The oncofetal protein, 5T4, is a suitable target for antibody-guided anti-cancer chemotherapy with calicheamicin.

The oncofetal protein, 5T4, is a tumor-associated protein displayed on the cell membrane of various carcinomas. This molecule is a promising target for anti-tumor vaccine development and for targeted therapy with staphylococcus exotoxin. The potential use of 5T4 as a target for antibody-guided chemotherapy has not been demonstrated. We report oncolytic efficacy and selectivity in vitro and in vivo with immuno-conjugates of calicheamicin (CM) and the anti-5T4 antibody, H8. CM is a potent cytotoxic drug that causes double strand breaks in DNA. Conjugates of CM and H8 were constructed with acid-labile as well as acid-stabile linkers. In vitro, when applied to monolayers of 5T4(+) cells, CM-conjugates targeting 5T4 were consistently more toxic than either free drug or a non-binding control CM-conjugate. This difference was less pronounced on 5T4-deficient cells. In vivo, four 5T4-positive subcutaneous tumor models were treated with conjugates. Efficacy was demonstrated by reduction of tumor growth relative to controls treated with drug vehicle. To evidence selectivity, the efficacy of the anti-5T4 conjugates was compared to the efficacy of H8, a mixture of H8 and calicheamicin, calicheamicin alone or calicheamicin conjugated to the anti-CD33 antibody, hP67.6. In addition, the efficacy and selectivity of an acid-labile conjugate of H8 was evaluated in an orthotopic model for 5T4(+) lung cancer. Increased survival following treatment was used as a parameter of efficacy. Calicheamicin conjugates of H8 were effective and selective in all the examined tumor models. Differences in efficacy between the acid-labile and acid-stabile conjugates depended on the investigated tumor model.

Expression and function of the leucine zipper protein Par-4 in apoptosis.

The prostate apoptosis response-4 (par-4) gene was identified by differential screening for genes that are upregulated when prostate cancer cells are induced to undergo apoptosis. The par-4 gene is induced by apoptotic signals but not by growth-arresting, necrotic, or growth-stimulatory signals. The deduced amino acid sequence of par-4 predicts a protein with a leucine zipper domain at its carboxy terminus. We have recently shown that the Par-4 protein binds, via its leucine zipper domain, to the zinc finger domain of Wilms' tumor protein WT1 (R. W. Johnstone et al., Mol. Cell. Biol. 16:6945-6956, 1996). In experiments aimed at determining the functional role of par-4 in apoptosis, an antisense par-4 oligomer abrogated par-4 expression and activator-driven apoptosis in rat prostate cancer cell line AT-3, suggesting that par-4 is required for apoptosis in these cells. Consistent with a functional role for par-4 in apoptosis, ectopic overexpression of par-4 in prostate cancer cell line PC-3 and melanoma cell line A375-C6 conferred supersensitivity to apoptotic stimuli. Transfection studies with deletion mutants of Par-4 revealed that full-length Par-4, but not mutants that lacked the leucine zipper domain of Par-4, conferred enhanced sensitivity to apoptotic stimuli. Most importantly, ectopic coexpression of the leucine zipper domain of Par-4 inhibited the ability of Par-4 to enhance apoptosis. Finally, ectopic expression of WT1 attenuated apoptosis, and coexpression of Par-4 but not a leucine zipperless mutant of Par-4 rescued the cells from the antiapoptotic effect of WT1. These findings suggest that the leucine zipper domain is required for the Par-4 protein to function in apoptosis.

Distinct patterns of E-cadherin CpG island methylation in papillary, follicular, Hurthle's cell, and poorly differentiated human thyroid carcinoma.

Expression of the invasion/metastasis suppressor, E-cadherin, is diminished or lost in thyroid carcinomas. Yet, mutational inactivation of E-cadherin is rare. Herein, we show that this loss is associated with hypermethylation of the E-cadherin 5' CpG island in a panel of human thyroid cancer cell lines. This aberrant methylation is evident in 83% of papillary thyroid carcinoma, 11% of follicular thyroid carcinoma, 40% of Hurthle's cell carcinoma, and 21% of poorly differentiated thyroid carcinomas. Contrary to previous reports, the majority of these poorly differentiated thyroid carcinomas express E-cadherin, but often within the cytoplasm rather than at the cell surface. Together, our data indicate that the invasion/metastasis suppressor function of E-cadherin is frequently compromised in human papillary, Hurthle's cell, and poorly differentiated thyroid carcinoma by epigenetic and biochemical events.

