Presence Of Tumor Stem Cells Research Articles

Meningioma originating from the superior petrosal vein without dural attachment: A case report

BACKGROUND Meningioma in the cerebellopontine angle (CPA) without dural attachment is extremely rare. We report a unique case of meningioma derived from the superior petrosal vein without dural attachment. CASE SUMMARY A 44-year-old right-handed woman presented with a two-month history of headache and tinnitus. Brain magnetic resonance imaging showed a well-defined contrast-enhancing lesion in the right CPA without a dural tail sign. Tumor resection was performed using a right retro sigmoid approach. A dural attachment was not seen at the tentorium or posterior surface of the petrous pyramid. The tumor was firmly adherent to the superior petrosal vein. The origin site was cauterized and resected with the preservation of the superior petrosal vein. A diagnosis of meningothelial meningioma was made. The patient’s headache and tinnitus gradually disappeared, and a recurrence was not observed five years after the surgery. CONCLUSION The rare occurrence of meningioma without dural attachment makes it difficult to determine dural attachment before surgery. The absence of dural attachment makes it easy to completely resect such tumors. Vessels related to tumors should be removed carefully, considering the possible presence of tumor stem cells in the microvessels.

Study on Correlation and Mechanism of Procollagen Lysine 2 Oxoglutarate 5 Dioxygenase on Stem Cell Characteristics and Vasculogenic Mimicry Formation in Esophageal Squamous Cell Carcinoma

In this study, we investigated the significance of the stem cell transcription factor PLOD2 in esophageal cancer to enhance our understanding of its occurrence, development, recurrence, metastasis, and potential treatment. We conducted the following experiments: (1) Immunohistochemistry revealed elevated PLOD2 protein levels in esophageal carcinoma compared to adjacent tissues, with higher levels associated with advanced disease stages and lower differentiation. (2) Immunofluorescence demonstrated increased PLOD2 expression in esophageal cancer cell lines TE-1 and Eca- 109, suggesting a connection to cell differentiation. (3) We successfully transfected fluorescently labeled PLOD2 siRNA into cells, achieving a transfection rate of 67.57% and silencing efficiency exceeding 80%. (4) Following transfection, we observed a decreased proliferation rate in PLOD2 siRNA-treated cells, indicating that PLOD2 reduction can slow esophageal cancer growth. These findings emphasize that PLOD2 plays a crucial role in esophageal cancer pathogenesis, particularly in low cell differentiation maintenance. It could serve as a promising target for inducing differentiation in esophageal cancer and evaluating its malignancy. The high PLOD2 expression in esophageal cancer cell lines suggests the presence of tumor stem cells. Moreover, reducing PLOD2 through RNAi technology slows cell proliferation, suggesting that inhibiting PLOD2 may offer a potential therapeutic approach for esophageal cancer treatment.

New approaches in 3D modeling of in vitro growth of primary cultures of malignant gliomas

