Tumor cells do not arise frequently

Summary A hypothesis greatly influencing thinking and experimental work in tumor immunology is that many tumor cells arise daily in an organism. However, relatively low numbers of tumours become clinically manifest. This discrepancy has intially led to the hypothesis that the great majority of these potential tumors is killed by immune surveillance mechanisms. After some time, however, serious objections were raised against this function of the immune system. When the interest in immune surveillance faded away, investigators started to study natural resistance against the de novo arising tumor cells. In this paper the data on natural resistance (and immune surveillance) are reviewed. These data lead to the conclusion than an efficient natural resistance that could kill many de novo arising tumor cells is lacking. Taken together the data suggest that no tumors arise when immune surveillance and/or natural resistance are absent. This implies that tumor cells probably do not arise frequently. Acceptance of this conclusion leads to a reappraisal of the role of immune surveillance and natural resistance against tumor cells. This reappraisal will mean a shift from (a) the hypothesized failsafe mechanisms to (b) mechanisms that may or may not kill rarely arising tumor cells, depending for instance on the antigenicity of the tumor cells and their sensitivity to tumoricidal mechanisms.     

Skeletal muscle differentiation potential of human adult bone marrow cells

Murine bone marrow (BM) cells have been shown to undergo myogenic differentiation and participate in muscle repair in different muscle regeneration models. In the present paper, we report on a subset of cells (CD45+/desmin+) with myogenic potential being present at very low frequencies in human adult BM. By a simple culture method, we were able to obtain in vitro multinucleated myotubes in up to 20% of the cultures. Myotubes were generated using both BM flushed from rib fragments obtained during thoracotomy and BM derived from iliac crest aspirates. Cells of the different adherent and non-adherent fractions expressed numerous muscle specific markers by immunocytochemistry, real-time RT-PCR, flow cytometry, and Western blot analyses. Moreover, direct injection of whole BM into the right tibialis anterior muscle of immunodeficient mice (NOD/RAG) that had previously been treated with cardiotoxin to induce muscle degeneration, showed a variable but significant level of human cell engraftment (from 0.06 to 0.26% Dys+/FISH+ fibers). These data suggest that cells with skeletal muscle differentiation potential are present in adult human BM can differentiate in vitro and give rise to myogenic cells in vivo in immunodeficient mice after muscle damage. Further improvements might allow new approaches to cell-mediated therapies for muscular diseases.     

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. Download video file.^{(65M, mp4)}     

Insulin cells of pancreas extend neurites but do not arise from the neuroectoderm.

It is generally believed that during mammalian embryogenesis neurons arise only from the ectodermal germ layer, while the other two germ layers, mesoderm and endoderm, give rise to connective tissue and gut, respectively. Pancreatic islet cells, however, may be an exception to this classical cell lineage derivation. These cells, of endodermal origin, can express several neuronal antigens in addition to the peptide hormones which regulate carbohydrate metabolism. This study sought to determine whether islet cells of adult mice, in addition to displaying biochemical homology to neurons, are also capable of extending neurites, the cytoplasmic elongations that are recognized as a hallmark of the neuronal phenotype. It was found that dissociated pancreatic islet cells can extend neurite-like processes when maintained in vitro and that these processes contain neurofilament, the intermediate filament protein specific to neurons. Islet cells maintained in vitro as explants, however, did not form neurites thereby indicating that normal histotypical contacts inhibit process formation. This observation may account for the absence of process elaboration by intact islets in vivo. These results demonstrate that cells derived from the endoderm share the ability to display a characteristic neuronal phenotype with neuroectodermal cells and, furthermore, that the expression of these traits is regulated by epigenetic cues.     

Chemically-induced cancers do not originate from bone marrow-derived cells

Background

The identification and characterization of cancer stem cells (CSCs) is imperative to understanding the mechanism of cancer pathogenesis. Growing evidence suggests that CSCs play critical roles in the development and progression of cancer. However, controversy exists as to whether CSCs arise from bone marrow-derived cells (BMDCs).

