Poster H3 - Triple-negative Breast Cancer Sensitization to Anti-metabolite Chemotherapy Utilizing PARP inhibitor Olaparib

Saturday, October 26, 2024

10:00 AM – 11:00 AM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Abigail Yoo

Undergraduate
Johns Hopkins University, United States

Co-Author(s)

Amanda Rakoski

PhD Student
Johns Hopkins University, United States

Primary Investigator(s)

Joshua C. Doloff, MA, PhD (he/him/his)

Assistant Professor
Johns Hopkins University, Depts. of Biomedical Engineering and Materials Science & Engineering, Translational Tissue Engineering Center (TTEC), and the Institute for NanoBioTechnology (INBT)
Baltimore, Maryland, United States

Introduction: Triple Negative Breast Cancer (TNBC) is known as one of the most aggressive breast cancer subtypes as it grows faster than typical invasive carcinoma, leading to poor prognosis by the time it is detected and an increased chance of recurrence even after elimination. Its lack of three key hormone receptors, estrogen, progesterone, and HER2 receptors eliminates the use of receptor-targeted therapies commonly used to treat breast cancer. The first line of treatment is the use of combination chemotherapies given at Maximum Tolerated Dosage (MTD) but this typically leads to higher toxicity and systemic side effects Therefore, we believe utilizing targeted drugs that can sensitize the tumor to attack by chemotherapy may be a way to lower the amount of chemotherapy needed overall. Here, we investigate olaparib (OLA) and gemcitabine (GEM) as a dual chemotherapy. OLA is an FDA-approved poly-ADP ribose polymerase inhibitor (PARPi) that prevents the repair of single-stranded DNA breakage by preventing PARP from replacing excised nucleotides in the DNA. GEM is an anti-metabolite that prevents cancerous cells from continuing mitosis and increasing apoptosis by taking the place of the nucleotide cytidine in the DNA during replication, leading to cell cycle arrest. We propose sensitizing TNBC to GEM by using OLA at lower doses than MTD while causing significant TNBC tumor cell death compared to the drug alone.

Materials and

Methods: To investigate the efficacy of these two drug therapies, we chose the TNBC cell line EO771 syngeneic to C57BL/6 immunocompetent mice. 24-well cell-culture-treated plates were plated with 100,000 EO771 cells per well. Crystal violet staining was used to visualize the amount of adherent live EO771 cells remaining after being treated with different concentrations (0, 10, or 30 nM) of GEM, OLA, or both. After 24 hours of incubation, all the treated wells were imaged using phase contrast microscopy. The stains were then solubilized using 70% ethanol to measure the absorbance of the stain at 550 nm wavelength, where there is a linear relationship between the amount of cells and absorbance. 6-well plates of EO771s were also set up with 500,000 cells per well was completed which we used to extract RNA and run qPCR to measure gene expression for genes associated with cell stress and cytokine signaling.

Results, Conclusions, and Discussions: We found that GEM at the 30 nM concentration, compared to GEM at 10 nM, was more effective in combination with OLA at 10 or 30 nM to induce increased cell death. The phase contrast images in Figure 1 denote how the E0771s continue to grow across the plate when no treatment is involved while combinations of GEM and OLA at nM concentrations cause cytotoxicity and cellular detachment from the plate. The significant difference is greatest between the positive control and the two treatments with high concentrations of GEM as shown in Figure 2. In Figure 2, GEM 30/OLA 10 and GEM 30/OLA 30 show the most significant cell death in response to combined chemotherapy. GEM 30/OLA 10 interestingly exhibits a slightly lower cell viability percentage than GEM 30/OLA 30. In addition to crystal violet assays and imaging, RNA from these plates was also collected. In Figure 3, TNF-α expression is observably the lowest in the GEM30/OLA10 treated wells. This suggests that GEM is effectively disrupting DNA synthesis. TNF-α promotes cancer cell invasion via MMP9 production, which remodels the extracellular matrix during metastasis. Lower concentrations of OLA and GEM can both be combined to decrease toxicity while allowing GEM to disrupt DNA synthesis. This implies that GEM is the key factor in significantly decreasing TNF-α expressions in the most effective combination therapies. OLA 30 concentrations in combination therapies resulted in higher levels of TNF-α while OLA 10 concentrations demonstrated lower levels of TNF-α. GEM30/OLA10 also showed the lowest percent of cell viability in crystal violet assays. This further emphasizes GEM’s ability to interfere with DNA synthesis by producing di/triphosphate metabolites. In the future, in vivo testing should be done to analyze the tumor microenvironment of the immune system. ELISA tests can be run on proteins of interest and the use of different cell viability tests such as MTT assays and XTT assays (which rely on cell metabolism to determine viability) should be considered to further validate the results from crystal violet.

Acknowledgements (Optional):