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.

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.

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.

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.

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.

Inhibition of collagenolytic activity relates to quantitative reduction of invasion in vitro in a c-Ha-ras transfected glial cell line.

The function of proteases in brain tumor invasion is currently not well established. For tumors of epithelial and fibromatous origin collagenase production can enhance the invasive capacity of cells to penetrate basement membranes. We showed previously that a c-Ha-ras transformed glial cell line (CxT24neo3) invaded hamster brain tissue in vivo. These cells were also capable of invading reconstituted basement membrane and embryonic chick hearts in vitro. Since the histopathology of CxT24neo3 tumors mimics that of glioblastoma multiforme in humans, CxT24neo3 was used as the model in vitro for this type of brain tumor. Presently, we detected by zymogram analysis a gelatinase that was secreted by CxT24neo3 and that had an apparent molecular mass of 62 kD. To verify whether gelatinase affected invasion in vitro of these glial cells we determined the efficacy of a substrate specific collagenase inhibitor on invasion in vitro. GM6001 is a synthetic polypeptide that specifically occupies the substrate binding sites of metalloprotease. Since this drug did not show cytotoxicity, its specificity for metalloprotease is a valuable tool to evaluate the physiological function of these enzymes on invasion. We found that treatment of CxT24neo3 with GM6001 reduced the fraction of invading CxT24neo3 cells through reconstituted basement membrane. These data suggest that metalloproteases can stimulate brain tumor invasion.

Immunohistochemical analysis of the proapoptotic protein Par-4 in normal rat tissues.

Prostate apoptosis response 4 (par-4) is a recently identified gene that encodes a transcription factor, Par-4, with a leucine zipper domain. Par-4 protein is constitutively expressed in various cell lines and is functionally required but not sufficient for apoptosis. Induction of Par-4 in cultured cells is found exclusively during apoptosis, and ectopic overexpression of Par-4 enhances the potency of apoptotic stimuli. Western or Northern blot analysis on mRNA or protein extracts, respectively, from rat organs revealed that the expression of Par-4 was ubiquitous and was not restricted to any specific organ(s). To further identify specific cell types that expressed Par-4, we performed an immunohistochemical analysis of the protein in paraffin-embedded sections of various organs from rats. Our findings indicated that consistent with its proapoptotic role, Par-4 is expressed in apoptotic granulosa cells of atretic ovarian follicles and in terminally differentiated cells, such as the cardiomyocytes, cerebellar Purkinje cells, and pyramidal cells of the hypothalamus. Moreover, testosterone ablation by castration of rats caused an early and transient induction of Par-4 in the ductal cells of the prostate that undergo apoptosis. By contrast, in tissues in which the cells could be visually differentiated from their mature counterparts, Par-4 expression was lowest in the mature cells. This was the case for epithelia of the mammary and the prostate gland in which the basal cells maintained higher protein levels of Par-4 than did the terminally differentiated ductal cells. Similarly, cells of the stratum corneum of the skin and cells on top of the duodenal villi stained less intensely for Par-4 as compared to the stem cells in the stratum basale and at the bottom of the crypts of Lieberkühn, respectively. It is possible that Par-4 has to be down-regulated for successful differentiation in these tissues. Taken together, the widespread expression of Par-4 in various adult cell types underscores the physiological importance of the protein. The observation of constitutive Par-4 expression in the stem cell compartments is inconsistent with the probability of apoptosis per se and can be extended to determine whether Par-4 plays a role in other cellular processes.

Reduction of translation initiation factor 4E decreases the malignancy of ras-transformed cloned rat embryo fibroblasts.

Expression of the T24ras oncogene induces malignancy (tumor growth, invasion and metastasis) in cloned rat embryo fibroblasts (CREF T24). In CREF T24, the rate of phosphorylation of eukaryotic translation initiation factor 4E (eIF-4E) is increased, resulting in increased protein synthesis rates. We have recently shown that reducing the protein levels of eIF-4E in CREF T24 (AS4E line) markedly decreases soft-agar colonization, increases tumor latency periods and increases tumor doubling times without significantly altering monolayer growth. In this study, cells with reduced eIF-4E had delayed and reduced invasiveness and decreased experimental metastasis. Furthermore, reduced eIF-4E levels correlated with decreased expression of the metastasis-associated 92-kDa collagenase type-IV and exon-6 variants of the CD44 adhesion molecule [CD44(6v)]. Reduced eIF-4E levels correlated inversely with increased levels of the putative metastasis-suppressor protein nm23. Cell lines established from AS4E tumors and lung metastases exhibited increased levels of eIF-4E protein and protein synthesis rates compared to the AS4E line. Tumor-derived AS4E had the shortened tumor latency periods of CREF T24 but displayed the slow tumor-growth rates of AS4E. Tumor-derived AS4E exhibited the metastatic capacity of CREF T24 controls. Furthermore, tumor- and lung-nodule-derived AS4E expressed levels of CD44 (6v) and the 92-kDa collagenase type IV comparable to CREF T24 and displayed reduced levels of nm23 relative to AS4E. These results demonstrate that eIF-4E is an important effector molecule involved in oncogenic p21ras-induced malignant transformation.