In 786-O/S cells, the high level of phosphorylation of MET and AXL was not affected by cabozantinib. Despite cabozantinib hampering the high constitutive phosphorylation of MET, the Caki-2 cells showed low sensitivity to cabozantinib, and this was independent of sunitinib pretreatment. In both sunitinib-resistant cell lines, cabozantinib increased Src-FAK activation and impeded mTOR expression. The modulation of ERK and AKT was cell-line-specific, mirroring the heterogeneity among the patients. Overall, the MET- and AXL-driven status did not affect cell responsiveness to cabozantinib in the second-line treatment. The activation of Src-FAK might counteract cabozantinib activity and contribute to tumor survival and may be considered an early indicator of therapy response.The tyrosine kinase inhibitor (TKI) cabozantinib might impede the growth of the sunitinib-resistant cell lines by targeting MET and AXL overexpression in metastatic renal cell carcinoma (mRCC). We studied the role of MET and AXL in the response to cabozantinib, particularly following long-term administration with sunitinib. Two sunitinib-resistant cell lines, 786-O/S and Caki-2/S, and the matching 786-O/WT and Caki-2/WT cells were exposed to cabozantinib. The drug response was cell-line-specific. The 786-O/S cells were less growth-inhibited by cabozantinib than 786-O/WT cells ( Renal cell carcinoma (RCC) is the seventh most common form of neoplasm in developed countries, accounting for approximately 3% of all diagnosed cancers worldwide ,2, with VHL) gene; chromosome remodeling by PBRM1, BAP1 and SETD2 genes; hyperactivated PI3K/AKT pathways; and mTOR alterations are the main molecular features [VHL elevates the stability of the hypoxia-inducible factor (HIF) transcription factor, which, in turn, tunes the expression of hypoxia-responsive genes, vascular endothelial growth factor (VEGF), platelet-derived growth factor beta (PDGF-β) and transforming growth factor (TGF-β) associated with both aberrant angiogenesis and tumor growth [VHL, along with hypoxia, can promote MET expression, which has also been associated with worse outcomes in RCC [At present, therapeutic approaches used to treat patients with metastatic RCC (mRCC) are based on specific molecular characteristics that play an essential role in disease development . In cleafeatures ,8,9,10. r growth ,12,13,14r growth ,16. Thisr growth ,18. Systr growth , which rr growth . Althougr growth ,22 and ir growth  for RCC r growth ,24. Hower growth  as a trar growth  that cour growth ,28,29 rar growth . In thiss in RCC ,31,32,33s in RCC , as wells in RCC . Whereass in RCC ,37, litts in RCC ,38. To ds in RCC  and whicIn the present study, we investigated the role and possible mechanism of MET and AXL activation in response to cabozantinib, particularly following the long-term administration of sunitinib to patients. Preclinical testing seldom considers second treatment with a TKI in tumor cell models that have experienced long-term exposure to first-line TKI, closely resembling the clinical setting. Therefore, we used two age-matched cell lines, i.e., the treatment-naïve (wild type) and corresponding sunitinib-treated cell lines, which were challenged with a single dose of sunitinib (3 µM) over four months (long-term treatment). We analyzed the effects of cabozantinib both alone and in sequence on cell survival and on the modulation of signaling transduction proteins that are responsible for cell proliferation, survival, and migration. This provides a better understanding of the molecular pathways related to drug responses that can be integrated with the data obtained from large clinical trials to improve the selection of patients for TKI-based sequence treatment.50) reached 80 and 95%, respectively, resulting in values significantly higher than those for the corresponding age-matched untreated 786-O/WT and Caki-2/WT cell lines . The drug reduced the cell viability of the 786-O/S cells to 50% (IC50) following exposure to 13 µM (±0.4), which is a greater reduction that that observed in 786-O/WT cells with an IC50 value of 10 µM (±0.6)   using a positive control that showed the maximum response to cabozantinib (786-O). We considered this value as a constant to constrain the baseline of the dose–response curve and determined the point on the curve where a 50% inhibition was observed. The absolute IC50 values were 14.5 µM (±1.50) and 13.6 µM (±1.05) for the Caki-2/WT and Caki-2/S cell lines, respectively   a. We als = 0.03) b. Therefki-2/WT) b. Overal50 values of cabozantinib in all cell lines, allowing us to treat the cells with the same drug concentration. Overall, the sunitinib-resistant 786-O/S cell lines displayed a four- and two-fold higher constitutive level of P-MET and P-AXL, respectively, as well as an enhanced phosphorylation rate (phosphorylate/pan forms) compared with the treatment-naïve 786-O/WT cell lines , in addition to inducing AXL phosphorylation without changing its total protein level. In contrast to the 786-O/WT cell line, the high constitutive phosphorylation level of MET and AXL was not induced further by cabozantinib treatment in the 786-O/S cells at the same time points b. ChangeFor the Caki-2 cell lines , the dosCabozantinib strongly deactivated the receptor after 4 h and throughout all the following time points of treatment in both cell lines. However, the molecular response to the drug did not correspond to the functional response (cell viability). Similar to the 786-O/S cell line, high expression and activation of the AXL receptor were related to sunitinib resistance occurring in Caki-2/S cell lines. Cabozantinib dephosphorylated P-AXL exclusively in sunitinib-resistant cells. Moreover, we detected a decrease in the total forms of MET and AXL in the sunitinib-resistant Caki-2/S cells, suggesting that cabozantinib inhibits the total expression of the two RTKs and not exclusively their activity. Based on these data, we inferred that only the modulation of P-AXL activity depended on the sunitinib > cabozantinib sequencing treatment. Following long-term sunitinib treatment, the specific signaling proteins P-FAK and BCL-2 presented seven- and five-fold higher protein levels, respectively, in the Caki-2/S cell line in comparison with the treatment-naïve Caki-2/WT cell line . The treThe aberrant activation of receptor tyrosine kinases MET and AXL, or MET amplification, is a major cause of the poor prognosis of RCC, as they are potent activators of cell proliferation and survival pathways and the development of bone metastases; most importantly, their activity helps cancer cells to bypass the growth-inhibitory effect of VEGFR-TKI sunitinib ,43,44,45VHL gene mutation was reported for both cell lines (https://www.depmap.org (release 22Q4 (accessed on 7 December 2022)) [The human RCC cell lines used in this study, 786-O/WT and Caki-2/WT, displayed a heterogeneous response to cabozantinib, with Caki-2 found to be less responsive to treatment. This result may have been caused by the different biological backgrounds of these cells, although a r 2022)) ,54. Cakir 2022)) ,56, whicr 2022)) . Based or 2022)) ,38,57, wr 2022))  as a resr 2022)) , as wellr 2022)) ,61. In tr 2022)) . These or 2022)) ,63 and rr 2022)) ,65. Recer 2022)) ,67, reprr 2022)) . Our datr 2022))  describir 2022)) ,71,72. Tr 2022)) , as wellr 2022)) . A recenr 2022)) . In addir 2022))  indicateOverall, the data presented in this study indicate that the high expression of the main target of cabozantinib, MET, does not reflect the drug-responsiveness property, as discussed in other studies. Considering that a viability assay, such as WST-1, evaluates the cell proliferation rate, cell size, metabolic rate and survival, it cannot be directly compared with the protein expression data. Nonetheless, we can argue that the cell-line-specific response to cabozantinib might be the result of the off-target effects of the drug. As a general observation, the variability occurring in cellular responses to cabozantinib between 786-O and Caki-2 cell lines suggests the preferential engagement of a specific downstream pathway. For instance, cabozantinib activated ERK and completely hampered AKT signaling at the T308 and S473 sites only in the Caki-2 cell line, and these events were independent of pretreatment with sunitinib. On the contrary, the cabozantinib treatment schedule  of the 786-O cell line may affect the AKT signaling process, which is one of the most important pathways involved in cell proliferation, survival and transformation occurring in RCC tumorigenesis ,76, as wOur results shed light on the in vitro effects of therapy sequencing for mRCCs. In summary, the upregulation of P-MET and P-AXL observed in our cell models did not suffice to sensitize cells to cabozantinib in the second-line treatment. The relationship between the cytotoxicity of cabozantinib, MET and AXL activities might depend on sunitinib pretreatment, drug incubation time and the cell phenotype. The data presented here also suggest a cell-line-dependent mode of action of cabozantinib, reflecting the heterogeneity among patients. Receptors MET and AXL might be activated by cabozantinib as a feedback mechanism only in 786-O. However, the sustained activation of the two receptors was preserved following prolonged drug exposure only in the sunitinib-resistant 786-O/S cell lines. On the contrary, the strong P-MET inhibition caused by cabozantinib was not affected by sunitinib pretreatment in the Caki-2 cell line, whereas P-AXL downregulation was affected by treatment with the sunitinib > cabozantinib sequence. In general, sunitinib pretreatment of both 786-O/S and Caki-2/S cells resulted in Src and FAK overexpression, ensuring a low sensitivity value for the second-line treatment.Sunitinib malate was purchased from Tocris Bioscience , and cabozantinib was purchased from TargetMol Chemicals Inc. . The compounds were dissolved in DMSO, stored as aliquots at −80 °C and thawed shortly before use. Sunitinib malate was used to prepare cell stocks of the sunitinib-resistant cell lines prior to the investigation of sequential TKI treatment.TP53 and PTEN and wild-type PBRM1) ) were cultivated in a 1:1 mixture of Dulbecco’s modified Eagle medium (DMEM) and RPMI-1640 , whereas the Caki-2 cells (mutant PBRM1 and wild-type PTEN and TP53)  were propagated in RPMI-1640, both containing 10% heat-inactivated fetal bovine serum (FBS). The cell growth conditions were set at 37 °C in a humid environment with 5% CO2. Exponentially growing cells were used for all experiments.For the current study, human clear cell RCC 786-O and Caki-2 cell lines were purchased from the American Type Culture Collection (ATCC) . These cell lines are both representative of subtype ccRCCs bearing a VHL mutation (the main feature of ccRCCs). The differences in the genetic backgrounds between the two cell lines were also reported, which helped us to include the patient heterogeneity effect in our study. The 786-O cell lines (mutant 50 (inhibitor concentration that causes 50% cell death) value