Histologic aspects of concomitant resistance induced by nonimmunogenic murine tumors.

Concomitant resistance to a second tumor implant was induced in both conventional and nude BALB/c mice by two nonimmunogenic syngeneic tumors of spontaneous origin, an epidermoid carcinoma and a lymphoid leukemia. In the secondary tumor, which was significantly inhibited by concomitant resistance, histologic examination revealed the presence of well-preserved tumor cells without any sign of necrosis and without any host cell infiltration, contrasting with classical immunologic rejection. Tumor cell proliferation as evaluated by the number of mitoses per high-power field was significantly inhibited in the secondary tumor as compared with the corresponding controls. No effect of concomitant resistance could be detected on primary tumor growth.

Invasive and metastatic potential of a v-Ha-ras-transformed human bronchial epithelial cell line.

The in vivo growth behavior and invasive potential of normal and "immortalized" human bronchial epithelial cells were studied by xenotransplantation procedures, an in vitro assay of invasiveness, and determinations of type IV collagenase activity and mRNA expression. BEAS-2B cells, immortalized after hybrid virus infection (adenovirus 12-simian virus 40), reconstituted a columnar epithelium when xenotransplanted into de-epithelialized rat tracheas transplanted sc into athymic BALB/c mice. A few adenomatous growths could be seen 16 weeks after transplantation. BZR cells, obtained by transfer of the v-Ha-ras oncogene into BEAS-2B cells, were tumorigenic in this xenotransplantation model. BZR-T33 cells, obtained from a tumor produced after injection of BZR cells, were also tumorigenic; however, they exhibited a shorter latent period. When these same cell lines were injected sc and iv into athymic BALB/c mice, BEAS-2B cells were not tumorigenic, and the BZR-T33 cells were more tumorigenic than the BZR cells. The incidence of spontaneous metastases after sc inoculation was zero for BEAS-2B cells, 33% for BZR cells, and 100% for BZR-T33 cells. Similar increasing values that correlated well with the data on in vivo growth were noted in the in vitro invasion assay, the collagenolytic ability, and the mRNA expression of type IV collagenase. Normal human bronchial epithelial cells showed the lowest values in all the assays. These progressive changes occurring in cells derived from the same parental line indicate that the presence of the v-Ha-ras oncogene in immortalized bronchial cells is associated with a full-fledged malignant phenotype, which is further enhanced by in vivo passaging.

Correlation between seric antitumor activity and concomitant resistance in mice bearing nonimmunogenic tumors.

Serum from mice bearing five weakly immunogenic or nonimmunogenic tumors inducing concomitant resistance exhibited a growth-inhibitory activity on in vitro proliferation of the tumor cells. This activity was proportional to the intensity of concomitant resistance and correlated with the capacity to restrain metastatic development. It was not attributable to cytotoxic antibodies, was relatively nonspecific, and operated through a cytostatic and reversible mechanism. All attempts to transfer antitumor resistance in vivo by serum inoculation have failed, but this could be attained by parabiosis. Physical and chemical serum treatments suggest that heat-, acid-, and alkali-resistant peptide(s) with molecular weights ranging from 1000 to 3000 could account for this inhibitory effect.

Expression of type IV collagenase and procollagen genes and its correlation with the tumorigenic, invasive, and metastatic abilities of oncogene-transformed human bronchial epithelial cells.

In a series of immortalized human bronchial epithelial cell lines continuing various oncogenes, the transcriptional levels of the type IV collagenase and the type IV procollagen genes were compared with the properties of invasiveness in vitro and tumorigenicity and metastatic ability in athymic nude mice. v-Ha-ras greatly enhanced invasion and metastasis, whereas v-Ki-ras, c-myc, and c-raf had lesser effects on these malignant phenotypes. In addition, cell lines derived from tumors obtained by injecting the original immortalized human bronchial epithelial cell lines into nude mice exhibited enhanced invasive and metastatic abilities and increased level of type IV collagenase mRNA when compared with the original immortalized human bronchial epithelial cell lines. Invasiveness and metastatic capacity correlated positively with expression of the type IV collagenase gene and negatively with the expression of the type IV procollagen gene, suggesting that these phenotypes are associated both with decreased production and increased dissolution of extracellular matrix.

Tumor necrosis can facilitate the appearance of metastases.

