Artemia), roundworms (nematodes), and tardigrades (water bears). Discovery of molecular protectants that allow each of these three animals to survive drying diversifies our understanding of desiccation tolerance, and convergent themes suggest mechanisms that may offer a general model for engineering desiccation tolerance in other contexts.Water is critical for the survival of most cells and organisms. Remarkably, a small number of multicellular animals are able to survive nearly complete drying. The phenomenon of anhydrobiosis, or life without water, has been of interest to researchers for over 300 years. In this review we discuss advances in our understanding of protectants and mechanisms of desiccation tolerance that have emerged from research in three anhydrobiotic invertebrates: brine shrimp ( The metabolism of life occurs in an aqueous environment, and the biological constituents of cells – DNA, RNA, protein, and membranes – are all unstable during desiccation. However, some animals defy this reasoning, surviving in the near-absence of water by entering a quiescent state and later resuming activity upon rehydration. Antonie van Leeuwenhoek first described reviving dehydrated “animalcules” from the sediment in his gutter in 1702 . Other iTo affirm that a being, whose animation has been suspended for an immoderate length of time, even for years, can, in virtue of certain conditions, be revived, has so singular and paradoxical an appearance, that reason finds it repugnant to admit the fact. But to produce an animal which has been stiff and motionless, withered, disfigured, and contracted, utterly incapable of any corporeal function… and, by a particular treatment, to make it renovate every action that it could perform before; …not only will it bear perfect conviction to the mind that it has come from a state which, if it was not death, certainly cannot be called life, but that it again lives as completely as before its animation was suspended or destroyed. Some animals in the world enjoy this wonderful prerogative , roundworms (with a focus on Caenorhabditis elegans), and tardigrades. Artemia, roundworms, and tardigrades all undergo substantial physical and physiological changes. Artemia are able to survive dehydration as diapausing embryos  that are exposed to desiccation conditions are able to survive as larvae or adults, whereas diapause . Tardigrdiapause . Thus, aArtemia, nematodes, and tardigrades have been somewhat complementary and revealed different aspects of the physiology and molecular details of desiccation resistance. In this review we explore the molecules and mechanisms of desiccation tolerance, highlight common themes, and suggest open questions whose answers will be instrumental to the advancement of our understanding of desiccation tolerance.The approaches researchers have employed in studying Artemia). Johannes Albertus Schlosser is credited with the discovery and initial description of Artemia ; however, when environmental conditions are poor, females will instead release gastrula-stage embryos (oviparous development) that diapause until conditions amenable to hatching and development are present  are a family of ATP-dependent chaperones essential for protein folding. Knockdown of one family member, Hsp70, in mpbellii . Knockdompbellii . J-domaimpbellii . Two J-drHsp40-2 . KnockdorHsp40-2 . DeveloprHsp40-2 . This tiArtemia  is a common mechanism contributing to anhydrobiosis in a variety of organisms. One of the most prominent examples of an IDP is the family of late embryogenesis abudant (LEA) proteins. LEA proteins were originally identified in plants, but subsequently have been found in multiple animal species, including Artemia . There aArtemia . Artemia animals . These L animals .Artemia embryos suggests a possible function in desiccation resistance. A common experimental strategy to test for LEA function has been to assess chaperone activity or protective capacity in heterologous or in vitro contexts. LEA proteins from Artemia are capable of protecting liposomes, stabilizing membranes, attenuating protein aggregation, and conferring resistance to osmotic and desiccation stresses  regulates expression of Artemin and the small heat shock proteins p26, Hsp21, and Hsp22  expression profiling to identify genes whose mRNA abundances change during desiccation and (2) disruption of candidate genes’ functions to identify genes that are required for desiccation tolerance. These approaches have identified molecules that suggest a striking diversity of physiological adaptations to desiccation, some prominent examples of which we discuss in the sections below.C. elegans, the upregulation of trehalose synthesis during gradual drying is essential for worms to survive desiccation  and the subsequent dephosphorylation of trehalose 6-phosphate by a protein encoded by gob-1    . Each ofgulated) . The iniscussed) . Despitean cells . LEA-1 ficcation .Aphelenchus avenae as a model, C. elegans LEA-1 , several glutathione peroxidases  and catalase (ctl-1) are required for desiccation survival, suggesting that upregulation of these genes  is an adaptive physiological response to gradual desiccation , Δ6 desaturase (fat-3), and Δ5 desaturase (fat-4). Of note, the ω3 desaturase, fat-1, does not impact desiccation survival, suggesting that arachidonic acid may play a particular role in desiccation resistance  must have mechanisms to recognize gradual loss of water and to induce the responses described above. Even animals like fruit flies that cannot survive total desiccation can sense and respond to humidity  in thermosensory neurons regulates hygrotaxis  channel gene, survival . TRP chagrotaxis . In C. egrotaxis . Additiogrotaxis . It is shsf-1 regulates the heat shock response and many chaperones, skn-1/Nrf2 is a master regulator of the oxidative stress response, and daf-16/FOXO transcriptionally regulates many genes involved in the glyoxylate shunt and trehalose synthesis during starvation , a patched related protein (DAF-6), and mitogen-activated protein kinase (MAPK) signaling . Specifiarvation . HoweverArtemia and tardigrades. More genes than discussed above play roles in desiccation tolerance. These genes   . HoweverAdorybiotus coronifer and those of the family Macrobiotidae have been shown to produce trehalose in response to desiccation  is considerably lower than the level of trehalose accumulation reported in Artemia (15%) or nematodes (∼13%)  . The appProtectants that function during desiccation must retain their protective properties even during conditions in which most proteins are prone to aggregation and dysfunction. Thus, protective proteins are likely to be resistant to aggregation and retain solubility even during extreme conditions if they are to interact with and preserve other cellular constituents. If protectants that function during desiccation are themselves resistant to severe dehydration stress, this may predispose them to withstand other stresses as well. To identify possible protectant proteins, one approach has been to determine the portion of the proteome that is highly stable and soluble even during stress. The most straightforward method for this has been to search for heat soluble proteins – those that can retain solubility even after transient exposure to near-boiling temperatures. Analysis of the tardigrade proteome has revealed such heat soluble proteins .Ramazzotius varieornatus, and homologs for some of the heat soluble proteins are also present in other tardigrade species including H. exemplaris and Milnesium tardigradum , mitochondrial abundant heat soluble (MAHS), and secretory abundant heat soluble (SAHS) proteins . The SAHdigradum . Furtherichtersi . Some CAichtersi . RNAi knichtersi . These eThe potentially broad function of TDPs piques interest in understanding the mechanisms by which they exert their protective effects. As with LEA proteins, the disordered nature of CAHS proteins and other TDPs goes against traditional intuition of structure-function analysis. However, CAHS proteins take on a glass form during desiccation, suggesting that disordered proteins in general may contribute to cellular vitrification – a mechanism we will discuss in more detail below .Artemia researchers, was used along with immunolocalization in tardigrades to determine the subcellular localization of a group 3 LEA protein of Ramazzottius varieornatus, RvLEAM. This LEA is mitochondrially localized, similar to AfrLEA3m  are also upregulated during dehydration  that would occur in an aqueous environment . A partiA glass is an amorphous, non-crystalline solid. Through the process of vitrification, biological glasses form and protect cells from damage during dehydration . During Artemia and tardigrades, suggest that glass formation is a widespread mechanism contributing to desiccation tolerance (Evidence for glassy solids in plants and animals, including olerance . While sThe term “molecular shield” was first proposed to describe the function of LEA proteins that may provide a protective shell that shields proteins during desiccation and prevent aggregation . The proWhile the water replacement hypothesis, vitrification, and molecular shielding are sometimes discussed as individual mechanisms for desiccation survival, they likely overlap and synergistically promote desiccation tolerance. This can be appreciated when individual molecular protectants contribute to more than one of these models. For example, LEA proteins contribute to each of the three models for protection, and trehalose plays a prominent role in both the water replacement hypothesis and vitrification . Thus, fArtemia, nematodes, and tardigrades has advanced our understanding of the protectants and mechanisms that contribute to desiccation survival. Comparisons of the types of protectants involved in desiccation tolerance in each animal converges on a few key molecules (see Artemia-specific protein, Artemin. Each organism has brought with it unique experimental advantages as well – Artemia have a unique life cycle and intergenerational diapause, C. elegans has provided a genetic model to greatly facilitate gene identification and functional studies, and tardigrades are emerging as another model for exceptional stress tolerance. A comparative organismal approach to the study of desiccation tolerance, using these organisms and others such as rotifers and midges, has enriched our understanding of mechanisms that multicellular organisms employ to survive desiccation, and no doubt will continue to do so.Our knowledge of the mechanisms of desiccation tolerance has been largely informed by research on a small number of anhydrobiotic organisms. Work on ules see . HoweverDesiccation tolerance is sometimes conceived as primarily a cellular survival strategy. While individual cells must synthesize the necessary protectants to survive, the coordination of the desiccation response in multicellular organisms presents an expanded set of challenges – to sense and respond appropriately to environmental cues that signal dehydration, and coordinate a response across tissues to allow the animal to survive. This necessitates exquisite spatial and temporal regulation. While some mechanisms by which this sensing and coordination are accomplished have been discussed, the basis of the intermediate regulatory steps between survival of an organism and survival of a cell remain largely unknown.in vivo experiments and drawing conclusions about protectants in their native context will help to distinguish between possible protectants and those that are essential. Still, part of the promise in studying mechanisms of desiccation tolerance is the application of discoveries to better preserve biomaterials. Therefore, discovery of new, uncharacterized protectants continues to be of interest. In over 300 years since the initial observation of desiccation survival, our understanding has grown considerably. Yet, in many ways the molecular basis of anhydrobiosis and mechanisms by which multicellular animals survive drying are only beginning to emerge. Continued research in Artemia, nematodes, and tardigrades in coming years will likely contribute significantly to our growing understanding of desiccation tolerance.In many cases, heterologous expression of proteins demonstrates the possible activity of protectants for desiccation survival. However, the endogenous functions of proteins are often poorly characterized. Conducting more JH drafted the original manuscript. All authors edited and revised the manuscript, approved the final version, and contributed to the conception of this review and discussion of its contents.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.


