Opg Report Where Tumor Detected

Fig. 8. Histological appearance of LNCaP tumors growing in adult human bone fragments implanted into NOD/SCID mice. Specimens obtained at 2 weeks (A and B), 4 weeks (C), 6 weeks (D and E), and 8 weeks (F) after LNCaP cell injection. (A) Metastatic foci were formed in bone marrow sinuses at 2 weeks after the inoculation of prostate cancer cells. Little initial response of bone marrow stromal cells was detected around the tumors (H&E, original magnification ×100). (B) Prostate-specific antigen (PSA)-positive tumor foci were often found in blood vessels (PSA stain, original magnification ×100). (C) Localized LNCaP tumors were observed in bone fragments at 4 weeks after injection of LNCaP cells. An initial mild response of bone marrow stromal cells was observed around tumor foci (H&E, original magnification ×40). (D) Intertrabecular bone metaplasia. Active bone formation by metaplasia of bone marrow stromal cells adjacent to tumor foci at 6 weeks after injection of LNCaP cells (H&E, original magnification ×100). (E) Apposi-tional bone formation. A minute focus of appositional new bone formation is seen on a trabecula of lamellar bone. The osteoid matrix shows intense eosinophilic staining (Yoshiki stain, original magnification ×100). (F) Reconstructive osteosclerosis in prostate cancer. The intramedullary space has been almost entirely replaced by tumor cells. Although this is a case of osteoblastic metastasis, an irregular cement line suggesting previous bone resorption can be observed (H&E, original magnification ×100). (Adapted with permission from ref. 99).

adult bone, Yonou et al. (99) and Tsingotjidou et al. (97) used adult bone as an environment for establishment of CaP bone metastases in immune-compromised mice. Yonou et al. (98,99) used non-obese diabetic/severe combined immunodeficient mice, adult human bone, and tail-vein injection of CaP cells. Three to 4 weeks after human bone implantation, PC-3 cells or LNCaP cells were injected. Sixty-five percent and 35% of animals, respectively, developed tumors in the grafted bones. The LNCaP cells colonized the bone marrow cavity, often exhibiting an osteoblastic response at the edges of metastatic foci, with new bone formation adjacent to mature lamellar bone (Fig. 8). Similar to the findings of Nemeth et al. (24), there was no colonization of human lung tissue implants. Tsingotjidou et al. (97) used human adult bone harvested from patients undergoing total joint arthroplasty, which was then implanted intramuscularly in the hind limbs of irradiated SCID mice. Tumor cells PC-3 and LAPC-4 were injected next to the bone 2 months after bone implantation. Both tumor types showed growth in this environment, with no detectable new bone formation, but significant osteolysis.

5.2.5. Intratibial Injection

Another approach for the generation of experimental CaP bone metastases consists of direct injections of CaP tumor cells into mouse bone (96,100-102). This technique was originally developed by Berlin et al. (103) in generating spontaneous metastases of osteosarcoma to lungs. We (96,102) have used CaP cells PC-3, LNCaP, and C4-2, as well as cells isolated from CaP xenografts LuCaP 23.1 and LuCaP 35, established in our laboratory, to characterize CaP/osseous models derived from the direct injection of tumor cells into tibiae of 4- to 6-week-old SCID mice. We have had considerable success with the intratibial injections. The general protocol, adapted from Berlin et al. (103), is to inject 1-2 x 105 CaP cells with a 26-gauge needle approx 3 mm into the proximal end of the tibia of 4-week-old SCID male mice. LuCaP 23.1, LNCaP, LuCaP 35, C4-2, and PC-3 cells injected into the tibia all exhibited tumor growth in the bone. PC-3 cells caused an osteolytic response, LNCaP and LuCaP 35 yielded mixed lesions, and LuCaP 23.1 yielded an osteoblastic response (Fig. 9), new bone development was evident throughout the lesions; tumor cells replaced all bone marrow, and extensive new bone formation was observed. We also examined the nature of the interactions of LuCaP 23.1 with bone at early, mid-phase, and late time-points of its growth in the bone environment. Figure 10 shows by radiography representative radiographs of early, mid-phase, and late tumor development. Histology of tibiae with prostate tumor cells was compared with samples of human CaP bone metastases. The LuCaP 23.1 and PC-3 tumored bones, respectively, showed many similarities to human samples of osteblastic and osteolytic bone metastasis of protate cancer (Fig. 11). We also used immunohistochemistry to evaluate the validity of these xenografts as models of human CaP bone metastasis. As in human specimens, we detected osteoprotegerin (OPG), receptor activator of nuclear factor kappa B, (RANKL), parathyroid hormone like protein (PTHrP), and endothelin-1 (ET-1) immunoreactivity in all CaP xenografts growing in the bone.

