45 replies to this topic

[kmoody]

Summary

In fall 2014, Longecity supported a crowd-funding initiative to assess the effects of c60oo on cancer using a model of acute myeloid leukemia. Briefly, CIEA NOGs (an immunocompromised mouse model) were administered 1x 10^7 Kg1a cells (a human AML cell line) by tail vein injection, and treated with either saline, olive oil, 2mg/kg c60oo, 4mg/kg c60oo, or 8mg/kg c60oo (n=5 for each group). All cause mortality, liver toxicity (as measured by AST and ALT levels), complete blood counts, and tumor burden (as measured by percent Kg1a in mouse peripheral blood) was evaluated.

 

 

Results

Complete blood counts revealed no significant findings. A representative readout is shown:

 

 

 

Liver toxicity was assessed by measuring serum levels of liver enzymes AST and ALT. No significant findings were noted. Representative results are shown:

 

Finally, all cause mortality was tracked over time. Raw data is shown. The initial death of the first olive oil mouse is attributed to complications associated with Kg1a injection, but has not been adjusted.

 

 

 

Discussion

As measured by median lifespan, c60oo appears to provide a dose-dependent protective effect against all-cause mortality in this model of acute myeloid leukemia. Tumor burden was evaluated by assessing peripheral blood by flow cytometry for the presence of Kg1a. Though effective at observing the presence or absence of Kg1a (preliminary studies showed that we could detect less than 1% Kg1a cells in mouse whole blood), we found that flow cytometric analysis of peripheral blood was an unreliable quantitative marker, and that because Kg1a initially localizes to the bone marrow, it was not detectable in peripheral blood until approximately 30 days post-administration. This suggests that future studies would benefit from the quantification of tumor burden by measuring Kg1a in bone marrow directly. This will be an important future metric because we are currently unable to determine whether the effects observed, if true, are mediated by direct cancer killing, retardation of cancer proliferation, or some sort of parenchymal protective effects. These questions need to be answered in future studies. While encouraging, it is important to note that this study was performed with a small cohort (n=5 per group), so additional studies are needed to confirm these findings. Further, cancer modeling in mice may not necessarily be representative of native processes in vivo, so care should be taken when attempting to extrapolate the results of this pilot study in the context of human pathology.

[Turnbuckle]

To be clear, this was a single oral dose of C60/EVOO dissolved in the Baati fashion?

[Metrodorus]

Why are the up-down lines on graph " All Cause Mortality Over Time" not vertical? This will, if I am not mistaken, introduce error, and push the data to the right of the graph. Is this an artifact of the software?

[Invariant]

Very encouraging! While the sample size is tiny, the results appear to point in the same direction as Baati's. The life extensions seen here is much smaller than what was seen in Baati, but that is to be expected for a cancer study. Here is the graph from the corrigendum to Baati et al. (note that the x-axis denotes months whereas kmoody's graph has days)

 

To be clear, this was a single oral dose of C60/EVOO dissolved in the Baati fashion?

 

Not quite; from the original research proposal:

 

 

The study will feature two control groups, one treated with phosphate buffered saline, and the other treated with olive oil. Low (1 mg/kg), medium (4 mg/kg), and high (8 mg/kg) treatment doses of c60oo will be administered to three experimental groups. For all conditions, treatment will occur every two days for six days, then once per week thereafter

 

C60oo is prepared as previously described [Baati et al.]. Briefly, 50mg c60 (purity >99.8%) from SES Research Corporation is dissolved in 10 mL virgin olive oil by stirring for 2 weeks at room temperature in the dark, then centrifuged at 5,000 G for 1 hour. The resulting supernatant is passed through a 0.25um porisity filter. Experimental animals will receive C60oo by intraperitoneal injection. Controls will receive either phosphate buffered saline or olive oil by intraperitoneal injection.

 

For comparison, here is what Baati et al. did in their chronic toxicity study:

 

 

The rats were housed three per cage and acclimated for 14 days, before dosing. Three groups of 6 rats (10 months old, weighing 465?31 g) were administered daily for one week, then weekly until the end of the second month and then every two weeks until the end of the 7th month, by gavages with 1 ml of water or olive oil or C60 dissolved in olive oil (0.8 mg/ml), respectively

 

 

@kmoody: it seems like there are quite a few details that people would love to hear (e.g.: how much oil was in each dose?). Will we see a more detailed writeup of the methods and results? It would also be nice to leave a message on the indiegogo page to make sure that the supporters see the results.