The PI 3-kinase (PI3K) pathway has been implicated as a target for melanoma therapy.Given the high degree of genetic heterogeneity in melanoma, we sought to understand the breadth of variation in PI3K signalling in the large NZM panel of early passage cell lines developed from metastatic melanomas.PIK3CA gene, and amplification of the PK3CG gene was common. Deletions in the PIK3R1 and PIK3R2 regulatory subunits were also relatively common. Notably, no genetic variants were seen in the PIK3CD gene despite p110δ being expressed in many of the lines. Genetic variants were detected in a number of genes that encode phosphatases regulating the PI3K signalling, with reductions in copy number common in PTEN, INPP4B, INPP5J, PHLLP1 and PHLLP2 genes. While the pan-PI3K inhibitor ZSTK474 attenuated cell growth in all the lines tested, isoform-selective inhibition of p110α and p110δ inhibited cell growth in only a subset of the lines and the inhibition was only partial. This suggests that functional redundancy exists between PI3K isoforms. Furthermore, while ZSTK474 was initially effective in melanoma cells with induced resistance to vemurafenib, a subset of these cell lines concurrently developed partial resistance to PI3K inhibition. Importantly, mTOR-selective or mTOR/PI3K dual inhibitors effectively inhibited cell growth in all the lines, including those already resistant to BRAF inhibitors and ZSTK474.We find the vast majority of lines show upregulation of this pathway, and this upregulation is achieved by a wide range of mechanisms. Expression of all class-IA PI3K isoforms was readily detected in these cell lines. A range of genetic changes in different components of the PI3K pathway was seen in different lines. Coding variants or amplification were identified in the Overall, this indicates a high degree of diversity in the way the PI3K pathway is activated in different melanoma cell lines and that mTOR is the most effective point for targeting the growth via the PI3K pathway across all of these cell lines.The online version contains supplementary material available at 10.1186/s12885-021-07826-4. BRAF [NRAS [NF1 [BRAF V600 mutations. However, this is almost invariably followed by resistance development within a few months [Two major breakthroughs have occurred recently in melanoma therapy. The identification of common mutations in BRAF , NRAS [2RAS [NF1 , 4 indicRAS [NF1 , 6 as a w months , 8. Morew months , 10.PIK3CA are also found in some melanomas [One focus of the efforts to develop strategies to overcome this resistance to treatment has been on the PI 3-kinase (PI3K) pathway , 8. Thiselanomas , 14. Botelanomas , 16, whielanomas , 18 and elanomas , 20.MTOR [The above findings have lead to interests in understanding the role PI3K inhibition might play in overcoming therapeutic resistance in melanoma. Indeed, it has been found that inhibition of PI3K can potentiate the effects of BRAF and MEK inhibitors in attenuating the growth of melanoma cells  and melaMTOR  and seveMTOR , 26 haveMTOR . Our stu2 for our cell culture studies on the basis that this level was more likely to be representative of conditions found in a tumour. Cells were passaged in Minimum Essential Medium α (MEMα) supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), and amphotericin B , insulin (5 μg/mL), transferrin (5 μg/mL), and sodium selenite , and 5% fetal bovine serum (FBS).The cell lines used were chosen from a panel of NZM early passage cell lines that had been developed in house from biopsies of metastatic melanoma samples . We firsDNA was prepared from NZM cell lines and analysed using Sequenom mass array Oncocarta panels for analysis of mutations of common oncogenes . Whole-eCells were seeded in 96-well plates (5000 cells/well) and after 24 h treated with inhibitors at a range of concentrations at which the drugs might be considered effective and selective. After 3 days of drug treatment, cell viability was determined using the sulforhodamine B (SRB) assay . ResultsWestern blotting was performed on lysates prepared from 75 NZM lines freely growing and harvested at 80–100% confluence. The protein isolation and immunoblotting procedures were performed as previously described from cells lysed in normal growth phase . EnhanceCell signaling inhibitors used were synthesized in house as previously described , 31 excePTEN deletions and a small number of PIK3CA mutations. However, our genetic analysis of the NZM cell lines indicates a more complex picture of the PI3K pathway’s genetic alteration in the NZM cell lines, with many of the lines having variants in more than one gene in this pathway  NZM52 (H1047R) and NZM91 (E545K)) validated by both Sequenom mass array and whole-exome sequencing . The majority of cell lines also express p101 indicating the signalling via class-IB PI3K is possible in these cells. Class II PI3Ks and Class-III PI3K were also detected in most of the cell lines  or PTEN loss at the protein level (17/75) expressed high basal levels of pAkt-Ser473, which is in agreement with previous studies [All lines exhibited strong expression of Akt1 and Akt2, which lie downstream of PI3K, even in the cell lines with copy number reduction of AKT genes  is also notable. However, p110δ expression levels, in fact, correlated inversely with sensitivity to IC87114 in growth assays.This study represents the most comprehensive investigation to date of the relationship between genetic variation in the PI3K related oncogenes and tumour suppressor genes and responses to drugs targeting PI3K. This is possible because of the availability of a large number of NZM melanoma cell lines to understand responses to the diverse genetic changes in components of the PI3K pathway. We find that inhibition of all class-I PI3K isoforms effectively blocked the growth of the NZM cells. Previous studies in other cell lines have suggested that responses to selective inhibitors of PI3K isoforms might be linked to specific genetic alterations in the PI3Ks and related genes. For example, p110α inhibitors have been found to be more effective in some cell lines with ications , 30. In ications . Previouications . Here wema lines . FurtherBRAF, NRAS or the TERT promoter as there was no clear correlation between these and the response of PI3K inhibitors. It is though notable that 2 PIK3CA mutant lines resistant to A66 also have KRAS mutations. PTEN loss was, in fact, a better predictor of resistance to A66, which is consistent with previous studies in other tumour types where knockdown on PIK3CA did not affect PI3K signalling in several PTEN null cancer lines [The lack of efficacy with individual isoform-selective inhibitors could be due to their effects being overridden by the dominant effects of other gene variants. This is unlikely to be the variants in er lines . Togetheer lines , 30.The findings above indicate that despite the increased risk of on-target toxicities, inhibitors targeting all class-IA PI3Ks would be required in strategies aimed at targeting the PI3K pathway in melanoma. Interestingly, although resistance to BRAF inhibition therapies can occur through different mechanisms such as reactivation of MAPK, PI3K, or TERT signalling, our data show that inhibition of all class-I PI3Ks significantly improves the efficacy of BRAF/MEK pathway inhibition, which is consistent with previous findings . We induOur results indicate a high degree of diversity in the way the PI3K pathway is activated in different melanomas and that mTOR is the most effective point for targeting the growth via the PI3K pathway across all of these cells.Additional file 1: Supplementary Table S1: Hotspot mutations of genes relating to the PI3K pathway in NZM cell lines.Additional file 2: Supplementary Table S2: Variants of genes encoding class I PI3K catalytic subunitsAdditional file 3: Supplementary Table S3: Variants of genes encoding class I PI3K regulatory subunitsAdditional file 4: Supplementary Table S4: Variants of genes encoding PI3K-related phosphatasesAdditional file 5: Supplementary Table S5: Variants of genes encoding mTOR and AKTAdditional file 6: Supplementary Table S6: Variants of genes encoding class II PI3KAdditional file 7: Supplementary Table S7: Variants of genes encoding class III PI3KAdditional file 8: Supplementary Table S8: Summary of the major genotypes and drug response of NZM cell linesAdditional file 9: Supplementary Fig. S1: Effect of oxygen tension in drug response. Melanoma cells were cultured in either 5% or 20% oxygen tension. The cells were seeded in 96-well plates (5000 cells/well) and treated 24 h later with vemurafenib, CI1040, ZSTK474, A66, IC87114 and BEZ235. Cell viability was determined using the sulforhodamine B (SRB) assay.Additional file 10: Supplementary Fig. S2: Expression at protein levels of PI3K pathway kinases in NZM cell lines.Additional file 11: Supplementary Fig. S3: Role of PI3Kβ and PI3Kγ in melanoma cell growth. Melanoma cells were seeded in 96-well plates (5000 cells/well) and treated 24 h later with inhibitors of (A) TGX221 or (B) AS252524 for 3 days. Cell viability was determined using the sulforhodamine B (SRB) assay. BRAF: BRAF-mutant cell lines; RAS: RAS-mutant cell lines; other: cell lines wild-type for both BRAF and RAS. Data were from 2 to 4 independent experiments performed in duplicates. Yellow areas represent the concentration ranges between the biochemical IC50 and 100xIC50 values of inhibitors for their specific targets; x-axis represents cell-based EC50 values; y-axis represents inhibition % at 100 μM of the corresponding inhibitors where 100% inhibition indicates complete stop of cell growth.Additional file 12: Supplementary Fig. S4: Effects of co-inhibition of the PI3Kα and PI3Kδ in melanoma cell growth. Melanoma cells were seeded in 96-well plates (5000 cells/well) and treated 24 h later with A66 alone, IC87114 alone, or combination of A66 and IC87114 for 3 days. Cell viability was determined using the sulforhodamine B (SRB) assay.Additional file 13: Supplementary Fig. S5: Protein expression of key signalling molecules in 6 parental BRAF-mutant NZM cell lines originally sensitive to vemurafenib, along with the drug resistant clones of these lines. All the lines were grown in presence of complete growth media as described in the methods. (PR: vemurafenib-resisistant cell lines. PZR: vemurafenib/ZSTK474 double-resistant cell lines).Additional file 14: Supplementary Fig. S6: The indicated NZM cell lines were seeded into 12-well plates (5 × 105 cells per well) in serum-free medium for 24 h. The cells were stimulated with 100 nM insulin and then treated with 1 μM vemurafenib (Vem 1 μM), 1 μM ZSTK474 (ZSTK474 1 μM), 1 μM linsitinib (Linsitinib 1 μM), or 1 μM BMS754807 (BMS 1 μM). The positive controls (+ Control) for the experiment were cells that were stimulated with 100 nM insulin but not treated with any drug, and the negative controls (− Control) were cells that were not stimulated with insulin and not treated with any drugs. The lysates were western blotted with the indicated antibodies in duplicates