The influence of the presence of adenovirus 5 E1a and E1b sequences on the pathology of rat embryonic fibroblasts transfected with activated c-Ha-ras and v-ras.

We compared the pathology of two groups of tumors following implantation of cells enmeshed in alginate beads into the syngeneic rat. The first group of tumors was generated by implanting alginate beads containing cloned embryonic fibroblasts (CREF) that were transfected with activated c-Ha-ras (T24) and v-ras (pH1) (CREF tumors). The second group was created by implantation of CREF cells that were transfected with E1a and E1b of wild type adenovirus type 5 prior to transfection with T24 and pH1 (Wt tumors). Alginate beads were implanted at three different sites in the rat, i.e. subcutaneous in the flank, subcutaneous in the tail and under the renal capsule. Tumorigenicity, invasiveness and metastatic capacity of the transfectant cell lines were determined. The tumor latency period (TLP), the doubling time of the tumors and the metastatic capacity of the cell lines depended on the site of implantation. Invasion was not influenced by site-dependency. Wt tumors were invasive and generally had longer TLP than the CREF tumors. Wt tumors did not metastasize to the lungs as opposed to CREF tumors. We concluded that the genetic background of Wt cells modulated the effect of ras transfection by stretching the TLP and by limiting the metastatic potential to the draining lymph nodes. Malignancy per se was not repressed since no differences in invasive capacity were noticed.

Increasing N-CAM-mediated cell-cell adhesion does not reduce invasion of RSV-transformed WC5 rat cerebellar cells.

The WC5 rat cerebellar cell line, infected with a Rous sarcoma virus (RSV) that is temperature-sensitive for pp60v-src transformation, expresses high levels of the neural cell adhesion molecule, N-CAM, when grown at the non-permissive temperature for pp60v-src activity. At the permissive temperature, N-CAM expression is 4- to 10-fold reduced and the cells aggregate poorly. To evaluate the effects of variations in N-CAM expression, we compared the invasive ability of transformed WC5 cells that express low levels of N-CAM with transformed cells in which N-CAM-mediated adhesion was restored. WC5 cells were transfected with expression vectors containing cDNAs encoding the 120 or 180 kDa forms of chicken N-CAM linked to constitutive promoters. Several permanently transfected lines that expressed chicken N-CAM at the cell surface were isolated. These cell lines showed enhanced aggregation at the permissive temperature relative to untransfected WC5 cells or cells transfected with control constructs. By comparing the ability of control and transfected WC5 cells to invade reconstituted extracellular matrix, we tested the effect of variations in N-CAM-mediated adhesion on invasion. Clones that expressed high levels of N-CAM showed invasion rates that were similar to control cells, indicating that increasing N-CAM-mediated adhesion does not inhibit the invasiveness of RSV-transformed WC5 cells.

Quantitative and qualitative differences in growth, invasion and lung colonization of an anaplastic and a papillary human thyroid cancer cell line in vitro and in vivo.

Invasion and metastasis remain major reasons for failure of anti-cancer therapy. Cell lines derived from human carcinomas are frequently used to investigate the molecular mechanisms that underlie invasion and metastasis. Unfortunately many of these cell lines do not retain the malignant characteristics of their parental tumors. We therefore conducted a series of experiments in vivo and in vitro to identify which aspects of malignancy of a papillary (NPA'87) and an anaplastic (DR090-1) thyroid carcinoma were consistent with the pathology of the parental tumor types. We evaluated tumor growth, invasion and metastasis of DRO90-1 and NPA'87 in vivo following inoculation of the tumor cells under the dermis, under the renal capsule and into the lateral tail vein of nude mice. This evaluation in vivo showed that the anaplastic carcinoma had a faster growth rate compared with the papillary carcinoma. Furthermore, the papillary carcinoma cells could destroy and infiltrate surrounding tissue but were not capable of extravasation and colonization of lung tissue. The anaplastic cells formed lung nodules following injection into the tail vein of nude mice. This lung colonizing capability of DRO90-1 correlated with their capacity to secrete an active 62 kDa gelatinase and to migrate through reconstituted basement membrane in vitro.

