Questions related to Tumor Biology
My experiment aims to make spheroids by sacrificial micromolding technique.
I am following the easy protocol:
"...The PDMS stamps were deposited onto a 250 μl drop of 3% liquid agarose within the wells of a 24-well plate. Agarose gelation occurred within minutes of placing the 24-well plate at room temperature..." Karen E. Samy, 2019.
The PDMS stamp has pillars with 120 um in diameter and 80 um in height according to the article.
However, the problem is, that during pull out of the PDMS stamp the bottoms or walls of agarose microwells are somehow stuck on the pillars, and they are pulled out with them. So, instead of having nice microwells, there are cones made out of pulled microwells, and the cells can't get inside and form a spheroid.
I have tried 3% agarose (according to protocol), 5% agarose, different timing, place the stamps with agarose to fridge or freezer for 10 - 20 minutes, used plasma on PDMS stamps to make them more hydrophilic and all these combinations together.
I am running out of any other ideas. Does anybody know, what could help my situation?
Unfortunately, the corresponding author hadn't answered.
The photo of the "microwells" is attached. In this picture, you see two successful microwells and poorly done the rest of them.
The investigation of suitable time point of T cell activation by specific antigen is the critical point to manage your real evaluation of T cell response of tumor antigens. Here, I am working on the Panc02 tumor lysate what is the experimental time points are useful to evaluate T cell activation response?
I'm considering implanting OT-1 mice with OVA expressing tumors (B16-OVA) or E.G7-OVA). Will these mice reject these tumors since a large fraction of their T cells are specific for OVA? Does anyone know of any references for this type of experiment? All studies I have seen use WT mice with adoptive transfer.
Also are there any safety concerns for the mice, such as massive inflammatory response? Thanks!
I am curious to know if it is possible to store tumors (or tumor tissues) isolated from mice at -4 degrees (frozen) in serum free RPMI or DMEM medium overnight or longer? I would like to FACS sort live cells later. The situation is such that I have to store the tissues overnight. Any insight will be helpful.
My lab is currently searching for a new human/mouse GLI2 antibody. We were using the sc-28674 from Santa Cruz and were quite satisfied with it, but it is no longer produced, so we tried the new sc-271786 from Santa Cruz and the ab26056 from abcam and both did not give satisfying results.
Many genetic changes can immortalized cancer cells including oncogene activation or tumor suppressor gene inactivation. For example telomerase activation is one way to immortalize cells.
But what is the difference between mechanism of immortality in normal stem cells (NSCs) vs cancer stem cells (CSCs)?
Is there any different mechanism for maintaining genome stability in NSCs and CSCs?
Is there any literature that anyone can provide?
hello,I am working on mouse tumor xenograft model of HepG2 (subcutaneous),now the tumor have began to grow,but i don't know the best time to initiate antibody treatment(the volume of tumor reach over 100mm3? if yes, why?if no,what? ) and the method to inject antibody(intraperitoneal or subcutaneous or caudal vein injection? which one is the best and why?),calculate the volume of tumor by V=(L*W2)/2 . I am a novice, I will be grateful for any reponses.some paper related is also good. thanks in advance.
I am trying to grow xenograft tumors with cells mixed with matrigel. I see that at the injection site, the gel sets and forms a bump. How do I measure the tumor growth accounting for the gel bump, that is while I am measuring tumor growth how do I differentiate between matrigel and tumors? I use caliper measurements for tumor growth.
How to calculate IC50 values to drugs that does not reach a 100% of inhibitory activity, for example? If I find a experimental value of 45% inhibition in the maximal concentration of solubility (for example 10uM), should I say that IC50 value is "higher than 50uM"? If one compound presents a inhibitory activity around ~60% at the highest concentration tested (total solubility), the 60% will be the "top" value to use to calculate IC50 ? thank you all!
