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Resistance to endocrine therapy is a major complication of luminal breast cancer and studies of the biological features of hormonal resistance are limited by the lack of adequate preclinical models. The aim of this study is to establish and characterize a panel of primary human luminal breast carcinoma xenografts, and to evaluate their response to endocrine therapies. Four hundred and twenty-three tumor fragments obtained directly from patients have been grafted in the interscapular fatpad of Swiss nude mice. After stable engraftment with estradiol supplementation, xenografted tumors have been validated by conventional pathology and immunohistochemistry examination, and additional molecular studies. In vivo tumor growth and response to different endocrine treatments were evaluated. We have engrafted 423 tumors including 314 ER+ tumors, and 8 new luminal breast cancer xenografts have been obtained (2.5%). Tumor take was much lower for luminal tumors than for non-luminal tumors (2.5 vs. 24.7%, P < 0.0001), and was associated with two independent criteria, i.e., ER status (P < 0.0001) and a high grade tumor (P = 0.05). Histological and immunohistochemical analyses performed on patient’s tumors and xenografts showed striking similarities in the tumor morphology as well as in the expression level of ER, PR, and HER2. Response to hormone therapy, evaluated in 6 luminal models, showed different sensitivities, thus exhibiting heterogeneity similar to what is observed in the clinic. We have established a panel of primary human luminal breast cancer xenografts, recapitulating the biological and clinical behaviors of patient tumors, and therefore suitable for further preclinical experiments.

Germline BRCA2 mutations in female carriers confer a cumulative breast cancer risk at age 70 years of 49% (95% CI: 40–57%), an ovarian cancer risk of 18% (95% CI: 13–23%), and a moderate increased risk of pancreatic cancer (Chen and Parmigiani, 2007). Although the available evidence is not sufficient to decisively conclude that the clinical outcome of women with BRCA1/2-associated breast cancer differs significantly from those of women with sporadic tumours, BRCA1-associated breast cancer often manifests adverse outcome features. Establishment of pre-clinical models, which accurately reflect the genetic and phenotypic features of primary tumours, and their response to treatment, is an important step in identifying novel therapeutic targets and testing new treatment modalities. New strategies may take advantage of the specific DNA repair defects inherent in BRCA-deficient cells, such as the defect in homologous recombination. In fact, most of the insights into the functions of the BRCA2 protein have included key insights from studies of mice by the use of gene targeting and from studies of altered mouse embryonic cells (Evers and Jonkers, 2006). BRCA2 has a key role in DNA double-strand break repair and cell-cycle control. BRCA2-related defects are associated with chromosomal abnormalities, a hallmark of the genomic instability that could foster tumourigenesis. Moreover, BRCA2 participates in the regulation of mitosis and cytokinesis that contribute to numerical chromosomal stability. Although conventional, non-conditional, mouse mutants might be used to model familial forms of cancer, they do not mimic sporadic tumourigenesis because the initiating mutation is present in all cells of the body, including those that constitute the tumour microenvironment. Moreover, embryonic lethality and development of non-epithelial tumours are another important limitation of genetically mutated Brca2 mice. Some murine Brca2 mutant mammary tumour models develop mammary tumours with histopathological features that are significantly different to their human counterparts. Although some studies report a strikingly similar histopathology in BRCA1 null breast tumours from mice and humans (Dennis, 1999; Xu et al, 1999), their relevance to the human situation remains to be demonstrated. The most used human BRCA2-mutated model is the CAPAN1 pancreatic cell line that is mainly used to understand drug resistance in BRCA1/2 mutation carrier, as well as in defining functionally important domains within BRCA2 (Edwards et al, 2008). To date, only one BRCA2-mutated breast cancer xenografts (MX1) is available, (Donawho et al, 2007) but its characterisation has not been described in detail. Genetic testing to identify BRCA1/2 mutations is widely available and commonly employed. As a result, increasing numbers of women are aware that they are mutation carriers at the time of their cancer diagnosis. Unfortunately, current knowledge is not sufficient to mandate specific local or systemic treatments that are tailored to BRCA1/2 mutation carriers. In fact, the available studies examining the issue of whether BRCA1/2-associated breast cancer should be treated differently from sporadically occurring, non-familial disease are almost exclusively retrospective and limited by small size and various ascertainment biases. Recently, inhibitors of the DNA repair proteins, poly(ADP-ribose) polymerase 1 and 2 (PARP1/2), have been shown to be selectively cytotoxic to tumour cells with BRCA1 or BRCA2 deficiency. Preclinical data, including that generated with a limited array of tumour cell line xenografts, suggest that PARP inhibitors can act as single agents to selectively kill cancers with BRCA1 or BRCA2 mutations, and phase I clinical trial results confirm that PARP inhibitors have some single-agent activity in cancers with BRCA1/2 mutations (Fong et al, 2009). However, little is known about the long-term effects of these drugs and it seems likely that some tumours may have de novo resistance or acquired resistance (Ashworth, 2008). Thus, definitive answers remain elusive, and preclinical evaluation of new targeted therapy is limited by the lack of suitable preclinical models (Robson, 2007a). Here, we report the establishment and characterisation of a novel xenograft, human breast cancer xenograft (HBCx-17) established from a breast cancer in a woman carrying a BRCA2 germline mutation. We show that this xenograft accurately reflects the genetic and the phenotypic features of the primary tumour, thus providing a new model to test new therapies for BRCA2-mutated patients.

