Low-level laser irradiation stimulated tumoricidal impacts and dose-dependent modulation of Wnt signaling in three-dimensional human colorectal cancer microspheres.

The high incidence of colorectal cancer (CRC) has been a big challenge in human medicine. Along with several available therapies, specific modalities, e.g., low-level laser irradiation (LLLI), have been introduced to achieve proper regenerative outcomes. Unfortunately, most studies have investigated the tumoricidal effects of LLLI on cancer cells using a 2D culture system with less comparable capacity to in vivo conditions. Here, the detrimental impacts of LLLI were studied in human CRC cells encapsulated inside alginate-gelatin (Alg-Gel) microspheres after 48 h. Alg (1% w/v)-Gel (1% w/v) microspheres were developed using CaCl₂-mediated ionic cross-linking. Physicochemical properties, including swelling and degradation rates, gel fraction, FTIR spectra, and SEM morphology, were examined. The mean diameter and cell distribution of HT-29 cell-laden microspheres were evaluated by bright-field microscopy. Cell membrane integrity and nitric oxide (NO) production were assessed using LDH-release and Griess assays 48 h after LLLI at fluences of 2 and 80 J/cm². Cell density was evaluated by hematoxylin-eosin staining, and the expression of Wnt-related genes was assessed by RT-qPCR. FTIR findings supported integration of Alg and Gel, and the composite showed measurable swelling, degradation, and gel-fraction characteristics together with a porous interconnected ultrastructure. The mean microsphere diameter was 305.85 ± 19.07 μm, and cells were distributed throughout the microspheres. The 80 J/cm² exposure was associated with increased LDH release, higher NO production, and lower HT-29 cell density (p < 0.05), while less LLLI intensity, 2 J/cm², showed a modest, assay-dependent cytoprotective pattern. RT-qPCR demonstrated a mixed transcriptional response involving both up- and down-regulation of Wnt-related genes; these data do not by themselves establish activation of a Wnt signaling pathway. LLLI can alter the dynamic growth of human CRC cells depending on laser intensity by influencing survival rate, nitrosative stress, and certain Wnt signaling genes. These data can help in the development of an accurate therapeutic protocol in the clinical setting.