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Keywords:

  • flow cytometry;
  • phospho-Stat1;
  • lung cancer;
  • malignant pleural effusion

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited
  8. APPENDIX
  9. Supporting Information

Single cell phospho-specific flow cytometry (SCPFC) enables the investigation of signaling network interactions and the categorization of disease outcome. While this method has been successfully used to study hematologic disorders, its application on solid tumors has not been examined. This study aimed to demonstrate the ability of SCPFC to detect dynamic changes of Tyrosine phospho-Stat1 (pStat1) in solid tumor models and in human tumor samples. In the human lung cancer cell line PC14PE6/AS2, the fluorescence intensity changes of pStat1 after IFN-γ stimulation were compatible to results obtained by Western blot analysis. In metastatic animal models, cancer cells from subcutaneous tumors, malignant ascites, and peritoneal tumors responded to IFN-γ. The pStat1 was activated in these cells after IFN-γ stimulation, with a 1.5- to 2.5-fold increase in fluorescence intensity compared to the unstimulated control. To examine the potential clinical application of SCPFC, cancer cells were collected from malignant pleural effusions (MPEs) of lung cancer patients to observe the activation of pStat1 after IFN-γ stimulation. Cell apoptosis after cisplatin treatment was evaluated by Annexin V staining, which showed that MPE cancer cells with higher pStat1 changes after IFN-γ stimulation were more resistant to cisplatin. In conclusion, there is a preliminary application of SCPFC to solid tumors and links to drug sensitivity are promising. © 2010 International Society for Advancement of Cytometry

Most human neoplasms have aberrant signal transduction elements. Understanding the structure and regulation of these elements will provide more insights into cancer treatment. Among these signaling networks, phospho-protein members of signaling cascades govern the initiation and regulation of proliferative signals within cells. It is proposed that the phosphorylation of key signaling molecules in response to particular stimuli correlates with certain mutations and the body's response to cancer treatment.

Using multi-parameter flow cytometry, single cell phospho-specific flow cytometry (SCPFC) has revealed that potentiated phospho-proteins in leukemia cells like Stats, p38, and Erk respond to several key stimuli, including IL-3, IFN-γ, GCSF, GM-CSF, and FL (flt3 ligand) (1). By recording the basal patterns and changes in activity of phospho-proteins, expression profiles have been correlated with a mutant form of Flt3 (fms-like tyrosine kinase 3) and with disease outcome. Unfortunately, although SCPFC is well demonstrated in hematologic oncology (2–4), its application in solid tumors has not been fully examined.

The signal transducer and activator of transcription (Stat) proteins are a family of transcription factors that mediate normal cell differentiation, tumor cell proliferation, and apoptosis. Previous studies have identified a suppression effect of Stat1 on oncogenesis, metastasis, and angiogenesis (5, 6). Recent studies show that Stat1 is associated with chemo-resistance and irradiation resistance (7, 8).

Lung cancer is the leading cause of cancer death in both men and women in the United States and in Europe (9). There has been no previous study that demonstrates the application of SCPFC on lung cancer. Moreover, there are two major obstacles in applying kinetic phospho-protein changes on lung cancers. First, solid tumor specimens must be processed to single cell suspensions, a procedure which may disturb cellular signals. Second, cells may experience “culture shock” and change their signaling network structure to adapt to the new environment ex vivo. Therefore, the subcutaneous and ascites animal model has been used to study the feasibility of applying this technology to solid tumors.

Lung cancer is frequently complicated by malignant pleural effusion (MPE) (10). Clinically, MPE is easily recurrent and difficult to manage (11, 12). This study aimed to illustrate that SCPFC is applicable to solid tumors and that the technique is promising for monitoring signaling changes in lung cancer cells and predicting tumor response to chemotherapy.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited
  8. APPENDIX
  9. Supporting Information

Cell Lines

The human lung adenocarcinoma cell line PC14PE6 was established from a metastatic animal model after intra-peritoneal (i.p.) injection with PC14PE6. PC14PE6/AS2 cells were cultured in MEN-α (Invitrogen-Gibco, Grand Island, NY) supplemented with 10% FBS and 1% antibiotic/anti-mycotic solution containing penicillin G, streptomycin, and amphotericin B (Invitrogen-Gibco). Human bronchial epithelial cell line NL-20, human lung adenocarcinoma cell line H1650 and A549 were obtained from the American Type Culture Collection (ATCC; Rockville, MD).

H1650, CL1-5 and NL-20 were cultured in RPMI 1640 (Invitrogen-Gibco) supplemented with 10% FBS and 1% antibiotic/anti-mycotic solution. A549 cells were grown in F12K (Invitrogen-Gibco) with 2 mM L-glutamine and 1.5 g l−1 sodium bicarbonate supplemented with 10% FBS and 1% antibiotic/anti-mycotic solution. The HONE1 (Human nasopharyngeal carcinoma, parental) was kept at RPMI 1640 supplemented with 5% FBS and 1% antibiotic/anti-mycotic solution containing penicillin G, streptomycin, and amphotericin B. The resistant sub-line HONE1-cis 6 (HONE1-derived cisplatin resistant) was maintained in 6 μM cisplatin medium.