CD44 is a marker of tumour-initiating cells and is upregulated in invasive breast carcinoma; however, its role in the cancer progression is unknown. Here, we show that antibody-mediated CD44-targeting in human breast cancer xenografts (HBCx) significantly reduces tumour growth and that this effect is associated to induction of growth-inhibiting factors. Moreover, treatment with this antibody prevents tumour relapse after chemotherapy-induced remission in a basal-like HBCx.

Resume Le cancer de la prostate est en frequence le deuxieme cancer chez l’homme. Souvent, initialement hormono-dependant, la perte de reponse aux traitements anti-androgeniques survient au cours de la progression tumorale, traduisant l’emergence d’un phenotype hormono-independant. Afin d’etudier les bases morphologiques, genetiques et moleculaires associees a la perte d’hormono-dependance, nous avons etabli un modele de xenogreffe d’adenocarcinome prostatique humain, PAC120, avec ses deux variants : hormono-dependant (HD) et hormono-independant (HID). La croissance de PAC120 HD peut etre inhibee par castration chirurgicale ou par administration d’un antagoniste de la LHRH, le FE200486 (Ferring, San Diego, CA). L’evolution vers l’hormono-independance est associee a une differenciation mucoide ou neuroendocrine, a la survenue d’alterations chromosomiques supplementaires et a des variations d’expression de genes. PAC120 est un nouveau modele de cancer de prostate qui offre l’opportunite d’etudier les mecanismes d’echappement hormonal et d’evaluer l’efficacite de nouveaux agents therapeutiques.

