For many cancer types, including colorectal carcinoma (CRC), combined modality treatments have shown to improve outcome, but are frequently associated with significant toxicity, illustrating the need for new therapeutic approaches. Based on preclinical data, TRAIL receptor agonists appeared to be promising agents for cancer therapy especially in combination with DNA damaging regimens. Here, we present the combination of the second-generation TRAIL receptor agonist APG-880 with radiation in a new and clinically relevant 3D model system.To investigate the effect of APG-880 in combination with radiation we performed short-term cytotoxicity and long-term clonogenic survival assays in established CRC cell lines, and in tumor organoids derived from colon cancer patients.APG-880 is a potent inducer of apoptosis in CRC cell lines and in patient-derived CRC organoids. Furthermore, a supra-additive effect on cytotoxicity was found when APG-880 and radiation were combined simultaneously, with combination indices around 0.7. Lastly, in the long-term survival assays, we demonstrated a radiosensitizing effect of APG-880 with dose enhancement factors between 1.3 and 1.5.In a new, clinically relevant CRC-organoid model system we demonstrated a more than additive combined effect between the second-generation TRAIL receptor agonist APG-880 and radiation. The highest incidences occur in Western countries Radiosensitizers are agents that increase the lethal effect of ionizing radiation and, ideally, achieve greater tumor cytotoxicity than would have been expected from the additive effect of each modality. Some radiosensitizers are used in the clinic already, while others are being studied for new indications. For example, non-specific agents such as cisplatin or 5-FU have shown to improve local control and overall survival when added to radiation therapy and are integrated in standard treatment protocols Since apoptosis is an important mechanism of radiation induced cell death, modulation of apoptosis sensitivity has been shown to increase tumor cell kill and relatively spare normal tissues, thereby enhancing therapeutic outcome in vivo, which is independent of cross-linking to Fc receptors A novel, second-generation TRAIL-receptor agonist has been engineered that simultaneously binds up to six TRAIL receptors, thereby improving the ability to form receptor clustering on cancer cells to induce superior anti-tumor efficacy C release The rational for combining TRAIL and radiation in our study, is that these agents activate the two distinct pathways leading to apoptosis in vivo efficacy suggesting that this second generation molecule may be superior to previously tested TRAs Unfortunately, TRAIL-based therapies have not yet led to improved clinical responses mainly because the first-generation TRAIL receptor agonists (TRAs) failed efficacy and did not meet clinical expectations as tested in phase I and II trials Second, the model systems used over the past years to test TRAIL efficacy were suboptimal in their capacity to predict clinical activity. Indeed, 2D cell culturing techniques are now considered structurally and functionally inferior to mimic cancer and are of limited use to predict successful clinical translation 22.1APG-880 stock solution (10.6 mg/ml) was provided by AbbVie , aliquoted in 2 μl portions and stored at −80 °C. Thawed samples were only used once.2.2Colon cancer derived cell lines HCT116 and HT29 were purchased from the American Type Culture Collection (ATCC). Jurkat cell line J16, were kindly provided by prof. dr. J. Borst . All cell lines were grown according to ATCC protocols.2.3Surgical specimens (in case of organoid cultures ITO17 and ITO60), or core needle biopsy material (in case of organoid culture ITO77), collected within a clinical trial at the NKI (NL48824.031.14) were dissected, stored in the central biobank and used for the establishment of organoids. For our experiments we used three different biobank-stored organoids from three different patients. After institutional approval, the biobank stored organoids ITO17, ITO60 and ITO77 were thawed and cultured according to the protocol earlier described Regarding the organoids used in this study, organoid ITO77 was derived from a peritoneal metastasis and organoids ITO17 and ITO60 originated from a primary colorectal carcinoma. Their mismatch repair (MMR) status is known: ITO17 and ITO60 are MMR proficient, while ITO77 is MMR deficient.2.4Cells were exposed to gamma rays from a Gammacell® 40 Exactor  at a dose rate of approximately 1 Gy/min.2.5Apoptosis was determined by staining cells with bis-benzimide or propidium iodide to detect morphological nuclear changes as described earlier 2.6Western blot analysis was performed to detect DR4 and DR5 protein. Cells were treated with radiation and/or APG-880, washed and lysed in Triton lysis buffer as described earlier 2.7DR4 and DR5 epitope expression was determined by flow cytometry as described by Verbrugge et al. 2.8To determine the cytotoxic potential of APG-880 in organoids we dissociated the organoids with TriplE Express  at day 1. We expanded the cells to form small organoids of comparable sizes, and treated them with a defined concentration range at day 4, then performed metabolic activity assays with the CellTiter‐Glo® 3D reagent kit  at day 10.2.9Single cells were plated and allowed to attach before treatment. Cells were irradiated and treated with the indicated amount of APG-880, or sham-treated, immediately after irradiation and cultured for at least 14 days to allow colony formation. Colonies were fixed and stained with 0.2% crystal violet/2.5% glutaraldehyde. Colonies consisting of 50 cells or more were counted. The surviving fraction of cells was calculated by normalizing plating efficiency values of the treated samples to the untreated controls.2.10In order to determine the clonogenic potential of organoids we dissociated growing organoids with TriplE Express  at day 1. We plated the organoid derived single cells in Geltrex LDEV-Free hESC-qualified Reduced Growth Factor Basement Membrane Matrix  and covered the matrix with growth medium as described above. Immediately after seeding the single cell suspension of the organoids, they were treated with the appropriate dose of radiation and/or concentration APG-880 and cultured for 14 days. Pictures of the colonies were taken with a cooled Hamamatsu ORCA R2 Black and White CCD-camera on a Zeiss AxioObserver microscope with a 10x/0.30 ECPlan-Neofluar Ph1 objective, run by ZEN2.3 Zeiss acquisition software. Analysis of the pictures and counting the number of organoids sized at least 50 cubic micrometer was performed with ImageJ. Organoids counts were done manually and double checked by an independent technician, in a blinded setting.2.111/(Dx)1 + (D)2/(Dx)2, and was previously applied by Zerp et al. The effects on cancer cells by ionizing radiation and APG-880 was characterized by calculating the Combination Index (CI) according to the classic isobologram equation described by Chou and Talalay 33.1First, we validated by Western blotting that DR4 and DR5 are expressed in HCT116 and HT-29 cell lines A. Patien3.2To confirm the apoptotic mode of cell death we first validated by Hoechst staining that APG-880 induces DNA condensation/fragmentation, a hallmark of apoptosis in HCT116 and HT29 cell lines and in CRC organoids . To quanDue to the 3D nature of the organoids we used a different quantification method that is presented with the results of the combination experiments in 3.3We next established the sensitivity of the colon carcinoma derived cell lines HCT116 and HT29 towards radiation and APG-880 in clonogenic survival assays, the gold standard for long-term cytotoxicity. HCT116 was more sensitive than HT29 for both treatment modalities A and B.F3.4When both treatment modalities were combined, we found a more than additive effect as quantified by calculating the combination index (CI) A and B. 3.5Because the 3D nature of the organoids hampered the Hoechst quantification method used in the cell lines, we applied a different quantification method for cell death/loss of viability, i.e. the cell titer glow 3D assay that is especially developed for measuring cell death in 3D cell culturing conditions C. The ce3.6In clonogenic survival assays in which APG-880 and radiation were combined, HT29 displayed the most effective decrease in clonogenicity at doses around 100 ng/ml. Above these concentrations APG-880 alone was too cytotoxic and at lower concentrations no reduction in radiation-induced clonogenicity was seen. In 37 of 1.5  and radiation in a clinically relevant organoid model system.We found that APG-880 and radiation show an enhanced combined effect in both short term and long term tumor cytotoxicity assays. The simultaneous activation of the intrinsic pathway by APG-880 and the extrinsic apoptotic pathway by radiation The CRC organoid model presented here was selected for its known high TRAIL receptor expression level. As expected, under control culturing conditions the expression levels of both DR4 and DR5 receptors were readily detectable. Furthermore, the CRC model is a relevant model for drug-radiation combinations as colorectal tumors are frequently treated with radiation alone, or in combination with conventional chemotherapy.in vivo setting was the insufficient clustering and inefficient DISC formation by TRAIL antibodies which likely prohibited effective pro-apoptotic signaling One of the main limitations of the first-generation TRAs in the The tumor organoid models that we use here mimic more closely the heterogeneity and structural similarities of growing tumors Taken together, we demonstrate here that in short-term as well as long-term assays, radiation combined with APG-880 causes an enhanced effect on tumor cell kill in CRC cell lines and in CRC patient-derived organoids. Importantly, this clinically relevant new CRC organoid model system can be leveraged to address mechanistic combination studies with TRAIL-R agonists to better define the role of the apoptotic pathway to radiation treatment sensitivity.This work was partially supported by a research grant from AbbVie Inc. The funding source had no role in the study design. AbbVie Inc. participated in the interpretation of data, review, and approval of this publication.