Background. The incidence of brain gliomas firmly occupies a leading position among all central nervous system tumors – 40–50 % of the cases detected, more than half of them are glioblastoma. Existing cell lines and cultivation methods do not reflect all the features of the three-dimensional (3D) organization of native glioblastoma. The use of temozolomide leads to the development of drug resistance and acute relapse, followed by a poor clinical outcome. The development of resistance is largely associated with the presence of tumor stem cells in the population and intratumoral heterogeneity. Obtaining 3D cultures from the primary material will allow us to save the stem cell pool and tumor-specific features.The study objective. Get a 3D model based on primary cell cultures, which allows you to save a heterogeneous population and the original phenotype of tumor cells.Materials and methods. We used U-87MG human glioma cells and GBM002 primary cell culture obtained from surgical material with a confirmed diagnosis of glioblastoma. Neurospheres were obtained from cell lines, the growth of which was monitored using the InCell Analyzer 6000 automatic cell analysis system. Flow cytometry was used to determine the CD133+ cell content. The expression of the receptor tyrosine kinases VEGFR1, VEGFR2 (endothelial growth factor type 1 and 2 receptors), FGFR2 (fibroblast growth factor receptor type 2) and the hypoxia marker HIF-1α (hypoxia inducible factor, 1α) in the neurospheres was evaluated using confocal microscopy.Results. GBM002 glioblastoma cells isolated from the surgical material formed neurospheres, while the number of CD133+ cells increased from 1–2 to 16–19 % compared with two-dimensional cultures. During long-term cultivation of cells with non-cytotoxic doses of temozolomide, it was found that such cells form smaller neurospheres compared to control cells. It was shown that the expression of receptor tyrosine kinases during cultivation of GBM002 glioblastoma cells in neurospheres differs from that in two-dimensional cultures. We found that in neurospheres, the expression of FGFR2 and VEGFR1, is significantly increased.Conclusion. 3D cultivation of primary cultures allows one to obtain a more heterogeneous population of tumor cells that reflects the spatial heterogeneity of cells, increase the pool of stem cells and recreate hypoxia conditions inside the brain micro-tumors.

Acute hypoxia induces upregulation of microRNA-210 expression in glioblastoma spheroids.

Tumor hypoxia and presence of tumor stem cells are related to therapeutic resistance and tumorigenicity in glioblastomas. The aim of the present study was therefore to identify microRNAs deregulated in acute hypoxia and to identify possible associated changes in stem cell markers. Glioblastoma spheroid cultures were grown in either 2 or 21% oxygen. Subsequently, miRNA profiling was performed and expression of ten stem cell markers was examined. MiRNA-210 was significantly upregulated in hypoxia in patient-derived spheroids. The stem cell markers displayed a complex regulatory pattern. MiRNA-210 appears to be upregulated in hypoxia in immature glioblastoma cells. This miRNA may represent a therapeutic target although it is not clear from the results whether this miRNA may be related to specific cancer stem cell functions.

Tumor stem cells in breast cancer

High mortality from breast cancer is associated with the high heterogeneity of tumor and the frequent recurrences of the pathological process, which are due to the presence of tumor stem cells. The review considers the biological properties of tumor stem cells, the molecular mechanisms of their regulation, interaction with the microenvironment, and their role in the heterogeneity of the morphological and clinical forms of breast cancer.

Clinical Relevance of CD45+/CD19- Stem-like Tumor Cells in Patients with Mantle Cell Lymphoma: A Single Center Experience

