Numerous methods have been developed for the fabrication of poly(ethylene glycol)-based hydrogel microstructures for drug-delivery and tissue-engineering applications. However, present methods focus on the fabrication of submicrometer scale hydrogel structures which have limited applications in creating larger tissue constructs, especially in recreating cancer tissue microenvironments. We aimed to establish a platform where cancer cells can be cultured in a three-dimensional (3D) environment, which closely replicates the native cancer microenvironment and facilitates efficient testing of anticancer drugs. This study demonstrated a novel surface tension-based fabrication technique for the generation of millimeter-scale hydrogel beads using a liquid-liquid dual phase system. The "hydrogel millibeads" obtained by this method were larger than previously reported, highly uniform in shape and size with better ease of size control and a high degree of consistency and reproducibility between batches. In addition, human breast cancer cells were encapsulated within these hydrogel constructs to generate "tumor millibeads", which were subsequently maintained in long-term 3D culture. Microscopic visualization using fluorescence imaging and microstructure analysis showed the morphology and uniform distribution of the cells within the 3D matrix and arrangement of cells with the surrounding scaffold material. Cell viability analysis revealed the creation of a core region of dead cells surrounded by healthy, viable cell layers at the periphery following long-term culture. These observations closely matched with those of native and in vivo tumors. Based on these results, this study established a rapidly reproducible surface tension-based fabrication technique for making spherical hydrogel millibeads and demonstrated the potential of this method in creating engineered 3D tumor tissues. It is envisioned that the developed hydrogel millibead system will facilitate the formation of physiologically relevant in vitro tumor models which will closely simulate the native tumor microenvironmental conditions and could enable future high-throughput testing of different anticancer drugs in preclinical trials.

Activation of pregnane X receptor (PXR) by clinical compounds during multidrug chemotherapy results in upregulation of the expression of PXR target genes, including cytochrome p450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1), leading to chemoresistance. It is possible to overcome the PXR‐mediated chemoresistance by downregulating the upregulated PXR target genes by inhibiting the activated PXR. However, a selective and less‐toxic PXR antagonist has yet to be developed. In this regard, a clinical anticancer drug, with selective PXR antagonistic activity at its less‐toxic concentrations, would be beneficial. We sought to determine whether belinostat, a clinically‐used histone deacetylase inhibitor, inhibits the PXR target gene expression at its clinically relevant plasma concentrations (< ~100 μM) in human hepatocytes (primary hepatocytes & hepatocells) and intestinal cells (LS174T colon cancer cells). Rifampicin, an agonist of human PXR, was used to activate PXR. Cell viability and CYQUANT cell proliferation assays were performed to determine cytotoxicity and cell proliferation, respectively. Quantitative RT‐PCR assays were conducted to study the gene expression. CYP3A4 p450‐Glo and Rhodamine‐123 intracellular accumulation assays were performed to determine the function of CYP3A4 and MDR1, respectively. Belinostat, at its unbound therapeutic plasma concentrations (< ~5 μM) did not affect the viability of LS174T cells and the hepatocytes. Belinostat (1 & 3 μM) not only inhibited rifampicin‐induced gene expression of CYP3A4 and MDR1, but also attenuated rifampicin‐induced activity of CYP3A4 and MDR1. However, belinostat alone did not affect CYP3A4 or MDR1 gene expression. These results suggest that belinostat does not affect the basal expression of PXR target genes but downregulates the upregulated PXR target genes by inhibiting the ligand‐activated PXR. Notably, belinostat, at its PXR inhibiting concentrations, decreased rifampicin‐induced proliferation of LS174T cells, suggesting that belinostat suppresses PXR‐mediated proliferation of the cancer cells. Interestingly, belinostat failed to inhibit rodent PXR agonist pregnenolone‐16 alpha‐carbonitrile (PCN)‐induced expression of CYP3A1 (the rat analog of human CYP3A4) in rat primary hepatocytes, suggesting that belinostat exhibits species‐specific inhibition of PXR at unbound plasma therapeutic concentrations. Taken together, these results are consistent with the conclusion that belinostat, at its less‐toxic and clinically relevant unbound plasma concentrations, inhibits the ligand‐activated human PXR target gene expression. Future studies will determine the mechanisms of belinostat inhibition of PXR, and belinostat sensitization of the cancer cells to chemotherapy drugs with PXR agonistic activity. Support or Funding Information The authors would like to thank Drs. Coleman, Schwartz, and Tao for sharing their research facilities. This work was supported by the Auburn University Research Initiative in Cancer Grant, Animal Health and Disease Research Grant...