## 3 Results and Discussion  
Here we present an important in vitro model for the evaluation of diagnostic tools and treatment protocols by combining HT29 spheroids grown with or without SA-MIPs. The first sets of HT29 spheroids were grown in conventional spheroid medium according to the Material and Methods section. After successful growth for up to 10 days, the spheroids were harvested, fixed with 4% formaldehyde, and stained with lectins MAL I, SNA, or SA-MIPs, respectively, as well as with DAPI (nuclei) and phalloidin (cytoskeleton) (Figure 1). The \(\uplambda\)-2,3- and \(\uplambda\)-2,6 SA expression of the HT29 cell line is determining the binding of MAL I and SNA, respectively, which was confirmed by flow cytometry analysis on single cell level. MAL I and SNA staining indicated an intact expression of \(\uplambda\)-2,3- and \(\uplambda\)-2,6 SA on the outer layer of the HT29 spheroids (Figure 1). MAL I was homogeneously distributed across the outer layer of the HT29 spheroids, whereas SNA was limited to certain patches of the HT29 spheroids. SA-MIPs have previously been shown to display a binding pattern comparable to that of SA-targeting lectins [9]. Here we demonstrated a direct binding of the SA-MIPs to the formaldehyde-fixed cells across the HT29 spheroids (Figure 1). Using a serum-free medium containing the appropriate growth factors, spheroids can be grown from established cell lines or from dissociated tissue cells. It is crucial that the cell environment is adjusted to mimic the in vivo conditions when performing 3D cell culture experiments in vitro. Spheroid cultures have become increasingly important tools, reproducing the in vivo cell environment and spheroid cultures can provide accurate data for various diseases [4]. Protocol development is particularly useful when working with more valuable patient samples in 3D, i.e., organoids. Additionally, by the utilization of spheroids and organoids, the reliance on animal models in drug discovery can be minimized offering more ethical and cost-effective alternatives for preclinical testing. The addition of MAL I and SNA to viable, growing HT29 spheroid cell cultures, caused cell death leading to incomplete spheroid growth. Plant lectins have been reported to be effective in targeting cancer cells and inducing cell death [19]. Specifically, MAL I and SNA have been shown to induce apoptosis in non-small cell lung cancer cells [19] or in ovarian cancer cells [20], respectively. In contrast to formaldehyde-fixed spheroids, the addition of SA-MIPs to viable HT29 spheroid cell cultures, resulted in a more pronounced SA-MIP staining pattern, with no detected effect on cell viability (Figure 2). In addition, the Z-stack images of viable SA-MIP-HT29 spheroids demonstrated detailed binding pattern of SA-MIPs. Most importantly, SA-MIPs were co-cultured with HT29 spheroids for 8-10 days without any signs of toxicity or cell death. Molecular imprinting relied on template-directed synthesis of polymers [7; 8; 11; 12; 21], and MIPs thus produced against different target molecules or molecular motifs had been used instead of antibodies against small non-immunogenic structures, including SA. MIPs are nowadays established in various applications and have proven to function well in drug delivery, cell signalling, sensors, assays, and imaging [11; 21; 22]. Tumor cells showed a de-regulated expression of SA as an immuno-suppressant to avoid recognition and elimination by the immune system, and hence it was important to target SA not only as a diagnostic tool [23]. Indeed, the importance of blocking SA in vivo in mice bearing metastatic lesions in the lung by using SA-blocking glycominetrics had been demonstrated [24]. Moreover, we had recently shown that SA-MIPs cultured in vitro with phagocytosis cells, macrophages, resulted in an attenuated induction of release of inflammatory cytokines [14]. Figure 1: HT29 cells were grown according to the described protocol for spheroids and left for 8–10 days in 37 \({}^{\circ}\)C and 5% CO\({}_{2}\).