The non-metastatic murine mammary adenocarcinoma M3 and its metastatic variant MM3 were used to evaluate the role of intratumoral necrosis in cell detachment and metastasis. Accelular extracts from necrotic areas of both tumors increased in vitro cellular detachment from M3 but not from MM3 fragments. Furthermore, the in vivo inoculation of the necrotic extracts within non-metastatic M3 tumors gave rise to pulmonary metastases. Histological studies revealed in M3 a central necrosis limited by an uninterrupted peripheral ring of well preserved cells, while in MM3 necrotic and non-necrotic areas alternated. It is concluded that the distribution of necrosis within the primary tumor by facilitating cell detachment is, at least in part, responsible for the development of metastases.

Expression of gelatinase/type IV collagenase in tumor necrosis correlates with cell detachment and tumor invasion.

We have previously observed that acellular extracts from necrotic areas (NE) of the non-metastatic murine mammary adenocarcinoma M3, enhance in vitro cell detachment and spontaneous lung metastases. In the present study, using different proteinase inhibitors along with NE, only the calcium chelator EDTA could significantly abrogate the enhanced cell detachment from M3 produced by NE. The typical cleavage products of type IV collagenase were detected inside the tumor necrotic area, mainly in association with necrobiotic cells, as evaluated by Western blot analysis and immunohistochemical assays. Zymography revealed the presence of 72- and 92-kDa gelatinase/type IV collagenase in NE. Moreover, NE increased the in vitro invasive ability of cultured M3 cells. The use of specific antibodies against both 72- and 92-kDa type IV collagenases in the invasion assay showed that only the latter was able to revert the enhanced invasiveness to the baseline. It can be concluded that tumor necrosis is an important source of gelatinase/type IV collagenase, mainly in its 92 kDa form, and plays a major role in tumor invasion.

Inhibitory effect of Lovastatin on spontaneous metastases derived from a rat lymphoma.

The HMGCoA reductase inhibitor Lovastatin (LOV) has previously shown to abrogate p21ras farnesylation, which is associated with invasive and metastatic abilities in many tumor models. Considering the scarcity of therapeutic resources against metastasis, our objective was to study LOV as an antimetastatic agent on L-TACB rat lymphoma, which as a syngeneic tumor model resembles more closely the situation in human cancer. We also aimed to analyze the effect of LOV on chemoinvasion, motility, metalloproteases secretion, angiogenic capacity, and adhesion to the reconstituted basement membrane Matrigel. Our results showed that LOV caused no effect on cell motility, metalloprotease secretion and neovascularization. Conversely, LOV produced a significant inhibition of invasiveness, which could be a consequence of an impaired cell adhesion to the basement membrane observed. These effects could explain, at least in part, the inhibitory action of LOV on L-TACB rat lymphoma metastases.

Mechanism of antimetastatic immunopotentiation by low-dose cyclophosphamide.

We have previously reported the antimetastatic effect of a single low-dose of cyclophosphamide (Cy) on L-TACB rat lymphoma. The phenomenon could be adoptively transferred in immunocompetent rats and is abolished in nude mice, facts for which an immunomodulatory explanation was proposed. The aim of this paper was to identify the mechanism(s) by which spleen cells from Cy-treated tumour-bearing rats could exert this antimetastatic activity. Conditioned media obtained by incubation of spleen cells from Cy-treated and non-treated tumour-bearing rats, under specific or non-specific stimulation, were assayed to evaluate their effect on lymphocyte proliferation. The production of transforming growth factor beta (TGF-beta), interleukin-10 (IL-10) and nitric oxide (NO) by conditioned media was also studied. The restoration of spleen lymphoproliferative responses to normal levels when exposed to media conditioned by splenocytes from Cy-treated tumour-bearing rats, together with a decreased production of suppressive cytokines TGF-beta, IL-10 and NO, suggest an enhancement of host antimetastatic immunity triggered by single low-dose Cy treatment.

Differential anthracycline sensitivity in two related human colon carcinoma cell lines expressing similar levels of P-glycoprotein.

Chemosensitivity of the human colon carcinoma HCT-15 cell line to 4'-epidoxorubicin proved to be 100-fold higher than that of its variant HCT-15 EDR. Confocal scanning microscopy showed significant less drug accumulation in HCT-15 EDR. A 2-fold increase in hsp27 expression was found in HCT-15 EDR, with no alteration in hsp70. The expression of the drug exporter Pgp was similar in both cell lines, despite the lower drug accumulation shown by HCT-15 EDR in respect to HCT-15. Other molecules implicated in the acquisition of enhanced chemoresistance or a more active Pgp variant present in HCT-15 EDR, could explain the phenomenon.