 

Links:

project idea thread: http://www.longecity...ration-in-vivo/

full research proposal: http://www.longecity...oposal2014A.pdf

crowdfunding page: https://www.indiegog...n-a-mouse-model

 

Edited by Invariant, 26 April 2015 - 11:21 PM.

[kmoody]

To be clear, this was a single oral dose of C60/EVOO dissolved in the Baati fashion?

See Invariant's reply. Our preparation of c60oo and administration is consistent with our original proposal.

 

Why are the up-down lines on graph " All Cause Mortality Over Time" not vertical? This will, if I am not mistaken, introduce error, and push the data to the right of the graph. Is this an artifact of the software?

This is an artifact of me just doing a quick graph in Excel and not yet constructing a proper mortality curve.

 

@kmoody: it seems like there are quite a few details that people would love to hear (e.g.: how much oil was in each dose?). Will we see a more detailed writeup of the methods and results? It would also be nice to leave a message on the indiegogo page to make sure that the supporters see the results.

Agreed on all points, and also thank you for fielding some of these other questions for me. Regarding your question, the doses were based on mouse weight so varied for each mouse at each time point. The upper limit of the range was ~45uL.

[Turnbuckle]

I see now that this wasn't oral but intraperitoneal injection, but I'm still not clear on how much oil the rats in the various groups were given. Was it the same in all except for the saline group, or did the olive oil dose vary?

[kmoody]

For each dosing, the olive oil control group received a volume equal to the largest dose by volume received by any of the c60oo mice. So the actual amount varied, but the general range was approximately 30-45uL depending on the day.

[Turnbuckle]

For each dosing, the olive oil control group received a volume equal to the largest dose by volume received by any of the c60oo mice. So the actual amount varied, but the general range was approximately 30-45uL depending on the day.

 

 

Doesn't that add a confounding variable, considering that phenol components of EVOO have known anti-cancer properties?

 

Virgin olive oil phenols inhibit proliferation of human promyelocytic leukemia cells (HL60) by inducing apoptosis and differentiation.

http://www.ncbi.nlm....pubmed/16484533

 

[tunt01]

Interesting results.  If you want someone to put the data in R, let me know.  

 

Who choose this mouse model (immuno compromised leukemia) and why?

Edited by prophets, 27 April 2015 - 01:24 PM.

[aribadabar]

These results are so baffling:

 

Saline controls fared much better than 4mg C60oo group, close to EVOO group. EVOO group is almost on par with 1mg C60 group.

 

If the 8mg group did best (signifying C60oo had protective effect) and the 1mg one finished second how the 4mg one fared the worst of all (if there is a supposed dose-response relationship)??

This doesn't correspond to the clearly delineated mortality curves in Baati between saline , EVOO and C60oo groups as posted above.

 

 

It looks like megadosing C60oo was a good idea and anything else is statistical noise (due to the small sample size) not to be relied upon for any conclusions.

[Mind]

Small n=not much statistical power. Larger studies cost more money (which is in short supply for much anti-aging research).

[Turnbuckle]

The EVOO control group looks so much worse that I wonder if the control oil wasn't filtered and thus contained pathogens that killed some of these immunocompromised mice. In fact, even filtered oil might become recontaminated unless care is taken.

[kmoody]

 

For each dosing, the olive oil control group received a volume equal to the largest dose by volume received by any of the c60oo mice. So the actual amount varied, but the general range was approximately 30-45uL depending on the day.

 

 

Doesn't that add a confounding variable, considering that phenol components of EVOO have known anti-cancer properties?

If the olive oil control performed well then yes, it could. However, these results are not consistent with a contribution from olive oil.

 

 

Who choose this mouse model (immuno compromised leukemia) and why?

I chose this model because, 1) low learning curve for my team (i.e. we could do this study for the community inexpensively), and 2) I prefer to use human cells whenever possible.

 

These results are so baffling:

 

Saline controls fared much better than 4mg C60oo group, close to EVOO group. EVOO group is almost on par with 1mg C60 group.

 

If the 8mg group did best (signifying C60oo had protective effect) and the 1mg one finished second how the 4mg one fared the worst of all (if there is a supposed dose-response relationship)??

This doesn't correspond to the clearly delineated mortality curves in Baati between saline , EVOO and C60oo groups as posted above.

