resistant HNSCC patient-derived cell lines. Many cell lines showed drug synergies between VEGFR, PIK3CA, AXL, AKT, and MET inhibition. Using MAPK synergies by co-targeting the AKT, MTOR, and PIK3CA has shown promise in other HNSCC studies.27 However, despite the observation of synergy between AXL/MET inhibition and trametinib combination treatment, there seemed to be a reduction in the overall AXL and MET expression in trametinib-treated cells, suggesting that the cells that are sensitive to trametinib may be the same cells that highly express AXL and MET . Alternatively, trametinib may lead to the down regulation of AXL and MET in the context of HNSCC.An additional area of interest is the Receptor Tyrosine Kinase (RTK) response in combination with Trametinib, many of which are upstream regulators of the PIK3CA and AKT pathways. AXL and MET have been suggested to increase resistance of MAPKi and have shown synergies with MAPKi in non-HNSCC models.28 By identifying the therapeutic window that yields the maximum synergistic benefit and minimum CI value, we identified optimum dosing regimens across many combination therapies (Table S1). Identification of optimal dosing ranges is critical due to off target effects of many of the inhibitors, as well as for limiting drug toxicity.29Interesting, the cell line demonstrating the most EMT, 10336, was only synergistic with FGFR inhibition.30Additionally, we show that response in vitro correlates to a decrease in tumor volume in vivo. This is especially promising due to the concordance between the primary tumor and the PDX established tumor. Furthermore, our data suggest that treatment durability may be improved by the addition of a synergistic agent, in this case glesatinib. Glesatinib targets MET, TIE-2, AXL, as well as VEGFR 1/2/3. It is unclear which of these signaling pathways may be responsible for a more durable response. Whereas trametinib decreased the concentration of both MET and AXL on the cellular surface in our cell lines shown by immunofluorescence , VEGFR1 showed significant increased affinity at 72 hours in the RPPA data. The implications of this finding remain to be elucidated; however, VEGFR1 as a plausible molecular mechanism behind this synergy and needs to be substantiated in further studies.31These exploratory RPPA studies identified many proteins upregulated in response to trametinib treatment that are targetable, including DDR1, LCK, MMP14, PAI-1, SOD, CD26, and FAK, that could be evaluated for future trametinib combination therapies. It was of interest to observe that trametinib-resistant lines had an increased adaptive proteomic response to trametinib treatment, including the activation of the MAPK pathway though increase of p-MEK1 and GAB2. The totality of these observations is suggestive of an adaptive response to trametinib allowing for resistant cell lines to survive MAPK inhibition. The primary culture conditions selected for epithelial HNSCC tumor cells without fibroblast or immune cells. Both a limitation and an advantage of this approach is that the RPPA data reflect the intrinsic epithelial tumor cells. While this allows us to dissect the intrinsic pathways of the patient’s individual HNSCC, it lacks the context of the tumor microenvironment. Recently, trametinib has been reported to enhance efficacy of PD-L1 inhibitors in HNSCC. Thus, besides combinations for intrinsic targeting, trametinib could be combined with already approved immunotherapies to increase treatment efficacy in HNSCC.While this study yielded several promising candidate drug combinations for trametinib, a major limitation remains biomarkers predicting who will and will not respond to trametinib or trametinib combination therapies. This is especially important considering that trametinib enhanced metabolism in a subset of primary cultures within our HNSCC patient cohort . In our 33 Further preclinical studies on these combinations in more complex immunocompetent models and on predictive biomarkers in larger cohorts is warranted to extend the therapeutic options of HNSCC patients by applying promising regimens in the clinic.A major limitation of the study is inability to address the potential complementary role of intrinsic and extrinsic targeting or how intrinsic pathways may affect the microenvironment or immunotherapy responses, given our focus on the epithelial tumor cell component in culture, and our use of an immunocompromised animal model for in vivo assessment of drug response.34 Primary tumor samples