ATXN7 gene. Patients with this disease suffer from a degeneration of their cerebellar Purkinje neurons and retinal photoreceptors that result in a progressive ataxia and loss of vision. As with many neurodegenerative diseases, studies of pathogenesis have been hindered by a lack of disease-relevant models. To this end, we have generated induced pluripotent stem cells (iPSCs) from a cohort of SCA7 patients in South Africa. First, we differentiated the SCA7 affected iPSCs into neurons which showed evidence of a transcriptional phenotype affecting components of STAGA (ATXN7 and KAT2A) and the heat shock protein pathway (DNAJA1 and HSP70). We then performed electrophysiology on the SCA7 iPSC-derived neurons and found that these cells show features of functional aberrations. Lastly, we were able to differentiate the SCA7 iPSCs into retinal photoreceptors that also showed similar transcriptional aberrations to the SCA7 neurons. Our findings give technical insights on how iPSC-derived neurons and photoreceptors can be derived from SCA7 patients and demonstrate that these cells express molecular and electrophysiological differences that may be indicative of impaired neuronal health. We hope that these findings will contribute towards the ongoing efforts to establish the cell-derived models of neurodegenerative diseases that are needed to develop patient-specific treatments.Spinocerebellar ataxia type 7 (SCA7) is an inherited neurodegenerative disease caused by a polyglutamine repeat expansion in the  ATXN7 gene. Since the translation of this CAG repeat leads to an expanded polyglutamine (polyQ) tract within the resultant protein, SCA7 is classified as a polyQ repeat disorder. Other diseases with a similar pathophysiology include five different SCAs , as well as Huntington disease, dentatorubral-pallidoluysian atrophy and spinal bulbar muscular atrophy  was approximated using the following equation, adapted from : n = (0.3063 x Length of major PCR product in base pairs)– 76.475bp. The major PCR product was defined as the product generating the highest fluorescent peak, as detected using the ABI 3130xl Genetic Analyzer.The length of the CAG repeat Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: PartlyReviewer #3: YesReviewer #4: YesReviewer #5: No**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: NoReviewer #2: YesReviewer #3: I Don't KnowReviewer #4: YesReviewer #5: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: NoReviewer #2: YesReviewer #3: YesReviewer #4: YesReviewer #5: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: YesReviewer #4: YesReviewer #5: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors describe the electrofisiological and molecular features in iPS derivated cells derived from 2 SCA7 patients and 1 control. Although, the results could be interesting the manuscript contains a lot of errors and lacks some fundamental data and results.1. Errors in references. In the page 7 the authors cite the reference 16 what is not their previous article and is not related to the text.2. Material and methods. Retinal differentiation lack some fundamental details. It is not clear until which day of differentiation toward retinal cells, the cells were maintained.3. The Supplementary Fig 2 should include the sequencing results to confirm the the disease genotype4. Page 7. The authors claim” the reprogramming Sendai virus, were confirmed by immunocytochemistry (antibodies listed in S1-2 Tables). The expression of selected pluripotency genes  was determined by quantitative PCR.All results related to confirmation of silencing of the virus  and expression of the endogenous pluripotent genes should be shown in manuscript.Also the shape of the colonies. The alkaline phosphate assay is missing of undifferentiated cells. Also panel should include differentiation capacity to three embryonic layers at least for 2 markers for each layer. And this all for 3 generated lines.Fingerprint analysis of generated lines is missing. Other pluripotency markers are missing for immune in S3 Figure.S3 B Hoecht staining is of low quality for the first figure.Immunocytochemistry has to be shown for all three lines (the best clon of each)It is not clear what C1, C2 and others mean. Different clones of the same patient and Control lines???It is strange that C2 and P2b does not express OCT2. Are they pluripotent? It is not clear why the authors continu the differentiation with iPS lines with no expression of pluripotent markers. Or the authors did not include tha data to confirm a full pluripotency of the generated lines.No data which representative iPS line is shown in S3The data is lacking.5. Results: Fig 1B the immune staining is low quality. The cells have no neuronal forms.Fig 1C It is strange that expression of PAX6 is so low and it is early neural marker.Fig 1 D n=2 for these kind of data is too low.6. Fig4. All cells are recoverin positive. Normally this protocol generates small yield of recoverin positive cells. It is no clear how many baches of differentiation protocol were used for statistical analysis.In conclusion, the manuscript does not have sufficient technical level for publication in PLOS ONE. Experiments, statistics, and other analyses are not performed to a high technical standard and are not described in sufficient detailReviewer #2: Manuscript by Burman, Watson, Smith et al. is a very interesting and valuable study of SCA7 pathology in patient-derived cell lines. These cell lines include iPSCs, reprogrammed from human fibroblasts, as well as cell lines obtained by neuronal differentiation. Neuronal cells were assessed for electrophysiological properties and examined for transcriptional aberrations. Cell lines were characterized comprehensively, both iPSCs and neuronal cell lines.My major concern is about the number of cell lines generated and analyzed. I do understand that generation and characterization of this kind of cell lines is difficult and often troublemaking. But the issue here is whether the use of this number of cell lines is enough for conclusions made. In numerous studies isogenic cell lines are used, which is more proper approach when only small number of cell lines can be used . I would find the results more convincing if the experiments were performed for more cell lines. For example the results presented in the Figure 3E, although the tendency of change is not consistent for SCA7 cell lines, is shown as statistically significant lowering of the measured parameter in Fig. 3F. The same refers to results presented in Fig. 3 G and H.First of all, it is not quite clear from the lines 107-108 and 127-128 how many control cell lines were generated. I see in Fig. S1 that two clones of control cell line were analyzed, but lines 127-128 could be rephrased to state this clearly.For results presented in Fig. 1D, n is 2 for control samples, but actually two clones of the same cell line were used. Were the results very consistent for these clones? If so treating them as two control cell lines is a bit misleading. If no – why?Lines 316-317: Again naming two clones of iPSC line as “two separated iPSC lines” is misleading.Other major comments:- Images in figure 1F for SCA7 and control cell line do not look like performed with identical settings. Also for better comparison they should be at the same magnification.- I cannot see reference to Fig. 1F in the text, whereas reference to Fig. 1E is only in the discussion. The same refers to Fig. 2 A-D. This is confusing.Minor comments:- Line 119: “quantitative RT-PCR” should be mentioned (RT – reverse transcription), instead of “quantitative PCR” - I found some essential references missing, like recent review papers: PMID: 31792895, 31432449; as well as experimental ones: 27999335, 31859031- Table S1: it is valuable also to mention catalogue numbers as some suppliers have several antibodies for specific protein- Fig. 1A: green word “NESTIN” is hardly visible when placed at the image. The same refers to Fig. 4 A-C.- Line 406: two patients can be named a “cohort”?Reviewer #3: Burman et al present the novel generation of patient-derived iPSC models of SCA7. Differentiation of iPSCs to neurons and photoreceptors enabled the finding of electrophysiological immaturity in SCA7 neurons and transcriptional alterations resulting from ATXN7 repeat expansions.The work is original and well carried out and the manuscript is very well written. I am supportive of its publication in PLOS ONE, with some minor consideration for the authors.1) Please can you clarify the number of batches of retinal cells used in Fig 4H (line 386). Is this 2 batches of patient cells relative to 1 control batch, or are both clones represented? I would say that two batches are minimum for this type of analysis.2) Please can you expand upon the iPSC culture conditions. Line 114. KOSR percentage, bFGF concentration. How were the colonies manually passaged?3) Line 150 – how many days of induction was performed before NSCs were produced? How many passages were NPCs cultured for before neural induction. Was this consistent between controls and patients? Neural and glial potential can shift with prolonged NSC passaging and maybe this is worth mentioning in the discussion?4) Consider a statistics section. I am unsure why Fig 2E used Chi-squared, Fig 3C used Kruskal Wallis and Fig 3E/G used ANOVA.MinorLine 342 please correct iPS to iPSCLine 365 please correct sentence, e.g. is “we asked” missing?Line 427 please correct sentence, e.g. is “are significant” missing?Reviewer #4: The manuscript by Burman et al. assesses molecular and electrophysiological features of neurons and retinal photoreceptor cells derived from induced pluripotent stem cells (iPSCs) that were reprogrammed from fibroblasts from two unrelated patients suffering from spinocerebellar ataxia type seven (SCA-7), and compared them with iPSC-derived cells from one healthy control donor. Authors report transcriptional aberrations in SCA-7 patient derived neural stem cells as well as photoreceptor cells, and differences in resting membrane potential and input resistance between control and SCA-7 patient derived neurons. Although the study is mainly descriptive due to the low number of subjects , it is scientifically sound and the results are relevant for future studies on iPSC-based phenotyping of neurodegenerative diseases.I recommend resolving only two very minor issues before publication:1. The references to some of the figures in the main text is incorrect, and – in those cases – does not correspond to the figure legends .2. The sentence in line 365 on p. 18 is incomplete.Reviewer #5: Within the manuscript Burman et al. generated iPSCs from two SCA7 patients and one control, differentiated iPSCs into iPSC-derived neurons and photoreceptors and analysed the molecular and electrophysiological properties of these cells.Unfortunately, the inaccurate and incomplete experimental setup does not allow to draw any final conclusions.This has mainly two major reasons:- The line to line variability independent from the disease background is to high to only use 2 control lines  and 2 patient lines. The authors claim that SCA7 patients show electrophysiological differences, although the electrophysiological results are variable and inconclusive towards a maturation phenotype.Timing of electrical maturation can greatly vary among different iPSC lines. Therefore, in order to compare control to diseased neurons, analysis of multiple electrophysiological parameters might be more predictive. To confirm a developmental functional phenotype analysis of multiple control and patient lines especially at different time points during the differentiation is absolutely essential.