May 05, 2023
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췌관 선암종(PDA)은 치료에 잘 저항하는 치명적인 질병입니다1,2. 이는 복잡한 종양 미세 환경3, 낮은 혈관성4 및 대사 이상5,6에 의해 부분적으로 매개됩니다. 변화된 신진대사가 종양 진행을 주도하지만 PDA가 영양분으로 사용하는 대사산물의 스펙트럼은 아직 거의 알려져 있지 않습니다. 여기에서 우리는 175개 이상의 대사산물이 영양 제한 하에서 21개 췌장 세포주의 대사 활동에 어떻게 영향을 미치는지 평가함으로써 포도당이 결핍된 상태에서 PDA의 연료로 우리딘을 확인했습니다. 우리딘 활용은 우리딘 유래 리보스를 해방시켜 중앙 탄소 대사에 연료를 공급함으로써 포도당 제한 PDA 세포에서 산화환원 균형, 생존 및 증식을 지원하는 우리딘 포스포릴라제 1(UPP1)의 발현과 밀접한 상관관계가 있습니다. PDA에서 UPP1은 KRAS-MAPK 신호에 의해 규제되며 영양 제한에 의해 강화됩니다. 일관되게 종양은 비종양 조직에 비해 높은 UPP1을 발현했으며 UPP1 발현은 PDA 환자 집단에서 낮은 생존율과 상관관계가 있었습니다. 우리딘은 종양 미세환경에서 이용 가능하며, 우리는 우리딘 유래 리보스가 종양에서 활발하게 이화된다는 것을 입증했습니다. 마지막으로, UPP1 결실은 PDA 세포가 우리딘을 사용하는 능력을 제한하고 면역 능력이 있는 마우스 모델에서 종양 성장을 둔화시켰습니다. 우리의 데이터는 우리딘 활용을 영양이 부족한 PDA 세포에서 중요한 보상 대사 과정으로 식별하여 PDA 치료를 위한 새로운 대사 축을 제안합니다.
PDA는 여전히 가장 치명적인 암 중 하나입니다1,2. PDA 종양 미세환경(TME)은 이러한 치사율의 주요 원인이며 풍부한 면역 세포 침윤, 간질 섬유아세포의 확장 및 세포외 기질의 관련 침착을 특징으로 합니다. 이로 인해 간질액 압력이 증가하고 세동맥과 모세혈관이 붕괴됩니다3,4,7. 이러한 현상은 총체적으로 낮은 산소 포화도, 치료 저항성, 대사 변화 및 세포 수준에서 종양 내 이질성에 기여합니다5,8,9. 이러한 영양소 및 산소 규제가 완화된 TME에서 살아남은 PDA 세포는 청소 및 이화 능력을 증가시키는 대사 적응을 나타냅니다. 또한 최근 연구에서는 세포외 기질, 면역 및 간질 유래 대사산물을 포함하여 PDA에 대한 종양 외부 영양원을 정의했습니다14,15,16. 이러한 연구를 통해 개별 영양소 입력이 밝혀졌지만, 이러한 많은 영양소 동인 및 메커니즘을 식별할 수 있는 포괄적인 검사는 이전에 수행되지 않았습니다.
영양이 부족한 PDA 세포에서 대사를 촉진하는 대사산물을 스크리닝하기 위해 우리는 19개의 인간 PDA 세포주와 2개의 불멸화 비악성 췌장 세포주(인간 췌장 성상세포 및 인간 췌장 네스틴 발현 세포)에 Biolog 표현형 스크리닝 플랫폼을 적용했습니다. (그림 1a). 우리는 영양소 제한 조건(0mM 포도당, 0.3mM 글루타민 및 5% 투석된 소태아혈청(FBS))에서 96웰 배열 형식으로 175개 이상의 영양소를 포착하고 대사하는 세포 능력을 평가하기 위해 스크린을 사용했습니다. 영양 패널에는 탄소 에너지와 질소 기질이 포함되었습니다 (보충 표 1). 대사 활성은 테트라졸륨 기반 염료의 감소, 세포 환원 전위의 판독을 약 3일 동안 15분마다 모니터링하여 평가되었습니다(그림 1a 및 확장 데이터 그림 1a). 영양소 소비 프로파일 분석을 통해 포도당이 없을 때 포도당 양성 대조군과 유사한 수준으로 활용되는 여러 대사 산물이 밝혀졌습니다(확장 데이터 그림 1b). 예를 들어, 아데노신, 유리딘 및 여러 당이 대부분의 세포주에서 활용되었습니다.
