Cell culture is an important and necessary process in drug discovery, cancer research, as well as stem cell study. Most cells are currently cultured using two-dimensional (2D) methods but new and improved methods that implement three-dimensional (3D) cell culturing techniques suggest compelling evidence that much more advanced experiments can be performed yielding valuable insights. When performing 3D cell culture experiments, the cell environment can be manipulated to mimic that of a cell in vivo and provide more accurate data about cell-to-cell interactions, tumor characteristics, drug discovery, metabolic profiling, stem cell research, and other types of diseases. Scaffold based techniques such as hydrogel-based support, polymeric hard material-based support, hydrophilic glass fiber, and organoids are employed, and each provide their own advantages and applications. Likewise, there are also scaffold free techniques used such as hanging drop microplates, magnetic levitation, and spheroid microplates with ultra-low attachment coating. 3D cell culture has the potential to provide alternative ways to study organ behavior via the use of organoids and is expected to eventually bridge the gap between 2D cell culture and animal models. The present review compares 2D cell culture to 3D cell culture, provides the details surrounding the different 3D culture techniques, as well as focuses on the present and future applications of 3D cell culture.

Metastasis is the leading cause of breast cancer-related deaths. Cancer-associated fibroblasts (CAFs), the predominant stromal cell type in the breast tumour microenvironment, may contribute to cancer progression through interaction with tumour cells. Nonetheless, little is known about the details of the underlying mechanism. Here we found that interaction of interleukin 32 (IL32) with integrin β3 (encoded by ITGB3; a member of the integrin family) mediating the cross-talk between CAFs and breast cancer cells plays a crucial role in CAF-induced breast tumour invasiveness. IL32, an 'RGD' motif-containing cytokine, was found to be abundantly expressed in CAFs. Integrin β3 turned out to be up-regulated in breast cancer cells during epithelial-mesenchymal transition (EMT). CAF-derived IL32 specifically bound to integrin β3 through the RGD motif, thus activating intracellular downstream p38 MAPK signalling in breast cancer cells. This signalling increased the expression of EMT markers (fibronectin, N-cadherin, and vimentin) and promoted tumour cell invasion. Counteracting IL32 activity, a knockdown of IL32 or integrin β3 led to specific inactivation of p38 MAPK signalling in tumour cells. Blockage of the p38 MAPK pathway also diminished IL32-induced expression of EMT markers and breast cancer cell invasion and metastasis. Thus, our data indicate that CAF-secreted IL32 promotes breast cancer cell invasion and metastasis via integrin β3-p38 MAPK signalling.

Tumor resistance to apoptosis and the immunosuppressive tumor microenvironment are two major contributors to poor therapeutic responses during cancer intervention. Pyroptosis, a lytic and inflammatory programmed cell death pathway distinct from apoptosis, has subsequently sparked notable interest among cancer researchers for its potential to be clinically harnessed and to address these problems. Recent evidence indicates that pyroptosis induction in tumor cells leads to a robust inflammatory response and marked tumor regression. Underlying its antitumor effect, pyroptosis is mediated by pore-forming gasdermin proteins that facilitate immune cell activation and infiltration through their release of pro-inflammatory cytokines and immunogenic material following cell rupture. Considering its inflammatory nature, however, aberrant pyroptosis may also be implicated in the formation of a tumor supportive microenvironment, as evidenced by the upregulation of gasdermin proteins in certain cancers. In this review, the molecular pathways leading to pyroptosis are introduced, followed by an overview of the seemingly entangled links between pyroptosis and cancer. We describe what is known regarding the impact of pyroptosis on anticancer immunity and give insight into the potential of harnessing pyroptosis as a tool and applying it to novel or existing anticancer strategies.

