Additional file 1 of Chemotherapy-induced CDA expression renders resistant non-small cell lung cancer cells sensitive to 5′-deoxy-5-fluorocytidine (5′-DFCR)
Creators: name: Gao, Yanyun
name: Zens, Philipp
name: Su, Min
name: Gemperli, Camila Anna
name: Yang, Haitang
name: Deng, Haibin
name: Yang, Zhang
name: Xu, Duo
name: Hall, Sean R. R.
name: Berezowska, Sabina
name: Dorn, Patrick
name: Peng, Ren-Wang
name: Schmid, Ralph Alexander
name: Wang, Wenxiang
name: Marti, Thomas Michael
Publisher: name: figshare
Year: 2021
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Hematology
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Plant Biology
subject: Virology
subject: Computational Biology

description: Additional file 1: Supplementary Figure S1. Mitochondrial metabolism inhibitors selected to target chemotherapy resistant para clone cells. A, C Inhibitors targeting different parts of mitochondrial metabolism IACS-010759 inhibiting Complex I of Electron Transport Chain, CPI-613 inhibiting pyruvate dehydrogenase ɑ1; Tigecycline, inhibiting mitochondrial ribosome 30S, blocking protein synthesis. B pyrimidine pathway related inhibitors, teriflunomide, DHODH inhibitor; tetrahydrouridine, CDA inhibitor; 5′-deoxy-5-fluorocytidine, cytidine analogue; D volcano curve based on RNA-seq data between A549 holo and para clone cells different genes related nucleotide synthesis were shown in the curve. Mitochondrial gene list (MitoCarta) was used in this curve. Supplementary Figure S2. Metabolic inhibitors selection to target chemotherapy resistant A549 para clone cells. Cell viability assay (APH assay), Cells were treated with different inhibitors for 6 days, Holo 1.1: A549 holoclone 1.1 cells; Para 3.7: A549 paraclone 3.7 cells. N = 3, two-way ANOVA was performed, * p &lt; 0.05, *** p &lt; 0.001, **** p &lt; 0.0001. Supplementary Figure S3. Silencing of CDA expression increased the fraction of cells featuring a hybrid-E/M status in the A549 cell line. A Analysis of EMT-related plasticity by flow cytometry in the A549 cell line 72 h after CDA siRNA transfection, Holo-, mero-, paraclone, and hybrid cells featured a CD90−/SOX2+, CD90−/SOX2−, CD90+/SOX2−, and CD90+/Sox2+ expression phenotype, respectively; B Relative quantification for each subpopulation after CDA siRNA knockdown, N = 2; C Cell cycle analysis of A549 cells after siCTRL and siCDA transfection, N = 2; D DNA damage analysis by quantification of ɣH2AX expression levels by flow cytometry, The level of ɣH2AX expression in A549 control cells was set as 10%, N = 2, two sided student’s t test was used, ns: no significant difference; E Cell morphology of A549 cells 72 h after transfection with siCTRL and siCDA, respectively (40×, total magnification). Supplementary Figure S4. High expression of CDA and TYMP associates with poor prognosis. A and B: Kaplan-Meier Plots, correlation between CDA, TYMP expression and prognosis of patients after chemotherapy treatment; C: Kaplan-Meier univariate survival analyses of overall survival of TCGA lung adenocarnoma patients(N = 502), Overall survival(C and D) is analyzed and plotted using the R ‘survival’ and ‘survminer’ packages. The p-value is calculated by the log-rank test. Supplementary Figure S5. Basal expression of CDA and TYMP in different cell lines and CDA and TYMP expression after chemotherapy in additional cell lines. A Basal expression level of CDA and TYMP in different cell lines used in the study; B, C Immunoblotting of CDA and TYMP in H 2009 cells after schedule chemotherapy treatment, single treatments and concomitant chemotherapy treatment; D Immunoblotting of CDA and TYMP expression in H 2228 cells after chemotherapy treatment; E,F Comparison analysis of CDA expression in KRAS WT (FALSE) and KRAS mutated (TRUE). One hundred eighty-nine lung cancer cell lines (E) and patient samples from TCGA (F) were used in the analysis. Supplementary figure S6. Quantification analysis of CDA and TYMP after treatment in different cell lines (Fig. 3). A-C Quantification analysis of CDA and TYMP expression in A549, H358, and H441 cell lines after schedule treatment, N = 2, two-sided student’s t test, (* p &lt; 0.05, **p &lt; 0.01); D-E Quantification analysis of CDA and TYMP expression in H358 and H441 cell lines after single MTA, cisplatin and combination treatment, N = 2, two-sided student’s t test (* p &lt; 0.05, **p &lt; 0.01); F-H Quantification analysis of CDA and TYMP expression in PC-9, H1993, and H3122 cell lines after schedule treatment, N = 2, two-sided student’s t test (* p &lt; 0.05, **p &lt; 0.01); I Quantification analysis of TYMP expression in the PC-9 cell line after single MTA, cisplatin and combination treatment, N = 2, two-sided student’s t test (* p &lt; 0.05); J and K Quantification analysis of CDA and TYMP expression in H1993 (J) and H3122 (K) cells after single MTA, cisplatin and combination treatment, N = 2, two-sided student’s t test (* p &lt; 0.05, **p &lt; 