I try to figure out some easy method to transfer spheroids from hanging drops from a petri dish lid into 96-well plates. So I had the idea to generate the tumor spheroids directly on the lid of a 96 well-plate directly instead of generating them on petri dish lids and to centrifuge the plate after spheroid initiation. Does that work to centrifuge the spheroids from the lid directly into the well?
We have BXPC-3 human pancreatic cell line which had mycoplsma contamination, we treated them with plasmocin for 2 weeks and now the morphology of the cells totally changed. They used to be polygonal and formed tight colonies at lower seeding densities, not they are spindle shaped and do not form colonies at all. We sent the DNA sample for authentication and it matched so there is no cross contamination with other cell lines and the mycoplasma result came negative, but the cells don't look like BXPC-3 anymore.
Please help us trouble shoot this issue.
Thank you very much!
Is anyone working on the pathological and/or molecular characterization of very aggressive breast cancer beyond "triple negative" definition?
checking billion of resources, apart from B16 cells, Im not able to find any clou for another murine melanoma cell line.
DOES ANYBODY KNOW SOMETHING or has an idea/tipp?
I would highly appreciate that!
(P.s: best would be, coming from any WT (e.g. BL6) background)
Hi, I am planning to isolate single cells from human lung tumour tissue. I am wondering what is the best way to enzymatically degrade the tissue to isolate MOST of the cells (since the population of interest is very rare and I want to avoid false negatives when I do FACS analysis). I came across multiple collagenase cocktails which have been reported in literature, but not sure which is the best way forward. Which is the best collagenase to work with?
Any leads would be appreciated. I don't want to optimise too much since these are precious patient samples. Thank you! :)
Many papers either do not specify the type of MatriGel (HC, GFR, phenol free, etc) or apparently do not use it for orthotopic implantation at all. Anyone use Cultrex as an alternative? Thanks in advance for the insight!
My research is about macrophages in breast cancer,so I have to separate TAM from tumor. I've tried several methods:mechanical digestion,enzymatic digestion.However,after making single cell suspensions from breast tumor,I don't know how to select TAM from cell suspensions.As the literatures say,1)magnetic bead-conjugated antibody cell isolation;2)density gradient separation;3) laser capture micro-dissection;4)FACS;
I hope you could give me some suggestions on separate and purify TAM from human breast tumor.Thanks a lot !!!
Currently I'm studying the cytotoxicity of metal complexes in 2D cell cultures, but at some point I would also like to go to tumor spheroids as they are closer to tumors in vivo. We use the SRB assay to determine cytotoxicity in 2D and in order to compare the results from the 2D and 3D culture I would like to use the same cytotoxicity assay. However I cannot seem to find any papers where the SRB assay is used to determine cytotoxicity in tumor spheroids. Can somebody tell me why this method is apparently not suitable to use in tumor spheroids?
We've been using the MC38 s.c. tumor model and continue to have issues with tumor ulceration. We have tried injecting in different locations, varying the cell number, varying the resuspension buffer (PBS, RPMI, matrigel), using different cell lots, etc. The cells are myco-free and have been MAP tested. This is not a tumor size issue as the ulcers appear in small tumors (~200 mm3).
Has anyone had success in reducing the ulceration frequency?
I have interest in monocyte-macrophage lineage and examining the behavior in the TME.
I know that MDSCs can be granulocytic or monocytic, but do monocytic MDSCs represent the same or an overlapping group as TAMs?
Would love both a verbal description differentiating their behavior and the flowcytometry markers used to distinguish/define them if possible.
Finding it very hard to distinguish from the literature due to inconsistencies in cell markers and terminology.
