Role of proteolytic enzymes in human prostate bone metastasis formation: in vivo and in vitro studies

Prostate cancers ability to invade and grow in bone marrow stroma is thought to be due in part to degradative enzymes. The formation of prostate skeletal metastases have been reproduced in vitro by growing co-cultures of prostatic epithelial cells in bone marrow stroma. Expression of urokinase plasminogen activator, matrix metalloproteinase 1 and 7 by prostatic epithelial cells were identified using immunocytochemistry. Also, in vivo tissue sections from human prostatic bone marrow metastases were stained. To establish the role of these enzymes on colony formation, inhibitory antibodies directed against urokinase plasminogen activator, matrix metalloproteinase 1 and matrix metalloproteinase 7 were added into primary prostatic epithelial cells and bone marrow stroma co-cultures. All prostatic epithelial cell cultures stained positively for matrix metalloproteinase 1, matrix metalloproteinase 7 and urokinase plasminogen activator. Generally prostatic epithelial cells derived from malignant tissues showed increased staining in comparison to epithelia derived from non-malignant tissue. In agreement with in vitro co-cultures, the in vivo tissue sections of prostate bone marrow metastases showed positive staining for all three enzymes. Inhibition studies demonstrated that blocking matrix metalloproteinase 1, matrix metalloproteinase 7 and urokinase plasminogen activator function reduced the median epithelial colony area significantly in bone marrow stroma co-cultures in vitro. Using a human ex-vivo model we have shown that matrix metalloproteinase 1, matrix metalloproteinase 7 and urokinase plasminogen activator play an important role in the establishment of prostatic epithelial cells within bone marrow.     

Prostatic carcinoma metastatic to bone: sensitivity and specificity of prostate-specific antigen and prostatic acid phosphatase in decalcified material

Decalcified bone marrow biopsies containing metastatic tumor from 36 patients were stained for prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) using the avidin biotin complex (ABC) immunoperoxidase technique. Of these patients, 22 had known prostate primaries, ten had known nonprostatic, and four female patients had unknown primaries. Prostate-specific antigen was identified in 86% (19/22) of the metastatic prostatic carcinomas. Prostatic acid phosphatase was present in only 36% (8/22). None of the patients with nonprostatic primaries or unknown primaries showed positive staining for either antigen (0/14). This study indicates that immunoperoxidase staining for PSA is very sensitive and specific in the diagnosis of metastatic prostate carcinoma, while PAP was less sensitive using decalcified bone marrow specimens. We believe that immunostaining with PSA should be of great value in diagnosis of prostatic carcinoma metastatic to the bone.     

Rat prostate adenocarcinoma cells disseminate to bone and adhere preferentially to bone marrow-derived endothelial cells.

Approximately 70% of patients with prostatic cancer develop bone metastases. Metastatic prostate adenocarcinomas are associated with high mortality rates and represent a leading cause of cancer-related deaths among males. To study the host-tumor interactions underlying the predilection of prostate cancer cells for skeletal bone, an experimental model was developed using rat Dunning carcinoma Mat-LyLu cells. Inoculations of these cells into the left ventricle of the heart led to the development of spinal metastases in 100% of inoculated animals. A subline of Mat-LyLu (Mat-LyLu-B5) was subsequently selected through the sequential inoculation of bone marrow-derived carcinoma cells into the left ventricle and was found to have an increased metastatic potential compared to the parental line. The possible role of tumor cell adhesion to host cells in the process of bone marrow colonization was then investigated in vitro using the metastatic line and primary cultures of rat bone marrow-derived stromal cells. It was found that the adhesion of the metastatic Mat-LyLu cells to a bone marrow stromal cell culture highly enriched for endothelial cells was significantly higher than the adhesion to other bone-derived cells, including nonendothelial bone marrow stromal cells (3.5x) and osteoblasts (1.7x). It was also significantly higher than the adhesion to rat fibroblasts (7x) and to hepatic endothelial cells (7.5x). The results suggest that the adhesion of prostate carcinoma cells to the bone marrow endothelium may play a role in their metastasis to bone.     

