Molecular Oncology - Science topic

Hi Glauco,

Are you aware of the Xena browser (https://xenabrowser.net/)? There are at least gene expression, mutation and methylation data per patient for the TCGA cohorts. Unfortunately I am not sure about the TE data, since I am not working with that but I think that Xena would be your best bet.

I am NOT a doctor but as compter science professional, little more AI provided detail:

In cases where there is nodular peritoneal thickening, ovarian cancer, and ascites, the source of ascites can be the result of various factors, including the presence of cancer cells in the peritoneal cavity.

CA-125 is a tumor marker that is often elevated in cases of ovarian cancer, although it can also be elevated in other conditions. The presence of high CA-125 levels, along with the imaging findings of nodular peritoneal thickening, suggests the possibility of peritoneal serosal carcinoma, which is a type of cancer that affects the lining of the abdominal cavity (peritoneum). Ovarian cancer can sometimes spread to the peritoneum, leading to peritoneal serosal carcinoma.

Ascites refers to the accumulation of fluid in the abdominal cavity. In the context of ovarian cancer, ascites can occur due to several reasons, including:

1. Peritoneal involvement: Cancer cells can spread to the peritoneum, leading to inflammation and the production of fluid.

2. Impaired lymphatic drainage: The presence of cancer can disrupt the normal flow of lymphatic fluid, leading to its accumulation in the abdomen.

3. Liver involvement: Advanced ovarian cancer can involve the liver, leading to impaired liver function and fluid accumulation.

Surgery, as you mentioned, is often a crucial component of the treatment plan for ovarian cancer. The specific surgical procedures performed can vary depending on the individual case, but they may include removal of the ovaries (oophorectomy), peritoneal biopsy, and collection of various samples for histological examination. These procedures aim to obtain a definitive diagnosis, determine the extent of the disease, and guide further treatment decisions.

Please consult qualified healthcare professionals who can provide personalized advice and guidance based on the specific details of your best friend's mother's case. They will be able to provide the most accurate information and help determine the most appropriate course of action.

Hope it helps

You can use DPPH assay and MTT assay

Hi Doaa, 

Our lab regularly uses DAI when using the DSS model and published on this a few years ago. 

In it, we make reference to how we evaluate DAI that includes bleeding (In this case, bleeding: 0 (no blood), 1 (Hemoccult positive), 2 (Hemoccult positive and visual pellet bleeding), and 4 (gross bleeding)). 

Hope this helps!

-Janice 

You could consider using p40. A diffuse (full layered) expression coupled with malignant features on histology favours a SCC cell from a normal keratinocyte.

I think only the basal cells shows p40 positivity in normal skin.

Dear Dr. Go J Yoshida,

the figures you added seem interesting to me, may I know the article reference please from which these figures are ??please

See a list in the link below. However, there may be additional genes and the criteria to include a gene in the list may not be stringent. Therefore, if a gene is not well-characterized with respect to an oncogene or tumor suppressor, additional studies may be needed before a gene can be included in the list.

Link: http://www.cancerquest.org/oncogene-table.html

Hi Lisa.

OK. In qRT-PCR, increased levels of GOI are indicated by a lower Delta-Ct between the GOI and the housekeeping gene (assuming of course that the treatment doesn't change the expression of the housekeeping gene - do you know this for sure?)

So - let's say that the DCt for IL-2 in T-cells is 10 in the absence of PHA stimulation and 6 when PHA stimulation is applied. Since 6<10, we know immediately that IL-2 mRNA levels are rising as a result of PHA stimulation. There is a difference of 4 Ct units, ~16x increase.

How do we get this from the DCt values?

DDCt = DCt(GOI) - DCt(REF)

           = 6 - 10

           =  -4

So, fold-change must be 2^-DDCt

here are a couple of links to look at:

and

Good Luck!

G

Eerola H, Heikkilä P, Tamminen A, Aittomäki K, Blomqvist C, Nevanlinna H. Relationship of patients’ age to histopathological features of breast tumours in BRCA1 and BRCA2 and mutation-negative breast cancer families. Breast Cancer Research. 2005;7(4):R465-R469. doi:10.1186/bcr1025

May be the attached articles could be of help.

