Angiogenesis is a hallmark phenomenon of solid tumors; it allows new vessels to grow into the tumor to bring in nutrients and at the same time drain lactic acid and other waste products. It also permits the tumor cells to leave the tumor mass and enter blood circulation, and hence, metastasis ensues. Typically, tumor vasculature is a disorganized network of vessels without clear definable arterioles, capillaries, and venules 9,10. Tumor vessels have varying diameter and shape with bulges and terminal ends 11. Their smooth muscle cells are scarce, endothelium lining discontinuous or absent, and basal lamina abnormal or lacking 12.
Our ultrastructural survey of the two types of glioma tumors showed that the tumor vessels have developed unique features that were the result of their interaction with tumor cell invasion and their co-option for the purposes of the tumor cells. These features have implications for aggressive invasion, metastasis, and unfavorable clinical prognosis. The survey has revealed the thickening of the VW and proliferation of the basement membrane, the presence of tumor cells within niches inside VW and their attachment to the luminal side of the VW, as well as the presence of tumor cells and lipid inclusions in the vessels lumina.
The tumor cells of both types had accomplished several tasks that encompassed a successful journeying to and penetration of the vessels as well as the use of the vessels as new colonization sites and conduits for metastasis. It appeared that the endothelial cells (ECs) initial reaction to the tumor was to increase the thickness of the VW, so instead of having a normal thin uniform VW, the tumor vessels had a very thick, pocketed, and proliferative basement membrane. The thick VW did not prevent the tumor cells from reaching the vessels’ lumina as the TEM images have shown (Figs. 1 & 2). Thus, the tumor cells of both glioma types had the capability of invasion and colonization of the tumor vessels as well as the displacement of ECs.
Additionally, glioma cells in the tumor vessels appear to deploy vascular mimicry (VM) once they have colonized the tumor vessels. VM by glioma cells was manifested by their attachment to the luminal side of VWs and tube formation (Figs. 1 & 2). Tumor cells use their β1 integrin subunit to adhere rapidly to the fibronectin of the VW extracellular matrix 13,14. VM is a recent concept describing tumor vascular pattern used by many tumors, particularly glioblastoma. The process of VM includes the transition of tumor cells into a vascular phenotype forming tube structure and expressing endothelial surface antigens such as VE-cadherin and EphA2 as well as periodic acid-Schiff (PAS) 15. The endothelial-like characteristics deployed by the tumor cells ensure their control of the tumor nutrient supply and their metabolic adaptation to resist the immune system assault and anti-angiogenic therapy. Tumor cells with such features are highly adaptable and possess a high metastatic potential. A better understanding of this process will permit the targeting of key steps in VM and provide better treatment options. Our documentation of the ultrastructural characteristics of VM is preliminary and additional research is required to fully elucidate the ontogeny of VM. Therefore, we described here only the pattern of tumor cells’ invasion and colonization of tumor vessels and their abnormal VW.
The enormous thickening of the VW caused by the proliferation of their basement membrane and its elastic layers is a newly observed feature that has not been reported on before. The absence of ECs, pericytes, smooth muscle cells, and sometimes basal lamina is an indication that the VW thickening was most likely carried out earlier before tumor cell invasion and the disappearance of ECs. There are reports that the tumor’s lactic acid could be involved in the loss of ECs due to its damaging action on VE-cadherin 16. The events preceding tumor cell invasion that led to the disappearance of the vascular cells are not fully resolved yet and they may take place much earlier during the incipient phases of tumor formation.
Gliomas are highly glycolytic tumors that convert glucose to lactic acid in high amounts to generate ATP and maintain macromolecule synthesis. Thus, in order to survive its own waste product, gliomas efflux lactic acid out of their cells through transmembrane transporters into the tumor microenvironment and drain it through tumor vessels to reduce microenvironment acidification and maintain physiological pH 17,18. Lactic acid is known to cause an increased vascular permeability through compromising the integrity of the EC monolayer by loosening their cell-cell adhesion, which subsequently induces the disassembly of the VW components 16.
