Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites.

The discovery of adipose-derived stromal cells (ASCs) has created many opportunities for the development of patient-specific cell-based replacement therapies. We have isolated multiple cell strains of ASCs from various anatomical sites (abdomen, arms/legs, breast, buttocks), indicating widespread distribution of ASCs throughout the body. Unfortunately, there exists a general lack of agreement in the literature as to their "stem cell" characteristics. We find that telomerase activity and expression of its catalytic subunit in ASCs are both below the levels of detection, independent of age and culturing conditions. ASCs also undergo telomere attrition and eventually senesce, while maintaining a stable karyotype without the development of spontaneous tumor-associated abnormalities. Using a set of cell surface markers that have been promoted to identify ASCs, we find that they failed to distinguish ASCs from normal fibroblasts, as both are positive for CD29, CD73 and CD105 and negative for CD14, CD31 and CD45. All of the ASC isolates are multipotent, capable of differentiating into osteocytes, chondrocytes and adipocytes, while fibroblasts show no differentiation potential. Our ASC strains also show elevated expression of genes associated with pluripotent cells, Oct-4, SOX2 and NANOG, when compared to fibroblasts and bone marrow-derived mesenchymal stem cells (BM-MSCs), although the levels were lower than induced pluripotent stem cells (iPS). Together, our data suggest that, while the cell surface profile of ASCs does not distinguish them from normal fibroblasts, their differentiation capacity and the expression of genes closely linked to pluripotency clearly define ASCs as multipotent stem cells, regardless of tissue isolation location.

CD4+ T cells inhibit the neu-specific CD8+ T-cell exhaustion during the priming phase of immune responses against breast cancer.

Studies conducted in animal model of infectious diseases or H-Y antigen model suggest a crucial role for CD4+ T cells in providing help for CD8+ T-cell memory responses. This concept suggests that inclusion of T helper epitopes in vaccine formulation will result in improved CD8+ T-cell responses. Although this concept has been applied to cancer vaccine design, the role of CD4+ T cells in the memory differentiation of CD8+ T cells and retention of their anti-tumor function have never been tested in breast cancer model. Using the FVB mouse model of neu-positive breast carcinoma we report for the first time that helpless T cells showed cytostatic or tumor inhibitory effects during primary tumor challenge whereas, helped T cells showed cytotoxic effects and resulted in complete tumor rejection. Such differential effects, in vivo, were associated with higher frequency of CD8+PD-L1+ and CD8+PD-1+ T cells in animals harboring helpless T cells as well as higher titer of IL-2 in the sera of animals harboring helped T cells. However, depletion of CD4+ T cells did not alter the ability of neu-specific CD8+ T cells to differentiate into memory cells and to retain their effector function against the tumor during recall challenge. These results suggest the inhibitory role of CD4+ T cells on CD8+ T-cell exhaustion without substantial effects on the differentiation of memory T cells during priming phase of the immune responses against breast cancer.

Elevated TRF2 in advanced breast cancers with short telomeres.

Telomere repeat binding factor 2 (TRF2) binds directly to telomeres and preserves the structural integrity of chromosome ends. In vitro models suggest that expression of TRF2 protein increases during mammary cancer progression. However, a recent study has reported that TRF2 mRNA levels tend to be lower in clinical specimens of malignant breast tissue. Here, we conduct the first large-scale investigation to assess the levels and cellular localization of the TRF2 protein in normal, pre-malignant and malignant breast tissues. Breast tissue arrays, containing normal, ductal carcinoma in situ (DCIS) and invasive carcinoma specimens, were used to assess the expression and localization of TRF2 protein. Telomere lengths were semi-quantitatively measured using a pantelomeric peptide nucleic acid probe. A mixed effects modeling approach was used to assess the relationship between TRF2 expression and telomeric signal scores across disease states or clinical staging. We demonstrate that TRF2 is exclusively nuclear with a trend toward lower expression with increased malignancy. More case-to-case variability of TRF2 immunostaining intensity was noted amongst the invasive carcinomas than the other disease groups. Invasive carcinomas also displayed variable telomere lengths while telomeres in normal mammary epithelium were generally longer. Statistical analyses revealed that increased TRF2 immunostaining intensity in invasive carcinomas is associated with shorter telomeres and shorter telomeres correlate with a higher TNM stage. All immortalized and cancer cell lines within the array displayed strong, nuclear TRF2 expression. Our data indicate that elevated expression of TRF2 is not a frequent occurrence during the transformation of breast cancer cells in vivo, but higher levels of this telomere-binding protein may be important for protecting advanced cancer cells with critically short telomeres. Our findings also reinforce the concept that serially propagated cancer cells, although tumor-derived, may not model all types of authentic tumors especially those demonstrating genetic heterogeneity.