ZD1839 (‘Iressa’) is an orally active, selective epidermal growth factor receptor–tyrosine kinase inhibitor (EGFR–TKI), which blocks signal transduction pathways implicated in proliferation and survival of cancer cells, and other host-dependent processes promoting cancer growth. Permanent downstream activation of the mitogen-activated protein kinase pathway can theoretically bypass the upstream block of epidermal growth factor receptor-dependent mitogen-activated protein kinase activation at the epidermal growth factor receptor level. We investigated the impact of epidermal growth factor receptor content, p53 status and mitogen-activated protein kinase signalling status on ZD1839 sensitivity in a panel of human tumour cell lines: seven head and neck cancer cell lines and two colon cancer cell lines (LoVo, HT29) with derivatives differing only by a specific modification in p53 status (LoVo p53 wt + p53 mut cells, HT29 p53 mut + p53 wt rescued cells). The antiproliferative activity of ZD1839 was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test. ZD1839 concentrations ranged from 0.2–200 μM (48 h exposure). Epidermal growth factor receptor expression, p53 status and p42/p44 (for testing a constitutively active mitogen-activated protein kinase pathway status) were determined by competition analysis (Scatchard plots), denaturing gradient cell electrophoresis and Western blot, respectively. Epidermal growth factor receptor levels ranged from 388 to 33794 fmol mg−1 protein, a range that is similar to that found in head and neck tumours. The IC50 values for cell sensitivity to ZD1839 ranged from 6 to 31 μM and a significant inverse correlation (P=0.022, r=0.82) between IC50 values and epidermal growth factor receptor levels was observed. There was no influence of p53 status on the sensitivity to ZD1839. In two head and neck cancer cell lines with comparably elevated epidermal growth factor receptor expression, a two-fold higher ZD1839 IC50 value was found for the one with a constitutively active mitogen-activated protein kinase. In conclusion, ZD1839 was active against cells with a range of epidermal growth factor receptor levels, although more so in cells with higher epidermal growth factor receptor expression. Activity was unaffected by p53 status, but was reduced in cells strongly dependent on epidermal growth factor receptor signalling in the presence of an intrinsically activated mitogen-activated protein kinase pathway. British Journal of Cancer (2002) 86, 1518–1523. DOI: 10.1038/sj/bjc/6600299 www.bjcancer.com © 2002 Cancer Research UK

Biological parameters influencing the response of human colorectal cancers (CRCs) to CPT-11, a topoisomerase 1 (top1) inhibitor, were investigated using a panel of nine CRCs xenografted into nude mice. CRC xenografts differed in their p53 status (wt or mut) and in their microsatellite instability phenotype (MSI+when altered). Five CRC xenografts were established from clinical samples. All five had a functional p53, two were MSI+and three were MSI–. Tumour-bearing nude mice were treated intraperitonealy (i.p.) with CPT-11. At 10 mg kg–1of CPT-11, four injections at 4-day intervals, four of the five xenografts responded to CPT-11 (growth delay of up to 10 days); the non-responder tumour was MSI−. At 40 mg kg−1of CPT-11, six injections at 4-day intervals, the five CRCs displayed variable but marked responses with complete regressions. In order to assess the role of p53 status in CPT-11 response, four CRC lines were used. HT29 cell line was MSI−/ Ala273-mutp53, its subclone HT29A3 being transfected by wtp53. LoVo cell line was MSI+/ wtp53, its subclone X17LoVo dominantly expressed Ala273-mutp53 after transfection. LoVo tumours (MSI+/ mutp53) were more sensitive than X17LoVo (MSI+/ mutp53. HT 29 tumours (MSI−Imutp53), were refractory to CPT-11 while HT29A3 tumours (MSI−/ wtp53) were sensitive, showing that wtp53 improves the drug-response in these MSI−tumours. Levels of mRNA expression of top1, fasR, TP53 and mdr1 were semi-quantified by reverse transcription polymerase chain reaction. None of these parameters correlated with CPT-11 response. Taken together, these observations indicate that MSI and p53 alterations could be associated with different CPT-11 sensitivities; MSI phenotype moderately influences the CPT-11 sensitivity, MSI+being more sensitive than MSI−CRC freshly obtained from patients, mutp53 status being associated with a poor response to CPT-11. © 2000 Cancer Research Campaign

It is generally accepted that primary tumors become heterogeneous as a consequence of tumor-cell genetic instability. Clonal dominance has been shown to occur in some experimental models allowing a subpopulation of cells to overgrow the primary heterogeneous tumor and to metastasize. Alternatively, interactions among coexisting tumor subpopulations may contribute to the emergence of a malignant invasive primary solid tumor. We asked the question whether emergence of carcinoma cells producing a growth/dissociating factor within a tumor cell population may be a determinant for tumor progression and for clonal dominance. To mimic such a situation, we have investigated the impact of tumor subpopulation heterogeneity in an in vivo model in which mixtures of carcinoma cells that differ in their ability to produce acidic fibroblast growth factor are injected into nude mice. Our data indicate that a growth-factor-producing cell subpopulation can confer increased tumorigenicity to an entire cell population and subsequently elicit a shorter delay for appearance of metastasis. A community effect via cell interactions may account for a heterogeneous tumor cell population rather than clonal dominance during progression of certain tumor types.

