Optimizing Small Cell Lung Cancer Treatment: Synergistic Effects of Chemo and Epigenetic Inhibitors

Transcription:

Hi, my name is Kavishi Udumullage, and I am a grade 12 student from Winnipeg, Manitoba. What if I told you that cancer treatment didn’t have to come at the cost of a patient’s quality of life?

Today, small-cell lung cancer remains one of the most aggressive and deadly cancers we know. The current reality is harsh: patients are treated with extremely high doses of chemotherapy, not because it’s ideal, but because it’s often the only way to slow the disease. The trade-off is severe toxicity, organ damage, and life-altering side effects.

I investigated small-cell lung cancer cells using combinations of two standard chemotherapy drugs, carboplatin and etoposide, paired with epigenetic inhibitors. Instead of increasing dosage, I explored whether drug synergy could amplify effectiveness at lower concentrations. Using LOEWE synergy analysis, I found strong drug synergy at specific concentrations, meaning the drugs worked better together.

In some cases, this reduced cancer cell viability by up to 50–60% using significantly lower doses.

What this suggests is a shift toward combination-based precision therapy, where we don’t just work harder, we attack smarter, reducing toxicity while maintaining effectiveness.

Thankyou

Introduction:

Lung cancer is the leading cause of cancerous deaths, with Small Cell Lung Cancer (SCLC) accounting for 15% (Rudin, 2021). Currently, people combating SCLC have limited access to successful treatment options, due to the highly aggressive division and spread of this cancer throughout the body’s healthy tissue. Lung cancer cases are linked to high levels of smoking, toxic chemical exposure, and genetic factors.

Chemotherapy for lung cancer uses two common drugs: Carboplatin and Etoposide. Both have significant mechanisms of action altering DNA structure and disrupt its replication process. They gain antitumor abilities to decrease the spread and inhibit the growth of lung cancer. Although effective, they are often administered at high dosages to fully treat the cancer, therefore given through cycles with recovery periods between treatments, causing patients to experience severe side effects like nerve damage, suppressed blood cell production, and hair loss. Highlighting why further study of targeted treatments is essential.

Epigenetic inhibitors are drugs designed to reverse abnormal gene expression in cancer cells by targeting enzymes responsible for epigenetic modifications. The two used in this project were Entinostat and Seclidemstat. Both disrupt cancer cell metabolism, alter gene expression, and suppress tumor growth, making cells more sensitive to chemotherapy.

Combining targeted treatments with conventional chemotherapy offers a more effective approach by attacking cancer through complementary mechanisms, reducing drug resistance.

Therefore, this project helps to determine whether combining chemotherapy drugs with epigenetic inhibitors reduces the dosage needed to kill SCLC while increasing effectiveness and minimizing harmful side effects.

Experiment Procedure:

1. H209 cells were received from a SCLC patient and were grown at 37°C for 3-5 days

2. Prepare a serial dilution of Drugs:

2.1: Use Dimethyl Sulfoxide to dissolve all drugs.

2.2: Prepare 7 test tubes with appropriate drug concentrations (Image 1)

2.3: Perform a 1:2 serial dilution (Image 1)

3. Counting the cells:

3.1: Collect a sample of H209 cells.

3.2: Mix the cell suspension, ensuring even distribution.

3.3: Mix a 1:1 ratio of the cell suspension and Trypan Blue, allowing the dye to stain dead cells.

3.4: Load 10-20µL into the automated cell counter chamber and run the process.

3.5: Note down the total cell count, viable and dead cells, and the concentration.

3.6: Calculate the number of cells needed for seeding.

4. Seed the cells → 100 µL of cells/well (approx. 20,000 cells/well, Image 2):

4.1: Label the 96-well plate with concentrations of Drug A and Drug B

4.2: Fill 64 wells with 100 µL of cells per well.

4.3: Fill each row with Drug A at different concentrations of the serial dilution.

4.4: Fill each column with Drug B at different concentrations.