The interaction between tumor cells and ECs rely mainly on VEGF–VEGF receptor signaling 19; however, the more detailed aspects of endothelial gene expression in GBM remain unclear 20, and could vary according to the tumor phases (see below). Vascular targeting by blocking VEGF signaling with bevacizumab has not enhanced overall survival in GBM patients 21. This is not surprising since our ultrastructural study showed that ECs were missing from tumor vessels in a much earlier stage and their place was occupied by antiangiogenic therapy-resistant tumor cells that co-opted the vessels for their own benefit. Therefore, our study points out that targeting the VEGF signaling pathway would no longer be effective. Furthermore, understanding the details of vessel invasion and colonization would enable us to target the specific mediators and receptors involved in tumor-EC interaction in order to stop tumor vessel modification and halt tumor cell metastasis.
As we have shown previously, lipid production is a salient feature of gliomas; lipid droplets and inclusions exist within tumor cells and the tumor microenvironment (Maraqah, in press). Lipid production is related to the hypoxic nature of the tumor 22. In regard to gliomas tumor vessels, we have observed lipid inclusions within the layers of the VW and in the vessels’ lumina. We concluded that the lipid inclusions in the tumor vessels were secreted by the invading tumor cells. Lipids in gliomas pose a serious clinical challenge due to their impact on tumor resistance to therapy. Layers of lipids are barriers to chemotherapy because they limit the entry of non-lipophilic medications and may act as a sink to lipophilic drugs; thus, preventing the medications from reaching the tumor cells in an effective dosage. This tumor feature emphasizes the need for designing lipophilic drugs for gliomas in order to improve drug delivery to tumor cells and achieve treatment success. Also, reducing the amount of lipids in the vessels or the tumor mass could enhance drug delivery. Furthermore, lipid quantitation could be explored as a diagnostic analyte for metastasis or recurrence.
Additionally, we observed that most of the viable tumor cells were located around and inside of the vessels and not inside the bulk of the tumor. No perivascular clasping groups of tumor cells or abluminal tube formations were seen as claimed by some authors 23; and no immune cells were visible either. Thus, the added absence of pericytes, smooth muscle cells, and ECs portends the loss of the brain-blood barrier.
In summary, the ultrastructural survey of gliomas has revealed the pattern of tumor cell invasion and colonization of tumor vessels, the tumor-related modifications that vessels undergo, and the pattern of lipid distribution in these vessels. Most of these features were not described before and they could be of clinical implications; therefore, further elucidation of the oncogenic steps of these features and their molecular mechanisms could bring about improved diagnostic and treatment options.
Table 1
Demographic of patients, “M” male, “F” female, “WT” wild type, “M” mutant,
Patient # | Sex | Age at Dx yr | IDH status | Location |
1 | F | 59 | WT | Frontal |
2 | F | 57 | WT | Frontal |
3 | F | 56 | WT | Frontal |
4 | F | 55 | WT | Thalamic |
5 | F | 26 | M | Frontal |
6 | F | 49 | M | Frontal |
7 | M | 54 | WT | Occipital |
8 | F | 57 | M | Frontal |
9 | M | 67 | WT | Temporal |
10 | F | 51 | WT | Frontal |
11 | F | 30 | M | Multifocal |
12 | F | 48 | WT | Temporal |
13 | F | 49 | M | Frontal |
14 | M | 22 | M | Temporal |
15 | M | 39 | WT | Frontal |
16 | M | 52 | WT | Frontal |
17 | F | 53 | WT | Parietal |
18 | M | 44 | M | Temporal |
19 | F | 63 | WT | Temporal |
20 | F | 35 | M | Frontal |
21 | M | 26 | M | Frontal |
22 | M | 58 | WT | Frontal |
23 | F | 30 | M | Frontal |
24 | F | 41 | WT | Parietal |
25 | F | 46 | WT | Temporal |
26 | F | 28 | M | Frontal |
27 | M | 56 | WT | Temporal |
28 | M | 50 | WT | Frontal |
29 | M | 60 | M | Parietal |
30 | M | 43 | WT | Frontal |