Differences In Tumorigenicity Research Articles

The clinical impact of soluble natural killer cell group 2-member D (NKG2D) receptor ligands on tumor tumorigenicity and anti-tumor immunity.

e14557 Background: NKG2D is an activating receptor expressed on all human natural killer (NK) cells and CD8 T-cells. It is widely accepted that human tumors shed soluble NKG2D ligands, such as soluble major histocompatibility complex-I chain-related molecule (sMIC), to evade immune activation. Harnessing the NKG2D/NKG2D-ligand axis has emerged as a promising avenue for immunotherapy. However, controversy exists over whether soluble NKG2D ligands are immunosuppressive or immunostimulatory. Methods: Cell lines for melanoma (B16F10), Lewis lung carcinoma (LLC1), and prostate cancer (TRAMP-C2, T-C2) were used as parental cell lines in this study. Derivative cell lines over-expressing the soluble human NKG2D ligand sMICB (B16F10-sMICB, TC2-sMICB and LLC-sMICB) were created. We inoculated these 3 syngeneic transplantable tumor models and their respective parental lines into mice and compared tumor incidence/growth over time. We also compared surface markers representative of cell stemness (SCA-1, CD166 and CD133) in mouse cancer cells between the sMICB-expressing tumor cells and their respective parental cell lines. We then characterized the tumor immune microenvironment by flow cytometry. Results: B16F10-sMICB mice demonstrated a significant delay in tumor development compared to control mice (23 vs. 6 days, p < 0.05). Over longer follow-up, rapid tumorigenesis still occurred in the experimental group, with a 50% tumor incidence by day 26. By contrast, the TRAMP-C2-sMICB mice demonstrated higher tumorigenic ability (p < 0.05). LLC-sMICB mice demonstrated no difference in tumorigenicity. Time to tumor onset was more strongly associated with expression of stemness-like markers rather than sMIC. Once tumors were established, they grew significantly more aggressively in the TRAMP-C2-sMICB, LLC-sMICB and B16F10-sMICB lines vs. their parental counterparts. sMICB expressing tumors contained significantly lower percentages of NK cells and CD8 T-cells in tumor infiltrates than their respective parental lines. NK cells and CD8 T Cells from sMICB-expressing tumors had an impaired capacity to produce interferon gamma. Conclusions: Expression of a soluble NKG2D ligand had minimal impact on tumor establishment but facilitated more aggressive growth of established tumors due to immune suppression. Soluble NKG2D ligands imprint negative anti-tumor immunity onto established tumors. Our data differentiated the impact of NKG2D ligands on tumor onset vs. tumor progression and reinforced the translational concept that soluble NKG2D ligands are strong therapeutic targets for future study.

Abstract 901: Sox2 and Oct4 activity identify CSC populations in ovarian cancer

Abstract High Grade Serous Ovarian Cancer is the most lethal gynecological cancer, mainly due to late-stage diagnosis and prevalence of chemotherapy-resistant disease. Tumors are comprised of heterogenous cancer cells, which makes targeted therapies challenging. Studies have identified a subset of cancer stem-like cells (CSCs) that, unlike bulk tumor cells, can evade chemotherapy and induce relapse. Cell surface markers such as CD133, CD44, CD117, or ALDH enzyme activity are typically used to identify and isolate CSCs; however, these markers are inconsistent across cell lines and patient samples making them unreliable for isolating CSCs. Consistent indicators of CSCs may be activity of stem cell transcription factors, such as SOX2, OCT4, and NANOG, which are known to promote long-term self-renewal and asymmetric division, processes required for tumor repopulation. We hypothesize that a reporter responding to SOX2 and OCT4 activity will represent a functionally relevant and reliable marker for identifying CSCs capable of enabling relapse. We have stably transduced a reporter (termed SORE6), which detects SOX2 and OCT4 activity, into OV90 and ACI23 ovarian cancer cells. SORE6+ cells, isolated using fluorescence assisted cell sorting (FACS), have 2 to 15-fold increases in SOX2/OCT4/NANOG transcripts, relative to SORE6- cells. Comparison of CSC markers in chemotherapy versus vehicle conditions suggests that SORE6 more consistently identifies the CSC population. Specifically, flow cytometry analysis of ACI23 cells showed a 2-fold increase in SORE6+ cells, 7-fold increase in CD117+ cells, and a 1.2-fold increase in CD133 cells in carboplatin treated cells relative to vehicle. For OV90, SORE6+ increases again by 2-fold, while CD117+ increases by 5-fold and CD133 increases by 1.5-fold. SORE6+ cells also showed significantly enhanced spheroid formation efficiency in low serum conditions and resistance to carboplatin and paclitaxel chemotherapies, relative to SORE6- cells. Finally, in vivo limiting dilution studies in mice showed a significant difference in tumorigenicity of SORE6+ cells at low dilutions in ACI23 cells, but not for OV90 cells. Flow cytometry analysis of disassociated tumors showed that tumors created using pure SORE6- cells remained SORE6-, while tumors created using pure SORE6+ cells maintained only 10% SORE6+, indicating the ability of SORE6+ cells to asymmetrically divide to create a heterogeneous tumor. In conclusion, these data suggest that the SORE6 reporter identifies a CSC population within ovarian cancer cell lines and could be a useful tool for consistent isolation of CSCs. Use of this reporter will enhance our understanding of mechanisms, such as SOX2 and OCT4 activity, that support chemoresistance and tumor repopulation. More reliable identification of CSCs will enable the design of therapies to overcome chemotherapy resistance and disease recurrence. Citation Format: Luisjesus S. Cruz, Mikella Robinson, Samuel F. Gilbert, Omar Lujano-Olazaba, Logan A. Alexander, Alex Horkowitz, Carrie D. House. Sox2 and Oct4 activity identify CSC populations in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 901.