Antibodies

The Stat1 antibody was obtained from BD Transduction Laboratories (San Diego, CA) while the pStat1-Y701 antibody was purchased from Cell Signal (Beverly, MA). Monoclonal β-actin antibody was obtained from Sigma-Aldrich (St Louis, MO) while the Alexa (Ax488) dye-conjugated pStat1-Y701 antibody, the PE-conjugated Annexin V antibody, and Alexa (Ax488) dye- and PE-conjugated isotype IgG antibodies were purchased from BD Pharmingen (San Diego, CA) for flow cytometry. PE-conjugated E-cadherin antibody was purchased from R&D Systems (Minneapolis, MN).

Animal, Subcutaneous, and Ascites Tumor Models

BALB/c nude mice 5 to 6 weeks old were obtained from the National Cheng-Kung Laboratory Animal Center, where they were maintained in a pathogen-free environment throughout the study. To generate subcutaneous tumors, 0.1 ml PC14PE6/AS2 cell suspensions (5 × 107 cells ml−1) were injected subcutaneously into their dorsal skin, where 0.5–1 cm3 subcutaneous tumors developed 4 weeks later. The mice were then sacrificed under pentobarbital anesthesia and the tumors were minced using sterilized scissors. Because the tumors were sticky and easily clumped, repeated dissection with pipette and 22-gauge needles was needed. After trypsinization, single cell suspensions were obtained by passing the digested solutions through a cell strainer with 40-μm nylon mesh (BD Falcon).

To generate the malignant ascites in the animal models, 0.1 ml PC14PE6/AS2 cell suspensions (1 × 107 ml) were injected into the peritoneal cavity of unanaesthetized mice. The mice developed peritoneal carcinomatosis with ascites after 2–4 weeks. They were then sacrificed and the ascites harvested.

The peritoneal tumors were dissected with 22-gauge needles and trypsin for 30 min. Single cell suspensions were obtained by passing the digested solutions through a cell strainer. Cancer cells from subcutaneous, ascites, and peritoneal tumors were collected and incubated at 37°C for at least 2 h in MEN-α medium before treatment.

Blood and Pleural Effusion Collection and Cancer Cell Purification from Malignant Pleural Effusions

Blood was obtained from a healthy volunteer. Pleural effusions from congestive heart failure (CHF) and malignant pleural effusions (MPE) from lung adenocarcinoma were obtained from patients who underwent thoracentesis or thoracotomy at the National Cheng-Kung Hospital (Tainan, Taiwan). The institutional review board approved the study and each patient provided written informed consent.

Lung cancer-associated MPE was verified by cytologic analysis of the pleural effusion or pleural biopsy. Effusions were collected in sterile tubes and centrifuged immediately at 4°C.

Tumor cells were separated from MPE-associated lymphocytes by serial gradient centrifugation with Histopaque 1077 and Percoll (Sigma-Aldrich, St Louis, MO) as described previously (13). Single cell suspensions were obtained by passing the cells through a falcon tube with a cell strainer (BD Falcon) and there was 70–90% purity of tumor cells in target fractions as determined by cytologic analysis. The cancer cells were resuspended and then cultured in RPMI 1640 medium (Invitrogen-Gibco) and maintained in a 37°C incubator for 2 h before treatment.

E-Cadherin Staining

For surface staining of E-cadherin, cancer cells from MPE were washed with staining buffer (isotonic solution of PBS supplemented with 0.5% BSA) and resuspended in staining buffer with optimal concentrations of PE-conjugated anti-E cadherin and IgG isotype control antibody. Samples were incubated for 30 min at 4°C. After incubation, the samples were washed and events were examined using FACS Calibur. The emission of PE was detected by channel FL2.

Western Blot Analysis

Lung cancer cell lines were plated in their respective growth media at 1 × 106 cells in 100-mm dishes and incubated in a humidified 37°C incubator in 95% air/5% CO2 atmosphere for 24 h before treatment. Cancer cells from mouse subcutaneous tumor, peritoneal tumor, ascites, and patients' MPE were resuspended in their respective growth medium at 1 × 106 cells in 100-mm dish for 2 h before treatment. Cancer cell lines and cancer cells from mouse and patients' MPE were then transferred to serum-free medium for 30 min before IFN-γ stimulation.

After starvation, the cells were treated with IFN-γ (PeproTech, London, UK) for 15 min. The samples were subjected to Western blot and flow cytometry analyses, respectively. For Western blot analysis, treated and untreated cells were pelleted and washed twice with ice-cold PBS. Cell lysates were obtained after adding the lysis buffer containing phosphatase inhibitors (1 mM Na3VO4 and 10 mM NaF) and protease inhibitors (Roche, Indianapolis, IN). These were then cleared by centrifugation at 14,000 rpm for 10 min.