5. Incubate the drug-treated cells for 96 hours (Image 2).

6. Add Alamar Blue to each well (25 µM/well).

7. Visualize the cells using a Fluorescence Reader.

8. Repeat this experiment for each drug combination.

9. Analysis was conducted using the Loewe synergy analysis

LOEWE analysis:

The LOEWE analysis evaluates whether drug combinations are synergistic, antagonistic, or additive using dA/DA + dB/DB = 1. Scores >10 (blue) indicate synergy, <0 (red) indicate antagonism, and 0–10 (yellow) represent additive effects, where drugs neither enhance nor interfere with each other

Results:

The synergy of drug combinations in Dose-response H209 cells was assessed using the LOEWE score, with varying results based on the drugs and concentrations tested. This method is used in drug interaction studies to determine whether two drugs act synergistically, antagonistically, or additively.

Synergistic combinations: x>10, appear as blue on the heatmap; drugs enhance each other's features

Additive combinations: 0<x>10, appear as yellow on the heatmap; drugs stay the same

Antagonistic combinations: x<0, appear as red on the heatmap; drugs are interfering with each other

Carboplatin & Entinostat (CE-3T, Figure 1):

The highest synergy was observed with 100nM of Carboplatin and 10nM of Entinostat (LOEWE score = 24), indicating a strong enhancement of each drug’s effectiveness.

This combination led to a 50% reduction in cell viability, suggesting effective inhibition of H209 cell growth.

Lower synergy scores were seen at higher concentrations (500-2000nM of Carboplatin and 10-30nM of Entinostat), where the drugs exhibited additive or antagonistic effects.

Etoposide & Entinostat (EE-3T, Figure 1):

The highest synergy was found with 100nM of Etoposide and 30nM of Entinostat (LOEWE score = 22), leading to a 40% reduction in cell viability.

The lowest synergy score (-5) occurred at 3000nM of Entinostat and 25nM of Etoposide, showing antagonism. Etoposide-Entinostat exhibited more instances of antagonism, particularly at higher concentrations.

Carboplatin & SP-2577 (CS-3T, Figure 2):

The highest synergy (LOEWE score = 17) was observed with 250nM of Carboplatin and 10nM of SP-2577, resulting in a 40% reduction in cell viability.

At higher concentrations (2000nM Carboplatin and 3000nM SP-2577), antagonism was observed. Carboplatin-SP2577 showed fewer instances of antagonism.

Etoposide & SP-2577 (ES-3T, Figure 2):

The highest synergy (LOEWE score = 19) was observed with 50nM of Etoposide and 50nM of SP-2577, leading to a 60% reduction in cell viability.

The lowest synergy score (-8) occurred at 25nM of Etoposide and 250nM of SP-2577, suggesting antagonism. Etoposide-SP2577 exhibited several instances of antagonism.

The highest synergy for each combination was observed at specific concentrations, with Carboplatin-Entinostat (CE) showing the most consistent enhancement of drug effectiveness. Other combinations, such as Etoposide-Entinostat and Carboplatin-SP2577, demonstrated antagonistic effects at certain concentrations, emphasizing the importance of concentration ratios in maximizing drug synergy.

Single drug curves analysis (Figure 3):

The drug curves show how the different drug doses of purely carboplatin and etoposide affect cell survival.

The IC50 value expresses the dose needed to kill half the cells; the lower the IC50 value, the stronger the drug is.

These curves allow us to compare our current results with drug combinations to how the single drug works.

Bar graphs 1 and 2:

Carboplatin used alone requires a higher dosage to kill 50% of the cancer cells in comparison to when using carboplatin in combination. The required dosage dropped significantly, while still maintaining the same effectiveness.

Bar graphs 3 and 4:

Similar results were shown with Etoposide; Etoposide used alone required a much higher dosage to reach 1C50-IC75 levels. While with the combination of epigenetic inhibitors, these levels were reached with a significant drop in drug dosage.

Conclusions:

The experimental results demonstrate that combining chemotherapy agents with epigenetic inhibitors significantly reduced the concentration required to achieve 50% cell viability (IC₅₀) in H209 cells. This reduction in effective dose, while maintaining therapeutic efficacy, indicates a positive synergistic interaction between the two drugs.

The observed synergy suggests that epigenetic modification enhances the sensitivity of cancer cells by altering gene expression patterns within the cells. A comparable effect between both carboplatin and etoposide was seen through multiple trials, proving repeatability of this experiment.

Highlighting that combining epigenetic inhibitors with standard chemotherapy drugs is a strategy to improve treatment outcomes, reduce drug resistance, and minimize systemic toxicity in small-cell lung cancer.

