Small Fraction Of Tumor Cells Research Articles

Abstract 260: CD44 isoform specificity in controlling breast tumor initiating cell properties.

Abstract Tumor initiating cells (TICs) constitute a small fraction of tumor cells that display stem cell characteristics, including the ability to self-renew and generate heterogeneous tumors. A CD44+/CD24- profile has been widely used to identify TICs in a variety of tumors. However, it has remained unknown whether CD44 is functionally required for TICs and whether a specific CD44 isoform is enriched in TICs. Here, we report that the CD44s isoform is the major splice isoform expressed in a CD44+/CD24- breast stem cell population. Functionally, CD44s is required for expression of a breast stem cell (BSC) gene signature and is essential for mammosphere formation. Modulation of CD44 alternative splicing by the splicing regulator ESRP1 prevents CD44s production and, in turn, inhibits mammosphere formation and expression of the BSC signature. Furthermore, CD44s is required for limiting numbers of TICs to form tumors in an animal model of breast cancer progression. Together, these data illustrate that the CD44s splice isoform is enriched in CD44+/CD24- breast stem cells and serves as an important regulator of stem cell properties. Our results also suggest that CD44s-dependent TIC properties can be controlled at the level of alternative splicing by splicing factors that regulate production of specific CD44 isoforms. Citation Format: Rhonda L. Brown, Sali Liu, Lauren M. Reinke, Chonghui Cheng. CD44 isoform specificity in controlling breast tumor initiating cell properties. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 260. doi:10.1158/1538-7445.AM2013-260 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 2364: Identification of therapy resistant pediatric brain tumor cells that have extraordinary survival and tumorigenic capabilities: The ultimate seed cells that drives the growth of autopsied tumor cells in mouse brains

Abstract Brain tumors are a leading cause of cancer related death in children. Little is known about the biology of recurrent or refractory pediatric CNS tumors, particularly those that are inoperable, such as hypothalamic or brainstem tumors. Since autopsy may be the only opportunity to obtain tumor tissue for inoperable or recurrent tumors, we have obtained postmortem tissue for the purpose of developing orthotopic xenografts. We hypothesized that a small fraction of tumor cells would survive the postmortem period of extreme hypoxia and starvation, maintain their tumorigenic capacity, leading to tumor formation in an appropriate in vivo microenvironment; and that these tumor cells may represent the ultimate seed cells that drive tumor recurrence and treatment failure. To identify this subpopulation of TREST cells, i.e. therapy resistant with extraordinary survival and tumorigenic capabilities, we collected 11 postmortem tumor samples, including 3 recurrent medulloblastomas (MBs),1 glioblastoma multiforme (GBM) and 7 diffuse intrinsic pontine glioma (DIPG), 5-72 hrs (mean 32 hrs) after the time of death. We examined tumor cell viability and their capacity of growth in vitro (neurospheres in serum-free medium and proliferation in FBS-based DMEM), analyzed the content of putative cancer stem cells, and determined their tumor forming capability in vivo in mouse brains. Our results showed that a small fraction (5-10%) of viable tumor cells exists in all the autopsied tumors, although most of them failed to proliferate in vitro. More importantly, we found that injection of unfractionated tumor cells into mouse brains resulted in the formation of xenograft tumors in 2 of 3 MBs (66.6%), 1/1 GBM and 5 of 7 DIPGs (71%). One of the medulloblastomas and the GBM model have been serially passaged in mouse brains 6 times, and two of the DIPGs in mouse brainstems 3 times. These tumors replicated the histopathological phenotypes of the corresponding patient tumors demonstrating diffuse invasion. Using a series of putative cancer stem cell markers (CD133, CD15, CD24/CD44, CD57 and CD117), we showed that CD133+ and CD15+ cells were enriched in the xenograft tumors. In conclusion, we have demonstrated that a small fraction of tumor cells, some of which expressed putative CSC markers, can survive a lengthy period of postmortem hypoxia/anoxia and starvation and maintain tumorigenic capabilities in mouse brains. Detailed analysis of these TREST cells, particularly with our unique panel of orthotopic xenograft mouse models, should provide novel insights about their biology and future development of new therapies specifically targeting the recurrent/late stage brain tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2364. doi:1538-7445.AM2012-2364

Abstract 3362: Tumor initiating cells are sensitive to paclitaxel encapsulated in polymeric nanoparticles