Protein concentration was determined via Bradford assay (Bio-Rad, Richmond, CA). Lysates were boiled for 5 min with sample buffer before separation on SDS-polyacrylamide gels. The proteins were transferred to a nitrocellulose filter (Millipore, Billerica, MA) in Tris-glycine buffer and run at 100 V for 1.5 h using an electro-blotter (Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were blocked by PBS buffer containing 5% nonfat milk before incubation with antibodies, while antibody binding was detected by electro-chemiluminescence (Amersham) according to the manufacturer's instructions. Alpha Ease FC software was used for densitometric analysis (Alpha Innotech, San Leandro, CA). The IDV (integrated density value) was calculated.

Single Cell Phospho-Specific Flow Cytometry

For SCPFC, treated and untreated cells were fixed with formaldehyde in a final concentration of 4% by directly mixing the culture medium with 8% formaldehyde. The formaldehyde was purchased from Merck (KGaA, Germany) and diluted with PBS. The cells were fixed with formaldehyde prior to permeabilization as in a previous study. The cells were fixed at room temperature for 10 min, and then pelletted by centrifugation at 1,500 rpm for 5 min and made permeable by resuspending in ice-cold methanol for 10 min at 4°C.

The sample was then directly analyzed by flow cytometry or stored at −20°C until analysis (within 4 weeks). The fixed cells were washed with staining buffer (PBS containing 1% BSA, Sigma-Aldrich, St Louis, MO) at 0.5–1 × 106 cell per 100 μl and resuspended in 100 μl staining buffer with Alexa dye-conjugated phospho-antibody (20 μl/1 × 106 cells). Samples were incubated for 30 min at room temperature (RT) and then washed with staining buffer and analyzed by flow cytometry.

Events were examined using FACS Calibur (Becton, Dickinson and Company, Franklin Lakes, NJ) and the emission of Alexa 488 was detected by channel FL1. Around 1–2 × 105 events were gathered per experiment.

Transfection with Small Interfering RNA (siRNA) Against Stat1

Oligonucleotides representing siRNA against human Stat1 expression (siRNA-Stat1) and scramble control oligo-nucleotides (siRNA-scramble control) were purchased from Invitrogen-Life Technologies (Carlsbad, CA). The siRNA sequences targeted Stat1: 5′-AUGAGUUCUAGGAUGCUUU CAAUCC-3′.

HONE1-cis 6 cells were seeded at 1 × 106 cells in 60-mm dish with 70–80% confluence before transfection. At the time of transfection, 1.25 μl containing siRNA oligonucleotides (siRNA-Stat1 or siRNA-control) and 4 μl lipofectamine™ 2000 reagent (Invitrogen-Life Technologies, Carlsbad, CA) were diluted in the final 250 μl of Opti-MEM® I (Gibco, Carlsbad, CA). Cells were transfected with lipofectamine + siRNA-Stat1, lipofectamine + siRNA-scramble control, or left untransfected for 3 h. The medium was then aspirated to terminate the transfection and fresh RPMI 1640 containing 10% FBS was added and incubated for 24 h. Cell lysates were sent to Western blot analysis and the transfected and untransfected cells were cultured in RPMI 1640 for apoptosis analysis.

Apoptosis Analysis After Cisplatin Treatment

For apoptosis analysis, cancer cells were cultured in medium with cisplatin for 24 h. These were then washed with ice-cold PBS and resuspended in Annexin V staining buffer (0.01 M HEPEA, pH 7.4, 0.14 M NaCl, and 2.5 mM CaCl2) with optimal concentration of PE-conjugated Annexin V antibody for 15 min. The samples were then washed and events were examined by FACS Calibur. The PE emission was detected by channel FL2.

Statistical Analysis

Data analysis was conducted using the WinMDI 2.9 software (Joseph Trotter, The Scripps Institute, La Jolla, CA) after gating for the designated population. Changes in pStat1 were calculated by evaluating the change in median fluorescence intensity (MFI) between treated and untreated cells. The extent of apoptosis was determined by the proportion of cells stained with Annexin V. Data were analyzed using Prism 4 (GraphPad Software for Science, San Diego, CA). Student's t test was used to determine differences between treated and untreated groups.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited
  8. APPENDIX
  9. Supporting Information

Stat1 Phosphorylation SCPFC Correlated with Western Blot Analysis of PC14PE6/AS2

To examine the application of SCPFC in lung cancer cells, PC14PE6/AS2 cells were treated with various concentrations of IFN-γ. The MFI (mean fluorescence intensity) had a 1.9-fold increase after stimulation with 0.31 ng ml−1 IFN-γ, 3.1-fold with 1.25 ng ml−1 IFN-γ, 6.1-fold with 5 ng ml−1 IFN-γ, and 6.3-fold with 20 ng ml−1 IFN-γ (Fig. 1A) compared to unstimulated control.