Methodology and Principal Findings

In the present study, n-nitrosodiethylamine (DEN) was used to induce tumor formation in female mice that received bone marrow from male mice. Tumor formation was induced in 20/26 mice, including 12 liver tumors, 6 lung tumors, 1 bladder tumor and 1 nasopharyngeal tumor. Through comparison of fluorescence in situ hybridization (FISH) results in corresponding areas from serial tumor sections stained with H&E, we determined that BMDCs were recruited to both tumor tissue and normal surrounding tissue at a very low frequency (0.2–1% in tumors and 0–0.3% in normal tissues). However, approximately 3–70% of cells in the tissues surrounding the tumor were BMDCs, and the percentage of BMDCs was highly associated with the inflammatory status of the tissue. In the present study, no evidence was found to support the existence of fusion cells formed form BMDCs and tissue-specific stem cells.

Conclusions

In summary, our data suggest that although BMDCs may contribute to tumor progression, they are unlike to contribute to tumor initiation.     

Development of T cells in vitro from precursors in mouse bone marrow

Bone marrow cells from 6- to 8-week-old athymic nude mice were depleted of nylon-wool adherent cells and cultured in vitro at low cell numbers (300 cells/well) in medium supplemented with a supernatant from a thymoma cell line. About 1% of cultured cells grew. Pooled cultures contained cells expressing CD3 (52%), CD4 (37%), CD8 (11%), Thy 1.2 (72%), MAC-1 (43%) and J11d (86%) but no cells expressing sIg. They also contained cells expressing mRNA for the alpha, beta, gamma, and delta chains of the T cell receptor as assessed with C region probes using a sensitive dot blot assay. These cells appear to develop from progenitors which are CD3-. When pooled Day 10 cultures were depleted of nylon-wool adherent cells, the remaining cells were nearly all J11d+, Thy 1.2+, MAC-1-, CD3+, and either CD4+CD8+; CD4+CD8-; CD4-CD8+, or CD4-CD8-; i.e., their surface marker patterns were reminiscent of those of thymocytes. We conclude that our culture system is enabling bone marrow precursors to commence differentiation down the T cell lineage in the absence of a thymic environment.     

Hepatotoxic doses of dioxin do not damage mouse bone marrow chromosomes

We report on a study of the cytogenetic and hepatotoxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Mice of the C57B1/6J (with high-affinity TCDD receptor) or DBA/2J (with low-affinity TCDD receptor) strains were given single intraperitoneal injections of 50, 100 or 150 micrograms of TCDD/kg body weight. At various times (8-48 h) after injection, we examined bone marrow cells for cytogenetic effects by performing structural aberration, sister-chromatid exchange, and micronucleus tests. 1 month after exposure, liver sections were studied for hepatotoxic effects. We found no evidence of chromosome damage by TCDD given in doses that cause liver damage in both strains of mice.     

In vitro generation of natural killer cells from SJL/J bone marrow precursors

The development of natural killer (NK) cells from undifferentiated bone marrow (BM) precursors of low-NK-reactive SJL/J mice was studied. Results indicate that BM cells of untreated mice are not able to generate NK effector cells in cultures supplemented with recombinant interleukin-2 (IL-2). On the other hand in the presence of IL-2, NK cells are generated in cultures of BM from mice pretreated with 5-fluorouracil (5-FU, 150 mg/kg iv 4 days before harvesting), a treatment which has been shown to eliminate more differentiated but spare less differentiated BM precursors. The 5-FU resistant BM progenitor is asialoGM1-, Thy.1+, Lyt.1- and Lyt.2-. The cells generated by culturing with IL-2 are asialoGM1+, Thy.1+, Lyt.5+, Lyt.1-, Lyt.2- and lyse only NK-susceptible targets. Generation of NK cells is blocked by addition of anti-IL-2 receptor (IL-2/r) antibodies. These studies demonstrate that it is possible to generate NK effectors from SJL/J BM cells by in vitro culturing with IL-2.     

Differentiation from precursors in normal bone marrow of spontaneously cytotoxic cells showing anti-self-MHC specificity