Patients diagnosed with basal-like breast cancer suffer from poor prognosis and limited treatment options. There is an urgent need to identify new targets that can benefit patients with basal-like and claudin-low (BL-CL) breast cancers. We screened fractions from our Marine Invertebrate Compound Library (MICL) to identify compounds that specifically target BL-CL breast cancers. We identified a previously unreported trisulfated sterol, i.e., topsentinol L trisulfate (TLT), which exhibited increased efficacy against BL-CL breast cancers relative to luminal/HER2+ breast cancer. Biochemical investigation of the effects of TLT on BL-CL cell lines revealed its ability to inhibit activation of AMP-activated protein kinase (AMPK) and checkpoint kinase 1 (CHK1) and to promote activation of p38. The importance of targeting AMPK and CHK1 in BL-CL cell lines was validated by treating a panel of breast cancer cell lines with known small molecule inhibitors of AMPK (dorsomorphin) and CHK1 (Ly2603618) and recording the increased effectiveness against BL-CL breast cancers as compared with luminal/HER2+ breast cancer. Finally, we generated a drug response gene-expression signature and projected it against a human tumor panel of 12 different cancer types to identify other cancer types sensitive to the compound. The TLT sensitivity gene-expression signature identified breast and bladder cancer as the most sensitive to TLT, while glioblastoma multiforme was the least sensitive. This stion data . Basal-lion data . Approxiion data . Howeverion data ,8.Interestingly, the claudin-low subtype shares some similarities in gene-expression features with the basal-like subtype such as low expression of HER2 and the luminal gene clusters, indicating genomic similarities between the two subtypes . MoreoveThe Marine Invertebrate Compound Library (MICL) is a unique resource that serves as a platform for discovery of novel small molecule-mediated biological activities in a variety of systems. The MICL is derived from an extensive collection of small-molecule natural products isolated from over 1200 unique marine organisms  collected from diverse locations around the world over the past twenty years ,14. NatuTopsentia sp. (PNG07-3-073) collected from Papua New Guinea , purified from a marine sponge w Guinea . TLT, asThe goal of this study was to isolate a novel inhibitor of BL-CL breast cancers, describe the pathways effectively blocked by the compound, and project treatment efficacy across other cancer types. We aimed at achieving this through a stepwise approach consisting of the following steps: (1) screening MICL for fractions that exhibited tumoricidal properties, (2) selecting candidate fractions that displayed effectiveness against BL-CL cell lines, (3) separating the active compounds in the fractions, (4) identifying the active compound with anti-BL-CL properties, (5) describing the cell signaling effects of the compound on BL-CL activity, (6) projecting TLT sensitivity across human tumors of various cancer types using a gene-expression signature A.Topsentia sponge PNG07-3-073 to exhibit anti-BL-CL activity  fractions from MICL against a panel of 16 cell lines. These fractions represent complex mixtures and served as a starting point to identify promising hits ,14. We sactivity B. This aactivity C. From tp = 0.247, Our next step was to proceed with the identification of the active compounds in the M6 subfraction to isolate any compounds inducing this response. We performed a scaled-up extraction of bulk PNG07-3-073 sponge and purified two compounds, i.e., isolate 1 and 2. One-dimensional (1D) nuclear magnetic resonance (NMR) analysis  identifip = 0.031, p = 0.024, p = 0.012, p = 0.003, Our next goal was to analyze and describe the signaling effects that are induced by TLT in cancer cells. For this purpose, we treated eight TLT-sensitive BL-CL cell lines with TLT or DMSO control and screened for changes in the phosphorylation of 217 proteins using a reverse-phase protein array (RPPA) . From thIn order to validate the observed effects on AMPK, p38, and CHK1 phosphorylation following TLT treatment in the RPPA screen, we analyzed phosphorylation levels by Western blotting. Downregulation of AMPK phosphorylation was most consistent across all four cell lines B. Reducep = 0.011, p = 0.001, p = 0.005) (Due to the consistency with which AMPK (either the protein itself or its downstream effector Raptor) and CHK1  phosphorylation are affected, as well as the recent discovery of CHK1 treatment efficacy against TNBC , we deci= 0.005) C. To con= 0.005) . Indeed,= 0.005) , furtherNext, we identified classes of solid tumors that were most sensitive to TLT using an unbiased computational approach. The gene-expression profiles of cancer cells are a valuable tool in the comprehension of transcriptional changes indicative of treatment. These profiles enable the identification of drug sensitivity across various cancer types. This approach has been used multiple times to show a pathway or drug sensitivity ,35,36. AWe have identified and described a previously uncharacterized trisulfated sterol that we have named topsentinol L trisulfate (TLT) that exhibited increased tumoricidal activity against BL-CL breast cancers. Interestingly, halistanol sulfate, another trisulfated sterol isolated from the same marine organism as TLT, did not exhibit similar selective activity against BL-CL breast cancers but was demonstrated to be more potent . This coFurthermore, we describe the treatment effect of TLT on BL-CL breast cancers, highlighting the particular changes in the phosphorylation of AMPK, CHK1, and p38. AMPK is a heterotrimeric serine/threonine kinase complex that is regulated by adenylate levels in the cell and functions as part of an evolutionarily conserved energy-sensing pathway ,42. The Another aspect of the inhibitory effects promoted by TLT treatment was the decreased phosphorylation of CHK1 leading to its inhibition. Upon cellular exposure to various genotoxic stresses, CHK1 is activated by ATR-mediated phosphorylation following DNA-damage leading to the phosphorylation of cdc25. CHK1 assumes the role of the major cell-cycle checkpoint kinase mediating S- and G2-arrest . In BL-CSimilarly, we have shown that treating BL-CL breast cancers with TLT leads to the significant activation of p38 ; p38 plaThe inhibitory effects of TLT on AMPK and CHK1 shed light on the potential therapeutic benefit of AMPK or CHK1 inhibition on BL-CL activity. However, additional work is required to validate these findings. Although dorsomorphin is a potent AMPK inhibitor, studies have shown this compound to exhibit high affinity towards other proteins such as BMP and inhibit several other kinases at a Km lower than AMPK , prompti2 in their respective media. The detailed description of the cell lines used for each screen is available in Cell lines were obtained from ATCC and plated at 1500–2000 cells/well in 384-well plates in 5% FBS  growth media and 1× antibiotic-antimycotic (Anti-Anti) . Cancer cell lines were cultured and maintained in a humidified environment at 37 °C and 5% COt-test < 0.05 with a positive average difference). Cell lines described as basal-like or claudin-low but being HER2+ were considered to be part of the luminal/HER2+ group. Liquid chromatography mass-spectrum fractionation of the anti-BL-CL fractions, following the MICL protocol [For Screen 1, 2778 HP20 fractions of marine organisms from MICL were screened at a single dose (~1.5 µg/mL) against a panel of 16 (9 lung and 7 breast) cancer cell lines to determine their antitumor properties. We selected 107 fractions for further evaluation based on one or more of the following criteria: (1) all fractions with a standard deviation in viability of greater than 0.325, (2) lung selective fractions (25% or less viable cells in 3 or more lung cancer cell lines and 2 or fewer breast cancer cell lines), (3) breast selective fractions (25% or less viable cells after treatment in 3 or more breast cancer cell lines and 2 or fewer lung cancer cell lines), (4) generally active non-universally toxic fractions (25% or less viability in a minimum of 5 and maximum of 13 cell lines), (5) relatively less active fractions (40% or less viability in a minimum of 9 and maximum of 13 cell lines). For our second screen, we added an additional 85 HP20 fractions from MICL that were not included in the first screen but based on the chemo-taxonomic judgment of potential chemical similarity to the 107 fractions. Then, these 192 HP20 fractions were assayed at a single dose (~1.5 µg/mL) against a panel of 35 breast cancer and 37 lung cancer cell lines. This identified breast-selective fractions  and fractions effective against BL-CL breast cancers  values were calculated from dose-response curve data by plotting on GraphPad Prism 6.01 and using the equation, Eight TLT-sensitive BL-CL cell lines  were treated separately with TLT or DMSO at a concentration of 105 µM  for 6 h, after which they were lysed, according to the method detailed in The same eight TLT-sensitive BL-CL cell lines that were used in the reverse-phase protein array assay were treated separately with TLT or DMSO for 6 h, after which total RNA was extracted using the RNeasy Mini Kit  with on-column digestion of the genomic DNA, as described in the manufacturer’s protocol. RNA sequencing was performed at the Huntsman Cancer Institute High Throughput Genomics Core Facility using 50-cycle, single-read sequencing (version 3) on an Illumina HiSeq instrument. To construct mRNA focused libraries from total RNA, the Illumina TruSeq RNA Sample Prep Kit (version 2) with oligo(dT) selection was used.p < 0.05) between the treated and control samples [To process the mRNA sequencing data, we used the TCGA mRNA-seq Pipeline . RNA seq samples ,52. One  samples . We pred samples  using th samples . We used samples . Prior t samples  to reducFour BL-CL cell lines sensitive to topsentinol L trisulfate  were treated with 105 µM of TLT in 5% FBS RPMI media and 1x Anti-Anti for 6 h. Proteins were extracted and Western blots were run with the following primary antibodies: β-actin ; β-tubulin ; pRaptor-S792 (#2083); pCdc25c-S216 ; pMAPKAPK-2-Thr334 ; p38-T180/Y182 ; pChk1-S317 ; pAMPKα-T172 . All antibodies were obtained from Cell Signaling Technology .t-test built into the Microsoft Excel program. Final statistical assessment was performed for the fractions from the sponge PNG07-3-073 by re-analyzing the statistical significance test based on the normality of the data. Gaussian distribution of the data was checked by using the following three tests built into the GraphPad Prism 6 software: the D’Agostino–Pearson omnibus test, the Shapiro–Wilk test, and the Kolmogorov–Smirnov test . Then, dot plots were created using GraphPad Prism 6.01 and a standard two-tailed Mann–Whitney U test was used, with the exception of the dot plot diagrams for halistanol sulfate, dorsomorphin, Ly2603618, and dorsomorphin + Ly2603618, where an unpaired t-test was used due to the normality of the data. To compare the difference of protein expression between treated cell lines and their DMSO controls, we used a two-tailed paired t-test.To identify the candidate fractions that were significantly more effective against BL-CL breast cancers than luminal/HER2+ breast cancer, a preliminary statistical analysis was performed using the unpaired two-sample equal variance Our work describes the projected efficacy of TLT against a variety of human tumors, highlighting the optimal effect of the compound against breast and bladder cancer. In a TCGA study characterizing the molecular landscape of urothelial bladder carcinoma, a p53 mutation rate of 49% was recorded in the samples tested , drawingIn this study, we discovered and identified a previously unreported sulfated sterol, TLT, and described its signaling effects on BL-CL activity. We described two potential therapeutic targets of BL-CL cell lines that can be exploited for the benefit of treatment efficacy. This work lays the groundwork necessary for the exploration of AMPK and CHK1 as potential targets of BL-CL treatment, with a need to further characterize and delineate the role of TLT as an investigational anti-BL-CL compound.