Abstract Tumor initiating cells (TICs) represent a small fraction of tumor cells, and are considered to be key drivers of tumor growth, drug resistance and recurrence. TICs are highly resistant to conventional chemotherapy, possibly due to overexpression of drug efflux transporters and other protective mechanisms. Because drug resistance and tumor relapse are significant problems, approaches to eliminate TICs are urgently needed. Recent studies show that tumor populations that are depleted of TICs demonstrate a return of the TIC sub-population 1 suggesting the possibility of de-differentiation of non-TICs to TICs. We hypothesized that sustained delivery of a cytotoxic agent to the tumor population could potentially reduce TIC fraction and consequently increase the therapeutic effectiveness of chemotherapy. To test this hypothesis, we encapsulated paclitaxel, a microtubule stabilizing agent, in PLGA nanoparticles formulated using an FDA-approved polymer, poly (lactide-co-glycolide). Our previous studies have shown nanoparticles can sustain the intracellular delivery of encapsulated drugs like paclitaxel 2. The effectiveness of nanoparticle-encapsulated paclitaxel in reducing TIC fraction was investigated by standard cancer stem cell assays- mammosphere formation and anchorage-independent growth on soft agar in MCF-7, an ER+ luminal breast cancer cell line. Results obtained in these studies show that drug encapsulated PLGA formulations effectively decreased mammosphere formation and soft-agar colony formation when compared to free drug treatment. In mammosphere assays, paclitaxel solution treatment did not significantly affect the mammosphere formation relative to untreated controls, whereas paclitaxel loaded PLGA nanoparticles treatment decreased the number of mammospheres to 50 ± 33% of untreated controls. In soft-agar colony formation assay, paclitaxel solution treatment increased the number of colonies to 122 ± 10% of untreated controls. In contrast to this, paclitaxel loaded PLGA nanoparticles treatment decreased the number of colonies to 66 ± 8% of untreated controls. Future studies will investigate the effectiveness of these formulations on TICs in mouse models of breast cancer. References 1 Gupta, P. B. et al., Cell 146 (4), 633; Chaffer, C. L. et al., Proc Natl Acad Sci U S A 108 (19), 7950. 2 Patil, Y. B. et al., Biomaterials 31 (2), 358. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3362. doi:1538-7445.AM2012-3362

Abstract 5205: Selectively high Wnt pathway activity in subpopulations of colon cancer cells and its significance for tumor initiation

Abstract Several cell surface markers that are reported to enrich for tumor-initiating colon cancer cells are presumed targets of the canonical Wnt/beta-catenin pathway. Furthermore, colon cancers typically express the Wnt-effector beta-catenin in a heterogeneous pattern, with strong nuclear expression detected only in a small fraction of tumor cells. We therefore hypothesized that high beta-catenin expression and Wnt pathway activity mark a tumorigenic subpopulation. To label viable tumor cells according to the level of Wnt pathway activity, we constructed lentiviral vectors that express GFP under control of an optimal, beta-catenin-responsive TCF/LEF1 promoter (TOP-GFP). We transduced primary colon cancer xenografts and two well characterized colon cancer cell lines with these vectors, used flow cytometry to isolate cell subpopulations with differential GFP expression, and examined these subpopulations for tumorigenicity and gene expression. High GFP expression accurately marked tumor cells with nuclear beta-catenin immunostaining in Caco2 but not SW1222 cells and in a fraction of primary colon cancer xenografts. Gene expression analysis in the concordant cases was consistent with increased Wnt-pathway activity within the GFP-high cell population, including high expression of the putative stem cell markers CD133, CD44 and LGR5. In SW1222 cells and some primary tumor xenografts, the GFP-high fraction correlated poorly with nuclear beta-catenin localization and GFP flow cytometry did not separate cells with a gene expression profile suggesting Wnt pathway activity. Among these various sources, only Caco2 cells showed increased tumorigenicity of GFP-high cells in immunodeficient mouse xenografts. The utility of TOP-GFP and related reporters to identify tumor-initiating colon cancer cells with high Wnt activity thus varies among primary human colorectal tumors and established cell lines. Furthermore, Wnt pathway activity might fluctuate substantially within individual tumor cells. We conclude that high Wnt activity among tumor cell subpopulations does not necessarily signify tumor-initiating potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5205. doi:10.1158/1538-7445.AM2011-5205

Cancer Stem Cell Niche: The Place to Be

Tumors are being increasingly perceived as abnormal organs that, in many respects, recapitulate the outgrowth and differentiation patterns of normal tissues. In line with this idea is the observation that only a small fraction of tumor cells is capable of initiating a new tumor. Because of the features that these cells share with somatic stem cells, they have been termed cancer stem cells (CSC). Normal stem cells reside in a "stem cell niche" that maintains them in a stem-like state. Recent data suggest that CSCs also rely on a similar niche, dubbed the "CSC niche," which controls their self-renewal and differentiation. Moreover, CSCs can be generated by the microenvironment through induction of CSC features in more differentiated tumor cells. In addition to a role in CSC maintenance, the microenvironment is hypothesized to be involved in metastasis by induction of the epithelial-mesenchymal transition, leading to dissemination and invasion of tumor cells. The localization of secondary tumors also seems to be orchestrated by the microenvironment, which is suggested to form a premetastatic niche. Thus, the microenvironment seems to be of crucial importance for primary tumor growth as well as metastasis formation. Combined with its role in the protection of CSCs against genotoxic insults, these data strongly put forward the niche as an important target for novel therapies.

