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  #1  
Old 06-01-2012, 04:08 PM
gdpawel gdpawel is offline
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Default Cell Lines vs Fresh Cells

The pathologist establishes a cell-line (immortalizes it) with your tumor cells. A cell-line is a product of immortal cells that are used for biological research. Cell-lines can perpetuate division indefinitely. Regular cells can only divide approximately 50 times.

Cell-lines are useful for experimentation in labs as they are always available to researchers as a product and do not require harvesting (acquiring of tissue from a host) every time cells are needed in the lab.

Problem is, cell-lines don't recapitulate drug response patterns which exist in the body. For drug selection, it is better to directly remove tumor microclusters straight from the body and immediately test them, before they change.

There is the issue about cell-lines vs fresh cells. Cell-lines have always played, and continue to play, an important role in drug screening and drug development.

The problem is that cell-lines do not predict for disease or patient specific drug effects. If you can kill ovarian cancer cell-lines with a given drug, it doesn't tell you anything about how the drug will work in real world, clinical ovarian cancer (real-world conditions). But you can learn certain things about general drug biology through the study of cell-lines.

As a general rule, studies from established cell-lines (tumor cells that are cultured and maniplated so that they continue to divide) have proved worthless as models to predict the activity of drugs in cancer. They are more misleading than helpful. An established cell-line is not reflective of the behavior of the fresh tumor samples (live samples derived from tumors) in primary culture, much less in the patient.

Established cell-lines have been a huge disappointment over the decades, with respect to their ability to correctly model the disease-specific activity of new drugs. What works in cell-lines do not often translate into human beings. You get different results when you test passaged cells compared to primary, fresh tumor.

Research on cell-lines is cheap compared to clinical trials on humans. One gets more accurate information when using intact RNA isolated from "fresh" tissue than from using degraded RNA, which is present in paraffin-fixed tissue.

My thoughts would be, do you want to utilize your tissue specimen for "drug selection" against "your" individual cancer cells or for mutation identification, to see if you are "potentially" susceptible to a certain mechanism of attack.

In the later, there are many laboratories that commercially offer analyses. These gene array methods can be automated and conducted comparatively cheaply.

In the former, some labs have not pursued genomics because cancer is more complex than its gene signature. Contained within the genes of each human is the information to create every protein, every enzyme, every lipid, every carbohydrate and all the organs and systems dependant upon their function.

What is not known is how all of those 25,000+ genes are regulated to produce the unique features that constitute us as human beings.

By studying human cellular behavior within the context of vascular, stromal and inflammatory elements, the functional profiling platform provides the closest approximation of human biology, short of possibly a clinical trial.

Cell Function Analysis

Batch Processing of tumor biopsies for cell markers

[url]http://cancerfocus.org/forum/showthread.php?t=3976
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Last edited by gdpawel : 09-25-2013 at 11:45 AM. Reason: additional info
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  #2  
Old 08-12-2012, 11:23 PM
gdpawel gdpawel is offline
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Default Will the Real Cancer Cell Please Stand Up?

In a recent article in Biotechniques, "Will the Real Cancer Cell Please Stand Up," it seems that cancer cells are not individual entities but networks. A harmonic oscillation develops between tumor, stroma, vasculature and cytokines. In this mix, the cancer cell is but one piece of the puzzle. The value of using cancer cell-lines for drug sensitivity investigation has recently come under fire.

According to research work at Baylor, some of the tumor promotion signals in the form of small interfering RNAs, may arise not from the cancer cells, but instead from the surrounding stroma. How then will even genomic analyses of cancer cells play out in the real world of human tumor biology and clinical response prediction?

Not very well, according to Dr. Robert Nagourney, Medical and Laboratory Director, Rational Therapeutics, Inc., Long Beach, California. The technology is so complex as to be beyond the ken of both patients and physicians alike. Thus, expertise is required and that expertise is provided by those engaged in the field. The utility of drug selection is beyond reproach.

Scientifically interesting technology has been brought to the market. It exists to meet an unmet need. What is lacking, however, is evidence. Not necessarily evidence in the rarefied Cochrane sense of idealized survival curves, nor even Level II evidence, but any evidence at all. Dr. Nagourney fears a lack of competent due diligence.