of sunitinib was determined by a dose–response assay performed over 72 h and measured by a WST-1 cell viability test, as described below. To generate sunitinib-resistant cells lines, 786-O and Caki-2 cell lines were exposed to the selective pressure of sunitinib at their respective IC50 values (3 µM) for approximately four months and defined as 786-O/S and Caki-2/S cells, respectively. During the development stage, age-matched untreated wild-type (WT) 786-O/WT and Caki-2/WT cells were also subcultivated. The sunitinib tolerance of 786-O/S and Caki-2/S was routinely assessed and compared with their WT counterparts with a WST-1 test. In addition, cell stocks collected from both cell phenotypes were regularly cryopreserved. A stable high-sunitinib tolerance was achieved within 34 passages of the cell culture. The cell stocks corresponding to this passage were thawed for the experiments conducted in this study. Prior to performing sequential treatment with cabozantinib, the sunitinib-resistant cells were cultivated as a monolayer in medium without sunitinib for five days, along with the corresponding age-matched wild-type cell lines.To assess the activity of cabozantinib in the second-line setting following failed sunitinib treatment, we referred to a panel of sunitinib-resistant cell lines previously generated at our institution (data not published). Briefly, the IC3 or 2 × 103/well, respectively, and allowed to adhere overnight at 37 °C. The cells were exposed to either DMSO  or increasing concentrations of cabozantinib (0.1–25 µM) for 72 h. Subsequently, a 10 µL/well of WST-1 solution  was added, and the cells were incubated further at 37 °C for 60 min. The optical density was measured at 450 nm and a reference of 620 nm using a microplate reader .A WST-1 assay was performed to evaluate cell survival rates in response to cabozantinib exposure. Exponentially growing cells were used for the experiments, as well as age-matched untreated wild-type and sunitinib-resistant cells. Briefly, the untreated wild-type 786-O/WT and Caki-2/WT cell lines and their corresponding sunitinib-resistant cells, 786-O/S and Caki-2/S, were seeded in a 96-well plate at a density of 1 × 105 cells/mL in a 60 mm Petri dish and allowed to adhere overnight. On the following day, the cells were left untreated  or were exposed to 12 µM cabozantinib. This concentration roughly corresponds to the average IC50 value of cabozantinib in all cell lines that were analyzed, allowing us to treat the cells with the same drug concentrations. A previously conducted preclinical study showed a rapid inhibitory effect of cabozantinib on receptor MET phosphorylation within 8 h [TM BCA protein assay kit . Subsequently, 20 µg of cell lysates was separated by SDS-PAGE  and transferred onto a methanol-activated polyvinylidene difluoride (PVDF) membrane  with a tank transfer system . The membranes were blocked in 5% blocking solution for 1 h at room temperature (RT). They were incubated further overnight with primary antibodies against PAN-MET, phospho-METY1234/5, PAN-AXL, phospho-AXLY779, phospho-ERK1/2T202/Y204, PAN-AKT, phosphor-AktT308, phosho-AktS473, phosphoS6S235/6, phosho-FAKY397 , phosphor-Src family, GAPDH  and BCL-2  under agitation at 4 °C overnight. The membranes were then washed three times in Tris-buffered saline and Tween 20 (TBST) and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies  for one hour at room temperature (RT). Antibody detection was performed using an ECL-plus HRP substrate kit . We performed a stripping and reprobing membrane protocol to visualize different proteins on the same membrane. Briefly, the membranes were agitated for 30 min in 25 mM glycine-hydrochloric acid (HCL), 1% sodium dodecyl sulfate (SDS) and pH 2 buffer at 50 °C. Subsequently, the blots were washed in 1X phosphate-buffered saline (PBS), equilibrated in Tris-buffered saline and 0.1% Tween 20® detergent (TBST) and incubated with 5% blocking solution prior to reprobing with another antibody.For analysis of receptor tyrosine kinases and downstream intracellular signaling, the wild-type and age-matched sunitinib-resistant cells were seeded at a density of 2 × 10thin 8 h . Therefo50 values were analyzed with a nonlinear regression model using GraphPad Prism version 9.2.0 for Windows, GraphPad Software , and the drc packagev3.0-1 [50. We calculated the concentration that provoked a response halfway between the blank  and a positive control (cells with a full inhibited response) using Graph Pad Prism® software. Analysis of variance (ANOVA) and Student’s t-test were applied for comparative analysis with IBM® SPSS® Statistics for Windows (Version 27.0). p-values of ≤0.05 were considered statistically significant. The data obtained from all the experiments are presented as mean ± standard error (SE) of biological replicates, each derived from technical replicates. Graphs were generated with GraphPad Prism or in Excel. Composite images for publication were generated with Inkscape . ImageJ 1.52a image analysis software  [Dose–response assays and ICgev3.0-1 . If the er 2022)  was usedOur in vitro study suggests that cabozantinib can efficiently primarily target P-MET and, to a limited extent, P-AXL in RCC. Despite a dose-dependent reduction in cellular viability following cabozantinib treatment, inferior drug sensitivity was observed in Caki-2/WT cells and sunitinib-resistant Caki-2/S and 786-O/S cell lines. Cabozantinib can induce intracellular signals involved in cell proliferation and survival processes and can also be considered an early indicator of response to therapy. The effect of cabozantinib on MET and MET-dependent signaling was highly variable between the two cell lines, highlighting the often-observed heterogeneity of patient sensitivity and responses to targeted therapy, which might complicate treatment standardization for all patients. Based on our analysis, we determined that the sequence of sunitinib followed by cabozantinib is not superior to first-line therapy with cabozantinib. In the second-line treatment, the MET- and AXL-driven status did not improve cell responsiveness to cabozantinib.


Human-relevant three-dimensional (3D) models of cerebral tissue can be invaluable tools to boost our understanding of the cellular mechanisms underlying brain pathophysiology. Nowadays, the accessibility, isolation and harvesting of human neural cells represents a bottleneck for obtaining reproducible and accurate models and gaining insights in the fields of oncology, neurodegenerative diseases and toxicology. In this scenario, given their low cost, ease of culture and reproducibility, neural cell lines constitute a key tool for developing usable and reliable models of the human brain. Here, we review the most recent advances in 3D constructs laden with neural cell lines, highlighting their advantages and limitations and their possible future applications. Despite considerable efforts, we still have a very limited understanding of how the brain works when it is healthy or sick. Non-invasive imaging methods performed in humans lack the spatial and temporal resolution to probe its microscopic anatomy and function. Thus, a simplified and accessible model of the human brain is urgently needed.Although animal models have provided a significant boost to neuroscience research and are still widely used, their limits have been extensively demonstrated. In fact, besides the ethical issues, studies have shown that the results of animal experiments often fail to translate into human clinical trials . MoreoveHuman neural cells cultured in highly controllable and monitorable environments have been widely used for investigating neurotoxicity, neuroprotection, drug screening and therapeutic assessment for both brain tumors and neurodegenerative diseases, e.g., Parkinson’s and Alzheimer’s ,8,9. PosSince soma are unrealistically flattened and neuronal axonal and dendritic outgrowth cannot occur in all directions in traditional monolayers , advanceThree-dimensional constructs have also been generated from neural cell lines and may provide a strategy for developing standardized models with better physiological relevance compared with traditional 2D  cultures. In this context, after an overview of the neural cell lines commonly used in the literature, we describe recent approaches exploiting cell lines for generating 3D models of brain tissue. Given the legislative and public urge to reduce the use of animals in scientific experiments, we suggest their re-evaluation in humane and human-relevant research, particularly for regulatory applications where standardized and reproducible inter-laboratory outcomes are crucial.To identify articles dealing with three-dimensional in vitro human-relevant models of brain tissue involving cell lines, we first conducted an analysis of the existing scientific literature. Web of Science was used with the query: (((neur* OR brain OR cereb*) AND model*) AND (neurosphere* OR 3d OR three-dimension*) AND human AND “cell* line*”). Only studies published from 2000 to 2022 were selected, thus identifying 304 original articles and 37 review papers.Oncology (18%) and science technology (18%) are the research areas where cellular models of human brain tissue are mainly involved . ScientiAfter this general inspection, a more precise analysis was carried out. Each abstract was read and assessed for inclusion according to whether its focus was on 3D models of brain tissue or neural tissue generated from immortalized human cell lines. Thus, a consistent number of papers was discarded because they focused on non-human models , in vivo models, models developed with induced pluripotent stem cells and primary cells, or papers describing models of tissues different from the brain . Thus, a total of 96 papers  was selected.Most of the cell lines used in 3D in vitro models in neuroscience research derive from tumor tissues, in particular glioblastoma and neuroblastoma. In the following paragraphs, we describe the studies identified with the literature search in which 3D in vitro models of brain tissue have been developed using cell lines derived from tumors and from healthy tissues.The cancer cell lines most exploited in cellular neuroscience research derive from glioblastoma  and neuroblastoma (35%). However, 3D models of brain tissue have also been generated using cells from embryonal carcinoma and medulloblastoma.Glioblastoma cell lines are used in 3D in vitro constructs for modeling gliomas, in particular glioblastoma multiforme (GBM), the most common malignant brain tumor with the poorest prognosis and survival ,27. MostAs detailed in Neuroblastoma is the most common extracranial solid tumor observed in childhood (less than five years old) ,45, origAs regards the applications, even for neuroblastoma cell lines, most of the efforts have been directed towards characterizing the effects of the microenvironment on cell behavior, e.g., in terms of proliferation, cell invasiveness and differentiation. However, it is interesting to note that SH-SY5Y cells have