High aerobic glycolysis is frequent in cancers. Glucose phosphorylation is under control of hexokinase which is found in the cytosol or bound to mitochondria. Glycolysis parameters (glucose, pyruvate and lactate) and hexokinase were evaluated in extracts of 15 human gliomas and of normal brain tissue. Extracts were run and analysed for glucose lactate and pyruvate content using a centrifugal automatic analyzer, Mitochondria fractions were separated from total extracts and hexokinase enzymatic activities were measured in both, using an original micro-method. Conditions of hexokinase assay were standardized in terms of substrate concentration and linearity. Mean hexokinase activity in gliomas was variable, 3 times lower than in normal tissues and mainly bound to mitochondria, although lactate/pyruvate ratios were found to be 3.5 to 5.4 times higher. Glycolytic profile of tumor tissues can be rapidly assayed and evalated glycolysis in tumors could constitute a basis for therapy using antiglycolytic strategies.

Small-cell lung carcinomas (SCLC) are highly responsive to various chemotherapies. However only a minority of patients benefit from long survival. SCLC patients treated at the Institut Gustave Roussy received a combined chemotherapy (CCAV) including cisplatin, cyclophosphamide (Cpa), Adriamycin (doxorubicin; Adm) and vepeside (VP16). We report here the intrinsic sensitivity of a small-cell lung carcinoma, designated SCLC-6, grafted in nude mice. This xenografted tumour was derived from an untreated patient. The CCAV regimen given to the patient donor of the tumour sample resulted in a complete response followed by recurrence and death, 8 months after the initial cure. The expression of P-glycoprotein encoded by the MDR1 gene was detected with the C219 antibody on the membrane of SCLC-6 tumour cells. When given to SCLC-6-tumour-bearing nude mice, CCAV induced a strong inhibition of tumour growth (84% of growth inhibition, 20 days after start of the treatment), but no cure. Intensification of CCAV doses did not improve the response. The efficacy of individual agents of the CCAV, given at maximal tolerated doses was analysed. Only cisplatin (10 mg/kg) and Cpa (3 x 50 mg/kg) inhibited SCLC-6 growth (79% and 100% inhibition respectively), VP16 (3 x 24 mg/kg) was poorly efficient (42%) and Adm (10 mg/kg) not at all. Two-drug combinations such as cisplatin plus VP16 or cisplatin plus Cpa inhibited tumour growth (81% and 70%, respectively). Curiously, the efficacy of Cpa, given in combination with cisplatin was less than that of Cpa alone. Repeated treatments with CCAV administered to mice at each in vivo passage of the tumour induced a loss of chemosensitivity, which was observed until the ninth passage. An improvement of the therapeutic response was obtained by adding a headline reverser of multi-drug resistance, verapamil (25 mg/kg), to CCAV (81% versus 63% inhibition). MDR1-related resistance appeared to play a role in the failure of SCLC-6 chemotherapy; frequent recurrences after treatment with cisplatin and Cpa, two drugs that are not recognized by the P-glycoprotein, indicated that other modes of resistance were simultaneously active.