The best medicine comes from treating the right patient with the right therapy at the right time. And while that seemingly simple prescription rings true, the path to the three Rs of medical treatment is anything but simple and straightforward—especially in treating the multiplicity of cancers. As scientists struggle to decode and decipher the mountain of cancer cell genomic data currently—or soon to be—available, knowing which data set correctly describes a tumor’s inner workings, is a huge challenge. Such is the case with research on established cancer cell-lines as opposed to cells derived directly from a patient’s tumor.

Cancer cell-lines date back to the 1950s, when the first line was established from a woman with cervical cancer named Henrietta Lacks. In the decades since, approximately 1000 cancer cell-lines have become essential tools for cancer cell biology and to test drugs. Indeed, almost every anti-cancer drug now in use gained early traction through testing on cancer cell-lines. But now some researchers are calling for new models that can assay primary cancer cells directly from patients to sketch a more realistic molecular portrait of primary tumors.

[url]http://www.biotechniques.com/news/Will-the-Real-Cancer-Cell-Please-Stand-Up/biotechniques-329467.html
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  #3  
Old 10-10-2012, 12:55 AM
gdpawel gdpawel is offline
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Default "Foundation Medicine" and the Big Barrier to Cancer Genomic Sequencing

According to Dr. Eric Topol, Director, Scripps Translational Science Institute, recent studies have highlighted the potential value of whole genome or exome sequencing to precisely guide therapy for patients with cancer. However, almost all samples today go into formalin-fixed, paraffin embedded (FFPE) blocks, which alters the DNA and makes sequencing quite compromised and difficult.

He told Medscape Connect that the company, Foundation Medicine, which works with formalin-fixed paraffin-embedded (FFPE) blocks, and gets about 250-300 genes, the exons or coding elements in those genes, and reads out any potential links to drugs. But the rate-liimiting step appears to be getting something beyond these paraffin blocks. This is, we could do better if we could use either fresh formalin-fixed or frozen tissue samples from a biopsy or surgical specimen.

Topol says the problem is that pathologists are seemingly quite ritualistic. They don't want to go to frozen samples, which would be the best for whole genome sequencing. We're just at the cusp of getting started with this type of limited, not even full exome sequencing, just a few hundred genes, but that isn't enough.

Rencent papers in multiple journals in Nature, Science, Nature Genetics and Cell have shown that with hundreds of tumor samples fully sequenced, no two cancers are the same and a lot of the action is not in the coding elements of the genes per se. Whole genome sequencing certainly appears to be an ideal path to pursue, but we can't do it with the fixed problems that we have with the way samples are handled today.

Topol thinks that maybe we could get fresh formalin-fixed samples, as those appear to be well-suited to whole genome sequencing, although this is still a somewhat bootstrapped situation, like the paraffin-embedded samples. It appears that the long those samples are embedded, the harder it is to get a reasonable sequence beyond very targeted regions.

There are no two cancer tissues that are the same on a molecular basis. There's quite a bit of heterogeneity within the samples and multiple sequencing could account for that. And we also want to anticipate recurrence, match up the right driver mutations and the backseat passenger mutations, whether or not there's needed immunotherapy; all those things that could be done if we could get the right information from the get go.

So Dr. Topol asks this: How are we going to move to a world with a clinic of the future where patients with cancer can get whole genome sequencing rapidly? That is, to have annotation and interpretation of the genome with a day, and have your therapy precisely guided genomically?

I only have to look at the above postings on this thread.

Gene Sequencing for Drug Selection?

Researchers have realized that cancer biology is driven by signaling pathways. Cells speak to each other and the messages they send are interpreted via intracellular pathways known as signal transduction. Many of these pathways are activated or deactivated by phosphorylations on select cellular proteins.

Sequencing the genome of cancer cells is explicitly based upon the assumption that the pathways - network of genes - of tumor cells can be known in sufficient detail to control cancer. Each cancer cell can be different and the cancer cells that are present change and evolve with time.

Although the theory behind inhibitor targeted therapy is appealing, the reality is more complex. Cancer cells often have many mutations in many different pathways, so even if one route is shut down by a targted treatment, the cancer cell may be able to use other routes.

In other words, cancer cells have "backup systems" that allow them to survive. The result is that the drug does not affect the tumor as expected. The cancer state is typically characterized by a signaling process that is unregulated and in a continuous state of activation.