Best regards

Robert

Hi Lairinzuali,

Well, the funny point is that your plant extract makes the enzyme activity you follow versus time to decrease then to increase, then to decrease again at a longer time of incubation.

A possible explanation is that, as suggested by Robert, a first component in your plant extract "quickly" inhibit the enzyme whom activity you look at. But this component does not act for long (why's that ... its stability??? do you heat the incubation mixture?). Then, another component, acting much less "quickly" begins to act at a longer time and, this time, being more stable, decrease largely yhe atcivity of the enzyme you look at.

A possible further explanation is that the second decrease, that appearing after the inetrmediate re-incrase of the enzyme you follow, is due to a component that is produced during your incubation by some enzyme of the tumor cells transforming a compound of your plant extract to a derivative that is toxic to the enzyme activity you look at. This is OF COURSE very interesting, this hypothesis paths the way to inetresting experments and results.

Best regards

Philippe

I do not know at all wether the attached article can be of help.

Best regards

Robert

Dear Gaurav,

You are perfectly right!

"Antiproliferative" and "cytotoxic" are used as synonymas while they are actually not!

When using a colorimetric assay (MTT, SRB, XTT, etx...) you cannot speak about "cytotoxicity" by only about "growth inhibitory effects" because a concentration that reduces by 50% the growth of your cell population of interest with your compounds of interest does not mean that 50% of the cells were killed. The possibility remains that indeed 50% of the cells were killed (cytotoxic effects), but also that 50% of the cells faced growth arrest (during the time of the test; cytostatic effects) or that 50% of the cells detached from the bottom of the flask (anti-adhesive (anti-metastatic?) effects), or a mix of these three biological processes.

You have to use the GI50, the LC50 and the TGI indices as validated by the US NCI (see their publicly available website) to make the difference between a cytotoxic (induced cell death) and a cytostatic (inducing "antiproliferative effects".

If you are using a colorimetric assay, simple observations with phase-contrast microscopy (that you are routinely used when you perform cell cultures) can more or less easily inform you about cytotoxic versus cytostatic effects.

You can also use vital dyes in addition to the colorimetric assay.

Hoping that it can be of help.

Robert

I realise this is an old thread - I'm having trouble getting hold of anti-mouse PD-1 (clone G4 ideally), would any one have access to this (and be willing to ship to Belfast, expenses paid!) or recommend a commercial alternative?

Thanks in advance!

Daudi cell line is a Burkitt lymphoma cell line that overxpresses c-Myc.

 Thanks for the answers. The phenomenon is that it can inhibits the growth of tomor cells in vitro, however, it promotes tumor growth in vivo. I am wondering if there is any good methods to check the differences and mechanisms.

Thank you Charitha. This is an interesting question.

I do not think their altered expression would cause cancer, but contribute to cancer potentiation, according to the evidences in colitis-associated colorectal cancer models that I have found.

It is hard to say whether their mutations were really relevant, but their regulators. However, as fas as they are very important metabolic modulators, it is very interesting to find out ways to detect promptly their alterations in any kind of cancer.

May be i might not have completely understood your question i would like to answer half portion of your question regarding ligand field that can allow reduction of Co (III) to Co(II) Cobalt(III) low spin t2g6 which is inert however high spin Co(III) with t2g4 eg2 configuration is not inert i suppose ligands which can build up high spin complexes with Co(III) should allow reduction of Co(III) to Co(II) moreover Co(III) is a hard metal ion while Co(II) is borderline hence ligands with softer donor site should allow reduction of Co(III) to Co(II).

Hope it half answers the question.  

Hi,

You can inject 4T1 cells from 5x10³ to 1x10⁵ in the up or low fat pad with volume 30ul to 50ul in Balb/c mice.  Then you can have lung metastasis and larger spleen ( 3-4 time weight (g) than normal mouse''s spleen.

