https://openworks.mdanderson.org/sumexp21/1158/thumbnail.jp

Ben Kacem, Mariam

Flint, David

Manandhar, Mandira

Martinus, David

Moran, Emma R.

Sawakuchi, Gabriel

Shaitelman, Simona

English

OpenWorks @ MD Anderson

0 Gy X-rays (5Gy) Proton (5Gy)0.000.020.040.060.08HCC1937Senescent cells (% total cells)✱✱✱ns0 Gy X-rays (5Gy) Proton (5Gy)0.000.020.040.060.08HCC1937-BRCASenescent cells (% total cells)0 Gy H2O2 X-rays (5Gy) Proton (5Gy)0.000.010.020.030.040.054T1Senescent cells (% total cells)Is proton radiation more effective than photon radiation at inducing senescence?Emma Moran, Mariam Ben Kacem, David Flint, David Martinus, Mandira Manandhar, Simona F. Shaitelman and Gabriel O. SawakuchiDepartment of Radiation Physics, The University of Texas  MD Anderson Cancer Center,  Houston, TXReferences(1) Breastcancer.org. (2021, February 4). U.S. breast cancer statistics.Breastcancer.org.https://www.breastcancer.org/symptoms/understand_bc/statistics.(2) National Cancer Institute at the National Institute of Health. (n.d.). BRCA geneMutations: Cancer risk and genetic testing fact sheet. National Cancer Insti tute.https://www.cancer.gov/about-cancer/causes-prevention/genetics/brca-fact-sheet.(3) Campisi, J., &amp; d'Adda di Fagagna, F. (n.d.). Cellular senescence: Whenbad things happen to good cells. Nature News.https://www.nature.com/articles/nrm2233.(4) Debacq-Chainiaux, F., Erusalimsky, J. D., Campisi, J., & Toussaint, O. (2009).Protocols to detect senescence-associated beta-galactosidase (sa-βgal) activity, abiomarker of senescent cells in culture and in vivo. Nature Protocols, 4(12), 1798–1806. https://doi.org/10.1038/nprot.2009.191Acknowledgements: This presentation is supported by the National Cancer Institute through the U54 CA096297/CA096300: UPR/MDACC Partnership for Excellence in Cancer Research Training Program. For further information, please contact Emma Moran at emoran1@mdanderson.orgMethods (continued)2) Flow Cytometry⁴• Plate cells in T12.5 flasks.• Irradiate with 5 Gy protons (9.9 keV/µm) and photons (6 MV X-rays) ortreat with H2O2 (200-400 µM) for 1-2h at 37ºC when cells are sub-confluent.• Allow 7 days for senescence to develop, then prepare cells for flow cytometry:• 1 hour incubation with bafilomycin A1 to get rid of background senescence (replicative senescence)• 2 hour incubation with C12FDG (5-Dodecanoylaminofluorescein Di-β-D-Galactopyranoside), a substrate of SA β-gal that fluoresces when cleaved•Run the samples in the flow cytometer Hypothesis We hypothesized that protons would cause greatersenescence than X-rays for the breast cancer cell lines4T1, HCC1937, and HCC1937-BRCA.Figure 3. The graphs above show the percent of senescent cellsfor 4T1 (A), HCC1937 (B), and HCC1937-BRCA (C) at varyingradiation types and doses. Each bar represents the meanof independent experiments (2 replicates for 4T1 and HCC1937-BRCAand 4 for HCC1937). Statistical analyses were performed on HCC1937only (one way Anova: * p<0.05: ** p<0.001 compared to 0 Gy).Results (continued) DiscussionWe are still working on optimizing the X-gal staining protocol. Thecurrent images are inconsistent and difficult to highlight differencesby microscopy. We are trying to obtain clearer images by usingglass coverslips and decreasing the seeding number.Further replicates of these experiments are currently beingconducted. Based on the current cytometry results, it appears thatprotons are more effective at inducing senescence for 4T1 but X-rays are more effective at inducing senescence for HCC1937-BRCA. We have significantly more senescent cells for HCC1937after radiation compared to 0 Gy but no differences after protonand X-rays groups.We expected protons would induce greater senescence becausethey are more massive than photons and have a higher linearenergy transfer (LET), resulting in more double strand breaks andthus greater replicative stress.After completing our data sets in vitro, we plan to designexperiments to evaluate our results in vivo. We hope that our invitro results are also reflected in vivo and that greater senescenceresults in better tumor control.BackgroundBreast Cancer is …• the most diagnosed type of cancer for women in the United States¹.• the 2nd most deadly type of cancer for women in the United States¹.BRCA = BReast CAncer genes• BRCA1 and BRCA2 encode proteins that repair damaged DNA,preventing cancer from developing – harmful mutations prevent thisprocess and cause cancer2.• Everyone inherits 1 copy of BRCA1 and BRCA2 from each parent -harmful BRCA mutations can also be inherited2.• We use 2 human breast cancer cell lines: HCC1937 (BRCA-mutated), HCC1937-BRCA (BRCA-restored), and 1 mouse breastcancer cell line: 4T1.Senescence = a state of permanent cell-cycle arrest3• Replicative senescence – caused by telomere shortening overmany replication cycles.• Stress-induced senescence – triggered by some type of externalstress on the cell (such as radiation).Radiotherapy• Ionizing radiation – protons or photons – damages DNA and canresult in apoptosis or senescence:Results: Flow Cytometry Figure 4. Images obtained from X-gal staining. The small dark spotsclustered within cells are the insoluble clue compounds released by X-gal (shown by the red arrows), and the cells that they are clusteredwithin are senescent cells (shown with blue arrows). Images are of 4T1irradiated with 0 Gy (A) and 10 Gy (B).Figure 1. Cytometry plots for non-irradiated 4T1 cells. Bounds on graph (A)isolate 4T1 cells (remove debris). Bounds on graph (B) select only living 4T1 cells.In graph (C), the top 2 quadrants contain senescent cells. For the non-irradiatedsample, senescence is set to 2%, and irradiated samples are analyzed relative tothe non-irradiated sample. Data represent one independent experiment.MethodsSenescence-associated β-galactosidase (SA β-gal) is a biomarker ofcellular senescence, and we used two methods of SA β-gal detectionto identify and quantify senescent cells.1) Histochemical Assay⁴• Plate cells in 6-well plates using glass coverslips.• Irradiate with 10 Gy protons (9.9 keV/µm) and photons (6 MV X-rays) when cells are sub-confluent.• Allow 7 days for senescence to develop, then stain cells with theInvitrogen β-gal Staining Kit, which uses:• X-gal = substrate of SA β-gal that releases an insolubleblue compound when cleaved. Senescent cells detectedby presence of blue spots.Results: X-gal Staining Created using BioRender(A) (B) (C)(A)(B)(C)(A) (B)Figure 2. Cytometry plots of 4T1 non-irradiated samples (red) overlaid with 5 Gy X-rays (A), and 5 Gy protons (B) in blue. Each bi-parametric representation(Size (FSC)/C12FDG (FL-1)) represents one independent experiment for at least5x105 living cells.(A) (B)

Is proton radiation more effective than photon radiation at inducing senescence?

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Is proton radiation more effective than photon radiation at inducing senescence?

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