been successfully exploited for neurodegenerative studies, and in particular Parkinson’s and Alzheimer’s diseases, accounting for 14% of the articles. Indeed, they are usually chosen for their catecholaminergic (though not strictly dopaminergic) neuronal properties , which tOnce differentiated, SH-SY5Y cells show the formation of neural processes and functional synapses, as well as the production of neuron-specific enzymes, neurotransmitters and neurotransmitter receptors ,75,76. IIn addition to glioblastoma and neuroblastoma cell lines, 3D human brain models have been generated using cells originating from embryonal carcinoma and medulloblastoma. Human pluripotent embryonal carcinoma NTera2 (NT2) cells are widely used for in vitro neurotoxicity studies thanks to their ability to differentiate into post-mitotic neurons after treatment with RA ,79. NT2-The UW228-3 cell line was established from human posterior fossa medulloblastoma . This liAlthough the majority of cell lines identified by the literature search had tumor origins, cell lines derived from healthy tissue can be used in 3D in vitro models of the human brain. Among these, the Lund human mesencephalic (LUHMES) line, which originates from the mesencephalon of a human subject, was established in 1998. The LHUMES neuron-like immortalized cells have been extensively characterized as a robust neuronal model suitable for neurodevelopmental studies, neurotoxicity and the modeling of brain diseases ,87. IndeNeural Progenitor Cells (NPCs) have a self-renewal capability and can give rise to healthy neural cell lineages. They can be obtained from iPSCs or derived directly from brain tissues ,89. In tOver the last few decades, numerous studies have highlighted the superiority of 3D cultures with respect to monolayers as they are able to better recapitulate the morphology and architecture of tissues and cells in their native environment, both in physiological and diseases conditions. 3D systems based on human neural cell lines exhibit specific cellular and molecular features which occur in vivo ,97 and sSuch 3D models can be realized using both scaffold-less and scaffold-based strategies ,84. The Several examples of alginate-based spheroids laden with the cell lines described in this review can be found in the literature, in combination with different cell adhesive materials such as gelatin and collagen ,64,70,93Although the results obtained with the generation of spheroids are promising, more systematic studies are still needed to exploit their versatility. For instance, 3D constructs may differ significantly in terms of mechanical and transport properties. This is mainly due to the high variability of the biomaterials used for fabricating them. Matrigel- and ECM-derived matrices are known to suffer from ‘batch syndrome’; on the other hand, alginate is more reproducible, but it is available in different molecular weights, and the protocols for obtaining spheroids involve different concentrations and crosslinking methods. Moreover, as alginate is a marine-plant-derived material, it lacks cell binding sites such as RGD motifs. For this reason, some attempts to combine alginate with cell adhesive materials, e.g., gelatin , can be The applications of the 3D in vitro models described in The development of 3D in vitro models mimicking the in vivo cell environment and behavior is also essential to obtain relevant results in toxicity testing ,110. A nA wide class of pathologies is included under the definition of neurodegenerative diseases, all of them characterized by the degeneration and death of neural cells ,112. TheThe ability to produce in vitro models with neural cells has been fundamental to advancing the understanding of the central nervous system’s (CNS) function at the microscale, as well as of the disease mechanisms underlying neuropathologies and neurotoxicity ,113. We From our literature search, the first thing that stands out is the heterogeneity of the studies reported and the fact that many of them do not carry on a systematic characterization of the cell lines or materials involved. Long-term culture often leads to the accumulation of mutations in such cells, resulting in outcomes which are difficult to reproduce in laboratories . We alsoThe regulatory testing of chemical substances to define their limits of safety for humans and the environment using in vitro methods requires a high degree of inter-lab reproducibility and throughput, with tightly defined experimental protocols. This is necessary for compliance with the Guidance on Good Cell Culture Practice (GCCP) standards . Risk anWe suggest that the 3D culture of neural cell lines should be improved to promote their use in safety and toxicity testing of chemicals at a regulatory level, leveraging the versatility of cells such as SH-SY5Y to exploit their full potential. Further investigations should be performed towards the standardization of protocols and towards the identification of supplements able to generate different classes of neurons in a controlled manner. Alternatively, since glial cells modulate neuron function and signaling, while neurons generate and propagate electrical and chemical signals , neurobl


Studies about radiation-induced human cataractogenesis are generally limited by (1) the poor number of epithelial lens cell lines available (likely because of the difficulties of cell sampling and amplification) and (2) the lack of reliable biomarkers of the radiation-induced aging process. We have developed a mechanistic model of the individual response to radiation based on the nucleoshuttling of the ATM protein (RIANS). Recently, in the frame of the RIANS model, we have shown that, to respond to permanent endo- and exogenous stress, the ATM protein progressively agglutinates around the nucleus attracted by overexpressed perinuclear ATM-substrate protein. As a result, perinuclear ATM crowns appear to be an interesting biomarker of aging. The radiobiological characterization of the two human epithelial lens cell lines available and the four porcine epithelial lens cell lines that we have established showed delayed RIANS. The BFSP2 protein, found specifically overexpressed around the lens cell nucleus and interacting with ATM, may be a specific ATM-substrate protein facilitating the formation of perinuclear ATM crowns in lens cells. The perinuclear ATM crowns were observed inasmuch as the number of culture passages is high. Interestingly, 2 Gy X-rays lead to the transient disappearance of the perinuclear ATM crowns. Altogether, our findings suggest a strong influence of the ATM protein in radiation-induced cataractogenesis. Only a few months after the discovery of X-rays, the first radiation-induced (RI) eye damage was observed in exposed laboratory animal models . Later, The lens is considered to be the most radiosensitive tissue among not only the ocular structures but also among all the organs ,5. HowevTo date, industrial and medical workers  rThere are two major limits in the quest for the basic mechanisms of cataractogenesis. First, the lack of availability of epithelial lens cell lines, notably human ones, reflects not only the difficulties of cell sampling to establish cell lines in humans but also the difficulties of amplifying this type of cell up to high cell culture passages to mimic aging. To date, two human epithelial cell lines are available: The untransformed HLEpiC cell line from ScienCell  and the transformed HLE-B3 cell line from ATCC . It is noteworthy that, as documented with skin fibroblasts, the transformation or immortalization of cell lines may introduce significant biases in the radiation response, and therefore, any radiobiological data from the transformed HLE-B3 cell lines may be interpreted with caution.Second, another difficulty in the cataractogenesis research area is the lack of reliable biomarkers for the RI aging process. Indeed, despite the considerable efforts provided by telomeres, telomerase activity, and residual DNA breaks, there is still no consensus about a molecular endpoint that would quantify a specific feature of aging .A model based on the RI nucleoshuttling of the ATM kinase protein (RIANS) has been proposed to quantify the risk linked to exposure to ionizing radiation ,24,25,26Two radioresistant skin fibroblast cell lines (1BR3 and MRC5) that originated from apparently healthy patients were used in this study as negative controls for radiosensitivity. These cell lines were purchased from the European collection of authenticated cell cultures  under the references, #90011801 and #05011802, respectively. Fibroblasts were routinely cultured as monolayers with Dulbecco’s Modified Eagle’s Minimum Medium (DMEM) , supplemented with 20% fetal calf serum, penicillin, and streptomycin.2+ and Mg2+, ScienCell), and then trypsinized with a trypsin/EDTA solution (ScienCell). The immortalized human lens epithelial cell line (HLE-B3) was purchased from the American Type Culture Collection  under the #CRL-11421 reference and was routinely cultured in the same conditions as indicated for human fibroblasts.The untransformed human lens epithelial cell line  and the untransformed human retinal pigment epithelial cell line  were purchased from ScienCell Research Laboratories . Note that Hamada’s group mentioned that ScienCell Research Laboratories proposed several lots of primary normal human lens epithelial cells derived from different donors  under the same name (HLEpiC) . Hence, One radioresistant porcine skin fibroblast cell line (PSF-1) that originated from a cutaneous biopsy from a butcher pig from a local slaughterhouse was used as a negative control for radiosensitivity. This biopsy was performed in the 1990s at Institut Gustave-Roussy  in agreement with the regulations of the French departments and the slaughterhouse veterinarians.2-independent serum . The eyes were cleaned for muscles and immersed for a few minutes in an antiseptic solution . The anterior segment of the bulb was removed, as well as the lens, the vitreous, and the retina. Lenses were collected in a saline solution at room temperature. Lenses were then placed on Petri dishes with their anterior side down. A small incision was made on the posterior capsule using sterile scalpels. The capsules were then opened and fixed on the Petri dishes using sterile forceps. The fibrous cellular mass was discarded, and only the capsules were kept  in agreement with the local regulatory department and the slaughterhouse veterinarians. This experimental step was performed with the assistance of T.M., and the procedure was described elsewhere . This prere kept .2+ and Mg2+), then trypsinized with TrypLE Express 1X solution (Gibco). All the experiments were performed with cells in the plateau phase of growth (95–99% in G0/G1). The cells used in the experiments were cultured at different passages (from passage 3 to passage 30).The capsules were incubated and cultured by following the same procedure as for fibroblasts, rinsed with PBS 1X (with Ca−1) [Irradiations were performed with a 6 MeV photon medical irradiator (SL 15 Philips) (dose rate: 6 Gy·min−1) .