PDF file - 77K, 1A, The c-Src inhibitor blocked E2 activated non-genomic pathway. MCF-7:5C cells were treated with vehicle (0.1% DMSO), E2 (10-9 mol/L), PP2 (5x10-6 mol/L), E2 (10-9 mol/L) plus PP2 (5x10-6 mol/L) respectively for 10 minutes and the cell lysates were harvested. Phosphorylated MAPK and c-Src were examined by immunoblotting with primary antibodies. Immunoblotting for total MAPK and c-Src were used for loading controls. 1B, E2 rapidly activated MAPK and c-Src. MCF-7:5C cells were treated with vehicle (0.1% EtOH) and E2 (10-9 mol/L) for different time points as indicated and the cell lysates were harvested. Phosphorylated MAPK and c-Src were examined by immunoblotting with primary antibodies. Immunoblotting for total MAPK and c-Src were used for loading controls. 1C, E2 stimulated c-Src after 24 hours treatment. MCF-7:5C cells were treated with vehicle (0.1% EtOH) and E2 (10-9 mol/L) for different time points as indicated and the cell lysates were harvested. Phosphorylated c-Src was examined by immunoblotting with primary antibody. Immunoblotting for total c-Src was used for loading control. 1D, Quantification of Annexin V binding assay. MCF-7:5C cells were treated with vehicle (0.1% DMSO), E2 (10-9 mol/L), 4-OHT (10-6 mol/L), E2 (10-9 mol/L) plus 4-OHT (10-6 mol/L), PP2 (5x10-6 mol/L), E2 (10-9 mol/L) plus PP2 (5x10-6 mol/L) respectively for 72 hours and the cells were harvested for Annexin V binding assay through flow cytometry. The percentage of Annexin V binding was compared with control. P<0.05, * compared with control. All the data shown were representative of at least three separate experiments with similar results.

(A) MCF-7:5C cells were treated with vehicle (0.1% EtOH; con) or E2 (10-9 mol/L) for 72 hours. Cells were harvested in TRIzol. IGF-1Rβ was quantitated by real-time RT-PCR. p<0.001, ** compared with control. (B) MCF-7:5C cells were treated with vehicle (0.1% DMSO; con) or AG1024 (5×10-6 mol/L) for 48 hours. Total-IGF-1Rβ and p-IGF-1Rβ were examined by Western blot.

MCF-7:5C cells were treated with vehicle (0.1% DMSO; con), E2 (10-9 mol/L), SP600125 (10-5 mol/L), and E2 (10-9 mol/L) plus SP600125 (10-5 mol/L) for 72 hours. Cells were harvested in TRIzol. Genes were quantitated by real-time RT-PCR. (A) PMAIP1, (B) PPP1R15A. p<0.001, ** compared with control.

(A) Activation of p38 by E2. MCF-7:5C cells were treated with E2 (10-9 mol/L), 4-OHT (10-6 mol/L), and E2 (10-9 mol/L) plus 4-OHT (10-6 mol/L) for the time points indicated. p-p38 was examined by Western blot. Total p38 was measured as loading control. (B) The p38 inhibitor could not block E2-induced apoptosis. MCF-7:5C cells were treated with vehicle (0.1% DMSO; con), E2 (10-9 mol/L), SB203580 (10-5 mol/L), and E2 (10-9 mol/L) plus SB203580 (10-5 mol/L) for 72 hours. Annexin V binding assay was used to detect apoptosis. p<0.001, ** compared with control.

PDF file - 75K, 3A, The 4-OHT completely blocked E2-induced growth inhibition. MCF-7:5C cells were treated with vehicle (0.1% EtOH), E2 (10-9 mol/L), 4-OHT (10-6 mol/L), E2 (10-9 mol/L) plus 4-OHT(10-6 mol/L) respectively for 7 days. Cells were harvested and total DNA was determined using a DNA fluorescence quantitation kit. P<0.001, ** compared with control. All the data shown were representative of at least three separate experiments with similar results. 3B, Classification of E2-induced apoptosis-related genes selected by RNA-seq in MCF-7:5C cells. MCF-7:5C cells were treated with different compounds as above for 72 hours. Cells were harvested in TRIzol for RNA-seq analysis as in Material and Methods.

