Intriguing observations. In the absence of additional information such as how cells were treated with blood, it is difficult to address your questions. Cell lines that you indicated, do well in culture (medium + 10% serum; 5% CO2). Assuming that cells were in blood, it is likely to change their carbon and energy source and induce stress. In response to stress, cells are predicted to die. However, this outcome can be reached by a number of reasons. Therefore, more information would be needed to interpret your observations. 

Hi Divaker, thanks for your response.

The MCF-7 and SKBR-3 cells were indeed kept in culture with media + 10% FBS, 5% CO2 in T75 flasks. They were trypsinized and added to 10% FBS containing media to neutralize the trypsin and then centrifuged at 150 x g for 5 min.

The cell pellets were then resuspended in serum-free media and viability assessed by Calcein-AM and/or trypan blue. Within 30 min, cells were spiked into normal whole blood (7.5 ml collected in EDTA vacutainers) and kept at RT.

I fully expected the spiked cells to die in whole blood, but I was surprised on how fast it happened. 

I have (inadvertently) left these epithelial cells in trypsin-EDTA for ~1 hour at RT, but they still survived.

Sorry, I forgot to add that spiked cells were isolated from whole blood using microfilters described in:

http://www.pnas.org/content/111/9/3514.full

and:

Article Cytometric characterization of Circulating Tumor Cells captu...

I am wondering about the immune reactions from the whole blood against "foreign" cells and proteins. From experimental metastasis assays with murine B16 melanoma cells we know that when i.v. injected into the blood stream of isogenic mice that most cells won't survive and, perhaps, 1 of 10,000 gives rise to a growing tumor in the lung. So, survival of cells in the blood (or blood stream) mght be very limeted. If I remember right, the citation is Fidler 1973.

It is possible that cancer cells having allogeneic antigens are recognized by the new blood components from another individual and got killed.

Hi Robert and Prasanta, thanks for your answers. There are papers on the metastasis of injected cells, but do you know of the survival of live cells spiked into normal blood samples?

I have heard anecdotal reports about cancer cells spiked into heparin vacutainer blood being more viable that cancer cells spiked into EDTA vacutainer blood.   

This is just my guess, i.e. please do not over-estimate my brainstorming here: EDTA binds and removes Calcium and other ions, especially from integrins and other cell adhesion receptors, therefore inactivating their binding capacity - and changing, perhaps, the survival or signaling pathways of tumor cells; another idea could be that leukocytes from the whole blood may not be able to support survival of the tumor cells, again, by interference of the binding. Heparin may have no harmful effect, but perhaps, may have also a supporting effect, binding to receptors of the tumor cells and providing other molecules (bound to the heparin) which support some survival signaling...

Many of such potential phenomena could be investigated with the technology I invented some time ago... AFM force measurements on a living cell or between two living mammalian cells, at the so-called single-molecule level (level of single pairs of adhesion receptors).

Conference Paper Single-molecule resolution of chemokine-mediated activation ...

Hi Robert, I am really impressed with your AMF technology that you developed and your papers! Kudos!

I do remember papers showing differences in AMF generated by cancer cells spiked into blood versus versus CTCs isolated from patient samples.

I do not have these publications ready, but were studies done using on CTCs using your technology? 

Hi Steingrimur, thank you for your nice and supporting words! I am really not aware of such specific papers you mentioned, but I was not aware about your field of study, so they may have escaped my attention before - I will check within the next few days; I remember only some papers on correlating elasticity of normal vs. cancerous tissues, or some research on niches of metastatic (stem) cells, but this is far away from your approach. It will be interesting to look for such papers.

Hi Robert, I just want to bring your attention to these papers from Google Scholar that claim to use AMF technique to  analyze CTCs. Are they using your techniques?

http://scholar.google.com/scholar?as_vis=1&q=%22circulating+tumor+cells%22+%22Atomic+force+microscopy%22&hl=en&as_sdt=1,2

Hi Steingrimur, thanks for pointing me to that paper, but from what I can read from the abstract, they use (as I had memorized) just "elasticity" measurements by AFM - in fact, they use AFM force measurements (i.e. also not producing microscopic pictures), but that is still very different from detecting specific adhesion (or de-adhesion) of single pairs of adhesion receptors between two living cells or between one living cell and a substrate (like an immobilized functional part of a counter-receptor). All the other analyses in that abstract (from my understanding) are based on genetic profiling, i.e. expression signatures. So, their approach may be helpful and straightforward to correlate or understand different types of elasticity in the tumor type they investigated, but I expect numerous different types of elasticity for every type of original tissue type. Of course, there should be very different types of elascticity, bone vs. brain, or fat tissue vs. muscle,  skin vs. stomach... maybe there is a real signature for metastatic cells; not sure if this is the right direction, but one will not know before doing such experiments. I never really went into elasticity measurements or stiffness of cells, and (in contrast to many others) I am not that enthusiastic about EMT or MET transitions of tumor cells - this appears to me a quite human and artificial view of things to explain something previously postulated... (but I could be very wrong here). I still wish to use my original approach and adapt it more in the field of real pharmacology to find the best treatments against cancer (and to understand the different physiology of different cells), but didn't get the funding or seed money, yet. Unfortunately, my approach is quite slow, far away from high-throughput technologies, therefore not many labs could reproduce such a complex approach (but most AFM labs just want to construct new apparatuses, not apply the technology to medical use).

