Nandini... What is your senior project?

What a great question! I'd like you all to reach way back in time... not too far... just to March. Remember my first post? It kind of explained what I was doing, but "kind of" doesn't cut it. Hopefully these next few paragraphs will give you all a fuller, better, and deeper understanding.

In my last post, I mentioned that the cellular response to the different radiation methods have yet to be investigated.

The University of Arizona Center for Applied Nanobioscience and Medicine has come of with a preliminary study to evaluate these responses.

The question: Is there a difference in cellular response between high single (1x10Gy) and fractionated dose (5x2Gy) radiation therapy?

The high single dose is used to model the hypofractionated regimen and the fractionated dose is used to model the conventional regimen. 

For this study we utilized the three prostate cancer cell lines: 22Rv1, DU145, and PC3. We conducted a gene analysis, clonogenic assay, and proliferation assay. 

The gene a analysis was the CLPA Assay and CE Analysis. 

The clonogenic assay was used to evaluate radioresistance and the proliferation assay was used to evaluate multiplication. 

Time flies when you're having fun!

As the senior project ends and my senior year comes to an end, I'd like to say congrats, thank you and till next time.

Congrats to all the seniors on getting through senior year, the research project, and going to college. These are all amazing achievements. Though my presence on your blogs has been scarce, I have been read little bits here and there when I can and they amaze me every time. You are all truly gifted people and will do amazing things in the years to come. 

Thank you to BASIS Phoenix, Ms. Anderson, Ms. Pajtas, Ms. Q, Dr. Lacombe, Dr. Zenhausern, my fellow seniors, my parents and anyone who helped with this project. This has been a truly amazing experiencing and I am glad that I participated. My knowledge and interest in bio medical engineering and oncology have greatly increased and my determination to achieve my goals and make an impact in this field has as well. The results of the study have been pleasing. We can conclude that there is a difference between single and fractionated dose radiation therapy. This conclusion allows the lab to expand research to a large screening. They will be conducting a proteomic and genomic assays once the cells have been irradiated. This will not occur until June. 

Till next time. If all bodes well, I hope to continue research (maybe in the same lab) over the summer and into college, so I will not be saying good bye to interning and my white lab coat any time soon. To all the seniors and BASIS Phoenix, there is still one month left so the times for goodbyes is not now. 

All in all, I have thoroughly enjoyed this opportunity and am thrilled that I took it. 

We about to do some puzzling!

For those of you who got the reference of my title, I have great respect and for those of you who didn't, I still have great respect, but also a lesson. If you would like to understand my reference, just google New Girl puzzling to experience the funniest 20 minutes of your life.

Now back to the main event.

We have all the puzzle pieces. By this I mean the data and analysis that we collected and completed.

Just to refresh everyone's memory:

What is this data really indicating? 

First off, each cell line, except for DU145 (is that an almond?), has a different gene expression profile between the cell treated with SD and those treated with FD. For 22Rv1, the genes that were differentially expressed between the two cell lines were PARP1, CDKN1A, DDB2, and TRIB2. While for PC3, the genes that were differentially expressed were DEDD, RPA1, and MDM. 

Additionally, the cell lines also exhibit different radioresistance status. For 22Rv1, the SD-treated cells were radiosensitive and the FD-treated cells were radioresistant. While for PC3, the SD-treated cells were radioresistant and the FD-treated cells were radiosensitive. 

This indicates that for each cell line radiation treatments induce different biological responses. 

Furthermore, this indicates the heterogeneity of the prostate tumor and the complexity of radiation treatment.

Whoa, big words.. must be something important!

You are correct! It is. 

These results show us the diversity of the prostate tumor. All the cell lines used in this study were from the prostate tumor and a tumor can consist of one, two, or all of these cell lines. Based on the results, this means that parts of a tumor may respond better to certain types of radiation treatments, while other parts will respond better to another type. This could lead to more personalized approach when treating patients with radiation therapy. 

One thing to keep in mind. There was a major limit on this study. The gene panel used was previously designed for leukocytes and bio-dosimetry. This means these genes were found to be responsive to radiation in white blood cells and respond to different total doses of radiation. This study was for prostate cancer cell lines and utilized the same total dose of 10Gy. 

Even though, the results of this study still indicate that there is a difference in biological response of the cell between the single dose and fractionated dose treatments. The lab will continue this reach by beginning large scale proteomic and genomic screenings. 

Proliferation Assay- Bet you can't proliferate like me.