CT-2584 (Cell Therapeutics).

CT-2584, an anticancer agent that inhibits phospholipid signaling, is under development by Cell Therapeutics Inc (CTI) as a potential treatment for various types of cancer. Phase II trials are underway for the treatment of prostate cancer and soft-tissue sarcoma [306617], [324290]. According to CIBC World Markets, completion of enrolment for these trials was expected in the fourth quarter of 2000. Furthermore, the initiation of phase II/III trials in combination with taxotere for the treatment of prostate cancer was anticipated in the second half of 2000, as were phase I/II trials in combination with cisplatin for the treatment of other cancers, including lung cancer [396582]. Results of a phase II study in patients with soft-tissue sarcomas evaluating pharmacokinetics, tolerance and therapeutic activity were presented at the 2000 American Society of Clinical Oncology (ASCO) meeting [367283]. Further data are expected to be presented at the ASCO meeting in May 2001 [396582]. Cell Therapeutics is seeking development and commercialization partners for CT-2584 [386398].

Inhibition of endothelial cell proliferation and tumor-induced angiogenesis by pentoxifylline.

PURPOSE: In this study we investigated the effect of pentoxifylline (PTX) on tumor-induced neovascularization as well as on different steps involved in the angiogenic process. METHODS: To assess angiogenesis inhibition. we injected intradermally (i.d.) 10 B16-F10 melanoma cells into C57BL/6J mice which were subsequently intraperitoneally (i.p.) inoculated with PTX or saline. On day 7 the number of blood vessels converging to the remnant of injected material was counted and the volumes of incipient tumors were calculated in each case. In vitro growth inhibition by PTX was evaluated in two different cell lines of endothelial origin and in human umbilical vein endothelial cells. Motility assays, as well as zymographic assays carried out to analyze gelatinolytic metalloproteinases and urokinase-type plasminogen activator, were performed in one of the endothelial cell lines. RESULTS: A significant inhibition of tumor-induced angiogenesis was observed in C57B1/6 mice i.p. inoculated with PTX, that paralleled reduced incipient tumor volumes. The endothelial cells derived from different sources were inhibited in a dose-response manner by PTX in vitro. Non-cytotoxic PTX concentrations assayed in one of the endothelial cell lines did not inhibit its in vitro cell motility nor its gelatinase secretion, but its low molecular weight urokinase-type plasminogen activator expression. CONCLUSIONS: Our findings suggest that the inhibitory effect of PTX on tumor angiogenesis is related to antiproliferative action on endothelial cells, as well as to down regulation of u-PA secreted by them.

Antiangiogenesis and apoptosis as mediators of concomitant tumor resistance induced by Calu-6, a human lung carcinoma cell line, in nude mice.

Concomitant resistance (CR), the phenomenon by which tumor-bearing hosts are able to inhibit secondary implants of the same tumor at distant sites of the body, has been previously observed by us and others in different murine tumor models. Here, we verified the generation of CR in nude mice by tumors induced by SC inoculation of Calu-6, a human lung carcinoma cell line. Histological analysis of secondary tumors subject to CR did not reveal macrophage infiltration nor cytotoxic signs. Although serum from tumor-bearing mice inhibited in vitro [3H]thymidine uptake by Calu-6 cells, no significant differences in [3H]thymidine labeling index of tumors implanted in the right flank of mice with and without a primary tumor in the left flank were detected. In our model, the presence of a primary tumor hindered remote tumor angiogenesis, as well as serum from tumor-bearing mice inhibited in vitro proliferation of an endothelial cell line derived from a murine hemangioendothelioma. Conversely, an enhancement of the apoptotic index was observed in secondary tumor implants carried out in tumor-bearing mice. The results reported herein show that human tumor cells are capable of inducing CR, and that this phenomenon would be a consequence of an impaired neovascularization as well as an increased programmed cell death at sites distant from the primary tumor.

[Antitumor concomitant immunity: a possible metastasis control mechanism]. / "Inmunidad" concomitante antitumoral: un posible mecanismo de control de las metástasis.

