Eradication of CD44-variant positive population in head and neck tumors through controlled intracellular navigation of cisplatin-loaded nanomedicines
Ming WangYutaka MiuraKenji TsuchihashiKazuki MiyanoOsamu NaganoMomoko YoshikawaAmi TanabeJunichiro MakinoYuki MochidaNobuhiro NishiyamaHideyuki SayaHoracio CabralKazunori Kataoka
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The cancer stem cell hypothesis suggests that tumors are initiated and maintained by cancer stem cells capable of self-renewal and differentiation into mature tumor cells. Recent studies have demonstrated the existence of cancer initiating cells in different solid tumors. In this study, we identified a subpopulation of CD44+ cells within the tumor of gastric cancer patients, which, upon treatment by chemotherapeutic agent 5-fluorouracil (5-FU), were markedly enriched. In vitro culture of isolated CD44+ subpopulation from gastric tumors by magnetic beads sorting led to formation of gastric spheroid colonies. These colonies retained CD44+ surface marker expression during culture, and were undifferentiated in nature. Subcutaneous injections of CD44+ gastric cancer cells conferred tumorigenicity in SCID mice. Moreover, implantation of CD44+ cells from these established tumors remained tumorigenic in successive passages. Using CD44+ cells isolated from the gastric cell lines AGS and SGC7901, similar results were obtained. Upon enrichment by 5-FU, CD44+ cells harbored increased ALDH expression as compared with CD44- cells. Our results demonstrated for the first time the existence of CD44+ cells within the tumors of gastric cancer patients that are endowed with stem cells properties, and also provide a plausible explanation for chemo-resistance frequently observed in gastric cancer patients. Such findings provide a basis for further studies on targeting this tumorigenic subpopulation for better treatment of gastric cancer patients.
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Cancer stem cells (CSCs)/tumor-initiating cells have been defined as a subset of tumor cells responsible for initiating and sustaining tumor development. Emerging evidence strongly supports the existence of CSCs in various solid tumors, but they have not yet been identified in human gallbladder carcinomas (GBC). In this study, we identified CSCs in primary GBC and in the cell line GBC-SD using the cell surface markers CD44 and CD133. The percentages of CD44+CD133+ cells were 1.76-3.05% in primary tumors and 40.29% in GBC-SD cells. These cells showed stem cell properties, including self-renewal, differentiation potential, and high tumorigenicity. In vitro culture experiments revealed that CD44+CD133+ GBC cells possessed a higher spheroid-colony forming ability in serum-free media than other subpopulations. When injected into nonobese diabetic/severe combined immunodeficient mice, these cells formed new tumors and generated CD44+CD133+, CD44-, and CD133- progeny. CD44+CD133+ cells also showed a high degree of chemoresistance, possibly due to upregulation of the breast cancer resistance protein (ABCG2) and the transcription factor Gli1 in these highly tumorigenic cells. These results suggest that the CD44+CD133+ population exhibited CSC-like characteristics and may thus provide a novel approach to the diagnosis and treatment of GBC.
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Abstract Introduction: Proprietary anticancer formulations based on the polysaccharide, hyaluronan (HA) are currently under development. The drugs utilize the unique physiochemical and biological properties of HA to entrain currently approved anticancer agent/s, where, after intravenous administration the HA moiety targets the drug complex to activated CD44 receptors which are over-expressed >95% of solid tumors. After extravasation into the tumor, the HA/drug complex forms a microembolism, increasing drug retention and tumor cell uptake, ultimately providing increased efficacy. The capacity to maintain tumor growth and resistance to therapy are associated with a small population of cells known as cancer stem cells (CSC). Current anticancer therapies focus on the eradication of proliferating cells, whereas CSCs have been demonstrated to be quiescent; often lacking hyperproliferation signals. CD44 is used as a reliable marker for colon cancer stem cells, therefore, through the high expression of this protein on both rapidly proliferating and CSCs there is strong rationale to use CD44 as a therapeutic target. In Phase ll clinical testing the anticancer drug, HA-Irinotecan has demonstrated superior efficacy in late-stage colon cancer patients but its effect on colon CSC is unknown. This study evaluated the efficacy of the CD44-targeted drug, HA-Irinotecan in the treatment of colon cancer CSC. Methodology: Two colon cancer cell lines were separated into CSC (CD44+CD133+ALDH+/−) and non-stem cell (CD44+CD133−ALDH+/−, CD44−CD133+ALDH+/−) sub-populations using FACS. Attachment and proliferation characteristics of the colon cancer sub-populations were established using real time electronic microsensory array assays. The activation status of CD44 was determined using a functional HA internalization assay. After confirming the CD44 activation status cell sub-populations were treated with HA-Irinotecan where efficacy was determined using clonogenic and microsensory array assays. Results: CD44s was the predominant CD44 isoform expressed on all CD44+ve colon cancer sub-populations where the CD44 was shown to be active via the active intenalisation and degradation of hyaluronan thereby providing rationale for CD44 as a therapeutic target. When CSC and non-CSC colon cancer sub-populations were treated the CD44-targeted drug, HA-Irinotecan, the EC50 was significantly reduced compared to drug alone. This increase in efficacy was negated when CD44−ve colon cancer sub-populations were treated with the CD44-targeted drug. Conclusions: This preliminary study has demonstrated that activated CD44 is a valuable therapeutic target in colon cancer. The treatment of human colon cancer with the CD44-targeted drug, HA-Irinotecan could provide a significant therapeutic advantage through eradication of all CD44+ve colon cancer populations including cancer stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4278.