Carcinogenicity risk associated with tacrolimus use in kidney transplant recipients: a systematic review and meta-analysis.

BackgroundCurrently, tacrolimus is the preferred anti-rejection therapy for kidney transplant recipients due to its greater protection against acute rejections compared to cyclosporin A (CsA). Despite the advantages of kidney transplantation, it has been associated with an increased incidence of de novo malignancies. Furthermore, a systematic review in 2005 revealed no statistical difference in tumorigenicity between tacrolimus and CsA. This report provides an up to date systematic review and evaluation of all relevant studies in the literature to determine the risk of malignancy in kidney transplant recipients exposed to tacrolimus.MethodsA systematic literature search was performed using the Medline (PubMed and Ovid), Embase, Clinical Trials, and Cochrane databases (from creation to May 2021). We performed a meta-analysis of 11 studies with 36,985 kidney transplant recipients that compared the tacrolimus group with the control group. Outcomes of this study were incidence of malignancies and skin cancer risk. Risk of Bias was assessed in terms of whether there was random sequence generation, allocation concealment, blinding, completeness of results, selective reporting, etc. This meta-analysis was performed in accordance with PRISMA guidelines.ResultsOf the 11 included studies, 8 were high quality studies, 1 was assessed as medium quality, and 2 were low quality studies. The results showed a significantly increased risk of overall malignancy associated with tacrolimus exposure compared to non-tacrolimus therapy [risk ratio (RR) =1.59; 95% confidence interval (CI): 1.19–2.11; P=0.002], and especially with sirolimus (SRL) (RR =2.58; 95% CI: 1.62–4.09; P<0.0001). The incidence of skin cancer was consistent with the overall study (RR =2.03; 95% CI: 1.25–3.28; P=0.004). However, there was no significant difference in the incidence of tumors between tacrolimus and cyclosporine A treatment (RR =1.12; 95% CI: 0.80–1.56; P=0.52), even in studies with long follow-up periods of more than 3 years.DiscussionThe data demonstrated that patients treated with tacrolimus had a higher risk of carcinogenicity compared to patients treated with SRL. However, patients treated with tacrolimus had a similar incidence of carcinogenicity compared to patients treated with CsA. Further clinical studies are warranted to confirm these findings.

Abstract 4941: Identification and isolation of ovarian cancer stem cell populations using a novel functional reporter may provide new insights into ovarian cancer cell dynamics and drug resistance

Abstract Background: Ovarian cancer is the most lethal gynecologic malignancy mainly due to a high prevalence of chemotherapy-resistant recurrent disease. This mechanism is unknown but recent studies have pointed to a small population of chemoresistant cancer stem cells (CSC) as the driver of these recurrences. Traditional surface markers of cancer stem cells (CD44, ALDH, CD117, etc.) have shown mixed success in identifying this subpopulation, with different cell lines and patient samples exhibiting dissimilar profiles. These markers are often related to the cell of origin and are not necessarily specific to CSCs. As such, a reporter that responds to functional features of stem cells would be beneficial in identifying the CSC subpopulation regardless of cell line and patient sample. Here we demonstrate that using a reporter to detect SOX2 and OCT4 overexpression (called SORE6) can identify the CSC subpopulation within ovarian cancer cell lines. Methods: The SORE6 reporter was stably transduced into OV90 and ACI23 ovarian cancer cell lines. The subpopulation of cells having high SOX2/OCT4 expression (SORE6+) were isolated using fluorescence assisted cell sorting (FACs) and evaluated for CSC characteristics relative to SORE6- cells. Gene expression, viability, spheroid formation capacity, and tumorigenicity was assessed for both SORE6+ and SORE6- cells. In vivo limiting dilution analyses were performed on athymic nude female mice using subcutaneous injection. Results: Compared to the bulk population which has low levels of SOX2/OCT4 expression (SORE6-), SORE6+ cells showed a 1.5-fold increase in SOX2/OCT4/NANOG transcript levels in ACI23 cells and 2 to 15-fold increase in these transcript levels in the OV90 cells. SORE6+ cells showed enhanced chemoresistance when treated with conventional chemotherapies for ovarian cancer, carboplatin and paclitaxel, although this difference was more dramatic in the OV90 cells (four-fold) relative to ACI23 (two-fold). These findings indicate that SOX2/OCT4 expression could contribute to drug resistance and clinical recurrence. SORE6+ cells also showed an increased capacity to form spheroids in low-serum conditions and again this difference was more dramatic in the OV90 cells. In vivo limiting dilution studies are underway but appear to show significant differences in tumorigenicity at low dilutions of ≤5,000 cells/mouse. Conclusion: The SORE6 reporter construct identifies CSC populations within ovarian cancer cell lines and may be a reliable tool for isolating CSCs that depend on SOX2 and OCT4 expression. Use of this reporter will enhance our understanding of mechanisms that support chemoresistance and enable the study of CSC dynamics within the tumor microenvironment, both of which have significant implications for ovarian cancer progression and treatment. Citation Format: Samuel F. Gilbert, Mikella Robinson, Logan A. Alexander, Omar Lujano-Olazaba, Jacqueline M. Lara, Jennifer A. Waters, Omid Patrus, Alex Horkowitz, Carrie D. House. Identification and isolation of ovarian cancer stem cell populations using a novel functional reporter may provide new insights into ovarian cancer cell dynamics and drug resistance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4941.