Cytologic detection of neuroblasts in the bone marrow

Cytological detection of bone marrow involvement by neuroblastoma is improved by sampling at ten sites because metastatic cells have a multifocal distribution. Examination of spread films from ten bone marrow aspirates has been compared to density separation of the pool of bone marrow aspirates followed by cytocentrifugation onto glass slides. Of 103 procedures performed, 3 positive results by concentration of the pool were found negative by examination of spread films from each bone marrow aspirate. Cytological examination of the pool of bone marrow aspirates after density separation is a simple, rapid and accurate technique for the detection of small aggregates of metastatic cells in patients with neuroblastoma. Additional immuno-enzymatic techniques may be used for the detection of isolated neuroblastoma cells.     

Immunohistochemical diagnosis of prostatic cancer with metastasis

An immunohistochemical method for detecting prostatic acid phosphatase is described for the diagnosis of metastatic prostatic carcinoma. The specific antiserum against prostatic acid phosphatase was prepared from rabbit by injection of acid phosphatase purified from seminal fluid. This method gives a selective staining of the cytoplasm of the glandular epithelial cells of prostatic tissue specimens on paraffin section. Most of the non-prostatic tissues were negative except for occasional weak staining in granulocytes, islet cells of pancreas, parietal cells of stomach, tubular epithelial cells of kidney, and liver cells. Also examined were 50 consecutive cases of metastatic tumor involving the bone marrow and 5 cases of metastatic prostatic carcinoma involving the lymph node or lung. All 20 cases with prostatic primary lesion showed positive staining. All other cases were negative, except 5 of the 14 cases of metastatic breast carcinoma in women showing weakly positive results. The method is fairly specific for identification of metastatic prostatic carcinoma. Occasional positive staining in breast tumor needs further study to establish whether the staining is due to the same isoenzyme or to certain cross immunoreactivity.     

The natural-killer-cell-associated HNK-1 (Leu-7) antibody reacts with hypertrophic and malignant prostatic epithelium

While using the natural killer (NK) cell-associated HNK-1 antibody in a panel of hematopoietic cell markers, the authors found that metastatic tumor cells in the bone marrow of a patient with disseminated prostatic carcinoma stained strongly with this antibody using an indirect immunofluorescence technique. More than 50% of cells from the patient's prostate also reacted with HNK-1. Subsequent study of frozen sections of prostate tissue from patients with benign prostatic hypertrophy (BPH) showed that HNK-1 reacted with prostatic epithelium and the contents of the glandular lumina. Two other markers associated with NK cells (OKM1 and OKT3) were not detected on nonneoplastic or neoplastic prostatic epithelial cells using a two-color immunofluorescence technique. Recent reports have shown that the HNK-1 antibody also detects an antigen on cells of neuroectodermal origin. The authors have concluded that HNK-1 also reacts with prognostic epithelium in patients with BPH and may be useful as another marker for metastatic carcinoma of the prostate.     

Preferential association of prostate cancer cells expressing prostate specific membrane antigen to bone marrow matrix

Prostate specific membrane antigen (PSMA) is a transmembrane glycoprotein expressed almost exclusively in prostatic epithelial cells. Expression of PSMA is elevated in prostate cancer, with levels closely correlated with disease grade. Although the highest levels of PSMA expression are associated with high-grade, hormone-refractory and metastatic prostate cancer, the significance of elevated PSMA expression in advanced prostate cancer has yet to be fully elucidated. We provide evidence that prostatic carcinoma cells expressing PSMA exhibit reduced motility and increased attachment when grown on a bone marrow matrix substrate. This phenomenon occurs via activation of focal adhesion kinase and provides the first evidence of a link between PSMA expression and prostate cancer metastasis to the bone.     

Bone marrow acid phosphatase by radioimmunoassay.