Human .GAMMA..DELTA. T Cells and Tumor Immunotherapy

Human Vgamma2Vdelta2 T cells recognize nonpeptide antigens derived from pathogenic microbes in a TCR-dependent manner, such as pyrophosphomonoester compounds from mycobacteria and malaria parasite and alkyl amines from Proteus, suggesting that this subset of gamma delta T cells is involved in infectious immunity. The precise recognition mechanism has been delineated using a site-directed mutagenesis strategy based on crystal structure of gamma delta TCR. On the other hand, several lines of evidence indicate that human gamma delta T cells are involved in tumor immunity. Although activated gamma delta T cells exhibit a cytolytic activity against most of tumor cells, only a small fraction of tumor cells, like Burkitt lymphoma cells and multiple myeloid cells, is recognized by human gamma delta T cells in a TCR-dependent manner. This implicates that human gamma delta T cells have two distinct pathways for anti-tumor immunity. One is a natural killer-like pathway and the other is a TCR-dependent pathway. Recently, it was shown that treatment of human tumor cells with nitrogen-containing bisphosphonates, therapeutic drugs for hypercalcemia in malignancy, generated antigenic structure on the surface of tumor cells, which could be recognized by human gamma delta T cells in a TCR-dependent manner. This tumor labeling system may lead to a novel strategy for cancer immunotherapy.

Forced expression of ΔN-TCF-1B in colon cancer derived cell lines is accompanied by the induction of CEACAM5/6 and mesothelin

The colon cancer cell lines HT29 and SW480 were transfected with an N-terminal β-catenin binding site-deficient high mobility group (HMG)-box T-cell factor 1 (ΔN-TCF-1) construct to identify differentially expressed genes. Oligonucleotide HG-U133A microarray expression profiling revealed increased mRNA levels of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 5, 6 and mesothelin in transfectants positive for nuclear ΔN-TCF-1B. Increased amounts of CEACAM5 (CEA) were detectable in membrane-associated compartments, particularly in cholesterol-enriched microdomains. Similarly, mesothelin was demonstrated as an uncleaved membrane-bound constituent. The identified markers were examined in specimens of 46 colorectal carcinomas (CRC) by immunohistochemistry. Patchy areas of increased CEACAM5/6 staining were seen at the tumour-host front in all samples studied. Twenty-eight (58%) of these cases showed over-expression of mesothelin in a small fraction of tumor cells displaying dedifferentiation and dissemination at the invasion front. We conclude that forced expression of ΔN-TCF-1B in HT29 and SW480 is associated with up-regulation of GPI-anchored adhesion molecules, which were assigned to the tumour-host front in CRC patients.

Overexpression of p53 Is Not a Feature of Ovarian Granulosa Cell Tumors

The p53 tumor suppressor gene has been extensively studied in various human tumors including epithelial ovarian cancers. However, little is known about the expression of this gene in ovarian granulosa cell tumors, the most common histologic type of sex cord–stromal tumors. We investigated whether overexpression of the p53 gene product occurs in this specific ovarian tumor. Nineteen patients with ovarian granulosa cell tumors were recruited in this study. Immunohistochemical staining for the p53 protein with monoclonal antibody PAb 1801 was performed in the paraffin-embedded tissue of each case to screen for p53 overexpression. Among the 19 ovarian granulosa cell tumors, there was only one well-differentiated tumor found to have nuclear immunoreactivity in a small fraction of tumor cells. Polymerase chain reaction–single-stranded conformation polymorphism was used to study the tumor showing focal p53 positivity, but no mobility shift was noted from exon 4 through exon 9 of the p53 gene. On the basis of this observation, we propose that alteration of the p53 tumor suppressor gene is not a common finding in ovarian granulosa cell tumors.

Virus-directed enzyme/prodrug therapy (VDEPT). Selectively engineering drug sensitivity into tumors.

A gene therapy approach has been described that generates a tumor-selective qualitative difference in the metabolic capability in tumor cells. This is the result of the selective expression of a nonmammalian enzyme in tumor cells. Selective expression is achieved by utilization of a chimeric gene composed of the TRS from a tumor-associated marker gene linked to the coding domain of a gene encoding a nonmammalian enzyme. We have described the application of this approach for the treatment of metastatic CRC. This approach involves creation of a chimeric gene composed of the CEA TRS linked to the coding domain of the CD gene. Selective expression of CD in the tumor cells will allow the selective conversion of the prodrug 5-FCyt to 5-FCyt in the tumor while sparing normal cells. Most importantly, delivery and expression of CD into a small fraction of tumor cells may be sufficient to achieve a significant antitumor effect.