The Enhanced metastatic potential of hepatocellular carcinoma (HCC) cells with sorafenib resistance

Acquired resistance towards sorafenib treatment was found in HCC patients, which results in poor prognosis. To investigate the enhanced metastatic potential of sorafenib resistance cells, sorafenib-resistant (SorR) cell lines were established by long-term exposure of the HCC cells to the maximum tolerated dose of sorafenib. Cell proliferation assay and qPCR of ABC transporter genes (ABCC1-3) were first performed to confirm the resistance of cells. Migration and invasion assays, and immunoblotting analysis on the expression of epithelial to mesenchymal transition (EMT) regulatory proteins were performed to study the metastatic potential of SorR cells. The expression of CD44 and CD133 were studied by flow cytometry and the gene expressions of pluripotency factors were studied by qPCR to demonstrate the enrichment of cancer stem cells (CSCs) in SorR cells. Control (CTL) and SorR cells were also injected orthotopically to the livers of NOD-SCID mice to investigate the development of lung metastasis. Increased expressions of ABCC1-3 were found in SorR cells. Enhanced migratory and invasive abilities of SorR cells were observed. The changes in expression of EMT regulatory proteins demonstrated an activation of the EMT process in SorR cells. Enriched proportion of CD44+ and CD44+CD133 + cells were also observed in SorR cells. All (8/8) mice injected with SorR cells demonstrated lung metastasis whereas only 1/8 mouse injected with CTL cells showed lung metastasis. HCC cells with sorafenib resistance demonstrated a higher metastatic potential, which may be due to the activated EMT process. Enriched CSCs were also demonstrated in the sorafenib resistant cells. This study suggests that advanced HCC patients with acquired sorafenib resistance may have enhanced tumor growth or distant metastasis, which raises the concern of long-term sorafenib treatment in advanced HCC patients who have developed resistance of sorafenib. © 2013 Chow et al.published_or_final_versio

Isolation and characterization of cancer stem cells in non-small cell lung cancer

Tumor heterogeneity has long been observed and recognized as a challenge to cancer therapy. The cancer stem cell (CSC) model is one of the hypotheses proposed to explain such a phenomenon. A specific cancer stem cell marker has not been determined for non-small cell lung cancers (NSCLC), preventing the definitive evaluation of whether the biology of NSCLC is governed by the CSC model. This study aimed to analyze the expression of candidate CSC markers and using the identified putative marker, to isolate CSC and determine the applicability of the CSC model in NSCLC. The expression or activities of four putative stem cell markers, CD24, CD44, CD133 and aldehyde dehydrogenase 1 (ALDH1) were measured by flow cytometry in eight NSCLC cell lines before and after chemotherapy for 24 hours. Markers with increased expression after treatment were considered potential CSC markers and used for isolating tumor cell subpopulations from the untreated cell lines by fluorescence-activated cell sorting (FACS). Confirmatory analyses using widely acceptable methodology were performed to test for CSC properties in the marker+ and marker- subpopulations. Isolated subpopulations were further characterized by functional and phenotypic studies. Flow cytometry showed amongst the 4 markers, only ALDH1 expression was significantly enhanced by chemotherapeutic treatment, suggesting ALDH1 could be a CSC marker. Untreated ALDH1+ cells formed significantly more and larger cell spheres in non-adherent, serum-free conditions than ALDH1- cells. Likewise, higher in vitro tumorigenic ability was observed in ALDH1+ subset using colony formation assay. Furthermore, a higher resistance to cytotoxic drugs was observed in ALDH1+ compared to ALDH1- cells. In vivo studies also showed ALDH1+ cells showed higher tumorigenicity than ALDH1- cells; as few as 2,500 ALDH1+ cells formed tumor in SCID mice which were serially transplantable to 2nd and 3rd recipients, while no tumor was formed from ALDH- cells with even ten times the number of cells. Also, expression analysis revealed higher expression of the pluripotency genes, OCT4, NANOG, BMI1 and SOX9, in ALDH1+ cells. In view of previous studies reporting CD44 as a CSC marker in lung cancer, double marker selection of putative CSC was performed to compare ALDH1+CD44+ and ALDH1-CD44+ subpopulations. Results of the spheroid body formation assay and cisplatin treatment experiments revealed the ALDH1+CD44+ subpopulation possessed higher self-renewal ability and chemo-resistance. Cell migration and invasion assays showed differences between the ALDH1+CD44+ and ALDH1- CD44+ subpopulations. The significance of these observations require further investigation. In conclusion, the result showed that ALDH1 could be a marker for NSCLC stem cells as evidenced by enhanced self-renewal and differentiation abilities in ALDH1+ subpopulation. Furthermore, this study observed the presence of at least two potential stem cell subpopulations in NSCLC cells with differential selfrenewal, chemotherapy resistance and cell mobility properties. Further investigations are required to validate these observations and to investigate the underlying mechanisms. Better understanding of these issues would help to solve the challenges brought by tumor heterogeneity in lung cancer therapy.published_or_final_versionPathologyMasterMaster of Philosoph