A double-antibody radioimmunoassay was developed and utilized to measure prostatic acid phosphatase in bone marrow aspirates. One hundred-eighteen patients with carcinoma of the prostate in various clinical stages, and fifty with benign prostatic hyperplasia were studied. In patients with carcinoma, levels of prostatic acid phosphatase in bone marrow aspirates were found to correlate well with increasing clinical stage of the disease. Determination of bone marrow prostatic acid phosphatase by radioimmunoassay may be a valuable adjunct to clinicopathologic staging of prostatic carcinoma.     

Effects of cytostatic drugs and 40.5 degrees C hyperthermia on human bone marrow progenitors (CFU-C) and human clonogenic tumor cells implanted into mice

Human spontaneous tumors after surgical resection were implanted into NMRI nude mice. Clonogenic cells from these tumors (4 malignant melanomas, 2 squamous cell carcinomas of the lung, and 1 small cell carcinoma of the lung) were cultured in a methylcellulose monolayer assay. Dose-response curves with 7 cytostatic drugs (doxorubicin, bleomycin, dactinomycin, cisplatin, vincristine, vinblastine, and melphalan) were assessed at 37 degrees C and after a 2-hour pulse at 40.5 degrees C. In addition, bone marrow cells (CFU-C) were plated in the same assay. Dose-response curves were assessed with the same drugs under the same conditions. Hyperthermic treatment without drugs did not alter the colony formation of tumor and bone marrow cells. Bone marrow samples from different donors showed homogeneous response patterns; the tumor probes revealed sensitivity patterns typical for each tumor. In 13 of 48 tumor-drug combinations a thermal enhancement of the drug effects was observed, whereas there was no significant difference between normothermic and hyperthermic cultures in the bone marrow cultures. A comparison of colony and bone marrow dose-response curves suggested that a hyperthermic enhancement can occur in single cases. However, this phenomenon seems to be due to individual properties of the tumor.     

Protective effects of human interleukin-1 on hematopoietic progenitor cells from L-phenylalanine mustard

The effects of interleukin-1 (IL-1) on protecting both human and murine bone marrow cells were studied using in vitro clonogenic assays, long-term bone marrow cultures and in vivo mouse studies. Incubation with 100 ng/ml human recombinant IL-1 beta for 20 hours prior to a one hour exposure to L-phenylalanine mustard (L-PAM) provided significant protection of bone marrow colony forming cells when compared to bone marrow cells not exposed to IL-1. Complete inhibition of colony formation was observed above 40 mu M L-PAM in the absence of IL-1 preincubation; whereas, colonies were still detectable in cultures which were initiated with IL-1-treated bone marrow cells. Similar results demonstrating greater protection with IL-1 incubation from L-PAM were seen when murine bone marrow cells were assayed for long-term culture-initiating cells. Furthermore, IL-1 protects long-term repopulating hematopoietic stem cells from L-PAM when studied using an in vivo irradiated mouse assay. In contrast, incubation with IL-1 does not protect colony formation by K562, KG-1 or HL-60 leukemic cell lines implying that protection by IL-1 may be selective. Finally, the protection observed by IL-1 preincubation could be abrogated by incubation with 50 mu M L-buthionine sulfoximine (BSO). This result indicates that IL-1 may increase the amount of glutathione in hematopoietic cells and be responsible for the observed protection from L-PAM.     

Disseminated prostatic carcinoma simulating primary lung cancer. Indications for immunodiagnostic studies

Recognition of disseminated adenocarcinomas potentially responsive to current treatment programs is an important objective in the management of cancer patients. Metastatic adenocarcinoma of the prostate gland is a malignant entity which often can be palliated effectively by hormonally based therapeutic strategies. In cases of metastatic prostate cancer presenting with typical clinical features, the correct diagnosis can be readily achieved, but in patients with less obvious presentations the diagnosis of prostatic carcinoma may be overlooked. In the current report, a group of elderly men presenting with a clinical syndrome resembling either metastatic primary adenocarcinoma of the lung or primary adenocarcinoma of the lung coexisting with prostate cancer were found in fact to have metastatic prostatic carcinoma as their sole disease process. In each case, cytologic characterization of clinically involved tissue specimens by the prostate specific antigen and prostatic acid phosphatase immunohistochemical markers enabled clinical investigators to arrive at the correct diagnosis. Other clinical features, such as a positive bone scan and an enlarged prostate gland on physical exam and/or radiographic studies were noted to be present in these patients. All the patients in the current series responded to hormonal treatment regimens once the diagnosis of metastatic prostate cancer had been established. In elderly male patients presenting with what appears to be primary adenocarcinoma of the lung, the diagnosis of metastatic prostate cancer should be considered and when necessary evaluated by the use of appropriate clinical and immunohistochemical studies.     

