Authors: (including presenting author): :
Wong CK(1), Lam Y(1), Lung TW(2), Mak WL(3)
Affiliation: :
(1)Hong Kong Metropolitan University, (2)Hospital Authority, Hong Kong (3)HealthBaby Biotech (Hong Kong) Co., Ltd
Keyword 1: :
Colorectal cancer
Keyword 2: :
SW620 cell line
Keyword 3: :
Pien Tze Huang
Keyword 4: :
PD-L1 expression
Keyword 5: :
Innovation in biomedical research training
Introduction: :
Biomedical science research training increasingly emphasizes practical laboratory skills that are safe, ethical, and accessible. Traditional teaching often relied on animal models or human tissue samples, which raised ethical concerns, required complex approvals, and posed biosafety risks. In contrast, established cancer cell lines provide a reproducible and ethically acceptable alternative for student training. Colorectal cancer (CRC) is the third most commonly diagnosed malignancy worldwide and the second leading cause of cancer-related deaths. Its progression involves immune evasion mechanisms, particularly through programmed death-ligand 1 (PD-L1), which suppresses T-cell activation. Understanding PD-L1 regulation is critical for both therapeutic development and biomedical education. Pien Tze Huang (PZH), a traditional Chinese medicine, has demonstrated anti-proliferative and immunomodulatory effects in several cancers. Investigating its impact on CRC cell lines offers scientific insights while providing students with an innovative research training platform. Using cell lines such as SW620, derived from metastatic colorectal adenocarcinoma, students can safely explore cancer biology and immune checkpoint regulation without ethical concerns associated with human or animal tissue use. This study demonstrates both the therapeutic potential of PZH and the value of cell line–based research training in biomedical science education.
Objectives: :
1. Scientific Objectives o Evaluate the anti-proliferative effects of PZH on CRC SW620 cells using colony formation assays. o Determine the impact of PZH on PD-L1 membranous expression through immunohistochemistry (IHC). o Assess whether PZH influences transcriptional regulation of PD-L1 and the PI3K/Akt/mTOR pathway using real-time quantitative PCR (qPCR). 2. Educational Objectives o Provide biomedical science students with hands-on research training in cell culture, drug treatment, histology, and molecular biology. o Demonstrate how cell lines serve as safe, reproducible, and ethically acceptable models. o Highlight the convenience of using established cell lines, which are readily accessible in teaching laboratories without invasive tissue collection or complex ethical approvals.
Methodology: :
Cell Line Model SW620 colorectal cancer cells, derived from a metastatic site, were chosen as a model for advanced CRC. Cell lines are safe under biosafety level 2 conditions, free from ethical concerns, and suitable for student training. Preparation of PZH Solution PZH capsules were aseptically processed, dissolved in PBS, sonicated, centrifuged, and filtered to obtain stock solutions. Working concentrations were prepared by dilution, training students in aseptic technique and solution preparation. Colony Formation Assay (CFA) SW620 cells were seeded in 6-well plates and treated with PZH (0–2000 µg/mL). After 24 hours, cells were incubated for 7 days to allow colony formation. Colonies were fixed, stained, and counted. Survival fraction (SF), plating efficiency (PE), IC₅₀, and total inhibitory concentration (TIC) were calculated, teaching students quantitative analysis of cell viability. Immunohistochemistry (IHC) PD-L1 membranous expression was assessed using the FDA-approved 22C3 antibody clone with DAB detection. Cell blocks were prepared, sectioned, and stained. Tumour proportion score (TPS) and staining intensity were evaluated microscopically, providing training in histological preparation and semi-quantitative scoring. RNA Extraction and qPCR Total RNA was extracted, quality checked, and reverse-transcribed into cDNA. Primers for GAPDH, PD-L1, PI3K, and mTOR were validated. Real-time qPCR quantified relative mRNA expression trained students in molecular biology and gene expression analysis. Statistical Analysis One-way ANOVA and t-tests were applied, with significance defined as p < 0.05. Students learned statistical interpretation of experimental data.
Result & Outcome: :
Anti-Proliferation Effect PZH inhibited SW620 colony formation in a dose-dependent manner. Survival fractions decreased significantly with increasing concentrations, with IC₅₀ at 1100 µg/mL and TIC at 1600 µg/mL. These results confirmed PZH’s anti-proliferative activity and provided students with a clear demonstration of dose-response analysis. PD-L1 Membranous Expression IHC revealed strong PD-L1 staining in untreated SW620 cells. PZH treatment reduced staining intensity in a dose-dependent manner. The TIC group showed significantly lower TPS (33% ± 8.29%) compared to controls (97.67% ± 2.05%). These findings demonstrated PZH’s ability to downregulate PD-L1 expression at the protein level, reinforcing the concept of immune checkpoint inhibition. Transcriptional Regulation qPCR analysis showed no significant changes in PD-L1, PI3K, or mTOR mRNA levels (p > 0.05). This suggests that PZH’s effects occur at the protein or post-translational level rather than through transcriptional regulation. Students learned the importance of integrating protein-level and gene-level analyses to fully understand drug mechanisms. Conclusion Pien Tze Huang (PZH) inhibits proliferation and reduces PD-L1 membranous expression in metastatic CRC SW620 cells in vitro. While transcriptional regulation of PD-L1 and PI3K/Akt/mTOR was not significantly altered, protein-level changes suggest post-translational mechanisms. From an educational perspective, this project illustrates an innovative biomedical science research training approach. By using established CRC cell lines, students were able to: • Safely perform advanced laboratory techniques without ethical concerns. • Gain hands-on experience in cell culture, drug treatment, histology, and molecular biology. • Access reproducible and convenient tissue models suitable for teaching laboratories. Cell lines provide a practical and ethical solution for biomedical research training, eliminating the need for animal models or invasive human tissue collection. This ensures students can explore cancer biology and therapeutic testing in a controlled, safe, and accessible environment. Future studies should expand training to include multiple CRC cell lines, longer treatment durations, and complementary protein-level assays such as Western blotting and flow cytometry. Incorporating these methods will provide students with a more comprehensive understanding of cancer biology and drug mechanisms. This study not only advances knowledge of PZH’s therapeutic potential but also establishes a model for innovative biomedical science education. By integrating cancer research with student training, it demonstrates how laboratory teaching can be both scientifically meaningful and ethically responsible, preparing the next generation of biomedical scientists with the skills and awareness needed for modern research.