- the characterisation of differentiated cells  is too inaccurate. Thereforem it is unclear if line to line differences only occur due to different compositions of cell types and/or different differentiation stages (cultivation artefact).Detailed comments:Electrophysiology:- In general there are nearly no differences between the control (C1a / C1b) and the matching patient line (P2b)Inconsistent data:298 - the spiking response of a cell to current injection can be used to determine the maturation stage of a differentiating neuron424 - Despite the presence of significant differences in spiking responses between the four cell lines, we did not observe a reliable trend between the control and patient derived neurons.- No differences of SCA7 neurons in neuronal maturity as determined by the occurrence of single and multiple action potentials 310 - The resting membrane potentials (Vm) of cells varied significantly across each cell line. The Vm derived from SCA7 patient lines was significantly more hyperpolarised compared to the control cell lines.- No explanation within the text on how resting membrane potential is relevant or indicative for impaired neuronal health or maturation.- Generally, increasingly hyperpolarized resting membrane potentials are a hallmark of maturation. In this respect, pooled SCA7 patient lines show a more mature phenotype.332 - Next, we performed voltage-clamp recordings of the neurons in order to measure the input resistance as well as the voltage-gated sodium and potassium currents . A lower input resistance is associated with neurite outgrowth and increased numbers of ion channels inserted into the plasma membrane during the process of neuronal maturation. (…). Cells derived from SCA7 patient lines had a significantly higher mean input resistance than cells from the control lines .- Generally, neurons change developmentally with a decrease in input resistance. In this respect, pooled SCA7 patient lines show a more immature phenotype341 -  Sodium and potassium currents in neurons differentiated from control and patient iPS cell lines360 - When pooled together, however, we noted that both the Max IK and Max INa were significantly smaller in the cell lines coming from SCA7 patients compared to the control cell lines.- High variance between patient lines, only significantly different from controls when pooled together- Increasing max sodium and potassium currents reflect the process of electrical maturation. In this regard, SCA7 patient lines show a more immature phenotypeExpression analysis:- Why were only NPCs used for the analysis in Fig. 1D? What about iPSC-derived neurons?- It is unclear how the qPCR results were normalized (two control lines?)- High variance in Fig. 4 D-G between the two control lines. How was this normalized in Fig. 4H?General comments:- In depth characterisation of iPSCs is missing - Where multiple housekeeping genes used for qPCR quantification?- p15, l282: Fig 2G-H: data not shown, Fig. 1?- The type of generated neurons is unclear - Which lines were used for the representative images of the ICC stainings?- p19, ll 375-376: Obvious difference: The expression of CRX in control and SCA7 cells seems to be different **********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  4 Dec 2020We thank the Editor and Reviewers for their thoughtful comments and suggestions. In the rebuttal below, we have addressed all the points with our responses shown in italics. We have also highlighted all changes to the original manuscript in yellow. Journal Requirements: 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfResponse: Noted, we have formatted our manuscript according to the PLoS ONE guidelines provided.  2. Thank you for including your ethics statement:'Ethics approval for the study was granted by the University of Cape Town (UCT) Faculty of Health Sciences Human Research Ethics Committee (HREC REF. 380/2009 and 434/2011), and was renewed annually, incorporating amendments to the project protocol where necessary. All methods were carried out in accordance with the guidelines approved by the Ethics Committee. Participants were recruited from the Neurogenetics clinic at Groote Schuur Hospital in Cape Town. Informed consent was obtained from all participants prior to their enrolment in the study.'Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified what type you obtained . If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.Response: The ethics statement has been amended to state that written informed consent was obtained by all participants. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript:'Funding for this work was provided by Ataxia UK, Commonwealth Scholarship Commission (UK), John Fell OUP Fund, National Research Foundation (South Africa), National Research Foundation , Medical Research Council (South Africa), Harry Crossley Foundation, Deutscher Akademischer Austausch Dienst, University of Cape Town Research Council, Wellcome Trust, Parkinson’s Disease UK, Medical Research Council UK, Blue Brain Project, and the James Martin 21st Century School.'We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:'The funders had no role in study design, data collection and analysis, decision topublish, or preparation of the manuscript.'Please include the updated Funding Statement in your cover letter. We will change the online submission form on your behalf.Response: Noted, any funding information has been removed from the Acknowledgements. The Funding Statement is included in the cover letter and can be updated as follows:‘Funding for this work was provided by Ataxia UK, Commonwealth Scholarship Commission (UK), John Fell OUP Fund, National Research Foundation (South Africa), National Research Foundation , Medical Research Council (South Africa), Harry Crossley Foundation, Deutscher Akademischer Austausch Dienst, University of Cape Town Research Council, Wellcome Trust, Parkinson’s Disease UK, Medical Research Council UK, Blue Brain Project, and the James Martin 21st Century School. Electrophysiology equipment was provided by the École Polytechnique Fédérale de Lausanne, Switzerland. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.’ Reviewer #1: Reviewer: The authors describe the electrofisiological and molecular features in iPS derivated cells derived from 2 SCA7 patients and 1 control. Although, the results could be interesting the manuscript contains a lot of errors and lacks some fundamental data and results.Response: We thank the reviewer for the effort put into appraising our manuscript. Reviewer: Errors in references. In the page 7 the authors cite the reference 16 what is not their previous article and is not related to the text.Response: Reference 16 is a general reference describing the establishment of fibroblast cultures from biopsy. The position of the reference in the text has been altered to reflect this.Reviewer: Material and methods. Retinal differentiation lack some fundamental details. It is not clear until which day of differentiation toward retinal cells, the cells were maintained. Response: The text has been amended to state that the retinal differentiation period was 30 days.Reviewer: The Supplementary Fig 2 should include the sequencing results to confirm the the disease genotypeResponse: CAG repeat sizing results are presented in Supplementary Figure 5.Reviewer: Page 7. The authors claim” the reprogramming Sendai virus, were confirmed by immunocytochemistry (antibodies listed in S1-2 Tables). The expression of selected pluripotency genes  was determined by quantitative PCR.All results related to confirmation of silencing of the virus  and expression of the endogenous pluripotent genes should be shown in manuscript. Also the shape of the colonies. The alkaline phosphate assay is missing of undifferentiated cells. Also panel should include differentiation capacity to three embryonic layers at least for 2 markers for each layer. And this all for 3 generated lines. Fingerprint analysis of generated lines is missing. Other pluripotency markers are missing for immune in S3 Figure. S3 B Hoecht staining is of low quality for the first figure. Immunocytochemistry has to be shown for all three lines (the best clon of each) It is not clear what C1, C2 and others mean. Different clones of the same patient and Control lines??? It is strange that C2 and P2b does not express OCT2. Are they pluripotent? It is not clear why the authors continu the differentiation with iPS lines with no expression of pluripotent markers. Or the authors did not include tha data to confirm a full pluripotency of the generated lines. No data which representative iPS line is shown in S3. The data is lacking.Response: We acknowledge that not all pluripotency techniques available were performed, however with clarification below we hope to alleviate the reviewers’ concerns to prove that the necessary characterizations were completed. In certain sections for this question we first refer to the reviewer’s comment and respond in italics:Reviewer: “All results related to confirmation of silencing of the virus  and expression of the endogenous pluripotent genes should be shown in manuscript.”Response: As the reviewer correctly indicates, one method of confirming the absence of non-integrating episomal Sendai virus can be shown by RT-PCR. However, this can equally be shown by IF; i.e. “This clearance rate is clone-dependent and can be confirmed by PCR or by anti-Sendai antibody” . Hence, representative images of clone X are shown in S3 Fig (see panel B). Expression of endogenous pluripotent genes as measured by qRT-PCR in each iPSC clone is shown in S3 Fig panels C-E. These are shown for both control clones (CONA and CONB) and the two patient clonal lines (47Q and 41Q). Expression of pluripotent genes as measured by IF evaluation of protein expression were further performed. Representative images are shown in S3 Fig panel A. Fingerprint analysis of iPSC lines is often used for confirmation that the final cell models are derived from the correct individual. References:Link to manufacturer's instructions:https://tools.thermofisher.com/content/sfs/brochures/CytoTune-iPS%202%200%20Sendai%20Reprogramming%20Kit%20FAQs.pdfLee HK, Morin P, Wells J, Hanlon EB, Xia W. Induced pluripotent stem cells (iPSCs) derived from frontotemporal dementia patient's peripheral blood mononuclear cells. Stem Cell Research. 