The C4-2 osseous model and its response to castration were also characterized (102). C4-2 tumors had a 100% take rate in bone and caused radiographically lytic expansile lesions. C4-2 cells decreased bone mineral density and bone volume of the injected tibiae vs normal tibiae. Castration caused a drop in serum PSA with a nadir at day 14, after which it began to rise again. Bone destruction in the tumorous tibiae of castrated animals was decreased by 15.9% vs tumorous tibiae of intact animals.

Simultaneously, Fisher et al. (101) published characterization of intratibial injections of PC-3, DU 145 and LNCaP CaP cells into athymic mice. In this report, PC-3 and DU 145 cells had a mixed sclerotic/lytic appearance and caused increases in the number of osteoclasts, whereas LNCaP did not form tumors in the bone environment.

Lee et al. (100) used LAPC9 and PC-3 in the tibial injection model. PC-3 caused osteolytic lesions with a large number of osteoclasts. In contrast, LAPC-9 caused osteoblastic lesions at 6 weeks, with osteoclasts detected only rarely. Greater osteoclast activity was detected at 8 weeks when the osteoblastic lesions were well-established (Fig. 12). Immunohistochemical analysis showed that PC-3 produced RANKL, interleukin (IL)-1, and tumor necrosis factor-a, which are associated with osteoclastogenesis. LAPC-9 cells produced no RANKL or IL-1, and minimal amounts of tumor necrosis factor-a, but large quantities of bone morphogenetic protein (BMP) 2, BMP 4, BMP 6, and IL-16, which are associated with bone formation.

Andresen et al. (104) injected CWR22 cells into tibiae of athymic rats. Both osteoblastic and osteolytic reactions were detected after 4 to 6 weeks, with the osteosclerotic reaction predominant. Radiological and histological evidence revealed osteosclerotic lesions with trabeculae of newly formed bone lined by active osteoblasts and surrounded by tumor cells. Near the end of the 7-week study, osteolytic bone lesions became more evident on X-rays.

Fig. 9. Characteristics of osseous prostate cancer (CaP) models. (A) Radiograph of LNCaP intratibial tumor showing osteolytic expansile lesion with soft-tissue abnormalities. (B) H&E of LNCaP intratibial tumor, demonstrating mixed lesion (eroded surface and thickened trabeculae). (C) Anti-prostate-specific antigen (PSA) stain of LNCaP in the tibia. (D) Radiograph of LuCaP 35 in the tibia showing osteolytic expansile lesion with soft-tissue abnormalities. (E) H&E of LuCaP 35 in the tibia demonstrating mixed lesion (eroded surface and thicker trabeculae). (F) Anti-PSA stain of LuCaP 35 in the tibia. (G) Radiograph of LuCaP 23.1 in the tibia showing osteoblastic growth. (H) H&E of LuCaP 23.1 in the tibia. (I) Anti-PSA stain of LuCaP 23.1 in the tibia. (J) Radiograph of PC-3 in the tibia showing osteolytic lesion with a large reduction in bone mass. (K) H&E of PC-3 in the tibia with an increase in osteoclasts lining the bone. (L) Anti-PSA IP stain of PC-3 in the tibia showing a lack of reactivity. (Adapted with permission from ref. 96).