were collected from the operating room in DMEM/F12 Media, supplemented with 2x antibiotic/antimitotic . Tissues were washed 3 times in fresh DMEM/F12 Media and minced before plating. Cells were cultured in DMEM/F12 Media , supplemented with 5% supplemented BCS (Hyclone), 1x antibiotic/antimitotic , 1.8 × 10–4 M adenine , 0.4 μg/mL hydrocortisone (Sigma), 1 × 10–10 M cholera enterotoxin (Sigma), 2 × 10–9 M triiodothyronine (Sigma), 5 μg/mL insulin (Sigma), and 10 μg/mL epidermal growth factor (Invitrogen). Fibroblasts were removed from primary cultures by differential trypsinization using Trypsin-EDTA (0.25%), phenol red . Frozen cultured cell lines were used within five passages of the original tumor.Primary tumor cell lines were obtained from patients treated at the OHSU Department of Otolaryngology for HNSCC from 2014 to 2019 . Cell culture methods have been previously described.At time of selection, sensitive cases that were <20% of the median IC50 values were selected for validation. Trametinib  was plated using the ECHO liquid handler in a 13, three-fold dilution series ranging from 10 μM to .01 nM. Cell lines 10004, 10292, 10336, 10326, and 20003 were randomized by column and plate order and plated in triplicate. Cells were assessed for metabolism after 72 hours of incubation at 37°C using atetrazolium-based metabolic rate assay  by measuring absorption at 490 nM using a Biotek Synergy H1 Microplate Reader. Values were then normalized to wells that received no drug. Dose response curves were created for area under the curve (AUC) analysis.Drug combinations were chosen based on preliminary combination data, drugs with specific targeted pathways, and targets from siRNA data. Trametinib-combination therapies were chosen for further validation on custom 7 × 7 drug combination matrices created using ECHO liquid handler. Trametinib started at 0.03 μM  and followed 6 three-fold dilutions. Cells were assessed for metabolism after 72 hours of incubation at 37°C using atetrazolium-based metabolic rate assay  by measuring absorption at 490 nM using a Biotek Synergy H1 Microplate Reader. Values were then normalized to wells that received no drug.36In parallel to the Bliss beta coefficients calculated by the SynScreen program, we evaluated synergy using the Chou-Talalay Combination Index (CI).Where 37 using a gradient-descent optimizer. Combination dose–response observations were initially modeled as a multidimensional logistic regression; however, after manual inspection, we noticed that the combination-agent dose–response surface exhibited complex behavior that the logistic model was not capable of explaining. To address this, we chose a non-parametric Gaussian process (GP) model.39 Manual inspection of the GP explained more variance in the dose-response without overfitting within the measured dose-range. The GP parameters were chosen using evidence lower bound (ELBO) and a gradient-descent optimizer.Single-agent dose–response curves were fitted with the Hill equationSeveral single-agent inhibitors did not create the desired effect (50% inhibition) within the measured concentration range. To address this, single agents that did not reach 50% inhibition were assigned the inequality:Where If the desired effect (50% inhibition) was not reached in combination screen, then no CI values were calculated.TM or Clarity MaxTM Western ECL Substrate  and imaged using a Bio-Rad ChemiDoc touch MP Imaging System. For all western blots the same blot was probed repeatedly.Patient-derived tumor cells were treated with cell lysis buffer , complete mini protease inhibitor mixture tablets , and PhosSTOP Phosphatase Inhibitor Cocktail Tablets . Lysates were spun at 8,000 xg for 10 minutes at 4°C, mixed 3:1 with 4x Laemmli Sample Buffer  with b-ME, and heated at 95°C for 5 minutes. Lysates were run on Criterion TGX Precast Midi Protein Gel , transferred to a polyvinylidene difluoride membrane , and blocked for 1 hour in TBS-T with 5% milk. Blots were probed overnight at 4°C with PERK (C33E10) Rabbit, Phospho-p44/42 MAPK (Erk1/2) Rabbit, Phospho-Akt (Ser473) (D9E) XP Rabbit, Akt (pan) (C67E7) Rabbit, or a-Tubulin (DM1A) Mouse . HRP conjugated secondary antibodies, anti-rabbit, or anti-mouse IgG were used for rabbit and mouse primary antibodies, respectively. Blots were developed using Clarity2/2.39,40 Mice were randomized using R, into vehicle, trametinib, and trametinib + glesatinib treatment groups. Treatment of 1 mg/kg/day trametinib  (n = 6) or 60 mg/kg/day glesatinib  