a, 영양 대사 스크리닝 분석 계획 및 PDA 세포주 및 종양의 유전자 발현과의 상관 관계. b, 스크리닝 데이터에서 우리딘 이화작용에 대한 표준화된 상대 대사 활성(RMA)과 독립적인 데이터세트17(16개 PDA 세포주)의 UPP1 mRNA 발현 데이터 사이의 Spearman 상관관계(r). UPP1-high 세포주는 굵은 글씨로 표시됩니다. c, 무글루코스 상태에서 3일 동안 1mM 우리딘을 보충한 후 PDA 세포주의 하위 집합에 대한 RMA. d, PDA 세포주의 하위 집합에서 UPP1 mRNA 발현의 정량적 PCR (qPCR) 검증. e, PDA 세포주에서 기본 UPP1 발현을 보여주는 면역블롯. 블롯은 유사한 결과를 보이는 세 가지 기술 복제를 대표합니다. f, e에서 강조된 8개의 PDA 세포주에서 e의 블롯의 단백질 농도계 분석과 UPP1 mRNA 발현 사이의 Spearman 상관관계(r). g, 영양 대사 화면에서 우리딘이 낮은 소비자와 비교하여 우리딘이 높은 소비자/대사자로 식별된 PDA 세포주에 의해 차등적으로 발현되는 상위 20개 유전자. 데이터 출처: 암 세포주 백과사전(CCLE). c, d의 데이터는 평균 ± sd입니다. 자세한 내용은 '통계 및 재현성' 방법 섹션을 참조하세요.
175 metabolites by 19 PDA cell lines and 2 non-PDA pancreatic cell lines was measured every 15 min for ~3 days (74.5 h) using the Biolog OmniLog device. The assay readout, RMA, was correlated with the expression level of metabolic genes in cell lines; human PDA data were used for subsequent analyses. Nutrient-deficient medium, no glucose, 0.3 mM glutamine and 5% dialysed FBS. c, n = 4 biologically independent samples per group. Statistical significance was measured by multiple unpaired two-tailed t-tests (two-stage step-up method) comparing RMA from cells in basal medium vs 1 mM uridine medium (both glucose-free), ****P < 0.0001. The experiments were performed twice with similar results. d, n = 4 biologically independent samples per cell line. The experiment was performed once./p> 0.9999; no glucose/uridine and 1 mM ribose, P = 0.3025; no glucose/uridine and 10 mM ribose, ****P < 0.0001). ASPC1 (comparison between no glucose/uridine and no glucose + 1 mM uridine, ****P < 0.0001; no glucose/uridine and 1 mM glucose/no uridine, ****P < 0.0001; no glucose/uridine and 0.1 mM ribose, P = 0.9974; no glucose/uridine and 1 mM ribose, *P = 0.0103; no glucose/uridine and 10 mM ribose, ****P < 0.0001). The experiment was performed once. b,c, n = 3 biologically independent samples. Statistical significance was measured using two-tailed unpaired t-test. Intracellular: comparison between no uridine and 1 mM uridine: ***P = 0.0005 (uridine), ****P < 0.0001 (uracil); extracellular: comparison between no uridine and 1 mM uridine: ****P < 0.0001 (uridine), **P = 0.008 (uracil). d–f, n = 3 biologically independent samples per cell line. ‘Others’ indicates M other than M+0 or M+5, where applicable. Bars shown for PATU8988S are same as the WT bars (where applicable) for that cell line in the Extended Data Fig. 5. Tracing experiments were performed twice in these cells with similar results. g, Number of samples: sub-Q, tumours from 3 mice injected on the left and right flanks; ortho, tumours from 4 mice. Mode of uridine injection is intratumoural for sub-Q and intraperitoneal for ortho. h, Median concentration of uridine = 24.1 µM; median concentration of uracil = 90.2 µM; n = 22 biologically independent TIF samples. i, Median concentration of glucose = 3.71 mM (plasma) and 0.63 mM (TIF). n = 8 biologically independent plasma samples and 8 TIF samples extracted from 8 tumour samples from same mice. These samples are from the control group of the study in Fig. 4a. Statistical significance was measured with two-tailed unpaired t-test with Welch's correction, ****P < 0.0001. j,k, j shows the mass isotopologue distribution in uridine and k shows in the indicated metabolites. n = 4 biologically independent samples per group per cell line. ‘Others’ indicates M other than M+0 or M+5, where applicable. Data in a–k are shown as mean ± s.d. The metabolomics experiments (b–k) were performed once./p> 0.9999 and P > 0.9999 for WT, 1A and 1B groups, respectively. The experiments were performed three times with similar results. c, n = 4 biologically independent samples per group per cell line. Statistical significance was measured using one-way ANOVA with Tukey's multiple comparisons test. PATU8988S, comparison between no uridine (−) and 1 mM uridine (+): ****P < 0.0001, P > 0.9999 and P = 0.9599 for WT, 1A and 1B groups, respectively. ASPC1, comparison between no uridine (−) and 1 mM uridine (+): ****P < 0.0001, P = 0.9977 and P = 0.6537 for WT, 1A and 1B groups, respectively. The experiments were performed twice with similar results. d, n = 3 biologically independent