Concomitant immunity (CI) is defined as the lack or retardation or proliferation of a secondary tumor implant at a distant site; it has been given an immunological interpretation. Our experiments were designed to investigate CI in association with spontaneous tumors with or without metastases. In BALB/c mice, various syngeneic tumors, of undetectable immunogenicity, induced CI, preventing the development of a secondary implant of the same and occasionally of another tumor, indicating some degree of nonspecificity. This CI could also be observed in young and adult nude mice with high and low NK level, and in those treated with silica, discarding and participation of T lymphocytes, NK cells and macrophages, respectively. The lack of development of the secondary implant was not due to tumor rejection--contrarily to observations in allogeneic systems with immunogenic tumor--but to cytostasis. This "dormant tumor" state is observed together with the absence of host cell infiltration. The creation of a local inflammatory reaction abrogated CI, resulting in rapid tumor growth. On the other hand, an inflammatory reaction created by a foreign body inhibited the development of a tumor implant in the contralateral flank and tumor growth could be inhibited by piroxicam; furthermore, the tumor itself diminished the inflammatory reaction created by a foreign body at a distant site. Considering metastases as natural secondary implants, two mammary adenocarcinomas with a common origin were compared, one with 0 and the other with 100% metastatic incidence. The non-metastatic tumor induced stronger and earlier CI against both tumors and prevented the development of experimental and spontaneous metastases; moreover; its surgical extirpation led to the appearance of lung metastases.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel function for platelet-derived growth factor D: induction of osteoclastic differentiation for intraosseous tumor growth.

Although increasing evidence suggests a critical role for platelet-derived growth factor (PDGF) receptor ß (ß-PDGFR) signaling in prostate cancer (PCa) progression, the precise roles of ß-PDGFR and PDGF isoform-specific cell signaling have not been delineated. Recently, we identified the PDGF-D isoform as a ligand for ß-PDGFR in PCa and showed that PDGF-D is activated by serine protease-mediated proteolytic removal of the CUB domain in a two-step process, yielding first a hemidimer (HD) and then a growth factor domain dimer. Herein, we demonstrate that the expression of PDGF-D in human PCa LNCaP cells leads to enhanced bone tumor growth and bone responses in immunodeficient mice. Histopathological analyses of bone tumors generated by PDGF-D-expressing LNCaP cells (LNCaP-PDGF-D) revealed osteolytic and osteoblastic responses similar to those observed in human PCa bone metastases. Importantly, we discovered a novel function of PDGF-D in the regulation of osteoclast differentiation, independent of the RANKL/RANK signaling axis. Although both PDGF-B and -D were able to activate ß-PDGFR, only PDGF-D was able to induce osteoclastic differentiation in vitro, and upregulate the expression and nuclear translocation of nuclear factor of activated T cells 1, a master transcription factor for osteoclastogenesis. Taken together, these results reveal a new function of PDGF-D as a regulator of osteoclastic differentiation, an activity critical for the establishment of skeletal metastatic deposit in PCa patients.

Secretion of gelatinases by human pancreatic cancer cell lines: lack of correlation with invasive ability.

Twelve immortalized human cell lines derived from primary or metastatic lesions from pancreatic carcinomas were studied with respect to their in vitro invasiveness and motility. Various levels of invasive capacity and chemotactic responses were found. Zymograms of cells conditioned media were carried out to determine the role of metalloproteinases in pancreatic cancer invasion. No correlations were found, however, between invasive capacity of pancreatic carcinoma cell lines and gelatinase secretion. Putative reasons for these findings are discussed.

Exposure to vinorelbine inhibits in vitro proliferation and invasiveness of transitional cell bladder carcinoma.