and 1 mg/kg/day of trametinib (n = 6) or vehicle (DMSO) control (n = 8) was delivered via oral gavage once tumors reached 350 mm3. Glesatinb treatment was lowered from 60 mg/kg/day (weeks 8–10) to 40 mg/kg/day (week 10), to 15 mg/kg/every-two-days (week 11), and finally down to 9 mg/kg/day every-two-days (week 13) after combination toxicity was observed. Three mice died during dose de-escalation in the combination group. All mouse cohort number changes are displayed in 3. Animals were housed within the same treatment group to avoid treatment contamination. Groups were non-blinded throughout the experiment and analyses. Graph pad prism was used to perform student’s t-tests and to generate figures. All studies were performed according to guidelines approved by OHSU Institutional Animal Care and Use Committee.20 female NOD SCID Gamma (NSG) mice (8–16 weeks old) were used for this study . Mice were inoculated with 2 million 10004 HNSCC cells in right flank, and measurements of tumor growth were taken daily. Tumors were measured, and volume was calculated by Length*WidthCells plated in 12-well vessels on collagen coated coverslips, fixed with 4% formalin solution at room temp for 15 minutes, washed twice with PBS, permeabilized in PBS + 0.1% TritonX. Cells were incubated overnight at 4°C with primary unconjugated rabbit cytokeratin 5 , mouse vimentin (abcam-ab8978), and Guinea pig cytokeratin 8/18  in blocking buffer composed of 2% BSA, 5% goat serum, and PBS. Cells were washed three times in PBS-T and incubated with mouse, rabbit, and goat secondary antibodies labeled with 488 GFP, 594 Texas, 647 Cy5, respectively, and diluted 1:300, along with a DAPI. Cells were washed twice with PBS-T and imaged with the 10x objective on the EVOS microscope. Image segmentation, storage, and data analyses were performed using Image J software.41 Cells were grown in 6-well dishes, and cell lysate was collected when cells reached confluence of 50–90%. Lysate buffer and SDS were purchased from MD Anderson. Cell lysate was immediately diluted to ~1 μg/μl and stored at −20°C prior to shipment to MD Anderson. Immune blots of excess lysate confirmed molecular response. Cells plated in triplicate in 96-well dish confirmed functional response after incubation for 72 hours using Promega MTS assay.Cell lysate was collected using RPPA lysis buffer provided by MD Anderson Cancer Center RPPA Core and following their collection protocols.https://www.mdanderson.org/research/research-resources/core-facilities/functional-proteomics-rppa-core/antibody-information-and-protocols.html. For all analyses, we considered the “validated” antibodies only, as defined by MD Anderson and used the normalized log2 values.41 We performed a differential affinity analysis comparing trametinib versus DMSO for both the 24-hour and 72-hour treatment groups. We used the Bioconductor package limma and defined an antibody to be significant if the FDR < .05 using the Benjamani-Hochberg correction.42 In addition, we performed differential affinity analysis comparing samples treated with DMSO and trametinib separately for resistant and sensitive patients. We used the Bioconductor package limma and defined an antibody to be significant if the FDR < .05 using the Benjamani-Hochberg correction.42The MD Anderson RPPA Core Facility performed the RPPA and processed all data. Full annotation of the antibodies can be found at the MD Anderson RPP Core Facility website: Oral and written patient consent was obtained under OHSU IRB protocol 10071, and work was performed under OHSU IRB protocol 809. All animal experimentation was approved by the Institutional Animal Care and Use Committee (IACUC) of OHSU protocol 1489.Click here for additional data file.


We further review recent advances in the understanding of GVHD as a complex immune phenomenon between donor and host immune systems, particularly in the early stages with mixed chimerism, and potential novel therapeutic approaches to prevent the development of GVHD.Immune checkpoint inhibitor therapies and allogeneic hematopoietic cell transplant  represent two distinct modalities that offer a chance for long-term cure in a diverse array of malignancies and have experienced many breakthroughs in recent years. Herein, we review the CD27-CD70 co-stimulatory pathway and its therapeutic potential in 1) combination with checkpoint inhibitor and other immune therapies and 2) its potential ability to serve as a novel approach in graft- However, despite the obvious life-saving