Role of humoral factors in granulopoiesis: Colony promoting factor (CPF) and its target “pre-CFU-C’ in long-term bone marrow culture

Supernatant of long-term bone marrow cultures contains colony promoting factor (CPF). The factor has no colony stimulating activity itself but augments granulocyte-macrophage colony formation in semi-solid culture of bone marrow cells in the presence of colony stimulating factor (CSF). CPF was specifically absorbed by bone marrow cells but not by thymocytes. Incubation of bone marrow cells with CPF alone resulted in the increase of the number of granulocyte-macrophage progenitor cells (CFUc). CPF-containing supernatant did not stimulate the proliferation of pluripotent stem cells (CFUs) but inhibited CFUs proliferation. These results suggest that CPF is a separate factor from CSF or from CFUs regulators, and that CSF-non-responsive cells (pre-CFUc) in the bone marrow mature into CSF-responsive CFUc by the stimulus of CPF.     

不同骨转移潜能人前列腺癌细胞亚株的筛选

背景与目的:前列腺癌具有很高的骨转移率,是导致患者死亡的主要原因,但是其机制仍不清楚。利用裸鼠肿瘤转移模型筛选人前列腺癌不同骨转移潜能的细胞亚株,通过分析其细胞生物学特性,将为今后探索前列腺癌骨转移机制提供模型。方法:采用人前列腺癌细胞系PC-3左心室注射法制作裸鼠肿瘤转移模型,分别获取骨和淋巴结转移灶内的肿瘤细胞,经体外培养及造模,筛选高、低骨转移潜能的细胞亚株。结果:筛选出具有高、低骨转移潜能的细胞亚株T3B和P24,其裸鼠体内骨转移率分别为8/10(80%)、1/10(10%),与母系PC-3细胞6/15(40%)比较差异有显著性(P<0.05);细胞生物学特性分析显示T3B、P24和PC-3的体外黏附率分别为112%±8%、80.3%±2.7%和77%±4%,侵袭能力分别为53±6个/视野、31±3个/视野和24±5个/视野,T3B的细胞黏附和侵袭能力均显著高于P24和PC-3(P<0.01),三者体外细胞增殖率、软琼脂克隆形成率、裸鼠皮下成瘤率及瘤体大小的差异无显著性(P>0.05)。结论:运用裸鼠肿瘤转移模型反复筛选,获得高(T3B)、低(P24)骨转移潜能的人前列腺癌PC-3细胞系亚株。     

Her-2/neu expression in prostate cancer: a dynamic process?

The clinical effects of targeting Her-2/neu in prostate carcinoma are not known. This study explores the feasibility of molecular profiling to determine the correlation between Her-2/neu expression and hormonal sensitivity. Patients with progressive androgen-independent prostate carcinoma were eligible to participate in the study. Her-2/neu expression was assessed on pretreatment tissue specimens and on bone marrow obtained in progressive androgen-independent disease. Her-2/neu expression was evaluated by immunohistochemistry and by fluorescence in situ hybridization in a consecutive series of 26 progressive androgen-independent prostate cancer patients. Twenty four bone marrow biopsy specimens and 16 prostate biopsies from 26 patients were analyzed. These biopsies were categorized by androgen sensitivity at the time of the biopsy. In total, 90% of specimens from bone marrow were Her-2/neu positive, and 10% of the specimens were Her-2/neu negative. Of the prostate biopsies, all were from patients with androgen-dependent disease. Three of 13 androgen-dependent prostate biopsies (23%) overexpressed Her-2/neu. Of the 10 tumor samples analyzed by fluorescence in situ hybridization, genomic amplification of the Her-2/neu locus was not detected in any of the metastatic prostate tumors. Her-2/neu expression varies with the clinical state of patients with prostate carcinoma: Accurate Her-2/neu profiling requires sampling metastatic tissue in patients with metastatic disease. Her-2/neu sampling from metastatic prostate carcinoma is not feasible until more reliable and practical methods can be developed.     