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Figure 1. The application of SCPFC in PC14PE6/AS2 cells. (A) Histograms and corresponding dot plots illustrate the fold changes in Stat1 phosphorylation in PC14PE6/AS2 cells with increasing amounts of IFN-γ treatment. (B) Western blot analyses show the corresponding Stat1 phosphorylation in PC14PE6/AS2 cells after IFN-γ treatment. (C) Maximum percentage in Western blotting and SCPFC are shown by calculating MFI (mean fluorescence intensity) and IDV (integrated density value) of each sample, respectively, with 20 ng ml−1 IFN-γ as maximum induction (i.e., MFIsample/MFI20 ng ml−1IFN-γ, IDVsample/IDV20 ng ml−1IFN-γ × 100).

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Induction of Stat1 phosphorylation was also demonstrated in Western blot analysis in a dose-dependent manner (Fig. 1B). The percentage of maximum induction in SCPFC and Western blot were calculated by dividing the MFI or integrated density value (IDV) of each sample by that of the maximally stimulated sample, as previously described (14). The percentage of maximum induction detected by SCPFC correlated well with those obtained by Western blot analysis (Fig. 1C).

Stat1 Phosphorylation SCPFC in Cancer Cells of Subcutaneous Tumors, Metastatic Peritoneal Tumors, and Malignant Ascites

To apply SCPFC to solid tumors in vivo, subcutaneous xenografts (Fig. 2A) were generated from PC14PE6/AS2 cells. The obtained cancer cells were subjected to analysis. PC14PE6/AS2 cells were also injected intra-peritoneally into nude mice and cancer cells that developed ascites and peritoneal tumors (Fig. 2B) were collected for further analysis. To exclude contamination of leukocytes and other stromal cells, fresh blood from healthy mice of the same age was collected as control.

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Figure 2. Representative images PC14PE6/AS2 derived subcutaneous and ascites animal model. (A) Tumor in nude mouse derived from PC14PE6/AS2 cells injected subcutaneously into 5-week old nude mice. (B) At 3 weeks after i.p. inoculation, the mice exhibited abdominal distention with bloody ascites (arrow head). Multiple peritoneal metastatic tumors were noted over the intestinal mesentery (arrow).

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The R1 region was composed of red blood cells and cell debris, which were small and had less granulation (Fig. 3A). The R2 region was composed of leukocytes, which were medium sized and had slightly increased granulation. The R3 region was primarily composed of cancer cells, which were larger and had the most obvious granulation. These cancer cells from the animal model showed the same morphologic characteristics (FSC and SSC) as the PC14PE6/AS2 cells from culture dishes. Although lower doses of IFN-γ (<5 ng ml−1) induced significant phosphorylation of Stat1 in the PC14PE6/AS2 cell line in vitro, changes in pStat1 at these lower doses were not detectable in the cancer cells derived in vivo. Therefore, the IFN-γ dosage used in the current study was at least 5 ng ml−1.

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Figure 3. SCPFC applied to the subcutaneous and ascites animal model. (A) The FSC (forward scatter) and SSC (side scatter) characteristics of blood from age-matched healthy mice and tumor cells from malignant ascites, peritoneal tumors, subcutaneous tumors, and PC14PE6/AS2 cells in vitro culture. (B) Histograms of fluorescence intensity changes in pStat1 in tumor cells after IFN-γ treatment. (C) Fold changes in MFI are calculated as the ratio of cancer cells treated with IFN-γ relative to the unstimulated control (**P < 0.01). (D) Western blot analyses show corresponding Stat1 phosphorylation after IFN-γ treatment in cancer cells from the peritoneal tumor.

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After appropriate gating, cancer cells from malignant ascites, peritoneal tumors, and subcutaneous tumors were analyzed. On IFN-γ stimulation, changes in pStat1 level in cancer cells from ascites were similar to those from subcutaneous tumors (Figs. 3B and 3C). Changes of pStat1 level after IFN-γ treatment in cancer cells from peritoneal carcinomatosis were more prominent than those from ascites or subcutaneous xenograft models. Fluorescence changes in pStat1 were 1.8-fold with 5 ng ml−1 IFN-γ, 2.4-fold with 20 ng ml−1 IFN-γ, and 2.9-fold with 80 ng ml−1 IFN-γ compared to unstimulated controls. Flow cytometry analysis correlated well with Western blot analysis (Fig. 3D).

SCPFC and Western Blot Analysis of IFN-γ-Induced Stat1 Phosphorylation in Cancer Cells

To evaluate the applicability of SCPFC in human lung cancer samples, MPE were collected from patients with lung adenocarcinoma. The cellular populations in MPE were heterogeneous, including leukocytes, mesothelial cells, and cancer cells. Blood samples were collected from healthy volunteers and pleural effusions from patients with congestive heart failure (CHF), which served as controls (Fig. 4A). The cancer cells were gated and analyzed separately. As in the animal model, the R1 region was composed of red blood cells and cell debris. The R2 region was primarily composed of leukocyte and mesothelial cells, while the R3 region was primarily composed of cancer cells.