Applications:

These findings are most useful for patients undergoing clinical trials who are diagnosed with SCLC. Because the combination of the epigenetic inhibitors with conventional chemotherapy drugs maintains effectiveness, with a decrease in dosage needed, it minimizes overall toxicity and enhances overall patient quality of life. Therefore, having a reduction in side effects that would occur. With the cycling through of these drug combinations, the body does not develop drug-resistant cells, thereby reducing the number of clinical trials the cancer patient would have to undergo.

Additionally, lowering the dosage of chemotherapy agents can significantly reduce overall treatment costs, allowing resources to be allocated to other aspects of cancer care.

These discoveries represent a promising advancement in the development of more effective and patient-centered cancer therapies.

Exploring additional drug combinations by pairing a single chemotherapy agent with multiple epigenetic inhibitors to observe potential synergistic combinations

Investigating the molecular activity that occurs with these drug combinations would allow for further advancements in this research

Expanding the different SCLC cell lines tested, as well as conducting in vivo studies, will help solidify the efficiency and safety of these combinations

Initiating early-phase clinical trials will be crucial to evaluating the potential of these findings in human patients

Exploring the integration of these combinations with other therapeutic drugs or regimens, such as immunotherapy, to develop more comprehensive/personalized treatment strategies

I express my sincere gratitude to Dr. Joel Pearson for his generous assistance in providing me with lab space at Pearson Labs, which enabled me to conduct this research over the past several months. His valuable guidance and support made this entire experience possible.

I would also like to extend my deepest appreciation to Nivitha Bhaskar for her incredible support, insightful feedback on the experimental design, and invaluable help with data collection. Thank you for your countless hours supervising and assisting me throughout this process. You have truly inspired me and significantly impacted my passion for science.

Lastly, I want to thank my family for their constant motivation and belief in my ability to complete this work. This project would not have been possible without you.

References

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American Cancer Society. (n.d.). Small cell lung cancer stages. https://www.cancer.org/cancer/types/lung-cancer/detection-diagnosis-staging/staging-sclc.html

American Cancer Society. (n.d.). What is lung cancer? https://www.cancer.org/cancer/types/lung-cancer/about/what-is.html

Canadian Cancer Society / Société canadienne du cancer. (n.d.). Lung and bronchus cancer statistics. https://cancer.ca/en/cancer-information/cancer-types/lung/statistics

Cleveland Clinic. (2024, May 1). What is epigenetics? https://my.clevelandclinic.org/health/articles/epigenetics

LUNGevity Foundation. (n.d.). Small cell lung cancer (SCLC). https://www.lungevity.org/lung-cancer-basics/types-of-lung-cancer/small-cell-lung-cancer-sclc

Mayo Clinic. (2024, April 30). Lung cancer. Mayo Foundation for Medical Education and Research. https://www.mayoclinic.org/diseases-conditions/lung-cancer/symptoms-causes/syc-20374620

National Cancer Institute. (n.d.). Carboplatin. https://www.cancer.gov/about-cancer/treatment/drugs/carboplatin

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National Cancer Institute. (n.d.). Small cell lung cancer treatment (PDQ®). https://www.cancer.gov/types/lung/hp/small-cell-lung-treatment-pdq

Rudin, C. M., et al. (2021, January 14). Small-cell lung cancer. Nature Reviews Disease Primers. https://pmc.ncbi.nlm.nih.gov/articles/PMC8177722/

Sebaugh, J. L. (2011). Guidelines for accurate EC50/IC50 estimation. Pharmaceutical Statistics, 10(2), 128–134.

Strober, W. (2015). Trypan blue exclusion test of cell viability. Current Protocols in Immunology. John Wiley & Sons.

Thermo Fisher Scientific. (n.d.). Alamar blue cell viability assay protocol. https://www.thermofisher.com

Image References

Alamy. (n.d.). The chemical structure of an anticancer drug carboplatin [Image]. https://www.alamy.com/the-chemical-structure-of-an-anticancer-drug-carboplatin-image491011307.html

Cancer Treatment Centers of America. (n.d.). Lung cancer types [Image]. https://www.cancercenter.com/cancer-types/lung-cancer/types

Cancer Biology & Medicine. (n.d.). [Lung cancer-related image] [Image]. https://www.cancerbiomed.org/content/19/8/1111

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