 

 

It looks like megadosing C60oo was a good idea and anything else is statistical noise (due to the small sample size) not to be relied upon for any conclusions.

A few things to keep in mind, 1) Baati provided lifespan data in rats whereas we did an aggressive cancer model in immunocompromised mice, so these are not comparable studies in any way, 2) the sample size is VERY low (n=5 per group), so modest effects are not going to be seen in this study, 3) we were looking to see if there was a dose dependent relationship (we never use words like "supposed to be" in science) and based on median survival (which is probably the best place to compare the groups in such a small study) we DO see a dose-dependent effect, and 4) for the sake of redundancy I will reiterate that the sample size is VERY low.

 

This data may tentatively suggest there could be value in taking slightly elevated doses of c60oo if you have AML. These results cannot be ubiquitously applied to all cancers, and the chronic effects of high level c60oo supplementation have not been studied in any model that I am aware of. You may speculate all you want, but be careful what definitive statements are drawn from this study.

 

The EVOO control group looks so much worse that I wonder if the control oil wasn't filtered and thus contained pathogens that killed some of these immunocompromised mice. In fact, even filtered oil might become recontaminated unless care is taken.

Control oil was filtered (prepared identically to c60oo except that c60oo was not present). Syringes for injection were drawn up in a certified laminar flow hood (aka aseptic environment). So I am doubtful that this is the case, though we did not explicitly test for it.

[AgeVivo]

Hello, I tried to compute some p_values. Nothing is statistically significant. Would it be worth/accessible repeating the experiment ? 

 

(Details: the only statistically significant difference I find is between 1 mg/kg and 8 mg/kg of c60oo, but this is extreme cherry picking [=wrong] and asking more globally [=good] if any curve differs from the others returns no statistical difference: R code:

library(Survival)

Saline=c(39,41,44,44,92);OO=c(2,26,31,73,99);CO1=c(20,34,54,82,100);CO4=c(34,34,57,70,70);CO8=c(72,74,83,88,105);#estimated from the graph

survdiff(Surv(c(Saline,CO8), (1:10)*0+1) ~ c((1:5)*0+1,(1:5)*0+2), rho=0);#logrank pvalue=0.173 > 0.05

survdiff(Surv(c(Saline,OO[2:5],CO8),(1:14)*0+1) ~ c((1:9)*0+1,(1:5)*0+2), rho=0);#pvalue=0.169 > 0.05

survdiff(Surv(c(Saline,OO[2:5],CO1,CO4,CO8),(1:24)*0+1) ~ c((1:5)*0+1,(2:5)*0+2,(1:5)*0+3,(1:5)*0+4,(1:5)*0+5),rho=0)#pvalue=0.32 > 0.05 : globallly speaking, nothing special),(1:14)*0+1

 

[kmoody]

Hello, I tried to compute some p_values. Nothing is statistically significant. Would it be worth/accessible repeating the experiment ? 

At n=5 per group that doesn't surprise me. If you do a power analysis how many mice would be needed per group to obtain statistical significance between the 8mg/kg and controls? Might be worth repeating... the hard work (method development) is done. Only cost would be some labor and consumables cost. We should also look at bone marrow to correlate tumor burden with lifespan outcomes. We attempted to do this by flow with peripheral blood but kg1a stays in the bone marrow until late in the disease so the data was very messy. Bone marrow would be better.

[niner]

The computed p's don't totally make sense to me.  Comparing saline and C08, for example, in 4 out of five datapoints, the C08 lifespan was practically double that of saline.  that certainly looks significant...  I don't know the inner workings of R's survdiff function, but the saline vs C08 case doesn't seem right.  I wonder what assumptions survdiff() makes?

 

Would it be hard to run the Kaplan-Meier estimator to see what that looks like?

[tunt01]

Is there any way we can get the raw data to run some KM stats? Thanks

[AgeVivo]

 If you do a power analysis how many mice would be needed per group to obtain statistical significance between the 8mg/kg and controls? Might be worth repeating... the hard work (method development) is done. 

Survival:

- If we make the assumption that indeed 8mg/kg doubles the lifespan (hypothesis), then indeed simply repeating once the experiment is largely sufficient.