Inhibition of normal human in vitro colony forming cells by cells from leukaemic patients.

Co-culture in agar of normal bone marrow cells from different individuals gave granulocyte macrophage colony counts that were expected from counts made when the marrows were cultured separately. Co-culture of normal marrow with normal peripheral blood leucocytes (which did not themselves give rise to colonies) caused inhibition of colony growth only when the ratio of peripheral blood to bone marrow cells was of the order of 4 : 1. Peripheral blood or bone marrow cells from 7 of 9 patients with acute myelomonocytic leukaemia, which did not give rise to colonies, caused a marked reduction in the number of colonies obtained from normal marrow cells when cultured with them. This inhibitory effect of leukaemic cells was found when ratios of leukaemic to normal cells were as low as 1 : 4. Additional evidence that the inhibition of normal colony formation was related to the leukaemic process was obtained from follow-up studies on one of the patients whose cells lost the capacity to inhibit normal colony formation during remission and became inhibitory again on relapse.     

The combined effects of Il-3, GM-CSF and G-CSF on the in vitro growth of myelodysplastic myeloid progenitor cells.

The decreased or absent in vitro colony formation in response to single recombinant haematopoietic growth factors has been reported previously. Here we report on the effects of the combination of interleukin 3 (Il-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte-CSF (G-CSF) and the effect of the conditioned medium of the giant tumour cell line (GCT-CM) on the proliferation of myelodysplastic (MDS) marrow myeloid progenitor cells and normal bone marrow (NBM) controls. Colony growth was most effectively sustained by GCT-CM and G-CSF in normal bone marrow (NBM) cultures. GM-CSF and Il-3 were less effective in inducing myeloid granulocytic colony growth, whereas the effects of Il-3 and GM-CSF were found to be approximately additive. The number of NBM granulocytic colonies induced by G-CSF and GCT-CM stimulation were comparable, whereas this granulocyte colony stimulating activity could be neutralized by anti-G-CSF antibodies. In addition GCT-CM was found to contain burst promoting activity, which could be neutralized by anti-Il-3 antibodies. Il-3 did not enhance the G-CSF activity in NBM cultures. No additive effect of stimulation with the combination of Il-3 and GM-CSF was observed in MDS marrow cultures, suggesting that these growth factors act on an identical progenitor cell population in MDS. G-CSF stimulated the growth of significantly lower colony numbers than GCT-CM, in contrast to NBM cultures. The decreased granulocytic colony formation of MDS marrow cells could clearly be enhanced by co-stimulation with Il-3. These results suggest that MDS myeloid progenitor cells require the exposure to both a pluripotent colony stimulating factor, like Il-3, and a lineage specific factor, like G-CSF, for optimal proliferation.     

Responsiveness of human prostate carcinoma bone tumors to interleukin-2 therapy in a mouse xenograft tumor model.

We have tested an immunotherapy approach for the treatment of metastatic prostate carcinoma using a bone tumor model. Human PC-3 prostate carcinoma tumor cells were heterotransplanted into the femur cavity of athymic Balb/c nude mice. Tumor cells replaced marrow cells in the bone cavity, invaded adjacent bone and muscle tissues, and formed a palpable tumor at the hip joint. PC-3/IF cell lines, generated from bone tumors by serial in vivo passages, grew with faster kinetics in the femur and metastasized to inguinal lymph nodes. Established tumors were treated with systemic interleukin-2 (IL-2) injections. IL-2 significantly inhibited the formation of palpable tumors and prolonged mouse survival at nontoxic low doses. Histologically IL-2 caused vascular damage and infiltration of polymorphonuclear cells and lymphocytes in the tumor as well as necrotic areas with apoptotic cells. These findings suggest destruction of tumor cells by systemic IL-2 therapy and IL-2 responsiveness of prostate carcinoma bone tumors.     

