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. 2016 May 19;165(5):1092-1105.
doi: 10.1016/j.cell.2016.04.009. Epub 2016 Apr 28.

"V体育官网入口" Effector T Cells Abrogate Stroma-Mediated Chemoresistance in Ovarian Cancer

Weimin Wang  1 , Ilona Kryczek  2 , Lubomír Dostál  3 , Heng Lin  2 , Lijun Tan  4 , Lili Zhao  5 , Fujia Lu  2 , Shuang Wei  2 , Tomasz Maj  2 , Dongjun Peng  2 , Gong He  4 , Linda Vatan  2 , Wojciech Szeliga  2 , Rork Kuick  5 , Jan Kotarski  6 , Rafał Tarkowski  6 , Yali Dou  7 , Ramandeep Rattan  8 , Adnan Munkarah  8 , J Rebecca Liu  9 , Weiping Zou  10
Affiliations

Effector T Cells Abrogate Stroma-Mediated Chemoresistance in Ovarian Cancer

Weimin Wang et al. Cell. .

Abstract

Effector T cells and fibroblasts are major components in the tumor microenvironment VSports手机版. The means through which these cellular interactions affect chemoresistance is unclear. Here, we show that fibroblasts diminish nuclear accumulation of platinum in ovarian cancer cells, resulting in resistance to platinum-based chemotherapy. We demonstrate that glutathione and cysteine released by fibroblasts contribute to this resistance. CD8(+) T cells abolish the resistance by altering glutathione and cystine metabolism in fibroblasts. CD8(+) T-cell-derived interferon (IFN)γ controls fibroblast glutathione and cysteine through upregulation of gamma-glutamyltransferases and transcriptional repression of system xc(-) cystine and glutamate antiporter via the JAK/STAT1 pathway. The presence of stromal fibroblasts and CD8(+) T cells is negatively and positively associated with ovarian cancer patient survival, respectively. Thus, our work uncovers a mode of action for effector T cells: they abrogate stromal-mediated chemoresistance. Capitalizing upon the interplay between chemotherapy and immunotherapy holds high potential for cancer treatment. .

