Noninvasive in vivo cell tracking using molecular imaging: A useful tool for developing mesenchymal stem cell-based cancer treatment

Mounting evidence has emphasized the potential of cell therapies in treating various diseases by restoring damaged tissues or replacing defective cells in the body. Cell therapies have become a strong therapeutic modality by applying noninvasive in vivo molecular imaging for examining complex cellular processes, understanding pathophysiological mechanisms of diseases, and evaluating the kinetics/dynamics of cell therapies. In particular, mesenchymal stem cells (MSCs) have shown promise in recent years as drug carriers for cancer treatment. They can also be labeled with different probes and tracked in vivo to assess the in vivo effect of administered cells, and to optimize therapy. The exact role of MSCs in oncologic diseases is not clear as MSCs have been shown to be involved in tumor progression and inhibition, and the exact interactions between MSCs and specific cancer microenvironments are not clear. In this review, a multitude of labeling approaches, imaging modalities, and the merits/demerits of each strategy are outlined. In addition, specific examples of the use of MSCs and in vivo imaging in cancer therapy are provided. Finally, present limitations and future outlooks in terms of the translation of different imaging approaches in clinics are discussed.     

Growth factor-receptor pathway interfering treatment by somatostatin analogs and suramin: Preclinical and clinical studies

Interference in growth factor mediated pathways is a new strategy in the treatment of cancer. Somatostatin analogs can inhibit hormone and growth factor secretion, while suramin can block the binding of several growth factors to their receptors. In addition, somatostatin analogs can cause direct growth inhibitory effects after binding to tumoral somatostatin receptors. We tested the efficacy and endocrine effects of chronic treatment with three somatostatin analogs (Sandostatin,^® RC-160 and CGP 15–425) or suramin in several tumor models and in patients with various types of cancer. Treatment with somatostatin analogs caused growth inhibition of breast cancer cells (MCF-7) in vitro, and of rat transplantable pancreatic (50–70% inhibition) and prostatic Dunning tumors (12% inhibition). No tumor growth inhibition was observed with respect to DMBA-induced rat mammary tumors, a transplantable color tumor and a rhabdomyosarcoma in rats. In 34 patients with metastatic pancreatic or gastrointestinal adenocarcinomas chronic Sandostatin treatment caused stable disease in 27% of the patients, but no objective remissions. Somatostatin receptors were found in the responding MCF-7 mammary tumor cells, rat pancreatic tumors and in 20–45% of human breast cancer specimens [J. Steroid Biochem. Molec. Biol. 37 (1990) 1073–1077], but not in rat DMBA-mammary tumors or in 10 human pancreatic adenocarcinomas. Suramin caused significant dose-dependent growth inhibition of human breast cancer cells in vitro and of rat pancreatic tumors in vivo in the presence of plasma levels up to 150 μg/ml. In a preliminary clinical study concerning 11 patients with various tumor types we observed significant hematological, biochemical, endocrine and clinical side effects, but no objective remissions in spite of relevant peak plasma suramin concentrations of 270–330 μg/ml. In conclusion: somatostatin analogs and suramin can cause growth inhibition of various experimental tumors in vitro and in vivo, but the clinical values has to be established for several types of cancer, especially with respect to suramin and suramin-like compounds.     

A mechanism for heterogeneous endothelial responses to flow in vivo and in vitro

Exposure of endothelium to a nominally uniform flow field in vivo and in vitrofrequently results in a heterogeneous distribution of individual cell responses. Extremes in response levels are often noted in neighboring cells. Such variations are important for the spatial interpretation of vascular responses to flow and for an understanding of mechanotransduction mechanisms at the level of single cells. We propose that variations of local forces defined by the cell surface geometry contribute to these differences. Atomic force microscopy measurements of cell surface topography in living endothelium both in vitro and in situ combined with computational fluid dynamics demonstrated large cell-to-cell variations in the distribution of flow-generated shear stresses at the endothelial luminal surface. The distribution of forces throughout the surface of individual cells of the monolayer was also found to vary considerably and to be defined by the surface geometry. We conclude that the endothelial three-dimensional surface geometry defines the detailed distribution of shear stresses and gradients at the single cell level, and that there are large variations in force magnitude and distribution between neighboring cells. The measurements support a topographic basis for differential endothelial responses to flow observed in vivo and in vitro. Included in these studies are the first preliminary measurements of the living endothelial cell surface in an intact artery.     