Fig. 9. Characteristics of osseous prostate cancer (CaP) models. (A) Radiograph of LNCaP intratibial tumor showing osteolytic expansile lesion with soft-tissue abnormalities. (B) H&E of LNCaP intratibial tumor, demonstrating mixed lesion (eroded surface and thickened trabeculae). (C) Anti-prostate-specific antigen (PSA) stain of LNCaP in the tibia. (D) Radiograph of LuCaP 35 in the tibia showing osteolytic expansile lesion with soft-tissue abnormalities. (E) H&E of LuCaP 35 in the tibia demonstrating mixed lesion (eroded surface and thicker trabeculae). (F) Anti-PSA stain of LuCaP 35 in the tibia. (G) Radiograph of LuCaP 23.1 in the tibia showing osteoblastic growth. (H) H&E of LuCaP 23.1 in the tibia. (I) Anti-PSA stain of LuCaP 23.1 in the tibia. (J) Radiograph of PC-3 in the tibia showing osteolytic lesion with a large reduction in bone mass. (K) H&E of PC-3 in the tibia with an increase in osteoclasts lining the bone. (L) Anti-PSA IP stain of PC-3 in the tibia showing a lack of reactivity. (Adapted with permission from ref. 96).

Fig. 10. Progression of LuCaP 23.1 lesions in the tibia. Representative radiographs of (A) LuCaP 23.1 in the early phase; (B) in the mid phase; and (C) in the late stage. (Adapted with permission from ref. 96).

Fig. 11. Goldner's staining of normal tibia, LuCaP 23.1 and PC-3 cells injected into tibia. (A) Normal mouse tibia; (B) LuCaP 23.1 cells injected into tibia; and (C) PC-3 cells injected into tibia; Goldner's staining; original magnification x6. (D-F) Examples of human bone samples of similar character; original magnification x6. (D) Normal human bone; bony trabeculae and bone marrow are present. (E) Human CaP osteoblastic lesion, with significant increase in mineralized bone (green area), replacement of bone marrow by tumor cells (grey areas). (F) Human CaP osteolytic lesion; most of the bony trabeculae have been lysed and the spaces filled with tumor cells. Green areas are remaining mineralized bone; tumor cells are grey. (Adapted with permission from ref. 96).

Fig. 11. Goldner's staining of normal tibia, LuCaP 23.1 and PC-3 cells injected into tibia. (A) Normal mouse tibia; (B) LuCaP 23.1 cells injected into tibia; and (C) PC-3 cells injected into tibia; Goldner's staining; original magnification x6. (D-F) Examples of human bone samples of similar character; original magnification x6. (D) Normal human bone; bony trabeculae and bone marrow are present. (E) Human CaP osteoblastic lesion, with significant increase in mineralized bone (green area), replacement of bone marrow by tumor cells (grey areas). (F) Human CaP osteolytic lesion; most of the bony trabeculae have been lysed and the spaces filled with tumor cells. Green areas are remaining mineralized bone; tumor cells are grey. (Adapted with permission from ref. 96).

Fig. 12. PC-3 and LAPC-9 in tibiae. (A) 1, (B) 2, (C) 4, (D) 6, and (E) 8 weeks after injection. Top row: radiographs of PC-3 injected tibias. Osteolytic lesions notable at 2 weeks, with complete destruction of the proximal tibia at 8 weeks. Middle row: radiographs of LAPC-9 injected tibias. Osteoblastic lesions notable at 6 and 8 weeks. Bottom row: Left: H&E of PC-3 bone interphase with osteoclast. Right: H&E of LAPC-9 in bone. (Adapted with permission from ref. 100).

Fig. 12. PC-3 and LAPC-9 in tibiae. (A) 1, (B) 2, (C) 4, (D) 6, and (E) 8 weeks after injection. Top row: radiographs of PC-3 injected tibias. Osteolytic lesions notable at 2 weeks, with complete destruction of the proximal tibia at 8 weeks. Middle row: radiographs of LAPC-9 injected tibias. Osteoblastic lesions notable at 6 and 8 weeks. Bottom row: Left: H&E of PC-3 bone interphase with osteoclast. Right: H&E of LAPC-9 in bone. (Adapted with permission from ref. 100).