Another assay we completed was the proliferation assay. This is exactly what it sounds like: an assay that tests rapid reproduction of the cells. We completed this experiment to determine if the type of radiation treatment had an effect on the proliferation of the cells.

Here are the results:

If you looked at these graphs and thought they all look the same, then ding ding ding. You are correct-ish. The results indicated that there is no difference in proliferation between the single dose and fractionated dose treatments in any of the cell lines.

Congrats! We have collected and analyszed all of the data.

YAYAY! We're free, free at last.

Not quite! We still have to figure out what it all means, but for this you.ll have to stay tuned.

Clonogenic Assay- How resistant are you?

The gene analysis is complete. There is a difference, but does it do?

That is where the clonogenic assay comes in. This might sound familiar and that's good because I have talked about them quite a bit. I never fully explained them (anyone else notice the trend... I'm working on it... I promise). The clonogenic assay is a method of testing radiorestance, or how resistant cells are to radiation. you start off by plating a number of cells on a 6-well plate (its a plastic rectangle with 6 wells just in case that was confusing) and then you irradiate them. For our experiment, we had six 6-well plate (for a total of 36 wells...Wow) and the irradiation treatments were from 0Gy - 10Gy. Duplicates of the three samples of the cell line were on each of the plates. This was completed for all the cell lines 4 times.

Here are the results:

Look at that all three cell lines are there this time. Those lines are cool... what do they do?

Solid question. Here's the answer (well almost): The lines indicate the radioresistance of the cells. On the y-axis, we have graphed the surviving factor, or the ratio of the colonies that have survived after treatment. This is the dependent variable for all you high-schoolers out there (that's me). On the x-axis, we graphed the radiation treatment (that's the independent variable). 

Some of you may be wondering how did all of these numbers were calculated. I appreciate your curiosity and applaud your interest, but to save confusion (and possibly effort) I will leave the explaining to the true experts. To find the full protocol, from irradiateng cells to interpreting data, click the link: http://www.nature.com/nprot/journal/v1/n5/full/nprot.2006.339.html. 

If you have read it, but still have questions, please ask. Now back to the show.

For 22Rv1, the cells that received the FD treatment exhibited greater radioresistance, meaning a greater number of colonies from that sample survived radiation. 

For DU145, there was no difference in radioresistance between the cells that received the two treatments. 

For PC3, the cells that received the SD treatment exhibited greater radioresistance. 

I don't mean to cut the party short, but that's it for the analysis of this data. Now before we begin figuring out what it all means there is one more thing to do. You guessed it! More data to analyze. 

Till next time, my friends!

Biostatistic Data- Bet you don't what that is!

Okay! We have compelted the CLPA analysis. Some genes were not expressed and some genes were expressed. Great, but what do we do now? Well, as I mentioned previously, to determine whether there is a significant difference in expression you must complete a biostatistic of your data.

What is that, you say? Here, I got you. Let me explain. Biostatisctics is the branch of statistics branch of statistics that handles the proper interpretation of data generated in health sciences such as biology and public health. Dr. Lacombe completed this analysis with the help of some software (thank you technology).

Here are the results:

Okay... 22Rv1... PC3... but wait one's missing. DU145, what happened?

Well, when running the biostatistic we were looking to see if any genes were deferentially expressed between the single and fractionated dose treatments. DU145, as seen by the lack of a graph, did not have any genes that were differentially expressed.

ATTENTION: This does not mean that DU145 is no longer an important part of the results. Not finding something has just as many implication as finding something.

Now back to the data.

As indicated by the graph above, in the 22Rv1 cell line there were four genes that were differentially expressed between the SD and FD. These genes were PARP1, CDKN1A, DDB2, and TRIB2.

For PC3, there were four genes that were differentially expressed between the two treatments. These genes were DEDD, RPA1, and MDM.

One thing to keep in mind: These that were differentially expressed between the two treatments were different between the cell lines. (Look back and check. It's pretty cool right.)

Here's the data! (well the first part of it)

The main event of this meet was the gene analysis. The differences in gene expression between the single dose and fractionated dose treatments is the main focus of this study.

The CLPA analysis and CE anlysis are the methods we used to analyze the genes. Here are the results:

I apologize for the lack of labels. Here's what you need to know:

• Starting from the left: the first three columns (0,SD, and FD) are 22Rv1
• The middle three (0, SD, and FD) are DU145
• The last three (0,SD, and FD) are PC3

That's great, but what does it mean?

Don't worry! I'm here to explain. 