samples per group. Statistical significance was measured using one-way ANOVA with Dunnett's multiple comparisons test. Comparison between WT and clonal cells 1A or 1B: ****P < 0.0001 (PATU8988S) and ***P = 0.0003 (ASPC1). Data are part of the metabolomics experiments shown in Extended Data Fig. 5a–c. The metabolomics experiment was performed once. e, n = 3 biologically independent samples per group. ‘Others’ indicates M other than M+0 or M+5, where applicable. Data are part of the metabolomics experiments shown in Extended Data Fig. 5e,h,j for ASPC1. The metabolomics experiment was performed once. f, Statistical significance was measured using two-tailed unpaired t-test with Welch's correction. Number of samples and statistical comparison: GSE62452 (NT, 61 vs PDA, 69, ***P = 0001), GSE71729 (middle: NT, 46 vs PDA, 145, *P = 0.0466), GSE71729 (right: primary, 145 vs liver met, PDA, 25, ****P < 0.0001). Box plot statistics: GSE42452 (NT: minimum = 3.582, maximum = 5.633, 25th percentile = 4.036, 75th percentile = 4.504, median = 4.262; PDA: minimum = 3.853, maximum = 5.989, 25th percentile = 4.37, 75th percentile = 4.843, median = 4.535); GSE71729 (NT: minimum = 2.18, maximum = 4.402, 25th percentile = 2.901, 75th percentile = 3.469, median = 3.139; PDA: minimum = 2.293, maximum = 4.725, 25th percentile = 3, 75th percentile = 3.657, median = 3.339); GSE71729 (primary: minimum = 2.293, maximum = 4.725, 25th percentile = 3, 75th percentile = 3.657, median = 3.339; liver metastasis: minimum = 3.306, maximum = 5.768, 25th percentile = 3.564, 75th percentile = 4.498, median = 4.023). g, Representative images from patient 1 of 3 tumour tissues. PanCK, pan-cytokeratin, stain indicates tumour cells. i, Number of samples: UPP1-low, 144; UPP1-high, 144. j, Number of samples: no alteration, 43; G12D, 42. Statistical significance was measured using two-tailed unpaired t-test with Welch's correction, **P = 0.0029. Box plot statistics: no alteration: minimum = 7.797, maximum = 10.66, median = 9.019, 25th percentile = 8.307, 75th percentile = 9.53; KRASG12D: minimum = 8.154, maximum = 11.3, median = 9.385, 25th percentile = 9.019, 75th percentile = 9.905. k, n = 3 biologically independent samples per cell line. Statistical significance was measured using two-tailed unpaired t-test. Comparison between Dox (−) and (+) in iKras* cell A9993: ***P = 0.0002; in iKras cell 8905: **P = 0088. The experiment was performed once. l, Vinculin is used as a loading control. The experiment was performed once. m, 3 biologically independent samples per group. Statistical significance was measured using two-tailed unpaired t-test. Comparison between cells cultured in uridine/glucose-containing medium with and without trametinib treatment: ****P < 0.0001; comparison between cells treated with and without trametinib in the presence of glucose but no uridine: ****P < 0.0001; comparison between cells treated with and without trametinib in the presence of uridine and no glucose: ****P < 0.0001; comparison between cells cultured with no uridine/glucose with and without trametinib treatment: ****P < 0.0001. The experiment was performed once. n, Vinculin is used as a loading control. The experiments were performed twice with similar results. o, Statistical significance was measured using one-way ANOVA with Tukey's multiple comparisons test. n = 4 biologically independent samples per group per cell line. PATU8988S (comparison between cells cultured with and without trametinib in the absence of uridine: ****P < 0.0001, and with uridine supplementation: ****P < 0.0001); DANG (comparison between cells cultured with and without trametinib in the absence of uridine: P = 0.9967, and with uridine supplementation: ****P = 0.0001); ASPC1 (comparison between cells cultured with and without trametinib in the absence of uridine: P = 0.9987, and with uridine supplementation: ***P = 0.0001. The experiment was performed once. Data in b–e,k,m,o are mean ± s.d./p> 25 tissues compared). c. Data obtained from the Human Protein Atlas (URL for ‘Normal’ - https://www.proteinatlas.org/ENSG00000183696-UPP1/tissue/pancreas; PDA – https://www.proteinatlas.org/ENSG00000183696-UPP1/pathology/pancreatic+cancer#img). d. Sample