Colonies containing spontaneously cytotoxic effector cells with specificity for target cells carrying self-MHC can be grown from normal mouse bone marrow (BM). BM was first depleted of nylon wool-adherent cells and was then cultured at low cell number (1 to 300 cells/culture) in multiple replicate microcultures in liquid culture medium containing supernatant from EL4 thymoma cells stimulated with PMA. Frequency of colony growth followed one-hit limiting dilution kinetics. Colonies contained lymphoid, myeloid, or both kinds of cells. About 5% of colonies contained self-specific cytotoxic effector cells. Analysis using the X chromosome-linked isoenzyme PGK-1 confirmed that colonies containing autoreactivity could be clonal. A factor other than IL 2, IL 3, or PMA appears to be required for the growth of autoreactive colonies. Similar colonies, both with and without autoreactive effector cells, could also be grown from the BM of athymic nude mice with frequencies and cytotoxic activities directly comparable to those found for normal BM. C.B-17 scid mice lack both B and T cells, apparently due to a block in the development of lymphoid stem cells. Colonies could be grown with comparable frequency from their BM, but these colonies lacked both lymphoid cells and spontaneous cytotoxic activity. Evidence is presented against the self-reactive effector cells being NK cells, macrophages, or mature T cells. It is speculated that they represent an early stage of the T cell differentiation pathway.     

Isolation and enrichment of skeletal muscle progenitor cells from mouse bone marrow

There is great interest in the therapeutic potential of non-hematopoietic stem cells obtained from bone marrow called mesenchymal stem cells (MSCs). Rare myogenic progenitor cells in MSC cultures have been shown to convert into skeletal muscle cells in vitro and also in vivo after transplantation of bone marrow into mice. To be clinically useful, however, isolation and expansion of myogenic progenitor cells is important to improve the efficacy of cell transplantation in generating normal skeletal muscle cells. We introduced into MSCs obtained from mouse bone marrow, a plasmid vector in which an antibiotic (Zeocin) resistance gene is driven by MyoD and Myf5 enhancer elements, which are selectively active in skeletal muscle progenitor cells. Myogenic precursor cells were then isolated by antibiotic selection, expanded in culture, and shown to differentiate appropriately into multinucleate myotubes in vitro. Our results show that using a genetic selection strategy, an enriched population of myogenic progenitor cells, which will be useful for cell transplantation therapies, can be isolated from MSCs.     

Mouse mesenchymal stem cells from bone marrow differentiate into smooth muscle cells by induction of plaque-derived smooth muscle cells

AimThe present study aimed to elucidate the mechanism by which bone marrow mesenchymal stem cells (BMSCs) differentiate into smooth muscle cells (SMCs) in atherosclerosis.Main methodsWe isolated mouse BMSCs and incubated them in conditioned medium from plaque-derived SMCs (SMC-CM) and analyzed growth factors from media. BMSCs were treated with different media and harvested at continuous time points for investigating the ability to differentiate toward SMCs. Next, BMSCs of green fluorescence protein (GFP) mice were transplanted into apolipoprotein E^?/? (apoE^?/?) mice fed on western type diet for 12 weeks. In vivo efficacy of BMSCs was investigated.Key findingsAfter being cultured using SMC-CM, hepatocyte growth factor (HGF) was abundantly secreted into the medium by BMSCs with time. BMSCs had increased expression of HGF receptor c-met and SMC-specific markers while they also displayed SMC characteristic ‘hill and valley-like’ appearance with an SMC ultra-structure including actin filaments and dense bodies. In vivo-grafted BMSCs aggravated atherosclerotic lesions and inflammation but ameliorated fibrosis in aorta while they displayed higher expression levels of c-met and early SMC-specific markers but not late-stage markers in aorta. They also demonstrated greater secretion of HGF in the aorta of apoE^?/? mice. Furthermore, when BMSCs were treated with HGF blocking antibody, they lost the ability to differentiate to SMCs.SignificanceHGF from local SMCs plays an important role for the differentiation of homing BMSCs.     

Skeletal muscle stem cells

Satellite cells are myogenic stem cells responsible for the post-natal growth, repair and maintenance of skeletal muscle. This review focuses on the basic biology of the satellite cell with emphasis on its role in muscle repair and parallels between embryonic myogenesis and muscle regeneration. Recent advances have altered the long-standing view of the satellite cell as a committed myogenic stem cell derived directly from the fetal myoblast. The experimental basis for this evolving perspective will be highlighted as will the relationship between the satellite cell and other newly discovered muscle stem cell populations. Finally, advances and prospects for cell-based therapies for muscular dystrophies will be addressed.     

Skeletal muscle stem cells

In this review we shall discuss recent publications on the heterogeneity of muscle stem cells, signaling pathways that affect their behaviour and regulatory mechanisms that underlie their myogenic fate, with reference to insights provided by work on skeletal muscle formation in the embryo as well as the adult, with the mouse as a model of reference.     