A similar experimental approach for the study of bone metastasis is injection of tumor cells into the femurs of SCID mice, as used by Navone et al. (35) and Pinthus et al. (105). Navone et al. used MDA PCa 2b established from bone metastases, and these cells resulted in an osteoblastic reaction. Pinthus et al. reported that WISH-PC2 produced osteolytic lesions with foci of osteoblastic activity when the cells were injected directly into the femur.

In summary, because of the paucity of models of spontaneous CaP bone metastases, new models have been developed to study interactions of CaP cells with the bone environment, using either human bone or mouse bone and various CaP cell lines. Informative morphological and biochemical findings corresponding to CaP growth in human bone have been observed using all of these models. However, because the bone metastases are experimentally induced by direct bone injection, these models cannot be used effectively for studies of early metastatic events, such as migration, and so on. This is an important limitation on the use of these models for the study of trafficking and migration of CaP, but the results reported herein suggest that this has little impact on the value of the models for investigation of the interactions between CaP and bone and testing of new therapeutic modalities. Another limitation of the existing models is that CaP cells commonly used for these studies result in modest new bone formation (C4-2, C4-2B, CWR22, and MDAPCa2b) or in osteolytic reactions (PC-3). There are only two xenografts, LuCaP 23.1 and LAPC-9, that produce osteoblastic reactions in the bone resembling those in human specimens. Unfortunately, neither of these xenografts grows in vitro, limiting the studies of characterization of phenotypic alterations and effects on signaling pathways.

5.3. Preclinical Use of Experimental Bone Metastasis Models

All of the above-described models of generating experimental bone metastases of CaP have been used to evaluate the effects of new therapeutics and to increase our understanding of the interactions of prostate tumor cells and the bone environment. What follows is a summary of studies from various groups showing the significant usefulness of these models in preclinical testing.

5.3.1. SCID-hu Model

There are multiple reports of use of the SCID-hu model. The first reported use of this model to evaluate new treatment modalities was published by Nemeth et al. (106). The authors showed that matrix metalloproteinase activity was associated with PC-3 cell proliferation in bone, and that daily treatment with batimastat, a broad-spectrum matrix metalloproteinase inhibitor, hampers osteolysis associated with the growth of these cells in the bone environment, as well as tumor growth. Cher et al. (107) used this model to show that expression of maspin, when overexpressed in DU 145 cells, caused inhibition of osteolysis, tumor growth, and angiogenesis. The model was also used to evaluate the effects of genistein on PC-3 bone metastases (108); the results showed that genistein inhibits growth of PC-3 cells in the bone environment. Nemeth et al. (109) also showed that inhibition of aM P -3 integrin reduced PC-3 cell proliferation in the SCID-hu model. Zhang et al. (110) blocked RANKL activity with soluble murine RANK-Fc (sRANK-Fc) to prevent progression of CaP cell growth in the bone environment. LuCaP 35 CaP cells, grown in the fetal bone and treated with sRANK-Fc, resulted in fewer osteoblastic lesions, lower bone mineral density, decreased serum PSA levels, and diminished tumor volume. In contrast, sRANK-Fc had no effect on subcutaneously implanted LuCaP 35 cells. The authors concluded that sRANK-Fc is an effective inhibitor of RANKL that diminishes progression of CaP growth in bone through inhibition of bone remodeling.

5.3.2. Adult Bone Model

Models using adult human bone were used by Goya et al. (111) and Yonou et al. (112). Goya et al.

(111) investigated whether a novel antibody directed against human IGF-I and IGF-II (KM1468) inhibits the development and progression of LNCaP bone tumors. KM1468 markedly and dose-dependently suppressed the development of new bone tumors and the progression of established tumor foci, and it also decreased serum PSA levels. These results indicate that the IGF signaling axis is a potential target for prevention and treatment of bone metastases arising from CaP. Yonou et al.

