In order to examine the role of cathepsin H in tumor development, we analyzed tumor progression in the RT2 model in the absence of this protease. Cathepsin H knockout mice have recently been generated and are viable and fertile with no gross phenotypes (Reinheckel and colleagues, manuscript in preparation). We generated cathepsin H null (Ctsh-/-) RT2 mice and first analyzed the frequency of angiogenic switching at 10.5 weeks of age, which is a measure of the ability of pre-neoplastic lesions to induce neo-angiogenesis in their quiescent vasculature. In fact, this is a rate-limiting step in the RT2 model and determines the ability of subsequent tumors to develop. Cathepsin H is expressed at this stage of tumor progression, as determined by immunofluorescence staining of pancreatic tissue from 10.5 week old wild-type (WT) RT2 mice (data not shown). The angiogenic islets that formed, identified grossly by their red, hemorrhagic appearance, were counted in Ctsh-/- RT2 mice and the number was compared to the heterozygous or WT RT2 littermates. In the WT RT2 group, the number of islets ranged from 33 to 69, with an average of 52; in contrast, deletion of one or both copies of cathepsin H reduced the angiogenic switching frequency by 35% and 32%, respectively (; P<0.01).
Deletion of cathepsin H (Ctsh) in RT2 mice leads to a reduction in angiogenic switching, tumor number and tumor volume
Progression though angiogenic switching is essential for subsequent tumor development since an adequate blood supply is necessary for the expansive tumor growth that follows. Given the observed defect in angiogenic switching in RT2 mice lacking one or both copies of Ctsh, we next analyzed tumor development in these mice. We aged a separate cohort of mice to the defined endpoint of 13.5 weeks and assessed the tumor burden and incidence. Tumors, defined as lesions with a minimal diameter of 1 mm, were macroscopically detected and counted. In WT RT2 animals, the number of tumors ranged from 3 to 20, with a mean of 11.24. In contrast the mean tumor number in Ctsh+/- RT2 mice was reduced by 29% (P<0.01) and a further reduction to 33% (P<0.01) was observed upon deletion of the second copy of Ctsh (). These results closely parallel the percent reduction in the number of angiogenic islets, suggesting that the inability of one third of all islets to undergo angiogenic switching led to a comparable decrease in subsequent tumor incidence. When cumulative tumor volume was assessed in these same animals, a significant reduction of 40% was observed in Ctsh null RT2 mice (; P<0.01). In contrast, tumor volume in Ctsh+/- RT2 mice was only slightly impaired, despite the substantial decrease in the number of tumors, suggesting that the resulting lesions are not as significantly impaired in growth as lesions in Ctsh null animals. As tumors in Ctsh+/- RT2 mice were similar in size to the WT littermate controls, their phenotypes were not investigated further.
We hypothesized that the decrease in tumor volume in the Ctsh deficient RT2 mice is due to a shift in the balance between proliferation and apoptosis rates, which collectively affect tumor growth. First, we analyzed the proliferation rates by quantifying the number of BrdU-positive cells and determined that while there was a trend towards a reduction in Ctsh deficient tumors compared to WT RT2 tumors (30% decrease), this was not statistically significant (). To assess the effect of cathepsin H deletion on apoptosis we analyzed the number of cleaved caspase 3-positive cells in tumors from Ctsh-/- RT2 mice and compared them to WT RT2 littermates. The apoptotic index was increased two-fold (P<0.05) in the homozygous knockouts (), which together with the trend towards reduced proliferation, likely accounts for the significant reduction in tumor growth.
Cathepsin H deletion affects the balance between apoptosis and proliferation in RT2 tumors
In addition to its role in apoptosis (Cirman et al., 2004
), several studies have suggested that cathepsin H is involved in invasion, with numerous clinical reports documenting increased cathepsin H expression in highly aggressive carcinomas (Berdowska, 2004
). Therefore, we sought to assess the role of cathepsin H in invasion in vivo
in the pancreatic islet cancer model. Pancreatic tissues from 13.5 week old RT2 mice were analyzed by hematoxylin and eosin (H&E) staining and the tumors were classified into three grades of increasing malignancy as previously described (Lopez and Hanahan, 2002
). Briefly, encapsulated tumors are relatively benign lesions with an intact collagen capsule and a clear separation between the tumor and the surrounding normal exocrine pancreas. On the contrary, invasive carcinomas have migrated into the surrounding normal tissue. In the case of microinvasive carcinomas (IC1) there are focal regions of invasion with adjacent margins, while frankly invasive tumors (IC2) have widespread invasion with no evidence of tumor margins and are also characterized by an increased nuclear to cytoplasmic ratio (Lopez and Hanahan, 2002
). Histological analysis of the spectrum of tumors revealed that while there was a slight decrease in the invasiveness of Ctsh-/-
tumors, with less microinvasive and more benign lesions, there was no difference in the number of highly invasive carcinomas that developed (). Deletion of one allele of cathepsin H
revealed an intermediate phenotype, which was also not statistically significant.