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Figure 4. The application of SCPFC in cancer cells from MPE of patients with lung adenocarcinoma. (A) FSC and SSC characteristics of blood from age-matched healthy volunteers, of cells derived from effusions of congestive heart failure patients, and of cancer cells from MPEs of lung cancer patients. (B) Dot plots of fluorescence intensity changes in E-cadherin intensity in different subgroups of the MPE relative to the isotype control. (C) Histogram of the difference of fluorescence intensity changes of pStat1 after IFN-γ treatment in cancer cells from MPE of Patients A and B. (D) Corresponding Western blot analysis shows changes of pStat1 after IFN-γ treatment in cancer cells from MPE.

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In a previous immuno-histochemisty study, there was higher expression of E-cadherin in cancer cells relative to mesothelial cells (15). E-cadherin was therefore used to validate gating. Its expression was higher in cells of the R3 region than those of the R2 and R1 regions (Fig. 4B). After treatment with IFN-γ, cancer cells from MPEs of different patients responded differently. For example, in Patient A, the intensity of pStat1 increased 2.0-fold by 5 ng ml−1 IFN-γ, 2.4-fold by 20 ng ml−1 IFN-γ, and 2.6-fold by 80 ng ml−1 IFN-γ relative to unstimulated controls. In Patient B, there was Stat1 activation only with higher doses of IFN-γ (1.8-fold increase in intensity with 80 ng ml−1 IFN-γ).

Western blot analysis of pStat1 in cancer cells from MPEs again correlated well with that of SCPFC (Fig. 4C). In the leukocytes, pStat1 increased modestly (<1.5-fold) with IFN-γ treatment (data was not shown). The clinico-pathologic characteristics of Patients A and B were listed in Table 1

Table 1. Characteristics of lung cancer patients with malignant pleural effusion (n = 8)
PatientSexAgeCell typeStage
AFemale80AdenocarcinomaIVb (bone)
BMale45AdenocarcinomaIVa
1Female54AdenocarcinomaIVa
2Female80AdenocarcinomaIVb (bone)
3Male63AdenocarcinomaIVa
4Male87AdenocarcinomaIVb (lung and bone)
5Male45AdenocarcinomaIVb (bone)
6Male76AdenocarcinomaIVa

Different Patterns of Stat1 Activation in Cancer Cells from MPE had Different Sensitivities to Cisplatin

Cisplatin was the main agent for treating advanced lung cancer (10). Because Stat1 activation might contribute to drug resistance of cancer cells (16, 17), the relationship between Stat1 activation after IFN-γ stimulation and cell sensitivity to cisplatin in cancer cells from MPEs was investigated. Cancer cells from MPEs of another six lung cancer patients were analyzed (Table 1, Patients 1–6). The MFI fold change in pStat1 after IFN-γ treatment (5 ng ml−1) separated the cancer cells into two groups. In Group 1 (n = 3), cancer cells had smaller changes in pStat1 after stimulation (<2-fold change, Fig. 5A). In Group 2 (n = 3), cancer cells had larger changes (>2-fold) in pStat1 after IFN-γ treatment.

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Figure 5. Different patterns of pStat1 change after IFN-γ treatment in cancer cells from MPEs display different sensitivities to cisplatin. (A) Fold changes in MFI of pStat1 after IFN-γ stimulation and corresponding changes in the proportion of apoptotic cells after cisplatin treatment (**P < 0.01; *P < 0.05). (B) Histograms show Stat1 phosphorylation and Annexin V staining of cancer cells from MPE of Cases 3 and 6. (C) Relationship between MFI fold changes of pStat1 after IFN-γ stimulation and change of apoptotic cell after cisplatin treatment (P < 0.01; R2 = 0.94).

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When treated with cisplatin (40 μM), the proportion of apoptotic cells measured by Annexin V staining markedly increased (19–22%) in the cancer cells of Group 1, but not in Group 2 (<5%). Changes of pStat1 with corresponding sensitivity to cisplatin in Cases 3 and 6 were shown in Figure 5B. In Case 3, pStat1 increased 1.3-fold by IFN-γ and the proportion of apoptotic cells markedly increased (22.1%) after cisplatin treatment. In contrast, pStat1 increased 2.8-fold by IFN-γ but the proportion of apoptotic cells did not increase in Case 6. The correlation between fold changes of pStat1 and changes of apoptotic proportion was statistically significant (Fig. 5C, R2 = 0.91). These suggested that cancer cells from MPE expressing higher levels of phosphorylated Stat1 after IFN-γ stimulation were more resistant to cisplatin.

Stat1 Activation was Higher in Cisplatin-Resistant Cancer Cell Line and Downregulation of Stat1 Reversed the Resistance

Though the above study provided a correlation between Stat1 phosphorylation and cisplatin sensitivity, the signification of Stat1 activation on cisplatin sensitivity remained unclear. The HONE1 (Human nasopharyngeal carcinoma, parental) and resistant sub-line HONE1-cis 6 (HONE1-derived cisplatin resistant) were used to address the question. The proportion of apoptotic cells after cisplatin treatment measured by Annexin V staining was significantly higher in HONE1 than in HONE-cis6 (Fig. 6A), while data confirmed that HONE-cis6 was more resistant to cisplatin than HONE1.