The hostile hypoxic microenvironment takes primary responsibility for the rapid expansion of breast cancer tumors. However, the underlying mechanism is not fully understood. Here, using RNA sequencing (RNA-seq) analysis, we identified a hypoxia-induced long noncoding RNA (lncRNA) KB-1980E6.3, which is aberrantly upregulated in clinical breast cancer tissues and closely correlated with poor prognosis of breast cancer patients. The enhanced lncRNA KB-1980E6.3 facilitates breast cancer stem cells (BCSCs) self-renewal and tumorigenesis under hypoxic microenvironment both in vitro and in vivo. Mechanistically, lncRNA KB-1980E6.3 recruited insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) to form a lncRNA KB-1980E6.3/IGF2BP1/c-Myc signaling axis that retained the stability of c-Myc mRNA through increasing binding of IGF2BP1 with m6A-modified c-Myc coding region instability determinant (CRD) mRNA. In conclusion, we confirm that lncRNA KB-1980E6.3 maintains the stemness of BCSCs through lncRNA KB-1980E6.3/IGF2BP1/c-Myc axis and suggest that disrupting this axis might provide a new therapeutic target for refractory hypoxic tumors.

The Ufm1 conjugation system is an ubiquitin-like modification system that consists of Ufm1, Uba5 (E1), Ufc1 (E2), and less defined E3 ligase(s) and targets. The biological importance of this system is highlighted by its essential role in embryogenesis and erythroid development, but the underlying mechanism is poorly understood. UFBP1 (Ufm1 binding protein 1, also known as DDRGK1, Dashurin and C20orf116) is a putative Ufm1 target, yet its exact physiological function and impact of its ufmylation remain largely undefined. In this study, we report that UFBP1 is indispensable for embryonic development and hematopoiesis. While germ-line deletion of UFBP1 caused defective erythroid development and embryonic lethality, somatic ablation of UFBP1 impaired adult hematopoiesis, resulting in pancytopenia and animal death. At the cellular level, UFBP1 deficiency led to elevated ER (endoplasmic reticulum) stress and activation of unfolded protein response (UPR), and consequently cell death of hematopoietic stem/progenitor cells. In addition, loss of UFBP1 suppressed expression of erythroid transcription factors GATA-1 and KLF1 and blocked erythroid differentiation from CFU-Es (colony forming unit-erythroid) to proerythroblasts. Interestingly, depletion of Uba5, a Ufm1 E1 enzyme, also caused elevation of ER stress and under-expression of erythroid transcription factors in erythroleukemia K562 cells. By contrast, knockdown of ASC1, a newly identified Ufm1 target that functions as a transcriptional co-activator of hormone receptors, led to down-regulation of erythroid transcription factors, but did not elevate basal ER stress. Furthermore, we found that ASC1 was associated with the promoters of GATA-1 and Klf1 in a UFBP1-dependent manner. Taken together, our findings suggest that UFBP1, along with ASC1 and other ufmylation components, play pleiotropic roles in regulation of hematopoietic cell survival and differentiation via modulating ER homeostasis and erythroid lineage-specific gene expression. Modulating the activity of this novel ubiquitin-like system may represent a novel approach to treat blood-related diseases such as anemia. Protein modification by Ubiquitin (Ub) and Ubiquitin-like proteins (Ubl) plays pivotal roles in a wide range of cellular functions and signaling pathways. The Ufm1 conjugation system is a novel ubiquitin-like system, yet its biological functions and working mechanism remains poorly understood. UFBP1 is a putative Ufm1 target that has been implicated in several signaling pathways but little is known regarding its in vivo function. In this report, by using multiple knockout mouse models, we demonstrate that UFBP1 is essential for murine development and blood cell development. While germ-line deletion of UFBP1 caused defective red blood cell development and embryonic lethality, somatic ablation of UFBP1 impaired production of mature red blood cells and other types of hematopoietic cells. We found that depletion of UFBP1 led to elevated stress in the endoplasmic reticulum that in turn caused cell death of hematopoietic stem cells. Furthermore, UFBP1 deficiency diminished expression of key transcription factors essential for red blood cell development. Taken together, our study provides strong genetic evidence for the essential role of UFBP1 as well as other components of the Ufm1 system in hematopoietic development. Therefore, the ufmylation pathway may represent a novel therapeutic target in treatment of blood diseases.

In vivo metastasis assays have traditionally been performed in mice, but the process is inefficient and costly. However, since zebrafish do not develop an adaptive immune system until 14 days post-fertilization, human cancer cells can survive and metastasize when transplanted into zebrafish larvae. Despite isolated reports, there has been no systematic evaluation of the robustness of this system to date.