Our previous findings that the deposition of fibrin is florid in the MPM of mice and in patients with MPM (3
), and that both TF and TFPI are expressed in human MPM tissue (3
), justify our focus on the role of TFPI in the pathogenesis of this disease. The increased expression of TF in tumor cells was associated with aspects of tumor progression, including angiogenesis, invasion, and metastasis (9
). Both TF and TFPI are readily detectable in virtually all forms of MPM, including the epithelioid and sarcomatous (spindle-shaped) variants that occur in human disease (3
). TFPI was reported to inhibit the growth of melanoma tumors in mice by more than 80% (13
). It also significantly reduced the formation of lung metastases (13
). Therefore, TFPI can influence the growth and propagation of some forms of malignancy. Although the role of TFPI in the pathogenesis of MPM was not, to our knowledge, previously studied, we hypothesized that TFPI could influence the progression of this tumor, and tested that possibility here.
TFPI occurs in two major forms. TFPI-1 is a TF inhibitor that is mainly responsible for inhibiting coagulation. Peritumoral injections of TFPI-1 in B16 melanoma tumors inhibited tumor growth (11
). Because of the prominent deposition of fibrin we previously observed in association with the development of REN tumors in mice and in patients with MPM (3
), we studied the effects of TFPI-1 on the progression of MPM. REN cells are well suited for these analyses, because they form aggressive tumors and they do not express TFPI. On the other hand, TFPI-2 was discovered and named after TFPI-1 because of the similarities in their structures. TFPI-2, however, is a serine protease inhibitor that mainly functions as a plasmin inhibitor, and it weakly inhibits the coagulation cascade (11
). Many studies were performed on TFPI-2 as an antiangiogenic and antimetastatic agent (11
). Our results show that TFPI-1 can regulate the aggressiveness of MPM in vitro
and in vivo
. In future studies, we will study the role of TFPI-2 in the growth and invasiveness of MPM, using the same approach deployed in this project.
In humans, MPM is strongly associated with aggressive local growth, late metastasis, pleural effusions, focal inflammation, and the deposition of fibrin (1
). These attributes were likewise found in the nude, athymic murine model we used. The deposition of fibrin was florid in tumors from the naive and EV groups, indicating that the model recapitulates this aspect of clinical MPM (3
). This finding is especially impressive, because the pleural cavities were washed with saline, which might have disrupted at least some of the fibrinous material that occurs in vivo
. Pleural effusions were also prevalent in the naive and EV mice. TFPI–MPM–bearing mice lacked pleural effusions, which paralleled the paucity of exophytic tumors and likely reflects the absence of pleural invasion. Interestingly, the pleural washes from mice challenged with TFPI-overexpressing REN cells demonstrated increased amounts of MPM cells that retained the characteristics of those seen within the tumors. In the majority of TFPI–REN mice, overt tumors did not form, but cells likely to be REN cells rather than reactive mesothelial cells were found in relative abundance within pleural washes. This result suggests that TFPI–REN cells did not readily form tumors in vivo
and that they persisted and may be prone either to associate with loosely, or dehisce from, the pleural surface.
The REN cell line was shown to be more aggressive than the MS-1 and M9K MPM cell lines that express TFPI (4
). Our in vitro
experiments indicate that the invasion and surface activity of TF in live cells was decreased when TFPI was transfected in the REN cell line. Our results further show that the transfection of TFPI into REN cells decreases tumor growth and invasion after injection in vivo
. Over the 25-day period allocated for tumor growth in vivo
, the naive and EV-generated tumors proved more invasive and aggressive than those of the TFPI cells. In lung CT images, lung volumes were significantly reduced by the formation of exophytic tumors. Pulmonary function testing revealed that the formation of exophytic tumors did not affect the compliance or elastance of the lungs, and did not detectably affect airway resistance. Elastance and compliance are physiologic indices of the expansive capacity and stiffness of the lung parenchyma, which were unchanged by the growth of exophytic tumors. These results suggest that lung volume, as measured with Microview software to create a three-dimensional image of the lungs, is a more sensitive index of tumor growth external to the murine lung. Our data suggest that the TFPI knock-in mice manifested relatively larger lung volume because their pleural tumor load was relatively reduced versus the EV or naive mice.
The results further show that the administration of TFPI–REN cells resulted in decreased tumor counts, volumes, and weights versus the control EV or naive REN tumor groups. Although the EV tumor counts were significantly different from those in naive-injected mice, tumor volumes and weights were comparable in the naive and EV groups. These findings suggest that the transfection of REN cells affects their ability to coalesce as exophytic tumors, but that overall tumor burden was not changed in the EV group. The data also show, however, that tumor burden was significantly increased in the EV and naive groups versus the TFPI knock-in REN group.
An increase was readily apparent in the invasiveness of naive and EV-expressing tumors versus the TFPI group. Further, naive and EV-expressing tumor invasion was characterized by focal chronic inflammation and an extensive deposition of collagen, mirroring the histologic findings that may occur during MPM in humans (1
). TFPI-expressing tumors were also found to be smaller, fewer in number, lacking in inflammatory cell infiltration, and not invasive. Tumor invasiveness correlated with the formation of sanguineous or bloody effusions and the increased formation of fibrinous neomatrix in the EV and naive groups. When grossly detectable, TFPI tumors were characterized by the formation of discrete, smaller tumors (in 4/10 mice) that presented no evidence of invasion in vivo
. Furthermore, increased TFPI was found in the pleural lavage of mice challenged with TFPI–REN cells, indicating that the injected cells continued to express TFPI throughout the 25-day course of the experiments. The trend toward tumor formation in these mice parallels the in vitro
trends we observed before the tumors were administered. Interestingly, the same trends were evident in tumor cells harvested and propagated from harvested tumors and then studied in vitro
. These pathophysiologic trends were consistent with the durable retention of the TFPI gene product in TFPI knock-in cells from tumor-bearing mice, and strongly suggest that the expression of TFPI was responsible for the effect.
MPM is refractory to conventional cancer treatments. Our data strongly implicate TFPI in controlling the growth of MPM, extending our previous finding that TFPI is expressed in clinical tumor samples from patients with MPM (18
). Because TFPI has already been used in clinical trials of patients with sepsis and pneumonia (19
), the findings assume potential translational applicability. Our observations suggest that the administration of exogenous TFPI could restrict the progression of MPM. Because TFPI is no longer available in the quantities needed for preclinical interventional administration, testing this postulate would entail the production and characterization of recombinant TFPI. In future preclinical analyses, we will test the ability of exogenous TFPI to slow or reverse the growth of established MPM.