Metastasis of C1300 and TBJ murine neuroblastomas correlates with expression of matrix metalloproteinases.

Because metalloproteinases, specifically type IV collagenases, may mediate metastasis, 72- and 92-kDa collagenase activities were evaluated in two murine neuroblastoma cell lines: non-metastatic C1300 and metastatic TBJ. Zymogram analysis demonstrated that 72- and 92-kDa collagenases were associated only with metastatic TBJ. Three human neuroblastoma cell lines were then evaluated by zymogram and Western blot analyses: 72-kDa collagenase was found in metastatic SK-N-SH and IMR-32, but not in SK-N-MC, which may be a peripheral neuroectodermal tumor and not a neuroblastoma. Therefore, 72- and 92-kDa collagenases may be markers of metastasis in neuroblastoma and may aid in differentiation from other small blue cell tumors.

Insulin-like growth factors (IGF) enhance three-dimensional (3D) growth of human glioblastomas.

Human glioblastomas (gliomas) are characterized as rapidly growing brain tumors which are highly invasive but rarely metastatic. Human gliomas synthesize and secrete increased levels of insulin-like growth factors (IGFs) as well as expressing increased numbers of IGF receptors when compared to normal brain tissue. These observations suggest the existence of an IGF-mediated autocrine mechanism for glioma growth regulation. The purpose of this study was to examine the effect of human recombinant IGF (hrIGF) treatment on the in vitro growth of human glioma monolayer and three-dimensional (3D) multicellular spheroid cultures. The data demonstrate that hrIGF-I treatment of glioma cell lines slightly enhanced tumor monolayer proliferation as measured by [(3)H]thymidine incorporation. In contrast, treatment of glioma spheroids with hrIGF-I or hrDes(1-3)IGF-I, the truncated brain form of IGF-I, dramatically enhanced 3D tumor growth with a 1.5-2-fold reduction in spheroid doubling time (FRSDT). In addition, IGF-treated glioma spheroids were more densely packed than spheroids grown in media alone with no observed necrosis. These data suggest that IGFs will dramatically enhance glioma proliferation when 3D cell-cell contact occurs. This observed enhancement suggests that IGFs both synthesized in the brain and systemically support rapid proliferation of gliomas in vivo.

Tumour invasion and metastasis: therapeutic implications?

Invasion and metastasis is the hallmark of tumour malignancy. Some aspects of invasion and metastasis with potential implications for tumour therapy are reviewed: the value of laboratory models; the acquisition of invasiveness and metastatic capability during carcinogenesis; the relationship between growth and invasion; clinical and experimental anti-invasive and antimetastatic agents. The value of joint experimental and clinical research is underscored.

Inhibition of invasion in vitro and stimulation of aggregation by 13-cis retinoic acid correlates with the clustering of N-CAM in c-Ha-ras transformed glial cells.

Invasion distinguishes malignant from benign primary brain tumors. The molecular mechanisms which permit malignant brain tumor cells to escape from the primary tumor mass and by which they can migrate through normal brain tissue are largely unknown. 13-cis retinoic acid (cRA) can induce morphological, biochemical and functional differentiation characteristics in various malignant tumors. Upon treatment of diffusely invasive hamster glial cells (CxT24neo3) with 30 mu M cRA, we found a significant reduction in cell proliferation in monolayer and spheroid cultures. cRA also inhibits invasion of CxT24neo3 through a reconstituted basement membrane (Matrigel(R)) in a dose dependent manner. Homotypic cell-cell adhesion, on the contrary, is stimulated in the absence of extracellular Ca++ by either treatment or pretreatment of CxT24neo3 with cRA. These phenotypic changes correlate with the induction of the clustering of the neural cell adhesion molecule: N-CAM at sites of cell-cell contact. This phenomenon is observed following immunohistochemical staining for N-CAM of CxT24neo3 cells that were treated with cRA in monolayer cultures. The relationship between reduction of proliferation and invasion in vitro and the increased homotypic cell-cell adhesion with clustering of N-CAM implicates N-CAM as a molecular effector molecule for reduction of malignancy by cRA.