BackgroundMantle cell lymphoma (MCL) is a rare but unique subtype of B-cell non-Hodgkin lymphoma (NHL) that is molecularly characterized by the chromosomal translocation, t(11;14)(q13:q32) leading to cyclin D1 overexpression. MCL is also characterized by heterogeneous clinical features from indolent to aggressive courses. The majority of patients with MCL initially show slowly progressing nature of disease, however, the treatment outcomes of conventional chemotherapy have been poor. As a result, most patients with MCL have shown repeated relapses ultimately resulting in death. Although upfront use of intensified chemotherapy regimens and active application of autologous stem cell transplantation as a consolidation treatment were introduced, MCL has still remained as one of deadly subtype of NHL because its prognosis is still worse than other NHL. Given this heterogeneity and incurability of MCL, the concept of tumor stem cells has been suggested to explain peculiar characteristics of MCL. Thus, previous in vitro studies reported a small fraction of tumor cells, CD45+/CD19- might reflect the presence of tumor stem cells and showed their resistance to chemotherapeutic agents as well as tumor formation ability. However, the clinical relevance of CD45+/CD19- stem-like cell population in bone marrow or other sites has rarely been studied and demonstrated in patients with MCL.MethodsBetween 2011 and 2013, we analyzed patients who were pathologically diagnosed with MCL. They all received systemic chemotherapy with curative intent including rituximab-containing chemotherapy. The flow cytometry for isolating CD45+/CD19- cell population was done with bone marrow aspirates or malignant effusion from patients at diagnosis and/or at the time of relapse. The follow-up evaluation of CD45+/CD19- cells was also done at the end of primary treatment. The in vitro experiments with B-cell lymphoma cell lines were performed to evaluate the association of CD45+/CD19- cell population with the resistance to chemotherapeutic agents. The percentage of CD45+/CD19- cells from surviving cells after doxorubicin treatment at the dosage of IC90 for 48 hours were compared with control group.ResultsTwenty-one patients were enrolled, and the median age was 67 years (range, 50–78 years). Eighteen patients had stage IV disease including 15 cases of bone marrow involvement. All patients’ sample was from bone marrow aspirates except one patient from malignant pleural effusion. The examination for CD45+/CD19- cells was done in six patients at diagnosis and the end of treatment whereas only single evaluation was done in the other patients at diagnosis or at the time of relapse. The median percentage of CD45+CD19- cells was 0.019% (range 0-1.8%) in all examined patients. The mean percentage of patients who relapsed or progressed (0.41%±0.69) was higher than that of patients at diagnosis (0.09%±0.11) or patients at the state of complete or partial response after the completion of chemotherapy (0.01%±0.01). The serial monitoring of three patients who relapsed or progressed showed the increased level of CD45+/CD19- cell population while patients who responded to primary treatment showed the decreased level of CD45+/CD19- cells at the end of treatment. The in vitro study with B-cell lymphoma cell lines, Toledo, Daudi, Raji and BJAB showed the increased fraction of CD19- cells among lymphoma cells resistant to doxorubicin compared to control cells. This increase of CD19- cells was correlated with the increase of ATP-binding cassette subfamily G2 (ABCG2) that was another marker reflecting the presence of tumor stem cells.ConclusionThe presence of CD45+/CD19- cell population was demonstrated in bone marrow aspirates of patients with MCL, and its level was correlated with clinical outcome including response to treatment and relapse or progression. Considering the association of CD45+/CD19- cells with resistance to treatment, the presence of this extremely small but significant stem-like fraction might be associated with treatment failure in patients with MCL. DisclosuresNo relevant conflicts of interest to declare.

Cell Budding from Normal Appearing Epithelia: A Predictor of Colorectal Cancer Metastasis?

Background: Colorectal carcinogenesis is believed to be a multi-stage process that originates with a localized adenoma, which linearly progresses to an intra-mucosal carcinoma, to an invasive lesion, and finally to metastatic cancer. This progression model is supported by tissue culture and animal model studies, but it is difficult to reconcile with several well-established observations, principally among these are that up to 25% of early stage (Stage I/II), node-negative colorectal cancer (CRC) develop distant metastasis, and that circulating CRC cells are undetectable in peripheral blood samples of up to 50% of patients with confirmed metastasis, but more than 30% of patients with no detectable metastasis exhibit such cells. The mechanism responsible for this diverse behavior is unknown, and there are no effective means to identify patients with pending, or who are at high risk for, developing metastatic CRC.Novel findings: Our previous studies of human breast and prostate cancer have shown that cancer invasion arises from the convergence of a tissue injury, the innate immune response to that injury, and the presence of tumor stem cells within tumor capsules at the site of the injury. Focal degeneration of a capsule due to age or disease attracts lymphocyte infiltration that degrades the degenerating capsules resulting in the formation of a focal disruption in the capsule, which selectively favors proliferating or “budding” of the underlying tumor stem cells. Our recent studies suggest that lymphocyte infiltration also triggers metastasis by disrupting the intercellular junctions and surface adhesion molecules within the proliferating cell buds causing their dissociation. Then, lymphocytes and tumor cells are conjoined through membrane fusion to form tumor-lymphocyte chimeras (TLCs) that allows the tumor stem cell to avail itself of the lymphocyte's natural ability to migrate and breach cell barriers in order to intravasate and to travel to distant organs. Our most recent studies of human CRC have detected nearly identical focal capsule disruptions, lymphocyte infiltration, budding cells, and the formation of TLCs. Our studies have further shown that age- and type-matched node-positive and -negative CRC have a significantly different morphological and immunohistochemical profile and that the majority of lymphatic ducts with disseminated cells are located within the mucosa adjacent to morphologically normal appearing epithelial structures that express a stem cell-related marker.New hypothesis: Based on these findings and the growth patterns of budding cells revealed by double immunohistochemistry, we further hypothesize that metastatic spread is an early event of carcinogenesis and that budding cells overlying focal capsule disruptions represent invasion- and metastasis-initiating cells that follow one of four pathways to progress: (1) to undergo extensive in situ proliferation leading to the formation of tumor nests that subsequently invade the submucosa, (2) to migrate with associated lymphocytes functioning as “seeds” to grow in new sites, (3) to migrate and intravasate into pre-existing vascular structures by forming TLCs, or (4) to intravasate into vascular structures that are generated by the budding cells themselves. We also propose that only node-positive cases harbor stem cells with the potential for multi-lineage differentiation and unique surface markers that permit intravasation.