Colorectal cancer (CRC) resistance to fluoropyrimidines and other inhibitors of thymidylate synthase (TS) is a serious clinical problem often associated with increased intracellular levels of TS. Since the tumour suppressor gene p53, which is mutated in 50% of CRC, regulates the expression of several genes, it may modulate TS activity, and changes in the status of p53 might be responsible for chemoresistance. Therefore, this study was aimed to investigate TS levels and sensitivity to TS inhibitors in wild-type (wt) and mutant (mt) p53 CRC cells, Lovo and WiDr, respectively, transfected with mt and wt p53. Lovo 175X2 cells (transfected with mt p53) were more resistant to 5-fluorouracil (5-FU; 2-fold), nolatrexed (3-fold), raltitrexed (3-fold) and pemetrexed (10-fold) in comparison with the wt p53 parental cells Lovo 92. Resistance was associated with an increase in TS protein expression and catalytic activity, which might be caused by the loss of the inhibitory effect on the activity of TS promoter or by the lack of TS mRNA degradation, as suggested by the reversal of TS expression to the levels of Lovo 92 cells by adding actinomycin. In contrast, Lovo li cells, characterized by functionally inactive p53, were 3-13-fold more sensitive to nolatrexed, raltitrexed and pemetrexed, and had a lower TS mRNA, protein expression and catalytic activity than Lovo 92. However, MDM-2 expression was significantly higher in Lovo li, while no significant differences were observed in Lovo 175X2 cells with respect to Lovo 92. Finally, mt p53 WiDr transfected with wt p53 were not significantly different from mt p53 WiDr cells with respect to sensitivity to TS inhibitors or TS levels. Altogether, these results indicate that changes in the status of p53, can differently alter sensitivity to TS inhibitors by affecting TS levels, depending on activity or cell line, and might explain the lack of clear correlation between mutations in p53 and clinical outcome after chemotherapy with TS inhibitors.

Androgen deprivation therapy has been used for decades in the treatment of prostate cancer (Huggins, 1967). However, although this treatment is initially very effective in these hormone-dependent cancers, they invariably become hormone-refractory and metastasise, leading to the death of patients. Recently, Feldman and Feldman (2001) reviewed possible mechanisms by which prostate cancer can escape androgen deprivation therapy. While the androgen receptor (AR) is activated by testosterone-induced phosphorylation in hormone-dependent prostate tumours, hormone-independent growth might be due to the activation of AR via a phosphorylation event induced by other growth factor receptors, such as HER1 (EGFR) and HER2 (Her2/neu) (Kwong and Hung, 1998; Yeh et al, 1999). In addition, several recent studies have linked the IGF-1 receptor pathway to the stimulation of the androgen-signalling pathway in hormone-refractory prostate cancer (Culig et al, 1994; Bubendorf et al, 1999). Hormone-independent growth of prostate tumours has also been associated with biological changes such as mucinous and neuroendocrine differentiation. Neuroendocrine differentiation has been observed in prostate cancer and has been correlated with tumoural aggressiveness, short survival and poor response to endocrine therapy (McWilliam et al, 1997), and was considered to be an early marker of progression toward hormone independence (Cohen et al, 1991; Di sant’Agnese and Cockett, 1996; Noordzij et al, 1996). Although true mucinous or colloid prostatic adenocarcinoma, with extensive mucin production, remains a rare entity (Epstein and Lieberman, 1985; Nagakura et al, 1986), production of neutral mucin as assessed by immunohistochemistry is found in up to 55% of prostate carcinomas (Sentinelli, 1993). A total of 19 different mucin genes (MUC1–MUC4, MUC5B, MUC5AC, MUC6–MUC18) have been identified to date and divided into two groups: those coding for membranous mucins such as MUC1, MUC3 and MUC4, and those coding for secreted mucins such as MUC2, MUC5AC, MUC5B and MUC6. Secreted mucins are glycoproteins constituting the major macromolecular component of mucus, while membrane-associated mucins contribute to epithelial cell–cell interactions. Their pattern of expression, especially for the secreted mucins, appears to be relatively tissue-specific. However, the distribution and type of mucin produced by normal prostatic tissue and prostatic carcinomas are not well documented (Daher et al, 1990), though focal mucin production in conventional prostatic adenocarcinomas has been recognised for many years (Pinder and McMahon, 1990). Mucin expression has also been observed in a few cases of prostatic intraepithelial neoplasia (PIN) (Sentinelli, 1993).