Butyrate alters the expression and activity of cell cycle components in anaplastic thyroid carcinoma cells.

Anaplastic thyroid carcinoma (ATC) is the most malignant and aggressive form of thyroid cancer. Most patients die within months of diagnosis, primarily due to the absence of effective chemotherapeutic strategies. Identifying alternative therapies is necessary to increase long-term survival. Butyrate elicits a number of responses from cancer cells both in vitro and in vivo including growth repression, cell cycle arrest, differentiation, and apoptosis. Even though many types of cancer cells have been studied, little is known of the response of ATC cells to this drug. In this study, we report that butyrate induces differential cell cycle arrest (arrest in G1 and G2/M phases) in an ATC cell line that correlates with changes in the expression, phosphorylation, and activity of key components of the cell cycle machinery. Exposure to butyrate increases the expression of the cyclin-dependent kinase inhibitors, p21/Cip1 and p27/Kip1, decreases the expression of cyclin A and cyclin B, inhibits the phosphorylation of the retinoblastoma protein (pRb), and decreases the activity of cdk1 and cdk2-associated kinases. These results suggest that butyrate may be useful in the clinical treatment of ATC.

Navitoclax (ABT-263) and bendamustine ± rituximab induce enhanced killing of non-Hodgkin's lymphoma tumours in vivo.

BACKGROUND AND PURPOSE: Bendamustine with or without rituximab provides an effective and more tolerable alternative to the polytherapy cyclophosphamide-doxorubicin-vincristine-prednisolone (CHOP) in the treatment of haematological tumours and is currently approved for the treatment of many haematological malignancies. Navitoclax (ABT-263) is a potent inhibitor of Bcl-2, Bcl-x(L) and Bcl-w, which has demonstrated efficacy in haematological tumours alone and in combination with other agents. This paper describes the in vivo efficacy of combining either bendamustine or bendamustine plus rituximab (BR) with navitoclax in xenograft models of non-Hodgkin's lymphoma EXPERIMENTAL APPROACH: Activity was tested in xenograft models of diffuse large B-cell lymphoma (DoHH-2, SuDHL-4), mantle cell lymphoma (Granta 519) and Burkitt's lymphoma (RAMOS). Activity was also monitored in a systemic model of Granta 519. KEY RESULTS: Navitoclax potentiated bendamustine activity in all cell lines tested. Bendamustine activated p53 in Granta 519 tumours, concurrent with activation of caspase 3. Navitoclax also improved responses to bendamustine-rituximab (BR) in a subset of tumours. CONCLUSIONS AND IMPLICATIONS: Navitoclax in combination with bendamustine and BR is a viable combination strategy for use in the clinic and demonstrated superior efficacy compared with previously reported data for navitoclax plus CHOP and rituximab-CHOP.

Invasion in vitro of malignant hamster brain tumor cells is influenced by the number of cells and the mode of malignant progression.

We investigated the capacity of two glial tumor cell lines (CxT24neo3 and CxT3Cl5) to invade through reconstituted basement membrane (Matrigel, MG). The purpose of our experiments was to establish whether the number of cells or the mode of malignant progression would quantitatively modify the invasion of a brain tumor cell population. To accomplish this goal, we used a vital-dye method to assess the fraction of cells that invaded through 30 micrograms MG coated on a polycarbonate filter (8 microns pore size). Our experiments demonstrated that the fraction of invasive CxT24neo3 and CxT3Cl5 cells in vitro reproducibly differed as a function of the number of initially seeded cells. This showed that invasion through MG was subject to quantitative changes caused by the number of cells present. Since CxT24neo3 and CxT3Cl5 became malignant by transfection with different oncogenes, the results also indicated that the type of quantitative change was influenced by the mode of malignant progression.

