Cancer Stem Cells

Yes, it opens up a lot of avenues to examine various chemos with various apoptotic regulators...!!

I am pleased to send you this link that probably help you with your search.

Virtually all differentiation assays have as part of their protocol, attachment of the stem cell population being studied, to a surface. Said surface is usually coated with a molecular substrate such as collagen 1 or many other types of extracellular matrix molecules. ESCs are a different subject. But regarding adult stem cells derived from solid organs with suspension culture isolation techniques, it is the lack of attachment that selects for the stem cell and for early progenitors, and attachment of said stem cell, especially as sphere, or as a single cell suspension, will immediately differentiate when the following are present, standard serum concentration, attachment to a substrate, and growth factors known to induce differentiation. Neural stem cells and Mesenchymal stem cells are the most commonly studied examples of differentiation assays. Many publications.

The cited textbook appears to be free online - and quite complete on many aspects of human and animal tumors, including some of the "biochemical" aspects. But I think one should not overemphasize any such biochemical differences, since the appear (to me) just as an epiphenomenon, which may not lead to useful therapies (the book describes the differences and doesn't imply that those could be used for therapies - perhaps the originator of the question here could think of using such a difference in treating cancer, which probably has been tried decades ago...). I didn't look too carefully into the book (it could have escaped to my attention), but I didn't see any discussion of "cancer stem cells" or its theory or concept, which may be helpful to understand, that "metastasis" not necessarily should be seen as "late event", but could be just the potential of the original cancer stem cell... And: biochemical differences of subpopulations or the great majority of cells within a tumor could not exist when it comes to comparing the cancer stem cell.

To summarise this simply, if the selective pressure on the tumour 'system' allows the survival of differentiated cells as in normal tissue and many early/low grade cancers, then stem cells/cancer stem cells will be rare. If you apply some sort of selective pressure to target/eradicate differentiated cells, or selectively promote survival of cells that undergo self renewal, then the stem cell-like cells will become more abundant. This is best illustrated in some Testicular Germ Cell Tumour cell lines, where CSC-like cells may predominate, and a similar argument may be applied to any cell line in the artificial environment of cell culture.

@Erik Haefner, sounds reasonable. And I think this question is very intriguing. I wonder if the adherent cells stick on the dish depending on gravity. Or the morphology of cells will keep the same?

Lung cancer or breast cancer

The simplest way to understand this is to consider that in this experiment, likely as in bone marrow transplants, the radiation is simply meant to knock out the stem cell compartment and create a graft bed the CB lin- cells could fill. If you do not create a graft bed, you are very unlikely to get a resultant engraftment of the introduced cell type.