Wilms' tumor 1 silencing decreases the viability and chemoresistance of glioblastoma cells in vitro: a potential role for IGF-1R de-repression.

Wilms' tumor 1 (WT1) is a transcription factor with a multitude of downstream targets that have wide-ranging effects in non-glioma cell lines. Though its expression in glioblastomas is now well-documented, the role of WT1 in these tumors remains poorly defined. We hypothesized that WT1 functions as an oncogene to enhance glioblastoma viability and chemoresistance. WT1's role was examined by studying the effect of WT1 silencing and overexpression on DNA damage, apoptosis and cell viability. Results indicated that WT1 silencing adversely affected glioblastoma viability, at times, in synergy with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and cisplatin. To investigate other mechanisms through which WT1 could affect viability, we measured cell cycle distribution, senescence, and autophagy. WT1 silencing had no effect on these processes. Lastly, we examined WT1 regulation of IGF-1R expression. Counterintuitively, upregulation of IGF-1R was evident after WT1 silencing. In conclusion, WT1 functions as a survival factor in glioblastomas, possibly through inhibition of IGF-1R expression.

Multipotent adipose stromal cells and breast cancer development: Think globally, act locally.

It has long been appreciated that stromal cells within the breast tumor microenvironment contribute to mammary carcinogenesis. However, to date, very little is known regarding the role of local adipose-derived stromal cells (ASCs) in the development of breast cancer. Based on pathological, epidemiological and experimental data, we postulate that breast-derived ASCs are unique mesenchymal stem-like cells that play a critical role in the development of breast cancer and discuss the global implications of this working model in terms of breast cancer prevention, early detection, and new targeted therapies.

Induction of nitric oxide synthase-dependent telomere shortening after functional inhibition of Hsp90 in human tumor cells.

In most cancer cells, the lengths of telomeres, the functional DNA-protein complexes located at chromosome ends, are maintained by the ribonucleoprotein telomerase. Hsp90 facilitates the assembly of telomerase and remains associated with the functional complex, implying a direct involvement of Hsp90 in telomere length regulation. In an effort to elucidate the effects of Hsp90 inhibition on function and viability of human prostate cancer cells, both pharmacological (radicicol) and genetic (small interfering RNA) approaches were utilized to target Hsp90. Depletion of functional Hsp90 caused dramatic telomere shortening followed by apoptosis. Of particular significance, these cells exhibit a high level of nitric oxide synthase (NOS)-dependent free radical production, and simultaneous treatment of cells with the NOS inhibitor L-NAME resulted in telomere elongation and prevention of apoptosis. In addition, we observe significant DNA damage assessed by telomere dysfunction, although in the absence of a classical DNA damage response. Overall, our data suggest a novel mechanism whereby inhibition of Hsp90 disrupts free radical homeostasis and contributes directly to telomere erosion, further implicating Hsp90 as a potential therapeutic target for cancer cells.

Recovery from stress is a function of age and telomere length.

Cells are constantly exposed to a wide variety of stimuli and must be able to mount appropriate physiological responses in order to maintain proper form and function. Cells from every organism have evolved highly conserved mechanisms to cope with environmental changes, including the widely studied heat shock response (HSR), which is induced by a variety of cellular stresses such as heavy metal ion exposure. It has long been known that as organisms and individual cells age, their ability to appropriately cope with environmental stress is attenuated. Here, we examine the ability of two heavy metal ions (ZnCl(2), SnCl(2)) to induce the HSR in human fibroblasts by assessing the expression of heat shock proteins (Hsp90, Hsp70, and p23) and the ability of the cells to recover over time. We demonstrate that the induction and recovery of chaperone levels is attenuated with age and that cells immortalized with the human telomerase reverse transcriptase component of the telomerase enzyme do not attenuate their HSR as their replicative age increases. Our data suggest that the recovery of normal human cells from an HSR is related in part to age and the cell's overall telomere length.

Upregulation of telomerase function during tissue regeneration.