γH2AX, -pATM, and -MRE11 and secondary antibodies [BFSP2 antibody  and the polyclonal anti-rabbit anti-FYCO1 antibody  were used at 1:100. Nuclei were counterstained with 4′,6′-Diamidino-2-phenylindole  antibodies [The immunofluorescence protocol was described elsewhere, together with the references for the primary anti-tibodies ,31,32,33tibodies ,31,32,332 in which R was defined as the average nucleus radius  allows the visualization of endogenous protein–protein interactions at the single-molecule level . The PLAThe procedures of the cell extracts and immunoblots are described elsewhere ,31,32,33A two-way ANOVA was used to compare numerical values, and Spearman’s test was used to compare the kinetic data. The foci kinetic data were fitted to the so-called Bodgi’s formula . Statistp < 0.01) while it was similar to that of the control in the HRPEpiC   micronuclei and cellular radiosensitivity ,41. Twenp > 0.5) . p < 0.05) (p > 0.5). These last findings suggest that the repair rate of DSB recognized by NHEJ can be considered normal  A. As abu < 0.05) A. At 24 d normal A. pATM immunofluorescence was applied to the same cell lines with the same irradiation scenario. No spontaneous pATM foci were observed in the three cell lines tested. In the radioresistant skin fibroblasts, the number of pATM foci scored 10 min post-irradiation was 40 ± 4 per cell, in agreement with previous studies [p < 0.05), supporting a delay in the RIANS. The number of pATM foci decreased progressively to a value non-significantly different from zero at 24 h post-irradiation  [p < 0.05)  ,43. Convd cells) ,43. From < 0.05) C. p < 0.0001) (p < 0.05). At 24 h post-irradiation, the HLE-B3 cell line showed a number of γH2AX foci significantly higher than the HLEpiC cell line  (p < 0.01)  A. These  0.0001) B, sugges < 0.01) C. Howeve < 0.01) D.A first set of trials (Set 1) was carried out after adult cataract surgery. The trials consisted of obtaining lens epithelial cells from different conditions: One clear lens, one moderate cataractous lens, and one dense cataractous lens. Set 1 procedure was based on phacoemulsification, allowing the fragmentation of the lens and a UV probe to aspirate the cataract. This resulted in the destruction of some lens, and the challenge was to get a critical number of viable and clonogenic cells. However, this procedure failed to provide clones.A second set of trials (Set 2) consisted of obtaining whole lens from adult enucleation procedures. Set 2 procedure was based on the extraction of the whole lens, thus preserving the cells of interest. However, most of the donors were adults older than 50, and, again, this procedure failed to provide clones. Another critical step of the procedure was that only one layer of epithelial cells is present on the anterior side of the lens capsule, and breaking the capsule can cause the detachment of the cells from each other, therefore reducing the chances of cell proliferation.Faced with the lack of availability of human lens epithelial cells, the low proliferating ability of the HLEpiC cell line, and the biases associated with the transformed HLE-B3 cell line, we attempted to establish human lens epithelial cell lines from adult eyes, and various procedures were considered:The technical difficulty of establishing human lens epithelial cell lines prompted us to investigate the radiation response of lens cells in some animal models. There was an opportunity to work on the porcine lens, and we therefore established four cell lines to document their radiation response by applying the same experimental conditions and techniques as those described above and in Materials and Methods. In order to compare the radiobiological data from the lens, we used historical data obtained from a porcine skin fibroblast, PSF-1, established in the 1990s.p < 0.01). These results support significant RI genomic instability in the porcine lens epithelial cell lines tested (The number of micronuclei assessed 24 h after 2 Gy X-rays in the PSF-1 and in the four porcine lens (CP) cell lines was examined. The four porcine cell lines tested showed a significantly high number of micronuclei in comparison with the skin PSF-1 cell line . The number of γH2AX foci scored 10 min after 2 Gy in the radioresistant control PSF1 was 71 ± 3 per cell, while the corresponding numbers of γH2AX foci in the four porcine lens epithelial cells were significantly lower  (p > 0.5), suggesting a normal repair rate of DSB recognized by NHEJ  A, sugges by NHEJ A. Altogep < 0.05)  B. These  < 0.05) B.With regard to the MRE11 foci, the PSF-1 cells showed a maximum of 5 MRE11 foci per cell 1 h post-irradiation and 3 MRE11 foci per cell 4 h post-irradiation. Three of the four CP cell lines tested showed a curvilinear increase in the number of MRE11 foci per cell at 4 h post-irradiation  C. These Our findings showed that the lens cells tested systematically elicit delayed RIANS with fewer γH2AX and pATM foci than expected. As evoked in the Introduction, the delay of RIANS is generally due to proteins overexpressed in the cytoplasm that are substrates of ATM phosphorylation  . We, theAs a second step, we focused on the BFSP2 protein (3 SQ and 2 TQ). We first examined BFSP2 expression by immunofluorescence. BFSP2 appeared to be mainly cytoplasmic, with some nuclear forms. Interestingly, we observed some perinuclear crowns of BFSP2 staining before irradiation, disappearing up to 1 h after irradiation, and then reappearing 4 h after irradiation .We thereafter investigated the BFSP2 expression in cytoplasmic protein extracts of the fibroblast radioresistant control cell line (MRC5) and of the four porcine lens epithelial cell lines  by applying immunoblots . Cell exp < 0.001). The influence of the irradiation on the number of PLA foci reflecting the ATM–BFSP2 complexes was cell line dependent. Since the RIANS is delayed in the porcine lens epithelial cell lines and since the BFSP2 protein was found to be very abundant in the cytoplasm, we hypothesized that some BFSP2–ATM complexes may exist. The PLA assay was applied to the CP cell lines . The PLARecently, in this journal, we have pointed out that fibroblasts from patients suffering from Alzheimer’s disease (AD) specifically show spontaneous perinuclear pATM crowns . The per12 = 4096 cells if one passage corresponds to one doubling time and from 1 to 224 = 16,777,216 cells if one passage corresponds to two doubling times. Altogether, these findings are consistent with our model of accelerating aging with the agglutination of ATM around the nucleus. Further investigations are, however, needed to relate these findings to the formation of pATM crowns with the BFSP2 protein that would be phosphorylated by ATM in an inner layer of the crown, as recently observed in AD cells with the APOE protein [The percentage of cells with perinuclear pATM crowns was plotted against the number of cell passages, and some individual differences appeared. Mathematically, such differences cannot be due to errors in cell seeding or passage number calculation. The highest regression coefficients were obtained when the data were fitted to exponential and power laws A. By con protein .In the context of the RIANS-aging model, exposure to ionizing radiation may lead to a transient monomerization of ATM and therefore to a temporary disappearance of the perinuclear pATM crown . In ordeThis work aims to document the radiation response of human epithelial lens cells. However, the radiobiological characterization of this type of cell remains limited by the lack of availability of human epithelial lens cell lines, notably explained by the difficulties in amplifying them to high cell culture passages. By applying the RIANS biomarkers  to the HLEpiC cell line from ScienCell , a significant delay appeared in RIANS, suggesting that this lens cell line elicits moderate but significant radiosensitivity. Furthermore, the MRE11 foci kinetics found in HELpiC cells showed an analogy with those obtained from skin cells from aging syndromes, with a maximal value reached at 24 h post-irradiation. The radiobiological features of the transformed B3 lens cell line led to the same conclusions. Hence, while we are aware that too few human lens cell lines have been tested, our findings and literature data converge on the fact that (1) the human lens is a radiosensitive tissue  and (2) In addition, we identified a potential X-protein specific to the lens, i.e., an overexpressed cytoplasmic ATM substrate whose perinuclear sub-localization may facilitate the progressive agglutination of ATM around the nucleus and the formation of the perinuclear pATM crown. Lastly, in porcine lens cells, the number of perinuclear pATM crowns reached its maximal value at cell culture passage 12, which supports a limited lifespan in cell culture for this type of cell. The identification of a lens-specific X-protein (BFSP2), that is perinuclear, supports once again that the lens is a radiosensitive and radiodegenerative tissue.-“radiosensitivity”: any RI clinical and cellular events attributable to cell death ;-“radiosusceptibility”: RI cancers or any RI event attributable to cell transformation;-“radiodegeneration”: any aspect of the response to ionizing radiation attributable to aging.The lens was considered one of the most radiosensitive tissues, such as the breast, thyroid, or skin ,47. HoweAs a consequence, the lens may be considered a radiosensitive and radiodegenerative but not radiosusceptible tissue (there is no report of cancer of the lens). Like with a number of genetic syndromes in our previous reports ,33,48,49Interestingly, by applying the same approach to the lens, we have identified at least one X-protein for the lens, the BSP2 protein, that shows perinuclear sublocalization and therefore may favor the formation of perinuclear pATM crowns in response to permanent stress. In the framework of the RIANS model, such proteins may be considered tissue-specific X-proteins, unlike the “syndromic” X-proteins defined in skin fibroblasts and specific to each human genetic disease. Hence, the lens appears to be a tissue whose radiosensitivity belongs to group II defined by the RIANS model  ,43 Figu.By combining the radiobiological characterizations accumulated with skin fibroblasts from genetic diseases associated with juvenile cataracts and those of the lens cells, the syndromic and lens-specific tissue X-proteins may contribute together to the sequestration of the ATM monomers in lens cells to explain both individual and tissue predispositions to aging. In other terms, the syndromic X-proteins may accelerate a process already “programmed” in the lens thanks to tissue X-proteins such as BFSP2 . Our finBy considering the perinuclear pATM crowns as specific biomarkers of aging , Figure 5 ATM monomers per Gy are produced in the cytoplasm [pATM antibodies, exposure to ionizing radiation may lead to the monomerization of the ATM dimers that compose the pATM crowns and therefore may cause