PDF file - 47K, 5A, The oxidative stress indicator HMOX1 expressed in wild-type MCF-7 cells after E2 treatment. Wild-type MCF-7 cells were cultured in E2 free medium for three days. Then, cells were plated in six-well plates. After one day, cells were treated with vehicle (0.1% EtOH) and E2 (10-9 mol/L) for different time points as indicated and the cells were harvested in TRIzol for real-time PCR. P<0.001, ** compared with control. 5B, The oxidative stress indicator HMOX1 expressed in long-term E2 deprived MCF-7:2A cells after E2 treatment. MCF-7:2A cells were plated in six-well plates. After one day, cells were treated with vehicle (0.1% EtOH) and E2 (10-9 mol/L) for different time points as indicated and the cells were harvested in TRIzol for real-time PCR. P<0.05, * compared with control. 5C, The oxidative stress indicator HMOX1 expressed in long-term E2 deprived MCF-7:5C cells after E2 treatment. MCF-7:5C cells were plated in six-well plates. After one day, cells were treated with vehicle (0.1% EtOH) and E2 (10-9 mol/L) for different time points as indicated and the cells were harvested in TRIzol for real-time PCR. P<0.05, * compared with control. P<0.001, ** compared with control.

PDF file - 104K, 2A, Activation of pS2 by different concentrations of estrogen dendrimer conjugate (EDC). MCF-7:5C cells were treated with vehicle (0.1% MeOH), different concentrations of EDC, E2, and empty dendrimer as indicated for 8 hours in triplicate. Cells were harvested in TRIzol for real-time PCR. 2B, Cell growth after EDC treatment. MCF-7:5C cells were treated with vehicle (0.1% MeOH), different concentrations of EDC, E2, and empty dendrimer as indicated for 7 days in triplicate. Cells were harvested and total DNA was determined using a DNA fluorescence quantitation kit. 2C, The c-Src inhibitor blocked EDC activated non-genomic pathway. MCF-7:5C cells were treated with vehicle (0.1% MeOH), EDC (10-8 mol/L), PP2 (5x10-6 mol/L), EDC (10-8 mol/L) plus PP2 (5x10-6 mol/L) respectively for 15 minutes and the cell lysates were harvested. Phosphorylated MAPK was examined by immunoblotting with primary antibody. Immunoblotting for total MAPK was used for loading control. 2D, EDC rapidly activated MAPK and c-Src in MCF-7:5C cells. MCF-7:5C cells were treated with vehicle (0.1% MeOH) and EDC (10-8 mol/L) for different time points as indicated and the cell lysates were harvested. Phosphorylated MAPK and c-Src were examined by immunoblotting with primary antibodies. Immunoblotting for total MAPK and c-Src were used for loading controls. 2E, EDC activated signaling pathways after 24 hours. MCF-7:5C cells were treated with vehicle (0.1% MeOH), EDC (10-8 mol/L), PP2 (5x10-6 mol/L), EDC (10-8 mol/L) plus PP2 (5x10-6 mol/L) respectively for 24 hours and 48 hours. Cell lysates were harvested. Phosphorylated MAPK, Akt, and c-Src were examined by immunoblotting with primary antibodies. Immunoblotting for total MAPK, Akt, and c-Src were used for loading controls.