Identification of the regulatory mechanism for conferring metastasis of CD26-expressing colorectal cancer stem cells

Cancer stem cells are a subpopulation of cells needed for cancer initiation and progression. Previous works have revealed CD26-expressing colorectal cancer (CRC) stem cells are not only endowed with tumor-initiating properties, but also capable of conferring metastasis. However, whether the CD26 molecule plays role in metastasis and the underlying mechanism by which CD26 may mediate metastasis remain unclear. This study aims to reveal the biology and the molecular characteristics of the CD26-expressing CRC stem cells. Here, by the gene manipulation experiment, we showed that CD26 molecule is a functional marker that confers metastasis as transient and stable knock-down of the CD26 molecule in the CRC stem cells resulted in reduced wound healing, migration and invasion abilities in vitro and the capability to generate metastatic liver nodules in vivo, respectively. With the use of genome-wide expression array and immuno-blotting analysis, Smad-dependent TGF-β signaling, orchestrated by the SMAD2, SMAD3 and SMAD4 molecules, was up-regulated and activated in the CD26 expressing colorectal CSCs. In addition, expressions of the SMAD2 and SMAD3 molecules were found to be positively correlated with the CD26 molecule in clinical samples by qPCR and immunohistochemistry studies. Furthermore, no metastasis through EMT could be achieved once the Smad-dependent TGF-β signaling was down-regulated in the CD26 expressing CRC stem cells, which suggested that Smad-dependent TGF-β signaling was necessary for CD26-expressing CRC stem cells to induce metastasis. Finally, our result showed that the Smad-dependent TGF-β signaling was regulated by the CD26 molecule possibly through the down-regulation of CAV1 protein. To conclude, our findings have not only revealed the functional role of CD26 molecule, but have also unveiled a linkage between the CD26 molecule and Smad-dependent TGF-β signaling. Further study of this connection may introduce a novel mechanism, through which CRC metastasis can be induced by this functional CD26 marker of CRC stem cells.published_or_final_versionSurgeryDoctoralDoctor of Philosoph

CD26 Induces Colorectal Cancer Angiogenesis and Metastasis through CAV1/MMP1 Signaling

CD26 has been reported as a marker for colorectal cancer stem cells endowed with tumor-initiating properties and capable of colorectal cancer (CRC) metastasis. In this study, we investigated the functional effect of CD26 on CRC angiogenesis and metastasis, and the potential underlying mechanism. The functional effects of CD26 overexpression or repression were determined by a wound healing experiment, and cell migration and invasion assays in vitro and in mouse models. Differentially expressed genes regulated by CD26 were identified by genome-wide mRNA expression array and validated by quantitative PCR. CD26 functionally regulated CRC cell migration and invasion in vitro and angiogenesis and metastasis in vivo. Genome-wide mRNA expression array and qPCR showed that MMP1 was up-regulated in CD26+ subpopulation, and a subsequent experiment demonstrated the regulatory effect of CD26 on MMP1 in CRC cell lines with CD26 repression or overexpression. Furthermore, overexpression of CAV1 abrogated the CD26-regulated MMP1 induction in CRC cell lines. This study demonstrated the functional roles of CD26 in inducing CRC migration, invasion, angiogenesis and metastasis and identified the potential involvement of MMP1 and CAV1 in such process. CD26 is an attractive therapeutic target for combating tumor progression to improve the prognosis of CRC patients

A Four-Gene Panel in Rectal Swab Samples as a Biomarker for Colorectal Cancer Screening