benefits of alloHCT, graft- disease , 2. The  disease . These iThe framework of classical acute GVHD occurring in the first 100 days of transplant due to alloreactivity driven by donor T-cells has more recently been supplanted by a more robust understanding involving the intricate interplay of donor and host immune cells with host tissue . The iniAnother realm of treatment modality in the arena of cancer therapy that has revolutionized the field has been the adoption of CPI therapies, which are now utilized in the treatment of a diverse array of advanced stage malignancies, from non-small cell lung cancer to classical Hodgkin’s lymphoma , 20. DesWhile CPI targeting agents derive their function by countering an inhibitory signal, an alternate and possibly synergistic approach has been agonizing T-cell stimulatory co-signaling pathways. Co-stimulatory pathways are broadly speaking, either part of the B7/CD28 or tumor necrosis factor (TNF) family . Clinicaversus-tumor (GVT) effect as there is a strong correlation between incidence and severity of GVHD and disease free survival  have been utilized in the development of both experimental and commercially available second generation chimeric antigen receptor T-cell (CAR-T) therapies leading to significantly greater activation, expansion, and persistence of CAR-T cells , 31. Morsurvival .Thus, it is critical to identify co-stimulatory pathways which when blocked decrease GVHD but do not interfere with GVT. One potential way to decrease the incidence of GVHD would be by inhibiting a co-stimulatory receptor thereby attenuating CD4+ and CD8+ cytotoxic T-cell activity. CD26 has been studied in both pre-clinical and clinical models, while the CD27-CD70 pathway has been studied extensively in pre-clinical murine models. In murine models, inhibition of CD26 [also known as dipeptidyl peptidase-4 (DPP4)] by a monoclonal antibody has been demonstrated to decrease GVHD incidence without compromising GVT . In a smHerein, we conduct an in-depth review of the CD27-CD70 pathway and its application in both GVHD attenuation following alloHCT and its use in the treatment of numerous malignancies in combination with CPI therapies.via Traf2 and Traf5 signaling and activation of the NF-κB pathway  and progenitor cells –40. CD27 pathway . The int pathway . However pathway . Althoug pathway , 49.Under pro-inflammatory conditions  CD27-CD70 activity is increased, leading to proliferation and survival of lymphocytes with multiple downstream effects . CD27-CDin vitro models, CD27 agonism of bone marrow progenitor cells decreased monocytic differentiation and overall inhibited leukocyte differentiation, while in competitive transplantation assays CD27 agonism decreased donor B and T lymphocytes, suggesting the CD27-CD70 pathway’s ability to influence hematopoiesis and immune cell differentiation , glioblastoma, melanoma, renal cell carcinoma, prostate adenocarcinoma, colorectal adenocarcinoma, and ovarian cancer  treated with varlilumab revealed that both CD4+ and CD8+ T-cells were stimulated , which was accompanied by upregulation of other co-stimulatory pathways  along with the inhibitory PD1 pathway , has resulted in varying degrees of objective clinical responses in a subset of cancer patients enrolled. This has included complete remission in Hodgkin’s lymphoma and partial responses in ovarian, colorectal, and squamous cell cancer of the head and neck see Table 1. pathway .CD27 agonism alone and with an PD1 checkpoint inhibitor has also been explored as a potential mechanism of increasing the efficacy of tumor-specific peptide vaccines by enhancing CD4+ helper T-cell and CD8+ cytotoxic T-cell response following vaccination , 74. CliWhile varlilumab has yet to obtain an FDA indicated approval for use, six clinical trials with varlilumab are actively recruiting patients with B and T-cell lymphomas, neurologic malignancies, melanoma, and non-small cell lung cancer Table 1.Traditionally, the prevailing thought behind the etiology of GVHD rested solely with donor immune cells, particularly T-cells becoming activated upon alloreactivity to host antigens. However, more recently, the complex interaction between donor and host immune systems leading to GVHD has been noted, particularly in the early stages of alloHCT, where a mixed chimerism exists , 76. WhiThe most commonly employed conditioning regimens in alloHCT are given with myeloablative or reduced intensity/non-myeloablative intensity consisting of a combination of myelotoxic chemotherapeutic agents with or without TBI –80. The Collectively, these findings suggest the importance of alterations in the bone marrow microenvironment following the noxious insult of the conditioning regimen leading to emergency hematopoiesis and the complex interaction of host and donor immune cells which may persist for many months following alloHCT, during the time acute GVHD is most likely to occur.via caspase-dependent T-cell apoptosis and upregulation of inhibitory immune checkpoint inhibitor pathways , R01HL135325 (XC).