Telomerase plays a primary role in the maintenance of telomeres in immortal, germ, and tumor cells in humans but is lacking in most somatic cells and tissues. However, many species, including fish and inbred mice, express telomerase in most cells and tissues. Little is known about the expression of telomerase in aquatic species, although the importance of telomerase for longevity has been suggested. We compared telomerase activity and telomere lengths among a broad range of tissues from aquatic species and found telomerase at significant levels in both long- and short-lived aquatic species, suggesting constitutive telomerase expression has an alternative function. Telomere lengths in these aquatic species were comparable to those observed in normal human tissues and cell strains. Given that a host of aquatic species with short life spans have telomerase and a tremendous capacity to regenerate, we tested the hypothesis that telomerase upregulation is important for tissue regeneration. During regeneration, telomerase activity was upregulated and telomere lengths are maintained with the shortest telomeres being elongated, indicating the importance for maintaining telomere length and integrity during tissue regeneration. Thus, the expression of telomerase in aquatic animals is likely not related to longevity but to their ability to regenerate injured tissue.

Overexpression of telomerase-associated chaperone proteins in prostatic intraepithelial neoplasia and carcinomas.

Over 90% of prostate cancers express telomerase activity. In an experimental model, hsp90 and p23, which are necessary for telomerase assembly and function, dramatically increase during tumorigenic conversion. We immunohistochemically analyzed 60 prostate carcinomas, 50 prostatic intraepithelial neoplasias (PIN) and 25 benign prostatic tissues to determine whether hsp90/p23 expression correlates with advancing stage and whether chaperone distribution overlaps with hTERT, the catalytic component of telomerase. Strong expression of hsp90/p23 was detected in approximately 95% of PIN and carcinomas without relationship to Gleason score. While hsp90/p23 immunostaining was predominantly diffuse and cytoplasmic, nuclear immunoreactivity was observed in several moderate-to-high grade carcinomas, and those carcinomas with nuclear chaperone staining exhibited detectable hTERT. Our data suggest enhanced chaperone-mediated telomerase assembly as a mechanism for increased activity in advanced prostate carcinomas, stable association between chaperones and telomerase in vivo, and utility for chaperone immunostaining to identify focal PIN in the context of widespread hyperplasia.

Ectopic telomerase expression inhibits neuronal differentiation of NT2 neural progenitor cells.

There is significant interest in the potential use of telomerase-immortalized cells in transplantation to replace neurons lost to neurodegenerative diseases and other central nervous system injuries. Neural progenitor cells (NPCs) transduced with human telomerase reverse transcriptase (hTERT), the catalytic component of telomerase, have the potential both to proliferate indefinitely in vitro and to respond to differentiation signals necessary for generating appropriate cells for transplantation. The purpose of this study was to evaluate the differentiation of neurons from NT2 cells, a model NPC cell line, following hTERT transduction. RT-PCR and telomerase activity data demonstrated that persistent exogenous hTERT expression significantly inhibited the differentiation of neurons from NT2 cells. Following retinoic acid induced differentiation, hTERT-NT2 cells produced only one fourth of the neurons generated by parental and vector-control cells. A differentiation-inhibiting effect of constitutive telomerase activity has not been reported previously in other hTERT-transduced progenitor cell lines, implying a unique role for telomerase in the proliferation and differentiation of NPCs that have tumorigenic potential. Elucidating the mechanism responsible for this effect may aid in understanding the potential role of telomerase activity in the tumorigenicity of NPCs, as well as in optimizing the production of safe, telomerase-engineered, transplantable neurons.

Evasion of a single-step, chemotherapy-induced senescence in breast cancer cells: implications for treatment response.

PURPOSE: The purpose of this study is to define the mechanistic basis for recovery of proliferative capacity in breast tumor cells after chemotherapy. Here, we test the hypothesis that evasion of senescence confers resistance to chemotherapeutic drugs and ionizing radiation. EXPERIMENTAL DESIGN: MCF-7 cells were treated with a single, clinically relevant dose (0.75-1.0 micromol/L) of Adriamycin. Two weeks following induction of senescence, clonal outgrowths were expanded and characterized in terms of senescence-associated beta-galactosidase activity, gene expression profiles (Affymetrix U95 probe sets, Affymetrix, Santa Clara, CA) with confirmatory Western analyses, and telomerase activity following a second drug treatment. Levels of intracellular Adriamycin, as well as cross-resistance to other therapeutic agents, were also determined to define the resistance phenotype. RESULTS: A senescence-resistant (SR) clone (clone 2) was identified that was largely refractory to both Adriamycin-induced and gamma-irradiation-induced senescence. Clone 2 continued to proliferate and maintain high levels of telomerase activity following a second drug treatment, when treated parental cells expressed very low levels of telomerase and many positive cell cycle regulators. SR clone 2 also expressed substantially more cdc-2 than parental cells and undetectable levels of MDR1, showed an intact p53 checkpoint and only a modestly lower level of intracellular drug accumulation, while exhibiting cross-resistance to other topoisomerase inhibitors. CONCLUSIONS: SR clone 2 is intrinsically resistant to DNA damage-induced senescence perhaps through an ability to prevent down-regulation of cdc-2. Telomerase is a marker of proliferative recovery for breast cancer cells after chemotherapy exposure. Evasion or escape from a single-step, drug-induced senescence may represent a unique and previously unrecognized drug-resistance phenotype.