PURPOSE: To study the effect of vinorelbine (VNR) on in vitro cell proliferation, invasiveness, cell adhesion to substrate, cell motility and metalloproteinase secretion of MB-49, a murine transitional cell carcinoma of the bladder (TCC). MATERIALS AND METHODS: The colorimetric MTS assay, which depends upon viable versus nonviable mitochondria, was used to evaluate the effect of graded concentrations of VNR on in vitro MB-49 cell growth. Chemoinvasion and cell motility were studied in TCC cells exposed for 24 hours to a noncytotoxic dose of VNR, through their ability to migrate across Matrigel-coated or Type IV collagen-coated 8-microns. pore filters. Zymographic studies in gelatin-embedded polyacrylamide gels were done to investigate gelatinolytic activity in conditioned media from treated and untreated MB-49 cells. RESULTS: Vinorelbine inhibited MB-49 cell growth in a dose-dependent manner (IC(50)40 ng./ml.). In vitro cell invasive capacity of MB-49 cells pretreated for 24 hours with VNR at noncytotoxic doses (1 and 10 ng./ml.) was significantly lower than that of untreated cells. The decreased invasion of VNR-treated cells was not accompanied by a diminished adhesion to Matrigel or type IV collagen nor by a significant reduced secretion of gelatinolytic metalloproteinases. Instead, motility of MB-49 cells exposed to noncytotoxic concentrations of VNR was inhibited in a dose-response fashion similar to that of invasion. CONCLUSION: Vinorelbine proved to be an effective drug to inhibit tumor cell growth and invasion in a transitional cell bladder carcinoma model. The results obtained would justify preclinical studies to evaluate the effectiveness of VNR as a potential treatment of TCC.

Multistage chemical carcinogenesis protocols produce spindle cell carcinomas of the mouse skin.

Spindle cell carcinomas were identified using polyacrylamide gel electrophoresis and immunoblotting of proteins extracted from paraffin-embedded tissue sections. Immunohistochemistry using rabbit monospecific antisera against the mouse 55 kd keratin polypeptide also identified these tumors. A group of 53 SENCAR mice initiated with 7,12-dimethylbenz[a]anthracene (DMBA) and promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA) yielded, after one year, four spindle cell carcinomas (0.07/mouse), whereas another group of 31 mice treated with a three-stage carcinogenesis protocol (initiation with DMBA and promotion for 10 weeks with TPA followed by 10 weeks of benzoyl peroxide) gave rise to six spindle cell carcinomas (0.19/mouse). The number of keratin-positive tumor cells and the intensity of the immunostain varied markedly, but all tumors expressed the 55 kd polypeptide. Although other carcinogens, mainly UV radiation, have been able to induce spindle cell tumors, the present data indicate that chemical carcinogenesis protocols are able to induce the formation of this highly malignant variant of skin carcinoma.

Benzoyl peroxide enhances the invasive ability of a mouse epidermal carcinoma cell line.

The effect of the skin tumor promoter benzoyl peroxide on the invasive potential of a murine squamous carcinoma cell line was investigated using an in vitro assay based on the capacity of cells to migrate through a porous filter coated with matrigel. Pre-treatment of the murine squamous carcinoma cell line CH72 with benzoyl peroxide added to the tissue culture medium increased the invasive capacity of these cells 5-8 times. No effects were observed on cells from primary cultures of normal murine epidermis. The simultaneous treatment of CH72 cells with benzoyl peroxide and the free radical scavengers, superoxide dismutase or CuSO4, prevented the increase and the values did not differ significantly from the baseline invasive potential. This suggests that activated oxygen species participate in the benzoyl peroxide-induced enhancement of the invasive capacity of these carcinoma cells.

Intravesical therapy with vinorelbine tartrate: antitumor activity in orthotopic murine cell carcinoma of the bladder.

OBJECTIVES: To ascertain intravesical vinorelbine tartrate (VNR) antitumor activity against MB-49, a murine transitional cell carcinoma of the bladder (TCC), in an in vivo setting. MATERIALS AND METHODS: C57B1/6J female mice were intravesically implanted with 5 x 10(4) MB-49 cells and treated locally with VNR. Tumor incidence and volume analyses, as well as survival studies were carried out. RESULTS: Tumor incidence was significantly lower in VNR-treated mice (48%, n = 23) than in controls (84%, n = 19), as evaluated sixteen days after MB-49 orthotopic inoculation. Intravesical tumor volume was also significantly smaller in treated mice respect to controls (median [range]: 0.5 [0.4 to 61.8] mm.3 versus 47.7 [4.2 to 179.7] mm.3 respectively, p < 0.001 Kruskal-Wallis test). Median survival duration of the animals treated with VNR was 68 [21 to 68] days, and was significantly greater (p = 0.01, Kruskal-Wallis test) than that of untreated controls (18 [16 to 20] days). CONCLUSION: Intravesical VNR treatment demonstrated an evident antitumor effect against the TCC model assayed. The results obtained suggest a potential use of VNR as intravesical treatment for superficial TCC following transurethral bladder tumor resection to prevent recurrence or retard tumor growth.