Extracellular vesicles (EVs), are important for intercellular communication in both physiological and pathological processes. To explore the potential of cancer derived EVs as disease biomarkers for diagnosis, monitoring, and treatment decision, it is necessary to thoroughly characterize their biomolecular content. The aim of the study was to characterize and compare the protein content of EVs derived from three different cancer cell lines in search of a specific molecular signature, with emphasis on proteins related to the carcinogenic process. Oral squamous cell carcinoma (OSCC), pancreatic ductal adenocarcinoma (PDAC) and melanoma brain metastasis cell lines were cultured in CELLine AD1000 flasks. EVs were isolated by ultrafiltration and size-exclusion chromatography and characterized. Next, the isolated EVs underwent liquid chromatography-mass spectrometry (LC-MS) analysis for protein identification. Functional enrichment analysis was performed for a more general overview of the biological processes involved. More than 600 different proteins were identified in EVs from each particular cell line. Here, 14%, 10%, and 24% of the identified proteins were unique in OSCC, PDAC, and melanoma vesicles, respectively. A specific protein profile was discovered for each cell line, e.g., EGFR in OSCC, Muc5AC in PDAC, and FN1 in melanoma vesicles. Nevertheless, 25% of all the identified proteins were common to all cell lines. Functional enrichment analysis linked the proteins in each data set to biological processes such as “biological adhesion”, “cell motility”, and “cellular component biogenesis”. EV proteomics discovered cancer-specific protein profiles, with proteins involved in processes promoting tumor progression. In addition, the biological processes associated to the melanoma-derived EVs were distinct from the ones linked to the EVs isolated from OSCC and PDAC. The malignancy specific biomolecular cues in EVs may have potential applications as diagnostic biomarkers and in therapy. Extracellular vesicles (EVs) are released by cells into the extracellular space and are classified according to their size and biogenesis , 2. AccoEVs are quite abundant in biofluids as they are continuously released by cells . In someAdditionally, since EVs contain signaling molecules, they are considered be a potential source of diagnostic biomarkers for the prediction of disease, as well as in disease monitoring and treatment decision making , 16. To In this study, we characterized the protein content of small EVs (diameters under 200 nm), most likely exosomes and small microvesicles. These were isolated by the combination of UF and SEC from cell culture supernatant of three distinct cancer cell lines: oral squamous cell carcinoma (OSCC), pancreatic ductal adenocarcinoma (PDAC), and melanoma, all with poor prognosis , 22. We 2 atmosphere incubator.Small EVs were isolated from the culture media of human oral squamous cell carcinoma (OSCC) , pancreatic ductal adenocarcinoma (PDAC) , and human melanoma brain metastasis . The cells were cultured as previously described . In ordeCell culture supernatants were collected once a week. After collection of the cell culture supernatant, the CELLine AD1000 flasks were washed with warm PBS. Cell culture supernatant and PBS were then pooled. Samples were centrifuged at 4000g for 5 min at room temperature, to remove cell debris and the supernatant was then centrifuged for another 45 min at 15000g, 20°C. Next, 15 ml of the 15000g supernatant was concentrated by UF using Amicon-Ultra 15 Centrifugal Filter Units (Merck Milipore) with a 50 kDa molecular weight cut-off (MWCO), to a final volume of 4 ml. Concentrates were loaded into 30 ml sepharose CL-2B  size-exclusion chromatography (SEC) columns. The concentrated cell culture supernatants were eluted through the columns with the continuous addition of filtered PBS. Eluates were collected by gravity in 20 fractions of 1 ml. For each fraction, protein quantification was determined by spectrophotometry (Absorbance 280nm) in a Nanodrop spectrophotometer (Thermofisher).®, Thermo-Fisher Scientific)), western blot (WB) ) and transmission electron microscopy (TEM), as described elsewhere [Following protein quantification, the EV enriched fractions were identified and pooled. Samples were characterized by nanoparticle tracking analysis (NTA), immunoaffinity capture , as described in detail in . BrieflyValidation of liquid chromatography-mass spectrometry (LC-MS)-based peptide and protein identifications was carried out using Scaffold_4.9.0 . Here protein threshold was set at 99.0% to ensure that only proteins with 99% of probability of being present were produced, and the minimum number of peptides was set to 2. Furthermore, the peptide threshold was set at 95%, the minimum probability to determine if a given spectrum identifies a peptide.Data from the vesicles isolated from E10, BxPC3, and H3 cell culture supernatants were analyzed and extracted individually from Scaffold. All datasets were screened and proteins that were not present in all 3 replicates within a cell line were not included for further analysis.http://www.funrich.org). In addition, the proteins identified in the preparations from E10, BxPC3, and H3 were compared with the Vesiclepedia database (http://microvesicles.org), a web-based open source compendium of biomolecules  found across all classes of EVs from published and unpublished studies [Comparison of the proteins from the different cell lines was carried out using the Functional Enrichment Analysis Tool (FunRich)  Bioinformatics Resources 6.8. The proteins were classified according to their gene ontology (GO) term and categorized into functional groups under the term “Biological processes” (GOTERM_BP_FAT). Next, the GO terms were grouped into “Functional annotation clusters”, where the terms are organized into groups based on the premise that genes with similar annotation are functionally related to each other . Functio10, 3.14x1010, and 2.22x1010 particles/ml for E10, BxPC3, and H3, respectively  analysis for identification and characterization of their protein content.Proteomic analysis of the vesicles isolated from cell culture supernatant from the three cell lines identified 678 different proteins from the E10 derived EVs, 618 proteins from the BxPC3 derived EVs, and 762 proteins from H3 derived EVs . InteresThe top 20 most highly expressed proteins in the EVs from each of the cell lines are listed in Thus, despite the presence of common proteins, the EVs isolated from the different cell lines showed an individual profile.When the proteins in our datasets were analyzed in view of the Hallmarks of cancer, we identified proteins with roles in the processes of angiogenesis, inflammation, cell proliferation, migration, immunity, and cell adhesion .The EVs from each of the cell lines demonstrated varied protein profiles related to these processes. For example, the panels of proteins associated with cell proliferation, migration, and adhesion were fairly extensive for both E10 and BxPC3. Of note is the presence of several members of the integrin family (IT), associated with cell proliferation, migration, and adhesion, such as ITGA2, ITGA3, ITGA6, ITGA5, ITGB1, and ITGB4 in the EVs derived from E10 and BxPC3. In contrast, only half of the cell proliferation-related proteins were found in EVs from the H3 cell line, most of them poorly expressed (with SC below 10). The number of proteins related to adhesion were also reduced in the H3 derived EVs. Here ITGB1, ITGA3, and ITGA2 were found in the vesicles from H3, the latter with fairly low SC.Overall, the protein content of the EVs isolated from the different cell lines carry signals that promote cancer traits. However, the EVs from the H3 melanoma cell line have a more distinct protein signature than the other two, with respect to both the type of proteins as well as their amount present.To better understand the roles and relationships of the proteins in the isolated EVs, Gene Ontology (GO) analysis was performed as described in the Materials and Methods section.In the dataset of proteins identified in the E10 vesicles, the 8 most enriched biological processes in the functional annotation cluster analysis  includedExtracellular vesicles carry complex biomolecular cues and thus, play an important role in the communication between cells. In this study we analyzed and categorized the protein content from EVs derived from oral squamous cell carcinoma (OSCC), pancreatic ductal adenocarcinoma (PDAC), and melanoma brain metastasis cell lines , all cancers with poor prognosis for the patient. Isolation of the EVs from the cell culture supernatants, prior to proteomic analysis, was carried out combining UF and SEC. The EVs were characterized by NTA, WB, immunoaffinity capture, and TEM.et al., indicates that SC of less than five is unrealiable in studies with a small number of replicates [Characterization of the protein content of the isolated vesicles by LC-MS identified a similar number of proteins for all preparations: 678 proteins for E10, 618 for BxPC3, and 762 for H3 . When coplicates , 38, 39.plicates . Neverthplicates . The graplicates .The proteins identified in the EVs of each cell line were ranked according to their average SC for further analysis. Due to the large datasets for each of the cell lines, the top 20 most abundant proteins were extracted . A charaIn summary, our results highlight that the EVs isolated from each of the different cancer cell lines have distinct protein profiles. Cell line-specific profiles already begins to emerge when comparing the 20 most adundant proteins. Here, proteins that seem to provide tumors with signals that promote its progression are found among EV-related proteins. These EV related proteins are present in the datasets from all three cell lines.The panel of proteins detected in the EVs from OSCC, PDAC, and melanoma cell lines  indicates that these vesicles can play roles in angiogenesis, inflammation, proliferation, migration, as well as in modulation of immunity, all representing Hallmarks of cancer. Within this selection of proteins , there wIn contrast, Muc5AC was found to be highly enriched in the BxPC3 derived EVs, while present in much lower amounts in the EVs from the other two cell lines. Muc5AC is a protein that stimulates angiogenesis in PDAC and might also suppress the immune response towards the tumor . In patiLastly, FN1 was abundant in the H3 derived EVs but detected in a smaller amount in the E10 derived