size, n: NT = 19, tumour = 408 (bladder cancer, TCGA); NT = 5, tumour = 154 (glioblastoma, TCGA); NT = 44, tumour = 520 (head and neck cancer, TCGA); NT = 59, tumour = 551 (lung cancer, TCGA); NT = 11, tumour = 184 (oesophageal cancer, TCGA); NT = 52, tumour = 497 (prostate cancer, TCGA); NT = 41, tumour = 452 (colon cancer); health colon mucosa = 50, distant colon = 98, tumour = 98 (colon cancer, GSE44076). NT – non-tumour/adjacent normal tissue. Data (a-b, f) shown as mean ± s.d. The experiments were performed three times with similar results. Box plot statistics – TCGA, bladder carcinoma (primary: minima = 5.83, maxima = 13.5, median = 9.77, 25th percentile = 9.015, 75th percentile = 10.47; normal: minima = 6.61, maxima = 12.43, median = 8.35, 26th percentile = 8.03, 75th percentile = 9.59); glioblastoma multiforme (primary: minima = 5.71, maxima = 11.84, median = 9.585, 25th percentile = 8.79, 75th percentile = 10.143; normal: minima = 7.04, maxima = 7.63, median = 7.4, 25th percentile = 7.36, 75th percentile = 7.61); head and neck squamous cell carcinoma (primary: minima = 6.59, maxima = 15.64, median = 10.75, 25th percentile = 9.787, 75th percentile = 11.565; normal: minima = 6.38, maxima = 13.73, median = 10.42, 25th percentile = 8.672, 75th percentile = 11.065); lung adenocarcinoma (primary: minima = 6.45, maxima = 13.44, median = 9.8, 25th percentile = 9.13, 75th percentile = 10.49; normal: minima = 8.3, maxima = 11.39, median = 9.3, 25th percentile = 8.945, 75th percentile = 9.93); esophageal carcinoma (primary: minima = 6.7, maxima = 13.08, median = 9.26, 25th percentile = 8.578, 75th percentile = 10.21; normal: minima = 6.17, maxima = 12.39, median = 7.62, 25th percentile = 6.7, 75th percentile = 8.26); prostate adenocarcinoma (primary: minima = 3.96, maxima = 9.69, median = 6.58, 25th percentile = 5.98, 75th percentile = 7.14; normal: minima = 4.56, maxima = 8.62, median = 6.97, 25th percentile = 6.447, 75th percentile = 7.24); colon cancer (primary: minima = 6.41, maxima = 12.96, median = 8.535, 25th percentile = 8.068, 75th percentile = 9.07; normal: minima = 7.76, maxima = 11.29, median = 9.57, 25th percentile = 9.09, 75th percentile = 9.92). Colon cancer (GSE44076, primary: minima = 4.564, maxima = 7.608, median = 5.917, 25th percentile = 5.487, 75th percentile = 6.405; normal: minima = 4.568, maxima = 9.154, median = 7.18, 25th percentile = 6.781, 75th percentile = 7.824; healthy colon mucosal cells: minima = 5.884, maxima = 8.279, median = 7.529, 25th percentile = 7.153, 75th percentile = 7.74). Statistical significance was tested using two-sided Wilcoxon or Kruskal-Wallis tests./p>0.9999). h. n = 3 biologically independent samples per group. Statistical significance was measured with one-way ANOVA with Tukey's multiple comparisons test. Comparisons between groups (from left to right): ****P < 0.0001, ****P < 0.0001, ****P < 0.0001 and ****P < 0.0001. The experiments (e, g, h) were performed once with similar results on UPP1 displayed by the three cell lines. i. n = 3 biologically independent samples per group. This blot was run on the same gel as Fig. 3n hence the first two columns (separated by a box) overlap between the two blots. j. Blots (c,i) are representative of two independent experiments; blot e experiment was done once. k. n = 3 biologically independent samples per group. The statistical significance (P < 0.05) was determined using limma package version 3.38.3 in R. l. Statistical significance was measured using one-way ANOVA with Tukey's multiple comparisons test. n = 4 biologically independent samples per group. PATU8988S (comparison between cells cultured with and without trametinib in the absence of uridine: ****P < 0.0001, and with uridine supplementation: ****P < 0.0001); DANG (comparison between cells cultured with and without trametinib in the absence of uridine: **P = 0.0055, and with uridine supplementation: ***P = 0.0009); ASPC1 (comparison between cells cultured with and without trametinib in the absence of uridine: P = not significant, and with uridine supplementation: ****P = 0.0006). Data (b, e, g-h, l) shown as mean ± s.d./p> 
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