The JNK inhibitor arrested cells at G2-phase. (A) MCF-7 cells were cultured in E2-free medium for three days. Then, MCF-7 cells were treated with vehicle (0.1% DMSO) or SP600125 (10-5 mol/L) for 48 hours. Cells were harvested for the analysis of cell cycle. p<0.05, * compared with control. (B) MCF-7:5C cells were treated with vehicle (0.1% DMSO) or SP600125 (10-5 mol/L) for 48 hours. Cells were harvested for the analysis of cell cycle. p<0.05, * compared with control. (C) MCF-7:2A cells were treated with vehicle (0.1% DMSO) or SP600125 (10-5 mol/L) for 48 hours. Cells were harvested for the analysis of cell cycles. p<0.05, * compared with control. (D) The JNK inhibitor completely blocked proliferation induced by E2 in MCF-7 cells. MCF-7 cells were cultured in E2-free medium for three days. Then, MCF-7 cells were treated with vehicle (0.1% DMSO), E2 (10-9 mol/L), SP600125 (10-5 mol/L), and E2 (10-9 mol/L) plus SP600125 (10-5 mol/L). Cells were harvested after 7 days treatment and cell viability was quantitated by determination of total DNA. p<0.05, * compared with control; p<0.001, ** compared with control.

MCF-7:5C cells were transfected with scrambled, PERK (A), IRE1α (B), and ATF6 (C) siRNAs for 72 hours. Then, cells were treated with vehicle (0.1% EtOH; con) or E2 (10-9 mol/L) for 72 hours. Annexin V binding assay was used to detect apoptosis. (D). Cell lysates were harvested after transfected with scrambled, PERK, IRE1α, and ATF6 siRNAs for 72 hours. IGF-1R levels were detected through Western blot. β-actin was measured as loading control.

PDF file - 82K, 6A, The c-Src inhibitor blocked tumor necrosis factor (TNF) super family genes induced by E2. MCF-7:5C cells were treated with vehicle (0.1% DMSO), E2 (10-9 mol/L), 4-OHT (10-6 mol/L), E2 (10-9 mol/L) plus 4-OHT (10-6 mol/L), PP2 (5x10-6 mol/L), E2 (10-9 mol/L) plus PP2 (5x10-6 mol/L) respectively for 72 hours. Cells were harvested in TRIzol. LTA (TNF super family member 1) gene was detected by real-time PCR. P<0.001, ** compared with control. 6B, LTB (TNF super family member 3) gene was detected by real-time PCR. P<0.001, ** compared with control. 6C, TNFα activated apoptotic pathways in MCF-7:5C cells. MCF-7:5C cells were treated with vehicle (H2O) and TNFα (5ng/mL) for 24 hours. Cell lysates were harvested. Pro-apoptotic pathways PARP and Caspase-9 were examined by immunoblotting with primary antibodies. Immunoblotting for beta-actin was detected for loading control. 6D, TNFα inhibited MCF-7:5C cell growth. MCF-7:5C cells were treated with vehicle (H2O) and TNFα (5ng/mL) for 7 days. Cells were harvested and total DNA was determined using a DNA fluorescence quantitation kit. P<0.001, ** compared with control. All the data shown were representative of at least three separate experiments with similar results.

Legends of supplementary Figures

Basal expression levels of stress-related genes in MCF-7:5C and MCF-7:2A cells.

PDF file - 53K, 7A, Inhibitors of Akt could not block growth inhibition induced by E2 in MCF-7:5C cells. MCF-7:5C cells were treated with vehicle (0.1% EtOH), E2 (10-9 mol/L), LY294002 (10-6mol/L), and E2 (10-9 mol/L) plus LY294002 (10-6mol/L) respectively. Cells were harvested after 7 days treatment and total DNA was determined using a DNA fluorescence quantitation kit. 7B, Inhibitor of MAPK could not block growth inhibition induced by E2 in MCF-7:5C cells. MCF-7:5C cells were treated with vehicle (0.1% EtOH), E2 (10-9 mol/L), U0126 (5x10-6 mol/L), and E2 (10-9 mol/L) plus U0126 (5x10-6 mol/L) respectively. Cells were harvested after 7 days treatment and total DNA was determined using a DNA fluorescence quantitation kit. All data shown were representative of at least three separate experiments with similar results.

PDF file - 59K, Sequences of Primers.