Invasion by WC5 rat cerebellar cells is independent of RSV-induced changes in growth and adhesion.

The WC5 rat cerebellar cell line, which is infected with a Rous sarcoma virus that is temperature-sensitive for pp60src transformation, shows temperature-dependent expression of the neural-cell-adhesion molecule (N-CAM) and glial fibrillary acidic protein (GFAP). We found that WC5 cells maintained at the non-permissive temperature in both monolayer cultures and spheroids are subject to density-dependent inhibition of growth, whereas cells maintained at the permissive temperature continued to grow. The movement of isolated WC5 cells at both temperatures was similar, while the migration of WC5 cells out of 3-dimensional aggregates was faster at the non-permissive temperature. We tested whether the RSV-induced changes affect the invasion of the WC5 cells in 2 in vitro assays: the chorio-allantoic-membrane assay and the chick-heart-fragment assay. In both assays, WC5 cells grown at either temperature were invasive. These results indicate that growth rate is unrelated to invasion and that loss of N-CAM-mediated cell-cell adhesion is not necessary for invasion.

A dominant-negative mutant of the platelet-derived growth factor A-chain increases survival of hamsters implanted intracerebrally with the highly invasive CxT24-neo3 glioblastoma cell.

Evidence is accumulating to suggest a role for PDGF in stimulating malignant growth in astrocytoma, although it has been obtained using model systems (growth in 2-dimensional cell culture, athymic nude mice) that do not assess the complex interactions of these tumors with normal brain tissue. In the current study, the highly invasive hamster glioblastoma cell line CxT24-neo3 was used as a model to study the role of platelet-derived growth factor (PDGF) in mediating malignant growth both in vitro and in vivo when implanted directly into the right lateral ventricle of the brain. Co-expression of PDGF B-chain mRNA and PDGF alpha-receptors was detected in these cells, indicating potential for autocrine activation of their growth. CxT24-neo3 cells transfected with wild-type and receptor binding-deficient forms of the PDGF A- and B-chains displayed alterations in their abilities to grow as three-dimensional spheroids, with overexpression of wild-type B-chain resulting in increased spheroid formation, but a decreased rate of spheroid growth. Influence of these PDGF polypeptides on tumor invasion and survival time in vivo was evaluated following implantation of these spheroids in the brain. While all hamsters implanted with control spheroids died within 21 d (average 17 d), those implanted with cells expressing the receptor binding-deficient A-chain survived for much greater periods of time (average 80 d). Modest increases in survival were also seen in cells stably expressing wild-type A-chain (25 d) and mutant B-chain (26 d) proteins. The present study suggests an important role of PDGF in mediating the malignant growth of the CxT24-neo3 cell line in cerebral cortex, possibly via paracrine interactions with normal cortical cell types (i.e., glia, neurons).

Numerical evaluation of the kidney invasion test.

We have implanted MO4 transformed mouse cells attached to a collagen matrix (artificial tumor) under the renal capsule of syngeneic mice. It was our purpose to evaluate whether or not the kidney invasion test (KIT) could be used for the determination in vivo of the invasive capacity of cultured cell populations. Invasion was expressed in terms of the proportion of the depth of the invasive part of the tumor to the total tumor thickness (invasion rate), both measured macroscopically after hemisection of the kidney. Macroscopic findings were confirmed by histology. For MO4 cells the invasion index changed in function of time after implantation. The relationship 'invasion index versus time after implantation' was demonstrated to be constant in 3 independent groups of mice. Therefore, we propose determination of the invasion rate versus time in the KIT as a method to compare the invasive capacity in vivo of different cell lines.

The effect of dibutyryl camp (dBcAMP) on morphological differentiation, growth and invasion in vitro of a hamster brain-tumor cell line: a comparative study of dBcAMP effects in 2- and 3-dimensional cultures.