Translating tissue culture results into animal models: the case of Salmonella typhimurium

Investigators use both in vitro and in vivo models to better understand infectious disease processes. Both models are extremely useful in research, but there exists a significant gap in complexity between the highly controlled reductionist in vitro systems and the largely undefined, but relevant variability encompassing in vivo animal models. In an effort to understand how Salmonella initiates disease at the intestinal epithelium, in vitro models have served a useful purpose in allowing investigators to identify molecular mechanisms responsible for Salmonella invasion of host cells and stimulation of host inflammatory responses. Identification of these molecular mechanisms has generated hypotheses that are now being tested using in vivo models. Translating the in vitro findings into the context of an animal model and subsequently to human disease remains a difficult challenge for any disease process.     

RU 58668: Further in vitro and in vivo pharmacological data related to its antitumoral activity

Previous studies with the pure antiestrogen RU 58668 showed that this compound proved to be highly antiproliferative in vitro, and to be the only antiestrogenic compound so far known to induce long-term regression of MCF-7 tumours implanted into nude mice. In order to obtain more insight into the therapeutic potential of this molecule, we performed a new set of experiments in vitro and in vivo in comparison with tamoxifen and/or ICI 182,780. In vitro, 1 nM RU 58668 induced an accumulation of MCF-7 cells in G0/G1 phases of the cell cycle within 48 h and, in contrast to trans-4-hydroxy-tamoxifen, blocked the invasiveness of ras-transfected MCF-7 cells into the chick embryo heart during the three weeks of co-culture. An in vivo dose-effect relationship study showed that RU 58668 induced a regression of MCF-7 tumour with as low a dose as 10 mg/kg/week, and that such an effect can not be obtained either with a sublethal dose of adriamycin or with ICI 182,780, (2–250 mg/kg/week). This reduction in the tumour volumes accords with histological modifications of the tumours, which showed a decrease in the ratio of epithelial cells over the tumoral mass, and with a concomitant decrease in their regrowth potential when reimplanted into naive nude mice. Taken together, these results suggest a promising usefulness for RU 58668 in the treatment of metastatic breast cancer in women.     

The role of cytokines in the regulation of Leydig cell P450c17 gene expression

Cytokines produced by immune-activated testicular interstitial macrophages (TIMs) may play a fundamental role in the local control mechanisms of testosterone biosynthesis in Leydig cells. We investigated whether in vivo immune-activation of TIMs can modulate Leydig cell steroidogenesis. To immune activate TIMs in vivo, mice were injected intraperitoneally (i.p.) with lipopolysaccharide (LPS, 6 mg/kg). TIMs and Leydig cells were purified for RNA analysis. LPS treatment resulted in a 47-fold increase in interleukin-1β (IL-1β) mRNA in TIMs. P450c17 mRNA levels in the Leydig cells from the same animals, decreased to less than 10% compared to control. The effect of LPS on IL-1β and P450c17 mRNA levels was reversible on both TIMs and Leydig cells, respectively. To determine if the effect of LPS on P450c17 was mediated by a possible decrease in pituitary LH secretion, mice were co-injected with LPS and hCG. Treatment with hCG did not change the effect observed with LPS alone, in TIMs or in Leydig cells. In vitro, LPS treatment of TIMs resulted in marked induction of IL-1β mRNA expression. In parallel, in vitro treatment of Leydig cells with recombinant IL-1 resulted in a dose-dependent inhibition of P450c17 mRNA expression and testosterone production. These data demonstrate that LPS treatment, in vivo and in vitro, induced IL-1 gene expression in TIMs, and that IL-1 inhibits P450c17 mRNA in vitro. Therefore, we suggest that immune-activation of TIMs might have caused the observed inhibition of P450c17 gene expression in Leydig cells in vivo.     

