The low rate of pericyte coverage in PC3/2G7 tumors compared to PC3 tumors is indicative of a more immature vascular system, and may contribute to the greater anti-angiogenic response of the PC3/2G7 tumors

The low rate of pericyte coverage in PC3/2G7 tumors compared to PC3 tumors is indicative of a more immature vascular system, and may contribute to the greater anti-angiogenic response of the PC3/2G7 tumors. Prolonged treatment of PC3/2G7 tumors with axitinib led to an apparent escape from anti-angiogenesis, marked by neo-vascularization and the resumption of tumor growth. proliferation and cell viability and a greater increase in apoptosis. The increased sensitivity of PC3/2G7 tumors to anti-angiogenesis Z-360 calcium salt (Nastorazepide calcium salt) indicates they are less tolerant of low vascularity and suggests they become addicted to their oxygen- and nutrient-rich environment. PC3/2G7 tumors showed strong up-regulation of the pro-angiogenic factors CCL2 and VEGFA compared to PC3 tumors, which Rabbit Polyclonal to TPH2 may contribute to their increased vascularity, and they have significantly lower endothelial cell pericyte coverage, which may contribute to their greater sensitivity to anti-angiogenesis. Interestingly, high levels of VEGF receptor-2 were expressed on PC3 but not PC3/2G7 tumor cells, which may contribute to the growth static response of PC3 tumors to VEGF-targeted anti-angiogenesis. Finally, prolonged anti-angiogenic treatment led to resumption of PC3/2G7 tumor growth and neo-vascularization, indicating these cancer stem-like cell-derived tumors can adapt and escape from anti-angiogenesis. 0.01, and *** 0.001. Results High vascularity PC3/2G7 tumor model PC3/2G7 is a clonal isolate from the human prostate cancer cell line PC3; it is one of several similar, independent clones derived from a sub-population of cancer stem-like cells present within the parental PC3 cell population and was isolated based on its characteristic holoclone morphology (33). Tumors derived from PC3/2G7 cells show significantly higher vascularity than parental PC3 cell-derived tumors, as indicated by immunostaining with anti-mouse CD31 antibody (Fig. 1A), and confirmed by the increased expression of CD31 and also VE-cadherin, a second marker of vascular endothelial cells (Fig. 1B, 1C). Tumor blood flow was substantially increased in PC3/2G7 tumors compared to PC3 tumors, as shown by Hoechst dye perfusion (Fig. 1D), indicating that the high-density PC3/2G7 blood vessels are functional. Immunostaining with anti-human CD31 was Z-360 calcium salt (Nastorazepide calcium salt) negative for both PC3 and PC3/2G7 tumors (c.f. normal human tonsil positive control; Supplemental Fig. 1). Thus, the dense blood vessels found in PC3/2G7 tumors are derived from host (mouse) endothelial cells and are not formed by differentiation of the (human) stem-like cells used to seed the PC3/2G7 tumors. Open in a separate window Figure 1 Vascularity of PC3/2G7 and PC3 tumor modelsA. Representative CD31 immunostained cryosections of PC3/2G7 and PC3 tumors showing high and low microvessel density, respectively (magnification, 10x). B. Quantification of CD31 immunostained PC3/2G7 and PC3 tumors (as in Z-360 calcium salt (Nastorazepide calcium salt) A) using NIH ImageJ software. Data are mean SE values based on stained cryosections from three different regions of n=7 PC3/2G7 tumors and n=6 PC3 tumors; **, (11), these findings indicate that the increased vascularity of PC3/2G7 tumors is VEGF receptor-dependent. Axitinib did not induce host toxicity in either tumor model, as judged by body weight measurements (data not shown). Open in a separate window Number 2 Anti-tumor activity of axitinib against Personal computer3/2G7 and Personal computer3 tumorsA. Effect of daily axitininb treatment (days 1C12) on tumor growth in male scid mice. Tumor quantities, mean SE, for n=10C14 tumor/group. BCE, Quantitative analysis of the effects of axitinib on: B, tumor microvessel denseness (CD31 staining); C, tumor cellularity (hematoxylin staining); D, tumor cell proliferation (PCNA staining); and E, apoptosis (TUNEL). Quantitation was identified using ImageJ. Data are mean SE ideals based on stained cryosections (magnification, 4.2) from three different regions of n = 4 tumors/group. Representative stained images are demonstrated in Supplemental Fig. 2. * (Fig. 2A) are indirect reactions to the loss of VEGF signaling, and are not due to direct Personal computer3/2G7 or Personal computer3 tumor cell cytotoxicity. Effect of sorafenib and DC101 on Personal computer3/2G7 and Personal computer3 tumors Next, we investigated whether the higher sensitivity of Personal computer3/2G7 tumors to axitinib is seen with two additional anti-angiogenic medicines, the multi-RTKI sorafenib (38) and the anti-VEGFR2 monoclonal antibody DC101, which blocks VEGF-induced receptor activation (9, 10). With both medicines, Personal computer3/2G7 tumor growth was inhibited more extensively and/or for a longer period of time than Personal computer3 tumors (Supplemental Figs. 4 and 5). Blood flow to Personal computer3/2G7 tumors was markedly decreased by both sorafenib and DC101, and a further decrease in the already low blood flow to Personal computer3 tumors was also apparent (Fig. 3A). The treated Personal computer3/2G7 tumors showed low microvessel denseness, with many blood vessels showing little internal volume in cross-sections, in contrast to the many large, open Z-360 calcium salt (Nastorazepide calcium salt) vessels found in untreated Personal computer3/2G7 tumors (Fig. 3B). The few blood vessels recognized in the sorafenib- and DC101-treated Personal computer3 tumors were much like or smaller than those in untreated Personal computer3 tumors. The very low vascularity of the treated Personal computer3/2G7 tumors was associated with more considerable tumor necrosis (fewer viable tumor areas), as.