them to disappear. However, exposure to ionizing radiation may not cause a definitive but a transient steric dispersion of the ATM monomers around the nucleus, and their local concentration may facilitate again the formation of ATM dimers after irradiation and therefore that of the pATM crowns. Hence, after irradiation and a “relaxation time” that would depend on the dose, the pATM crowns may reappear. In our conditions, the relaxation time deduced from our preliminary data was found to be between 24 h and 48 h. Exposure to ionizing radiation not only adds more DNA breaks in the nucleus but also triggers the monomerization of the ATM proteins that help recognize them. It has been suggested that, while about 40 DSB are induced per Gy per fibroblast, about 10ytoplasm . Since t5 ATM monomers. For the formation of the first monolayer, let us consider an average nucleus radius of lens cells of 5–10 μm, an average surface of the nuclear membrane of about 800 μm2, and an average number of nuclear pores of about 1000. The formation of a pATM crown monolayer may therefore require at least the agglutination of about 200 ATM monomers per nuclear pore and 250 ATM monomers per μm2. In order to relate our findings to RI cataractogenesis, further experiments are needed to link the number of cells with pATM crowns, the number of cells undergoing senescence, and the in vivo occurrence of cataracts. However, an interesting notion emerges from our findings: The predisposition to accelerated aging and to early cataracts that would be reflected by a non-negligible number of cells with pATM crowns at the lowest passages. Interestingly, Obviously, different scenarios of irradiation may fill such requirements  and are the subject of calculations in progress. Altogether, our findings support that all the epithelial lens cells tested, whether human or porcine, show a delay in the radiation-induced ATM nucleoshuttling, late MRE11 foci, and perinuclear ATM crowns, as already observed in cells from aging syndromes . In the


Functional genetic screens to uncover tumor-intrinsic nodes of immune resistance have uncovered numerous mechanisms by which tumors evade our immune system. However, due to technical limitations, tumor heterogeneity is imperfectly captured with many of these analyses. Here, we provide an overview of the nature and sources of heterogeneity that are relevant for tumor-immune interactions. We argue that this heterogeneity may actually contribute to the discovery of novel mechanisms of immune evasion, given a sufficiently large and heterogeneous set of input data. Taking advantage of tumor cell heterogeneity, we provide proof-of-concept analyses of mechanisms of TNF resistance. Thus, consideration of tumor heterogeneity is imperative to increase our understanding of immune resistance mechanisms. The reason that screens have largely been limited to single cell lines in publications is a technical one: to ensure maintenance of library complexity , and thus fidelity and confidence of the hits identified, a(n extremely) large number of cells need to be used in such screens, making the inclusion of multiple cell lines labor-intensive. Despite this limitation, their success and fidelity were demonstrated by virtue of their identification of common pathways by several groups. By and large they comprise the TNF, IFNγ, antigen presentation and autophagy pathways .APOBEC3 activity in late-stage cancers which promotes the stochastic mutation of the tumor genome  hypoxic tissues, such as melanoma, have heightened expression of cIAP1. These tumors therefore display enhanced resistance against TNF . Extendisitivity , 36.+ T cell responses antigen burden and immune resistance mechanisms genomic, transcriptomic and proteomic omics data. At the same time, the screening data should be derived from both in vitro and in vivo screens from as many genetic backgrounds as possible [reviewed in -specific dependencies. Aside from these functional genetic screens, the cell lines used in these studies have also been extensively characterized, including the collection of RNA, DNA, epigenetic, metabolic and drug-sensitivity metrics –72. The Hi cell lines specifically  and RNF31 . By performing this analysis, we could find factors whose ablation reduces viability of TNFnd RNF31 .TRAF2 and BIRC2 to establish resistance to TNF in different tumor cell lines. While it had been difficult to fully comprehend this differential sensitivity before, given that TRAF2 and cIAP1 are thought to signal in a linear fashion, we could now make transcriptomic comparisons between TNFHi cell lines in which both TRAF2 and BIRC2 sensitize, those in which solely BIRC2 knockout sensitizes, those in which solely TRAF2 knockout sensitizes, or those cell lines in which neither the loss of TRAF2 nor the loss of BIRC2 reduces the viability of the affected cell line ? Therefore, and as mentioned above, the true complexity of tumor-immune interactions, and forms and mechanisms of heterogeneity at play require more data to be integrated in these models. Firstly, and perhaps most easy to accomplish, the field should invest in performing more tumor : T cell screens, to complement those that have already been reported in key publications in the recent past , 75, 80.s (as in , 13, in DV performed the analyses. DV and DP wrote the manuscript. All authors contributed to the article and approved the submitted version.


Our dataset has a great potential for re-use given the recent surge in interest surrounding the role of CIN in breast cancer. The large size of the dataset, coupled with the diversity of the cell types represented, provides numerous possibilities for future comparisons.These data show the relative amount of chromosomal instability (CIN) in a diverse array of human breast cell types, including non-transformed mammary epithelial cells as well as cancer cell lines. Additional data is also provided from human embryonic and mesenchymal stem cells. To produce this dataset, we compared a published chromosomal instability gene signature against publicly available datasets containing gene expression information for each cell. We then analyzed these data with the Python GSEAPY software package to provide a CIN enrichment score. These data are useful for comparing the relative amounts of CIN in different breast cell types. This includes cells representing the major clinical (ER/PR Specifications Table1•While CIN is a defining hallmark of cancer, little is known about its relationship with stemness. High levels of CIN are associated with breast cancer evolution and metastasis •These data add value to our original research article by allowing us to assess potential associations between CIN levels and stem-like breast cancer cells and draw important conclusions regarding the impact of CIN on tumor initiation.•The CIN enrichment scores provided in this dataset are useful for comparing the relative amounts of CIN present in different non-transformed and cancer cell lines from the breast.•This dataset may be useful to anyone associated with breast cancer research. CIN is a hallmark of cancer and is well described to play a role in tumor evolution, especially in regards to therapeutic resistance •These data can be used/reused for further insights into associations between CIN levels and numerous additional variables, including cell-of-origin, mutational status, subtype, gene expression, etc.2The deposited dataset consists of two distinct Excel spreadsheets Breast cancer cell lines https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE50470HCC38 sorted cells https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA750073We compared these datasets to the CIN signature by first converting FASTQ files to gene-expression matrices and processing so that only the data were retained. Scores were obtained for each cell type by performing single sample gene set enrichment analysis (GSEA) with Python GSEAPY Library software, and the resulting data were processed into Excel format.The resulting spreadsheets are labelled and described as follows:Breast cell lines – Excel spreadsheet listing the CIN enrichment scores for an assortment of breast cancer cell lines as well as non-transformed human cells. Cell names are listed across the top of each column with the corresponding ES and NES scores underneath.HCC38 sorted breast cancer cells – Excel spreadsheet containing the CIN enrichment scores for HCC38 breast cancer cells sorted according to their cell surface EpCAM (Ep) and integrin αvβ3 (b3) status from three independent experiments. Sorted cells from each experiment were divided into four categories: EpCAM low (lo) versus high (hi) and αvβ3 positive (pos) versus negative (neg). Each cell type belonging to a particular experiment was given a unique Run number (SRR) listed across the top of each column. For quick reference, the sorted cell type is included underneath, followed by the ES and NES scores.3Publicly available RNA sequencing data from breast cancer and normal mammary cell lines was obtained from NCBI GEO (GSE50470), while data from sorted HCC38 cell populations was downloaded from the NCBI Sequence Read Archive (PRJNA750073). FASTQs were converted to gene-expression matrices and the files were processed to remove all the header information and only retain the data. The CIN gene signature was acquired from Bakhoum et al. https://gseapy.readthedocs.io/en/latest/). First, input files were read using Python's Pandas library and joined with each other using the ID & amp columns before deleting any unnecessary columns. Ensemble Gene IDs were mapped to their HGNC Symbols using Python's BioMart API (https://pypi.org/project/biomart). Any Ensemble ID which did not have a corresponding HGNC Symbol was dropped. Once we obtained the data frame having HGNC Symbols as rows, samples as columns, and their feature counts as values in all rows, this data frame, along with the CIN gene set was passed to the Single Sample GSEA Python library. The final data comprised 36,866 rows and 106 columns before feeding it into GSEAPY. To determine the enrichment scores (ES), we applied Single Sample GSEA to the final data frame. The experiment was repeated with a normalized version of the data frame, but the normalized enrichment scores (NES) were identical to the ES. GSEAPY output was then processed into Excel format and saved as final results files.To generate the CIN scores for each cell type, we analyzed data with the Python GSEAPY Library (None.We confirm that the authors have read and follow the ethical requirements for publication in Data in Brief and that the current work does not involve human subjects, animal experiments, or any data collected from social media platforms.Shahnawaz A. Baba: Conceptualization, Methodology, Writing – original draft. Shreyas Labhsetwar: Methodology, Formal analysis, Software. Richard Klemke: Methodology, Formal analysis, Software. Jay S. Desgrosellier: Conceptualization, Methodology, Formal analysis, Visualization, Investigation, Supervision, Funding acquisition.


