Colorectal cancer (CRC) is a heterogeneous disease grouping a variety of subtypes with different histological features, mutational status, and therapeutic response. Unlike conventional colorectal adenocarcinoma (CAC), serrated adenocarcinoma (SAC) is a less common subtype associated with a poorer prognosis, a higher frequency of KRAS and BRAF mutations and overexpression of fascin. Fascin is an F-actin bundling protein significantly linked to invasion and metastasis, which makes it a promising target in therapy with fascin inhibitors. Patient-derived organoids (PDOs) provide an accurate model for drug screening, as they replicate the three-dimensional structure of the original tissue. This study aims to evaluate the effect of anti-fascin drugs on PDOs derived from CRC with varying molecular and histological characteristics, as well as different levels of fascin expression.

PDOs were established from primary tumor tissue of six CRC patients with different subtypes (CAC, SAC, medullary carcinoma - MEC, or mucinous carcinoma - MAC) and cultivated in 3D Matrigel matrix and medium designed for intestinal organoids. Fascin expression, mutational status (WT, KRAS/BRAF-mutated), and microsatellite stability (microsatellite stable - MSS, microsatellite unstable - MSI) were assessed using ddPCR, MACE-seq, and immunohistochemistry. To evaluate the effect of anti-fascin compounds, PDOs were treated with increasing concentrations of imipramine and NP-G2 (0.1–200 μM) for 5 days, followed by a viability assay to determine the IC50 values.

The molecular characterization revealed that all patients diagnosed with non-conventional carcinoma (Patient 4: SAC, BRAF, MSI; Patient 5: MEC, BRAF, MSI; Patient 6: MAC, KRAS, MSS) carried oncogene mutations. When comparing patients diagnosed with CAC (Patient 1–3), a stronger effect of imipramine was observed in the presence of KRAS mutation in Patient 3 (KRAS, MSS, 279.3 tpm, IC50 IMI = 14 µM) compared to the WT status in Patient 1 (WT, MSS, 361 tpm, IC50 IMI = 50 µM) and Patient 2 (WT, MSS, 22.57 tpm, IC50 IMI = 42 µM). Higher fascin expression correlated with a stronger effect of NP-G2, as seen when comparing Patient 1 (IC50 NP-G2 = 12 µM) and Patient 2 (IC50 NP-G2 = 46 µM). NP-G2 generally showed lower IC50 values (ranging from 12 µM to 46 µM, median = 19 µM) compared to the non-specific inhibitor imipramine (ranging from 14 µM to 74 µM, median = 46 µM).

This study confirms the potential of using PDOs in drug screening, as it was possible to establish a functional system for evaluating fascin inhibition. Both anti-fascin compounds demonstrated the ability to inhibit cell growth and viability, especially in cases with KRAS/BRAF mutation which are not candidates for anti-EGFR therapy. While some differences in drug effects seem to be related to molecular and histological characteristics, a larger sample size would be necessary to validate these findings more profoundly.