Thanks Wanda (Miles), glad to be of help, and thanks also to Guruprasad Gopalakrishnan and Joachim Pimiskern for your comments. You are correct that kynurenine and the kynurenine pathway (KP) exhibit "Janus-like" or dual modes of activity depending on the context, with a positive role in: infection control, pregnancy, transplantation acceptance, autoimmunity (especially in non-obese diabetes and systemic lupus erythematosus (SLE)), and allergy control among others, and a decidedly negative role in: malignancies (especially endometrial, colorectal, lung, esophageal squamaous cell and vulvar squamous cell carcinoma’s , and in virtually all brain tumors, among other cancers). In addition, kynurenine pathway dysregulation plays a significant role in non-malignancies such as Alzheimer’s disease, schizophrenia, depression, Huntington and Parkinson disease, and age-related cataracts. SOME CAUTION Trials therefore must exert caution to avoid potential secondary / reflexive adverse effects of kynurenine pathway (KP), and IDO / TDO, inhibition, part of which can be accomplished by deployment of highly selective rather than broad-spectrum inhibitors. To date however it should be noted clinical trials have not yet uncovered significant adverse events although we should continue to avoid what I would designate at risk populations such as SLE patients, pregnant women, or any patient with active or at-risk infection, until mature data provide highly targeted selective inhibition strategies. KYNURENINE PATHWAY (KP) INHIBITION IN CLINICAL TRIALS Now, as to therapeutic goals, let me sketch these out within primarily the oncology context, although progress is emerging in non-malignancy disorders too: In dealing with kynurenine pathway (KP) research, I divide cancers into two large groups depending on whether they are driven by the IDO versus the TDO component of the kynurenine pathway (right now, the third component I noted above, namely ACMSD, is no as yet being pursued fully). Remember that IDO and TDO respond to different activating stimuli, with IDO but not TDO the expression of IDO - but not TDO - being upregulated following pro-inflammatory cytokine challenge. So the groups are either: (1) the class of IDO-inhibitor targeting cancers such as Non Small-cell Lung Cancer (NSCLC) and Chronic Lymphocytic Leukemia (CLL), or (2) the class of TDO-inhibitor targeting cancers such as malignant gloimas. But there are I should note cancers that are both IDO- and TDO-driven such as Head and Neck Cancers (HNC) in which we would speculate that concurrently inhibition of both IDO and TDO might prove of benefit. There is more research activity in the IDO-inhibitor targeting class: it's known that IDO directly suppresses the effector T cell proliferation and differentiation, while also markedly enhances the suppressor activity of regulatory T cells (Tregs), suggesting that IDO-inhibitor drugs can both: (1) enhance anti-tumor immune responses making them attractive for anti-tumor vaccines and other immunotherapy, and (2) they are also synergistic with many chemotherapeutic drugs, so that chemotherapeutic synergy is another exploitable strategy. An example of the first IDO inhibitor strategy, namely of enhancing anti-tumor immune response, is the IDOvaccine Trial (NCT01219348) recruiting in Denmark. An example of the second IDO inhibitor strategy is the IDO MBC Trial (NCT01792050) which is exploring the potential synergy of the IDO inhibitor Indoximod when used in combination with docetaxel (Taxotere) chemotherapy for the treatment of metastatic breast cancer (MBC); the trial is in-progress at the H. Lee Moffitt Cancer Center and Research Institute in Florida. One intriguing trial is the IDO/Survivin Peptide Vaccine Trial (NCT01543464) which combines both approaches - IDO anti-tumor immune response enhancement and chemotherapeutic synergy - by combining the IDO/Survivin Peptide Vaccine with temozolomide (TMZ) in treating metastatic malignant melanoma, now recruiting in Denmark. And the INCB024360 EOC Trial (NCT01685255) takes the direct antitumor approach, comparing the IDO inhibitor INCB024360 (from Incyte Corporation) to tamoxifen in epithelial ovarian cancer (EOC), and is now recruiting in multiple U.S. locations and in Canada. In addition, 1-D-MT Inhibitor Trial (NCT00567931) uses 1-methyl-D-tryptophan (1-D-MT, also called D-1MT) which is an IDO inhibitor, in the treatment of patients with non-resectable metastatic or refractory solid tumors; now recruiting in Florida and Virginia. OTHER APPLICATIONS, AND THE EMERGENCE OF NATURAL KP INHIBITORS As to TDO (tryptophan 2,3-dioxygenase) inhibitors, the Belgium team of Luc Pilotte and colleagues (Proc Natl Acad Sci U S A. 2012) have completed a proof-of-concept preclinical study of the reversal of tumoral immune resistance via a TDO inhibitor. My own research in integrative oncology find the green tea component EGCG behaves as a natural IDO inhibitor and this has been confirmed by Kengo Ogawa and colleagues in Japan [Oncol Letters 2012] where EGCG may exert antitumor effects on colorectal cancer (CRC) in part by inhibition of IDO expression and function via the suppression of STAT1 activation, adding more evidence that EGCG may serve as a potential natural antitumor immunotherapy agent for the chemoprevention and treatment of CRC, findings that are motivated by the fact that high IDO expression levels of IDO are associated with disease progression and poor overall survival in CRC patients, as demonstrated by Liesbeth Ferdinande and colleagues in Belgium (Br J Cancer 2012). In addition, Sophie Chen and colleagues in the UK [Biochem Biophys Res Commun. 2012] have demonstrated that flavone phytochemicals apigenin, baicalein, chrysin, and wogonin can also provide anti-cancer and neuro-protective activity via IDO inhibition, thus also serving as natural IDO inhibitors. And Helen Ball's team at the University of Sydney [Bakmiwewa et al., Bioorg Med Chem Lett. 2012] has recently that certain proton pump inhibitors (PPIs) can serve as selective IDO inhibitors, as can certain COX-2 inhibitors. What this suggests is that certain selective and demonstrably safe natural KP/IDO/TDO inhibitors can be cautiously exploited until clinical trials complete and commercially viable agents enter the marketplace (highly standardized EGCG for example, or a combination of EGCG + celecoxib serving as a dual-IDO selective inhibitor complex that may prove of benefit in advanced cancers or otherwise unsalvageable malignant stages where research has already confirmed the absence of appreciable harm). And within the next 18 months we will experience something of an explosion of research and clinical trials further refining our understanding of the complexities and nuances of kynurenine pathway in cancers and in non-malignant disorders. Constantine Kaniklidis Director of Medical Research, No Surrender Breast Cancer Foundation (NSBCF) European Association for Cancer Research (EACR)