Hi Robert, I really appreciate your insights!

I think you are right about using AFM on cell receptors (like integrins) that are connected to cytoskeletal fibers.

I have an attached my JBC paper on the (supposed) high affinity of integrins for the RGD sequence of vitronectin. --SPOILER ALERT-- There is no high affinity binding of avb3, avb5 to vitronectin RGD. The "high affinity activated" integrin binding to the RGD is totally bogus. "High affinity binding" of these integrins to vitronectin RGD are mediated through their cytoskeletal attachments. 

But anyway, lots of groups have been using your AFM technology on circulating tumor cells. I am not a fan because they use all different receptors.

PS, your reservations about EMT and MET transitions of tumor cells are well founded. We have manuscripts in the pipeline to prove this.

Article The Contributions of Integrin Affinity and Integrin-Cytoskel...

Hi Steingrimur, thank you for your paper and your good points. Binding to the cytoskeleton is also important for those rolling/homing-associated integrins I found in my metastasis model looking for the metastatic stem cell when I was in California; those do not recognize RGD containing proteins or receptors, but may act similar to your favourized integrins. I am not aware of many groups really using my AFM approach for circulating tumor cells - but will check again. The reason why I developed this AFM method was a very different receptor... but still could not get the seed capital or collaboration, or professorship.

Hi Robert. I feel your pain. I was an Asst. Prof in SOM Maryland, but I was not funded so now  I am now working as a contractor.

Anyway, there are a lot of papers using your AFM technique on CTCs.  I just want to know they are they legit?

http://scholar.google.com/scholar?hl=en&as_sdt=0,21&q=%22circulating+tumor+cells%22+%22atomic+force+microscopy%22

Hi Steingrimur. Thanks for clarifying how you searched. There must be something wrong  with this search leading to over 500 papers on "circulating tumor cells" and "atomic force microscopy". I tried several similar searches  (CTC AFM) on PubMed.gov and found just one. I did not check the over 500, just the first page. It seems there are many labs and companies using different approaches to collect CTCs. Sometimes they check their material surfaces with an AFM, but not the cell (as far as I could screen some papers). To the best of my knowledge to date, I am the first to measure the physiologic activation of any integrin by any chemokine on any cell type - and there is no significant number of papers or labs to use this technology (as far as I know) to the medical potential; this is due to the nature of medical researchers - it takes too long to get such a sophisticated technology working to this resolution - and the nature of the physicist - it is just too boring for a good scientist to apply the technology in medicine instead of developing a new microscopy or technology. I recently helped writing a related, but small business NIH grant on this topic, but it was only close to get funded. Nevertheless, the tumor part appeared to be well accepted...

Hi Robert, there is a huge difference whether you search with " " or without on Google Scholar

The " "  on Google Scholar searches for the exact phrase. Let me give you an example:

Typing in circulating tumor cells on Google scholar gives you about 2 million hits because the  search engine recognizes all the words and ranks them equally

http://scholar.google.com/scholar?hl=en&q=circulating+tumor+cells&btnG=&as_sdt=1%2C21&as_sdtp=

Type in "circulating tumor cells" in Google scholar and you get ~35K hits;

http://scholar.google.com/scholar?q=%22circulating+tumor+cells%22&btnG=&hl=en&as_sdt=0%2C21

Type in "circulating tumor cells" "Eibl" and you get 80 results:

http://scholar.google.com/scholar?q=%22circulating+tumor+cells%22+%22eibl%22&btnG=&hl=en&as_sdt=0%2C21

I believe you are being ripped off. 

Hi Steingrimur, I am absolutely aware about using "..." "...", and your search leading to 596 articles is functionally OK, but does not give you the scientific results you expect. There are papers using AFM just as elasticity measurements, others used AFM for checking their surface of anything else, their instruments, surfaces, but not cells. For my method it is not that important to use CTCs, that is your field; so show me any good paper closely related to my research... and not plagiarizing it.

Hi Robert, I respect you and your research. Please believe me, I did not mean to  sound condescending!  If you think so, then I deeply apologize! 

All I am saying is that groups are using your technology to make claims about CTCs and their properties:

 https://www.researchgate.net/profile/Chun_Liang_Chen/publication/234031871_Single-cell_analysis_of_circulating_tumor_cells_identifies_cumulative_expression_patterns_of_EMT-related_genes_in_metastatic_prostate_cancer/links/00b49538f959313883000000.pdf

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3830628/

http://scholar.google.com/scholar?q=%22circulating+tumor+cells%22+%22atomic+force%22+%22emt%22&btnG=&hl=en&as_sdt=0%2C21

Article Single-cell Analysis of Circulating Tumor Cells Identifies C...