2015; 15(2): 325-27. doi: 10.1016/j.scr.2015.07.004Reviewer: “Also panel should include differentiation capacity to three embryonic layers at least for 2 markers for each layer.”Response: We thank the reviewer for pointing out the omissions. Whilst we did not complete double labelling, we followed prior art as shown in other publications  and have inserted a new supplementary figure and added a caption to the manuscript . Reference: https://doi.org/10.1371/journal.pone.0087388Hartfield EM, Yamasaki-Mann M, Ribeiro Fernandes HJ, Vowles J, James WS, Cowley SA, et al. (2014) Physiological Characterisation of Human iPS-Derived Dopaminergic Neurons. PLoS ONE 9(2): e87388. Reviewer: “Different clones of the same patient and Control lines???Response: We agree that the naming of the iPSC lines could be misleading and therefore have renamed them as follows: PSCs from an affected patient and her affected daughter were used to generate two patient derived lines from each respective individual:� 47Q� 41QiPSCs from an unaffected sibling genetically confirmed to have a normal allele size control was used to generate two clones:� CONA� CONB This has been corrected in the text and the figures and is specifically highlighted in S1 Fig. As per the methods section, “Primary dermal fibroblast cultures were established from punch skin biopsies (16) taken from the inner forearm of two unrelated SCA7 patients (41Q and 47Q) and an unaffected control individual ”. As we had samples from two SCA7 patients but only dermal fibroblasts from one control individual we generated two clones from the control to minimise differences in epigenetic reprogramming Reviewer: “It is strange that C2 and P2b does not express OCT2. Are they pluripotent? It is not clear why the authors continu the differentiation with iPS lines with no expression of pluripotent markers.”Response: As per our previous comment above, all the iPSC clones generated do express OCT4 (which we believe the reviewer is speaking to) to show pluripotency in S3 Fig in addition to the additional pluripotency data we show as outlined above. If we understand the reviewer correctly, they may be referring to Fig 1C where we show that neural progenitors from these clones no longer express OCT4 to indicate they have lost pluripotency and are moving towards PAX6 neural progenitor cells as indicated in the figure legend. Reviewer: “No data which representative iPS line is shown in S3.”Response: iPSC clone P1 (now 47Q) is shown as a representative image in S5 Fig. Reviewer: Fig 1B the immune staining is low quality. The cells have no neuronal forms.Fig 1C It is strange that expression of PAX6 is so low and it is early neural marker.Fig 1 D n=2 for these kind of data is too low.Response: These are not fully differentiated neurons, and more closely resemble neuroepithelial cells in culture. In each case, n = 2 biological replicates, performed in technical triplicate. The text has been amended to reflect this.Reviewer: Fig4. All cells are recoverin positive. Normally this protocol generates small yield of recoverin positive cells. It is no clear how many baches of differentiation protocol were used for statistical analysis.Response: The methods section has been updated to include the following description: “Each patient and control line underwent two rounds of differentiation, with each time point analysed in biological duplicate.” Reviewer #2: Reviewer: Manuscript by Burman, Watson, Smith et al. is a very interesting and valuable study of SCA7 pathology in patient-derived cell lines. These cell lines include iPSCs, reprogrammed from human fibroblasts, as well as cell lines obtained by neuronal differentiation. Neuronal cells were assessed for electrophysiological properties and examined for transcriptional aberrations. Cell lines were characterized comprehensively, both iPSCs and neuronal cell lines.Response: We thank the reviewer for the thoughtful and encouraging comments. Reviewer: My major concern is about the number of cell lines generated and analyzed. I do understand that generation and characterization of this kind of cell lines is difficult and often troublemaking. But the issue here is whether the use of this number of cell lines is enough for conclusions made. In numerous studies isogenic cell lines are used, which is more proper approach when only small number of cell lines can be used . I would find the results more convincing if the experiments were performed for more cell lines. For example the results presented in the Figure 3E, although the tendency of change is not consistent for SCA7 cell lines, is shown as statistically significant lowering of the measured parameter in Fig. 3F. The same refers to results presented in Fig. 3 G and H.Response: The reviewer raises an important point. Indeed, we considered this as a strategy, however generating isogenic lines from CAG triplet repeat disorders is extremely challenging. For example, Dastidar et al (2018) were able to excise the CTG repeat expansion in myotonic dystrophy patient-derived iPSCs. Due to the sequence homology between the wild-type and mutant alleles, the subsequent cells were shown to include CRISPR-mediated targets on the wild-type allele. Whilst this did not impact the protein coding region of the DMPK gene because of the 3’UTR location of the triplet repeat, the same strategy targeting the disease-causing repeat in ataxin-7 would likely render the wild-type protein non-functional via a frameshift. Furthermore, the precise correction of 42 to 12 CAG repeats is incredibly technically challenging given the repetitive nature required of the repair template. Thus we were restricted to patient derived iPSC lines. Whilst these are not isogenic they do retain significantly similar background genetics, as they are all generated from the same immediate family, minimising (to a degree) the extensive differences between unrelated individuals. In addition, whilst SCA7 patients are more common in South Africa, this remains a rare genetic disease and we were limited to two patients. To acknowledge the reviewer’ concern we have added the following to the text in the discussion, “It remains, however, technically challenging to target the disease-causing repeat in ataxin-7 and retain the endogenous regulatory landscape. Due to the sequence homology of the wild -type allele, such a strategy would likely render the wild-type protein non-functional via a frameshift.”Reviewer: First of all, it is not quite clear from the lines 107-108 and 127-128 how many control cell lines were generated. I see in Fig. S1 that two clones of control cell line were analyzed, but lines 127-128 could be rephrased to state this clearly.Response: We have amended the methods section lines 116 and 117 to clarify this and to ensure it is clear throughout the manuscript. For results presented in Fig. 1D, n is 2 for control samples, but actually two clones of the same cell line were used. Were the results very consistent for these clones? If so treating them as two control cell lines is a bit misleading. If no – why?Lines 316-317: Again, naming two clones of iPSC line as “two separated iPSC lines” is misleading.Response: We have amended the methods section to clarify this and to ensure it is clear throughout the manuscript. Reviewer: Images in figure 1F for SCA7 and control cell line do not look like performed with identical settings. Also for better comparison they should be at the same magnification.Response: We thank the reviewer for noting this. We have corrected the image to include the same area imaged with the correct (same) magnification.Reviewer: I cannot see reference to Fig. 1F in the text, whereas reference to Fig. 1E is only in the discussion. The same refers to Fig. 2 A-D. This is confusing.Response: We amended the text to ensure all figures are referenced correctly. Reviewer: Line 119: “quantitative RT-PCR” should be mentioned (RT – reverse transcription), instead of “quantitative PCR” .Response: The text has been edited accordingly.Reviewer: I found some essential references missing, like recent review papers: PMID: 31792895, 31432449; as well as experimental ones: 27999335, 31859031 Response: We thank the reviewer for making us aware of these references. We have included them in the following section of the introduction: “More recently, dysfunction in cell metabolism has been proposed as an additional pathogenic mechanism . As with many neurodegenerative conditions, research into the molecular pathogenesis of SCA7 has been hindered by a lack of suitable models of human disease progression. This is particularly relevant in cases where the genomic context of the mutation may have an impact on gene function and might prove useful for therapeutic development. There does, however, seem to be increasing momentum in this area with new models being proposed and refined (10-12).”Reviewer: Table S1: it is valuable also to mention catalogue numbers as some suppliers have several antibodies for specific proteinResponse: These have been added. Reviewer: Fig. 1A: green word “NESTIN” is hardly visible when placed at the image. The same refers to Fig. 4 A-C.Response: We have adjusted the colours of the labels to a lighter shade so that they stand more clearly against the darker images. Reviewer: Line 406: two patients can be named a “cohort”?Response: We agree with the Reviewer that the term ‘cohort’ is inappropriate when including only two patients. In the context of our study, however, we refer the South African cohort as being a group of more than two patients who have been diagnosed with SCA7. From this cohort of patients, only two patients were available and willing to participate in this study.  