Abstract 2133: 24R,25(OH)2D3 is differentially tumorigenic in ER+ and ER- breast cancer

Abstract Vitamin D3 (VD3) has long been associated with improved breast cancer prognoses. However, recent clinical trials examining the efficacy of VD3 supplementation on patient prognoses have been largely inconclusive. One reason for this may be that most clinical trials ignore the complex VD3 metabolome and focus solely on 25(OH)D3, the circulating form of VD3. Our study focuses on 24R,25(OH)2D3 (24,25D), a naturally-occurring metabolite of VD3. The aim of the present study was to examine the effect of 2425D on breast cancer tumorigenicity, and to determine if this effect is dependent on estrogen receptor expression in vitro and in vivo.In vivo, MCF7 (an estrogen receptor positive [ER+] breast cancer cell [BCC] line) or HCC38 (ER-) BCCs were implanted in adult female NOD SCID gamma IL2R mice. Mice were supplemented with 24,25D (0, 25, or 100 ng/ IP injection 3X/week). Tumor growth was monitored throughout the study. In vitro, both cells were treated with 0-100nM 24,25D for 15 min or 24h and assayed for phospholipase D (PLD) activity, proliferation (DNA synthesis), apoptosis (BAX, BCL2, p53, TUNEL), epithelial-to-mesenchymal transition (EMT) (SNAI1, MMP1, ERBB2), migration (scratch test), and metastasis (CXCR4/CXCL12, OPG/RANKL). Inhibitors to PLD, caveolin-1, and palmitoylation were used to examine the mechanism of 24,25D. Data were analyzed with one or two-way ANOVA with Tukey’s post-tests.In vivo, mice with ER+ tumors given 24,25D showed decreased tumor burden, metastasis, and increased survival as compared to vehicle-treated controls. Conversely, mice with ER- tumors given 24,25D showed a dose-dependent increase in tumor burden compared to controls. In vitro, both ER+ and ER- cells showed increased proliferation at 24 hours after exposure to 24,25D for 15min but not after exposure for 24h. In ER+ cells, 2425D increased apoptosis and decreased EMT, migration, metastasis, and PLD activity; while in ER- cells, the opposite trend was observed, with 24,25D decreasing apoptosis and increasing EMT, migration, metastatic markers, and PLD activity. The proliferative and apoptotic effects of 24,25D in both cell lines were inhibited by pre-treatment with inhibitors to PLD, caveolin-1, and palmitolyation.24,25D had opposing effects on ER+ and ER- tumor burden in vivo, suggesting that 24,25D is differentially tumorigenic, and observed differences in tumorigenicity are dependent on the expression of ER. In vitro, this ER-associated differential effect was confirmed, with 24,25D decreasing tumorigenic markers in ER+ BCCs and enhancing them in ER- BCCs. Furthermore, 24,25D appears to act at the membrane through a PLD-dependent caveolae-associated mechanism using a palmitoylated transmembrane receptor(s) in both cell types. This study indicates the important role of 24,25D in breast cancer and suggests that ER-status may be an important prognostic marker to consider before prescribing vitamin D3 supplementation to breast cancer patients. Citation Format: Anjali Verma, David J. Cohen, Chandana Muktipaty, Thomas W. Jacobs, Jennifer Koblinski, Barbara D. Boyan, Zvi Schwartz. 24R,25(OH)2D3 is differentially tumorigenic in ER+ and ER- breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2133.

Replication Study: Wnt activity defines colon cancer stem cells and is regulated by the microenvironment.

As part of the Reproducibility Project: Cancer Biology we published a Registered Report (Evans et al., 2015), that described how we intended to replicate selected experiments from the paper 'Wnt activity defines colon cancer stem cells and is regulated by the microenvironment' (Vermeulen et al., 2010). Here, we report the results. Using three independent primary spheroidal colon cancer cultures that expressed a Wnt reporter construct we observed high Wnt activity was associated with the cell surface markers CD133, CD166, and CD29, but not CD24 and CD44, while the original study found all five markers were correlated with high Wnt activity (Figure 2F; Vermeulen et al., 2010). Clonogenicity was highest in cells with high Wnt activity and clonogenic potential of cells with low Wnt activity were increased by myofibroblast-secreted factors, including HGF. While the effects were in the same direction as the original study (Figure 6D; Vermeulen et al., 2010) whether statistical significance was reached among the different conditions varied. When tested in vivo, we did not find a difference in tumorigenicity between high and low Wnt activity, while the original study found cells with high Wnt activity were more effective in inducing tumors (Figure 7E; Vermeulen et al., 2010). Tumorigenicity, however, was increased with myofibroblast-secreted factors, which was in the same direction as the original study (Figure 7E; Vermeulen et al., 2010), but not statistically significant. Finally, we report meta-analyses for each results where possible.

A glycoproteomic approach to identify novel glycomarkers for cancer stem cells

Most cancers consist of heterogeneous populations of cells with substantial differences in tumorigenicity. Cells that possess self-renewal and tumor-initiating properties are often called cancer stem cells (CSCs). Since CSCs underlie tumor recurrence and metastasis and are resistant to current anti-cancer therapies, novel therapeutic strategies to efficiently target this subset of cells are needed. Aberrant glycosylation is one of the hallmarks of cancer. Many cancer-associated glycans have been reported to be involved in tumor progression and metastasis, and are used as tumor markers. Over the past several years, we have identified characteristic glycans on CSCs by utilizing recent advances in glycoproteomic technologies. In this review, we would like to summarize a series of our recent studies and discuss possible applications of glycomarkers for CSCs.