In chemotherapy selection, molecular profiling examines a single process within the cell or a relatively small number of processes. All a gene mutation study can tell is whether or not the cells are potentially susceptible to a mechanism of attack. The aim is to tell if there is a theoretical predisposition to drug response.

It doesn't tell you the effectiveness of one drug (or combination) or any other drug which may target this in the individual. There are many pathways to altered cellular function. Functional Profiling measures the end result of pathway activation or deactivation to predict whether patients will actually respond (clinical responders).

It measures what happens at the end, rather than the status of the individual pathway, by assessing the activity of a drug (or combinations) upon combined effect of all cellular processes, using combined metabolic and morphologic endpoints, at the cell population level, measuring the interaction of the entire genome.

Translational science: past, present, and future

[url]http://www.biotechniques.com/multimedia/archive/00003/BTN_A_000112749_O_3671a.pdf

Note: Foundation Medicine is not any different than Caris Diagnostics in Phoenix (now Miraca Life Sciences), beyond testing for standard pathology "targets" such as ER, PR, Her2, EGFR mutations, KRAS, BRAF. They aren't worth much for the sorts of chemotherapy which is used in 95% of all cancers and useless with respect to drug combinations. While fresh tissue is very dear and hard to come by, function trumps structure, in terms of potency and robustness of information provided than using archival paraffin blocks.
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Last edited by gdpawel : 05-11-2013 at 02:09 AM. Reason: added url address
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  #4  
Old 01-06-2013, 09:58 PM
gdpawel gdpawel is offline
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Default Cancer cell-lines: fact and fantasy

Cancer cell-lines are used in many biomedical research laboratories. Why, then, are they often described as unrepresentative of the cells from which they were derived?

There are a multitude of definitions for each tissue culture term. This perspective follows the definitions of the terminology committee of the Society for In Vitro Biology (formerly the American Tissue Culture Association).

Primary culture

Produced by growing cells from tissue taken directly from an individual.

Cell-line

A primary culture becomes a cell-line when it is transferred into the next culture vessel. For adherent cultures, the cells are detached using a protease, such as trypsin, and/or a chelating agent, such as EDTA, and subdivided this process is known as passaging. For cells that grow in suspension, the culture is split into new culture vessels. Unless specialized culture conditions are used, within a few passages a relatively uniform population of proliferative cells is selected. This population is probably representative of the cells that divide when the tissue of origin is wounded, and will carry on growing until the end of the natural proliferative lifespan is reached and senescence occurs. As long as the cells proliferate, they show little or no evidence of tissue-specific differentiation. However, given the appropriate signals, they can regenerate a functional tissue.

Immortal cell line

Normal human cells have a limited lifespan in culture and almost never spontaneously immortalize. Consequently cell-lines can only be used over a limited period until they senesce. Most human cancers express telomerase, but either cannot be cultured or undergo senescence. To delay senescence, the lifespan can be extended by transfection with viral genes. The products of the viral genes sequester proteins such as p53 and Rb, allowing the cells to continue dividing for more passages. The cultures still senesce (this period is described as 'crisis'), but if one is patient, in some cultures the occasional cell will acquire the mutation(s) that make it immortal and sometimes tumorigenic. Cell immortalization and carcinogenesis have much in common.

Conditionally immortalized cell-lines

The advantages of immortal cell-lines (a constant supply of almost identical cells) can be achieved, without the disadvantage of transforming the cells into the equivalent of cancer cells, by using conditional immortalization with a temperature-sensitive mutant of the viral gene. For example, one mutant of the SV40 T-antigen is functional at 33 C, but conformationally inactive at 39 C. Cells conditionally immortalized with this construct grow exponentially at the permissive temperature (33 C), but stop dividing and can express tissue-specific features at the non-permissive temperature (39 C). However, there is often a degree of 'leakiness', where dividing cells escape and grow at the non-permissive temperature.

Continuous cancer cell-lines

Generally, it is only highly aggressive cancers that have accumulated the genetic changes necessary for unlimited growth in vitro that spontaneously become continuous cell-lines. Cancer cell-lines tend to be grown in commercial tissue culture medium that contains fetal calf serum, under which the main selection pressure is for proliferative cells.