Hi Michael,

It is a interesting topic Michael. PRC1 leads to monoubiquitination of H2AK119 not methylation in CpG islands. There have been many groups who have knocked out or Knocked Down specific PcGs (both PRC1 and PRC2) to identify the target most affected, but there isn't a particular gene that's affected. Different groups have got different protein owing to different starting materials (mES, hES, MSCs). It can be said that some key transcription factors can be used as read out of PRC1 or PRC2 activity, but it will depend on cell type to be used. 

Regards,

Prasad.

 Ｉｆ you would not like to ignore the anti-tumor immune response, I strongly recommend that you should use the autograft experiments, in which murine tumor cells such as 4T1 breast cancer cells or B16 melanoma cancer cells established from mice are injected to B6 WT mice in terms of immune system. We have previously published the work focusing on CD44 variant function in the metastasis against redox stress in the pre-metastatic niche (Nat Commun. 2012 Jun 6;3:883).

Ultimately, there has to be functional data showing that a particular mutation can transform cells. But realistically, we will never be able to test all of the potential drivers in all types of tumor in that way.

From a genomics perspective, the key question is whether a particular basepair/gene/locus undergoes somatic changs at a greater rate than chance would predict. If it does, then we can suppose that change confers a selective advantage and is likely a driver.

Current "state of the art" models like MutSig for coding changes:

and GISTIC for copy number changes:

attempt to do just this. These approaches are not perfect but I think they've helped us take a step forward in separating drivers from passengers.

Very interesting discussion, I would just like to add besides the discussions above, one may also keep in mind the PI3K, MEK, AKT mutations that may be involved and molecules of other alternative or additional  pathways that may be involved in resistant metastatic colorectal carcnomas. The article below may be of help.

Hi Brandon,

Could you kindly explain in more deatails!

Regards.

At least in mouse f4/80 antibody recognizes macrophages.

Hello Christine

Thank you so much for such great information. I will follow as your suggestions.

Good luck.

if you are using micrococcal nuclease or another endonuclease, the first thing that one could think observing the second picture is that the enzyme did not work: in the new picture there seem to be only high molecular weight DNA and small fragmented RNA. Next time you should have a control (DNA not digested) to see if there are differences between digested and not digested. Good luck

Hi again

Yes sure Antonio. You have been very supportive. Thanks

Some molecular techniques can be used to identify clonality for example in Multiple Myeloma Ig gene rearrangements is used as an indicator. If all cells in a colony have the same rearrangement pattern they should be grown clonally. I think this is important because CSCs should recapitulate the disease on their own, from one cell origin. If a tumor bulk is polyclonal, it could be a mixture of different cells divided a couple times and that does not mean these cells are totally clonogenic, in my opinion.

Dear Aneel,

First of all, I would like to tell you that it is possible to perform this kind of analysis with such a low no. of samples. Actually you want to measure differential gene expression (DGE) which is a quantitative experimental set-up for RNA-seq. Here biological replicates are important and 3 vs 3 will be enough to perform this kind of statistics which we call Explorative or as discovery setting. However, latter on you need to validate your findings on larger cohort which you already know.

I have to admit the we don't work on Lymphoid cancer, so I can't offer any possibilities of collaboration. But my point is to clear your doubt about sample size for RNA-seq...I would say go for it...

Hope this would help you...good luck for successful collaboration

CD34 is pan-endothelial marker, CD31 stains mature endothelial cells, CD105 and VEGFR2 are suitable for identification of newly formed blood vessels. However, I think that the only way to differentiate arterioles, capillaries and venules is according to their morphology.

Eventually, you could try double staining with endothelial marker and vascular smooth muscle cell marker, like for example, alpha smooth muscle actin.

Arterioles have 1 to 3 layers of smooth muscle cells and they will be CD34+ and alpha SMA+.

Capillaries, in general, have pericytes that stain with alpha SMA. Nevertheless, sinusoidal capillaries have very few if any pericytes, so they will probably stain CD34+ and alpha SMA-.

Postcapillary venules have pericytes, but their morphology is different than morphology of arteriole.

I would prefer plain morphology. Even if you apply double staining, the hematopoetic cells in bone marrow could be alpha SMA positive and that could cause problem in the interpretation of the results.