Abstract 2279: The oncogenic role of TRPM7 in cell proliferation and invasion of U87MG glioma cells

Abstract Background: We have previously reported that suppression of TRPM7 channels inhibit proliferation, migration, and invasion of malignant human glioma cells (CNS Neuroscience &amp; Therapeutics, in press) using A172 glioma cell line and brain tissue of human glioma. By studying glioma cancer stem cells derived from A172 cells, we further found that TRPM7 channels regulate glioma stem cell growth/proliferation through STAT3 and Notch signaling pathways (Cellular Signaling, 2014; 26: 2773-2781). However, the genetic difference of in vitro cell lines mirrors the diversity of individual patient cases. For instance, Ke et al reported that the differences in tumorigenicity among the different glioma cell lines were associated with the expression levels of bFGF and/or VEGF, two potent angiogenic factors in gliomas (Clin Cancer Res 2000; 6: 2562-2572). Interestingly, A172 cells produce a high-level bFGF, while U87MG cells secrete a significantly higher level of VEGF than other glioma cells. To determine whether our previous findings are not limited to A172 cells, we examined the role of TRPM7 channels in aforementioned malignant features of U87MG cells as well. We hypothesize that TRPM7 promotes proliferation and invasion in U87MG glioma cells. Methods: 1) We examined TRPM7-like current using patch-clamp technique in U87MG glioma cells. 2) We examined the TRPM7 mRNA and protein expression using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot respectively. 3) We determined the effects of TRPM7 on glioma cell proliferation and invasion using MTT assay and transwell invasion assay respectively by downregulating TRPM7 expression using siTRPM7 and inhibiting TRPM7 function using 2-APB, a non-specific pharmacological inhibitor. Results: 1) Electrophysiological recording showed a TRPM7-like current induced by Ca2+ and Mg2+ removal, which suggested that TRPM7 channels also exist in U87MG cells. 2) PCR and Western blot showed that U87MG cells express TRPM7 mRNA and protein. 3) Treatment of U87MG cells with siTRPM7 significantly reduced TRPM7 expression. 4) The cell proliferation and invasion were significantly reduced when treated with siTRPM7 and 2-APB which indicate that TRPM7 promotes growth and invasion of U87MG cells. 5) Inhibition of TRPM7 activity blocked calcium induced cell proliferation further indicating that TRPM7 activity is required for cell proliferation. Conclusion: TRPM7 promotes proliferation and invasion of U87MG glioma cells. TRPM7 may play a generalized role in glioma initiation and progression and might be an important drug target of glioma. (This work is supported by R01NS066027, NIMHD S21MD000101, U54NS08932, and AHA 0840132.) Citation Format: Mingli Liu. The oncogenic role of TRPM7 in cell proliferation and invasion of U87MG glioma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2279. doi:10.1158/1538-7445.AM2015-2279

Enhanced tumorigenicity by mitochondrial DNA mild mutations.

To understand how mitochondria are involved in malignant transformation we have generated a collection of transmitochondrial cybrid cell lines on the same nuclear background (143B) but with mutant mitochondrial DNA (mtDNA) variants with different degrees of pathogenicity. These include the severe mutation in the tRNALys gene, m.8363G>A, and the three milder yet prevalent Leber's hereditary optic neuropathy (LHON) mutations in the MT-ND1 (m.3460G>A), MT-ND4 (m.11778G>A) and MT-ND6 (m.14484T>C) mitochondrial genes. We found that 143B ρ0 cells devoid of mtDNA and cybrids harboring wild type mtDNA or that causing severe mitochondrial dysfunction do not produce tumors when injected in nude mice. By contrast cybrids containing mild mutant mtDNAs exhibit different tumorigenic capacities, depending on OXPHOS dysfunction.The differences in tumorigenicity correlate with an enhanced resistance to apoptosis and high levels of NOX expression. However, the final capacity of the different cybrid cell lines to generate tumors is most likely a consequence of a complex array of pro-oncogenic and anti-oncogenic factors associated with mitochondrial dysfunction.Our results demonstrate the essential role of mtDNA in tumorigenesis and explain the numerous and varied mtDNA mutations found in human tumors, most of which give rise to mild mitochondrial dysfunction.

Differential in vivo tumorigenicity of distinct subpopulations from a luminal-like breast cancer xenograft.

