https://openworks.mdanderson.org/sumexp23/1119/thumbnail.jp

Amero, Paola

Kavarthapu, Meghana

Lopez-Berestein, Gabriel

Rodriguez-Aguayo, Cristian

Szymanowska, Anna

English

OpenWorks @ MD Anderson

Wound Healing Assay: ID8 and BR-Luc were plated onto 6-well plates for 24 h beforetransfection with IL-27Rα siRNA or control siRNA. After transfection, cells were incubated untilthey reached 100% confluence. Each well was vertically scratched using P200 pipet tip, andcellular debris was removed by washing with PBS. Images were captured at 0, 6, 12, 24, and48 hours depending on cell line. Migration area was calculated by measuring the total distancethat transfected cells migrated and is expressed as percentage migration.Matrigel Invasion Assay: Transwells (Fischer) were coated with Matrigel (BD Biosciences)onto a 6-well plate. ID8 and BR-Luc cells transfected with control siRNA or IL-27Rα siRNAwere suspended in serum-free medium and added into the upper Matrigel-coated transwells.Medium with 10% FBS was added to the lower wells. The plates were incubated for 48h. Thecells were then stained with the HEMA 3-stain system (Fischer) and were counted by usingImageJ software.Tube Formation Assay: 10 μL of Matrigel (BD Biosciences) were added to each well of a15-well angiogenesis plate and incubated for 1hr. RF-24 cells were seeded and transfectedwith either control or IL-27Rα siRNA for. 50 μL of cell resuspension was added onto theMatrigel. The plates were incubated, and tube formation was observed at 16 hours.AbstractIn this study, we utilized a novel explainable artificial intelligence (XAI) approach to identifyInterleukin-27 Receptor α (IL-27Rα) as a potential therapeutic target for high grade serousovarian cancer (HGSC) patients based on the Cancer Genome Atlas (TCGA) dataset.High expression of IL-27Rα was found to be significantly correlated with poorer survivalin HGSC patients. To investigate its therapeutic potential, we silenced IL-27Rα in murineHGSC cells lines using siRNA and assessed its impact on tumor proliferation, migration,invasion, and angiogenesis. Our results demonstrated that IL-27Rα siRNA effectivelyreduced its expression, as confirmed by Western Blot analysis, and significantly attenuatedproliferation by clonogenic assay, migration by wound healing assay, and invasion assay,and angiogenesis by tube formation. These findings support the potential of targetingIL-27Rα as a promising strategy for improving outcomes in HGSC.ConclusionWe have shown that IL-27Rα is associated with poor survival in patients with HGSC.Silencing of IL-27Rα using siRNA in murine HGSC cells reduced colony formation,migration, invasion and tube formation. IL-27Rα is possible therapeutic target for HGSCpatients.HypothesisSilencing IL-27Rα expression leads to anti-tumor activity in murine HGSC cells.AcknowledgementsThis work was supported by the University Outreach Summer Program andthe Lopez-Berestein Laboratory at the MD Anderson Cancer Center.Targeting Interleukin-27 Receptor α in Murine HGSC CellsMeghana Kavarthapu 1,2, Anna Szymanowska 2, Paola Amero 2 , Cristian Rodriguez-Aguayo 2, Gabriel Lopez-Berestein 2 Nova Southeastern University 1, MD Anderson Cancer Center Department of Experimental Therapeutics 2MethodsTCGA Analysis: TCGA Dataset was used to generate a Kaplan-Meyer survival curveusing the database of HGSC patients (GEPIA).Cell Lines: Murine HGSC cell lines ID8 and BR-Luc were cultured in Dulbecco modifiedEagle High Glucose Medium and supplemented with 10% FBS.ID8 was additionally supplemented with 1% penicillin/streptomycin, 0.05% Insulin ST,while BR-Luc with 0.1% gentamicin. Cells were incubated at 37ºC at 5% CO2.Human endothelial cell line RF24 were cultured in MEM medium with 10% FBS, sodiumpyruvate, nonessential amino acids, minimal essential medium vitamins, and glutamine.siRNA Transfection: The IL-27Rα siRNA was synthesized by Sigma-Aldrich. Cellswere transfected with IL-27Rα siRNA or non-silencing control siRNA. All siRNAtransfections were carried out with HiPerfect (Qiagen) according to the manufacturer'srecommended protocol.Western Blot Analysis: Cells were collected and lysed in RIPA buffer with proteaseand phosphatase inhibitors. Protein was quantified using BCA analysis and sampleswere run on 4-15% polyacrylamide SDS gel (Bio-Rad). Protein was transferred topolyvinyl difluoride membrane (Millipore) and blocked for 1h in 6% milk. Primaryantibodies rat IL-27Rα (R&D Systems), mouse β-actin (Cell Signaling Technology),and rabbit p-SRC (Invitrogen) were prepared in 5% milk. Membranes were incubatedovernight at 4ºC with respective antibody. Secondary antibodies anti-rat IgG HRP(ThermoFisher), anti-mouse IgG HRP (Cell Signaling Technology), and anti-rabbit IgGHRP (Cell Signaling Technology), were prepared in 5% milk and membranes wereincubated for 