We next wanted to investigate whether cathepsin H plays a role in tumor angiogenesis as one explanation for the observed decrease in tumor growth in the Ctsh null RT2 mice is that there is an insufficient or poorly developed blood vasculature in the lesions. Moreover, we observed defects in angiogenic switching in Ctsh-/- RT2 animals (), suggesting that cathepsin H might have pro-angiogenic functions. Pancreatic tissue from 13.5 week old WT or Ctsh null RT2 mice was stained with an antibody recognizing the endothelial specific marker CD31 and several different parameters of the tumor vasculature were analyzed.
A reduction in vessels was observed in Ctsh-/- RT2 tumors, with a 32% decrease in area covered by CD31-positive structures relative to the control group (; P<0.01). To further characterize the angiogenic defects in Ctsh-/-RT2 tumors, we perfused mice with a FITC-conjugated lectin, which binds to the luminal surface of endothelial cells thus labeling the vessels that are connected to the circulation. Measurement of the area of lectin covering the tumor vasculature showed that there was a 59% decrease in lectin perfusion in the Ctsh-/- RT2 tumors compared to WT, a reduction that was greater than the overall decrease in CD31 positive area (; P<0.05). Together these data suggested that some of the vessels that were present in Ctsh-/- RT2 tumors are not connected to the circulation and are therefore non-functional. To determine if this was indeed the case, we analyzed the co-localization of the lectin signal with that for CD31. We found that there was a reduction in the percentage of functional vessels in Ctsh deficient tumors and there was a corresponding increase in the number of CD31-positive vessels not filled with lectin compared to WT RT2 tumors.
Cathepsin H has important roles in regulating tumor angiogenesis
One factor that can affect vessel integrity is their ability to recruit pericytes, which are a key component of the vasculature and can regulate vessel stability, maturation and function. While in normal vessels pericytes are in tight association with endothelial cells, in tumors pericytes are less abundant and more loosely attached to endothelial cells, with cytoplasmic processes that can extend away from the vasculature (Baluk et al., 2005
). Co-staining of CD31 with the pericyte marker NG2 revealed a reduction in pericyte coverage of the blood vessels, calculated by the area of overlap between CD31 and NG2-positive areas, in Ctsh
null tumors compared to WT RT2 controls (; P<0.01). Analysis of the ratio of NG2-positive area normalized to the vessel area revealed that there is no difference between WT and Ctsh-/-
tumors in their ability to recruit pericytes in the vicinity of CD31-positive cells (). Rather the reduction in coverage was due to a defect in subsequent pericyte integration (calculated by the ratio of CD31+NG2+ area relative to CD31-NG2+) into the blood vessels in tumors lacking Ctsh
Given the angiogenic defects in the Ctsh-/- tumors at both the pre-neoplastic as well as tumor stage, we next wanted to determine the cellular source of this protease as expression by endothelial cells would suggest a cell-intrinsic effect for cathepsin H. However, immunofluorescence staining of WT RT2 tumors with cell type-specific antibodies revealed that cathepsin H is not expressed by CD31-positive endothelial cells, although many of the cathepsin H-positive cells are localized in close proximity to the vasculature (). Given the close interactions between endothelial cells and pericytes and the ability of mural cells to affect vessel integrity, we then investigated cathepsin H expression by pericytes by co-staining tissues with NG2; however, again no co-localization was observed (). Since certain inflammatory cells can also modulate tumor angiogenesis, we next stained RT2 tumors with antibodies against antigens present on neutrophils and macrophages. While there was no co-localization between cathepsin H protein and the neutrophil-specific antibody 7/4 (), this analysis revealed that the majority of cathepsin H expression is macrophage derived (). Cathepsin H expression by macrophages was verified using a second marker for this cell type, Iba1, and co-staining with CD31 showed that cathepsin H is often expressed by the macrophages in close proximity to the blood vessels ().
Analysis of cathepsin H expression in WT RT2 tumors