Intratumor heterogeneity caused by genetic, phenotypic or functional differences between cancer cell subpopulations is a considerable clinical challenge. Understanding subpopulation dynamics is therefore central for both optimization of existing therapy and for development of new treatment. The aim of this study was to isolate subpopulations from a primary tumor and by comparing molecular characteristics of these subpopulations, find explanations to their differing tumorigenicity. Cell subpopulations from two patient derived in vivo models of primary breast cancer, ER+ and ER-, were identified. EpCAM+ cells from the ER+ model gave rise to tumors independently of stroma cell support. The tumorigenic fraction was further divided based on SSEA-4 and CD24 expression. Both markers were expressed in ER+ breast cancer biopsies. FAC-sorted cells based on EpCAM, SSEA-4 and CD24 expression were subsequently tested for differences in functionality by in vivo tumorigenicity assay. Three out of four subpopulations of cells were tumorigenic and showed variable ability to recapitulate the marker expression of the original tumor. Whole genome expression analysis of the sorted populations disclosed high similarity in the transcriptional profiles between the tumorigenic populations. Comparing the non-tumorigenic vs the tumorigenic populations, 44 transcripts were, however, significantly differentially expressed. A subset of these, 26 identified and named genes, highly expressed in the non-tumorigenic population, predicted longer overall survival (N = 737, p<0.0001) and distant metastasis free survival (DMFS) (N = 1379, p<0.0001) when performing Kaplan-Meier survival analysis using the GOBO online database. The 26 gene set correlated with longer DMFS in multiple breast cancer subgroups. Copy number profiling revealed no aberrations that could explain the observed differences in tumorigenicity. This study emphasizes the functional variability among cell populations that are otherwise genomically similar, and that the risk of breast cancer recurrence can only be eliminated if the tumorigenic abilities in multiple cancer cell subpopulations are inhibited.

Abstract 1981: Drug-tolerant gastric cancer cell subpopulation enriched by 5-fluorouracil acquires malignant phenotype

Abstract Background: Post-operative adjuvant chemotherapy for gastric cancer is a treatment for suppressing the growth of invisible cancer cells, but substantial numbers of patients experience recurrence despite of this therapy. We attempted to clarify the mechanism of cancer relapse after chemotherapy using 5-FU tolerant human gastric cancer cell line, MKN45. Methods: Through a stepwise dose escalation of 5-fluorouracil (5-FU) for one year, a 5-FU-tolerant cell subpopulation, MKN45T, was established from a human gastric cancer cell line, MKN45. Nucleotide variation was screened with a panel of 46 cancer-associated genes, and western blot analyses were performed to examine whether known proteins were involved in the acquisition of the drug-tolerant phenotype. For molecular profiling, cancer cell subpopulations emerging as colonies in the presence of anticancer drugs including 5-FU, cisplatin, and docetaxel, “reverse-phase” protein arrays (RPPAs) were produced with the drug-tolerant 480 colonies. Subcutaneous or orthotopic xenografts of MKN45 and MKN45T to immunodeficient mice were performed to examine tumorigenicity. Results: Western blot analysis revealed that most of protein expression levels were not visibly different between MKN45 and MKN45T, except that p53 showed slight reduction in MKN45T. Analysis of 46 cancer-associated genes revealed that all gene variations were identical in both MKN45 and MKN45T, suggesting that gene mutations had a limited effect on the acquisition of the drug-tolerant phenotype. RPPA analysis revealed that colonies from MKN45T exhibited a protein level increase in a 5-FU concentration-dependent manner in Atg5, Atg7, and PI3K, suggesting a functional association between drug-tolerance and autophagy/glucose metabolism. An orthotopic xenograft to the stomach demonstrated tumors in the stomach (9 out of 9, 100%), and lymph node and liver metastases only in MKN45T at a high frequency (6 out of 9, 67%). No difference in tumorigenicity was observed when MKN45 and MKN45T were transplanted subcutaneously. The orthotopic xenograft of MKN45T followed by administration of 5-FU (30mg/kg/day) for five post-xenograft days suppressed metastasis in 4 out of 5 mice (80%) at 42 post-xenograft days, indicating that chemotherapy is particularly effective if it is performed before initiating tumor formation. Conclusion: Chemotherapy does not only reduce tumor size, but may also be selecting for tumors that are resistant to the chemotherapy. Our clinical experiences are well-explained by this observation, as relapse after chemotherapy is often chemotherapy resistant. Citation Format: Kaoru Ishida, Satoshi S. Nishizuka, Kohei Kume, Mamoru Nukatsuka, Kei Sato, Fumitaka Endo, Hirokatsu Katagiri, Takashi Kobunai, Teiji Takechi, Keisuke Koeda, Go Wakabayashi. Drug-tolerant gastric cancer cell subpopulation enriched by 5-fluorouracil acquires malignant phenotype. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1981. doi:10.1158/1538-7445.AM2014-1981