I am currently trying to establish cell line for canine cutaneous epithelial tumors (hair follicles in origin). For primary cultures, I followed the standard protocol using 10% FBS in DMEM containing 1%Penicillin-Streptomycin. I subcultured it using the same media as above. At P3, I performed immunocytochemistry and found that most of my tumor cells were strongly positive for vimentin and nestin but showed very pale positivity for pan-cytokeratin. The secondary cell line did not survived until the fifth passage (I tried twice but the cell stop growing and started dying at around 5th passage). The DMEM contained: 4.5 g/L D-glucose, L-Glutamine and 110 mg/L Sodium Pyruvate. I had look into some journals reporting the establishment of squamous cell carcinoma cell line which used 3T3 Feeder Layer, cholera toxin and hydrocortisone. I would like to ask if anyone can share their experience in culturing tumor cell lines of epithelial in origin and the basic growth supplements and suitable media required for successful cultivation of epithelial tumors. Thanks a lot !
Neurotransmitters have been shown to drive tumor progression in the prostate glands and the stomach. In particular, it has recently been shown that acetylcholine promotes proliferation of Lgr5-positive cancer stem cells via the modulation of YAP-dependent canonical Wnt/beta-catenin signal pathway. Then the secretion of NGF occurs. This forms the positive feedback machinery. Is there any clinical method to demonstrate the denervation in the tumor tissues?
I am setting up an experimental metastatic model of Lewis lung carcinoma by intravenous injection of LL/2 cells into the tail vein. I am looking for ways to quantify tumor burden in the lungs.
I understand that using Luciferase/fluorescence expressing cell line is one way to detect tumor cells in the lungs, however, I am looking for something more specific, perhaps a marker expressed in these tumor cells which would increase with tumor progression (For e.g. for B16F10 cells, I am using a melanocyte specific gene and study tumor burden using RT-PCR).
Thanks for your suggestions!
We have been trying to develop 4T1 breast tumor model in balb/c mice. We have been injected 10^6 4T1 cells suspended in 100 uL PBS into the mammary fad pad region, subcutaneously. After 10 days we see mass, in site of injection. The size of mass is approximately 5mm ×5 mm. After 20-days mass is completely regressed in some mice and in some other the size of mass aggressively diminished. in pathological examination, some of the small mass have tumor properties and are malignant. if these mass are tumor what are the causes of restricted or abolished growth of them? why these cell line can induce small tumors in some balb/c mice but not in all of them?
I am working with adaptively transferring leukocytes in tumor-bearing mice. So I am taking the cells from the donor animals, label them with fluorescent dyes and inject them back to the recipients. However, because of all the unnecessary damages that labelling of the cells in vitro causes, I am looking for a way to label the neutrophils in vivo.
Currently I am planing to label the monocytes in vivo so I am also thinking to label the neutrophils as well in different settings.
Moreover, I wondering when I label the circulating blood monocytes in vivo how many monocytes I can label and how many of them actually migrate to the tumor xenograft!
I am looking for immunocompromised mice to grow human tumor xenografts in. I am potentially interested at looking at macrophage function in these tumors. Is there any mouse model that will give me the best of both worlds by allowing my tumors to engraft while having normal macrophage numbers/activity/function ?
Hello, what I want to ask: How do you decide which area of the tumour ist the best for counting? How do you go on with, if there in one slide are areas without any signals, for example good signals in the periphere and no signals in the center ? How do you interpretate, if there are only green ( control ) signals , is it loss of 1p or 19q, or will we have to do it again ?
I have found mixed messages in published articles around the likelihood that a primary (non-metastatic) vertebral tumor (i.e. extradural tumor) is benign.
"Metastatic tumors are most common (97%) tumors of the spine." Ciftdemir et al (2016) W J Orthopedics 7:109-116
"Primary extradural tumors of the spine are rare and constitute approximately 4% of all spine tumors." Lam et al (2014) SNI 5:S373-S375
"Benign tumors such as meningiomas and neurofibromas account for 55 to 65 percent of all primary spinal tumors... Metastatic spinal tumors are the most common type of malignant lesions of the spine, accounting for an estimated 70 percent of all spinal tumors." AANS (http://www.aans.org/Patient%20Information/Conditions%20and%20Treatments/Spinal%20Tumors.aspx)
What is the truth? Do metastatic spinal tumors make up 70% or 97% of spinal tumors? What is the likelihood that a primary vertebral (extradural) tumor is benign? 20% or as little as 01.2%? This difference is far from trivial.