Reviewer #3: Burman et al present the novel generation of patient-derived iPSC models of SCA7. Differentiation of iPSCs to neurons and photoreceptors enabled the finding of electrophysiological immaturity in SCA7 neurons and transcriptional alterations resulting from ATXN7 repeat expansions.The work is original and well carried out and the manuscript is very well written. I am supportive of its publication in PLOS ONE, with some minor consideration for the authors.Response: We thank the reviewer for the encouraging remarks. Reviewer: Please can you clarify the number of batches of retinal cells used in Fig 4H (line 386). Is this 2 batches of patient cells relative to 1 control batch, or are both clones represented? I would say that two batches are minimum for this type of analysis.Response: The data in this figure includes the cell lines from both patients (P1 and P2b now changed to 47Q and 41Q) relative to one of the control lines (C1a now CONA). A comparison against both control lines was not deemed necessary as there were shown to have similar expression profiles. We have included this clarification in the figure caption. Reviewer: Please can you expand upon the iPSC culture conditions. Line 114. KOSR percentage, bFGF concentration. How were the colonies manually passaged?Response: We have corrected the following in the text: One week after infection, the cells were transferred to mitomycin C-inactivated feeder cells in stem cell medium . Half of the medium was refreshed every day and colonies were expanded by manual passaging (dissection of colonies using needles) on feeder layers as previously described .Reviewer: Line 150 – how many days of induction was performed before NSCs were produced? How many passages were NPCs cultured for before neural induction. Was this consistent between controls and patients? Neural and glial potential can shift with prolonged NSC passaging and maybe this is worth mentioning in the discussion?Response: NPCs were observed in culture after seven days of differentiation, and neurite outgrowth was also typically observed after approximately one week of switching NPCs to neuronal differentiation media. The text has been edited to reflect this. Every attempt was made to treat patient and control lines consistently, to ensure accurate comparisons could be made.Reviewer: Consider a statistics section. I am unsure why Fig 2E used Chi-squared, Fig 3C used Kruskal Wallis and Fig 3E/G used ANOVA.Response: We have included a more detailed description on the choice of statistical tests in the ‘Electrophysiology’ part of the Methods section. To answers the Reviewer’s comments directly: 1) In Fig. 2E a Chi-squared test was used to assess for differences in the proportion or fraction (%) of cells in each category of cell firing across the different cell lines. 2) In Fig. 3C we realise that we incorrectly wrote Kruskal Wallis instead of ANOVA. This was a typographical error and we confirm that an ANOVA test was used. We have corrected this in the text. The ANOVA is the statistical test of choice as it allows us to identify differences across all cell lines. Minor:Line 342 please correct iPS to iPSC Line 365 please correct sentence, e.g. is “we asked” missing?Line 427 please correct sentence, e.g. is “are significant” missing?Response: We thank the reviewer and have corrected these minor comments.  Reviewer #4: The manuscript by Burman et al. assesses molecular and electrophysiological features of neurons and retinal photoreceptor cells derived from induced pluripotent stem cells (iPSCs) that were reprogrammed from fibroblasts from two unrelated patients suffering from spinocerebellar ataxia type seven (SCA-7), and compared them with iPSC-derived cells from one healthy control donor. Authors report transcriptional aberrations in SCA-7 patient derived neural stem cells as well as photoreceptor cells, and differences in resting membrane potential and input resistance between control and SCA-7 patient derived neurons. Although the study is mainly descriptive due to the low number of subjects , it is scientifically sound and the results are relevant for future studies on iPSC-based phenotyping of neurodegenerative diseases. I recommend resolving only two very minor issues before publication: Reviewer: The references to some of the figures in the main text is incorrect, and – in those cases – does not correspond to the figure legends .Response: We have corrected the text to ensure all figures are referenced correctly. Reviewer: The sentence in line 365 on p. 18 is incomplete.Response: We thank the reviewer and have corrected. Reviewer #5: Within the manuscript Burman et al. generated iPSCs from two SCA7 patients and one control, differentiated iPSCs into iPSC-derived neurons and photoreceptors and analysed the molecular and electrophysiological properties of these cells. Unfortunately, the inaccurate and incomplete experimental setup does not allow to draw any final conclusions.Response: We thank the reviewer for the effort put into appraising our manuscript. This has mainly two major reasons:The line to line variability independent from the disease background is to high to only use 2 control lines  and 2 patient lines. The authors claim that SCA7 patients show electrophysiological differences, although the electrophysiological results are variable and inconclusive towards a maturation phenotype. Timing of electrical maturation can greatly vary among different iPSC lines. Therefore, in order to compare control to diseased neurons, analysis of multiple electrophysiological parameters might be more predictive. To confirm a developmental functional phenotype analysis of multiple control and patient lines especially at different time points during the differentiation is absolutely essential.The characterisation of differentiated cells  is too inaccurate. Therefore, it is unclear if line to line differences only occur due to different compositions of cell types and/or different differentiation stages (cultivation artefact).Response: We thank the reviewer for these comments. We accept and fully acknowledge that the limited number of cell lines and large variability make it difficult to make firm conclusions from our data. We are completely transparent about this and have aimed to address these issues to the best of our ability and within our limited capacity. We do, however, believe that our study does provide useful experimental insights to those who are working in this field. Detailed comments:Electrophysiology: Reviewer: In general there are nearly no differences between the control (C1a / C1b) and the matching patient line (P2b)Response: We acknowledge that there are limited difference between the patient and control lines. We are transparent about this and explain that this is likely due to the limited number of cell lines. Reviewer: 298 - the spiking response of a cell to current injection can be used to determine the maturation stage of a differentiating neuronResponse: We followed a similar approach used by other studies which use spiking response to assess neuronal maturation in different stem cell lines. Specific references include: Moore AR, Filipovic R, Mo Z, Rasband MN, Zecevic N, Antic SD. Electrical excitability of early neurons in the human cerebral cortex during the second trimester of gestation. Cerebral Cortex. 2008;19(8):1795-805.Perrier AL, Tabar V, Barberi T, Rubio ME, Bruses J, Topf N, et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(34):12543-8.Vazin T, Becker KG, Chen J, Spivak CE, Lupica CR, Zhang Y, et al. A novel combination of factors, termed SPIE, which promotes dopaminergic neuron differentiation from human embryonic stem cells. PloS one. 2009;4(8):e6606.Belinsky GS, Moore AR, Short SM, Rich MT, Antic SD. Physiological properties of neurons derived from human embryonic stem cells using a dibutyryl cyclic AMP-based protocol. Stem cells and development. 2011;20(10):1733-46.Reviewer: 424 - Despite the presence of significant differences in spiking responses between the four cell lines, we did not observe a reliable trend between the control and patient derived neurons.Response: As above, we are transparent about the limited differences between the control and patient derived neurons we observed. Reviewer: No differences of SCA7 neurons in neuronal maturity as determined by the occurrence of single and multiple action potentials Response: As above, we are transparent about the limited differences between the control and patient derived neurons. Reviewer: 310 - The resting membrane potentials (Vm) of cells varied significantly across each cell line. The Vm derived from SCA7 patient lines was significantly more hyperpolarised compared to the control cell lines.Response: We acknowledge that there was significantly variability across the different cell lines with this being more apparent in some parameters compared to others. Reviewer: No explanation within the text on how resting membrane potential is relevant or indicative for impaired neuronal health or maturation. Generally, increasingly hyperpolarized resting membrane potentials are a hallmark of maturation. In this respect, pooled SCA7 patient lines show a more mature phenotype.332 - Next, we performed voltage-clamp recordings of the neurons in order to measure the input resistance as well as the voltage-gated sodium and potassium currents . A lower input resistance is associated with neurite outgrowth and increased numbers of ion channels inserted into the plasma membrane during the process of neuronal maturation. (…). Cells derived from SCA7 patient lines had a significantly higher mean input resistance than cells from the control lines . Generally, neurons change developmentally with a decrease in input resistance. In this respect, pooled SCA7 patient lines show a more immature phenotypeResponse: A neuron’s resting membrane potential (Vm) affects its excitability. Neurons with more hyperpolarised potentials are less excitable . Disease processes have been associated with either depolarised  or hyperpolarised Vm . Typically, however, a more hyperpolarised Vm is associated with neurons that are more mature. To highlight this point, we have added the following to our discussion: “A more hyperpolarised membrane potential is usually associated with neuronal maturity whist the high input resistance is usually associated with neuronal immaturity. These conflicting findings will need to be further explored in future studies to provide a more conclusive understanding as to how SCA7 specific changes cause changes in neuronal maturation or drive alternative functional alterations. We can, however, postulate that the more hyperpolarised membrane potential in the patient cells may be indicative of a reduction in the excitability of neurons containing mutant ATXN7 in response to synaptic input.”References:https://doi.org/10.1016/j.expneurol.2006.03.029Jeub M, Herbst M, Spauschus A, Fleischer H, Klockgether T, Wuellner U, Evert BO. Potassium channel dysfunction and depolarized resting membrane potential in a cell model of SCA3. Experimental neurology. 