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Both our previous study and other reports have suggested that CD133, originally classified as a hematopoietic stem cell marker, could be used for enrichment of cancer stem cells (CSCs) in human hepatocellular carcinoma (HCC). It was also noted that not all of CD133(+) cells were representative of CSCs. Further identification and characterization of CSCs or tumor-initiating cells in HCC are necessary to better understand hepatocarcinogenesis. In present study, we demonstrated that CSC phenotype could be precisely defined by co-expression of CD133 and CD44 cell surface markers. CD133(+)CD44(+) HCC cells showed stem cell properties, including extensive proliferation, self-renewal, and differentiation into the bulk of cancer cells. In vivo xenograft experiments revealed that, actually, the highly tumorigenic capacity of CD133(+) cells as previously described was primarily attributed to CD133(+)CD44(+) cell subpopulation, instead of their CD133(+)CD44(-) counterparts. Moreover, cells double-positive for CD133 and CD44 exhibited preferential expression of some stem cell-associated genes and were more resistant to chemotherapeutic agents due to the upregulation of ATP-binding cassette (ABC) superfamily transporters, including ABCB1, ABCC1, and ABCG2, further supporting these cells as HCC cell origin. Our findings suggest that CD133(+)CD44(+) cells might represent true cancer stem/progenitor cells in HCC, which could allow a better understanding of HCC initiation and progression, as well as establish a precise target for the development of more effective therapies.
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Objective: To compare the carcinogenic abilities of CD133(+)CD44(+) laryngeal cancer stem cells and general laryngeal cancer stem cells and to identify the mechanism underlying the action of miRNAs. Methods: Solid tumor-derived laryngeal carcinoma stem cells and Hep-2-derived laryngeal carcinoma stem cells were cultured, and CD133(+)CD44(+) laryngeal cancer stem cells were sorted by flow cytometry. Boden chamber invasion assay, cell migration assay and tumor formation assay were then performed to compare the invasion, migration and tumorigenic abilities of CD133(+)CD44(+) laryngeal cancer stem cells and general laryngeal cancer stem cells. And then, miRNAs isolated from two laryngeal cancer stem cells were detected and analysed with miRNA chip. Results: (1)In Boyden chamber invasion assay, the cell invasion rate of CD133(+)CD44(+) laryngeal cancer stem cells was obviously higher (80.2%±2.3% vs. 63.9%±3.2%, t=5.011, P=0.027); (2)CD133(+)CD44(+) laryngeal cancer stem cells also had higher mobility in cell migration assay (82.9%±1.1% vs. 70.9%±0.6%, t=4.514, P=0.031); (3)In tumor formation assay, the tumor formation rate of CD133(+)CD44(+) laryngeal cancer stem cells was also higher (80% vs. 50%). What's more, we identified 15 miRNAs that were significantly upregulated in CD133(+)CD44(+) laryngeal cancer stem cells and 3 miRNAs that were significantly downregulated in CD133(+)CD44(+) laryngeal cancer stem cells, compared with normal laryngeal cancer stem cells. Conclusions: CD133(+)CD44(+) laryngeal cancer stem cells have stronger invasion, migration and tumorigenic abilities compared with normal laryngeal cancer stem cells, and the difference of miRNAs' expression is one of the possible causes.目的： 比较CD133(+)CD44(+)喉鳞状细胞癌（简称喉鳞癌）干细胞与一般喉鳞癌干细胞的致癌能力，并探讨微小RNA(miRNA）在其中发挥的作用。 方法： 培养实体瘤来源的喉鳞癌干细胞和Hep-2来源的喉鳞癌干细胞，通过流式细胞仪分选出CD133(+)CD44(+)喉鳞癌干细胞，通过Boyden小室侵袭实验、细胞迁移实验和裸鼠成瘤实验比较CD133(+)CD44(+)喉鳞癌干细胞与未筛选的喉鳞癌干细胞的侵袭、迁移和成瘤能力，然后提取两种喉鳞癌干细胞的总miRNA，通过丹麦Exiqon公司的miRNA芯片进行检测和分析比较。 结果： （1）在Boyden小室侵袭实验中，CD133(+)CD44(+)喉鳞癌干细胞与未筛选的喉鳞癌干细胞比较，侵袭细胞更多，细胞侵袭率也更高（80.2%±2.3%比63.9%±3.2%，t=5.011，P=0.027）；（2）在细胞迁移实验中，CD133(+)CD44(+)喉鳞癌干细胞比未筛选的喉鳞癌干细胞具有更高的迁移率（82.9%±1.1%比70.9%±0.6%，t=4.514，P=0.031）；（3）在动物成瘤实验中，CD133(+)CD44(+)喉鳞癌干细胞比未筛选的喉鳞癌干细胞成瘤需要注射的细胞数少（1×10(3)个比1×10(5)个），成瘤率更高（80%比50%）。另外，用miRNA芯片检测CD133(+)CD44(+)喉鳞癌干细胞和未筛选的喉鳞癌干细胞的miRNA水平后发现，在CD133(+)CD44(+)喉鳞癌干细胞中，有15条miRNA水平明显较高，有3条miRNA水平明显较低。 结论： CD133(+)CD44(+)喉鳞癌干细胞具有更强的侵袭、迁移和成瘤能力，miRNAs的差异表达是其中的一个可能原因。.