Cancer cells transferred by blood from one organ to another within the metastatic cancer patient. This means that tumor cells are freely moving in the blood during the metastatic phase. If this cancer patient donates blood to another, there is a possibility that the cancer cells transferred to the recipient especially if the recipient is immunocompromised is this TRUE or NOT.
Although gall bladder and bile ducts are lined by similar biliary epithelium, what is the reason for such a aggressive behavior and biology of gall bladder cancer as compared to cholangiocarcinoma?
I would like to do a tumor burden follow-up by measuring circulating cell-free DNA (ccfDNA) at different time points in a mouse experiment (by treating or not mice after tumor initiation).
Does someone have a protocol to amplify DNA as I can draw only 100-200µl of blood/week in mice.
I have a protocol to isolate DNA (QIAmp DNA blood mini kit) from plasma.
Thanks in advance!
Does anybody have an experience in difference between PET / CT scanning of mice tumors with a scanner designed for animals and humans?
e.g. Continuous trauma in one quadrant of the mouth of five cases causes a tumour to grow in that very quadrant, while for another five cases the tumour grew from a seperate quadrant than the one where trauma happened. So if I want to establish that the site of exposure is correlated to the site of tumour or vice versa, how do I do it ?
I already tried manually dissociation as well as GentleMACS but both are not working well with my frozen breast tumor tissue. I would like to do flow-sorting of single tumor cells afterwards! Thanks for all comments!
Macrophage can engulf tumor cell , in some case to kill it but sometimes it's a good way to help tumor cell to live and metastasis. So, How to distinguish a macrophage engulfed tumor cell is dead , alive or dormant?
I was looking in the literature for prostate cancer C4-2 cells (CRPC) mouse xenografts that used enzalutamide, but I wasn't able to find any such papers. Could any of you help me with this? Thanks in advance.
I'm working on melanoma tumors from mouse and I want to extract proteins from it. I have a protocol where the detergent used is 10% Brij.
Is the concentration too high ? Or is it normal because it's from the tumor and not from cells ?
Thanks for your help
Can someone suggest a genetically modified spontaneous mammary tumor /chemical induced carcinogenesis model where genetic modification should result in significant alteration in glucose metabolism in mammary epithelium!
I want to obtain tumor supernatant by culturing cancer cells in FBS free media. Right now my cells are in 10% FBS. Should I change the media directly to 0% FBS or reduce it gradually like 10%, 5%, 1%, then 0% so that the cells can adjust well in reduced serum? Please suggest.
I am estimating the mutation rates (mutations/Mb) in a series liver tumors. How can I estimate the confidence interval of each mutation rate? I tried using the binomial test (binom.test function in R) but it is quite computation intensive...
Any better idea?
Thanks you very much in advance,
Patients suffered from insulinoma have clinical feature of hypoglycemia.Insulinoma secrets insulin without control.What's the possible mechanism?
We are setting up a tumor model, injecting B16F10 into B6. Many people use simply PBS for the injection. I wonder if there is a preferred site of injection (flank, back, scruff, belly)? We are concerned that upon injection into the flank, the PBS solution does not stay put long enough (e.g. bleb seems to disperse fairly quickly).
Apart from the subcutaneous tumour, is the lung tumour also primary or metastatic? If metastatic, does it still represent actual lung tumour?
Stereotactic Intracranial injections are extremely time consuming (Can only do 10-12 animals in a full day), are expensive- requires purchase of drill, dissecting scope, stereotactic apparatus, automatic fluid injector. Since many papers use different brain locations for injection of tumour cells and there is still significant error due to brain curvature etc., the exact location of the injections may not be crucial.