2006;201(1):182-92. doi: https://doi.org/10.1093/brain/awf041Kiernan MC, Guglielmi JM, Kaji R, Murray NM, Bostock H. Evidence for axonal membrane hyperpolarization in multifocal motor neuropathy with conduction block. Brain. 2002 Mar 1;125(3):664-75. doi: Reviewer: 341 -  Sodium and potassium currents in neurons differentiated from control and patient iPS cell lines. 360 - When pooled together, however, we noted that both the Max IK and Max INa were significantly smaller in the cell lines coming from SCA7 patients compared to the control cell lines. High variance between patient lines, only significantly different from controls when pooled togetherResponse: We acknowledge that there are significant differences between patient lines and for this reason the pooled data (control vs SCA7) is misleading. For this reason, we have opted to remove the pooled data from the figures and text. We now report the cell lines individually and report on observed trends whilst highlighting that these would need be further explored in future studies. Reviewer: Increasing max sodium and potassium currents reflect the process of electrical maturation. In this regard, SCA7 patient lines show a more immature phenotypeResponse: We are hesitant to draw strong conclusions from this data given the variability of the patient lines. Expression analysis:Reviewer: Why were only NPCs used for the analysis in Fig. 1D? What about iPSC-derived neurons?Response: The low cell numbers and heterogeneity of mature neuronal cultures proved a challenge to obtaining significant biological material to perform reliable expression analysis. Hence, the decision was made to perform this analysis on the relatively homogeneous NPC cultures, with the view that these cells may yield insights into the early cellular dysfunction underlying SCA7 pathology.Reviewer: It is unclear how the qPCR results were normalized (two control lines?)Response: Results are shown relative to the mean of the two control lines (set as 1), and normalised to the housekeeping gene, β-actin.Reviewer: High variance in Fig. 4 D-G between the two control lines. How was this normalized in Fig. 4H?Response: As above, Results are shown relative to the mean of the two control lines (set as 1), and normalised to the housekeeping gene, β-actin.General comments:Reviewer: In depth characterisation of iPSCs is missing Response: We followed prior art as shown in other publications  and have inserted a new supplementary figure  Reference: https://doi.org/10.1371/journal.pone.0087388Hartfield EM, Yamasaki-Mann M, Ribeiro Fernandes HJ, Vowles J, James WS, Cowley SA, et al. (2014) Physiological Characterisation of Human iPS-Derived Dopaminergic Neurons. PLoS ONE 9(2): e87388. Reviewer: Where multiple housekeeping genes used for qPCR quantification? Response: Only β-actin was used as a housekeeping gene, as its levels were observed not to vary according to disease state. Reviewer: p15, l282: Fig 2G-H: data not shown, Fig. 1?Response: This was a typographical error and has been corrected. The figures are now correctly referenced. Reviewer: The type of generated neurons is unclear Response: Fig 1F shows that a proportion of the neurons stain positive for GABA. However, as this was intended as a pilot study on a small number of patients and controls, extensive phenotyping of the neuronal population was not conducted.Reviewer: Which lines were used for the representative images of the ICC stainings?Response: For Fig 1, CONA and 41Q were used as representative control and patient lines, respectively. Pluripotency staining images in S4 Fig. used CONB.Reviewer: p19, ll 375-376: Obvious difference: The expression of CRX in control and SCA7 cells seems to be different Response: Whilst representative images have been selected for the figure, no consistent differences in expression were noted across the differentiation experiments. 30 Dec 2020Pécs, HungaryDecember 28, 2020PONE-D-20-27215R1Molecular and electrophysiological features of spinocerebellar ataxia type seven in induced pluripotent stem cellsPLOS ONEDear Dr. Burman,Thank you for submitting your manuscript (R1 version) to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised by Reviewer #2 and Reviewer #5, listed below.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Feb 13 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at Please include the following items when submitting your revised manuscript:A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsIf applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: We look forward to receiving your revised manuscript.Kind regards,Joseph Najbauer, Ph.D.Academic EditorPLOS ONE[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #2: (No Response)Reviewer #3: All comments have been addressedReviewer #4: All comments have been addressedReviewer #5: (No Response)**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: PartlyReviewer #3: YesReviewer #4: YesReviewer #5: No**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesReviewer #3: YesReviewer #4: YesReviewer #5: No**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: NoReviewer #3: YesReviewer #4: YesReviewer #5: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: YesReviewer #4: YesReviewer #5: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors addressed properly all my concerns. They replied correctly.Reviewer #2: The majority of my comments was sufficiently addressed, except for the comment concerning interpretation of the results based on two SCA7 cell lines analyzed.First of all, I am not convinced by explanation for not generating isogenic cell lines by comparison with DMPK gene. I know that the CRISPR/Cas-9-based strategy is difficult to be applied for repeated tracts and its application for this study would take a long time. Still, there are studies like PMID: 28238795 and 32182692 which show application of such strategy for huntingtin, where CAG repeat tract is also present in ORF. It can be referred and commented instead.Unfortunately, in attached files I could not access current version of Fig. 3. (zip files contained only main figures 1 and 4) which was changed as I see in this figure legend and the interpretation.Reviewer #3: The authors have satisfactorily addressed my comments.I recommend that this manuscript is appropriate for publication.Reviewer #4: (No Response)Reviewer #5: Unfortunately the revised manuscript "Molecular and electrophysiological features of spinocerebellar ataxia type seven in induced pluripotent stem cells" does not lead to any improvement in either of the two main critical points.1.) Since the line to line variability is too high, no conclusive results can be obtained.2.) Even more important, the identity of differentiated cell types  is still more or less unknown. As the authors stated in their response: "The low cell numbers and heterogeneity of mature neuronal culturesproved a challenge to obtaining significant biological material to perform reliable expression analysis." If even such an analysis was not possible, then the results of the electrophysiological recordings are inappropriate. The maturity and the cellular composition/identity of differentiated neurons determines the electrophysiological properties. Therefore, it can not be excluded that the "features of functional aberrations" in SCA7 iPSC-derived neurons are a cell culture artefact rather than a consequence of the mutation.Without any additional experiments to prove the identity of differentiated neurons  and photoreceptors no conclusive findings can be formulated.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  16 Jan 2021We thank the Editor and Reviewers for their thoughtful comments and suggestions. In the rebuttal below, we have addressed all the points with our responses shown in italics. We have also highlighted all changes to the original manuscript in yellow. Reviewer #2:Reviewer: The majority of my comments was sufficiently addressed, except for the comment concerning interpretation of the results based on two SCA7 cell lines analyzed.First of all, I am not convinced by explanation for not generating isogenic cell lines by comparison with DMPK gene. I know that the CRISPR/Cas-9-based strategy is difficult to be applied for repeated tracts and its application for this study would take a long time. Still, there are studies like PMID: 28238795 and 32182692 which show application of such strategy for huntingtin, where CAG repeat tract is also present in ORF. It can be referred and commented instead.Response: We thank the reviewer for these comments and agree with what has been suggested. We have revised the manuscript to expand our discussion on future experiments and the application of CRISPR/Cas-9-based strategies (see lines 548-557). We also thank the Reviewer for the references they have suggested which we have now included in our manuscript. Reviewer: Unfortunately, in attached files I could not access current version of Fig. 3. (zip files contained only main figures 1 and 4) which was changed as I see in this figure legend and the interpretation. Response: We apologise to the Reviewer for this technical glitch. We can confirm that all the Figures were successfully uploaded during the resubmission. We have ensured that all Figures are again uploaded successfully upon submission of this revision. Reviewer #5:Reviewer: Unfortunately the revised manuscript "Molecular and electrophysiological features of spinocerebellar ataxia type seven in induced pluripotent stem cells" does not lead to any improvement in either of the two main critical points: 1.) Since the line to line variability is too high, no conclusive results can be obtained.2.) Even more important, the identity of differentiated cell types  is still more or less unknown. As the authors stated in their response: "The low cell numbers and heterogeneity of mature neuronal culturesproved a challenge to obtaining significant biological material to perform reliable expression analysis." If even such an analysis was not possible, then the results of the electrophysiological recordings are inappropriate. The maturity and the cellular composition/identity of differentiated neurons determines the electrophysiological properties. Therefore, it cannot be excluded that the "features of functional