Nature Reviews Molecular Cell Biology 1, 233-236 (December 2000) doi:10.1038/35043102
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  #5  
Old 05-19-2013, 11:16 AM
gdpawel gdpawel is offline
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Default The Cancer Clinic of the Future?

Eric Topol, M.D.
Director of the Scripps Translational Science Institute
Editor-in-Chief of Medscape Genomic Medicine

When we have an individual presenting for a new diagnosis of cancer, we have to move away from fine-needle aspiration and minimal tissue; we need real tissue to be able to process it properly. Not only do we need the formalin-fixed paraffin-embedded (FFPE) specimen, but we also need another type of FF -- that is, flash-frozen specimens so that we can then whole-genome sequence this tissue.

Now, when that is done at the primary diagnosis and done within hours and analyzed with the appropriate software algorithms, we could get the driver mutations nailed within 24 hours from the diagnosis. This can set up remarkably precise therapy that can be given to the patient on the basis of that individual's tumor. There are no 2 different cancers that are the same anywhere. Just like there are no 2 individuals who have the same DNA, that's the same for a tumor.

One of the issues that we have to confront is that there's a lot of intratumor heterogeneity. We need multiple samples to sequence from the tumor, and if there's already a metastatic lesion, we need a sample of that as well. Multiple sequencing, frozen tissue, genome-driven guided therapy -- right from the get-go -- is what we need. That's not what we have today, but that's where we can go in the future of cancer genomic medicine. It's really an exciting opportunity. It has to be validated.

The cancer drugs that are used today are remarkably expensive, and what's fascinating is to see -- and this is a recurrent theme -- is that a drug being used, for example, for renal carcinoma can also be used for leukemia. There was a classic 3-part article on the front page of the New York Times that exemplified some of the stories along those lines.

It's a story about mutations -- a war on mutations, not a war on cancer -- and this type of cancer clinic in the future can take us there but there's going to have to be a whole different look with respect to the way that we take samples of the tumor. We need much more tissue, and to use frozen tissue so that we don't have to bootstrap the FFPE (that paraffin-embedded specimen) and only get a couple of hundred genes or coding elements, but in fact get a whole genome from the flash-frozen specimen. That's really important, and we have to move in that direction -- get more tissue in order to account for the heterogeneity that we know exists. And we have to do deep sequencing of that frozen tissue in order to get the driver mutations identified, and also be able to anticipate where relapses can occur downstream.

That is precision therapy. This exemplifies the future of cancer genomic medicine, and it will be really interesting to see how that plays out in these cancer clinics of the future.

References

Mukherjee S. The Emperor of All Maladies: A Biography of Cancer. New York: Scribner; 2010. The 2011 Pulitzer Prize Winners: General Nonfiction. [url]http://www.pulitzer.org/works/2011-General-Nonfiction

University of Texas MD Anderson Center. Moon Shots program. [url]http://cancermoonshots.org/

Kolata G. In treatment for leukemia, glimpses of the future. New York Times. July 7, 2012. [url]http://www.nytimes.com/2012/07/08/health/in-gene-sequencing-treatment-for-leukemia-glimpses-of-the-future.html?pagewanted=all&_r=0

Citation: Topol on the Cancer Clinic of the Future. Medscape. Apr 17, 2013.
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  #6  
Old 02-25-2014, 11:20 AM
gdpawel gdpawel is offline
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Default Frozen cells instead of paraffin-fixed cell lines?

If tumor specimens are stored frozen instead of in paraffin wax, can these still be used for functional cytometric profiling?

If you re-warmed these specimens, would they have changed like they would in paraffin cell lines?

When you snap freeze cells, the water inside of them swells, breaking them open upon thawing. So snap frozen tissue is OK for "molecular profiling," but it's not OK for cell culture studies (functional cytometric profiling).

What needs to be done is to cryopreserve (as opposed to merely "freeze") the cells in culture medium containing 10% DMSO at a rate of one degree celsius per minute. This makes the cell membranes pliable and, when the water inside the cells swells, the cell membranes stretch, but don't break.

When cells cyropreserved in this way are thawed carefully, the remain viable and can be suitable for cell culture testing. They cryopreserve "leftover" tumor cells whenever they are able.
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