- If you want to statistically prove that OO, 1 and 4 mg/kg do not affect the lifespan by more than say 20%, then we need 25 mice per those groups, but I don't think that's what we want

- I would suggest to repeat exactly the same experiment with N=5 but while adding 6 and 10 mg/kg. I am sure you had such a view in mind

With the later, in terms of stats we could then

i) group 8-10 or 6-8-10 results (depending on what consecutive doses appear to have large survivals): logrank between Saline+OO and C6-6/8/10 (*)

ii) estimate a simple dose-response effect (eg proportional hazard cox statistical analysis)

 

Bone marrow:

Do you have an estimate of the variability you have? With rules like "multiplying N by 4 should divide the variability (standard-error of the estimation) by 2" you can estimate the required N. If the bone marrow taken from the same mice that do the survival test? I would naively think that it is better to first redo the survival experiment [as for now we only have vague results that might be confirmed or not and that such a survival experiment is not so long to perform now] and then consider the bone marrow approach. But you may have constrains of time/people/funding that lead to some other choice.

 

Once that (*) or another design of the experiment and its stastistical analysis is chosen,

it is possible to check/adjust/quantify/optimize the power analysis and the N to choose in a very concrete way:

- to simulate the experiment 100k times (eg in R or Excel) with an underlying theoretical survival function of the mice: and to check that

- i) if all groups have in fact the same underlying risks, then in less than 5% of the simulations we have a false positive

- ii) if say 6 8 10 mg/kg respectively increase the lifespan by 30% 70% 100% measure in what portion of the simulations ("statistical power") the logrank test finds that indeed the mice with 6/8/10 live longer than Saline+OO.

I describe the technique so that you/someone does it/learns the technical, but I can do it: let me know.

 

The computed p's don't totally make sense to me.  Comparing saline and C08, for example, in 4 out of five datapoints, the C08 lifespan was practically double that of saline.  that certainly looks significant...  I don't know the inner workings of R's survdiff function, but the saline vs C08 case doesn't seem right.  I wonder what assumptions survdiff() makes?

Hi niner, survdiff(, rho=0) is the logrank test, quite the basic golden standard as you surely know

 

A. More explanation on what I did.

If you look at the code above you will notice that I actually showed 3 tests:

i) 8mg/kg vs saline, because that's what one immediately wonders. // Here a Wilcoxon test survdiff(, rho=1) would provide a better pvalue because the curves are nicely decayed but still >5% and that's quite cherry picking the test based on the results..

ii) a general test of whether it would be strange that all groups have actually the same risks. Answer: no. pvalue=0.33, with various statistical tests. That test itself should be enough

iii) all versus 8 mg/kg: still not statistically significant. It is somewhat an introduction to the following:

 

B. The "supercentenarian" possibility:

If you know "for quite sure" that someone (n=1) is 115 year old, then you can say with a high probability that the person has something special. This is because we have so much data of lifespans that we can somehow model the extremes and find that it is clearly an outlier (low p_value).

Similarly, **If** the protocol has been used many times in the lab and if we know the obtained control curves harldy ever survive more than 70 days **THEN** we can include that past knowledge in the analysis to robustify the control and perhaps consider that 8 mg/kg is statistically significant. That's a question to @Kmoody I guess.

 

(note: in the above code there is a typo: "library(survival)": small s)

 

Is there any way we can get the raw data to run some KM stats? Thanks

Hi prophets, until you have the answer I've estimated the data from the graph: Saline=c(39,41,44,44,92);OO=c(2,26,31,73,99);CO1=c(20,34,54,82,100);CO4=c(34,34,57,70,70);CO8=c(72,74,83,88,105);

[bixbyte]

 

 

For each dosing, the olive oil control group received a volume equal to the largest dose by volume received by any of the c60oo mice. So the actual amount varied, but the general range was approximately 30-45uL depending on the day.

 

 

Doesn't that add a confounding variable, considering that phenol components of EVOO have known anti-cancer properties?

If the olive oil control performed well then yes, it could. However, these results are not consistent with a contribution from olive oil.

 

 

Who choose this mouse model (immuno compromised leukemia) and why?

I chose this model because, 1) low learning curve for my team (i.e. we could do this study for the community inexpensively), and 2) I prefer to use human cells whenever possible.

 

These results are so baffling:

 

Saline controls fared much better than 4mg C60oo group, close to EVOO group. EVOO group is almost on par with 1mg C60 group.

 

If the 8mg group did best (signifying C60oo had protective effect) and the 1mg one finished second how the 4mg one fared the worst of all (if there is a supposed dose-response relationship)??