aberrations" in SCA7 iPSC-derived neurons are a cell culture artefact rather than a consequence of the mutation. Without any additional experiments to prove the identity of differentiated neurons  and photoreceptors no conclusive findings can be formulated. Response: We thank the Reviewer for their critical appraisal of our study. We agree with the Reviewer that given the significant variability between cell lines, it is not appropriate to make conclusive statements from the findings. We have, however, avoided from doing so and have instead aimed to present our results as they are whilst fully acknowledging the limitations. In this revision, we have ensured that the tone of our manuscript does not mislead the reader into thinking that our findings are conclusive and have again highlighted the limitations. In addition, we clearly highlight what future research is needed to take the work forward. We do believe our paper does still provide technical insights that may prove useful to others especially those considering embarking on developing or refining iPSC models of SCA7. While we agree that the experiments you suggest to prove the identity of the differentiated cell are warranted, at present this is not possible. This is largely due to the significant negative impact the ongoing COVID-19 pandemic has had on our research efforts as well as the ongoing restrictions it is having on our capacity.Attachmentburman et al Response to Reviewers Jan 2021.docxSubmitted filename: Click here for additional data file. 8 Feb 2021Pécs, HungaryFebruary 7, 2021Molecular and electrophysiological features of spinocerebellar ataxia type seven in induced pluripotent stem cellsPONE-D-20-27215R2Dear Dr. Burman,We’re pleased to inform you that your manuscript (R2 version) has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Joseph Najbauer, Ph.D.Academic EditorPLOS ONE------------------------------------------------------Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #2: All comments have been addressedReviewer #5: (No Response)**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: YesReviewer #5: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: YesReviewer #5: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #2: YesReviewer #5: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #2: YesReviewer #5: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #2: (No Response)Reviewer #5: I fully understand that under the current COVID 19-crisis it is difficult to perform the necessary experiments to address my two main points of criticism .I still believe that analyzing patients to controls without knowing the identity and potential differences of the differentiated cell type is a major error which makes any interpretation of the existing data difficult.Nevertheless this study may be a good starting point for the establishment of a patient-derived model to investigate pathogenic mechanism in SCA7.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #2: NoReviewer #5: No 15 Feb 2021PONE-D-20-27215R2 Molecular and electrophysiological features of spinocerebellar ataxia type seven in induced pluripotent stem cells Dear Dr. Burman:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. 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Background: PTEN-deficient tumors are dependent on PI3Kβ activity, making PI3Kβ a compelling target. We evaluated the efficacy of PI3Kβ inhibitor AZD8186 on tumors with PTEN loss.Results:In vitro cell viability assay and immunoblotting demonstrated that PTEN loss was significantly correlated with AZD8186 sensitivity in triple negative breast cancer (TNBC) cell lines. Colony formation assay confirmed sensitivity of PTEN-deficient cell lines to AZD8186. AZD8186 inhibited PI3K signaling in PTEN loss TNBC cells. AZD8186 in combination with paclitaxel, eribulin had synergistic effects on growth inhibition in PTEN loss cells. AZD8186 promoted apoptosis in PTEN loss cells which was synergized by paclitaxel. In vivo, AZD8186 had limited activity as a single agent, but enhanced antitumor activity when combined with paclitaxel in MDA-MB-436 and MDA-MB-468 cell-line xenografts. AZD8186 significantly enhanced antitumor efficacy of anti-PD1 antibodies in the PTEN-deficient BP murine melanoma xenograft model, but not in the PTEN-wild-type CT26 xenograft model.Methods:In vitro, cell proliferation and colony formation assays were performed to determine cell sensitivity to AZD8186. Immunoblotting was performed to assess PTEN expression and PI3K signaling activity. FACS was performed to evaluate apoptosis. In vivo, antitumor efficacy of AZD8186 and its combinations were evaluated.Conclusions: AZD8186 has single agent efficacy in PTEN-deficient TNBC cell lines in vitro, but has limited single agent efficacy in vivo. However, AZD8186 has enhanced efficacy when combined with paclitaxel and anti-PD1 in vivo. Further study is needed to determine optimal combination therapies for PTEN-deficient solid tumors. Phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway is an important regulator of many physiological cellular processes that promote differentiation, proliferation and survival of a normal cell . In cancet al demonstrated that PTEN-deficient tumors are dependent on PI3Kβ catalytic isoform activity [In vitro, they revealed significant growth inhibition of PTEN-deficient tumors by depleting PIK3CB which encodes PI3Kβ, while no such growth inhibition effect was shown in corresponding PTEN-deficient tumors with downregulation of PIK3CA or PIK3CD encoding PI3Kα and PI3Kδ, respectively. The pathway inactivation and subsequent growth inhibition as a result of downregulation of PI3Kβ isoform were confirmed in vivo. Thus, PI3Kβ isoform is the driver of abnormal proliferation in PTEN-null cancers, and as such, PI3Kβ is a promising target for therapy in PTEN-deficient TNBC.PI3K family is composed of four classes  of intracellular signal transducer enzymes capable of phosphorylating phosphatidylinositol. PI3K-p110α, PI3K-p110β, PI3K-p110γ, and PI3K-p110δ  are the members of class I. Recently, Wee AZD8186 is a selective and potent small-molecule inhibitor of PI3Kβ, with additional activity against PI3Kδ isoform –14. Its et al. reported that PTEN loss increased PD-L1 expression in TNBC [Mittendorf  in TNBC . In mela in TNBC . Further in TNBC .In vivo confirmation of in vitro identified combinations was performed. Further, the antitumor efficacy of AZD8186 in combination with anti-PD1 inhibitor was evaluated in syngeneic mouse models of varying PTEN status and tumor types.We sought to determine the antitumor efficacy of AZD8186 as a single agent and in combination with standard chemotherapeutic agents in TNBC cell lines with varying PTEN status. PTEN mutation that did not result in loss of PTEN protein.To evaluate the antitumor efficacy of AZD8186 in TNBC, we tested AZD8186 sensitivity in a panel of 10 TNBC cell lines with varying PTEN genotypes. First, we examined expression of PI3Kβ, the target of AZD8186, in these cell lines. Immunoblotting showed that all these TNBC cell lines, including the PTEN null cell lines, expressed PI3Kβ at various levels . Westernin vitro studies [50 less than 1 μM are sensitive to AZD8186. Cell lines were treated for 72 hours with DMSO or AZD8186. Cell growth was measured using SRB colorimetric assay. IC50 calculated by dose-response isobologram curves was used to evaluate sensitivity. The results showed that out of ten TNBC cell lines, three  were sensitive to AZD8186 with IC50 of 31, 358, and 899 nM, respectively   . Togethep = 0.037 and 0.0002, respectively)  , 3B-1. Octively) , 3B-1. Tctively) . Normalictively) , 3B-4. Ictively) , 3B-4. Ictively) , but AZDctively) , 3B-4.The effect of AZD8186 on cell-cycle progression was evaluated in MDA-MB-436 and MDA-MB-468 cell lines. The cells were treated with AZD8186 at 1 μM or DMSO for 48 hours and subsequently harvested. Percentage of cells in G1, S and G2-M phases of the cell cycle were determined by flow cytometry using propidium iodide. We found that AZD8186 did not induce significant changes in the percentage of each of cell cycle phases , 3C-2.50 was calculated for single agent treatment alone and the combination. Combination index (CI) values were then calculated using the Chou-Talalay method, where a CI value <0.8 indicates synergism, 0.8 to 1.2 indicates addition and a CI greater than 1.2 indicates antagonism [To determine the effects of AZD8186 in combination with standard chemotherapy, we selected three chemotherapeutic agents commonly used for breast cancer therapy: paclitaxel (microtubule stabilizer), carboplatin  and eribulin (microtubule inhibitor). MDA-MB-436, MDA-MB-468, Sum-159, and MFM-223 cells were treated with serial concentration dilutions of AZD8186 in combination with serial dilutions of the three various chemotherapy agents. After 72 hours of treatment, growth inhibition was assessed with SRB assay, and ICtagonism , 22. Thep = 0.006 . The above four cell lines were treated with AZD8186 at 2 μM, or paclitaxel at 5 nM, or both for 3 days. The results showed that AZD8186 moderately induced annexin V-positive apoptotic cells in both PTEN-loss MDA-MB-436 and MDA-MB-468 cells, but not in non-PTEN-loss cells . However = 0.006 . We did in vivo efficacy of AZD8186 in combination with paclitaxel, MDA-MB-436 and MDA-MB-468 cells were implanted subcutaneously into the mammary fat pad of athymic nude mice. Mice bearing xenografts were treated with either vehicle, AZD8186 (50 mg/kg daily), paclitaxel  or AZD8186 + paclitaxel. Dynamic tumor volume measurement showed that compared to the vehicle control groups where xenograft tumors progressively grew within the experimental span, AZD8186 did not cause significant tumor growth inhibition as a single agent in both cell line models . Further, this combination achieved a substantial stabilization of the growth rate compared to vehicle control with a T/C ratio 0.33 (tumor volume of Treatment group / tumor volume of Control group × % at the end time point).To test the e models , 5B. HowIn vivo antitumor efficacy of AZD8186 in combination with RPM1-14  was evaluated in a syngeneic PTEN-deficient mouse cell model, BP (murine melanoma). Cells were implanted subcutaneously into BALB/c mice. When tumors reached at least 100 mm3, mice were treated with either vehicle, AZD8186 (50 mg/kg daily), anti-PD1  or AZD8186 + anti-PD1. We found that at day 14 of treatment, while AZD8186 and anti-PD1 alone moderately reduced tumor volume with T/C ratio 84.4% and 74.7%, respectively, combination treatment demonstrated significantly