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. 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Bone metastases are typically associated with a short-term prognosis. Photodynamic therapy (PDT) emerges as a promising alternative treatment for targeting metastatic lesions. In this study we investigated the effect of 5-aminolevulinic acid-mediated PDT treatment on both primary and human bone metastatic cancer cell lines. We found that human cell lines have different sensitivity to the same doses and exposure of 5-ALA PDT resulting in two different cell fates, apoptosis or senescence, depending on the extent of the cellular damage. As such, PDT has potential applicability in bone metastases of invasive ductal carcinoma.Bone is a frequent site of metastases, being typically associated with a short-term prognosis in affected patients. Photodynamic therapy (PDT) emerges as a promising alternative treatment for controlling malignant disease that can directly target interstitial metastatic lesions. The aim of this study was to assess the effect induced by PDT treatment on both primary (giant cell bone tumor) and human bone metastatic cancer cell lines . After 24 h post light delivery (blue light-wavelength 436 nm) with 5-aminolevulinic acid, the effect on cellular migration, viability, apoptosis, and senescence were assessed. Our results showed that bone metastasis derived from breast cancer reacted with an inhibition of cell migration coupled with reduced viability and signs of apoptosis such as nuclei fragmentation following PDT exposure. A limited effect in terms of cellular viability inhibition was observed for the cells of giant cell bone tumors. In contrast, bone metastasis derived from renal carcinoma followed a different fate—cells were characterized by senescent features, without a notable effect on cell migration or viability. Collectively, our study illustrates that PDT could act as a successful therapy concept for local tumor control in some entities of bone metastases. The incidence of bone metastases in patients with advanced tumor disease has increased in recent decades. After the lung and liver, bone is the third most affected organ by tumor metastases . The cliPhotodynamic therapy (PDT) has been emerging as a promising way to treat and control malignant diseases ,6,7. TheDespite the fact that PDT is used in brain ,17, lungIn this study, we aimed to investigate the effects of PDT treatment on both primary and human metastatic cancer cell lines. Particularly, we investigated both qualitatively and quantitatively the effect of PDT exposure with 5-ALA on cellular migration/invasiveness potential, viability, apoptosis, and senescence growth arrest.Two primary bone metastases cell lines and one primary bone tumor cell line were subjected to investigations. Cell lines originating from primary bone metastases were as follows: ”MAM”—a cell line derived from bone metastases of renal cell carcinoma, and ”MAC”- bone metastases of invasive ductal breast carcinoma. Cell lines originating from a primary bone tumor, “17-1012”—giant cell tumor of bone, were also employed. All cell lines were kindly provided by Dr. Sabine Schleicher from the University Children’s Hospital Tuebingen. The patients  had given their written informed consent to the scientific analysis and cell line establishment from tissue samples. Full Institutional and ethical approval was obtained before the commencement of the study (project no. 008/2014BO2). As controls, bone marrow-derived mesenchymal stem cells (MSC) were isolated as previously described  at the Uv/v) fetal bovine serum  and 2 mM L-glutamine  and MAC and MSC cells in Dulbecco’s modified Eagle’s medium (DMEM) with GlutaMAX, 4.5 g/L D-glucose  supplemented with 10% (v/v) FBS . Cells were cultured at 37 °C in a humidified atmosphere with 5% CO2.Cell lines: MAM, 17-1012 were cultured in RPMI  supplemented with 10% .4 cells—MAC, 1 × 104 cells—MAM, 3 × 104—17-1012, and 2.5 × 104—MSCs cells