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Figures

Figure 1
Figure 1. Fibroblasts Induce Cancer Platinum Resistance
(A–D) Effect of fibroblasts on cisplatin resistance to OC8 (A and B) or A2780 (C and D) in vivo. Tumor cells or tumor plus fibroblasts were inoculated subcutaneously into mice and treated with or without cisplatin every 3 days for three cycles. Tumor volume was monitored (A and C) (mean ± SEM; n = 5). Apoptosis was determined by TUNEL staining. Representative images and TUNEL-positive cells (percentage of control) are shown (B and D). Scale bar, 40 μm. Mean ± SD; n = 10 random fields from three sections. *p < 0.05. (E) Cisplatin-induced apoptosis on GFP+ tumor cells in the mixed co-culture of A2780-GFP and fibroblasts. Representative FACS data are shown (left). Mean ± SD, n = 3. *p < 0.05. (F–H) Cisplatin-induced apoptosis in A2780 (F), OC8 (G), or NIH: OVCAR3 (H) co-cultured with fibroblasts from different ovarian cancer patients in the Transwell. Mean ± SD; n = 3, *p < 0.05. (I and J) Cisplatin-induced apoptosis on A2780 (I) or OC8 (J) cultured in fibroblast medium. Mean ± SD; n = 3, *p < 0.05. See also Figure S1 and Table S2.
Figure 2
Figure 2. CD8+ T Cells Abolish Fibroblast-Induced Platinum Resistance via IFNγ
(A and B) Effect of CD8+ T cells on fibroblast-induced platinum resistance. A2780 and fibroblast were cultured with the supernatant from activated CD8+ T cells (A). Fibroblasts were primed with CD8+ T cell supernatants and cultured with tumor cells in the presence of cisplatin (B). Tumor cell apoptosis was determined by Annexin V staining. Mean ± SD; n = 3, *p < 0.05. (C) IFNγ released by activated CD8+ T cells. Mean ± SD; n = 3. (D–G) Effect of IFNγ on fibroblast-induced platinum resistance. Fibroblasts were primed with IFNγ (D and E) or with CD8+ T cells supernatant in the presence of anti-IFNγ R1 (F) or JAK inhibitor I (G) and subsequently cultured with tumor cells in the Transwell. Cisplatin-induced tumor cell apoptosis was determined. Mean ± SD; n = 3, *p < 0.05. (H) Effect of IFNγ on fibroblast-induced cisplatin resistance to OC8 in vivo. OC8 and fibroblasts were inoculated subcutaneously into NSG mice and then treated with IFNγ, cisplatin, or IFNγ + cisplatin for three cycles. Tumor volume was monitored (mean ± SEM, n = 5). *p < 0.05. See also Figure S2.
Figure 3
Figure 3. Fibroblasts Reduce the Accumulation of Cisplatin in Cancer Cells
(A) Cisplatin-induced γH2AX in cancer cells cultured with fibroblasts was detected by western blotting. Fibroblasts were from four patients. One of three experiments is shown. (B) Cisplatin-induced γH2AX in vivo was determined in A2780 tissues and representative images are shown. Scale bar, 50 μm. (C–E) Effect of fibroblasts on cisplatin intracellular content in tumor cells. A2780 (C and D) or OC8 (E) were cultured with fibroblasts in the presence of different concentrations of cisplatin (C) or 10 μg/ml cisplatin (D and E). Platinum content in the whole tumor cells was measured by ICP-MS in triplicate (mean ± SD). *p < 0.05. (F and G) Effect of fibroblasts on cisplatin DNA content in A2780 (F) or OC8 (G). Platinum content in the genomic DNA was measured by ICP-MS in triplicate (mean ± SD). *p < 0.05. (H) Dot blot assay of cisplatin-DNA adduct in genomic DNA of OC8 cultured with fibroblasts in the presence of cisplatin. (I) Effect of fibroblasts on cisplatin content in tumor cells in vivo. GFP+ OC8 were enriched from xenograft tumor tissues and platinum content was measured by ICP-MS (n = 5 or 6). *p < 0.05. (J) Effect of IFNγ blockade on tumor cisplatin content in vivo. GFP+ OC8 and fibroblasts were inoculated into NSG mice with CD8+ T cells. The mice were treated with cisplatin and anti-human IFNγ blocking antibody or control antibody. GFP+ OC8 were enriched and platinum content was measured by ICP-MS (n = 4). *p < 0.05. See also Figure S3.
Figure 4
Figure 4. Fibroblasts Elevate Cancer Intracellular GSH Conferring Platinum Resistance
(A and B) Intracellular GSH level in A2780 (A) or OC8 (B) co-cultured with fibroblasts. Mean ± SD, n = 3, *p < 0.05. (C) Intracellular GSH level in A2780 cultured in control or fibroblast medium. Mean ± SD, n = 3, *p < 0.05. (D) Intracellular GSH level in A2780 treated with 200 μM NAC for 6 hr. GSH was measured in triplicate (mean ± SD). *p < 0.05. (E and F) Effects of NAC on cisplatin-induced γH2AX (E) and cisplatin content (F) in A2780. Pro-caspase 9 and γH2AX were detected by western blotting (E). Platinum content in genomic DNA of A2780 was measured by ICP-MS in triplicates (mean ± SD) (F). *p < 0.05. (G) Intracellular GSH level in A2780 treated with 6 μM BSO for 6 hr. GSH was measured in triplicates (mean ± SD). *p < 0.05. (H and I) Platinum content in genomic DNA (H) or cisplatin-induced apoptosis (I) of A2780. A2780 were pretreated with 6 μM BSO for 6 hr. Mean ± SD, n = 3, *p < 0.05. (J and K) Effects of GCLC knockdown on intracellular GSH level (J) and cisplatin-induced apoptosis (K) in A2780. GSH was measured and normalized with total protein. Apoptosis was determined by Annexin V staining. Mean ± SD; n = 3, *p < 0.05. (L and M) Effect of GSH monoester on cisplatin resistance in vivo. Mice bearing A2780 ovarian cancer were treated with PBS (control) or GSH monoester for 8 hr and followed with cisplatin treatment for 24 hr. Platinum content in tumor tissue was measured by ICP-MS (L). For tumor volume monitoring, mice were treated with GSH monoester and cisplatin for three cycles (M) (mean ± SEM, n = 5 tumors/group). *p < 0.05. See also Figure S4.
Figure 5
Figure 5. Fibroblasts Release GSH and Cysteine Conferring to Cisplatin Resistance