Possible mechanisms of action of lithium on augmentation of in vitro spontaneous myeloid colony formation.

To understand the possible mechanisms of lithium carbonate-induced neutrophilia, the in vitro effect on human myeloid progenitor cells was examined. A significant increase in spontaneous colony formation (15 of 24 experiments) was observed with the addition of lithium. Increased colony formation seldom occurred when human placental conditioned media as a source of colony-stimulating activity (CSA) was simultaneously added to the cultures. Further data suggest that lithium requires an adherent marrow cell population for this action and that increases in CSA-containing cultures may be due to suboptimal CSA concentrations. Lithium was shown to release CSA from marrow cells and adherent cell population prepared from human bone marrow. Lithium possibly increases spontaneous human myeloid colony development indirectly through CSA release by adherent cells.     

Rat Prostate Tumor Cells Progress in the Bone Microenvironment to a Highly Aggressive Phenotype

Prostate cancer generally metastasizes to bone, and most patients have tumor cells in their bone marrow already at diagnosis. Tumor cells at the metastatic site may therefore progress in parallel with those in the primary tumor. Androgen deprivation therapy is often the first-line treatment for clinically detectable prostate cancer bone metastases. Although the treatment is effective, most metastases progress to a castration-resistant and lethal state. To examine metastatic progression in the bone microenvironment, we implanted androgen-sensitive, androgen receptor–positive, and relatively slow-growing Dunning G (G) rat prostate tumor cells into the tibial bone marrow of fully immune-competent Copenhagen rats. We show that tumor establishment in the bone marrow was reduced compared with the prostate, and whereas androgen deprivation did not affect tumor establishment or growth in the bone, this was markedly reduced in the prostate. Moreover, we found that, with time, G tumor cells in the bone microenvironment progress to a more aggressive phenotype with increased growth rate, reduced androgen sensitivity, and increased metastatic capacity. Tumor cells in the bone marrow encounter lower androgen levels and a higher degree of hypoxia than at the primary site, which may cause high selective pressures and eventually contribute to the development of a new and highly aggressive tumor cell phenotype. It is therefore important to specifically study progression in bone metastases. This tumor model could be used to increase our understanding of how tumor cells adapt in the bone microenvironment and may subsequently improve therapy strategies for prostate metastases in bone.Abbreviations: PC, prostate cancer; DTCs, disseminated tumor cells; BrdU, bromodeoxyuridine; DHT, dihydrotestosterone; T, testosterone; FBS, fetal bovine serum; AR, androgen receptor; CSC, cancer stem cells     

Granulocytopoietic precursor cells and regulatory factors in irradiated human bone marrow

The effects of irradiation on committed granulopoietic progenitor cells (CFU-C) and granulopoietic humoral regulators were studied in 17 X-ray treated patients with carcinoma of the breast or of the prostate. After 45 or 70 Gy, the CFU-C decreased to 14 +/- 10% in irradiated areas. This remaining CFU-C population probably reflects a combination of radioresistance and survival in situ, and migration of CFU-C from protected marrow areas. The frequency of peripheral blood CFU-C did not change after irradiation. Endogenous colony stimulating activity (CSA), i.e., release of CSA within the marrow cell population, in post-irradiation bone marrow persisted on roughly the same level, indicating a fairly low radiosensitivity of the CSA-producing cells in the bone marrow. Colony stimulating activity in peripheral blood cells and serum remained unchanged, but there were great interindividual variations, and some of the patients with hypercellularity and CFU-C increase in nonirradiated marrow areas also had increased CSA. Serum lipoprotein inhibitors were higher in the post-treatment patients than in healthy control patients.