Cyytokeratin-specific immuohistochemical stains have been used in histopathology for quite sometime to aid diagnosis, for example to discern if cells are indeed epithelial in origin, or in the case of breast cancer ,whether they are of the luminal or basal-like subtypes. Recently overlap has been discovered between some members with the polar opposite molecular subtypes in breast cancer - luminal and basal-like. However, when compared to the gold standard definition of molecular subtypes using gene expression microarray expression signatures (either Intrinsic gene list or PAM50), they have not been found to be specific enough as luminal cytokeratins are expressed in basal-like breast cancers as well, and not all basal-like breast cancers express basal cytokeratins. Thus cytokeratin expression patterns in practice cannot be considered to be characteristic or pathognomonic of either subtype, but rather can be said to be highly associated with each entity. Basal-like cytokeratins DO possess prognostic significance, but less than more specific biomarkers for basal-like breast cancer such as FOXC1, which have been shown to be superior prognostic markers on the basis of comparing prognostic models using either cytokeratins or FOXC1 [Ann Surg Oncol. 2011 Dec;18(13):3839-47. doi: 10.1245/s10434-011-1657-8. Epub 2011 Mar 18.

Basal-like breast cancer defined by FOXC1 expression offers superior prognostic value: a retrospective immunohistochemical study. Ray PS, Bagaria SP, Wang J, Shamonki JM, Ye X, Sim MS, Steen S, Qu Y, Cui X, Giuliano AE].

There may be specific cell markers to identify metastatic cancer cells and these would be important to identify. Notably, circulating tumor cells have an altered cytoskeleton and microtentacles that protect from deformation caused by shear stress. Interestingly, both carcinogenic and non carcinogenic circulating breast cells have microtentacles but those in tumorigenic cells are extra long and motile compared to those in non-tumorigenic cells (Cancer Res. 68, 5678-88, 2008; Cancer Res. 70, 8127-37, 2010). Microtentacles, which are formed by microtubules and also may contain intermediate filaments, are required for efficient attachment and act in the metastatic spread of cancer. Interestingly the common chemotherapeutic paclitaxel, which acts by stabilizing microtubules, strengthens the microtentacles resulting in increased attachment of circulating tumor cells to secondary sites (Breast Cancer Res Treat. 121, 65-78 (2010) so this type of one highly successful therapy for primary tumors may set the stage for a greatly increased risk of metastasis. The observation that ~90% of all cancer deaths are caused by metastatic spread of the primary tumor underscores the importance of this question: the need to understand circulating tumor cells and how they find favorable attachment sites and what their mechanisms of reattachment may be.

Warburg effect is one the halmarks of cancer. There are basically 3 facts you should know:

1) Most energy produced by cancerous cells is via glycolysis, followed by lactic acid fermentation; essentially anaerobic respiration as opposed to traditional aerobic respiration in non cancerous cells.

2) the rate at which respiration is occurring is much faster for cancerous cells than non-cancerous cells.

3) Low oxygen content in tumors

This should be a start for you

The only cells that can't form cancer AFAIK are the ones that can't ever proliferate. Erythrocytes are so differentiated that they loose their nucleus. Therefore, without any genes, cancer can't emerge from them. Their precursors however, which possess a nucleus, can develop cancer.

Some cells form cancer more often than others. Different parts of the body are exposed to different concentrations of various carcinogens. Tissues have different proliferative potential, some cells do not proliferate physiologically (cardiomyocytes), while others proliferate like crazy (epithelium). The more "breaks" are limiting cell's life cycle, the more barriers need to be overcome by mutations in protooncogenes, suppressors, and apoptotic genes to start uncontrolled cell division and eventually lead to cancer.

In a German article research by Werner Paulus is

mentioned. According to the article there are several

factors:

- some genes influence the cytoskeleton

- some genes influence transmission of

signals from the surrounding of the cell

- some genes regulate the nucleus

Other literature, maybe relevant:

http://cancerres.aacrjournals.org/content/73/5/1536.short

Regards,

Joachim (not a medic)

Dear All

Please also note that EXPERT OPINIONS are also cordially invited from leaders in molecular Oncology. I have already received 3 Expert Opinions for the Thematic Issue.

