I. Embryonal vasculature formation recapitulated in transgenic mammary tumor spheroids implanted pseudo-orthotopicly into mouse dorsal skin fold: the organoblasts concept

Abstract

Inadequate understanding of cancer biology is a problem. This work focused on cellular mechanisms of tumor vascularization. According to earlier studies, the tumor vasculature derives from host endothelial cells (angiogenesis) or their precursors of bone marrow origin circulating in the blood (neo-vasculogenesis) unlike in embryos. In this study, we observed the neo-vasculature form in multiple ways from local precursor cells. Recapitulation of primitive as well as advanced embryonal stages of vasculature formation followed co-implantation of avascular ( in vitro cultured) N202 breast tumor spheroids and homologous tissue grafts into mouse dorsal skin chambers. Ultrastructural and immunocytochemical analysis of tissue sections exposed the interactions between the tumor and the graft tissue stem cells. It revealed details of vasculature morphogenesis not seen before in either tumors or embryos. A gradual increase in complexity of the vascular morphogenesis at the tumor site reflected a range of steps in ontogenic evolution of the differentiating cells. Malignant- and surgical injury repair-related tissue growth prompted local cells to initiate extramedullar erythropoiesis and vascular patterning. The new findings included: interdependence between the extramedullar hematopoiesis and assembly of new vessels (both from the locally differentiating precursors); nucleo-cytoplasmic conversion (karyolysis) as the mechanism of erythroblast enucleation; the role of megakaryocytes and platelets in vascular pattern formation before emergence of endothelial cells; lineage relationships between hematopoietic and endothelial cells; the role of extracellular calmyrin in tissue morphogenesis; and calmyrite, a new ultrastructural entity associated with anaerobic energy metabolism. The central role of the extramedullar erythropoiesis in the formation of new vasculature (blood and vessels) emerged here as part of the tissue building process including the lymphatic system and nerves, and suggests a cellular mechanism for instigating variable properties of endothelial surfaces in different organs. Those findings are consistent with the organoblasts concept, previously discussed in a study on childhood tumors, and have implications for tissue definition.

Bevacizumab Modulation of the Interaction Between the MCF-7 Cell Line and the Chick Embryo Chorioallantoic Membrane

“…Witkiewicz et al demonstrated that not only vessels but also blood formed de novo at the location of growing tissue, not just in bone marrow and spleen (23). We confirmed this process in the MCF-7/CAM model, by demonstrating that inside the newly formed capillary-like structures, hematopoietic progenitors were identified.…”

III. Cellular ultrastructures in situ as key to understanding tumor energy metabolism: biological significance of the Warburg effect

“…Formation of vasculature in the avascular tumor spheroids has been shown to depend in our model on the presence of the homologous tissue graft which results in faster tumor growth 5 , 6 . However locally, the phenomena described for the ectopically implanted tumors (Figure 1–3 in 6 ) also occurred in the presence of the graft, i.e.…”

“…Here the old controversy on the respiratory impairment in cancer is addressed from the perspective of tissue biology rather than cell biology, following recent findings regarding cellular mechanisms of tumor vasculature formation 5 , 6 . The new perspective emerged from putting in vitro grown mammary tumor spheroids back into the tissue context and increasing the resolution of morphological analysis in situ to the ultrastructural level which enabled evaluation of subtle differences in the physiological status of individual cells.…”

“…Before implantation, tumor spheroids were getting all the energy they needed from properly buffered culture medium in an optimized atmospheric environment and they formed no vessels. After pseudo-orthotopic implantation, the tumors induced stem cells of homologous tissue graft to form neo-vasculature for them 5 . The ectopic implantation put tumors in a critical situation because none of the first two options were available; the spheroids had to fend for themselves 6 .…”

“…The less efficient pathway (glycolysis) occurs in cytoplasm and can be reproduced in vitro . Without specific protein markers, no particular glycolysis-associated structure is visualized by transmission electron microscopy (TEM) although in erythrosomes, known for using glycolysis to generate ATP 10 , abundance of calmyrites suggested that those complexes might be involved 5 . When not needed, mitochondria become degraded.…”