Protection by α-tocopherol but not ascorbic acid from hydrogen peroxide induced cell death in normal human breast epithelial cells in culture

-Tocopherol and ascorbic acid have been suggested to play a role in breast cancer prevention due to their antioxidative capacity. Increased exposure to endogenous and exogenous sex steroids is a known risk factor for breast cancer. We have studied the effects of -tocopherol and ascorbic acid on hydrogen peroxide induced cell death in sex hormone treated normal breast epithelial cells in culture. We found that -tocopherol but not ascorbic acid alone protected the cells. The effect of -tocopherol increased when ascorbic acid was added to the cultures. The hydrogen peroxide degradation rate decreased in cultures treated with -tocopherol alone and in combination with ascorbic acid compared to cells grown in medium or with ascorbic acid only. Oestradiol and progesterone treatment did not influence the results. Possible beneficial effects of combining various antioxidants, endogenous as well as exogenous, on human breast tissue need to be investigated further both in vivo and in vitro.     

Intracrinology of estrogens and androgens in breast carcinoma

Intratumoral metabolism and synthesis of biologically active steroids such as estradiol and 5-dihydrotestosterone as a result of interactions of various enzymes are considered to play very important roles in the pathogenesis and development of hormone-dependent breast carcinoma. Among these enzymes involved in estrogen metabolism, intratumoral aromatase play an important role in converting androgens to estrogens in situ from serum and serving as the source of estrogens, especially in postmenopausal patients with breast carcinoma. However, other enzymes such as 17β-hydroxysteroid dehydrogenase (17β-HSD) isozymes, estrogen sulfatase (STS), and estrogen sulfotransferase, which contribute to in situ availability of biologically active estrogens, also play pivotal roles in this intratumoral estrogen production above. Androgen action on human breast carcinoma has not been well-studied but are considered important not only in hormonal regulation but also other biological features of carcinoma cells. Intracrine mechanisms also play important roles in androgen actions on human breast carcinoma cells. Among the enzymes involved in biologically active androgen metabolism and/or synthesis, both 17β-hydroxysteroid dehydrogenase type 5 (17βHSD5; conversion from circulating androstenedione to testosterone) and 5-reductase (5Red; reduction of testosterone to DHT (5-dihydrotestosterone) were expressed in breast carcinoma tissues, and in situ production of DHT has been proposed in human breast cancer tissues. However, intracrine mechanisms of androgens as well as their biological or clinical significance in the patients with breast cancer have not been fully elucidated in contrast to those in estrogens.     

Interaction of glucocorticoid analogues with the human glucocorticoid receptor

Transient co-transfection of receptor cDNA and suitable reporter genes was used to study human glucocorticoid receptor (hGR) function in a neutral mammalian cell background. A variety of natural and synthetic steroids were analyzed for their ability to activate gene expression through the hGR and to bind to extracts of cells expressing the hGR cDNA. There was very good correlation between these two in vitro parameters for these compounds. Furthermore, correlation of these data with reported in vivo anti-inflammatory potencies was surprisingly close, with two exceptions. The in vitro data suggest an explanation for the discrepant compounds, consistent with published data on their metabolic fate in vivo. The co-transfection assay has utility as a quantitative predictor of in vivo glucocorticoid pharmacology.     

Enterolactone and estradiol inhibit each other''s proliferative effect on MCF-7 breast cancer cells in culture

