Directed capillary ingrowth has long been considered synonymous with tumor vascularization. However, the vasculature of primary tumors and metastases is not necessarily formed by endothelial cell sprouting; instead, malignant tumors can acquire blood vessels via alternative vascularization mechanisms, such as intussusceptive microvascular growth, vessel co-option, and glomeruloid angiogenesis. Importantly, in response to anti-angiogenic therapies, malignant tumors can switch from one vascularization mechanism to another. In this article, we briefly review the biological features of these mechanisms and discuss on their significance in medical oncology.

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Monolayer cultures, the less standard three-dimensional (3D) culturing systems, and xenografts are the main tools used in current basic and drug development studies of cancer research. The aim of biofabrication is to design and construct a more representative in vivo 3D environment, replacing two-dimensional (2D) cell cultures. Here, we aim to provide a complex comparative analysis of 2D and 3D spheroid culturing, and 3D bioprinted and xenografted breast cancer models. We established a protocol to produce alginate-based hydrogel bioink for 3D bioprinting and the long-term culturing of tumour cells in vitro. Cell proliferation and tumourigenicity were assessed with various tests. Additionally, the results of rapamycin, doxycycline and doxorubicin monotreatments and combinations were also compared. The sensitivity and protein expression profile of 3D bioprinted tissue-mimetic scaffolds showed the highest similarity to the less drug-sensitive xenograft models. Several metabolic protein expressions were examined, and the in situ tissue heterogeneity representing the characteristics of human breast cancers was also verified in 3D bioprinted and cultured tissue-mimetic structures. Our results provide additional steps in the direction of representing in vivo 3D situations in in vitro studies. Future use of these models could help to reduce the number of animal experiments and increase the success rate of clinical phase trials.

Background and aimsDuctular reaction is a standard component of fibrotic liver tissue but its function is largely unknown. It is supposed to interact with the matrix producing myofibroblasts and compensate the declining regenerative capacity of hepatocytes. The relationship between the extent of fibrosis—ductular reaction, proliferative activity of hepatocytes and ductular reaction were studied sequentially in experimental hepatic fibrosis models.MethodsLiver fibrosis/cirrhosis was induced in wild type and TGFβ overproducing transgenic mice by carbon tetrachloride and thioacetamide administration. The effect of thioacetamide was modulated by treatment with imatinib and erlotinib. The extent of ductular reaction and fibrosis was measured by morphometry following cytokeratin 19 immunofluorescent labeling and Picro Sirius staining respectively. The proliferative activity of hepatocytes and ductular reaction was evaluated by BrdU incorporation. The temporal distribution of the parameters was followed and compared within and between different experimental groups.ResultsThere was a strong significant correlation between the extent of fibrosis and ductular reaction in each experimental group. Although imatinib and erlotinib temporarily decreased fibrosis this effect later disappeared. We could not observe negative correlation between the proliferation of hepatocytes and ductular reaction in any of the investigated models.ConclusionsThe stringent connection between ductular reaction and fibrosis, which cannot be influenced by any of our treatment regimens, suggests that there is a close mutual interaction between them instead of a unidirectional causal relationship. Our results confirm a close connection between DR and fibrogenesis. However, since the two parameters changed together we could not establish a causal relationship and were unable to reveal which was the primary event. The lack of inverse correlation between the proliferation of hepatocytes and ductular reaction questions that ductular reaction can compensate for the failing regenerative activity of hepatocytes. No evidences support the persistent antifibrotic property of imatinib or erlotinib.

The hepatic stem cells reside periportally forming the canals of Hering in normal liver. They can be identified by their unique immunophenotype in rat. The oval cells, the progenies of stem cells invade deep the liver parenchyma after activation and differentiate into focally arranged small-and eventually trabecularly ordered regular hepatocytes. We have observed that upon the completion of intense oval cell reactions narrow ductular structures are present in the parenchyma, we propose to call them parenchymal ductules. These parenchymal ductules have the same immunophenotype [cytokeratin (CK)7 -/CK19 + /alpha-fetoprotein (AFP) -/delta-like protein (DLK) -] as the resting stem cells of the canals of Hering, but different from them reside scattered in the parenchyma. In our present experiments, we have investigated in an in vivo functional assay if the presence of these parenchymal ductules has any impact on a progenitor cell driven regeneration process. Parenchymal ductules were induced either by an established model of oval cell induction consisting of the administration of necrogenic dose of carbontetrachloride to 2-acetaminofluorene pretreated rats (AAF/CCl 4 ) or a large necrogenic dose of diethylnitrosamine (DEN). The oval cells expanded faster and the foci evolved earlier after repeated injury in the livers with preexistent parenchymal ductules. When the animals were left to survive for one more year increased liver tumor formation was observed exclusively in the DEN treated rats. Thus, repeated oval cell reactions are not necessarily carcinogenic. We conclude that the expansion of hepatic stem cell compartment conceptually can be used to facilitate liver regeneration without an increased risk of tumorigenesis.