was seeded into each 2-chamber culture inserts of 6-well plates with opaque F-bottom . After 24 h incubation at 37 °C, 5% CO2, the insets were removed, and the culture medium was replaced with a new one containing either 1 mM or 1.5 mM of 5-aminolaevulinic acid (5-ALA) photosensitizer. The cells were subjected to PDT exposure—blue light  in a continuous output mode for predefined time frames of 300 s and 2000 s. Cells that were not subjected to PDT exposure served as controls.An amount of 2 × 10w/v) paraformaldehyde in phosphate-buffered saline  for 30 min, and stained with 0.5% (w/v) Coomassie Brilliant Blue R250  in 100% (v/v) methanol  for 30 min. The migration of cells into gaps was visualized and photographed using an inverse phase-contrast microscope  and quantified using the open-source software ImageJ . The experiments were repeated three time for each cell type and exposure condition.Following PDT exposure, the cells (see section: “PDT exposure with 5-aminolaevulinic acid”) were fixed with 4% . Principally, cell viability determination was based on the mitochondrial conversion of MTS to soluble formazan, indicative of the number of viable cells [4 cells for each cell type) were supplemented with 50 μL of a 2 mg/mL solution of MTS in complete media and incubated for 90 min at 37 °C, 5% CO2. The absorbance was measured with an EL800 microplate reader  at a wavelength of 490 nm. Each experiment was conducted in triplicates and was repeated three times.The cellular viability after PDT treatment was assessed using a -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) MTS colorimetric assay (Promega CellTiter 96le cells . Followiv/v) methanol  for 10 min. Afterward, cells were washed with PBS and permeabilized with 0.2% (v/v) Triton X-100  in PBS. Nuclear staining was performed with 1% (w/v) 4′,6-diamidino-2-phenylindole  in PBS for 5 min. As a green-fluorescent cytosolic stain, 500 nm of CellTracker™ Green  was added onto the cells for 30 min. Fluorescence-stained cells were visualized with a Carl Zeiss Observer Z1 fluorescence microscope . Staurosporine  treated cells were included as a positive control  [A qualitative nuclear morphology assessment was done as previously described . Brieflyidation) . Each ex4 cells for each cell type) were subjected to a quantitative senescence-associated ß-galactosidase assay  following the manufacturer’s instructions. The absorbance was measured with a GM3510 fluorescence microplate reader  at 360 nm (Excitation)/465 nm (Emission). Three independent measurements were performed for each cell type and exposure condition.In order to investigate if PDT exposure triggers a state of stable cell cycle arrest, following PDT irradiation the seeded cells  and graphically displayed as bar diagrams. As the seeding density was adjusted for the migration assay in order to observe an effect, due to different seeding densities we refrained from performing statistical analysis. For the quantitative assays, comparison between experimental results was performed by one-way analysis of variance and t-test for independent samples as a post hoc test. Alpha adjustment based on a significance level of 0.05 was performed using the Bonferroni method. Statistical analysis was performed with SPSS Statistics 22 . As the seeding density was adjusted for the migration assay in order to observe an effect, due to different seeding densities, we refrained from performing statistical analysis.The MSC-control group showed no effect after 5-ALA PDT irradiation . In contA decrease in cell density, reduction in cytoplasm, and a smaller, altered morphology were observed for 17-1012 and MAC cells F,J,G,K wp = 0.009) and 1.5 mM (p = 0.002) at a 2000 s exposure time when compared with their corresponding controls. Contrastingly, the 17-1012 cell line exhibited an increase in viability  at an exposure of 300 s, followed by a decrease at 2000 s of exposure time (p = 0.257). The MAM cell line showed an increasing tendency in viability for both exposure times and 5-ALA concentrations . Irrespective of the exposure time and photosensitizer concentration, the number of viable MSCs remained unaltered and no notable change was observed.To investigate the effect on cellular viability after PDT exposure with 5-ALA, an MTS assay was conducted on all cell lines. Independent of the exposure time, for both of the employed 5-ALA concentrations (1 mM and 1.5 mM) a similar and comparable tendency for all of the cell lines was observed . A statiChanges in nuclear