1h at 20ºC. Bound antibodies were visualized using chemiluminescentand colorimetric detection.Clonogenic Assay: Cells were plated onto 6-well plates with 2000 cells per well andthen transfected with control siRNA or IL-27Rα siRNA after 24hr. Plates were incubatedfor 5-6 days . After incubation, the colonies were washed with PBS, stained with crystalviolet, washed until excess crystal violet was removed, and quantified using ImageJ.Preliminary DataIntroductionHGSC has the highest mortality rate among gynecological cancers with 152,000 deathsout of 300,000 confirmed cases each year1. The large mortality of HGSC can beattributed to the lack of symptoms until it has progressed to the later stages. Peritonealspread is the leading cause of death from gynecologic cancers and is associated withabdominal obstruction, intestine, blood vessel obstruction. HGSC first metastasizes to thepelvic organs and blood (Stage 2), then the abdomen (Stage 3), and then beyond (Stage4).Artificial Intelligence (AI) is a set of algorithms that are capable of learning without humaninput and can be used to make new predictions through machine learning2. AI was usedto determine relationships between HGSC development and the tumor microenvironmentby using the TCGA dataset to identify potential therapeutic targets. An ExplainableArtificial Intelligence (XAI) model was generated to interpret the results. According to theXAI model, IL-27Rα is a potential therapeutic target in HGSC patients because the highexpression is associated with a lower 5-year survival rate.Interleukin-27 Receptor (IL-27R) is a heterodimeric cytokine receptor composed ofInterleukein-27 Receptor α (IL-27Rα) and glycoprotein 1303. IL-27R is shown to displaypro-tumor activity. It induces the expression of immune regulatory molecules such as IDOand PD-L1, which leads to immune suppression through decreased T cell response andfunction, and eventually to tumor cell survival and proliferation.Silencing IL-27Rα may be a possible approach to increase survival rates among HGSCpatients.References1) Zhang, R., et al., Molecular Biomarkers for the Early Detection of Ovarian Cancer. Int JMol Sci, 2022. 23(19)..2) Chakraborty, D., et al., Explainable Artificial Intelligence Reveals Novel Insight intoTumor Microenvironment Conditions Linked with Better Prognosis in Patients with BreastCancer. Cancers (Basel), 2021. 13.3) Caveney, N.A., et al., Structure of the IL-27 quaternary receptor signaling complex.Elife, 2022. 11.4) Chakraborty, D., et al., Discovering genetic biomarkers for targeted cancer therapeutics with eXplainable AI. bioRxiv, 2023: p. 2023.07.24.550346.5) Data was generated by the TCGA Research Network at: “https://www.cancer.gov/tcga.”Figure 1. Shapley values graph explaining gene expression in HGSC patients usingXAI model4.IE FBR-Luc ID8(O) Silencing of IL-27Rα in RF-24 tube formation assay (P) and quantification of angiogenesisSilencing IL-27Rα expression leads to decreased invasion in murineHGSCRF-24Silencing IL-27Rα expression inhibited angiogenesis in endothelialcellsResultsHigh IL-27Rα expression is correlated with lower survival rates of HGSCpatientsFigure 2. (A) IL-27Rα is overexpressed in HGSC tissue compared to normal tissue.(B) High expression of IL-27Rα is associated with lower survival rate in HGSC patients5.A BSilencing IL-27Rα by siRNA leads to decreased expression of IL-27Rα inID8 murine HGSCFigure 3. (A) Silencing of IL-27Rα by Western Blot and (B) Densitometric analysis of  IL-27Rα expression of Western Blotβ-actinIL-27Rα1.00 0.81 0.60(IL-27Rα/β-actin)/NT IL-27RαSilencing IL-27Rα expression leads to decreased migration in murineHGSCObjectives• To determine the effect of silencing IL-27Rα in murine HGSC cells• To examine the effect of silencing IL-27Rα on tumor migration, invasion, and colonyformation in murine HGSC cells and angiogenesis in endothelial cellsDBCAsiIL-27RαNT siControlID8BR-LucSilencing IL-27Rα expression leads to decreased proliferation in murineHGSCFigure 4. (A and B) Silencing of IL-27Rα in BR-Luc and ID8 clonogenic assays (C and D) and quantification of proliferationβ-actinp-SRC1.00 0.61 0.33 1.00 0.43 0.18(p-SRC/β-actin)/NT p-SRCG HJFigure 5. (E and F) Silencing of IL-27Rα in BR-Luc and ID8 wound healing assays (G and H) andquantification of migration (I) Silencing of IL-27Rα by Western Blot and (J) Densitometric analysis ofp-SRC expression of Western BlotNTsiControlsiIL-27RαK LM NID8BR-LucFigure 6. (K and L) Silencing of IL-27Rα in BR-Luc and ID8 Matrigel invasion assays (M and N) andquantification of invasionBAPNT siControl siIL-27RαO

Targeting Interleukin-27 Receptor α in Murine HGSC Cells

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1119&context=sumexp23

Targeting Interleukin-27 Receptor α in Murine HGSC Cells

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