0.01). Supplementary Figure S7. Cell response comparison after three different schedule treatment strategies. A Colony morphology comparison after different treatments, A549 cell line: colony morphology and colony formation assay at recovery day 21 and 27; B Representative image of counting senescent and normal cells, ‘1’ in red represents a healthy cell, while ‘2’ in blue represents a senescent cell; C Comparison of senescent cell ratio after three different schedule treatment regimens, N = 3. Supplementary Figure S8. gating strategy for cell cycle analysis and DNA damage. A-C gating strategies for single cells; D-E: Gating strategy to distinguish small and large cells by gating FSC-SSC High and FSC-SSC low populations; F-G ɣH2AX high gate, set the gate ɣH2AX-High of untreated sample to 10% as basal level. H Comparison of FSC-SSC high population in H358 cells after different treatment strategies, N = 2. on RD11, two-sided student’s t test was performed to compare untreated group with the other groups, * p &lt; 0.05, *** p &lt; 0.001. On RD18, two-sides student’s t test was performed to compare the 5′-DFCR only group with the other three groups, * p &lt; 0.05, ns: no significant difference. 2-way ANOVA was performed to compare the three groups Schedule, Schedule + 5′-DFCR D1, and Schedule + 5′-DFCR RD2, ns: no significant difference. Supplementary Figure S9. Cell cycle distribution of three different treatment strategies. Cell cycle distribution was analysed in F/S-low and F/S-high cells Group, respectively. ‘5’-DFCR at recovery day 2′ increased long-term S-phase and G2/M-phase arrest induced by schedule-dependent treatment. Data are represented as mean ± SD (N = 2). Supplementary Figure S10. Gating strategy for analyzing ɣH2AX and CDA expression levels within different EMT state. A-C: gating strategies for single cells; D: Gating for EMT state including epithelial, meshenchymal, hybrid, and negative; E, F: Under the gate ‘single cells’, CDA-High and ɣH2AX-High were gated, while roughly 10% was set for untreated H358 cells. G, H: From each status of EMT, the same gates ‘CDA-High’ (E) and ‘ɣH2AX-High’ (F) were used to evaluate CDA and ɣH2AX expression levels within different EMT state; I, H: Percentages of ‘ɣH2AX-High’ and ‘CDA-High’ in hybrid-EMT status at different time points, after different regimen treatment. High levels of ɣH2AX and CDA were shown in the hybrid-EMT status. N = 2, 2-way ANOVA was performed, ns: no significant difference. Supplementary Figure S11. Schedule+ 5′-DFCR RD2 regimen delayed the decrease of CDA and ɣH2AX expression levels. A-B: Total CDA and ɣH2AX expression over time after three regimen treatments, including schedule, schedule + 5′-DFCR D0, and schedule + 5′-DFCR RD2, N = 2, two-way ANOVA was performed, * p &lt; 0.05, ns: no significant difference; C,D: Untreated H 358 cells at Hybrid-EMT harbored higher expression of CDA and ɣH2AX, compared with other EMT state, N = 2, one-way ANOVA was performed to compare mesenchymal, hybrid, and negative with the epithelial population, ** p &lt; 0.01, *** p &lt; 0.001; E, F: On recovery day 26 (RD26) after treatment of different regimens, CDA and ɣ H2AX levels of H 358 cells at different EMT states, N = 2, one way ANOVA was performed to compare mesenchymal, hybrid, and negative with the epithelial population, * p &lt; 0.05. Supplementary Figure S12. DNA damage persistence during recovery phase after chemotherapy treatment in different cell lines A Immunoblotting of CDA, EMT markers and DNA damage marker p-H2AX expression in H 2009 cells after chemotherapy treatment; B-C Immunoblotting of CDA, TYMP and p-H2AX expression in H358 and H441 cells after chemotherapy treatment; D: morphology change H358 cells after MTA and cisplatin combination chemotherapy treatment at recovery day 5 (40×, total magnification). Supplementary Figure S13. No correlation of CDA expression an clinico-pathologic characteristics. A: No correlation of CDA expression and the pT stage of the tumors (109 samples included, Spearman correlation, p = 0.893); B: No correlation of CDA expression and the pN stage of the tumors, all primary resected samples were excluded due to a minimum pN2 (49 samples included, Spearman correlation p = 0.792). Supplementary Figure S14. Representative immunohistochemical CDA staining in patient tissues and A549 cells. A-D: Intensity scoring of CDA expression: A strong; B moderate; C weak; D negative sample; E-F Establishment of CDA staining protocol with A549 holo and para clone cells. Supplementary Figure S15. Heterogeneous CDA expression in patient tumor tissues. A Heterogeneity within the same TMA core, B-C: Heterogeneity in two different TMA cores of the same case; D, G: Heterogeneous CDA expression in two different cases; E, F: heterogeneity in two different TM -cores of the same case; H: Number of homogeneous and heterogeneous CDA-staining patterns in 109 patient tumor tissues (Fisher’s exact p = 0.228).