EVs. Furthermore, a dramatic drop in the FN1 content was found in the BxPC3 derived EVs. FN1 plays a role in cell adhesion, migration, and differentiation . Furtheret al the levels of integrin expression in exosomes derived from melanoma brain metastasis are down regulated [et al in saliva from OSCC patients  [Differences among the cell lines were also noticeable regarding members of the integrin family. The members of this family of cell adhesion receptors are relevant in cancer processes such as cell migration and invasion , 83. In egulated . Moreovement C3) . Similarment C3) , 87. ThiOverall, the EV content reflects the characteristics of each cell line. Additionally, the cell line specific EV protein combinations point to the cellular phenotypical characteristics described as Hallmarks of cancer. Furthermore, the proteins identified have been detected in clinical cancer tissues in other studies, indicating that these may be relevant as diagnostic biomarkers for these cancers.Functional enrichment analysis was performed using DAVID annotation clustering. Here, we identified the most enriched biological processes regulated by the hundreds of proteins identified in the EVs from each cell line . We founThe EVs derived from the three cancer cell lines also had some biological processes in common. “Interspecies interaction between organisms”, related to viral reproduction is one example. This process reflects the similarity of EVs and viruses even though EV biogenesis is a naturally occurring biological process while viruses hijack the cellular machinery for its propagation . In addiTogether, these findings reveal that the EVs analyzed in our study contain proteins that can regulate biological processes towards a cancer phenotype, in a manner that is characteristic for each of the cell lines.In conclusion, in the present study we used LC-MS to characterize the protein content of small EVs derived from OSCC, PDAC, and melanoma brain metastasis cell lines. More than 600 proteins were identified in the EVs of each cell line. Analysis of the datasets revealed cell-specific protein panels. In short, EVs derived from E10 had a high expression of EGFR, ITGB4, and NT5E, the EVs isolated from BxPC3 were enriched in mucins , and the most abundant proteins in the EVs from the H3 cell line included HMCN1, HSP8A, and APOE. Furthermore, screening of the protein datasets for proteins involved in the Hallmarks of cancer also exposed cell-specific protein panels. Here, proteins associated with the promotion of processes such as angiogenesis, proliferation, migration, or adhesion were found to be present in the EVs derived from all three cell lines. This is relevant due to the role of EVs in the modulation of the cells in the tumor microenvironment  into a more permissive setting for tumor progression. The list of proteins involved, for example, in cell proliferation was quite extensive in E10 and BxPC3 in comparison to H3 where only half of the proteins were found and most of them poorly expressed. Functional enrichment analysis by gene ontology (GO) was carried out to extract a broader biological significance. Here, processes such as “biological adhesion”, “cell motility”, and “cellular component biogenesis”, all associated with aspects of cancer, were found. However, the processes identified from the proteins from the H3 derived EVs were distinct from the ones in the datasets from E10 and BxPC3, revealing an underlying difference between the cell lines. This study focuses on the protein content of small EVs, and does not explore other classes of biomolecules such as RNAs. Nevertheless, the protein lists generated and analyzed here may provide important information regarding the complex biomolecular cues EVs carry. In a clinical setting, biofluids from cancer patients will include a higher number of cancer-derived EVs in addition to non-cancer-related EVs. This will complicate the collection and examination of spacific caner-derived EVs. Hence, we believe it is important to start with a broad analytical approach that may assist in pinpointing the cancer-derived EVs in biofluids.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Transforming growth factor-beta (TGFβ1) is considered as a master regulator for many intracellular signaling pathways, including proliferation, differentiation and death, both in health and disease. It further represents an oncogenic factor in advanced tumors allowing cancer cells to be more invasive and prone to move into the metastatic process. This finding has received great attention for discovering new therapeutic molecules against the TGFβ1 pathway. Among many TGFβ1 inhibitors, peptides (P17 and P144) were designed to block the TGFβ1 pathway. However, their therapeutic applications have limited use, due to lack of selection for their targets and their possible recognition by the immune system and further due to their potential cytotoxicity on healthy cells. Besides that, P144 is a highly hydrophobic molecule with less dissolution even in organic solution. Here, we aimed to overcome the dissolution of P144, as well as design nano-delivery strategies to protect normal cells, to increase cellular penetration and to raise the targeted therapy of both P17 and P144. Peptides were encapsulated in moieties of polymer hybrid protein. Their assembly was investigated by TEM, microplate spectrum analysis and fluorescence microscopy. SMAD phosphorylation was analyzed by Western blot as a hallmark of their biological efficiency. The results showed that the encapsulation of P17 and P144 might improve their potential therapeutic applications.  Transforming growth factor (TGFβ) is a secreted cytokine, having the ability to regulate and control cell proliferation, migration, differentiation and cytoskeleton morphology ,2,3. ApaAdditionally, the surface of the protein–peptide complex was further functionalized by folic-acid-attached carboxymethyl cellulose CMC; . Folic aThe suppliers of the chemicals were as follows. Carboxy methylcellulose (CMC) was purchased from Fluka, Sigma-Aldrich , phosphate-buffered saline (PBS) tablets of pH 7.3 were purchased from Oxoid Limited Basingstoke ; ethanol from Baker Analyzed, Fisher Scientific, ; bovine serum albumin (BSA), folic acid (FA), crystal violet, 40,6-diamidino-2-phenylindole dihydrochloride (DAPI), paraformaldehyde, N-ethyl-N0- (3 dimethylaminopropyl)carbodiimide hydrochloride (EDAC), N-hydroxy succinimide (NHS), dimethyl sulfoxide (DMSO) from Sigma-Aldrich .A 1 mg amount of sonicated P144 and suspended P17 was mixed into BSA solution (50 µg/50 mL). Then, the mixture will be grafted by carboxymethyl cellulose inside sterilized glass vials for 30 min under rotation by a magnetic stirrer in the presence of EDAC/NHS. The complex was further coated by 5 mL of 50 µg/50 mL protamine and the stirrer was continued into addition for 10 min. Afterward, the mixture was furthermore covered by folic-acid-attached carboxymethyl cellulose and the rotation was completed in an additional 15 min. Un-reacted materials were removed by using a dialysis bag against distilled water for 48 h. The water was changed several times. Then, the final product was lyophilized at a freeze-drying machine while the material was kept at −20 °C until use.A 10 µL volume of nanoparticle suspension was deposited on the copper grid and air-dried before measurement. Copper grids sputtered with carbon films were used to support the sample. High-resolution TEM images of nanoparticles were analyzed by a Hitachi HT 7700 operating  at 100 kV, coupled with a GATAN camera ORIUS SC600 ) with a resolution of 7 megapixels. The GATAN camera is controlled by Digital Micrograph  The absorbance of folic acid and rhodamine was measured by using a multiplate reader at the option of UV Absorbance Spectrophotometer in multiplate wells. A 500 µL mL volume of fabricated nanoparticles was scanned at a range of 300–600 nm .The electrophoretic mobility of samples was determined by photon correlation spectroscopy by using a Zeta Nano Sizer . All measurements were performed at 25 °C. Five following measurements were taken for analysis; each was run five times with a delay of 5 s after each measurement ..10]. ThiTo investigate their properties with fluorophore conjugation and to follow their cellular uptake, the anti-TGFβ inhibitory peptides (P17 and P144) were labeled by rhodamine RG6; G 31]. R. R31]. RBoth peptides (P17 and 144) were suspended in PBS at pH 7.2 and then P144 was sonicated by ultrasonicator. The processing time was adjusted for 15 min under amplitude five (intensity). The processing time is further divided into three steps each one has taken 5 min. Under magnetic stirrer, the sonicated P144 and suspended P17 were then integrated into BSA moieties in the presence of carbodiimide and n-hydroxy succinimide . The reaThe rhodamine-labeled non-encapsulated peptides were accumulated successfully inside the cytoplasm as shown by fluorescent emission of TRITC channel (red spots). On the other hand, the green color was shown by using the FITC channel, indicating that peptides are able to emit green intensity after their doping with R6G G,H. SimiThe results indicate that there is a good distribution of adsorbed peptides inside cytoplasm after their encapsulation compared to those used with no encapsulation. Similarly, free capsules were localized inside cytoplasm after their cellular adsorption . On the DAPI  is a specific fluorophore for a nucleic acid because it is able to bind preferentially to adenine thymine base pair and also to phosphate groups of DNA ,38,39,40On the contrary, the Hep3B and SNU449 exposed to non-encapsulated peptides exhibited no more changes in their morphology. However, a slightly condensed nucleation was shown .Crystal violet cell proliferation assay is a colorimetric method based on the use of crystal violet as a basic dye with avidity to nuclear structures. After binding and solubilization of the crystal violet, optical density measurements of extracted dye provide a measure of the relative number of viable cells. This test has the advantage that it is less time consuming, easier to perform and more objective .Encapsulated peptides (P17 and P144) have an antiproliferative influence on human liver cancer cell lines (SNU449 and Hep3B) as shown by proliferation assay. Both encapsulated peptides (P17 and P144) inhibited the proliferation of human liver cancer cell lines in vitro in a dose-dependent manner. This inhibition was substantial in both cell lines after treatment, with significant