A cell line (HEW) from embryos of haddock (Melanogrammus aeglefinius) and its capacity to tolerate environmental extremes.

Cell lines can be useful experimental tools for studying marine fish, which are often difficult to routinely obtain and maintain in the laboratory. As few cell lines are available from coldwater marine fish, cultures were initiated from late gastrula embryos of haddock (Melanogrammus aeglefinus) in Leibovitz's L-15 with fetal bovine serum (FBS). From one culture, a cell line (HEW) emerged that has been grown for close to 100 population doublings, was heteroploid, and expressed telomerase activity, all of which suggest HEW is immortal. Growth occurred only if FBS was present and was optimal at 12 to 18 degrees C. Usually most cells had an epithelial-like morphology, but under some conditions, cells drew up into round central bodies from which radiated cytoplasmic extensions with multiple branches. These neural-like cells appeared within a few hours of cultures being placed at 28 degrees C or being switch to a simple salt solution (SSS). At 28 degrees C, cells died within 24 h. In SSS, HEW cells survived as a monolayer for at least 7 days. The sensitivity of HEW cells to morphological change and their capacity to withstand starvation should make them useful for investigating cellular responses to environmental stresses.

Telomerase as a target for cancer immunotherapy.

Telomerase is the ribonucleoprotein that enables cancer and stem cells to maintain their telomeres, resulting in unlimited proliferative potential. The catalytic component of telomerase in humans, hTERT, is upregulated in nearly 90% of all cancers, making it the most widely expressed marker of malignancy. With the exception of germ cells and stem cells, hTERT is undetectable in somatic human tissues. Together, these properties make telomerase a leading candidate for cancer therapy. Various therapies have been tested in tissue culture and in mouse models utilizing genetic, pharmacological, and immunological approaches. The purpose of this review is to critically examine the role of hTERT in cancer immunotherapy by providing a comparison of the current experiments and a proposal for an innovative method utilizing DNA vaccination.

Telomerase protects cancer-prone human cells from chromosomal instability and spontaneous immortalization.

Studies were conducted to directly test whether the introduction of telomerase protects cancer-prone human mammary epithelial cells from chromosomal instability and spontaneous immortalization. Using a model for Li Fraumeni Syndrome (LFS), infection of human telomerase resulted in maintenance of telomere lengths, extension of in vitro lifespan, and prevention of spontaneous immortalization. In stark contrast to the spontaneously immortalized LFS cells, cells expressing ectopic telomerase displayed a remarkably stable karyotype and even after >150 population doublings, did not express endogenous telomerase. Since the hTERT-infected and spontaneously immortal LFS cells, like the parental cells, exhibit loss of p53 function, our data suggests that telomere shortening is the primary driving force for the genomic instability characteristic of LFS cells, while p53 inactivation is necessary for triggering the spontaneous immortalization event. Collectively, our data indicate that exogenous telomerase prevents chromosomal instability and spontaneous immortalization of LFS cells, suggesting a unique protective role for telomerase in the progression to immortalization.

Retroviral-mediated expression of telomerase in normal human cells provides a selective growth advantage.

Retroviral infection of hTERT, the catalytic component of telomerase, into BJ fibroblasts (population doubling 28) resulted in reconstitution of telomerase activity, telomere maintenance, and extension of in vitro lifespan. The hTERT-infected cells also exhibited increased growth rate and colony forming efficiency relative to controls, while remaining contact-inhibited and maintaining a p53-mediated damage response following gamma-irradiation. All single cell-derived BJ-hTERT clones grew faster than the hTERT mass cultures and maintained telomeres; however, neither telomerase activity levels nor mean telomere length correlated with the growth rate. Introduction of hTERT rescued aged BJ fibroblasts from senescence via a telomere-dependent mechanism and provided renewed proliferative potential. Collectively, our data indicate that both early and late in the cellular lifespan of human cells, ectopic expression of telomerase using a retroviral system provides a growth advantage while maintaining normal cellular characteristics.

Real-time quantitative analysis of telomerase activity in breast tumor specimens using a highly specific and sensitive fluorescent-based assay.