298 IDENTIFICATION AND CHARACTERIZATION OF CANCER STEM CELL OF RENAL CELL CARCINOMA CELL LINES

You have accessJournal of UrologyKidney Cancer: Basic Research (II)1 Apr 2013298 IDENTIFICATION AND CHARACTERIZATION OF CANCER STEM CELL OF RENAL CELL CARCINOMA CELL LINES Kosuke Ueda, Sachiko Ogasawara, Jun Akiba, Masamichi Nakayama, Sakiko Sanada, Shigetaka Suekane, Masanori Noguchi, Kei Matsuoka, and Hirohisa Yano Kosuke UedaKosuke Ueda Kurume, Japan More articles by this author , Sachiko OgasawaraSachiko Ogasawara Kurume, Japan More articles by this author , Jun AkibaJun Akiba Kurume, Japan More articles by this author , Masamichi NakayamaMasamichi Nakayama Kurume, Japan More articles by this author , Sakiko SanadaSakiko Sanada Kurume, Japan More articles by this author , Shigetaka SuekaneShigetaka Suekane Kurume, Japan More articles by this author , Masanori NoguchiMasanori Noguchi Kurume, Japan More articles by this author , Kei MatsuokaKei Matsuoka Kurume, Japan More articles by this author , and Hirohisa YanoHirohisa Yano Kurume, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1682AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Evidence for existence of cancer stem cells (CSCs) has been reported in several malignant tumors. The population of CD105 is reported to be one of the CSC markers in renal cell carcinoma (RCC). However, there are a few reports examining the CSCs in human RCC. In our study, we isolated side population (SP) cells from the human RCC cell lines and systematically identified the CSC characteristics of the SP cells. Moreover, we examined the malignant potential with CSC like biological features of ALDH1-positive cells. METHODS We used two RCC cell lines derived from metastatic RCC (ACHN) and primary RCC (KRC/Y). The SP and Non-SP(NSP) cells were isolated from the two RCC cell lines by FACS Aria 2. We examined proliferative ability, drug resistance, clonoginity, sphere forming ability in serum-free medium in vitro and tumorigenicity in vivo between SP and NSP cells. We examined ALDH1 activity and sphere forming ability in ALDH1 expression. Moreover, we analyzed the expression of ALDH1 after treatment with Sorafenib or IFNa and exposing hypoxia. RESULTS The SP cell rates in ACHN and KRC/Y were 1.4% and 1.7%, respectively. SP cells of KRC/Y had higher cell proliferative ability and clonogenity in vitro. Although SP cells in KRC/Y expressed higher CD105-positive cell rate than NSP cells (SP vs NSP: 24.6% vs 4.6%), there was no difference in tumorigenicity in vivo. On the other hand, there was no significant difference in the cell growth rate and colony formation between SP and NSP cells in ACHN. However, the SP cells in ACHN had higher sphere forming ability and IFNa resistance in vitro and had higher tumor growth ability in vivo. The mRNA expression levels of stemness genes, HIF1a and VEGFA in isolated SP and Non-SP cells showed no significant difference. Whereas, the SP cells in ACHN expressed higher ALDH1-positive cells, which was widely known as one of the representative CSC markers, compared with NSP cells (SP vs NSP: 32.7% vs 14.6%). Furthermore, ALDH1-positive cell had higher sphere forming ability than ALDH1-negative cells. ALDH1-positive cells rate, especially treated with sorafenib or exposed in hypoxic condition, was much higher than in the control. CONCLUSIONS Cells with the features of CSC may be involved in SP cell fraction, especially in ALDH1-positive SP cell fraction. SP cells in ACHN not only overexpress ALDH1 activity but also have more resistance to the various conventional treatment for RCC. These findings may provide new insights for future CSC research and clinical anti-cancer therapy for RCC. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e121 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kosuke Ueda Kurume, Japan More articles by this author Sachiko Ogasawara Kurume, Japan More articles by this author Jun Akiba Kurume, Japan More articles by this author Masamichi Nakayama Kurume, Japan More articles by this author Sakiko Sanada Kurume, Japan More articles by this author Shigetaka Suekane Kurume, Japan More articles by this author Masanori Noguchi Kurume, Japan More articles by this author Kei Matsuoka Kurume, Japan More articles by this author Hirohisa Yano Kurume, Japan More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Phenotypic characterization of mammosphere-forming cells from the human MA-11 breast carcinoma cell line

The phenotypic diversity of breast carcinoma may be explained by the existence of a sub-population of breast cancer cells, endowed with stem cell-like properties and gene expression profiles, able to differentiate along different pathways. A stem cell-like population of CD44 +CD24 −/low breast cancer cells was originally identified using cells from metastatic pleural effusions of breast carcinoma patients. We have previously reported that upon in vitro culture as mammospheres under stem cell-like conditions, human MA-11 breast carcinoma cells acquired increased tumorigenicity and lost CD24 expression compared with the parental cell line. We now report that upon passage of MA-11 mammospheres into serum-supplemented cultures, CD24 expression was restored; the rapid increase in CD24 expression was consistent with up-regulation of the antigen, and not with in vitro selection of CD24 + cells. In tumors derived from subcutaneous injection of MA-11 mammospheres in athymic nude mice, 76.1 ± 9.7% of cells expressed CD24, vs. 0.5 ± 1% in MA-11 cells dissociated from mammospheres before injection. The tumorigenicity of sorted CD44 +CD24 − and CD44 +CD24 high MA-11 cells was equal. Single cell-sorted CD24 − and CD24 high MA-11 gave rise in vitro to cell populations with heterogeneous CD24 expression. Also, subcutaneous tumors derived from sorted CD24 − sub-populations and single-cell clones had levels of CD24 expression similar to the unsorted cells. To investigate whether the high expression of CD24 contributed to the tumorigenic potential of MA-11 cells, we silenced CD24 by shRNA. CD24 silencing (95%) resulted in no difference in tumorigenicity upon s.c. injection in athymic nude mice compared with mock-transduced MA-11 cells. Since CD24 silencing was maintained in vivo, our data suggest that the level of expression of CD24 is associated with but does not contribute to tumorigenicity. We then compared the molecular profile of the mammospheres with the adherent cell fraction. Gene expression profiling revealed that the increased tumorigenicity of MA-11 mammospheres was associated with changes in 10 signal transduction pathways, including MAP kinase, Notch and Wnt, and increased expression of aldehyde dehydrogenase, a cancer-initiating cell-associated marker. Our data demonstrate that (i) the level of CD24 expression is neither a stable feature of mammosphere-forming cells nor confers tumorigenic potential to MA-11 cells; (ii) cancer-initiating cell-enriched MA-11 mammospheres have activated specific signal transduction pathways, potential targets for anti-breast cancer therapy.