This doesn't correspond to the clearly delineated mortality curves in Baati between saline , EVOO and C60oo groups as posted above.

 

 

It looks like megadosing C60oo was a good idea and anything else is statistical noise (due to the small sample size) not to be relied upon for any conclusions.

A few things to keep in mind, 1) Baati provided lifespan data in rats whereas we did an aggressive cancer model in immunocompromised mice, so these are not comparable studies in any way, 2) the sample size is VERY low (n=5 per group), so modest effects are not going to be seen in this study, 3) we were looking to see if there was a dose dependent relationship (we never use words like "supposed to be" in science) and based on median survival (which is probably the best place to compare the groups in such a small study) we DO see a dose-dependent effect, and 4) for the sake of redundancy I will reiterate that the sample size is VERY low.

 

This data may tentatively suggest there could be value in taking slightly elevated doses of c60oo if you have AML. These results cannot be ubiquitously applied to all cancers, and the chronic effects of high level c60oo supplementation have not been studied in any model that I am aware of. You may speculate all you want, but be careful what definitive statements are drawn from this study.

 

The EVOO control group looks so much worse that I wonder if the control oil wasn't filtered and thus contained pathogens that killed some of these immunocompromised mice. In fact, even filtered oil might become recontaminated unless care is taken.

Control oil was filtered (prepared identically to c60oo except that c60oo was not present). Syringes for injection were drawn up in a certified laminar flow hood (aka aseptic environment). So I am doubtful that this is the case, though we did not explicitly test for it.

 

 

 

I am starting to believe it, that if test subjects have cancer C60 has little or no benefit.

C60 in Olive Oil has been shown to possess prophylactic properties.

Whatever is the efficacy, it appears to be working in the spleen.

[aribadabar]

To AgeVivo, niner, Turnbuckle and the other experienced researchers:

 

Can one scientifically draw any definitive conclusions given:

 - the perplexing results

 - small sample size

 - the genetically immunocompromised (SCID) type of mice used in the experiment ( i.e. isn't it too big of a burden for the (genetically crippled) test animal and C60oo to overcome/retard a cancer progression with such odds stacked against them)

 - I.P. injection instead of oral gavage was the used method of delivery (to somewhat approximate the Baati study design)

 

Serious question.

 

Thanks!

[niner]

Can one scientifically draw any definitive conclusions given:

 - the perplexing results

 - small sample size

 - the genetically immunocompromised (SCID) type of mice used in the experiment ( i.e. isn't it too big of a burden for the (genetically crippled) test animal and C60oo to overcome/retard a cancer progression with such odds stacked against them)

 - I.P. injection instead of oral gavage was the used method of delivery (to somewhat approximate the Baati study design)

 

Well, I don't think the results are all that perplexing, just noisy.   Also, the two lower doses didn't show much effect in this model.   If the sample size were larger, then the noise wouldn't be so much of a problem.  

 

If you want to look at a human cancer line, then you have to have an immunocompromised host.  A syngeneic model, where the cancer cells were genetically the same as the host, would be better in many regards, but that would rule out using human cells.   It's true that SCID mice can't fight a cancer quite as well as a normal mouse, but we're looking for the effect of the treatment.  The controls and the treatment group all had the same immunologic background.

 

I.P. should be fine, based on the acute tox part of Baati's study, where i.p. was used to good effect.  The i.p. dosing was actually better than oral, in their hands.  It's less stressful to the animals, not to mention the experimenter.

[kmoody]

I am starting to believe it, that if test subjects have cancer C60 has little or no benefit.

C60 in Olive Oil has been shown to possess prophylactic properties.

Whatever is the efficacy, it appears to be working in the spleen.

 

I do not think we can speculate as to mechanism of action because we did not perform any sort of histopath or biodistribution to see where c60oo ended up and what it did there.

 

To AgeVivo, niner, Turnbuckle and the other experienced researchers:

 

Can one scientifically draw any definitive conclusions given:

 - the perplexing results

 - small sample size

 - the genetically immunocompromised (SCID) type of mice used in the experiment ( i.e. isn't it too big of a burden for the (genetically crippled) test animal and C60oo to overcome/retard a cancer progression with such odds stacked against them)

 - I.P. injection instead of oral gavage was the used method of delivery (to somewhat approximate the Baati study design)

 

Serious question.

 

Thanks!