Interleukin-1 augments the interleukin-2-dependent generation of natural killer cells from bone marrow precursors

We have studied the possible role of interleukin-1 (IL-1) on the interleukin-2 (IL-2) dependent development of mouse natural killer (NK) cells from primitive bone marrow (BM) precursors. Results indicate that both IL-1 alpha and IL-1 beta (1-10 U/ml) are able to stimulate the generation of NK cells from 5-fluorouracil (5-FU) resistant BM progenitors. These precursor cells are asialoGM1-, Thy-1+, Lyt-1- and Lyt-2-. Effector cells generated by culturing with IL-2 (40 U/ml) and IL-1 (5 U/ml) are Thy-1+, asialoGM1+, Lyt-5+, Lyt-1-, Lyt-2- and lyse only NK-susceptible targets. Generation of NK cells is blocked by addition of anti-IL-2/r. These data indicate that IL-1 may play a role in the generation of mature NK cells from undifferentiated BM precursors.     

Cancer cells arise from bacteria

Background

The origin of cancer cells is the most fundamental yet unresolved problem in cancer research. Cancer cells are thought to be transformed from the normal cells. However, recent studies reveal that the primary cancer cells (PCCs) for cancer initiation and secondary cancer cells (SCCs) for cancer progression are formed in but not transformed from the senescent normal and cancer cells, respectively. Nevertheless, the cellular mechanism of PCCs/SCCs formation is unclear. Here, based on the evidences (1) the nascent PCCs/SCCs are small and organelle-less resembling bacteria; (2) our finding that the cyanobacterium TDX16 acquires its algal host DNA and turns into a new alga TDX16-DE by de novo organelle biogenesis, and (3) PCCs/SCCs formations share striking similarities with TDX16 development and transition, we propose the bacterial origin of cancer cells (BOCC).

Presentation of the hypothesis

The intracellular bacteria take up the DNAs of the senescent/necrotic normal cells/PCCs and then develop into PCCs/SCCs by hybridizing the acquired DNAs with their own ones and expressing the hybrid genomes.

Testing the hypothesis

BOCC can be confirmed by testing BOCC-based predictions, such as normal cells with no intracellular bacteria can not “transform” into cancer cells in any conditions.

Implications of the hypothesis

According to BOCC theory: (1) cancer cells are new single-celled eukaryotes, which is why the hallmarks of cancer are mostly the characteristics of protists; (2) genetic changes and instabilities are not the causes, but the consequences of cancer cell formation; and (3) the common role of carcinogens, infectious agents and relating factors is inducing or related to cellular senescence rather than mutations. Therefore, BOCC theory provides new rationale and direction for cancer research, prevention and therapy.

     

Exogenous bone marrow cells do not rescue non-irradiated mice from acute renal tubular damage caused by HgCl2, despite establishment of chimaerism and cell proliferation in bone marrow and spleen

Abstract.   Objective : Various studies have shown that bone marrow stem cells can rescue mice from acute renal tubular damage under a conditioning advantage (irradiation or cisplatin treatment) favouring donor cell engraftment and regeneration; however, it is not known whether bone marrow cells (BMCs) can contribute to repair of acute tubular damage in the absence of a selection pressure for the donor cells. The aim of this study was to examine this possibility. Materials and methods : Ten-week-old female mice were assigned into control non-irradiated animals having only vehicle treatment, HgCl₂-treated non-irradiated mice, HgCl₂-treated non-irradiated mice infused with male BMCs 1 day after HgCl₂, and vehicle-treated mice with male BMCs. Tritiated thymidine was given 1 h before animal killing. Results : Donor BMCs could not alleviate non-irradiated mice from acute tubular damage caused by HgCl₂, deduced by no reduction in serum urea nitrogen combined with negligible cell engraftment. However, donor BMCs could home to the bone marrow and spleen and display proliferative activity. This is the first report to show that despite no preparative myeloablation of recipients, engrafted donor BMCs can synthesize DNA in the bone marrow and spleen. Conclusions : Exogenous BMCs do not rescue non-irradiated mice from acute renal tubular damage caused by HgCl₂, despite establishment of chimerism and cell proliferation in bone marrow and spleen.     