Mammosphere Culture of Established Cell Lines Does Not Enrich for a More Tumorigenic Breast Cancer Stem Cell Population.

Abstract Background:The cancer stem cell model suggests that tumors arise from and are maintained by a relatively small proportion of cells, termed cancer stem cells. According to this model, only these cells have the capacity for self-renewal and the ability to repopulate the tumor bulk by giving rise to the phenotypically heterogeneous populations of cells that comprise the primary tumor. Evidence to support the cancer stem cell model has been provided by isolation of subpopulations of cells on the basis of differential expression of cell surface markers, followed by functional transplantation into animal models. These studies reveal that only a small subset of CD44+/CD24-/low cancer cells retain the ability to form new tumors after transplantation in immunodeficient mice, consistent with the cancer stem cell model. These results indicate that it may be necessary to target and eliminate cancer stem cells in order to eradicate tumors.Material and Methods:Previous studies have suggested that only breast stem cells have the capacity to proliferate in suspension as spherical colonies termed mammospheres, and that mammosphere derived breast cancer cells are CD44+/CD24-/low and able to form tumors that reproduce the heterogeneity of the original primary tumor when injected into the mammary fat pad of immunodeficient mice. This indicates that mammospheres are comprised of breast cancer stem cells. To identify and characterize breast cancer stem cells more completely, the mammosphere culture system was used to isolate and propagate breast cancer stem cells from three basal breast cancer cell lines (BT549, MDA231, MDA436) and three luminal breast cancer cell lines (MCF7, SKBR3, T47D). The tumorigenicity of the cultured mammospheres and parental cell lines was examined using dilution studies in immunodeficient mice and gene expression profiles were studied by microarray analysis.Results and Discussion:No significant differences in tumorigenicity were observed between breast cancer cells cultured as mammospheres versus standard culture conditions. Furthermore, no consistent differences in gene expression profiles were observed between mammosphere and parental cells across the panel of cancer cell lines. These results suggest that mammosphere culture is not capable of significantly enriching for a more tumorigenic breast cancer stem cell population from established breast cancer cell lines. This may indicate that cancer subpopulations that exist in primary tumors are lost during the long term culture of breast cancer cell lines and that these systems may not be a suitable model for breast cancer stem cell studies. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 502.

Molecular cytogenetic characterization of human breast cancer cell line MDA-MB-468 and its variant 468LN, which displays aggressive lymphatic metastasis

Two human breast carcinoma cell lines, MDA-MB-468 and its variant MDA-MB-468LN, which displays aggressive lymphatic metastasis, were investigated for molecular cytogenetic characteristics using G-banding, spectral karyotyping, and fluorescence in situ hybridization (FISH). Both cell lines have multiple chromosome aberrations, but differ in the types of rearrangements and their breakpoints. The MDA-MB-468 karyotype identified in the present study differs from that previously reported, which suggests that this cell line is unstable. Neither cell line exhibited amplification of ERBB2 (alias HER-2) by FISH. MDA-MB-468 cells have a modal number of 60, with 42 types of aberrations: 11 numerical and 31 structural. 468LN cells have a modal number of 55, with 37 types of aberrations: 10 numerical and 27 structural. The most common aberrations in MDA-MB-468 cells were der(5)t(5;16), i(7)(p10), i(18)(p10), der(19)t(2;19), del(6)(q23), and der(10)t(1;10). The most common aberrations in 468LN cells were der(18)t(10;18), +5, der(1;7)(q10;q10), der(19)t(2;18;19), and der(20)t(20;21). This cytogenetic result is consistent with previous findings that showed differences in tumorigenicity and metastatic capability of these two cell lines and indicates that 468LN is a new cell line distinctive from MDA-MB-468. We hypothesize that the cytogenetic changes in 468LN may be related to its new biological characteristics. Knowledge of the chromosome aberrations and breakpoints identified could be useful for further genetic and epigenetic studies of breast cancer.

Effect of Age on Pulmonary Metastases and Immunotherapy in Young and Middle-aged Mice

We used B16-F10 (B16) melanoma tumor cells and syngeneic C57BL/6 (B6) mice as a study model for pulmonary metastases, to better understand whether or not there exist differences in tumorigenicity and in the effectiveness of immunotherapy as a function of host age (1-, 3-, 12- and 24-month-old). Intravenous injection of B16 melanoma cells were administered to B6 mice of different ages with/without interleukin (IL)-2 and IL-12 daily treatment. Tumor growth, splenocyte function, serum cytokines (IL-10, interferon-gamma, vascular endothelial growth factor) and survival were compared. The study showed that, without IL-2 and IL-12 treatment, middle-aged mice suffering from pulmonary metastases had fewer pulmonary metastases and better survival than younger mice suffering from pulmonary metastases. Three days' IL-2 plus IL-12 treatment could not prolong mice survival, but prolonged treatment significantly improved the survival of both the younger and older tumor-bearing mice, especially the older mice, despite the fact that the younger mice had a better serum cytokine and splenocyte cellular immune response to cytokine treatment. The young B6 mice that suffered from B16 pulmonary metastases had a poorer prognosis than the middle-aged mice. Short-term IL-2 plus IL-12 treatment is ineffective in prolonging survival, and a longer duration of treatment is needed. This kind of immunotherapy was effective in both the young and middle-aged mice, but it was more effective in the middle-aged mice.