The use of agents that stimulate cancer cells to differentiate is proposed as a potential approach to the treatment of malignancy. To evaluate the effects of a differentiation inducer on morphology, growth and invasion in vitro of brain-tumor cells, a diffusely invasive hamster glial cell line (CxT3C15) was treated with ImM dibutyryl cyclic adenosine monophosphate (dBcAMP). The efficacy of dBcAMP was tested in monolayer cultures, 3-dimensional static cultures (i.e., spheroids) and confrontation cultures with an embryonic chick heart. CxT3C15 cells exhibited increased numbers of long cellular processes (morphological differentiation) following treatment of monolayer cultures with ImM dBcAMP. One mM dBcAMP also altered the macroscopic and ultrastructural morphology of CxT3C15 grown as spheroids. These alterations were: (i) a fast transition of rough to smooth morphology macroscopically, and (ii) fading of the cell borders concomitant with the disappearance of cell-membrane excrescences, as seen by scanning electron microscopy. Exponential growth of CxT3C15 in monolayers was not changed following treatment with ImM dBcAMP. Treatment of CxT3C15 spheroids with the same dose of dBcAMP caused a reduction of relative volume increase (30-40%). Invasion of CxT3C15 in an embryonic chick heart in vitro was not altered after addition (prior to or at the time of co-culture) of ImM dBcAMP to the co-cultures. These results indicate that invasion of CxT3C15 is not necessarily linked to morphological differentiation or moderated by reduced proliferation.

Spontaneous acquisition of tumorigenicity and invasiveness by mouse lens explant cells during culture in vitro.

The lens of the eye is one of the rare organs in which tumors do not occur spontaneously. It therefore appeared to us that lens cells would not present the background of spontaneous transformation toward malignancy found with many other cell cultures. We have cultured C3H/HeA mouse lens explant (MLE) cells for 70 wk and analyzed changes in malignancy-related phenotypes in function of the number of passages. In vitro, we studied morphology, colony forming efficiency on tissue culture plastic substrate (CFEtc) and in soft agar, population doubling time, saturation density, and invasiveness into precultured chick heart fragments. In vivo, tumorigenicity, invasion, and metastasis were analyzed after injection of cell suspensions subcutaneously and intraperitoneally, after implantation of cells aggregated to collagen sponges under the renal capsule and after implantation of cell aggregates subcutaneously into the tail and into the pinna. The CFEtc, population doubling time, and saturation density increased as the number of passages of culture in vitro increased, but colony formation in soft agar was never observed. MLE cells till passage 16 were not invasive in vitro, but hereafter consistently were found to be invasive. After about 17 passages, corresponding to 25 wk of culture, MLE cells acquired the capacity to form tumors in syngeneic mice. These tumors were invasive but metastases were not observed. We concluded that MLE cells acquired in an apparently spontaneous way a number of malignancy-related phenotypes, without, however, reaching the stage of metastasis.

Qualitative and quantitative analysis of tumour invasion in vivo and in vitro.

Qualitative and quantitative methods for the analysis of invasion in 'natural' and in experimental tumours in vivo and in vitro are reviewed. In human tumours the functional consequences of invasion were evaluated histologically through staging on the basis of depths of invasion and through the presence of tumour cells inside vessels. Antibodies against components of the basement membrane have facilitated the definition of minimal invasion. With new probes derived from oncogene research the search for molecular differences between invasive and non-invasive parts of the tumour has begun. Since the same methods as those used for analysis of natural tumours also apply to experimental tumours in vivo, the major advantage of the latter is the possibility of manipulation. We have described a new mesenterium assay that may permit the selection of invasive cells from non-invasive ones in transfection experiments. Invasion relative to growth as a function of time was quantified in the kidney invasion test. In three-dimensional confrontations between embryonic chick heart fragments and invasive cells, we have used both a subjective grading and a qualitative computer-assisted image analysis of serial histological sections to score invasion. In two-dimensional confrontations supplementary methods could be applied, since such confrontations permitted direct observations on living cultures. In a variety of natural and experimental tumours, ultrastructural analysis, transmigration in two-compartment chambers, and release of metabolic label have demonstrated the role of motility and of lytic activity in tumour invasion.