differences from 10 μg/mL to 200 μg/mL in treated cells . SimilarSMADs are a group of encoded homologs mammalian protein identified by Small (Sma) and Mothers against dpp (Mad) genes that act as intracellular transcriptional factors . SMAD2 aTGFβ1 inhibitory peptides have attracted much interest recently because of their ability to block the TGFβ1 signaling pathway , due to The protein–peptide assembly was further functionalized by protamine sulfate. This provides a novel strategy in terms of α helix titration resulting in turbidity . ArgininApart from cellular adsorption, the DAPI staining noticeably revealed nuclear morphological changes in both SNU449 and Hep3B human liver cancer cell lines in terms of nuclear fragmentation, nuclear hypotrophy, nuclear condensation and cell structure loss. Accordingly, the present results proved significantly the potential efficient therapy of both encapsulated peptides against the analyzed cell lines. Similar to our results, P144 and P17 treatment resulted in significant inhibition at lung cancer cell lines  respectiIn conclusion, the encapsulation of TGFβ inhibitory peptides (P17 and P144) has shown potential therapeutic inhibition to basal phospho-SMAD as a result of TGF-β pathway regulation. It has proved clearly their ability to inhibit the autocrine TGFβ pathway and basal nuclear SMAD. However, SNU449 is one of the cell lines that is more responsive to those encapsulated by P17 and P144 is a mesenchymal, invasive HCC cell line that produces TGFβ autocrinally and is unresponsive to its suppressor effects. Additionally, rhodamine-labeled peptides enable researchers to follow nearly the localization of peptides inside cytoplasm after their cellular uptake. In the current studies and our previous studies indicated that encapsulation of TGFβ signaling pathway inhibitors may facilitate their delivery and overcome their cytotoxicity. Additionally, folic acid functionalized drug delivery systems enable them to reach and accumulate inside cancer cells ,60,61,62


Nano-scale zero-valent iron (nZVI) is among the most common nanoparticles widely used for the treatment of various environmental contaminants. However, little is known about the combined effects of nano-zero-valent iron (nZVI) and other soil amendments on soil remediation and plant performance. For the first time, we studied the remediation of Cr(VI)-contaminated soil using bare nZVI (B-nZVI) and starch-supported nZVI (S-nZVI) in combination with either biochar (BC) or humic acid (HA), and the consequent effects on plant growth and Cr accumulation. Both S-nZVI and B-nZVI decreased the contents of Cr(VI) and available Cr in soil, but increased available Fe content, with S-nZVI generally showing more pronounced effects at a higher dose (1000 mg/kg). B-nZVI exerted no inhibition and even stimulation on plant growth, but 1000 mg/kg S-nZVI produced significant phytotoxicity, resulting in decreased plant growth, low chlorophyll content in leaves, and excessive accumulation of Fe in roots. Each nZVI decreased shoot and root Cr concentrations. BC and HA produced synergistic effects with nZVI on Cr(VI) removal from soil, but HA decreased soil pH and increased the availability of Cr and Fe, implying a potential environmental risk. Addition of BC or HA did not alter the effects of either nZVI on plant growth. In conclusion, combined application of 100 mg/kg nZVI and BC could be an ideal strategy for the remediation of soil contaminated with Cr(VI), whereas high-dose S-nZVI and HA are not recommended in the remediation of agricultural soils for crop production or in the phytostabilization of Cr(VI). Chromium (Cr) can enter the environment via a variety of human activities, including mining, smelting, and electroplating, leading to potential contamination to soil, surface water, or groundwater ,2,3. AmoRecently, nanoremediation based on nano zero-valent iron (nZVI) has attracted increasing attention due to its environmental friendliness, high efficiency, and wide applications . nZVI haBiochar (BC) and humic acid (HA) are both among amendments that are widely used in soil remediation. As a type of carbon-rich material with rich porous structures, various functional groups, a high pH and a high cation exchange capacity, BC generally possesses a high adsorption capacity that potentially contributes to the immobilization of heavy metals ,27, inclConsidering the above context, combined use of nZVI with soil amendments such as BC and HA may produce better remediation effects for Cr(VI)-contaminated soil. We hypothesize that combined application of BC or HA may enhance nZVI’s performance in Cr(VI) remediation, and decrease the possible phytotoxicity induced by nZVI. Here, using bare-nZVI and starch-stabilized nZVI as targets, we conducted a pot culture experiment to assess the effects of nZVI in combination with BC or HA on Cr(VI) removal from soil and the growth and accumulation of Cr in mung bean. The results may provide evidence for combined applications of nZVI with other amendments and phytoremediation approaches. Cr-free and Cr-contaminated soils were selected for this study. The Cr-free soil was collected from a local farmland in Qingdao, China , and the Cr-contaminated soil was sampled from the Cr-containing slag heap site of Qingdao Red Star Chemical Plant . The detailed characteristics of the two kinds of soil samples were described in our previous studies ,36. The 2/g  was purchased from Beijing Deke Daojin Science and Technology Co., Ltd. , with following properties: purity 99.9%, average particle size 30–50 nm and specific surface area 40–60 m2/g, C 86.15%, H 3.17%, N 0.29%, S 0.51%, O 7.36%, Cr 32.45 ng/g, zeta potential −26 mV and average particle size 9469.6 nm. SEM images and FTIR spectra of the BC are shown in Pine shoots-derived biochar (BC) was obtained from Qingdao Biochar Environmental Biological Engineering Co., Ltd., Qingdao, China, with the following properties: pH 9.64, average specific surface area 139.4 mHumic acid (HA) was obtained from Tianjin Guangfu Fine Chemical Research Institute, Tianjin, China. The purity was >99% and the pH was 5.92. The HA contained ≥90% fulvic acid, 10% ash, 8% water, 0.3% Fe and <1% insoluble matter. w/w)). Each treatment had four replicates. An appropriate amount of nZVI was mixed thoroughly into soil to achieve the target doses. Next, 1% (w/w) of BC or HA was mixed into the soil samples. Seeds of mung bean (Vigna radiata) from a local market in Shangqiu in Henan Province, China were used for the pot culture experiment. The experiment was conducted according to the procedure described previously [We designed a trifactorial experiment, including (1) two types of nZVI ; (2) three nZVI doses ; and (3) two doses of BC or HA  of the shoots and roots were measured respectively after cleaning. The dry weights (DWs) were determined after oven-drying the fresh materials at 70 °C for 24 h. On the date of harvesting, fresh leaves were sampled for measurement of chlorophyll content. The contents of chlorophyll a and b were determined using an ultraviolet spectrophotometer  after extracting the pigments from the leaves in a 95% ethanol solution . Total c3. Cr concentration in the solution was estimated with an inductively coupled plasma mass spectrometer . Fe concentration in the solution was determined with an atomic absorption spectrophotometer .The dried shoot (0.5 g) and root (0.2 g) samples were ground and then digested using HNOTo observe the phytotoxic symptoms and iron element distribution at the cell level, the microscopic structures were observed according to the detailed procedure described in . Briefly2, pH 7.3) [w/v) was determined using a pH meter .The post-harvest soil from each pot was mixed thoroughly and 50-g soil samples were taken for analysis of soil properties. Available Fe and Cr concentrations were determined using FAAS after extracting using DTPA solution  . The conp < 0.05. All figures were generated using OriginPro9 , and the results are presented as means ± standard deviation (SD). One-way analysis of variance and multi-way analysis of variance were conducted with a 5% probability level using SPSS 23.0 . Significance among treatments was compared using a Duncan’s multiple range test at As shown in Three-way ANOVA results revealed that nZVI type, dosage, use of BC or HA, and type of nZVI interaction (nZVI-BC or nZVI-HA) all had significant impacts on soil Cr(VI) concentrations, with better effects of S-nZVI than B-nZVI, as well as synergistic interactions between nZVI and both BC and HA . In comparison with the control, B-nZVI and S-nZVI both significantly decreased DTPA-extractable Cr concentrations in soil, but there were no significant differences between two nZVI types and two doses a. AdditiF = 1237.1) mostly influenced DTPA-extractable Fe, and significantly interacted with nZVI type and dosage  significantly increased DTPA-extractable Fe concentrations in soil, particularly at higher doses, but S-nZVI always had a stronger promoting effect than B-nZVI . Addition of BC alone decreased DTPA-extractable Fe concentrations in soil, but HA increased them. Compared to nZVI alone, combined treatments with HA or BC had higher DTPA-extractable Fe concentrations in soil, with HA producing more promoting effects than BC. Three-way ANOVA results showed that HA alone  in the soil. More interestingly, both BC and HA were effective in the removal of Cr(VI) from soil, both alone or in comibation with nZVI . However2+, Zn2+  and Cr-contaminated soil assisted by BC or HA and the consequent phytotoxicity. Overall, both S-nZVI and B-nZVI were effective in Cr(VI) removal from soil, although the effects were more pronounced for S-nZVI at a higher dose. Combined applications with BC or HA further enhanced the remediation effects of both nZVI, but HA increased the availability of Fe and Cr in soil. Both types of nZVI decreased Cr accumulation in mung bean seedlings, but only S-nZVI displayed “high-dose inhibition effects” on plant growth, suggesting that B-nZVI is more environmentally friendly for plants than S-nZVI when applied at a high dose. Taking plant performance, Cr(VI) removal and Fe release altogether, 100 mg/kg of B-nZVI or S-nZVI in combination with BC is determined to be an ideal remediation strategy. Given that high-dose S-nZVI can cause phytotoxicity, and HA promotes the increased release of available Fe and Cr, hence S-nZVI and HA are not recommended in the remediation of agricultural soils for crop production and phytostabilization of Cr(VI).