(112) evaluated whether blockade of osteoclastogenesis by OPG inhibits the development of new bone tumors and the progression of established osteoblastic bone tumors. In this study, OPG reduced the number of osteoclasts and the size of the tumors at the bone sites, but it had no effect on the local growth of subcutaneous LNCaP tumors. These findings demonstrate that osteoclasts play an important role in bone tumors of CaP, and that OPG decreases the LNCaP CaP burden selectively in bone and prevents the development of new lesions. This suggests that inhibition of osteoclastic bone resorption may be an effective therapy for the treatment of CaP that has colonized bone. Similar findings using intratibial injections have been published by Zhang et al. (113) and Kiefer et al. (114) (see Section 5.3.3).

5.3.3. Direct Injection Into Mouse Bone

Our efforts to treat cancer have focused on compounds that affect growth and apoptosis of tumor cells. Compounds that modulate the host organ microenvironment may provide additional benefits in cancer treatment. Because literature data suggest that increased osteolysis is a key component of CaP bone metastasis, there has been interest in whether inhibitors of osteolysis could slow the growth of these lesions. Zhang et al. (113) and Kiefer et al. (114) evaluated the effects of OPG, an inhibitor of osteoclastogenesis, on growth of prostate tumor cells in mouse tibiae. Zhang et al. (113) used C4-2 cells that cause a moderate osteoblastic reaction, and, in our study, we used our highly osteoblastic xenograft, LuCaP 23.1. Both studies showed that OPG administration inhibited CaP-induced osteoclastogenesis and tumor growth. OPG may, therefore, be beneficial to patients with advanced CaP bone metastases. Using the intratibial model with C4-2 cells overexpressing OPG, we have also shown that OPG may be at least partially responsible for the osteoblastic character of most CaP bone lesions (115).

Bisphosphonates are potent inhibitors of osteolysis, and a new-generation bisphosphonate, zoledronic acid, has been tested as a potential treatment for osteolytic and osteoblastic CaP tumors (116,117). Zoledronic acid inhibits osteoclastogenesis and, therefore, modulates the bone environment, and it may also have direct antitumor effects. Lee et al. (116) used osteoblastic LAPC-9 and osteolytic PC-3 cells injected into the tibiae to evaluate the effects of zoledronic acid. Zoledronic acid decreased the number of osteoclasts in PC-3 experimental metastases in bone, but it was not effective in halting blastic lesions of LAPC-9. LAPC-9 lesions were formed even when the number of osteoclasts was significantly reduced. In our study, Corey et al. (116), we used osteoblastic LuCaP 23.1 and osteolytic PC-3 cells in the intratibia model. Growth of osteoblastic and osteolytic lesions was significantly inhibited by administration of zoledronic acid. Both of these studies suggest that the anti-osteolytic activity and the antitumor effects of zoledronic acid could benefit CaP patients with bone metastases. Also, Burton et al. (118) used green fluorescent protein imaging to monitor skeletal progression of PC-3 cells injected into the tibia. Subsequently, these authors used this model to evaluate the effects of the bisphosphonate pamidronate on the growth of osteolytic PC-3 bone lesions. As with zoledronic acid, they observed that pamidronate inhibited growth of PC-3 in the bone environment.

Fidler's group has also used the tibial injection model to study the effects of potential new therapeutics on CaP bone metastases (119-122). This group blocked the platelet-derived growth factor receptor (PDGF-R) and the epidermal growth factor receptor (EGF-R) alone or in combination with paclitaxel. PC3-MM2, a metastatic subline of PC-3 cells with higher propensity for bone, was used in these studies. Uehara et al. (119) blocked PDGF signaling by the tyrosine kinase inhibitor, STI571, with or without paclitaxel. They observed reductions in tumor incidence and size in animals treated with STI571, and more pronounced inhibition of tumor growth in animals treated with the combination of STI571 plus paclitaxel. Kim et al. (120) examined the effects of EGF-R signaling blockade by PKI166, an EGF-R tyrosine kinase inhibitor, alone or in combination with paclitaxel. Administration of PKI166 or the combination reduced the incidence and size of bone tumors and destruction of bone. In subsequent studies, Kim et al. (121) used this model to evaluate the combination treatment with CaP bone metastases. Combination therapy using PKI166, STI571, and paclitaxel induced a high level of apoptosis in tumor cells and vascular endothelial cells within the tumor, along with inhibition of tumor growth in bone. In aggregate, these data demonstrate that blockade of EGF-R and PDGF-R