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Figure 6. Annexin V staining and intracellular pStat1 staining of HONE1 and HONE1-cis 6 cells. (A) The proportions of apoptotic HONE1 and HONE1-cis 6 cells after treatment with various doses of cisplatin (**P < 0.01). (B) MFI changes in pStat1 in HONE1 and HONE1-cis 6 cells after IFN-γ treatment (**P < 0.01).

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After treatment of IFN-γ, the MFI (mean fluorescence intensity) increased by 1.7-fold with 5 ng ml−1 IFN-γ, by 2.1-fold with 20 ng ml−1 IFN-γ, and by 2.2-fold with 80 ng ml−1 IFN-γ (Fig. 6B) compared to unstimulated control in HONE1 cells. As for HONE1-cis 6, the MFI had a 1.9-fold increase after stimulation with 5 ng ml−1 IFN-γ, 2.5-fold with 20 ng ml−1 IFN-γ, and 2.8-fold with 80 ng ml−1 IFN-γ compared to unstimulated control. Therefore, the induction of Stat1 phosphorylation after IFN-γ stimulation was higher in HONE1-cis 6 than in parental HONE1 cell. Fold changes of p-Stat1 by IFN-γ stimulation in the two cells were similar by Western blot analyses (Supporting Information Fig. 1).

To further demonstrate the significance of Stat1 activation on cisplatin resistance, small interfering RNA was used against Stat1 (siRNA-Stat1) to knockdown Stat1 expression in HONE1-cis 6 cells. The transfection of siRNA-Stat1 into cells suppressed the expression of Stat1 as assayed by Western blot. There was no change of Stat1 expression in scramble and untransfected control cells (Fig. 7A). Various dosage of cisplatin, the proportion of apoptotic cells measured by Annexin V staining was higher in siRNA-Stat1 transfected cells than in the scramble control (Figs. 7B and 7C). Taken together, Stat1 activation correlated well with cellular sensitivity to cisplatin treatment. This could be predicted by short-term IFN-γ treatment and revealed by SCPFC method.

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Figure 7. The effect of siRNA-Stat1 on HONE1-cis 6 cells sensitivity to cisplatin. (A) Western blot analyses show the expression of Stat1 of HONE-cis 6 cells untranfected or transfected with siRNA-scramble control, siRNA-Stat1, for 3 h. (B) Histograms show Annexin V staining of HONE1-cis 6 transfected with siRNA-scramble control or siRNA-Stat1 after treatment with various doses of cisplatin. (C) Proportions of apoptotic HONE1-cis 6 (siRNA- scramble control and siRNA-Stat1) cells after treatment with various doses of cisplatin (**P < 0.01).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited
  8. APPENDIX
  9. Supporting Information

This study applied single cell phospho-specific flow cytometry (SCPFC) to lung cancer lines, subcutaneous and ascites animal models, and human tumor samples. It first demonstrated the dose-dependent change of Stat1 phosphorylation after IFN-γ treatment in PC14PE6/AS2 cells detected by SCPFC, which correlated well with Western blot analysis. The platform was also applied to normal bronchial cell and other cancer lung cell lines (Supporting Information, Figs. 2–4). Similarly, IFN-γ-induced Stat1 phosphorylation was detected by SCPFC in cancer cells from subcutaneous xenograft, malignant ascites, and peritoneal tumors of nude mice. The method was applied to detect Stat1 activation by IFN-γ in cancer cells from MPEs of lung cancer patients. The sensitivity of cancer cells from MPE in response to IFN-γ, as measured by pStat1 expression, was associated with cellular sensitivity to cisplatin. This was the first study to reveal the potential of application of SCPFC to solid tumors.

Previous studies have demonstrated the use of SCPFC on hematologic disorders such as leukemia (2, 3) or lymphoma (18). The applicability of this technique on solid tumors, however, has not been previously demonstrated. In this study, SCPFC has been applied to two animal models, the subcutaneous and ascites animal models. In the subcutaneous xenografts, cancer cells have been collected from tumors generated by injecting PC14PE6/AS2 cells into nude mice subcutaneously. In the ascites tumor model, PC14PE6/AS2 cells have been injected into nude mice i.p., thereby enabling the harvest of cancer cells from peritoneal tumors and malignant ascites for further analysis.

This study shows that SCPFC can detect IFN-γ-induced Stat1 phosphorylation in cancer cells of these two animal models. The results correlate well with those of Western blot assays. Intriguingly, patterns of Stat1 activation by IFN-γ in cancer cells from malignant ascites and peritoneal tumor cells are similar, indicating that the behavior of tumor cells in malignant effusions may be used to represent that of tumor cell seeding in the peritoneum or pleura. Thus, it is meaningful to apply SCPFC to analyze cancer cells from MPEs of lung cancer patients. Such application offers several advantages. First, only a small number of cancer cells are needed (19). Second, thoracocentesis, used to obtain malignant plural effusion, is less invasive. Third, MPE cells may offer information about the signaling pathways utilized in the primary tumor. Fourth, recurrent MPEs can be used to study sequential changes in signaling over the course of tumor progression. Lastly, SCPFC can be used to investigate several signaling pathways at a time.