 

I agree on all points with niner. This is precisely why the same question needs to be asked many times over in different models. Our model was good because it allowed us to test human cells. However, you correctly point out that there are shortcomings with this "genetically crippled" model.

 

Regarding stats (from previous posts), we have not yet finished them in house and are working on that now. So I reserve comment about whether the results are statistically significant or not.

[Turnbuckle]

 

Whatever is the efficacy, it appears to be working in the spleen.

 

 

 

If enhancing the activity of the spleen increased lifespan, then removing it should do the opposite. However, studies show that people with splenectomies have the same life expectancy, thus while C60 may show up in the spleen, that is not important to longevity.

[bixbyte]

 

 

Whatever is the efficacy, it appears to be working in the spleen.

 

 

 

If enhancing the activity of the spleen increased lifespan, then removing it should do the opposite. However, studies show that people with splenectomies have the same life expectancy, thus while C60 may show up in the spleen, that is not important to longevity.

 

 

 

https://answers.yaho...21234914AAHb50J

[bixbyte]

 

I am starting to believe it, that if test subjects have cancer C60 has little or no benefit.

C60 in Olive Oil has been shown to possess prophylactic properties.

Whatever is the efficacy, it appears to be working in the spleen.

 

I do not think we can speculate as to mechanism of action because we did not perform any sort of histopath or biodistribution to see where c60oo ended up and what it did there.

 

 

 

 

So if you had treated the tests subjects with the C60 first for a while and then inoculated the test subjects with the cancer cell line secondly the tests might have displayed different results?

Edited by bixbyte, 05 May 2015 - 01:57 PM.

[kmoody]

 

So if you had treated the tests subjects with the C60 first for a while and then inoculated the test subjects with the cancer cell line secondly the tests might have displayed different results?

 

Sure, this could cause different results. Not sure whether or not it would, but I can imagine a world where it does.

[bixbyte]

 

 

So if you had treated the tests subjects with the C60 first for a while and then inoculated the test subjects with the cancer cell line secondly the tests might have displayed different results?

 

Sure, this could cause different results. Not sure whether or not it would, but I can imagine a world where it does.

 

 

The same exact experiment and the test subjects are first have fullerenes fully in their bodies and then invade their bodies with cancer.

In in your experiment you introduced cancer and then dosed the test subjects with fullerenes?

I would have given the tests the fighting chance first.

Cancer is not the same as cabon tet poisoning.

But, Respect on your experiments. 

[kmoody]

The same exact experiment and the test subjects are first have fullerenes fully in their bodies and then invade their bodies with cancer.

In in your experiment you introduced cancer and then dosed the test subjects with fullerenes?

I would have given the tests the fighting chance first.

Cancer is not the same as cabon tet poisoning.

But, Respect on your experiments. 

 

You bring up an interesting point here. Of course, this particular experiment was not intended to look at whether or not c60 can play a role in preventing cancer. Recall that our original question was whether c60 promoted, inhibited, or had no impact on proliferation of human cancer, since most work up until this point has been in rodents. Niner correctly pointed out that human and rodent cancers often behave differently, and there were plausible mechanisms by which c60 could be good or bad in the context of human cancer. This is what we wanted to investigate, especially since so many people supplement with c60.

 

I fully agree with you that a preventative model would be worthy of study. In fact, I have had to point out to several people that our results do not suggest people who supplement with c60oo should increase to 8mg/kg or more for an anti-cancer effect. I would expect, as you do, that lower doses may be effective at preventing cancer from occurring in the first place. And I definitely agree that further research looking into that question would be worthwhile.

[Mind]

Sorry for being a little off topic here, but many people are suggesting variations on this study (or baati). If we find scrounge up some $$$, I am sure Ichor would be prepared to start a new round of experiments quickly.

[bixbyte]

Sorry for being a little off topic here, but many people are suggesting variations on this study (or baati). If we find scrounge up some $$$, I am sure Ichor would be prepared to start a new round of experiments quickly.

 

How Much $$$?

Dozen rats or Mice divided each in two groups X 12 = 24 Rats or Mice

Group One C60 dosed 2 mg/per Kilogram for one month and control group dosed on ordinary Saline

Then we introduce same exact human cancer line to all the rats / mouse with a known longevity for controls 

Wait for them to pass away and record the times of death and Chart it

Nothing fancy  

Just a simple experiment.

I personally think a member of SENS could have performed this test with the millions they have in donations.