Haloferax mediterranei bacterioruberin-rich carotenoid extracts (BRCE) (0–100 µg/ml) in six BC cell lines, representative of the intrinsic phenotypes and a healthy mammary epithelium cell line. Cell index values were obtained using xCELLigence RTCA System. Furthermore, cell diameter, viability, and concentration were measured at 12 h, 24 h, and 30 h. We found that BC cells were selectively affected by BRCE . After 30 h, the population of BC cells exposed to 100 µg/ml was 11.7–64.6% of the control (p = 0.0001–0.0009). Triple-negative cells were significantly affected [MDA-MB-231  and MDA-MB-468 ]. Cell size was also reduced after 30 h treatment in 3.8 (± 0.1) µm and 3.3 (± 0.02) µm for SK-BR-3 (p < 0.0001) and MDA-MB-468 (p < 0.0001), respectively. In conclusion, Hfx. mediterranei BRCE exerts a cytotoxic effect on BC cell lines representative of all studied intrinsic subtypes. Furthermore, results obtained for MDA-MB-231 and MDA-MB-468 are very promising, considering the aggressive behaviour of the triple-negative BC subtype.Oxidative stress has been linked to the onset and progression of different neoplasia. Antioxidants might help prevent it by modulating biochemical processes involved in cell proliferation. Here, the aim was to evaluate the in vitro cytotoxic effect of  These microorganisms synthesize a rare C50 carotenoid called bacterioruberin (BR) and its derivatives monoanhydrobacterioruberin (MABR) and bisanhydrobacterioruberin (BABR)3. In addition, although to a lesser extent, they also synthesize astaxanthin, zeaxanthin, lycopene, and β-carotene5. Haloferax mediterranei is particularly relevant among haloarchaea since it has been used as a model organism for studying numerous pathways9. Furthermore, its carotenoid production can be easily enhanced by altering cell culture conditions15.Halophilic archaea or haloarchaea are extremophilic microorganisms that require a hypersaline environment to thrive16. Nevertheless, C50 carotenoid properties have not been addressed lately21. Haloarchaeal carotenoids have excellent antioxidant activity28, but there is still scarce information concerning their potential effects on human health. Recent research has revealed that haloarchaeal carotenoids exerted in vitro anticancer activity in a few colorectal, breast, liver, and cervical cancer cell lines20. However, much research needs to be done to support these preliminary results and draw a firm conclusion.Carotenoids are well-known for their health-beneficial properties, including antioxidant, antiproliferative, antitumoral, and immunomodulatory activities29. Breast tumours can be classified into four subtypes according to the presence or absence of hormone receptors (estrogen (ER) and progesterone receptors (PR)) and the overexpression of human epidermal growth factor receptor 2 (HER2) in combination with a set of clinical features. The four subtypes were named luminal A (ER+ PR+ HER2−), luminal B (ER+ PR HER2±), HER2-enriched (ER− PR− HER2+), and triple-negative (ER− PR− HER2−) BC31. Despite the advanced BC treatments in recent years, there is still a need for bioactive compounds that can help in the prevention and/or treatment of this pathology. Hence, this in vitro work aimed to determine if a Hfx. mediterranei BRCE was cytotoxic to BC cell lines representative of each subtype plus a mammary epithelium cell line as a control to evaluate the cytotoxicity in healthy tissue. In addition, we investigated the effect of the BRCE treatment on total viable cell number, cell adhesion, and cell diameter. Thus, this study contributes to the knowledge of the potential therapeutic applications of haloarchaeal carotenoids in BC.Breast cancer (BC) is still the leading cause of death (7%) in women worldwideHfx. mediterranei R-4 (ATCC33500) was grown in a complex medium containing 12.5% (w/v) of inorganic salts32, 0.5% (w/v) yeast extract  and 1.5% (w/v) d(+)-Glucose anhydrous BioChemica . The pH was buffered using 30 mM Tris  and adjusted to a pH of 7.3. Growth conditions included 36.5 °C and shaking at 170 rpm in a shaking incubator  based on the data reported by Montero-Lobato and Giani et al.11. First, cells were incubated as described elsewhere12. Then, cells were centrifuged at 7800 rpm for 30 min to remove the supernatant and were washed twice with a 10% (w/v) inorganic salts solution, plus the last wash with distilled water to remove all remnants of salt and induced cell lysis. Cell pellets were kept at − 20 °C until further use.Panreac AppliChem, Panreac Quimica, Barcelona, Spain) was added to the cell pellets in a ratio of 1 ml of acetone per 10 ml cell culture10. After that, a 4 °C overnight incubation and later centrifugation  were necessary to obtain the BRCE. As a result, BR concentration was calculated as follows10:Pure acetone of HPLC grade . Then, a mammalian cell culture media (DMEM (Dulbecco's modified Eagle's medium) F-12 (1:1) with l-glutamine and 15 mM HEPES  supplemented with 10% fetal bovine serum (FBS) , and 1% penicillin (50 U/ml) and streptomycin (50 mg/ml)  was added to the BRCE up to a final concentration of 150 µg/ml. Sonication with an ultrasonic probe (Branson SFX 550)  was required to ensure total solubilization. Previous assays were carried out to optimize sonication time. Therefore, both the solubilization and stability of the pigments were guaranteed. The samples were kept on ice, and the sonication conditions with microtip were pulsed on-time of 20 s, off-time of 40 s, amplitude of 20%, and a total duration of 4 min. The sonication cycle was repeated with off-times of 5 min between cycles to ensure the cooling of the sample. Filtration using 0.2 µm filters was carried out to guarantee sterilization of the solution before cell treatment.Considering that carotenoids were solubilized in acetone, we aimed to replace this organic solvent with a solution compatible with human cells so that in evaluating the anticancer activity, we could ensure that the pigments caused the effects observed. Therefore, after BR quantification, acetone was removed by evaporation with a centrifugal vacuum concentrator system  medium supplemented with 10% FBS  and 1% penicillin–streptomycin . DMEM F-12 was selected as culture media for all cell lines to reduce variability and to ensure the nutrition requirements of all cell lines, considering that it is one of the most enriched available culture media33. All cell lines were incubated at 37 °C in a humified atmosphere with 5% CO2.One healthy epithelial mammary cell line (184A1) and six human BC cell lines representative of each intrinsic subtype were used in this study : Luminal A (MCF-7 and T-47D), Luminal B (BT-474), HER2-enriched (SK-BR-3), and triple-negative (MDA-MB-468 and MDA-MB-231). Since no analytical studies were carried out to confirm the absence of trace quantities of acetone, 184A1 was used as a control to evaluate the effect of the treatment on non-tumour cells and to normalize the results when compared with tumour cells. They were all grown in a monolayer and maintained as adherent cell cultures in DMEM F-12 with stable Agilent Technologies, San Diego, CA) was used to evaluate cell adhesion over time34. The equipment took measurements every 15 min for 100 h to ensure the achievement of the complete curve.xCELLigence real-time RTCA DP Instrument   using the external computer system RTCA software Pro 2.3.4 . First, background reading was done with DMEM F-12  culture media. Next, cells were seeded to the 16 well microplates . All steps were performed under sterile conditions. Firstly, 25,000, 50,000, and 75,000 cells were seeded to determine the seeding density for the rest of the experiments. The objective was to determine an initial cell density allowing later measurements at different stages of cell growth  in 96-well plates  and DMEM F-12 was used as the positive and negative control, respectively.For this experiment, xCELLigence RTCA was set up according to the manufacturer's instructions. The instrument was placed inside a humidified COCapricorn Scientific; Ebsdorfergrund, Germany). Total cell number, viability, and diameter were analyzed using a CASY OLS cell counter and analyzer (CASY TTT) . Cell adherence was assessed using xCELLigence Real-Time Cell Analysis System. Half maximal inhibitory concentration (IC50) for the total viable cells was calculated using GraphPad 7 Software . In addition, the selectivity index (SI), defined as the ratio of IC50 for normal cells to that for BC cell lines, was calculated to evaluate the toxicity of the BRCE studied against normal cells and to predict their therapeutic potential.Normal mammary epithelium and BC cells were seeded in 96-well cell culture plates with a density of 50,000 cells/well. After 12 h (to ensure complete cell attachment), culture media was removed, and serial concentrations ranging from 10 to 100 µg/ml BRCE were added. DMSO and DMEM F-12 were used as the positive and negative control, respectively. After 12 h, 24 h, and 30 h of treatment, cells grown in each plate well were harvested using trypsin–EDTA (0.05%)  using a 10 ×/0.25 objective.Cell images were acquired with an optical microscope . The differences were considered statistically significant at p-values < 0.05*, < 0.005**, < 0.0005***, < 0.0001****. All experiments were repeated at least three times unless otherwise indicated.Data are expressed as the mean ± standard deviation (SD). Statistical significance was calculated by ANOVA (followed by Dunnet's test for multiple comparisons) analysis using GraphPad 7 software (Hfx. mediterranei BRCE (10–100 µg/ml) on the adhesion of human epithelial mammary and BC cell lines was studied using the xCELLigence RTCA system.In this experiment, the effect of 34. For this reason, this equipment was also used to establish the optimal cell quantity to seed for the subsequent experiments.The curves generated by the cell index representation can give us information about the proliferation dynamics of each cell lineFigure An initial cell density of 50,000 cells was chosen to keep homogeneity because, in most cell lines, this density ensured that three measurements could be made at three stages of growth . However, this number of cells in the BT-474 cell line was excessive for proper measurements, as shown in Fig. In Fig. 50 values at 30 h (Table p < 0.0001).When considering the different intrinsic subtypes, the IC h Table  indicate50 of 184A1 cells could not be calculated since 75% inhibition was obtained with the highest concentration tested (100 µg/ml). Consequently, this concentration was used as IC50 without a better option. Thus, the SI is an underestimation of the real potential value. An SI > 1.0 is considered a favourable value since it indicates a drug with higher efficacy against tumour cells than against normal cells35. Hfx. mediterranei carotenoids present an SI > 2 in MCF-7 and BT-474 cell lines (Table p < 0.0001) was obtained for MDA-MB-231 and MDA-MB-468 cell lines representing triple-negative subtype. Furthermore, the highest concentration tested led to an approximately 60–70% reduction of cell index in these cell lines.A SI was estimated to determine the cytotoxicity of the compound to the normal cells Table . The IC5Of note, perturbance in all curves is observed at 12 h due to plate removal from the equipment to add treatment.p < 0.0001), 1.3 (± 0.2) µm (T-47D) (p = 0.006), 1.5 (± 0.1) µm (BT-474 25K) (p = 0.0006), 1.8 (± 0.2) µm (SK-BR-3) (p < 0.0001), 1.2 (± 0.3) µm (MDA-MB-468) (p = 0.02) and 1.5 (± 0.2) µm (MDA-MB-231) (p = 0.0005) at the highest concentration (100 µg/ml) , 3.8 (± 0.1) µm (SK-BR-3) (p < 0.0001), 3.3 (± 0.02) µm (MDA-MB-468) (p < 0.0001), 1.8 (± 0.1) µm (MDA-MB-231) (p < 0.0001), and except for 1.3 (± 0.1) µm (T-47D) (p = 0.0005), and 1.4 (± 0.3) µm (BT-474 25K) (p = 0.0001), whose diameters remained constant.Regarding human mammary epithelial cells (184A1), after 30 h treatment, their diameter did not seem to be affected by the treatment, with an average diameter of 18.9 (± 0.2) µm  and MCF-7 (55.4 (± 4.3)%) (p < 0.0001), followed by SK-BR-3 (62.3 (± 1.1)%) (p < 0.0001) and MDA-MB-231 (69.9 (± 0.7)%) (p < 0.0001). After 30 h, T-47D viability dropped to 54.9 (± 2.0)% (p < 0.0001), joining the previously mentioned