PDF file - 76K, The c-Src inhibitor blocked apoptosis-related genes induced by E2. MCF-7:5C cells were treated with vehicle (0.1% DMSO), E2 (10-9 mol/L), 4-OHT (10-6 mol/L), E2 (10-9 mol/L) plus 4-OHT(10-6 mol/L), PP2 (5x10-6 mol/L), E2 (10-9 mol/L) plus PP2 (5x10-6 mol/L) respectively for 72 hours. Cells were harvested in TRIzol for real-time PCR. 4A, PPP1R15A (GADD34) gene. 4B, BCL2L11 (Bim) gene. 4C, NUAK2 gene. 4D, PMAIP1 (Noxa) gene. P<0.001, ** compared with control. All the data shown were representative of at least three separate experiments with similar results.

(A) The phosphorylation of Akt was blocked by the inhibitors of IGF-1R and PI3K. MCF-7:5C cells were treated with vehicle (0.1% DMSO; con), E2 (10-9 mol/L), AG1024 (5×10-6 mol/L), E2 (10-9 mol/L) plus AG1024 (5×10-6 mol/L), LY294002 (10-6 mol/L), and E2 (10-9 mol/L) plus LY294002 (10-6 mol/L) for 48 hours. p-Akt was examined by Western blot. Total Akt was measured as loading control. (B) Effects of the c-Src inhibitor on the expression of Akt. MCF-7:5C cells were treated with vehicle (0.1% DMSO; con), E2 (10-9 mol/L), PP2 (5×10-6 mol/L), and E2 (10-9 mol/L) plus PP2 (5×10-6 mol/L) for different time points indicated. p-Akt and total-Akt were examined by Western blot. β-actin was measured as loading control.

These abstracts were presented at the 2017 annual meeting of the UK In Vitro Toxicology Society (IVTS). The meeting was hosted at the Senate House in London, UK on November 23–24, 2017. The main session topics included hepatotoxicity; dermal and barrier toxicity; IVIVE, exposure and non-mammalian; cardiotoxicity; neurotoxicity; and genotoxicity.

Prostate cancer remains one of the most common cancers diagnosed in men and one of the leading causes of cancer death in men. Tumor development and progression have been shown to be highly influenced not simply by the genetic makeup of a cell, but by its surrounding stroma, particularly fibroblasts. It has been demonstrated that prostate cancer-associated fibroblasts (CAFs, which are located marginal to the prostate tumor), differ from prostate normal-associated fibroblast (NAFs, which are located distal to the prostate tumor), on their contribution to tumor progression. However, human prostate cancer in-vitro model systems have focused largely on prostate cancer epithelial cells exclusively. A need exists for a more physiologically relevant human cell model system to study prostate cancer progression within the context of its tumor microenvironment. In this study, we utilized prostate cancer-associated fibroblasts (CAFs), prostate normal-associated fibroblasts (NAFs) and normal prostate epithelial (PrE) cells; all three lines were immortalized by hTERT (human telomerase reverse transcriptase) alone and they were continuously passaged for at least 15 passages without any indications of a decrease in growth rate. All cell lines express appropriate specific cell lineage markers for either fibroblasts or epithelial cells. Fibroblasts expressed TE7 and alpha smooth muscle actin (a-SMA), while prostate epithelial cells expressed cytokeratin 5, low levels of prostate specific antigen (PSA) and high levels of p63 throughout their continuous passage; all characteristics in accord with their primary cell counterparts. Next, cell proliferation was measured for various prostate-derived epithelial cells under the influence of CAFs and NAFs cells. Normal prostate epithelial cell proliferation was inhibited and produced a visible morphological change in the cells in the presence of CAFs or CAF-conditioned medium. Meanwhile, the effects of stromal cells on prostate cancer cells was cell line dependent, demonstrating both promotion and inhibition of growth of selected cancer cells. Surprisingly, both CAFs and NAFs promoted cancer cell growth, but CAFs promoted a greater increase in cell proliferation than NAFs in some cancer cell lines. This study demonstrates that these three hTERT immortalized cells from human prostate are a valuable model system for the study of prostate cancer cell progression and tumor micro environment studies. Citation Format: Luis G. Rodriguez, Russell E. McDaniel, Xiangshan Zhao, Chaozhong Zou. An authenticated in vitro model for prostate microenvironment studies utilizing prostate epithelial cells and stromal-derived cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2115.