I agree with Francis - the best way is to use a "realistic" concentration of drug based on clinical measurements (if available) and wait for resistant clones to grow out. This is tedious - depending on the combination of cell line and drug you are using it can take months - but from my own experience and from talking to other people who have done this, cell lines generated by selecting with a "lethal" dose may develop different mechanisms of resistance compared to those selected via escalating doses. I would add that some cell lines are so sensitive to certain drugs that you will have no choice but to drop the dose below the max. plasma concentration. Doing a basic GI50 type of analysis (using a cell viability/proliferation assay) will help you find the right concentration of drug, as others have suggested. Good luck.

I think the cancer cells are clever enough to distinguish each other, enemy or friend, but how we can teach the cancer cells turnover?

Paolo:

On systematic review, I found the evidence aggregated to date gives us two human clinical trials of dabrafenib. As for vemurafenib, we have only preclinical data but there are some trials in progress. In both cases, BRAF K601E mutation in the advanced melanoma setting as a target was a strict inclusion requirement. My review findings are summarized below:

Dabrafenib (GSK2118436)

In a phase I/II trial [1] of this highly potent and selective ATP competitive BRAF inhibitor, of nine melanoma patients with V600K BRAF mutations, four had objective responses. Note however that four of five patients with BRAFK601E progressed during the first restaging. What this suggests is that only patients with BRAF V600K mutations are likely to respond to dabrafenib treatment.

Similarly, in another dabrafenib study [2], three patients experienced a partial response, but both of the patients with BRAFK601E progressed after first restaging, again suggesting dabrafenib response is restricted to BRAF mutations at the 600 position.

Vemurafenib (PLX4032 OR RG7204; commercially: ZELBORAF)

In a preclinical study [3] of 17 melanoma cell lines, vemurafenib (PLX4032/RG7204) was a potent inhibitor of proliferation in those expressing BRAFV600E but not BRAFWT, but note that it also potently inhibited proliferation of melanoma cell lines expressing other codon 600 BRAF mutations (including V600D, V600K, and V600R), and most importantly, also moderately inhibited proliferation of the WM1789 melanoma cell line expressing BRAFK601E. Should human clinical trial data support this preclinical finding, vemurafenib may show clinically relevant inhibition of BRAFK601E mutation of advanced melanoma.

Lessons Learned

Of these two BRAF inhibitors specifically for inhibition of advanced melanoma BRAFK601E mutation, human clinical data fails to provide sufficient support for dabrafenib, and although vemurafenib currently lacks supporting human clinical data, preclinical cell data is suggestive of potential activity, awaiting trial confirmation.

Review Methodology

Parameterized search was conducted over PUBMED, EMBASE, CAMBASE, AMED, Scirus, CENTRAL, Ovid, and Terrko (University of Helsinki), without language or date restrictions, and updated again current as of date of review [April/2013], with systematic reviews and meta-analyses extracted separately. Search was expanded in parallel to include just-in-time (JIT) medical feed sources as returned from Terkko. A further "broad-spectrum" science search using Scirus (410+ million entry database) was then deployed for resources not included in PUBMED and elsewhere. Unpublished studies were located via contextual search, and relevant dissertations were located via NTLTD (Networked Digital Library of Theses and Dissertations) and OpenThesis. Sources in languages foreign to this reviewer were translated by language translation software.

References

1. Falchook GS, Long GV, Kurzrock R, et al. Dabrafenib in patients with melanoma, untreated brain metastases, and other solid tumours: a phase 1 dose-escalation trial. Lancet. 2012;379:1893–1901.

2. Kefford R, Arkenau H, Brown MP, et al., “Phase I/II study of GSK2118436, a selective inhibitor of oncogenic mutant BRAF kinase, in patients with metastatic melanoma and other solid tumors,” Journal of Clinical Oncology, vol. 28, abstract 8503, 2010.

3. Yang H, Higgins B, Kolinsky K, et al. RG7204 (PLX4032), a selective BRAFV600E inhibitor, displays potent antitumor activity in preclinical melanoma models. Cancer Res 2010 Jul 1; 70(13):5518-27.

Constantine Kaniklidis

European Association for Cancer Research (EACR)