Natural products are popular insights for researchers to investigate promising anti-cancer agents since some of these substances have lesser adverse effects restricting the treatment than traditional chemotherapeutic agents. A well-known monoterpene Carvacrol, widely consumed in Mediterranean cuisine and lower risks of cancer, has efficient anticancer effects. However, the mechanism of action is yet to be discovered.  The investigation aims to illuminate a new perceptive in the role of this substance on colorectal cancer treatment, by the means of differences in a well-defined range of soluble factors. Carvacrol effect on both HT-29 and HCT-116 cell lines was evaluated on proliferation and the IC50 values were calculated by the RTCA xCELLigence device. Then MAGPIX assay was performed to obtain the changes in soluble factors of the cell lines.  The Multiplexing assay suggests some of these factors were altered in favor of surviving and proliferation in aggressive cell line HCT-116 whereas they were altered against these characters in HT-29, were correlated with the increased IC50 concentration of HCT-116 in carvacrol treatment.  The current study indicates that differences in the levels of these soluble factors could modulate the anticancer effect related to carvacrol. Colon cancer has become a major public health issue with 1,849,518 new cases in 2018. 10.2% of all cancer deaths in the same year were caused by this malign disease which brings it to the second rank in cancer related deaths [1]. Several factors  affect the development of this malignancy [2]. Nowadays, the most accepted therapies are surgery, radiotherapy, and chemotherapy such as 5-fluorouracil and oxaliplatin . However, all these procedures have their own difficulties, the adverse effects related to systemic chemotherapy are much restrictive due to their mortality. Since some of the natural products as chemotherapeutic agents indicate lower side effects on patients, they are promising anticancer agents and safer sources of cancer treatments. Moreover, such phytochemicals are also related with chemopreventive agents because of their eﬀiciency and low toxicity. Therefore, the scientists focus on evaluating phytochemicals with multitargeted potentials for eﬀective chemoprevention [5].Terpenes (C10H16) illustrated enormous effectiveness as chemopreventive agent in the last years however the mechanism of action is not clear yet . They involve roughly 25,000 chemical structures which are used in pharmaceutical industries [6]. Carvacrol is also a member of this family. The substance presents in various sources that are extensively tested with enormous biological activities [7]. Mediterraneans regularly consume this product with oregano spice that presents a lesser risk of colorectal cancer compared to the other local cuisines [8]. Nowadays, it is well-known that the carvacrol in this spice is the one reason that the substance has preventive properties. It basically protects mammalian cells against DNA strand breaks along with rosmarinic acid . Moreover, several studies point out that carvacrol illustrates further anticancer effects such as genotoxic, cytotoxic, and proapoptotic activity to cancer cells, in a dose-dependent manner. Additionally, it has also an important preventive role in cell invasion by down regulating of matrix metalloprotease 2 and 9 expressions [11].Tumor microenvironments involve various cell types and extracellular matrix substances in the niche of cancer such as soluble factors. The soluble factors which could be related to cell proliferation are granulocyte-macrophage colony-stimulating factor (GM-CSF), leptin, prolactin and soluble vascular endothelial growth factor receptor-2 (sVEGFR2) evaluated [12]. Prolactin may involve in the genetic and molecular mechanisms for regulating the proliferation and growth of neoplastic and normal epithelial cells in the gastrointestinal tract [13]. Moreover, leptin is crucial for colorectal cancer (CRC) growth in obese patients [14]. On the other hand, sVEGFR2 involves the extracellular domains of the receptor but lacks the tyrosine kinase domain [15]. GM-CSF has an important role in hematopoiesis and immune modulation. GM-CSF also stimulates the progression of immune-independent tumors by inducing tumor growth, metastasis, and promoting tumor microenvironments. Hence several studies suggest that this factor triggers some effects on tumor progression [16].Since these soluble factors manage the tumor microenvironment and the behavior of cancer as mentioned previously, the present study investigates the changes in these soluble factors when HT-29 and HCT-116 colorectal adenocarcinoma cell lines are treated with carvacrol in a dose-dependent manner. We suggest that the results could lead us one step further to discover the mechanism of action of carvacrol through the soluble factors.Human colorectal carcinoma, HCT-116 (ATCC CCL-247) and HT-29 (ATCC HTB-38) cell lines were purchased from American Type Culture Collection (ATCC) . They were cultured in McCoy’s-5a modified medium  supplemented with 1% penicillin/streptomycin  and 10% fetal bovine serum . Cells were incubated at 37 °C in a humidified 5% CO2 incubator.The antiproliferative effect of carvacrol  has been studied on HCT-116 and HT-29 colorectal adenocarcinoma cell lines. The cell lines were seeded in a triplicate in 96-well plate . Cell viability was tested after 48 h of incubation with 25–200 µM carvacrol via xCELLigence real-time cell analysis instrument , San Diego, California, USA).Colorectal adenocarcinoma cell lines, HCT-116, and HT-29 were treated with carvacrol at IC50 values individually. Both carvacrol treated and nontreated control groups were seeded into wells and run in triplicate. The fluorescent signal was measured by a CCD imager and the concentrations of the analytes were determined by MAGPIX xPONENT software. The results were normalized to total protein concentration estimated with Bradford assay.SPSS version 24.0  was used for all analyses. Willcoxon signed ranks nonparametric test was used to determine the significance between the application of different doses of carvacrol on different cell lines individually and its control group. The data in the Figure are represented as the mean ± standard error of the mean. In the study, all tests were performed in triplicate. The level of statistical significance was set at P < 0.05 .The current investigation draws attention to the carvacrol effect in proliferation and also points out its mechanism of action through the alterations in the level of soluble factors in colorectal cancer cell lines with different phenotypes. The methodology indicates the IC50 values for carvacrol and follows through the MAGPIX multiplexing assay to illuminate the changes in soluble factors in response to IC50 values of carvacrol on HCT-116 and HT-29 colorectal cancer cell lines.The in vitro proliferation activity doses of carvacrol on HCT-116 and HT-29 cell lines were measured by using the xCELLigence real-time cell analysis instrument. After 48 h of incubation with carvacrol, IC50 values were found as 92 µM for HCT-116 and 42 µM for HT-29 cell lines. The results are illustrated in Figure.The soluble factors were analyzed to evaluate the cellular response leading to cytotoxicity in both cell lines and the difference between the response intensity to carvacrol. The measurements in samples were achieved by MAGPIX multiplexing assay. The Table which shows the normalized data illuminates that the secretion of several soluble factors related to growth and survival such as prolactin, leptin, and GM-CSF were increased in carvacrol treated HCT-116 while they were decreased in carvacrol treated HT-29 comparing to nontreated control groups. Prolactin, leptin, and GM-CSF concentrations were measured as 57.5, 9, and 82 pg/mL in HCT-116 and, 147, 17.74, and 104 pg/mL in HT-29 with no carvacrol concentration. These values raised in concentration to 117, 23, 105 pg/mL , in response to 92 µM and the IC50 value of carvacrol, respectively. However, they decreased to 53.49, 14.49, and 96 pg/mL concentrations  compared to the control concentrations in HT-29 treated with 42 µM of the phyto-compound. However, proliferative and survival factor TGF-α decreased in both cell lines, correlated with earlier proliferation assay. This factor was estimated as 67 pg/mL for HCT-116 and 106.2 pg/mL for HT-29. Both values diminished where the concentration for HCT-116 treated with 92 µM substance was 24 pg/mL (P = 0.028) and 26 pg/mL (P = 0.109) for HT-29 treated with 42 µM of carvacrol.The present study focused on the effect of carvacrol in proliferation and also illuminated its mechanism of action through the change in the level of soluble factors in colorectal cancer cell lines with different phenotypes. Both of these cell lines are treated with different doses of carvacrol which are 25 µM, 50 µM, 100 µM, 200 µM in xCELLigence real-time cell analysis instrument which measures the proliferation once per 15 min. The results signify that the proliferation levels of both cell lines decreased while the concentration level of carvacrol increased. A previous study points out that a carvacrol rich essential oil of origanumacutidens has also significant anti-proliferative effect against the HT-29 cell line. The most active concentration of this oil has been found as 100 μg/mL [17]. Moreover, similar to our results, Kai Fana et al. have studied the antiproliferative effect of carvacrol on HCT-116 and they detected the IC50 dose as 544.4 µM which is an even higher concentration comparing to our result [11]. Even though carvacrol has a significant antiproliferative effect on both of these cell lines in the dose-manner, our results indicate that it is more efficient against HT-29 since IC50 value of HCT-116 doubles the other line.The data of proliferation assay on the carvacrol effect point out that the phyto-compound has a significant role in both HCT-116 and HT-29 cell lines. However, the intensity of the cytotoxic responses is different since a more concentrated dose is required in HCT-116. Considering this result, the cellular response was evaluated by the means of secreted soluble factors via multiplexing assay (Table). The normalized data illuminate that the secretion of several soluble factors such as prolactin, leptin, and GM-CSF were increased in carvacrol treated HCT-116 while they were decreased in carvacrol treated HT-29 comparing to nontreated control groups. Considering this mitogenic effect, survival character was more abundant in HCT-116 whereas it reduced in HT-29 in response to the phyto-compound. Therefore, HCT-116 could be evaluated more resistant and aggressive in carvacrol treatment. Nevertheless, a significant decrease in proliferative and survival protein TGF-α and the increase in antigrowth factors VEGFR2 were correlated with the total cytotoxic effect in the final response.The literature mostly confirms the results which we have obtained in the current study. The sVEGFR2 fragments, transfected to tumor cells, are secreted to the extracellular matrix and inhibit angiogenesis in vivo [18]. Another soluble factor increased in HCT-116, prolactin is actively involved in tumorigenesis in several cancers. The current research also points out that the prolactin level of HT-29 is dramatically lower compared to the control group. On the other hand, the level of this factor is enormously enhanced in the HCT-116 cell line. Moreover, the scientists investigated in improvement cures to control tumor growth via reducing the prolactin production [19]. The data introduced in the current study illuminates the levels of prolactin increased in HCT-116 as a result of favoring survival and proliferation, whereas it was decreased in HT-29. Therefore this growth-favored factor indicates that it is possible to enhance carvacrol treatment. In addition, the expression level of leptin was measured in lower levels in the HT-29 cell line compared to its control group. However, HCT-116 has an elevated level of this soluble factor. Since, previous reports suggest that leptin is overexpressed in various types of cancer cells and plays a role in the development and progression of a variety of malignancies including colon cancer. Our current study also reveals another reason why HCT-116 cell line was more resilient than HT-29 against this compound . GM-CSF was another growth factor that decreased in HT-29 whereas it increased in HCT-116. In addition to the earlier studies indicating that neutralizing GM-CSF reduced the proliferation, angiogenesis, and colonic epithelial cells (CECs) in neoplasia, our data point out that this factor is also an accessory after the fact along with leptin and prolactin [22].In conclusion, different colorectal cancer cell lines respond in distinct ways to this phytochemical compound. On one hand, the antiproliferative feature of carvacrol has been shown in both cell lines, on the other hand the HCT-116 response is more resistant to carvacrol treatment compared to HT-29 cell line. On the basis of present data, our results revealed that the alteration in levels of soluble factors in the HCT-116 cell line enhances the ability to proliferate and survive. Even though some of these factors slightly favored to tumor growth for HCT-116, carvacrol affects both cell lines as an antiproliferative agent. Therefore this study also suggests that some soluble factors could be drug targets to enhance the effectiveness of carvacrol. We sincerely thank Gizem Çalıbaşı Koçal, Tuğba Uysal, Mahdi Akbarpour, and Ece Çakıroğlu for their contribution in laboratory work.