In earlier studies it has been shown that women with breast cancer and at risk for breast cancer have low excretion of urinary mammalian lignans (enterolactone and enterodiol) mainly due to low intake of whole-grain products and other fiber-rich foods. It is well known that estradiol (E2) has proliferative effects on estrogen dependent cancer cells and that antiestrogens inhibit this effect. To elucidate whether enterolactone (Enl) has antiestrogenic properties we studied, using MCF-7 breast cancer cells in culture, the in vitro effect of relatively low concentrations of Enl added both alone and in combination with E2. E2 (1 nmol/l) and Enl (0.5–2 μmol/l) separately stimulated the proliferation of MCF-7 cells, but their combination always resulted in lower stimulation than any of them alone, or the combined compounds had no stimulatory effect at all compared to the control. Higher concentrations above 10 μmol/l of Enl inhibited significantly the growth of the cells suggesting a toxic effect. The lignan was very rapidly conjugated to its monosulfate. It is suggested that one possible mechanism by which Enl may affect the growth of these estrogen sensitive cells is by competition of Enl and its sulfate with the estrogens for sulfokinases and sulfatases involved in estrogen metabolism in the cells. It is concluded that Enl inhibits E2-stimulated MCF-7 breast cancer cell growth in vitro, and vice versa. The concentrations of Enl needed for the elimination of the proliferative effect of E2 are physiologic and similar to those used in corresponding experiments utilizing tamoxifen.     

Coronavirus infection of polarized epithelial cells

Epithelial cells are the first host cells to be infected by incoming coronaviruses. Recent observations in vitro show that coronaviruses are released from a specific side of these polarized cells, and this polarized release might be important for the spread of the infection in vivo. Mechanisms for the directional sorting of coronaviruses might be similar to those governing the polar release of secretory proteins.     

Control of aromatase in breast cancer cells and its importance for tumor growth

Three approaches have been taken to elucidate further the biological importance of intratumoural aromatase activity. (i) MCF₇ and T47D hormone-dependent breast cancer cell lines both showed detectable aromatase activity in vitro. The up-regulation of this by TGF indicates the possible existence of an autocrine growth stimulatory loop involving aromatase. (ii) Both tamoxifen and cytotoxic chemotherapy caused the suppression of aromatase activity in breast carcinomas in vivo. Aromatase activity prior to treatment did not predict for clinical response to tamoxifen. (iii) Transfection of aromatase into MCF₇ cells led to their growth being stimulated by low doses of androgens and this was inhibited by the aromatase inhibitor CGS 16949A.     

乳腺癌细胞QSOX1的CRISPR/Cas9基因编辑及其对增殖侵袭的影响研究*

目的:乳腺癌细胞的恶性增殖和易于侵袭转移特性与其对患者的危害直接相关,因此,探究其产生的分子机制,对其有效防治具有重要意义。静息巯基氧化酶-1(QSOX1)是巯基氧化酶家族成员之一,有研究证明其对细胞内蛋白质折叠过程中二硫键形成及细胞外基质的形成发挥重要作用。由于QSOX1在乳腺癌和胰腺癌等多种癌细胞中过表达,将探索QSOX1对乳腺癌细胞过度增殖和侵袭转移方面的可能作用。方法:通过利用CRISPR/Cas9技术构建QSOX1基因敲除和敲入的乳腺癌细胞模型,检测分析QSOX1对乳腺癌细胞MCF-7的分裂增殖、侵袭迁移能力等方面的影响。结果:利用CRISPR/Cas9基因编辑技术成功构建了QSOX1基因敲除和敲入的乳腺癌MCF-7细胞株,其与对照野生型组细胞相比,QSOX1基因敲除株的增殖能力显著下降,癌细胞在体外的迁移和侵袭能力受到明显抑制;而QSOX1基因敲入株的增殖能力和体外迁移侵袭能力却明显有提高。结论:初步揭示了QSOX1在癌症发生与发展中的作用,为进一步阐明其作用的分子机制和设计靶向药物奠定了重要基础。     

Insight into generation of induced mesenchymal stem cells from induced pluripotent cells

Mesenchymal stem cells(MSCs)have the potential for use in cell-based regenerative therapies.Currently,hundreds of clinical trials are using MSCs for the treatment of various diseases.However,MSCs are low in number in adult tissues;they show heterogeneity depending upon the cell source and exhibit limited proliferative potential and early senescence in in vitro cultures.These factors negatively impact the regenerative potential of MSCs and therefore restrict their use for clinical applications.As a result,novel methods to generate induced MSCs(iMSCs)from induced pluripotent stem cells have been explored.The development and optimization of protocols for generation of iMSCs from induced pluripotent stem cells is necessary to evaluate their regenerative potential in vivo and in vitro.In addition,it is important to compare iMSCs with primary MSCs(isolated from adult tissues)in terms of their safety and efficacy.Careful investigation of the properties of iMSCs in vitro and their long term behavior in animals is important for their translation from bench to bedside.     