Introduction Tumour development begins with mutational changes to the genetic makeup of a cell; tumour progression is not solely determined by the mutated cell, but also by the tumour’s microenvironment. Prostate cancer, a leading cancer diagnosed in men, has been determined to be highly influenced by its surrounding stroma, particularly fibroblasts. It has been demonstrated that cancer-associated prostate fibroblasts (CAFs) differ from normal-associated prostate fibroblasts (NAFs). However, human prostate cancer model systems have focused largely on prostate cancer epithelial cells. Currently, a need exists for a more physiologically relevant human cell model system to study prostate cancer progression within the context of its tumour microenvironment. Material and methods In this study, we characterised three prostate-derived cells: CAFs, NAFs, and prostate epithelial cells (PrEs); all three lines were immortalised by human telomerase reverse transcriptase (hTERT) alone and selected with puromycin. The selected clones grew continuously in culture for more than 40 population doublings in our hands. Results and discussions Our data shows that the hTERT-immortalised CAFs proliferate faster than the NAFs; in addition, both CAFs and NAFs express fibroblast markers such as TE7 and alpha smooth muscle actin (a-SMA), while neither cell line expresses epithelial markers such as CK14. Both CAFs and NAFs also express elevated levels of a-SMA upon TGF-b stimulation. All three prostate-derived cells weakly express the prostate specific marker AR, and show similar markers staining after long time passaging. Importantly, conditioned media collected from CAFs promotes tumour cell growth better than NAF conditioned media. Conclusion In conclusion, CAFs, NAFs, and immortalised PrEs may provide a very valuable model system for the study of prostate cancer cell progression and tumour microenvironment studies.

Abstract Tumor development begins with mutational changes to the genetic makeup of a cell, but its progression is not solely determined by the mutated cell, but also by the tumor’s microenvironment. Prostate cancer, a leading cancer diagnosed in men, has been shown to be highly influenced by its surrounding stroma, particularly fibroblasts. It has been demonstrated that cancer-associated prostate fibroblast (CAFs) differ from normal-associated prostate fibroblast (NAFs). However, human prostate cancer model systems have focused largely on prostate cancer epithelial cells. Currently, a need exists for a more physiologically relevant human cell model system to study prostate cancer progression within the context of its tumor microenvironment. In this study, we characterized three prostate-derived cells: prostate cancer-associated fibroblast (CAFs), prostate normal-associated fibroblast (NAFs) and prostate cancer epithelial (PrE) cells; all three lines were immortalized by hTERT (human telomerase reverse transcriptase) alone, and have been continuously passaged for more than 40 PDL in our hands. Our data shows that the hTERT immortalized CAFs proliferate faster than the NAFs; in addition, both CAFs and NAFs express fibroblast markers such as TE7 and alpha smooth muscle actin (α-SMA), while neither cell line expresses epithelial marker such as CK14. Both CAFs and NAFs also express elevated levels of α-SMA upon TGF-β stimulation. All three prostate-derived cells express the prostate specific marker AR, and show similar markers staining after long time passaging. Importantly, conditioned media collected from CAFs promotes tumor cell growth better than NAF conditioned media. In conclusion, CAFs, NAFs, and immortalized prostate cancer epithelium may provide a very valuable model system for the study of prostate cancer cell progression and tumor microenvironment studies. Citation Format: Luis G. Rodriguez, Russell E. McDaniel, Xiangshan Zhao, Elizabeth Turner, Christopher Annesi, Chaozhong Zou. Characterization of hTERT-immortalized prostate-derived stromal and epithelial cells: An authentic in vitro model for tumor microenvironment studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4948. doi:10.1158/1538-7445.AM2017-4948