The IFN-γ-induced phosphorylation of Stat1 may have other biological applications. In prostate tumor cells, Stat1 expression is heightened in docetaxel-resistant cancer cells (17). Transfection of Stat1-specific siRNA into docetaxel-resistant cells results in re-sensitization to docetaxel. Aside from the role of Stat1 in cisplatin resistance, micro-array analysis of an ovarian cancer cell line reveals an inverse relationship between increased Stat1 expression and drug sensitivity. Transfection of full-length Stat1 into drug-sensitive cancer cells has led to cisplatin resistance (16). However, the associated mechanisms remain unexplored.

In this study, cancer cells from different patient MPEs have different sensitivities to IFN-γ stimulation. The activation of Stat1 by lower doses of IFN-γ predicts cancer cell vulnerability to cisplatin. It also demonstrates that pStat1 expression is higher after IFN-γ stimulation in HONE1-cis 6 (HONE1-derived cisplatin resistant) than in parental HONE1 cell.

The importance of Stat1 on cisplatin resistance is further verified by the increase of apoptosis after knocking down Stat1 expression by Stat1 siRNA in HONE1-cis 6 cells. In different lung cancer cell lines, including CL1-5, A549, and H1650, the reverse correlation between cisplatin sensitivity and pStat1 expression after IFN-γ stimulation (Supporting Information Figs. 3 and 4) is also demonstrated, suggesting that these interesting findings are not limited to a particular cell population. Though the results are promising, the patient cohort is small. Further studies are needed to validate the application.

A previous study has found a discrepancy of activation of signal proteins between cell line and primary patient samples (1). Although the expression of mutant, activated Flt3 in leukemia cell lines has been observed to activate Stat5 (20), the basal level of Stat5 phosphorylation is statistically indistinguishable in Flt3 mutant and wild-type patient-derived AML blasts (21). It is proposed that the changes of phosphorylation of key signals after short-term stimulation with growth factor or cytokine are more informative than the basal level of phosphorylation. Therefore, instead of studying the basal level of Stat1 phosphorylation, this study investigated dynamic change of pStat1 after IFN-γ stimulation after isolating cancer cells from MPE and culturing them in the short-term. As such, the correlation between cisplatin sensitivity and pStat1 activation after IFN-γ stimulation is demonstrated.

In conclusion, SCPFC can detect changes of Stat1 phosphorylation after IFN-γ treatment in cancer cells from lung cancer cell lines, animal tumor models, and MPEs of lung cancer patients. The SCPFC technique is applicable to solid tumors and offers a potential clinical role in predicting the drug sensitivity of cancer cells.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited
  8. APPENDIX
  9. Supporting Information

The authors thank Dr. Fidler (MD Anderson Cancer Center, Houston, TX) for the human lung adenocarcinoma cell line PC14PE6, Dr. Pan-Chyr Yang (National Taiwan University College of Medicine, Taiwan) for the human lung adenocarcinoma cell line CL 1-5, and Dr. Jang-Yang Chang (National Institute of Cancer Research, National Health Research Institutes, Taiwan) for the HONE1 (Human Nasopharyngeal Carcinoma, parental) and HONE1-cis 6 (HONE1-derived cisplatin resistant) cell lines.

Literature Cited

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited
  8. APPENDIX
  9. Supporting Information
  • 1
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APPENDIX

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited
  8. APPENDIX
  9. Supporting Information

Isolation and Preparation of Cancer Cells from Animal and MPE Samples

  • 1
    The tumors from animal samples are minced using a sterilized scissors. MPE cancer cells are isolated by serial gradient centrifugation with Histopaque 1077 and Percoll.
  • 2
    Repeat dissecting the cancer cells with 1,000-ml pipette and 22-gauge needles. The step is more useful for preventing cell clumping than vigorously vortexing alone.
  • 3
    Centrifuge 5 min at 1,500 rpm, room temperature, and then decant the supernatant.
  • 4
    Add 2 ml trypsin (depend on the sediment amount) and incubate for the 5 min in a 37°C, 5% CO2 incubator.
  • 5
    Repeat Step 2 and then filter the dissociated cell solution through a 40-μm cell strainer.
  • 6
    Add 5-ml serum containing medium to stop the effect of trypsin.
  • 7
    Centrifuge 5 min at 1,500 rpm, at room temperature, and then decant the supernatant.
  • 8
    Resuspend the cells in serum containing medium at the concentration of 1 × 106 cells ml−1. Place in a 50-ml polypropylene tube or a 100-mm ultra-low-attachment dish (Corning, NY) and allow the cells resting 2 h in a 37°C, 5% CO2 incubator. Higher cell concentration or smaller polypropylene tube may induce cell adherence and clumping.