Abstract 443: Effects of ACNU on U87 monolayer cultures and spheroids are dependent on hypoxia

Abstract Tumor hypoxia and the presence of tumor stem cells are features related to glioblastoma tumorigenicity and malignancy. Moreover, chemotherapy seems to have limited effects on tumor stem cells. In the present study the aim was to investigate this further using U87 monolayer cultures, but also the more in vivo like multicellular spheroids. We investigated the effect of nimustine (ACNU) on U87 monolayer cultures and spheroids cultured in 1 % and 21 % oxygen. A WST-1-based cytotoxicity assay and a clonogenic assay were used for detection of effects of ACNU on monolayer cultures. Moreover, spheroids were exposed to ACNU and the effect evaluated by a clonogenic assay and immunohistochemical staining on spheroid paraffin sections using markers of apoptosis (caspase-3, TUNEL), stem cell related markers (CD133, podoplanin, Bmi-1, nestin) and the proliferation marker (Ki-67). We found hypoxia to reduce the effect of ACNU on U87 monolayer cultures and spheroids in both WST-1 and clonogenic assays. In spheroids, this was confirmed by caspase-3 and TUNEL staining. Furthermore, stem cell markers were preserved to a higher extent in hypoxia. However, this preservation was not seen with the proliferation marker Ki-67. In conclusion, the present study underlines the role of tumor hypoxia in chemoresistance, suggesting that the more immature features related to tumor stem cells may contribute to this. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 443.

Abstract 4269: Heterogeneity of glioblastoma stem and non-stem cells in the expression of caspase-8 and response to TRAIL-induced apoptosis