Meanwhile, guide screw injections are considerably faster and cheaper- only requires the drill, the accessory screw pieces and perhaps a dissecting microscope. It would also be much more practical for the sake of performing injections in large cohorts. This technique has been used in many high impact publications, particularly Zhang, 2015 in Nature (Exosomes PTEN Brain metastasis paper).
The flaw with this technique are that they are less precise than stereotactic injections, and this may lead to larger variation in tumour growth between animals. However, for the above stated reasons, I am wondering if it is worth the gamble.
Does anyone have experience with the guide screw technique, or both techniques that can provide input?
I know the ideal thickness for this staining is usually half of it, but I already have cut my samples on the vibratome and kept them frozen. So now I would like to perform a H&E staining and I would appreciate any suggestion to optimize the staining resolution for that thickness. Or if there is any other staining protocol to localize tumor area on those samples, also might be helpful.
Many thanks in advance,
I am trying to isolate T cells from human glioblastoma for FACS and Sequencing analysis. I tried digestion with collagenase and DNAse and subsequent seperation by 100%75% Ficoll gradient, however the purity is very poor and I would like to improve. Can anyone share experiences or protocols? Thank you!!
I have prepared tumour spheroids with U87 MG cells and I am trying to get fluorescence images using confocal microscope. However when I placed tumour spheroids between cover slips and microscope slides, I think they get squeezed and I lose the 3D shape of the spheroids. Do you have any suggestion to prevent this? Thank you.
I am studying novel immuno therapeutic strategies in cancer and have had trouble in establishing a reliable system to study combination therapy with PD-1 blockade. I am wondering if anyone has had success with such a model. I have tried B16 tumors and EL4 tumors. Thank you.
These cell lines along with available xenografts and other factors along with other biomarkers ( if you have any it would be appreciated) will be used to build a preclinical model for high risk Wilms tumors. The development of assays specific to this model will result in the ability to do novel cell line and animal testing which could potentially lead to clinical trials with new compounds for children with these disease.
We have observed high TIM-3 expression on Tregs in a TC-1 tumor and am wondering if they are somehow functional and would TIM-3 blockade have an effect on Tregs' suppressive effects?
MANY AUTHORS HAVE REPORTED DIFFERENT INCIDENCE AND TUMOR LATENCY FOR RAT MAMMARY TUMORS. CAN WE USE BOTH CARCINOGEN AT DIFFERENT TIME INTERVAL TO INDUCE TUMORS FAST WITH BETTER INCIDENCE?
I have found that cbioportal is a good website to study molecular expression level of certain proteins in certain cancer cell lines. Unfortunately, some cell lines are not in the database. Where can I find information on those cell lines? I am working on esophageal cancer cell lines. I need to get cancer genomic data on kyse50, kyse 110, TE2, TE3. Which websites are suitable for this purpose?
I have some mouse tumors that I am going to harvest. The vet who performs the surgery will place them in an isotonic saline solution, which they will be in for a brief time (< 1 hour) before I can get them back to the lab. I have read many protocols but I still don't know which is best. Right now I see my main assays being H&E, so what I care most about is preserving the morphology and tissue structure. How long could they be left in the saline solution in the fridge at 4 °C? I assume though that they would be better frozen as soon as possible. I will embed them in tissue freezing medium for cryosectioning. Is it better to store tissues by themselves in -80 °C or first embed them in tissue freezing medium. How important is the freezing rate depending on whether the tissue is embedded in tissue freezing medium or not? The cryomicrotome system that I use has a small area on it that gets down to around -60 °C. If I place the specimen in a plastic mold or a metal mold and freeze it in that small area, is that fast enough? What about just placing it in the -80 °C freezer to freeze? Finally, are snap-freezing protocols or sucrose cryoprotection protocols something I should look into in conjunction with tissue freezing medium embedding? Thank you.