cell lines. In contrast, BT-474 (25K) viability was only reduced by 22.7 (± 3.0)% (p < 0.0001). Interestingly, although affected, cell viability was not reduced below 50% in most cases. In consequence, IC50 values were not calculated for this section.After 12 h treatment, BC cell viability was already diminished at 100 µg/ml. The most affected cell lines were MDA-MB-468 (40.4 (± 0.4)%)  except BT-474 (p = 0.0103), whose cell population was smaller as the concentration of BRCE increased.Considering the significant results obtained in the xCELLigence curves of the effect of the haloarchaeal BRCE treatment but the relatively low decrease in viability, cell counting was used to monitor the proliferation of BC cell lines. Figure p = 0.002), 29.4 (± 0.7)% (T-47D) (p < 0.0001), 11.7 (± 0.4)% (BT-474) (p < 0.0001), 64.6 (± 7.2)% (SK-BR-3) (p = 0.0009), 19.6 (± 0.9)% (MDA-MB-231) (p < 0.0001), 45.5 (± 1.6)% (MDA-MB-468) (p = 0.0002), of the control without treatment. Table However, with prolonged exposure to the treatment 24–30 h), the population of viable cells was significantly reduced. After 30 h, the cell population exposed to 100 µg/ml was 57.9 (± 0.9)% (MCF-7)  was not significantly affected by the treatment regarding cell size, viability, adhesion, and concentration.34. Apart from that, the innovative technology of xCELLigence was crucial for obtaining continuous measurements of cell adhesion rather than a single-time quantification.One of the best advantages of xCELLigence RTCA system technology is that it allows observing growth responses to treatments on cell lines in real-time without chemicals and dyes as in other conventional cytotoxicity analyses34. Cell index depends on cell number, morphology, size, and attachment strength.This system measures the electrons that flow between the gold microelectrodes placed at the bottom of the microtiter plate using an electrically conductive solution, such as a cell culture medium. Once cells have adhered to the plate, the electron flow between electrodes and the conductive solution is blocked. The equipment translates this impedance into a unit called cell index that allows monitoring and quantifying cell proliferation and attachment20. However, the effect on cell adhesion had not been addressed before this work. Luminal A and luminal B were more sensitive to the treatment than the rest of the subtypes. Luminal A is the most frequently diagnosed subtype, representing between 50 and 60% of cases36. Despite this, Luminal A tumours generally imply a good patient prognosis since strategies that block estrogen receptor function can be used37. Although these treatments are usually effective, over time, tumours evolve and might become resistant to these hormone-based drugs38. The fact that these carotenoids can act selectively on cancer cells is very promising. Luminal B usually behaves more aggressively than Luminal A tumours since they are associated with a higher probability of relapse and metastasis40.The most affected cell lines in terms of cell index were BT-474 , MCF-7, and T-47D. The sensitivity of the MCF-7 cell line to haloarchaeal carotenoids has been recently reported41. The particularity of triple-negative tumours is that, given their phenotype, they do not respond to endocrine or molecular targeted therapy42. Consequently, they can only be treated with chemotherapy, resulting in poor outcomes. Therefore, developing or discovering new drugs or coadjuvants is necessary.The results obtained for triple-negative cell lines are also relevant, considering the aggressiveness that these tumours usually present, with a mortality rate of 40%35. This study demonstrates that BRCE from Haloferax mediterranei exerts a selective effect in all tested breast cancer cell lines representative of the four different intrinsic subtypes. These results agree with the selective effect of haloarchaeal carotenoids. Previous research reported that Natrialba pigments present an SI > 2, higher than the SI for the chemotherapeutic drug 5-fluorouracil (SI < 1.7)20.A selective effect is crucial for effective cancer treatment to exert high antitumor activity and minimal toxicity to normal tissues. An SI > 1.0 indicates that a compound has higher efficacy against tumour cells than against normal cellsThe observed decrease in cell index indicates a reduction in the detected adhesion of the cells to the plates. However, from these data is difficult to conclude if it is due to reduced cell proliferation, cell death, or a change in the morphology of the cells. For this reason, the diameter, viability, and total number of viable cells per ml were studied in parallel.47 and none on carotenoids than those using more standardized methods, such as MTT52. On that account, this makes comparing results with previous research more difficult.Although the advantages of using the xCELLigence technology are numerous, one limitation shall be considered in this field. Since it is a relatively new technique, there are fewer studies on natural compoundsHfx. mediterranei BRCE on BC cells. Cell diameters were significantly reduced, and morphology alterations were detected in all tested BC cell lines exposed to the BRCE treatment. This effect was also reported for HepG2 cells testing extract from another haloarchaeon and morphological modifications22. In addition, the loss of the standard shape has been previously reported in the MCF-7 cell line in response to haloarchaeal carotenoids20.The analysis of viable cell diameter, cell viability, and the total number of viable cells was essential to determine the effect of Hfx. mediterranei BRCE could affect BC cell viability, following the few studies previously published. Our results showed that BRCE treatment hindered cell viability, but it did not reduce it below 50% in most cases. MDA-MB-468 and MCF-7 were the most affected cell lines. The sensitivity of the MCF-7 cell line to haloarchaeal carotenoids has been recently reported with an IC50 value of 21 µg/ml20.One of our main objectives in this study was to determine if Halogeometricum limi (0.72 µg/ml) and Haloplanus vescus (0.045–0.72 µg/ml) exerted anticancer activity on HepG2 cells, leading to a decrease of 23% and 27% viability, respectively21. Recent research indicated that Natrialba sp. M6 pigments could reduce the viability of different cancer cell lines with concentrations ranging between 21.18 and 38.24 µg/ml20. The differences observed between studies might be related to the composition of the extract since it has been recently reported how the culture conditions and the species influence it12.Many studies have explored the antiproliferative activities of carotenoids, but few are focused on haloarchaeal ones. Results reported by Hou and Cui coincide with the results here displayed since they described that carotenoid extracts from 0/G1 cell cycle arrest apoptosis induction53, which was in agreement with a similar effect on MDA-MB-23154. This research on lycopene also demonstrated that it could inhibit protein kinase B (Akt) and mammalian target of rapamycin (mTOR) activation, although the mechanism remains unclear. Recent research has described the potentiating effect of fucoxanthin combined with doxorubicin, a chemotherapeutic drug55. Further research will be necessary to elucidate the mechanism of action behind the impact of haloarchaeal carotenoids on BC cell lines.The sensitivity of the triple-negative cell line MDA-MB-468 should be highlighted. An earlier study has reported this responsiveness to lycopene due to GNatrialba sp. M6 carotenoids20. In addition, lutein has been described to selectively target cancer cells by exerting a pro-oxidant effect since it increases ROS generation in triple-negative cells. In contrast, normal cells remain unaffected56.The normal mammary cell line 184A1 was not significantly affected by the treatment in terms of cell size, viability, adhesion, and concentration, which is in line with previous researchers reporting similar selective effects on cancer cell lines using .Regarding Luminal A cell lines (MCF-7 and T-47D), cell adhesion, viability, and viable cell concentration were altered significantly in both cell lines, whereas cell diameter was differentially influenced. The Luminal subtype seems to be sensitive to haloarchaeal carotenoid treatment+ PR− HER2+). Therefore, more studies would be needed to explore the effects of this treatment also on cell lines representing Luminal B without HER2 overexpression (ER+ PR− HER2−).Similarly, the Luminal B cell line (BT-474) was one of the most affected cell lines since the cell index values differed clearly from the control, probably due to a delay in growth since viability was not strongly influenced, whereas the cell concentration was. However, BT-474 belongs to those Luminal B tumours with overexpression of HER2  are encouraging. Cell adhesion was significantly impaired due to the several variables studied in this article. The diameter, viability, and cell concentration were significantly diminished in both cell lines at the highest concentrations. MDA-MB-468 was more sensitive to treatment than MDA-MB-231, which makes sense considering that MDA-MB-231 is a Basal B cell line with features of claudin-low triple-negative tumours, which usually behave more aggressively than Basal A triple-negative tumours, from which MDA-MB-468 cell line is representative−/low/CD44+, cell surface markers, have been described for this cell line, implying stem cell properties59. However, despite this, both cell lines responded favourably, opening the door for further studies in this area.CD2420. In addition, lycopene suppresses cell proliferation, possibly through p53 and Bax mRNA upregulation60, and can modulate the gap junction intercellular communication in BC61.The molecular mechanisms involved in the effect of haloarchaeal carotenoids in BC cell lines are not precise yet. However, some researchers have described that carotenoids from halophilic archaea might exert an apoptotic influence via caspase activation. They can also inhibit MMP-9 protease, which is involved in invasion, angiogenesis, and metastasis in cancerHfx. mediterranei cell culture conditions might influence carotenoid extracts composition, thus, altering their antioxidant, antiglycemic, and antilipidemic properties12. This study explores the antiproliferative activity of a Hfx. mediterranei carotenoid extract obtained from a cell culture under certain conditions (12.5% SW (w/v), 1.5% glucose, 36.5 °C). For that reason, further studies are required to determine if variations in the composition of BRCE due to culture conditions also affect the antiproliferative effect. Currently, very little information is available about the pharmacological applications that haloarchaeal carotenoids might have. This study provides for the first time the effect of BRCE on different BC cell lines, representative of the four main types of BC tumours. This work presents new insights into the biological activity and potential biomedical applications of complex haloarchaeal carotenoids. The experiments presented here confirmed that Haloferax mediterranei BRCE was cytotoxic to all tested BC cell lines representative of each BC subtype.It is also worth mentioning that recent research has discussed how In contrast, normal mammary tissue cells were not sensitive to the treatment. One of the main limitations of this work is that the contribution of the individual carotenoids conforming the extract to its antiproliferative activity is yet unknown. Consequently, more experimental evidence is required to answer the questions about these natural compounds' potential health benefits and mechanism of action.In conclusion, we have demonstrated that BRCE exerts selective antiproliferative and cytotoxic effects on BC cell lines representative of each intrinsic subtype. Notably, its effect on triple-negative cell line viability is promising. Therefore, considering the aggressiveness and the lack of targeted treatments for this intrinsic subtype, BRCE may be postulated as a potential therapeutic strategy. Finally, our study provides insights into potential applications of haloarchaeal carotenoids and enables advances in understanding its effects in vitro.