The panel on the bottom is a legend. ( For everyone who thought of Drake when they read that sentence...same). The green (0) indicates that the gene is not expressed  and the red indicates that the gene is over expressed. 

For the three cell lines, there is a similar gene profile. The genes are expressed and the gradient expression is relatively similar. 

There are 6 genes that are not expressed in any of the prostate cancer cell lines. This is indicated by the light green on the graph. These genes are CD27, CEBPB, PRIM1, BCL11B, PTRCAP, and LAPTM5. 

There are three genes that are over-expressed in the three cell lines. This is indicated by the red or black on the graph. These three genes are RFC4, PARP1, and HIST1H3D. 

On the graph, there is also an indication of some differences in expression between the single dose and fractionated dose treatments for some genes. To determine if these differences are significant, we must do a biostastic. 

What's that you say?

Well stay tuned to find out!

Radiation Therapy (RT): What is it really?

As the amount of work in the lab dies down and the senior project comes to an end, I realize that I have never truly explained (at least in one post) the background of what I am doing and what I am truly doing. Since my posts have been mostly about my experience and day to day in the lab I am changing it up. These next few posts will be information on what I am doing and the results of what I have done.

To start off, a little background.

Radiation therapy. What is it?

Radiation therapy(RT) utilizes high doses of radiation to kill cancer cells and prevent them from spreading. It may be external or internal. External radiation is delivered through a linear accelerator while internal radiation is delivered from a radioactive substance a physician places in your body. This study focus on external radiation.

RT is extremely important to the treatment of cancer prostate patients (really all cancer patients, but since this study is on prostate cancer let's focus on that). 40-60% of prostate cancer patients receive RT as a treatment.

Conventional radiation is delivered in fractions of 1.8-2Gy per fraction. Patients will receive 35-40 fractions in total.

Recently, improvements in radiators and technology used in radiation therapy has allowed for the advent of hypofractionation such as stereotactic RT. This allows for higher doses and fewer fractions, meaning a patient will receive a dose of 7-10Gy per fraction and a total of only 5 fractions.

You might be wondering whether there really is a difference between these two therapies.

Well, there have been several clinical studies to evaluate the difference between the conventional and hypofractionated regimen on patients' outcomes. This includes things such as tumor resistance and overall survival.

Even though, the cellular responses have not been investigated and are still unknown.

This is where we come in. We meaning University of Arizona and me (for three months at least).

It's been so long!

Hello, everybody! (Imagine that was said in Gru's voice.)

It has been a while since I posted, which I apologize for, and a lot has happened, both in and out of the lab.

First off, I got college decisions. March was a month of Madness for college basketball players and seniors all over the country. Even though not all the decisions are what I hoped they would be I am happy and excited with the results. Also, CONGRATS to all the seniors! Even if you did not get into your number one, you all got into amazing places and should be proud! Wherever you go you will do amazing things!


Secondly, the lab has experienced some changes. The amount of work to be done in the cell culture room has greatly declined because Dr Lacombe has throw away the cells and begin with fresh cells for the new proteomic  assay. This has given me time to work on data analysis, which there has been plenty of. Luckily, with the help of my trusty friend excel, I was able to knock it all out in a matter of days. Two to be exact.

Lastly, there are far more problems to encounter in the lab than I realized. When doing labs in school, everything is though out and planned for us. The teacher and the students, if they are really paying attention, know what to expect. Very few real, lab-threatening mistakes are made. But in the real lab it's different and when mistakes are made the consequences are greater.

 For example, in the last clonogenic assay we made (  I will do a much more detailed post about it later) the cells did not get distributed the way hey should have been. There were too many cells in wells where there should have been almost none.

Another example: For the proteomic assay, the new cells have to be irradiated. Dr. Lacombe has been trying for the past two weeks to set up a time with the Mayo Clinic, but due to problems they are facing we are unable to get them irradiated as of now. This has a direct impact on the timeline that Dr. Lacombe had worked out to complete the experiments.

Finally, if anyone remembers, there are two parts to this study. One is determining the cellular response of three different cell lines to different radiation therapies and the second is building a radioresistant cell line to determine biomarkers of radiation. For the second study we have been using just the PC3 cell line and the cells have been very difficult to make radioresistant. The other day, while we were discussing the results of the experiments, Dr. lacombe informed me that he had read an article that said the PC3 cell line was known to be radioresisant already. No wonder it's been so difficult. Now, he is looking into possible changing the cell line he uses in the second study.

All in all, these past few weeks have been long, but exciting. Congrats again to everyone!