Dexamethasone influences human clock gene expression in bronchial epithelium and peripheral blood mononuclear cells in vitro

We determined whether human peripheral blood mononuclear cells (PBMCs) could be used to analyze clock genes by studying their mRNA expressions in human bronchial epithelium (BEAS-2B) and PBMCs following stimulation by the glucocorticoid homologue dexamethasone (DEX) in vitro. PBMCs were obtained at 10:00 h from two diurnally active (∼07:00 to 23:00 h) healthy volunteers and were evaluated for hPer1 mRNA expression following DEX stimulation in vitro using real time-PCR analysis. DEX stimulation of human BEAS-2B cells and PBMCs in vitro led to a remarkable increase of hPer1 mRNA. The glucocorticoid rapidly affected the expression of hPer1 mRNA in PBMCs, suggesting that human PBMCs may be a useful surrogate marker for the investigation of drug effects on clock genes.     

Inhibition of cell growth by overexpression of manganese superoxide dismutase (MnSOD) in human pancreatic carcinoma

Manganese superoxide dismutase (MnSOD) levels have been found to be low in human pancreatic cancer [Pancreas26, (2003), 23] and human pancreatic cancer cell lines [Cancer Res.63, (2003), 1297] when compared to normal human pancreas. We hypothesized that stable overexpression of pancreatic cancer cells with MnSOD cDNA would alter the malignant phenotype. MIA PaCa-2 cells were stably transfected with a pcDNA3 plasmid containing sense human MnSOD cDNA or containing no MnSOD insert by using the lipofectAMINE method. G418-resistant colonies were isolated, grown and maintained. Overexpression of MnSOD was confirmed in two selected clones with a 2-4-fold increase in MnSOD immunoreactive protein. Compared with the parental and neo control cells, the MnSOD-overexpressing clones had decreased growth rates, growth in soft agar and plating efficiency in vitro, while in vivo, the MnSOD-overexpressing clones had slower growth in nude mice. These results suggest that MnSOD may be a tumor suppressor gene in human pancreatic cancer.     

Effect of aging on behaviour of mesenchymal stem cells

Organs whose source is the mesoderm lineage contain a subpopulation of stem cells that are able to differentiate among mesodermal derivatives (chondrocytes, osteocytes, adipocytes). This subpopulation of adult stem cells, called “mesenchymal stem cells” or “mesenchymal stromal cells (MSCs)”, contributes directly to the homeostatic maintenance of their organs; hence, their senescence could be very deleterious for human bodily functions. MSCs are easily isolated and amenable their expansion in vitro because of the research demanding to test them in many diverse clinical indications. All of these works are shown by the rapidly expanding literature that includes many in vivo animal models. We do not have an in-depth understanding of mechanisms that induce cellular senescence, and to further clarify the consequences of the senescence process in MSCs, some hints may be derived from the study of cellular behaviour in vivo and in vitro, autophagy, mitochondrial stress and exosomal activity. In this particular work, we decided to review these biological features in the literature on MSC senescence over the last three years.     

Vitamin D: A modulator of cell proliferation and differentiation

1,25-Dihydroxyvitamin D₃, [1,25(OH)₂D₃], the biologically most active metabolite of vitamin D₃, is involved in the regulation of calcium homeostasis and bone metabolism. Recently, receptors for 1,25(OH)₂D₃ have also been shown in cells and tissues not directly related to calcium homeostasis. Experimental data obtained with leukemic and cancer cell lines, both in vitro and in vivo, showed the effects of 1,25(OH)₂D₃ on cell differentiation and proliferation. However, high doses of the sterol have to be used to observe these effects. Additional studies are needed to establish whether 1,25(OH)₂D₃ or suitable analogues have a therapeutic potential in malignant diseases without unacceptable toxicity like the development of hypercalcemia.