https://openworks.mdanderson.org/sumexp22/1084/thumbnail.jp

Garza, Nathalie A

Machado, Alyssa M.

Machado, Annette A.

Marszalek, Ethan D.

Meyers, Booke A.

English

OpenWorks @ MD Anderson

BackgroundObjectives.ConclusionsResultsTitle of the Poster Presentation Goes HereAuthors of the Poster Presentation Goes HereInstitutional and/or Graduate School of Biomedical Sciences Affiliation Goes Here Materials and MethodssiRNA experiments:• CCNA2 and Non-targeting controls(NTC) siRNAs were acquired fromDharmacon.• siRNA transfections were performed using DharmaFect formulation tooptimize seeding density for 10 cm plates.• To induce Cyclin A2 protein, HCT116 (human colorectal carcinoma celllines) were treated with aphidicolin at 10uM for 16hrs and 30uM for 4hours and released for 4 hours.• Cells were collected 72 hours later for knock down validation usingwestern blots and cytoblocks.• HCT 116 cell line was used for generation of cyctoblocks given itscommon availability and easiness to transfect.Western Blotting• Cells were lysed and protein concentration was determined via DCProtein Concentration Protocol (BioRad Lab#5000116) as specified.• Absorbance was read using PheraStar reader.• CCNA2, Non-targeting controls, and treated lysates were loaded andrun-on ice- at 60V for 15 minutes and 180V for 40 more minutes.• Gel was transferred via iBlot system, incubated with antibodiesovernight and developed using LI-COR secondaries and read with LI-COR reader (Odyssey CLX).IHC Optimization and 6-color multiplex CDK-Cyclin panel.• Antibodies were first optimized by IHC using positive and negativecontrols, using serial 2-fold dilutions, then optimized in Opal IF usingserial 4-fold dilutions.• The multiplex panel was ordered based on the affinity and pattern ofeach stain, using a serial OPAL protocol.• This process was repeated to include CycE1 and CDK2. CDK1 andHLA (Human Leukocyte Antigen) were developed using anti-mouseand anti-rabbit fluorescent secondaries, respectively.Title of the Poster Presentation Go s HereAuthors of th  Poster Presentation Go s HereInstitutional and/or Graduate Scho l of Biomedical Sciences Affiliation Goes Here Developing an IF panel to examine Cyclin and CDK interactions in a Pancreatic Adenocarcinoma Patient Derived Xenograph.Nathalie Garza¹ , Ethan Marszalek2, Alyssa Machado2, Brooke Meyers3, Annette Machado3¹ Partnership for Careers in Cancer Science and Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas 770302 Summer Internship, TRACTION, The University of Texas MD Anderson Cancer Center, Houston, Texas 770303 TRACTION, The University of Texas MD Anderson Cancer Center, Houston, Texas 77030Cyclins and cyclin-dependent kinases are known for controlling cell-cycle progression through various phases of the cell division cycle.Cyclins coordinate cell division by binding to their associated cdks andmaintain genomic integrity by tightly controlling these processes. Incancer, cellular proliferation becomes dysregulated, implicating bothcyclins and cdks. Complexes of therapeutic interest are Cyclin E1(CCNE1) and CDK2 that complexes during late G1/S phase and CyclinA2 (CCNA2) and CDK1/2 that are essential for S-phase progression.• Validate specific Cyclin A2 antibody• Develop a multiplex Cyclin-CDK immunofluorescent panel.• Observe how Cyclins (CCNA2, CCNE1) interact with cyclin-dependentkinases (CDK2) in pancreatic adenocarcinoma patient derivedxenograph.Figure 2. Immunoblot showing siRNA CCNA2 knock-down efficiency with different cell densities. KD1 wasused to create cytoblocks for further IHC validation.Figure 3. Immunohistochemistry ofHCT116 cells probing Cyclin A2. (A)shows a Non-targeting control (wild-type, low positive expression) (B) issiCCNA2 (negative expression). (C)and (D) show treated HCT116aphidicolin cells with 10uM and 30 uMrespectively (over-expression).Figures 5. Dilution optimization of Opal simplex in PDAC PDX models.Figure 1. Depicts the cell-cycle phases and their associated CDK/cyclin complexes (InternationalJournal of Molecular Sciences, The Emerging Role of Cyclin-Dependent Kinases (CDKs) inPancreatic Ductal Adenocarcinoma)Figure 4. Confirmed optimization of PDAC PDX model is shown by bothlow and high CCNA2 expression.A BC DA B• Multiplex panel of Cyclins and CDKs (complexes) was optimized and developed.• Cyclin and CDK phenotypes were observed and quantified.• Subcellular localization of each cyclin-cdk complex shows baseline state..• Panel will help in identifying possible biomarkers for CDK inhibitors. References1) Balbina Garcia-Reyes, Anna Laura Kretz, Jan-Philipp Ruff, Silvia Von Karstedt, Andreas Hillenbrand, Uwe Knippschild, Doris Henne-Bruns, Johannes Lemke. (2018). The Emerging Role of Cyclin-Dependent Kinases (CDKs) in Pancreatic Ductal Adenocarcinoma. MPDI Open Access Journals. Figure 1. https://www.mdpi.com/1422-0067/19/10/3219/htm2) Neil Johnson, Geoffrey Shapiro. (2010) Cyclin-dependent kinases (cdks) and the DNA damage response: rationale for cdk inhibitor–chemotherapy combinations as an anticancer strategy for solid tumors. NIH Public Access. 1-12 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3957489/3) Bridget Hughesa, Julia Sidorovab, Jherek Swangera, Raymond Monnat, Jr., and Bruce Clurmana. (2013). Essential role for Cdk2 inhibitory phosphorylation during replication stress revealed by a human Cdk2 knockin mutation. Proceedings of the National Academy of Sciences. https://www.pnas.org/doi/10.1073/pnas.1302927110HSP90ActinCyclinA255kD90kD42kDGraph 2. Percent Positive Cyclin & CDKcomplexes. CDK1 and CDK2 partneredwith Cyclin E1 more than Cyclin A2 onaverage. low levels of Cyclin A2 in thePDACPDX model used for the panel.Graph 1. Percent positive cells ofcdk2+, CycE1+, CycA2+, and CDK1in PDAC PDX.Figure 6. Multiplex panel PATX PDX model with CDK2, CycE1, CycA2, CDK1, and HLA biomarkerdetection.1.2.CDK2 RatioCycE1 RatioCycA2 RatioCDK1 Ratio0.00.51.01.52.0Multiplex Cyto:Nuc RatioGraph 3. Quantification analysis of Cytoplasmto Nuclear ratio for Multiplex panel showsCDK1 and Cyclin A2 to have the highest ratioindicating they are more cytoplasmic.

Developing an IF panel to examine Cyclin and CDK interactions in a Pancreatic Adenocarcinoma Patient Derived Xenograph.

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1084&context=sumexp22

Developing an IF panel to examine Cyclin and CDK interactions in a Pancreatic Adenocarcinoma Patient Derived Xenograph.

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