Background: The dysregulation of gene expression is one of the key molecular features of colorectal cancer (CRC) development. This study aimed to investigate whether such dysregulation is reflected in rectal swab specimens of CRC patients and to evaluate its potential as a non-invasive approach for screening. Methods: We compared the expression level of 14 CRC-associated genes in tumor and adjacent non-tumor tissue of CRC patients and examined the correlation of their levels in tissue with paired rectal swab specimens. The level of these 14 genes in rectal swab specimens was compared among patients with CRC or polyp and control subjects, and the diagnostic potential of each dysregulated gene and the gene panel were evaluated. Results: The expression of CXCR2, SAA, COX1, PPARδ, PPARγ, Groγ, IL8, p21, c-myc, CD44 and CSF1 was significantly higher in CRC, and there was a significant correlation in the levels of most of them between the CRC and rectal swab specimens. In the training study, we showed that CD44, IL8, CXCR2 and c-myc levels were significantly higher in the rectal swab specimens of the CRC patients. Such result was confirmed in the validation study. A panel of these four genes was developed, and ROC analysis showed that this four-gene panel could identify CRC patients with an AUC value of 0.83 and identify overall polyp and precancerous adenoma patients with AUC values of 0.6522 and 0.7322, respectively. Finally, the predictive study showed that the four-gene panel demonstrated sensitivities of 63.6%, 76.9% and 88.9% in identifying overall polyp, precancerous adenoma and CRC patients, respectively, whereas the specificity for normal subjects was 72.2%. Conclusion: The expression of CRC-associated genes in rectal swab specimens reflects the dysregulation status in colorectal tissue, and the four-gene panel is a potential non-invasive biomarker for early precancerous adenoma and CRC screening

Enriched CSCs subpopulation in SorR cells.

<p>A) Harvested CTL and SorR cells derived from PLC/PRF/5, MHCC97L and HepG2 cells were stained with CD44 (FITC) and CD133 (APC) antibodies and 20000 cells were assessed by flow cytometry. The percentage of cells was indicated in each quadrant. B) Total RNA from CTL and SorR cells were extracted to perform the qPCR analysis of Lin28, Oct4, Nanog, Msi1 and SOX2. Data are presented as means ± SD from three independent experiments. *p<0.05 vs. CTL cells by one-way ANOVA.</p

Higher metastatic potential of SorR cells in an orthotopic model.

<p>CTL and SorR cells derived from PLC/PRF/5 cells were injected under the capsule of the left liver lobe. A) Under anesthesia, bioluminance signal produced by the injected cells were measured to study the tumor size at week 2, 4 and 6. B) Mice were sacrificed at week 6, bioluminance signal from primary tumor were detected to quantify the tumor size. C) Lung were isolated and bioluminance signal demonstrated the presence of injected cells which represents lung metastasis. D) Representing IHC staining of Ki-67 (left panel) and H&E staining (right panel) the lung sections obtained from CTL and SorR group (magnification: 100x and 400x). E) Representing H&E staining (first row), IHC staining of Ki-67 (second row) and IHC staining of CD44 (third row) of adjacent liver and primary tumor obtained from CTL and SorR group (magnification: 400x). F) The scoring of IHC staining of CD44 and Ki67 based on the percentage and intensity of the positively stained cells under high power (400x) microscopy was performed. Data are presented as means ± SD from 8 mice in each group. *p<0.05 vs. CTL cells by one-way ANOVA.</p

Enhanced cellular migration and invasion with activated EMT process of SorR cells.

<p>CTL and SorR cells derived from PLC/PRF/5, MHCC97L and HepG2 cells were plated in top chambers to perform the migration and invasion assay. A) Representing images of the migrated cells under a phase-contrast microscopy (magnification: 100x) were shown in the left panel and the number of migrated cells was counted and presented in the right panel. B) Representing images of the invaded cells under a phase-contrast microscopy (magnification: 100x) were shown in the left panel and the number of invaded cells was counted and presented in the right panel. Data are presented as means ± SD from three independent experiments. *p<0.05 vs. CTL cells by one-way ANOVA. C) Immunoblotting analysis demonstrated the change in total protein expression of E-cadherin, N-cadherin, Vimentin and Snail. The expression level of β-actin was used as loading control. D) Immunoblotting analysis demonstrated the change in protein expression of β-catenin, Smad2 and Smad3 from the nuclear fraction. The expression of nuclear matrix protein p84 was used as loading control.</p

Establishment of SorR cells using HCC cell lines.

<p>PLC/PRF/5, MHCC97L and HepG2 cells were cultured at maximal tolerated dose of sorafenib to obtain the CTL and SorR cells derived from each cell line. A) CTL and SorR cells were cultured at 0–14 µM sorafenib and MTT assay was performed 72 hours after treatment. B) Total RNA from CTL and SorR cells were extracted to perform the qPCR analysis of ABCC1, ABCC2 and ABCC3. C) Representing images of CTL and SorR cells under a phase-contrast microscopy (magnification: 400x). D) CTL and SorR cells were stained with phalloidin (red) and counterstained by DAPI (blue). Representing images of CTL and SorR cells under a fluorescence microscopy (magnification: 400x). Cellular protrusions were indicated by arrows. Data are presented as means ± SD from three independent experiments. *p<0.05 vs. CTL cells by one-way ANOVA.</p

IC₅₀ of CTL and SorR cells towards sorafenib treatment.

<p>IC₅₀ of CTL and SorR cells towards sorafenib treatment.</p