(A and B) Cisplatin-induced apoptosis (A) and intracellular GSH (B) in A2780. Fibroblast medium was filtered into two fractions, >3 kDa and <3 kDa. Tumor cells were cultured with unfiltered or filtered fibroblast medium in the presence of cisplatin. Apoptosis and intracellular GSH in A2780 cells were measured. Mean ± SD; n = 3, *p < 0.05. (C and D) Total thiol (C) and GSH (D) in fresh medium, A2780 medium, and fibroblast medium. Mean ± SD, n = 3. (E) Cysteine, Cys-Gly, and γ-Cys-Glu concentrations in fresh medium, A2780 medium, and fibroblast medium. Mean ± SD, n = 3. (F–K) Effects of exogenous GSH (F–H) and cysteine (I–K) on intracellular GSH level (F and I), cisplatin content (G and J), and cisplatin-induced apoptosis (H and K) in A2780. A2780 were pretreated with 50 μM GSH or 100 μM cysteine for 6 hr and followed with cisplatin treatment. Mean ± SD; n = 3, *p < 0.05. (L–N) Effects of cystine deficiency on fibroblast-generated thiol (L), GSH (M), and fibroblast-mediated tumor protection (N). Total thiol (L) and GSH (M) were measured in medium that were generated in cystine complete (Cystine+) or cystine free (Cystine−) culture. Mean ± SD; n = 3. A2780 were incubated in Cystine+ or Cystine− fibroblast medium for 6 hr and followed with cisplatin treatment. For Cystine− medium, 100 μM cystine was supplemented before A2780 were exposed to the medium. Tumor apoptosis was determined by Annexin V staining (N). Mean ± SD; n = 3, *p < 0.05. See also Figure S5.
Figure 6
Figure 6. Effector CD8+ T Cell-Derived IFNγ Reduces GSH and Cysteine in Fibroblasts
(A and B) Total thiol (A) and GSH (B) concentrations in samples. Fibroblasts were primed with 5 ng/ml IFNγ. Mean ± SD; n = 3, *p < 0.05. (C) Real-time PCR quantification of GGT mRNAs in IFNγ-treated fibroblasts. Data are presented as fold change relative to control group, n = 3, mean ± SD. (D) GGT enzymatic activity in fibroblasts after 48 hr of IFNγ treatment. Data are presented as fold change relative to control (mean ± SD), n = 3, *p < 0.05. (E) Role of GGT5 knockdown in IFNγ-mediated protective effect. Fibroblasts expressing scramble shRNA or shGGT5 were primed with IFNγ. A2780 were cultured with these fibroblasts and cisplatin-induced tumor apoptosis was determined. n = 3, mean ± SD. *p < 0.05. (F) Quantification of cysteine and cystine concentrations in fibroblast medium by LC-MS. Fibroblasts from three different patients were treated with IFNγ. *p < 0.05. (G) Effect of IFNγ and CD8+ T cell supernatants on 14C-Cystine uptake by fibroblasts. Mean ± SD, n = 3. *p < 0.05. (H and I) Platinum content (H) and γH2AX level (I) in A2780 cultured with IFNγ-primed fibroblasts in the presence of cisplatin. The intracellular platinum was measured by ICP-MS. Mean ± SD n = 3, *p < 0.05. Tumor γH2AX was detected by western blotting. (J) Representative immunoblots of xCT and SLC3A2 in fibroblasts treated with IFNγ for 24 hr. (K) Effect of xCT knockdown on fibroblast-mediated cisplatin resistance. A2780 were cultured with fibroblasts expressing scramble shRNA or shxCT, and followed with cisplatin treatment. Tumor apoptosis was determined by Annexin V staining. n = 3, mean ± SD. *p < 0.05. The knockdown efficiency of shxCT was determined by western blotting. (L) Real-time PCR assays of mRNAs in fibroblasts treated with IFNγ for different time points. Data are presented as fold change relative to control (mean ± SD), n = 3. (M) Real-time PCR assays of xCT pre-mRNA in above fibroblasts. xCT pre-mRNA was analyzed with two different primer pairs and normalized to GAPDH pre-mRNA. Mean ± SD, n = 3. (N) ChIP of RNA Pol II in fibroblasts treated with or without IFNγ. RNA Pol II binding to xCT TSS and intragenic regions (+900 and +6 k) was quantified by qPCR. Results are expressed as the fold changes in site occupancy over control fibroblasts. Mean ± SD from two fibroblasts samples. (O) IFNγ-induced STAT1 phosphorylation and xCT downregulation in fibroblasts were determined by western blotting. (P) Graphics map showing the positions of primers used to quantify potential STAT1 binding sites around xCT promoter region. Lower panel shows UCSC genome browser tracks of the xCT promoter region with data for STAT1 ChIP-seq. (Q) ChIP of STAT1 in IFNγ-treated fibroblasts. The binding of STAT1 to the three GAS regions around xCT promoter was determined. Results are expressed as the fold enrichment over IgG. Mean ± SD from two fibroblasts samples. (R) Effect of xCT promoter GAS elements on luciferase activity. Fibroblasts were transfected with luciferase reporter constructs containing full-length xCT promoter (GAS1+2+3) or individual GAS elements. Luciferase activity was assessed after IFNγ treatment and presented as the relative activity compared with controls (mean ± SD). n = 3, *p < 0.05. See also Figure S6.
Figure 7
Figure 7. Fibroblasts and Stromal CD8+ T Cells Clinically Impact Chemoresistance
(A) Impact of chemotherapeutic responses on patient outcome. Patients were divided into platinum-resistant and platinum-sensitive groups. The Kaplan-Meier curve of overall survival is shown (p < 0.0001, n = 178). (B) Impact of stromal fibroblasts on patient outcome. Patients were divided into high and low stromal fibroblast groups. The Kaplan-Meier curve of overall survival is shown (p = 0.0026, n = 176). (C) Association between the levels of stromal fibroblasts and platinum response. The proportion of stromal fibroblasts is shown in chemoresistant and chemosensitive patients (chi-square = 15.02, p = 0.0001). (D) Impact of stromal CD8+ T cells on patient outcome. Patients were divided into high and low groups. The Kaplan-Meier curve of overall survival is shown (p < 0.0001, n = 176). (E) Correlation between the levels of stromal CD8+ T cells and platinum response in patients with high stromal fibroblasts. The proportion of stromal CD8+ T cells is shown in chemoresistant and chemosensitive patients (Fisher’s exact test, p = 0.0298). See also Figure S7 and Tables S3, S4, S5, and S6.
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