Telomerase activity has been associated with almost 90% of malignant human cancers from a variety of tissue sources, making it one of the most prominent molecular cancer markers known to date. As such, telomerase has become a very attractive diagnostic and therapeutic target. The advent of the telomeric repeat amplification protocol (TRAP) has allowed for the semiquantitative detection of telomerase from limiting sample amounts. Both the standard TRAP assay and a real-time assay using Amplifluor technology with primers designed specifically for telomerase activity amplification were used to quantitatively assess telomerase activity in primary tumors and tumor-derived cell lines. We have adapted the recently developed TRAPeze XL telomerase detection kit (Intergen, Gaithersburg, MD) for use with real-time polymerase chain reaction for more accurate quantification and high-throughput capabilities. In doing so, the reliability, assay time, and accuracy of quantitation have all been dramatically improved. A comparison of the quantitative analysis for the standard TRAP assay versus the real-time assay using 19 breast tumors revealed telomerase quantitation and standardization using the real-time assay was superior to the standard assay. Our data suggest that this assay will be useful for clinical and research studies involving detection of telomerase activity as it relates to cancer diagnosis.

Electrospinning adipose tissue-derived extracellular matrix for adipose stem cell culture.

Basement membrane-rich extracellular matrices, particularly murine sarcoma-derived Matrigel, play important roles in regenerative medicine research, exhibiting marked cellular responses in vitro and in vivo, although with limited clinical applications. We find that a human-derived matrix from lipoaspirate fat, a tissue rich in basement membrane components, can be fabricated by electrospinning and used to support cell culture. We describe practical applications and purification of extracellular matrix (ECM) from adipose tissue (At-ECM) and its use in electrospinning scaffolds and adipose stem cell (ASC) culture. The matrix composition of this purified and electrospun At-ECM was assessed histochemically for basement membrane, connective tissue, collagen, elastic fibers/elastin, glycoprotein, and proteoglycans. Each histochemical stain was positive in fat tissue, purified At-ECM, and electrospun At-ECM, and to some extent positive in a 10:90 blend with polydioxanone (PDO). We also show that electrospun At-ECM, alone and blended with PDO, supports ASC attachment and growth, suggesting that electrospun At-ECM scaffolds support ASC cultivation. These studies show that At-ECM can be isolated and electrospun as a basement membrane-rich tissue engineering matrix capable of supporting stem cells, providing the groundwork for an array of future regenerative medicine advances.

Mesenchymal stem cells in mammary adipose tissue stimulate progression of breast cancer resembling the basal-type.

Data are accumulating to support a role for adipose-derived mesenchymal stem cells (MSCs) in breast cancer progression; however, to date most studies have relied on adipose MSCs from non-breast sources. There is a particular need to investigate the role of adipose MSCs in the pathogenesis of basal-like breast cancer, which develops at a disproportionate rate in pre-menopausal African-American women with a gain in adiposity. The aim of this study was to better understand how breast adipose MSCs (bMSCs) contribute to the progression of basal-like breast cancers by relying on isogenic HMT-3255 S3 (pre-invasive) and T4-2 (invasive) human cells that upon transplantation into nude mice resemble this tumor subtype. In vitro results suggested that bMSCs may contribute to breast cancer progression in multiple ways. bMSCs readily penetrate extracellular matrix components in part through their expression of matrix metalloproteinases 1 and 3, promote the invasion of T4-2 cells and efficiently chemoattract endothelial cells via a bFGF-independent, VEGF-A-dependent manner. As mixed xenografts, bMSCs stimulated the growth, invasion and desmoplasia of T4-2 tumors, yet these resident stem cells showed no observable effect on the progression of pre-invasive S3 cells. While bMSCs form vessel-like structures within Matrigel both in vitro and in vivo and chemoattract endothelial cells, there appeared to be no difference between T4-2/bMSC mixed xenografts and T4-2 xenografts with regard to intra- or peri-tumoral vascularity. Collectively, our data suggest that bMSCs may contribute to the progression of basal-like breast cancers by stimulating growth and invasion but not vasculogenesis or angiogenesis.

Isolating adipose-derived mesenchymal stem cells from lipoaspirate blood and saline fraction.

Isolation of adipose-derived stem cells (ASCs) typically involves 8+ hours of intense effort, requiring specialized equipment and reagents. Here, we present an improved technique for isolating viable populations of mesenchymal stem cells from lipoaspirate saline fractions within 30 minutes. Importantly, the cells exhibit remarkable similarities to those obtained using the traditional isolation protocols, in terms of their multipotent differentiation potential and immunophenotype. Reducing the acquisition time of ASCs is critical for advancing regenerative medicine therapeutics, and our approach provides rapid and simple techniques for enhanced isolation and expansion of patient-derived mesenchymal stem cells.