Thank you all...

Search pubmed...YAP OVEREXPRESSION IN MCF10cells

Blebbishields are the step before cancer stem cell spheres but after the blebbing stage of apoptosis(it is not yet a generalized concept for all cancer stem cells because some cancer stem cells may not form spheres). The sphere forming cancer stem cells might have a blebbishield stage followed by apoptosis, but before sphere formation. If you have cells that are capable of sphere formation in 2D (not in soft-agar but in regular tissue culture) you may do a time-lapse microscopy to see the initiation of spheres by apoptotic cells through blebbishields. Based on our results, yes, the cancer stem cells are capable of forming spheres after apoptosis through blebbishield stage hence the CSC poulation will survive after apoptosis (growth is a different issue here...)

Yes, of course known as MET in the target organ where metastasis develops from micro-metastasis

cancer stem cells is, stem cell that acquire cancer character or cancer cell which acquire a stem cell properties, so any dividing cell may have this identification and character

Both Media can be apply but upon my experience it a bit different. For example, in colorectal cancer serum free medium is better, in the case of Brain tumor, medium containing serum is preferable however is not obligatory. Moreover, establishing a protocol is different from a lab to another lab. So I suggest that you do not rely completely on the protocols mentioned in the papers though the principle is the same in all protocols.

I am working with thyroid cancer cell lines and I isolated like-cancer stem cells from them, I used the sphere formation assay and my cells grown in suspension like spheres and I was able to pass the spheres for several generations, to support the self-renewal. You can look this paper published by Setoguchi T et al, 2004 Cell cicle and Gene expression profiling of cancer stem cells in the Du145 prostate cancer cell line by Chen W, Wang GM. 2012 Oncology letters. Paola

I 'm working with thyroid cancer lines and i isolated thyroshere. I'm using to disgregate spheres StemproAccutase (invitrogen) 7 min at 37C and it works very well, I help disgregation with pipet and finally I have good single cells suspension with 90-95% viability.

I didnt get what are you looking for? However I will tell u some assays that might help you if thats what you were looking for. use cd44+/CD24(low) markers in FACS , U can also use ALDH markers in FACS , SP markers or u can purify by mammosphere formation culture. Or u can use combination of these. To identify if what you have is CSCs you need to inject them into SCID/NOD mice and see if it forms tumors. You also need to do a differentiation assay to confirm that your cells have both properties of stem cells. Just enter these terms into pubmed with term 'breast cancer stem cells and you will find many papers'. You may like to go to RGCB website and see work of Dr. Priya Srinivas , she has work going on in her lab on BCSCs.

If the antigen is cell surface like CD133 then staining is best done without fixation. Cells can be fixed later on if you need to store the cells or if other intracellular antigens are to be stained as well.

I met the same question, when I withdraw the mitogen, and induce the neurosphere into neurons with RA, but only 10% cells can transfer into NF positive cells. So I have to do the primary culture from E17 embryos.

To Rob Streeper: thank you for referencing a nice paper here!