greater antitumor efficacy with T/C ratio 26.2%, than vehicle (p < 0.001), or AZD8186 (p = 0.002), or anti-PD1 (p = 0.003) 01) , combination treatment was associated with significantly higher percentage of CD8+ cells than vehicle control (p = 0.001), or AZD8186 alone (p = 0.01), or anti-PD1 alone (p = 0.014) (003) 01) . Bliss a= 0.014) .p = 0.001). However, when anti-PD1 was combined with AZD8186, it demonstrated significantly greater antitumor efficacy than single AZD8186 (p < 0.0001), or anti-PD1 (p = 0.0011), or vehicle control (p = 0.0002), with a T/C ratio 9.2% , anti-PD1 , paclitaxel , or various combinations between these agents as indicated in tio 9.2% . These ltio 9.2% . All thrtio 9.2% . These stio 9.2%  and by mtio 9.2% .On the other hand, no significantly enhanced antitumor efficacy was appreciated in all the combination cohorts in the PTEN-wild-type CT26 model . Intereset al reported that up to 30% of breast cancer patients have PTEN loss [TNBC is defined as tumors lacking hormone receptors (estrogen and progesterone) and HER2 expression and represents approximately 15–20% of all breast cancer patients and is associated with a poor prognosis . PatientTEN loss . DeregulTEN loss . Therefoin vitro evaluation of AZD8186 demonstrated selective sensitivity in PTEN-deficient TNBC cell lines, as evidenced by selective inhibition of proliferation and PI3K signaling, and stimulation of apoptosis in these cells. However, in vivo evaluation demonstrated limited single agent efficacy of AZD8186 on tumor growth inhibition at a clinically achievable dose in PTEN-deficient cell line xenografts noted to be sensitive in vitro. No tumor regression was observed in either TNBC model.Our There is little information about factors that limit single agent clinical efficacy of AZD8186. Although PI3Kβ is the primary PI3K isoform involved in many cases of tumorigenesis that are driven by PTEN loss, studies have shown that depending on the tissue type and pathology, both PI3Kα and PI3Kβ may be involved –29. PrevIn vitro combination assays in the TNBC cell lines revealed that combinations of AZD8186 with chemotherapy agents have enhanced inhibition efficacy on cell proliferation and cell apoptosis. These data suggest that chemotherapeutic agents could be effective partners for AZD8186 in TNBC therapy. Moreover, our in vivo TNBC tumor models demonstrated a strong therapeutic synergy of combination between AZD8186 and paclitaxel. These finding are consistent with the results of a previous study where AZD8186 synergized with microtubule inhibitor docetaxel on tumor growth inhibition in xenograft of a PTEN-deficient cell line, HCC-70 breast cancer cells [Given the limited efficacy, we sought to seek effective combination approaches in AZD8186 therapeutics in TNBC tumors. First, we explored combination of AZD8186 with chemotherapeutic agents. er cells . HoweverIn addition to PI3Kβ inhibition, AZD8186 also has activity against PI3Kδ. Although PI3Kδ does not function as a pivotal driver in PTEN-deficient tumors , inhibitin vivo data demonstrated that the combination produced a strong synergistic antitumor efficacy compared to AZD8186 or anti-PD1 alone, and resulted in tumor regression in PTEN-deficient BP model, while no increased antitumor efficacy of the combination therapy was appreciated in PTEN-WT CT26 model. Next, we sought to elucidate the mechanisms underlying such a combination efficacy. Previously, PTEN loss was found to be correlated with decreased T cell tumor infiltration, and loss of PTEN inhibits T cell-mediated tumor killing in preclinical models [Our l models . Therefoin vitro, with modest single agent efficacy in vivo. Furthermore, AZD8186 enhanced the antitumor efficacy of paclitaxel but stable and progressive disease were noted with this combination in immunosuppressed models. In immunocompetent models, AZD8186 in combination with anti-PD1 resulted in tumor regression in PTEN-deficient BP tumor. We realize that while there appears to be an association of AZD8186 sensitivity to PTEN loss, a cause effect relationship can only be speculated on. In summary, although further insights are needed into the mechanisms of activity of these combinations, the combination of AZD8186 with taxanes and with anti-PD1 agents hold promise for the treatment of PTEN-deficient solid tumors.In conclusion, these results provide preclinical evidence of antitumor efficacy of AZD8186 in PTEN-deficient solid tumors. AZD8186 has single agent efficacy in PTEN-deficient TNBC cell lines 2.Breast cancer cell lines BT-20, BT-549, HCC-1806, HCC-1937, HCC-38, MDA-MB-231, MDA-MB-436, and MDA-MB-468 were obtained from American Type Culture Collection (ATCC). Breast cancer cell line MFM-223 was purchased from Sigma. Sum-159 breast cancer cell line generation was previously described . All thein vitro studies AZD8186 and AZD5363 were obtained from Selleck Chemicals. For in vivo studies AZD8186 was obtained from AstraZeneca as a generous gift. Anti-mouse PD1 monoclonal antibody (clone: RPM1-14) was purchased from BioXcell. For in vitro studies, all drugs were dissolved in DMSO.Paclitaxel and carboplatin were obtained from Selleck Chemicals. Eribulin was acquired from D Anderson Cancer Center’s pharmacy . For Immunoblotting antibodies purchased from Cell Signaling Technology (CST) include anti-phospho-AKT (S473) (#4060), anti-AKT (#9272), anti-phospho-S6K (T389) (#9234), anti-S6K (#9202), anti-phospho-PRAS40(T246) (#2997), and PRAS40 (#2610). Anti-β-actin antibody (#A5441) was purchased from Sigma. Secondary antibodies Goat-anti-Rabbit-Alexa Fluor-680 (#A21076) and Goat-anti-Mouse-Dyligh-800 (#610145-121) were purchased from Life Tech and Rockland Immunochemicals, respectively.50) and combination index (CI) were determined from dose-response curves generated using GraphPad Prism v6.05 software. All experiments were repeated at least three times.Cells were seeded in 96-well plates at densities of 5000 to 10000 cells per well depending on growth characteristics of each cell line. After adhering overnight, titrating concentrations of the designated drug were added to the wells in triplicates and incubated at 37° C for 72 hours. Anti-proliferative activity was evaluated by sulforhodamine B (SRB) assay. The half maximal inhibitory concentration  for two weeks. The culture medium was changed every seven days. The colonies were then fixed in 10% formalin and stained with 0.05% crystal violet in 25% methanol. Percent surface area was measured using NIH ImageJ v.1.48 software.Cells were plated at a density of 2 × 10Cells were washed with cold PBS and lysed in Laemmli buffer. The protein was quantified using Pierce BCA protein assay Kit (ThermoFisher) before loading to the gel. After SDS-PAGE, the protein was transferred to a 0.2 μm nitrocellulose membrane (Bio-Rad Laboratories). Membranes were blocked with 0.1% casein in TBS. Immunoblotting was performed with the following antibodies: PTEN, PI3Kβ, AKT, phospho-AKT, S6K, phospho-S6K, PRAS40, phospho-PRAS40, and β-actin. The immunoblots were visualized using the Odyssey IR imaging system (Li-Cor Biosciences). Representative blots of at least 2 independent experiments are shown.Cells were plated and allowed to attach to the petri dish overnight. The following day, cells were treated with DMSO or AZD8186 in triplicates. After 72 hours, floating and attached cells were collected. DNA content was determined in flow cytometry using propidium iodide (Roche) following manufacturer’s protocol. Apoptosis was identified by using the Annexin V apoptosis kit (Roche) according to the manufacturer’s protocol. Samples were analyzed by flow cytometry at The Flow Cytometry and Cellular Imaging Core Facility at MD Anderson Cancer Center.Four TNBC cell lines MDA-MB-436, MDA-MB-468, Sum-159 and MFM-223 were treated with each drug at six concentrations (5× dilution), starting the highest concentration at 25 μM for AZD8186, 100 nM for paclitaxel, 5 nM for eribulin, and 100 μM for carboplatin, and with drug combinations at each six concentrations, for 72 hours. Sulforhodamine B staining was performed to measure cell viability rate. CI was calculated using Chou and Talalay’s combination model based on IC50s of single drug treatment and combination treatment. CI values: <0.8: synergism; 0.8 – 1.2: addition; and >1.2: antagonism.in vivo experiments, AZD8186 was prepared in 10% DMSO/60% tri-ethylene glycol (TEG)/30% water, and paclitaxel was diluted to appropriate volume in PBS prior to administering to mice. RPM1-14  was diluted in appropriate volume of InVivoPure pH7.0 dilution buffer. Female athymic nude mice were used for inoculation of human breast cancer cell lines MDA-MB-436 or MDA-MB-468. BALB/c mice were used for murine cell lines BP (BRAF and PTEN mutant melanoma) and CT26 (PTEN wild-type colon carcinoma). 5 × 106 cells were subcutaneously inoculated into the mammary fat pad. Drug treatments were started once tumors reached at least 100 mm3. AZD8186 was orally administrated at 50 mg/kg daily; paclitaxel was intravenously injected at 10 mg/kg, once a week; Anti-PD1 was administrated by intraperitoneal injection at 200 μg, twice a week. Each treatment group has five mice. Tumor volume (TV) was measured at different time points using the formula: TV (mm3) = ((width)2 × length)/2. T/C ratio at the end time point was calculated using formula: TV of treatment group / TV of control group × %. Combination Index (CI) for tumor inhibition was calculated using Bliss Independence combination model [For on model –36. Form6 cells per tumor were then incubated with fluorescence-conjugated anti-mouse CD16/CD32 antibody (2.4G2) in PBS with 2% of each bovine, murine, rat, hamster, and rabbit serum at 4° C for 10 min [Tumors were dissected, manually dissociated, and digested enzymatically with Liberase TL (Roche) and DNase I (Roche) in RPMI-1640 for 30 min at 37° C. Digested tumors were then run through 70 μm filters into RPMI-1640 supplemented with 10% FBS, sodium pyruvate, β-mercaptoethanol, and penicillin and streptomycin. Single cell suspensions were then purified on a Histopaque-1119 (Sigma-Aldrich) gradient centrifuged at 2000 rpm for 20 min at room temperature [in vitro studies, Student t-test was performed to compare between two groups, while 1-way ANOVA followed by Tukey multiple comparison tests was performed to compare multiple groups. Association between PTEN protein status and AZD8186 sensitivity was tested with Fisher’s exact test. For the in vivo study, two-way ANOVA tests followed by Tukey for multiplicities. Data was presented as mean ± SEM.For