Integrating Studies on Carcinogenic Risk of Carbon Black: Epidemiology, Animal Exposures, and Mechanism of Action

We sought to address the toxicology literature on carbon black (CB) since 1996, when IARC reclassified CB from group 3 to group 2B. We reviewed epidemiology and laboratory studies from 1996 to 2006, focusing on new analyses of worker populations, on species differences in tumorigenicity of poorly soluble particles, and on the role of particle-bound organics in tumorigenicity. Some epidemiology studies have reported positive associations between cancer risk and worker's possible exposure to CB, but larger studies, in more highly exposed populations, have not shown consistent patterns of either elevated risk or dose-response. High levels of inhaled CB were linked with rat lung tumors in 1996, but today scientists increasingly recognize that rats exhibit a unique lung tumor response to all inert inhaled particles that is unlikely to be relevant to humans. On mechanism of action, new reports have continued to show that CB has a high surface area of elemental carbon, and a low quantity of organic material, which is poorly bioavailable. Overall, the new epidemiological evidence decreases concerns for cancer risk compared with pre-1996 evidence. Laboratory studies support a conclusion that the mechanism of tumorigenicity of CB in rats is no different from that of any poorly soluble particle, ie, toxicity results from the particle overload per se, and not from the particles' chemistry. Thus, research published after 1996 has not identified an increase in support for CB cancer risk, but rather, points to limited and inadequate evidence for carcinogenicity.

Blockade of Transforming Growth Factor-β Signaling Suppresses Progression of Androgen-Independent Human Prostate Cancer in Nude Mice

We investigated the role of transforming growth factor-beta (TGF-beta) signaling in the growth and metastasis of PC-3MM2 human prostate cancer cells. Highly metastatic PC-3MM2 human prostate cancer cells were engineered to constitutively overexpress a dominant-negative type II TGF-beta receptor (DNR). Transfection of DNR had minimal direct effects on cell growth and attenuated TGF-beta-induced cell growth inhibition and TGF-beta1 production. There were no discernable differences in tumorigenicity (tumor incidence) among PC-3MM2 variants when the cells were implanted into the prostates of nude mice. Growth rate and metastatic incidence of DNR-engineered PC-3MM2 cells, however, were significantly reduced. Most cells in the control tumors were positively stained by an antibody to proliferation cell nuclear antigen and very few cells were stained by terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL). In sharp contrast, tumors formed by PC-3MM2-DNR cells contained fewer proliferation cell nuclear antigen-positive cells and many more TUNEL-positive cells. Staining with antibody against CD31 showed that control tumors contained more blood vessels than PC-3MM2-DNR tumors. Expression of interleukin-8 (IL-8) in tumors formed by PC-3MM2 cells was significantly reduced as revealed by both Northern blotting and ELISA. Finally, transfection of antisense IL-8 cDNA significantly reduced IL-8 production by PC-3MM2 cells and antisense IL-8-transfected PC-3MM2 cells grew slower in comparison with parental and control vector-transfected cells. Taken together, our data suggest that TGF-beta signaling, by regulating IL-8 expression in tumor cells and hence tumor angiogenesis, is critical for progressive growth of PC-3MM2 cells in the prostate of nude mice.

Analysis of gene expression in the K562-n high tumorigenitic human leukemia cell line

The human leukemia K562—n cell line displays much higher tumorigenic actively in nude mice compared with its parental K562 cell line. The molecular mechanism of the differences in tumorigenicity between K562-n and K562 in nude mice was examined. The differences in gene expression between K562 and K562-n cells were analyzed by using cDNA microarrays. Among the12,800 genes examined, there was a significant difference in expression of 139 genes between K562—n and K562 cells. Eighty—five of these genes have been registered in the GeneBank and 54 are unknown. The genes accessible from the GeneBank include: 1) oncogenes and tumor—supressor genes; 2) genes related to transcription regulation, the cell cycle and apoptosis; 3) genes related to the cytoskeleton and cytokinetics; 4) genes related to metabolism and transport; 5) genes related to immune function. There were also some differently expressed genes with mixed functions. There are many genes differentially expressed between K562—n and K562 cells The high tumorigenicity of the human leukemia K562—n cell line in nude mice might be related to its specific gene-expression profile.

A novel role for the coxsackie adenovirus receptor in mediating tumor formation by lung cancer cells.

The Coxsackie Adenovirus Receptor (CAR) has primarily been studied in its role as the initial cell surface attachment receptor for Coxsackie and group C adenoviruses. Recent reports suggest that CAR mediates homotypic intercellular adhesion as part of the tight and/or adherens junction. Thus, CAR is well positioned to participate in intercellular interactions and signaling. Using an antisense (AS)-CAR plasmid vector, we silenced surface CAR expression in lung cancer cells that possessed a high basal expression of this molecule and monitored the resultant tumorigenesis. AS-CAR transfectants exhibit a profound loss in the ability to generate xenografts in scid/scid mice. The emergence of delayed-onset tumors in animals that received injection with AS-CAR transfectants correlates with the resurfacing of CAR expression, suggesting that such expression and tumor emergence are temporally related. To study the mechanism underlying the differences in tumorigenicity, control and AS-CAR cells were compared in terms of their in vitro growth potential. Whereas only subtle differences in the proliferative capacity of the two populations were evident when assayed with growth on plastic, significant differences became apparent when one compared the relative ability of these populations to form colonies in soft agar. These data indicate that silencing surface CAR expression abrogates xenograft tumorigenesis in vivo and colony formation in vitro and invoke the novel possibility that CAR expression is needed for the efficient formation of tumors by a subset of lung cancer cells.