Dehalobacterium and Dehalogenimonas, and nZVI as a treatment train to detoxify chlorinated methanes , and 1,2-DCA. First, we showed that CF (500 μM) was fully degraded by 1 g/L nZVI to DCM as a major by-product, which was susceptible to fermentation by Dehalobacterium to innocuous products. Our results indicate that soluble compounds released by nZVI might cause an inhibitory impact on Dehalobacterium activity, avoiding DCM depletion. The DCM dechlorination activity was recovered when transferred to a fresh medium without nZVI. The increase in H2 production and pH was discarded as potential inhibitors. Similarly, a Dehalogenimonas-containing culture was unable to dichloroeliminate 1,2-DCA when exposed to 1 g/L nZVI, but dechlorinating activity was also recovered when transferred to nZVI-free media. The recovery of the dechlorinating activity of Dehalobacterium and Dehalogenimonas suggests that combination of nZVI and bioremediation techniques can be feasible under field conditions where dilution processes can alleviate the impact of the potential inhibitory soluble compounds.Nanoscale zero-valent iron (nZVI) has the potential to degrade a diversity of chlorinated compounds, and it is widely used for remediation of contaminated groundwaters. However, some frequently detected contaminants such as dichloromethane (DCM) and 1,2-dichloroethane  have shown nearly no reactivity with nZVI. Here, we tested the feasibility of combining anaerobic dechlorinating bacteria, The online version contains supplementary material available at 10.1007/s11356-023-25376-z. At the heart of this process are membrane-bound reductive dehalogenases, which use chlorinated compounds as electron acceptors to generate cellular energy while using H2 as electron donor for organohalide respiration  aqueous solution and a 0.9 M ferrous chloride (III)  aqueous solution. Both solutions were prepared using ultrapure water (Milli-Q). Water used to prepare the FeCl3 solution was previously deoxygenated by bubbling with N2. Then, the NaBH4 solution was added dropwise to the FeCl3 solution while stirring at 200 rpm and bubbling with N2. Then, nZVI was washed three times using anaerobic ultrapure water, and it was finally stored in a serum bottle and sealed with Teflon-coated butyl rubber aluminum crimp caps until use to keep anaerobic conditions.Particles of nZVI were synthesized as described elsewhere  estuary sediments and slurry samples collected from a membrane bioreactor operating in a centralized industrial wastewater treatment plant, respectively. Both cultures were cultivated for more than 5 years in an anaerobic defined medium described elsewhere  and Na2S·9H2O and L-cysteine (0.2 mM each) as reducing agents. Dehalobacterium cultures also contained tungsten (22.8 μM) and selenium (24.2 μM). Yeast extract (200 mg/L) and acetate (5 mM) were added to the Dehalobacterium and Dehalogenimonas cultures, respectively. Microcosms containing Dehalobacterium were gassed with N2 (0.4 bar overpressure), and Dehalogenimonas cultures were gassed with N2/CO2  and H2 (added to an overpressure of 0.4 bar) unless otherwise stated. Both media were buffered with bicarbonate solution (0.01 M). When indicated, either Tris-HCl  or HEPES (60 mM) was added as buffer solutions.The cultures containing Dehalobacterium and Dehalogenimonas were inoculated by transferring 3 mL and 5 mL from cultures during the exponential degradation phase of DCM and 1,2-DCA, respectively. The microcosms containing nZVI were not gassed with H2 but with N2/CO2 . Biotic controls were cultivated under static conditions; biotic-abiotic treatments and abiotic controls were incubated under shaking conditions (150 rpm) with the aim of maintaining the nanoparticles in suspension. All treatments were maintained at 25 °C in the dark.Sixty-five mL of the anaerobic medium described above was added into 100-mL serum bottles and sealed with Teflon-coated butyl rubber septa aluminum crimp caps. When indicated, some of the medium components were omitted. At least three treatments were included for each experiment: (i) abiotic controls containing the anaerobic defined medium and nZVI, (ii) biotic controls containing the anaerobic defined medium and the corresponding bacterium, and (iii) biotic-abiotic treatments containing the anaerobic defined medium, nZVI, and the corresponding bacterium. Each microcosm was amended with either 1,2-DCA, CF or DCM . In the biotic treatments, hexamethyldisilazane.The morphological features of nZVI and the interaction between cells and nZVI particles were analyzed with a Zeiss Merlin Field-Emission Scanning Electron Microscope (SEM). Samples were prepared as follows: a drop from the sample was placed in silicon chips, where it was then fixed with 2.5% glutaraldehyde in phosphate buffer 0.1 M for 2 h at 4 °C, post-fixed with 1% osmium tetroxide with 0.8% potassium ferrocyanide for 2 h, and dehydrated in increasing concentrations of ethanol . Finally, it was chemically dried with L32 ratio, which corresponds to a defined oxidation state of iron according to Tan et al.  to measure the oxidation state of nZVI. A FEI Tecnai G2 F20 HR(S) was used. This TEM was equipped with a Gatan Image Filter (GIF) Quantum SE963 operated at 200 kV to obtain energy electron loss spectroscopy (EELS) analysis. This analysis defines an n et al. . Accordicis-1,2-dichloroethene, vinyl chloride, chloroethane, methane, ethene, and 1,2-DCA, the initial oven temperature was 35 °C; it was held for 3 min and then ramped at 10 °C/min to 240 °C, which was held for 4 min  fitted with an Agilent DB-624 column (30 m × 0.32 mm with 0.25 μm film thickness) and a flame ionization detector. Helium was used as the carrier gas (0.9 mL/min), and the injector and detector temperatures were set at 250 and 300 °C, respectively. To analyze CF, propene, We first studied the abiotic dechlorination of CF, DCM, and 1,2-DCA by nZVI and the effect of omitting vitamins (V−) and sulfur-cysteine (S−) from the complete medium (CM) described in the “nZVI synthesis and bacterial cultures” section.Based on previous reports, CF reacted with nZVI producing mainly DCM (Song and Carraway − or bicarbonate (present in our culture medium) can deactivate reaction sites in the nZVI surface , and DCM was detected as a byproduct at very low concentrations (≤ 10 μM) after 115 days Fig. , B. In mcis-1,2-dichloroethene, and 1,2-DCA  and 1,2-DCA (500 μM) was assessed using 1 g nZVI/L. Consistent with previous reports  by a Dehalobacterium was not observed with either 0.5 or 1 g nZVI/L, while biotic microcosms fully fermented DCM in 4 days , which could favor the decomposition of functional groups in proteins and lipopolysaccharides from cell membranes  compared with cultures without nZVI (4.9 ± 0.1) , 8.5, and 9 without nZVI. Degradation of DCM in cultures at pH 7 and 8.5 occurred at similar degradation rates after a lag phase of 6 days, but cultures at pH 9 exhibited an extended lag phase of 18 days before DCM was fully consumed  Fig. . To deteDehalobacterium activity, we buffered the Dehalobacterium-containing cultures with 25 mM Tris-HCl (pH = 7.5) and amended 1 g nZVI/L, but degradation of DCM was not observed for 12 days although pH was 8.2 ± 0.3 . It provides evidence that H2 accumulated because of water hydrolysis by nZVI even though hydrogenotrophs were present in the bacterial consortium  found inhibitory for DCM fermentation in Dehalobacterium , ii) particles of nZVI were removed from microcosms with no addition of H2 , and iii) particles of nZVI were not removed (control). All treatments were amended with 2000 μM DCM after CF depletion.We prepared nine abiotic-biotic microcosms containing y 7 Fig. , three d2 concentration was 4 ± 1 mM  , and complete degradation of DCM was observed after a lag phase of 14 ± 3 days in microcosms derived from treatment “–nZVI, –H2” Fig. , indicatDehalobacterium. Some strategies to attenuate such inhibitory effect include the surface modification of nanoparticles with surface stabilizers such as surfactants or polyelectrolytes, which slow down nZVI reaction rates, the production of H2, the release of ROS and iron ions, and avoid contact between nZVI particles and cells   Fig.  and D annm) Fig.  and E. Anm) Fig. . At the nm) Fig.  and F.FiL32 ratio values  biotic microcosms (control + H2), ii) biotic microcosms without H2 (control -H2), iii) abiotic-biotic microcosms containing 0.5 g nZVI/L, and iv) abiotic-biotic microcosms containing 1 g nZVI/L. Both control treatments removed 1,2-DCA at the same rate with the concomitant production of ethene, while those microcosms containing nZVI did not show 1,2-DCA depletion despite the circumneutral pH and the high production of H2 that could be used as electron donor for Dehalogenimonas .As shown in Fig. nas Fig. . Therefonas Fig. . This isnas Fig. , indicatDehalogenimonas activity by nZVI, our results are not necessarily transferable to field studies. The high complexity of the soil and groundwater composition could avoid the direct contact between nanoparticles and cells while providing optimum conditions for ORB growth .Despite the inhibition of Dehalobacterium and Dehalogenimonas was inhibited at 1 g nZVI/L, which was the optimal concentration of nZVI found to fully remove CF (500 μM). Further experiments discarded that changes in both pH and partial pressure of H2 provoked by nZVI exerted inhibition on Dehalobacterium. The fact that both Dehalogenimonas and Dehalobacterium recovered the dechlorinating capability after being transferred to nZVI-free medium pointed to soluble compounds released by particles as potential inhibitors. These findings have implication for remediation strategies aiming to combine both abiotic and biotic treatments to decontaminate aquifers with mixtures of chlorinated compounds. In particular, our study suggests that factors such as the buffering capacity of groundwaters and dilution processes can play crucial roles in the viability of this combined strategy in bioremediation treatments.Our laboratory tests demonstrated that dechlorinating activity of ESM 1(DOCX 236 kb)