An Exploratory in vitro Study of Cytotoxicity and Cytokine Profiling of Cytokine-Induced Killer (CIK) Cells Against HCT-15 Colorectal Cancer (CRC) Cells

Background: Cytokine-induced killer (CIK) cells are a promising adoptive immunotherapy due to their non-major histocompatibility complex (MHC)-restricted cytotoxicity. However, their cytokine-mediated response against CRC remains poorly defined. Methods: In this exploratory study, CIK cells were generated from three healthy donors and one metastatic CRC patient. Phenotypes were assessed by flow cytometry. Cytotoxicity against the HCT-15 CRC cell line was evaluated at multiple effector-to-target (E:T) ratios (1:5 to 40:1). Combination treatment with 5-fluorouracil (5-FU) and cytokine profiling using a 105-plex array were performed using CIK cells from donor 2 and donor 3, respectively. Results: CIK cells derived from one CRC patient exhibited phenotypic characteristics (CD3⁺CD8⁺ 85.91%, CD3⁺CD56⁺ 27.81%) comparable to the range observed in three healthy donors. Co-culture with CIK cells from donor 1 resulted in reduced HCT-15 viability at higher E:T ratios, with viability decreasing to 8.98 ± 0.61% and 8.08 ± 2.00% at 20:1 and 40:1, respectively. In combination experiments, HCT-15 viability was reduced to 1.02 ± 1.01% at an E:T ratio of 20:1 when co-cultured with CIK from donor 2 and treated with 5-FU. Similar reductions were observed with CIK cells from donor 3 and the CRC patient, reaching 2.60 ± 5.86% and 0.0% at 20:1, and 0.0% at 40:1 ratio for both donors. CIK cells from all donors showed dose-dependent reduction in cell viability against HCT-15 cells. Cytokine profiling of CIK from donor 3 identified changes in immune-related factors, including upregulation of interferon gamma-inducible chemokines (CXCL9 and CXCL10), alongside downregulation of immunosuppressive mediators such as TIM-3 and VEGF. Conclusion: This exploratory study demonstrates that functional CIK cells can be generated from healthy individuals and CRC patients, with observed cytotoxic activity against HCT-15 cells. The findings suggest potential immunomodulatory effects and support further investigation of CIK cells in combination strategies; however, larger patient cohorts and functional validation studies are required.