Supplemental Table 1 Molecular Profile of the SCLC Cell Line Panel
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1 Supplemental Table 1 Molecular Profile of the SCLC Cell Line Panel p53 RB Myc Cell Line Mutation A Mutation A Amplification B COR-L103 C p.y234c p.d584e L-Myc NCI-H526 p.s33_splice None N-Myc NCI-H1048 p.r273c p.r552* None NCI-H524 p.t155n p.s576* C-Myc DMS114 None None C-Myc DMS79 p.g279fs p.g801* None NCI-H146 None p.q850* None A Mutation status according to Cancer Cell Line Encyclopedia B Myc amplification according to (1), (2) and/or Cancer Cell Line Encyclopedia and confirmed by in house qpcr analysis. C COR-L103 not recorded in CCLE, data reported is from COR-L24 which was isolated from the same patient (2). Polanski et al. MCT1 Inhibition in Small Cell Lung Cancer Page 1 of 14
2 Supplemental Table 2 Patient Characteristics at Diagnosis Cohort with clinical data Characteristic [n (%)] A Clinical characteristics Median age (range) 61 (35-83) Gender Male 33 (57) Female 25 (43) Clinical stage Limited 47 (81) Extensive 7 (12) Unknown 4 (7) Smoking history Current 19 (33) Ex 29 (50) Never 1 (2) Unknown 9 (15) Laboratory values Hemoglobin g/l (n=51) Median (range) 132 (94-158) Leukocyte 10 9 /L (n=51) Median (range) 9.7 (3-22) Platelets 10 9 /L (n=51) Median (range) 302 (72-631) Serum sodium mmol/l (n=51) Median (range) 136 ( ) Serum LDH B IU/L (n=35) Median (range) 518 ( ) A total n = 58 unless specified B LDH, lactate dehydrogenase Polanski et al. MCT1 Inhibition in Small Cell Lung Cancer Page 2 of 14
3 Supplemental Figure 1 Structure of AZD3965 Polanski et al. MCT1 Inhibition in Small Cell Lung Cancer Page 3 of 14
4 Supplemental Figure 2 MCT1 overexpression in NCI-H1048 cells increased resistance to AZD3965. NCI-H1048 cells were engineered to overexpress MCT1 when treated with doxycycline (Dox). (A) MCT1 expression was assessed in hypoxic parental NCI-H1048 or hypoxic NCI-H1048 MCT1 cells treated with vehicle (no Dox) or Dox for 72 hours by western blotting. Blots are representative of three independent experiments. (B) NCI-H1048 MCT1 cells were cultured under hypoxia and treated with either vehicle (no Dox open circles) or Dox (closed squares) and the indicated concentration of AZD3965. Cellular biomass was assessed using the SRB assay. Graph represents mean of three independent experiments ± SEM. Polanski et al. MCT1 Inhibition in Small Cell Lung Cancer Page 4 of 14
5 Supplemental Figure 3 Effect of AZD3965 on cell cycle progression. Representative cell cycle histograms which are quantified in Figure 3A. Polanski et al. MCT1 Inhibition in Small Cell Lung Cancer Page 5 of 14
6 Supplemental Figure 4 AZD3965 did not Induce Autophagy. Normoxic and hypoxic COR-L103, NCI-H1048, DMS114 and DMS79 cells were treated with 8 nm AZD3965 and/or 50 µm chloroquine (CQ) for 24 hours. The level of LC3 was assessed by western blotting and tubulin blotting was performed as a loading control. Blots are representative images of three independent experiments. Polanski et al. MCT1 Inhibition in Small Cell Lung Cancer Page 6 of 14
7 Supplemental Figure 5 MCT4 expression correlated with AZD3965 efficacy in hypoxia. (A) Cells were cultured under hypoxic conditions for 72 hours and the lysates assayed for MCT4 expression by western blotting. Tubulin is used as a loading control. (B) Ratio of MCT4:Tubulin from A vs. fraction unaffected by 8 nm AZD3965 in hypoxia (reported in Table 1). p value calculated by Spearman nonparametric correlation test. Polanski et al. MCT1 Inhibition in Small Cell Lung Cancer Page 7 of 14
8 Supplemental Figure 6 Representative images of COR-L103 xenograft tumour. Examples of staining with MCT1, MCT4 or isotype control antibody of a COR-L103 xenograft tumour. Scale bar = 100µM. Polanski et al. MCT1 Inhibition in Small Cell Lung Cancer Page 8 of 14
9 Supplemental Figure 7 Validation of MCT1 and MCT4 IHC staining. (A) MDA- MB-231, SKOV3 and K562 cells were chosen as cell lines with varying MCT1 and MCT4 expression by western blotting. (B) IHC staining of FFPE cell pellets with anti- MCT1 and MCT4 antibodies demonstrated membrane staining in the expected cell lines confirming the suitability of the antibodies for IHC analysis. Scale bar = 100µM. Polanski et al. MCT1 Inhibition in Small Cell Lung Cancer Page 9 of 14
10 Supplemental Figure 8 Representative images of TMA IHC. (A) Examples of staining with MCT1, MCT4 or CA IX antibody. Scale bar = 50µM. (B) Examples of TMA cores stained with MCT1, MCT4 or CA IX antibody which were scored as indicated. Scale bar = 500µM. Polanski et al. MCT1 Inhibition in Small Cell Lung Cancer Page 10 of 14
11 Supplemental Figure 9 Scorer correlations and staining distributions for MCT1, MCT4 and CA IX. (A) Mean score of intensity x extent for each patient reported by observer 1 vs. observer 2. Dotted line represents the 1:1 line and solid line linear regression fit of data (with appropriate R 2 value). p-value calculated by Pearson correlation test. (B) Distribution of mean score of intensity x extent for each patient reported by observer 1. Polanski et al. MCT1 Inhibition in Small Cell Lung Cancer Page 11 of 14
12 Supplemental Figure 10 Positive correlation between MCT4 and CA IX staining. (A) Examples of cores stained for MCT1, MCT4 and CA IX showing colocalisation of MCT4 and CA IX staining; scale bar = 200 µm. (B) Mean score of intensity x extent for each patient after staining with MCT4 vs. CA IX. Line represents linear regression fit of data (with corresponding R 2 value). p-value calculated by Pearson correlation test. Polanski et al. MCT1 Inhibition in Small Cell Lung Cancer Page 12 of 14
13 Supplemental Figure 11 Effect of CA IX expression, stage and serum LDH on overall survival. Kaplan-Meier plots of patient survival stratified by low (less than median) and high (greater than median) expression of CA IX (A), stage (B) and serum lactate dehydrogenase (LDH) levels (C). p-values calculated by log-rank test. Polanski et al. MCT1 Inhibition in Small Cell Lung Cancer Page 13 of 14
14 Supplemental Refernces 1. Johnson BE, Russell E, Simmons AM, Phelps R, Steinberg SM, Ihde DC, et al. MYC family DNA amplification in 126 tumor cell lines from patients with small cell lung cancer. J Cell Biochem Suppl. 1996;24: Ibson JM, Waters JJ, Twentyman PR, Bleehen NM, Rabbitts PH. Oncogene amplification and chromosomal abnormalities in small cell lung cancer. J Cell Biochem. 1987;33: Polanski et al. MCT1 Inhibition in Small Cell Lung Cancer Page 14 of 14
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