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Hepatocellular carcinoma (HCC) is a type of liver cancer, in which CD44 isoforms have been proposed as markers to identify cancer stem cells (CSCs). However, it is unclear what characteristics are associated with CSCs that exclusively express CD44 isoforms. The objective of the present study was to determine the expression of CD44 isoforms and their properties in CSCs. Analysis of transcriptomic data from HCC patient samples identified CD44v8-10 as a potential marker in HCC. In SNU-423 cells, CD44 expression was detected in over 99% of cells, and two CD44 isoforms, namely, CD44std and CD44v9, were identified in this cell line. CD44 subpopulations, including both CD44v9+ (CD44v9) and CD44v9- (CD44std) cells, were obtained by purification using a magnetic cell separation kit for human CD44v9+ cancer stem cells. CD44v9 cells showed greater potential for colony and spheroid formation, whereas CD44std cells demonstrated significant migration and invasion capabilities. These findings suggested that CD44std and CD44v9 may be used to identify features in CSC populations and provide insights into their roles in HCC.
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Despite significant advances in the use of diagnosis and therapy to treat head and neck squamous cell carcinoma (HNSCC), the prognosis has improved only marginally in the last decades. Thus, there is an enormous need for better understanding of tumor biology and reversely novel immunotherapeutic approaches. It is becoming increasingly obvious that stem cells play an important role in tumor development and progression. The identity of these cells and the underlying cellular and molecular mechanisms are mostly unknown in HNSCC to date.Solid HNSCC tumors, as well as permanent HNSCC cell lines, were analyzed by flow cytometry concerning the expression of different putative stem cell marker proteins.Distinct populations of CD44 expressing potential stem cells could be identified in solid tumors of HNSCC patients with strong individual deviations. Surprisingly, the potential stem cell marker CD44 was found to be constitutively expressed on the surface of all the permanent HNSCC cell lines analyzed.CD44+ 'tumor stem cells' may play a key role in the establishment of permanent HNSCC cell lines, selecting especially robust cell entities that might drive the progression and metastasis of HNSCC. Individual analysis of 'tumor stem cell' markers will be an important tool for innovative therapies and for determining the prognosis of patients with HNSCC.
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Cancer stem cells are one of the most promising targets for cancer treatment. In head and neck cancer, CD44 has been regarded as a cancer stem cell marker. However, apart from the standard CD44 (CD44s), many other variants of CD44 exist, and their specific roles are still unknown. Radiation resistance is a known characteristic of cancer stem cells. Therefore, we irradiated five head and neck squamous cell carcinoma cell lines and investigated whether the expression patterns of CD44 variants changed. Common results showed that the number of CD44s-positive cells decreased and that of CD44 variant 9 (CD44v9)-positive cells increased. This suggested that CD44v9 is useful as a marker for cancer stem cells and may be a target for therapy.
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Background/Aim: Cancer stem cells (CSCs) are considered to be one of the causes of tumor recurrence after chemotherapy. The purpose of our study was to isolate CSCs from human colorectal cancer cell (CRC) lines. Materials and Methods: Nine CRC lines were screened based on the expression level of potential CSC markers to identify putative CSCs. Tumor formation capacity in immunodeficient mice was compared with that of their counterparts. Stemness, differentiation potency and sensitivity to 5-fluorouracil (5-FU), in vitro, were also assessed. Microarray analysis was used to characterize the features of the putative CSCs. Results: COLO 201 cells were separated into two populations based on CD44 expression. CD44 positive (CD44+) cells showed significantly higher tumor formation capacity than CD44− cells in immunodeficient mice. CD44+ cells also possessed stemness properties and lower sensitivity to 5-FU in vitro. Moreover, cancer stemness and chemoresistance-related genes were highly up-regulated in CD44+ cells. Conclusion: CD44+ COLO 201 cells possessed the features of CSCs; therefore, the present CSC model could serve as a valuable tool to accelerate CSC research.
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