morphology as a hallmark for apoptosis were also examined qualitatively utilizing fluorescence microscopy. DAPI and cytosolic staining were used to visualize the changes in the nucleus and formation of apoptotic bodies—important hallmarks of apoptosis. It could be noted that after 2000 s of PDT exposure with 1.5 mM of 5-ALA, MAC, MAM, and 17-1012 cell lines . NuclearCellular senescence or cellular aging is characterized by a diminished replicative capacity coupled with altered functionality within the cell . To clarCurrently, radiotherapy is the gold standard in the treatment of metastases; however, it only offers a limited capacity for tissue repair and is related to severe adverse effects. Alternate approaches for treating tumors and metastases are urgently needed and one such potential candidate is the combination of photosensitizer, 5-aminolevulinic acid (5-ALA), and photodynamic therapy (PDT); however, the data on its safety and effectiveness in the treatment of metastatic lesions is limited. 5-ALA PDT offers several advantages, such as low toxicity, fast clearance, and rapid conversion to porphyrin.In this study, we assessed 5-ALA PDT treatment against different human cell lines, primary giant cell tumor of bone and bone metastases of invasive ductal carcinoma, renal carcinomas, as well as MSC (control group). We found that different human cell lines have distinct 5-ALA-PDT-sensitivities. Exposure of cells to 5-ALA PDT proved particularly effective in decreasing the migration potential, viability, cell proliferation, and density in bone metastases of invasive ductal carcinoma. For cells derived from giant cell tumor of bone, 5-ALA PDT irradiation triggered a notable decrease in migration potential, and cell density. No notable effect was observed in bone metastases of renal carcinoma and MSC group. The reduction effect, was, in general, more pronounced with a longer PDT exposure time and higher 5-ALA concentration suggesting a dose and exposure-dependent effect consistent with the previous research ,32. NumeIn fact, the cell line with lower sensitivity to 5-ALA PDT, that is, bone metastases of renal carcinoma (MAM), actually went into the state of cellular senescence caused by 5-ALA PDT exposure, and no therapeutic effect was observed. Senescence-induced cell lines showed no significant changes in cell migration even after prolonged PDT irradiation, and in fact exhibited increased viability even after a long exposure time. Thus, different sensitivity to the same doses and exposure of 5-ALA and PDT results in a completely different cell fate. While cellular senescence may be a beneficial strategy preventing the growth of the tumor short-term, cells remain metabolically active, affecting neighboring cells and stimulating cancer-promoting mechanisms ,38,39,40In bone metastases of the renal carcinoma cell line, 5-ALA PDT-induced senescence could be attributed to a too-low light dose, as the cellular decision between apoptosis/necrosis and senescence partially depends on it ,43. Our Even though nuclear morphologic changes such as chromosome condensation and fragmentation are considered hallmarks of apoptosis , DNA fraOne of the key advantages of PDT is that it can be used either before or after chemotherapy, radiotherapy, or surgery, without compromising these therapeutic modalities and the adverse effects of chemotherapy or radiation do not affect sensitivity to PDT. Several in vitro studies have shown the synergistic benefit of PDT and ionizing radiation in several pathologies ,50,51,52Human cell lines have different sensitivity to the same doses and exposure of 5-ALA PDT, resulting in two different cell fates apoptosis or senescence, depending on the extent of the cellular damage. We found that PDT has potential applicability in bone metastases of invasive ductal carcinoma. While no possible therapeutic effect was observed in bone metastases of renal carcinoma, a limited effect on primary bone tumors (giant cell bone tumor) is conceivable.While our study offers an interesting new perspective to the in vitro effects of 5-ALA PDT irradiation on metastatic cell lines, the approach implies a laborious work. For these reasons, only three independent experiments for each cell line were finally conducted. This aspect should be kept in mind when interpreting the statistical results, however, the measured tendency should not be affected. Additionally, it has to be noted that cellular heterogeneity in tumor cells is a well-established phenomenon ,54 consi