Biological characteristics of foam cell formation in smooth muscle cells derived from bone marrow stem cells

Bone marrow mesenchymal stem cells (BMSC) can differentiate into diverse cell types, including adipogenic, osteogenic, chondrogenic and myogenic lineages. There are lots of BMSC accumulated in atherosclerosis vessels and differentiate into VSMC. However, it is unclear whether VSMC originated from BMSC (BMSC-SMC) could remodel the vessel in new tunica intima or promote the pathogenesis of atherosclerosis. In this study, BMSC were differentiated into VSMC in response to the transforming growth factor β (TGF-β) and shown to express a number of VSMC markers, such as α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain1 (SM-MHC1). BMSC-SMC became foam cells after treatment with 80 mg/L ox-LDL for 72 hours. Ox-LDL could upregulate scavenger receptor class A (SR-A) but downregulate the ATP-binding cassette transporter A1 (ABCA1) and caveolin-1 protein expression, suggesting that modulating relative protein activity contributes to smooth muscle foam cell formation in BMSC-SMC. Furthermore, we found that BMSC-SMC have some biological characteristics that are similar to VSMC, such as the ability of proliferation and secretion of extracellular matrix, but, at the same time, retain some biological characteristics of BMSC, such as a high level of migration. These results suggest that BMSC-SMC could be induced to foam cells and be involved in the development of atherosclerosis.     

Establishment of myoid cells from bone marrow

A peculiar adherent cell clone (R613BM) was established under muscle tissue free conditions from bone marrow of a Wistar rat. The cloned cell line was able to form myofibrils and expressed nicotinic acetylcholine receptors specific for skeletal muscles. The muscle specific characteristics have been maintained consistently for more than five years. These results suggest that bone marrow contains a precursor cell which has the potency to differentiate into muscle cells.     

Pre-B cells in mouse bone marrow: in vitro maturation of peanut agglutinin binding B lymphocyte precursors separated from bone marrow by fluorescence-activated cell sorting

Peanut agglutinin (PNA) binding by mouse bone marrow cells and fractionation by the fluorescence-activated cell sorter have previously been shown to separate high concentrations of pre-B cells, as identified by cytoplasmic mu-chains (c mu). PNA+ and PNA- marrow cell fractions have now been assayed for the presence of functional pre-B cells able to generate mature B cells in culture, as defined by three criteria, the appearance of cell surface mu-chains (s mu), immunoglobulin secretion in response to bacterial lipopolysaccharides, and B cell colony formation. Small PNA+ cell fractions contained pre-B cells that developed into mature B lymphocytes in 1/2 to 1 day but did not sustain B cell production. Large PNA+ cells included pre-B cells that gave rise to mature B lymphocytes after an interval of 1 1/2 to 3 days and were able to sustain B cell genesis in vitro for at least 3 to 5 days thereafter. PNA- cell fractions contained mature B cells but lacked pre-B cell activity. The results demonstrate that PNA binding allows the separation of functional subsets of pre-B cells from bone marrow and that the three in vitro assays used in this study are closely comparable with one another as functional pre-B cell criteria. The findings suggest correlations between functional assays, c mu expression, PNA receptors, and cell size in characterizing stages of pre-B cell development.     

Interleukin 3 is a major negative regulator of the generation of natural killer cells from bone marrow precursors

We have established a bone marrow culture system in which mature natural killer (NK) cells can be generated from inactive precursors by interleukin 2. Recombinant interleukin 3 (IL 3) almost completely blocked the induction of NK cells in this culture system as judged by cytotoxic activity, as well as appearance of cells with NK phenotype. The dose-response curve for inhibition of the generation of NK activity with IL 3 parallelled the growth promoting activity on the strictly IL 3-dependent cell line L/B. The effect of IL 3 was selective for the precursor stage of the NK cell, because mature NK cells were not affected by culture with IL 3 for the same period of time. Moreover, the effect of IL 3 was confined to the first 24 hr of culture, indicating an effect on an early stage of NK cell differentiation. IL 3 did not increase the small normally occurring NK-sensitive population in bone marrow, and did not affect the activity of a variant cytotoxic cell with specificity for adherent target cells, the natural cytotoxic cell. Concomitantly with downregulation of NK cell generation, IL 3 induced strong proliferation in the bone marrow cultures and an increase in the percentage of cells expressing the T cell marker Thy-1. A model for regulation of NK cells based on competition of growth factors for target cells with a common progenitor is discussed.