Head and neck squamous cell carcinomas (HNSCC) resulting from human papillomavirus (HPV) are increasing in incidence but demonstrate significantly better treatment response than HNSCC from other causes such as tobacco and alcohol. This study sought to identify differences in HNSCC, intrinsic to HPV status, in their response to radiation dose. Previously unexamined changes in radio-responsiveness following fractionated X-ray irradiation were compared between HPV positive and negative statuses of HNSCC. Six HNSCC cell lines, 3 of each HPV status, were investigated for radiosensitivity by clonogenic assay and modelled by response as a function of dose. Generational cultures of each cell line were developed to follow changes in radiosensitivity after repeated irradiations simulating fractionated radiation therapy. As a group, the HPV positive cell lines were more radiosensitive, but with changes following repeated fractions of dose, and modelling of response as a function of dose, both statuses displayed large radiobiological heterogeneity. These findings challenge current radiobiological assumptions of head and neck cancers as early responding tissue to radiation and may go some way in explaining difficulties reaching consensus in stratification of treatment by HPV status. Consequently, results from this study do not support stratifying radiation therapy by HPV status. Head and neck cancers are heterogeneous, occurring from the oral and nasal cavities to the larynx and pharyngeal areas where about 95% are squamous cell carcinomas (HNSCC) of the mucosal epithelium . Importan is the number of fractions and d is dose per fraction.The use of clonogenic assays to determine radiosensitivity remains a gold standard in radiation biology. It determines cellular radiation sensitivity by surviving fractions (SF) of clonogens in cell cultures as a function of radiation dose delivered ,14. This (>2 Gy) . The LQM (>2 Gy) ; i.e., t (>2 Gy) ,18. This (>2 Gy) :(2)BED=nSome radiobiological studies of HNSCC report HPV positive cancers to be more radiosensitive than HPV negative cancers which is consistent with clinical observations of better responses to radiotherapy ,21,22. HAs such, the aim of this study was to go beyond the single measure of radiosensitivity and compare changes in intrinsic HNSCC cellular radio-responsiveness according to HPV status, following repeated fractional radiation doses, thus simulating the clinical scenario in radiation therapy. To facilitate this, “generational cell cultures” were developed from HNSCC cell lines in the current work to investigate post-irradiation changes in radio-responsiveness.This is the first-time HNSCC cell lines were investigated to compare treatment response following fractionated irradiation, enabling comparison of the radiobiology intrinsic to HPV status. Evaluations of response to radiation dose are made by the SF of cell population and by α/β ratios for each cell line. Data on changing radio-responsiveness following multiple dose fractions presents a further dimension to the radiobiology of these cell lines. This study illustrates the complexity of using HPV status for predicting response to irradiation.® CRL-3241TM) and UPCI-SCC-090 (ATCC® CRL-3239TM) were sourced from ATCC . To reflect characteristic mutational status of the HPV groups, the chosen HPV negative cell lines have TP53 mutations and HPV positive retain wild type expression. Cell lines were grown in Dulbecco’s Modified Eagle’s Medium with 4500 mg/L glucose  10% fetal bovine serum (FBS), 10 mM HEPES, 100 U/mL penicillin and 0.1 mg/mL streptomycin . Cell cultures were grown in T25 flasks  and incubated at 37 °C in humidified atmosphere with 5% CO2.Three cell lines representing each HPV status were selected. UM-SCC-47, UPCI-SCC-154, and UPCI-SCC-090 are HPV positive. UM-SCC-17a, UM-SCC-22a and UM-SCC-1 are HPV negative . UM-SCC-3). For clonogenic assays, one flask of each cell culture was irradiated to an absorbed radiation dose of 0.5, 1.0, 2.0, 4.0 and 6.0 Gy, respectively. A 6th flask was sham irradiated as control. An additional flask was irradiated to 4 Gy for growing the next generation of cells to simulate fractionated radiation therapy (see below).Cells were irradiated as a monolayer in T25 flasks using an RS2000 X-ray cabinet irradiator  using a 160 kV, 25 mA X-ray beam with 9.1 mm Al half value layer . The beahttps://imagej.nih.gov/ij/).Following irradiation, cells were trypsinized and counted prior to plating in 6 well plates  previously coated with extracellular matrix . One plate (6 repeat assays) for each dose point was used, with 1.5 mL of a 1:1 mix of culture media and conditioned media (filtered media from 1st generation cell cultures) per well, then incubated 10 to 14 days. Resulting colonies were fixed with 1:5 acetic acid/methanol solution before staining with 0.5% crystal violet. Clonogens were identified microscopically by colonies >50 cells and counted using ImageJ software v.1.51j   were re-cultured to grow the next  generation of that cell line for subsequent irradiation and clonogenic assay . Four ge® v8.2.1 . SF as a function of absorbed dose, was determined from colony numbers (6 repeats per irradiation dose) by calculating the plating efficiency (PE) of controls and normalizing colony numbers as a function of dose to PE.Data from colony counting was analyzed in Microsoft Excel and PrismComparing SF for each dose between HPV positive and negative cell line groups demonstrated significantly greater survival at each dose for the HPV negative cell line group in the first 2 generations and in the absorbed dose range of 0.5–2.0 Gy for the 3rd and 4th generations. The most significant differences in radiosensitivity among cell line groups defined by HPV status was at 1.0 and 2.0 Gy absorbed dose. Survival of HPV negative cells for 1.0 Gy was approximately 2-fold greater than HPV positive cells and 4-fold at 2 Gy absorbed dose. There was little overall change in SF differences according to HPV status over the 4 generations .Comparison of the dose to 50% lethality (LD50) by HPV status, also demonstrated greater radiosensitivity in the HPV positive cell lines. Although not statistically significant, the difference in sensitivity was evident for all generations of cell lines shown in Only the HPV negative HNSCC cells as a group, displayed an increased SF for dose where the 2nd generation increased SF over the 1st generation by 18.5%, 16.5% and 58.5% for 1.0, 2.0 and 4.0 Gy absorbed doses respectively. Both HPV groups then demonstrated diminishing SF after the second generation . Cell li2 of α/β ratios please see LQ modelling of parameters α, β and resulting ratios, exhibited highly varied values. The α and β ranges were similar irrespective of HPV status . For con−1 for UPCI-SCC-090 2nd generation, to 1.80 Gy−1 for UPCI-SCC-154 4th generation  as a function of dose. With increasing dose, multiple hit cell killing accelerates as a quadratic function, described by the parameter β (Gy−2). The significance of these parameters, and in particular their ratio α/β, lies in the prediction of response to radiation and optimization of fractionation schedules for a total dose. The α/β is the most useful radiobiological parameter in radiotherapy [2I statistic, where an 2I measure >75% is considered highly heterogeneous. The I2 for head and neck cancers was 87%, consistent with the diverse results we have observed in vitro. Both the tumor site and histology are of major influence on the cancer’s α/β and given the variable nature of these aspects in HNSCC, the observed heterogeneity may not be surprising [Radiosensitivity, as determined by SF as a function of absorbed dose, generally increased in all cell lines with subsequent generations. Two points of exception however, were the 2nd generations of UM-SCC-17a and UM-SCC-22a (both HPV negative cell lines) which showed increased radioresistance following 4 Gy irradiation, before the 3rd and 4th generations became more radiosensitive. Determining the intrinsic radiosensitivity of tumor cells is elemental to modelling for prediction of response and dose optimization. Cancers may display change in response to therapy during the course of treatment ,34,35,36otherapy . This isrprising . Additiorprising  looking rprising . LimitatAll cell lines displayed increasing radiosensitivity with repeated 4 Gy radiation dose fractions except the 2nd generations of UM-SCC-17a and UM-SCC-22a which showed a transient increase in radioresistance.Comparison of SF as a function of dose between cells of the 2 HPV groups found that HPV positive cells were significantly more radiosensitive across all generations.Broad heterogeneity was found for the α and β parameters from LQ modelling across all 6 cell lines without any distinct association found according to HPV status.The inherent α/β for each of the 6 cell lines, and the changes in this ratio with 4 Gy radiation dose fractions, did not describe any fractional sensitivity among HNSCC cell lines according to HPV status.Although the study found, similar to previous reports, that HPV positive cell lines as a group are more radiosensitive, we also observed broad heterogeneity in radiosensitivity irrespective of HPV group, illustrating the difficulty in stratifying patients for radiotherapy by HPV status. Specific findings from the study are:These observations do not support stratifying radiation therapy by HPV status. Moreover, these findings suggest that factors other than intrinsic radiosensitivity contribute more substantially to the observed differences in treatment responsiveness between HNSCC patients based on HPV status. Future work to measure radiosensitivity in progressive response to fractionation would require development of in vivo studies to assess the contribution of exogenous factors such as hypoxia, microenvironment, and immunology on response to radiation.


This document contains all abstracts of the 20th European Conference on Eye Movements, August 18-22, 2019, in Alicante, SpainVideo stream "Glimpses at 20th ECEM":https://vimeo.com/user43478756