Abstract BACKGROUND: Recent studies suggest that tumor stem cells (TSCs) possess self-renewal property and thus are responsible to tumor growth and resistance to treatments. In this study, we examined how glioblastoma TSCs and non-stem cells respond to the treatment of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). METHODS AND RESULTS: TSCs-enriched neurospheres (NSCs) were generated from the earlier passages of primary cultures of glioblastomas surgically removed from patients. The NSCs were grown in serum-free medium containing epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF-2). Immunoflorescent staining of neurospheres revealed the presence of TSCs, as identified by nestin and CD133 positive cells. CD133+ cells were sorted from the NSC with CD133 antibody labeled magnetic microbeads and flow cytometry using fluorochrome-labeled CD133 antibody revealed approximately 7% and 73% CD133+ cells in the NSCs and sorted CD133+ cell preparation. The NSC and CD133+ cell population were characterized for the following TSC properties: the self-renewing capacity was established by neurosphere formation assay; the tumorigenic capacity was demonstrated by xenograft formation in NOD-SCID mouse brain; and the multipotent property was shown in differentiation assay in the generation of neurons as identified by neuronal marker Tuj1 and astrocytes as marked by glial fibrilary acid protein (GFAP). Of 8 sets of NSCs and CD133+ cells examined thus far, one set was sensitive to TRAIL-induced apoptosis, as evident by a bromide-based colorimetric cell viability assay and Caspase-Glo®-8 and −3/7 activity assay. Of the remaining 7 sets of TRAIL resistant NSC and CD133+ cell population, one set showed the lack of expression of caspase-8, as demonstrated by western blot. Fluorescence in situ hybridization (FISH) further revealed the loss of chromosome locus 2q33-34 of caspase-8 gene. In addition, multiple clones were generated from the TRAIL-resistant primary culture in serum culture condition and showed heterogeneity in the expression of caspase-8, as revealed by western blot, and the response to TRAIL-induced apoptosis, as shown by cleavage of caspases. G-banding karyotying analysis of chromosomes revealed heterogeneity of chromosomal alterations among TSC-enriched NSC and CD133+ population and non-stem serum-grown clones. CONCLUSION: This study establishes the heterogeneity of glioblastoma TSC enriched NSC and CD133+ cell population and non-stem serum-grown clones in the genomic status of caspase-8 locus and response to TRAIL-induced apoptosis. Genomic loss of caspase-8 in the stem and non-stem cells results in their resistance to TRAIL treatment. 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 4269.

Alloreactive NK-Cells Cure Tumor Stem Cell Containing Breast Cancer.

Abstract 4082Poster Board III-1017 BackgroundPatients with metastasized solid tumors can not be cured by current standard treatment options. One hypothesis is that slow cycling chemo-resistant tumor stem cells give rise to new tumors after cytoreductive treatment, ultimately leading to chemoresistant tumors. Acute myeloid leukemia tumor stem cells can be killed by alloreactive T- or NK-cells, as patients have a higher survival chance after allogeneic transplantation. In the past we published the curative effectiveness of allogeneic spleen and bone marrow transplantation in breast cancer bearing mice. Our current aim was to study if either alloreactive T- or NK-cells are able to cure these mice. In addition we wanted to know if the tumors contain slow cycling tumor stem cells. MethodsThe 4T1 breast cancer cell line was cultured under standard conditions. Fifty thousand 4T1 cells were injected s.c. in the flank of CB6F1 (H-2b/d) mice. At day 12 mice were treated with 2× 2Gy total body irradiation and 200 mg/kg cyclophosphamide (CY), followed by transplantation of spleen and bone marrow cells. B6CBAF1 (H-2b/k) mice were used as allogeneic donors. These donors harbour alloreactive NK-cells towards CB6F1 recipients as determined by an in vivo NK-cell alloreactivity assay. NK-cell or T-cell depleted grafts were obtained from donors injected with anti-AsialoGM1 or a combination of anti-CD4 and anti-CD8 respectively some days before spleen and bone marrow harvest. Tumor stem cells were defined as slow cycling (i.e. label retaining) Hoechst extruding cells. 4T1 tumor cells were labelled with Vybrant CM-Dil (Dil) and then injected s.c. in CB6F1 mice. Fourteen days later tumors were excised. Part of the tumor was prepared as single cell suspension. Subsequently the presence of Dil retaining tumor stem cells was determined by fluorescence microscopy. The other part of the tumor was snap frozen and studied by histochemistry and fluorescence microscopy. For in vitro cytotoxicity testing 4T1 cells were again labelled with Dil and then seeded in 96 well plates at 30 cells/well. Chemotherapeutics were added in the different plates: 4-OH-CY (the active metabolite), cisplatine and doxorubicin, in concentrations far above the lowest maximal lethal dose as determined in MTT assays. Wells were checked weekly for growth and presence of tumor stem cells. Hoechst staining was used in these cultures to underscore that Dil retaining chemorefractory cells are indeed stem cells. [Display omitted] ResultsNinety percent of CB6F1 mice injected with 4T1 breast cancer cells developed a tumor growing progressively to a size that made it necessary to sacrifice them. Growth in mice treated with radio- and chemotherapy only or additionally with CB6F1 spleen and bone marrow cells was delayed for 10 days compared with tumor growth in untreated mice, but the incidence was equally high (90% and 80% respectively). In contrast, but in concord with our previous published results, only 10% of 4T1 breast cancer bearing mice transplanted with haploidentical spleen and bone marrow cells did show progressive tumor growth (follow-up 99 days at the time of abstract submission, p<0.004 log-rank test)). Similarly, mice transplanted with spleen and BM cells from T-cell depleted donors did not develop tumors at all (p<0.004 log-rank test), in contrast to recipients of in vivo NK-cell depleted grafts (90% tumor incidence).Label retaining tumor stem cells are unmistakably present in tumors at the day of transplantation, evidenced by the presence of a minute number of Dil retaining cells in single cell suspension of the tumor and in tumor tissue sections. More direct evidence for chemo-resistance of 4T1 tumor stem cells is derived by results of in vitro co-culture experiments. When Dil labelled 4T1 cells are plated in 30 cells/well concentration, approx. 25% of the wells contain one or two label retaining cells while the other wells comprise non-label retaining cells only. After incubation with chemotherapeuticals growth is observed only in wells containing a label retaining cell. These label retaining cells are also Hoechst negative. ConclusionsThis report provides the first evidence that tumor stem cells can be eliminated in vivo by alloreactive NK cells resulting in cure of chemorefractory breast cancer. It is also shown in vitro that only tumor stem cell containing tumor colonies resist the effect of various chemotherapeuticals commonly used in oncology as they do in vivo. These results strongly encourage the exploration of clinical alloreactive NK therapy in patients with metastasized solid tumors. Disclosures:No relevant conflicts of interest to declare.

