Accelerated and Improved Vascular Maturity after Transplantation of Testicular Tissue in Hydrogels Supplemented with VEGF- and PDGF-Loaded Nanoparticles.

Avascular transplantation of frozen-thawed testicular tissue fragments represents a potential future technique for fertility restoration in boys with cancer. A significant loss of spermatogonia was observed in xeno-transplants of human tissue most likely due to the hypoxic period before revascularization. To reduce the effect of hypoxia-reoxygenation injuries, several options have already been explored, like encapsulation in alginate hydrogel and supplementation with nanoparticles delivering a necrosis inhibitor (NECINH) or VEGF. While these approaches improved short-term (5 days) vascular surfaces in grafts, neovessels were not maintained up to 21 days; i.e., the time needed for achieving vessel stabilization. To better support tissue grafts, nanoparticles loaded with VEGF, PDGF and NECINH were developed. Testicular tissue fragments from 4-5-week-old mice were encapsulated in calcium-alginate hydrogels, either non-supplemented (control) or supplemented with drug-loaded nanoparticles (VEGF-nanoparticles; VEGF-nanoparticles + PDGF-nanoparticles; NECINH-nanoparticles; VEGF-nanoparticles + NECINH-nanoparticles; and VEGF-nanoparticles + PDGF-nanoparticles + NECINH-nanoparticles) before auto-transplantation. Grafts were recovered after 5 or 21 days for analyses of tissue integrity (hematoxylin-eosin staining), spermatogonial survival (immuno-histo-chemistry for promyelocytic leukemia zinc finger) and vascularization (immuno-histo-chemistry for α-smooth muscle actin and CD-31). Our results showed that a combination of VEGF and PDGF nanoparticles increased vascular maturity and induced a faster maturation of vascular structures in grafts.

Metformin enhances T lymphocyte anti-tumor immunity by increasing the infiltration via vessel normalization.

Abnormal tumor vasculature blocks the extravasation of T lymphocytes into the tumor, thereby suppressing anti-tumor immunity. Recently, metformin has been shown to affect tumor vasculature and enhance T lymphocyte anti-tumor immunity. However, whether or how metformin affects T lymphocyte anti-tumor immunity via a vascular mechanism remains poorly understood. Herein, we show that a large number of CD8+ lymphocytes gathered in the peri-tumoral region, while very few infiltrated the tumor. Metformin administration increased the expression of anti-tumor immunity-associated genes and the number of tumor-infiltrating CD8+ lymphocytes. Injection of CD8 but not CD4 neutralization antibody into tumor-bearing mice significantly abrogated the anti-tumor effect of metformin. Critically, CD8+ lymphocytes were found to pass through the wall of perfused vessel. Further results of immunofluorescent staining showed that metformin greatly elevated tumor perfusion, which was accompanied by increased vascular maturity in the intratumoral region (ITR) but not peritumoral region (PTR). These findings provide evidence for the vascular mechanism involved in metformin-induced enhancement of T lymphocyte anti-tumor immunity. By remodeling the abnormal tumor vasculature, also called vessel normalization metformin increases vascular maturity and tumor perfusion, thus allowing more CD8+ lymphocytes to infiltrate the tumor.

Effects of compound Kushen injection on pathology and angiogenesis of tumor tissues.

Effects of compound Kushen injection on pathology and angiogenesis of tumor tissues were investigated. Forty nude mice were used to establish the liver cancer model of nude mice, and were divided into model group (n=10), low-dose compound Kushen injection group (n=10), medium-dose compound Kushen injection group (n=10) and high-dose compound Kushen injection group (n=10). When the tumor volume reached 0.5 cm3, 200, 400 and 600 µl of compound Kushen were injected into the mice of the low-, medium- and high-dose compound Kushen injection groups, respectively, for 3 consecutive days; while 400 µl normal saline were injected into the mice of the model group. At 9 days after treatment, the mice were sacrificed, and the tumor was taken and weighed. The tumor inhibition rate was calculated, α-smooth muscle actin (α-SMA) and cluster of differentiation 31 (CD31) were detected via immunohistochemistry, and the vascular maturity index (VMI) and microvessel density (MVD) were also detected. With the increase of compound Kushen injection dose, the tumor mass was decreased significantly (P<0.05), and the tumor inhibition rate was obviously increased (P<0.05). In the model group, the nuclei were large and deeply stained, and there were many mitotic figures, and more small blood vessels could be seen. In the three Kushen injection groups, the number of mitotic figures was slightly decreased, and the vascular distribution was reduced. With the increase of compound Kushen injection dose, MVD of transplanted tumor was decreased significantly, but VMI was increased significantly (P<0.05). Compound Kushen injection can reduce the angiogenesis in tumor tissues and play a role in inhibiting tumor growth. Therefore, anti-angiogenesis may be one of the important mechanisms of compound Kushen injection in inhibiting tumor growth.