IFN-γ Treatment, Fixation, and Permeabilization

  • 1
    After resting, centrifuge for 5 min at 1,500 rpm, at room temperature, and then decant the supernatant.
  • 2
    Resuspend cancer cells with serum-free medium and transfer them into a 50-ml polypropylene tube (4 ml/per tube) or into a 100-mm ultra-low-attachment dish (8 ml/per dish) and incubate in a 37°C, 5% CO2 incubator for 30 min. Four tubes or dishes are needed at least for each experiment:
  • 3
    Add serum-free medium with or without IFN-γ (1 ml per tube or 2 ml per dish) and incubate in a 37°C, 5% CO2 incubator for 15 min.
  • 4
    Fix the cells by adding freshly prepared 8% PFA (5 ml per tube or 10 ml per dish) to a final concentration of 4%, vortex to mix the cells, and then incubate 10 min at room temperature. Large amount of PFA decrease the formation of cell clumps.
  • 5
    Centrifuge for 5 min at 1,500 rpm, at room temperature, and then decant the supernatant.
  • 6
    Resuspend the cells thoroughly in the residual supernatant volume by vigorously vortexing and pipetting. Add 1 ml of cold (4°C) methanol to each tube, and then transfer to the eppendorf and vortex to mix. Incubate cells at 4°C for 20 min.
  • 7
    The sample can be then directly analyzed by flow cytometry or stored at −20°C for later analysis (within 4 weeks).

Intracellular Staining

  • 1
    The fixed cells are centrifuge for 3 min at 6,000 rpm and washed with 1 ml staining buffer (PBS containing 1% BSA) at least for three times to avoid the precipitation of BSA by methanol.
  • 2
    Decant the supernatant and add 100 μl staining buffer to 1st tube, 80 μl staining buffer + 20 μl Ax-488 IgG isotype control Ab to 2nd tube, 80 μl staining buffer + 20 μl Ax-488-anti-pStat1Y701 Ab to 3rd tube and 4th tube separately, pipet for four to five times and incubate cells for 30 min at room temperature in the dark. Vigorous vortex should be avoided because this procedure will cause the adherence of cell to the cover of eppendorf and affect total cell number.
  • 3
    Centrifuge for 3 min at 6,000 rpm, decant the supernatant and then wash the cells with 100 μl staining buffer to discard unbinding antibody.
  • 4
    Centrifuge for 3 min at 6,000rpm, decant the supernatant and then resuspend the cells with 500 μl staining buffer and transfer to FACS tubes.
  • 5
    Analyze the results by FACS Calibur (the emission of Ax-488 is detected by channel FL1). Around 1–2 × 105 events were gathered for each experiment. The software CellQuest is applied to adjust the forward scatter (FSC) and side scatter (SSC). Adjust the FSC Amp Gain, the SSC Voltage, and the FSC Threshold to appropriately display the scatter properties of the sample. The 1st tube (blank tube) is used to adjust the fluorescent background. The success of intracellular staining is confirmed by the elevated Ax-488 fluorescent intensity of 2nd tube (AX-488-IgG isotype control Ab), and 3rd tube (Ax-488-anti-pStat1Y701 Ab) compared to the blank tube. The instrument setting of FSC, SSC, and FL1 is recorded and applied to the same cancer cell line.

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Literature Cited
  8. APPENDIX
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
CYTOA_20965_sm_SuppFig1.tif6230KSupporting Information Figure 1. Stat1 phosphorylation change after IFN-γ treatment in HONE1 and HONE1-cis 6 cells. Western blot analyses show Stat1 phosphorylation change after IFN-γ stimulation in HONE1 and HONE1-cis 6 cells.
CYTOA_20965_sm_SuppFig2.tif20791KSupporting Information Figure 2. The application of SCPFC in normal bronchial cell-NL-20. (A) Histograms and dot plots show fold changes of Stat1 phosphorylation after IFN-γ treatment. (B) Western blot analyses show corresponding Stat1 phosphorylation change in NL-20 cells after IFN-γ treatment.
CYTOA_20965_sm_SuppFig3.tif20480KSupporting Information Figure 3. Annexin V staining and intracellular pStat1 staining of CL1-5, A549 and H1650. (A) Histograms show pStat1 changes after IFN-γ stimulation and the corresponding changes in the proportion of apoptotic cells after cisplatin treatment in CL1-5, A549, and H1650. (B) Fold changes in MFI of pStat1 after IFN-γ stimulation and the corresponding changes in the proportion of apoptotic cells after cisplatin treatment.
CYTOA_20965_sm_SuppFig4.tif15729KSupporting Information Figure 4. Stat1 phosphorylation change after IFN-γ treatment in CL1-5, A549, and H1650. Western blot analyses show Stat1 phosphorylation changes after IFN-γ stimulation in HONE1 and HONE1-cis 6 cells.

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