Cancer stem cells: its existence, proliferation and differentiation in an adenoid cystic carcinoma cell line

To investigate the evidences of the presence of tumor stem cells and its impact on the tumorigenesis of adenoid cystic carcinoma cell (ACC)-2 cell line by analyzing the biologic characteristics of different sub-clones of adenoid cystic carcinoma cell line. In vitro individual cell culture was employed to observe the proliferating character of ACC-2 cells. The expression of CD44(+) and CD24(-) of ACC-2 cells were investigated by immunohistochemical. Immunomagnetic isolation of different phenotype of ACC-2 cells, followed by cell culture, was used to study the proliferating abilities of different clusters of the cell line. The hetero-transplanted tumor mold was established using BALB/C nude mice by subcutaneous injection of tumor cells. The tumorigenic and differentiating properties of the different cluster were investigated. Only 4.41% of cultured ACC-2 cell had ability of division, proliferation and establishment of cell clone. CD44(+)-CD24(-) cluster accounted for about 8.1% of total ACC-2 cells, among which, 25.71% cells could divide and proliferate. All of CD44 and CD44(+)-CD24(+) cells were failure to be eternal alive in the condition of in vitro individual cell culture. According to the results of in vivo tumorigenic study, the minimal cell quantity to develop a subcutaneous transplanted tumor by CD44(+)-CD24(-) cells was 1 x 10(3), where as the needed cell amount were 1 x 10(5) and 1 x 10(4) as to non-isolated ACC-2 cells and CD44(+) cells, respectively. The CD44(-) and CD44(+)-CD24(+) did not develop transplanted tumors. CD44(+)-CD24(-) ACC-2 cell could differentiate into cells of other phenotypes. CD44(+)-CD24(-) ACC-2 cells consist of a very small portion of all ACC-2 cells (about 4%). They have remarkable proliferating ability and can bear special phenotypes, The tumorogenic ability of CD44(+)-CD24(-) cells are stronger than that of CD44(+) and non-isolated ACC-2 cells. Eliminating of this cluster from ACC-2 would actually deprive the tumorogenic ability of the cell line.