phosphorylation significantly suppresses experimental human CaP bone metastasis, and, when combined with paclitaxel, the therapy is even more effective, indicating significant potential of these treatments against CaP bone metastasis. Finally, because of the heterogeneity of CaP metastasis and the necessity of attacking different pathways to eliminate the advanced disease, Kim et al. (122) set out to determine whether systemic administration of zoledronic acid could prevent bone lysis and inhibit progression of PC3-MM2 cells growth in the bone. Zoledronic acid inhibited bone lysis, but did not inhibit the growth of PC-3MM2 cells. Systemic administration of zoledronic acid together with STI571 and paclitaxel yielded significant inhibition of bone lysis and decreases in tumor incidence and tumor growth. These results indicate the potential benefits of a combination of a bisphosphonate with a protein tyrosine kinase inhibitor and a cell-cycle blocking drug for the treatment of CaP bone metastasis.

Gamradt et al. (123) used the tibial model to determine whether COX-2 plays a role in the bone formation observed in osteoblastic CaP metastases, using cell lines that produce either osteoblastic lesions (LAPC-9) or osteolytic lesions (PC-3, negative control). Administration of SC-58236, a COX-2-specific inhibitor, significantly reduced the size of osteoblastic lesions after LAPC-9 injection. In contrast, large osteolytic lesions were seen in both control and SC-58236-treated animals after PC-3 cell injections. These findings suggest that progression of osteoblastic metastases induced by injection of human CaP cells may be reduced or delayed by COX-2 inhibitors.

Fizazzi et al. (124) directly injected osteoblastic MDA PCa 2b and osteolytic PC-3 CaP cells into the femurs of mice to assess the activity of docetaxel in combination with hormonal therapy on experimental CaP bone metastases. Docetaxel exhibited strong antitumor effects on both osteolytic and osteoblastic lesions. These results provide a strong preclinical rationale for the clinical use of docetaxel in treatment of both locally advanced and disseminated CaP.

Zhang et al. (110) used C4-2B cells injected into mouse tibiae to establish a model for detection of prostate tumor cells in the bone environment. The C4-2B cells had been stably transfected with the RANKL promoter driving the luciferase gene. Animals with established tumors were treated with transforming growth factor-p, and bioluminescence was measured. The measurements demonstrated an increase in intraosseous tumor size over time that correlated with serum PSA levels. These observations provide a novel method to use in exploring the biology of CaP.

5.3.4. Cardiac Injection

We found only one published report of intracardiac injections of CaP cells to generate bone metastases as a model for testing new treatment modalities (125). In this report, Sun et al. injected PC-3 cells labeled with luciferase into the left cardiac ventricle of athymic mice, and evaluated the effects of SDF-1/CXCR4 blockade by neutralizing antibodies. The results of this study showed that SDF-1/ CXCR4 blockade decreased the number of bone lesions, suggesting that this receptor plays a role in "capturing" CaP cells within the bone marrow. Moreover, a parallel study using the intratibial injection model showed that SDF-1/CXCR4 blockade inhibits growth of these cells in the bone environment.

In summary, experimental models of CaP bone metastasis have given rise to a range of important findings. For example, these models have shown that alteration of the bone environment by administration of compounds inhibiting osteolysis can affect tumor growth. Another general finding of considerable use is that because CaP is nearly always heterogeneous, multimodal therapy often exhibits superior antitumor activities These findings can be used directly in designing clinical trials to attack human CaP more effectively.

Was this article helpful?

0 0

Post a comment