Update (11/12/2015). The undergraduate student who performed the cryosectioning made the mistake of freezing some of the initial samples straight after removal from the saline solution at -80 C. We examined both mouse tumor and mouse spleen. For the pre-frozen samples, we decided to do fixation followed by cryoprotection in 15% and 30% sucrose on those samples anyways, finally embedding in OCT compound. The spleen had many tears, especially at 10 micron thickness, specifically near the center. A 20 micron thickness gave us the best results, with much less tearing and even some whole slices. The tumors appeared to hold up better despite the initial freezing compared to the spleens. 10 microns did give us more smaller tears running through the tumors, and 20 microns again gave us the best.
With the fresh samples, they cut great, but it seemed that they started to crack a short time after they were adhered to the slide. Is there anything we can do to avoid this?
Some literature mentions that 4T1 is sensitive to recombinant human TRAIL whereas some articles mention that recombinant human TRAIL cannot affect mice tumors since they don't have death receptors necessary for TRAIL actions.
If the relative sphere number with each passage is dropping in a cell that is overexposing a gene associated with increased tumorogenicity, does that mean that a stable overexposing cell line has not been formed?
I'm searching for a breast cancer cell line in which Her2 and uPAR are both over-expressed. Does anyone know about any? Thanks in advance
Tumor contains heterogeneous population of cells with different mutations. So, is there any possibility of existence of ‘NRAS mutated-BRAF-WT’ cells in the tumor/cancer lesions of BRAFV600E (Mutant BRAF) positive patients? If so, then how do the NRAS mutated-BRAF-WT cells respond to the inhibitors for BRAF in tumor? What signaling(s) one could expect during combination therapy in such situations? It would be a great help if you could comment, emphasizing some published or unpublished work related to my questions. Thank you.
Our lab uses frequently uses matrigel for intraperitoneal injection of ovarian/endometrial tumour cells. However, there is no literature I can find that describes the use of matrigel with IP injections, and it is only recommended for orthotopic and subcutaneous injections.
Intraperitoneal injection is more like a metastatic model and so it seems as though the formation of a matrigel plug is counter-intuitive.
Should I stick to just using PBS? Or should it not really matter?
Thank you for all your answers so far! I would like to add that with gynecological cancers, direct spread to the peritoneal cavity is very common; metastasis via the blood stream not so much. That's why we inject tumour cells intraperitoneally.
I did an experiment with 6 mice using 5x105 and 10x6 cells , ( 3 mice each condition), and both cell concentration were rejected in 15 days.
I used matrigel
Cell+matrigel + 50ul
Should I use more cells? How much more?
Does anyone know why these PC12 cells of one picture look a little rough on their surfaces compared to the other one? They look slightly different from usual and I can't figure out why..Humidity issue maybe? I used the same media for both pictures.
Any ideas? Thank you!
How to measure tumor size in corel pictures. Suppose I have mice pictures with tumors and these tumors are separated from body part. The problem in the quantification of tumor size as by visual and corel measurement not looks correct. Someone have an idea how to quantify these tumors? Thanks in advance.
Kindly suggest the alternate for Propylene glycol which is commonly used as a vehicle control in the experiments (Genotoxicity and antitumor studies) pertaining to EAC models..and also explain why propylene glycol is the most preferred vehicle control for comparison studies done using chemotherapeutic agents.
I have attached an article for better understanding..
When staining both fixed (4.0% PFA in PBS) and permeabilized (1.0% EDTA in PBS) 3D multicellular tumour spheroids (MCTS) with phalloidin for confocal imaging, I get quite unexpected results. Though it may seem that diffusion rate could limit the staining efficiency at the core of the MCTS, but I get reverse results - the core is stained perfectly, but outer rim cells (counterstained with DAPI) seem not to be stained at all (only DAPI appears). Has anyone faced similar problem? Is it probable that PBS washing steps remove phalloidin bound to actin? I would be grateful for any suggestions.
I am going to isolate TAMs from some murine models. As I searched, it seems that MACS from miltenyi biotec has been used by a great number of researchers for isolating TAMs but theoretically Easy sep from stem cell has its own advantag