In order to develop preclinical models of malignant astrocytomas and oligodendrogliomas, a series of 54 resected gliomas (37 from oligodendroglial lineage and 17 from astrocytic lineage) were xenografted subcutaneously into nude mice. Molecular alterations commonly observed in gliomas subtypes, including LOH 1p and 1q, LOH 19q, LOH 10p and 10q, LOH 9p, TP53 and PTEN mutations, EGFR amplification, CDKN2A homozygous deletion and telomerase reactivation were systematically screened in the original and xenografted tumours. In all, 23 gliomas grew in nude mice. The most anaplastic tumours were selected as shown by pathological and molecular studies of the original tumour as well as shorter survival in patients whose tumours were successfully grafted. Comparison between the two growth profiles showed that 10q LOH and EGFR amplification gave a tumorigenic advantage. With a few exceptions, the genetic pattern was remarkably stable before and after growth in nude mice. These results suggest that subcutaneous xenografts are useful and reproducible models to analyse the molecular profile of malignant astrocytoma and oligodendroglioma. This represents the first step to improve our understanding of the correlations between molecular alterations and response to standard or experimental therapies.

Metastasis is defined by the capacity of tumoral cells to leave the primary tumor, migrate and become implanted in a distant organ, then to proliferate, thus forming new tumoral sites. Metastatic dissemination is related to the local invasiveness of the tumoral cells in tissues. This invasiveness is characterized by the property of these cells to penetrate and migrate into the normal tissues surrounding the seeded site. Metastasis characterizes malignant tumors [28], although certain normal cells exhibit the same capacity to leave their tissue of origin. Such cell migration phenomena are observed, for example, in the embryo during the migration of cells to the thymus and, in adults, during the course of an immunizing reaction with the migration and proliferation of lymphocytes and monocytes in lymph nodes. However, the latter types of physiological or pathophysiological events, which culminate in the proliferation of normal cells in particular sites, obey strict signals to stop proliferation. In most cases, these signals involve the differentiation or the apoptosis of the cells in question. Tumoral cells escape these controls and proliferate in the affected organs, forming often multiple secondary sites of development.

The antitumour activity of vinflunine, 20',20'-dichloro-3',4'-dihydrovinorelbine, a fluorinated Vinca alkaloid obtained by reaction in superacid media, was evaluated in comparison with vinorelbine against a series of subcutaneously-implanted human tumour xenografts. The tumours studied were established from bladder (BXF1299), pancreas (PAXF546), kidney (RXF944LX), colon (DLD-1, HT-29, TC37), central nervous system (SF-295), small cell lung (NCI-H69) and prostate (PC-3). Vinflunine or vinorelbine was administered as four weekly intraperitoneal treatments, within dose ranges of 5-80 or 0.63-10 mg/kg/injection, respectively. The overall antitumour activity of vinflunine was superior to that of vinorelbine. Vinflunine showed high activity against RXF944LX and NCI-H69 xenografts and moderate activity against PAXF546, PC-3 and TC37 tumours, achieving an overall response of 64%. This contrasts with a 27% response with vinorelbine, which proved only moderately active against RXF944LX and TC37 xenografts. These results confirm and extend our previous report of the broad spectrum of in vivo antitumour activity of vinflunine and reinforce its potential as a valuable addition to current chemotherapeutic agents.

Small cell lung carcinomas (SCLC) are characterised by chemosensitivity to diverse antitumoral compounds. However, responses are transitory and relapses are commonly observed. We examined the ability of verapamil, a reverser of P-glycoprotein (Pgp)-related resistance, to improve the efficacy of CyCAV combined chemotherapy (Cy, cyclophosphamide (CPA); C, cisplatin (CDDP); A, doxorubicin (ADM);V, etoposide (VP16)), as currently administered to SCLC patients at Institut Gustave-Roussy, France, and adapted to the treatment of nude mice implanted with these tumours. Although Pgp encoded by the MDR1 (multidrug resistance) gene is not the only mechanism for multidrug resistance (MDR), and not all drugs included in this regimen are recognised by Pgp, we anticipated a therapeutic benefit. Four different SCLC lines, expressing the MDR1 gene and recently grafted into nude mice, were used. SCLC-75, SCLC-6 and SCLC-41 originated from untreated patients, and SCLC-74T was derived from a patient treated with a combination of ADM, CPA and VP16. SCLC-41T and SCLC-6T tumours were used after having undergone, respectively, five and nine cycles of in vivo passage and CyCAV treatment of the tumour-bearing nude mice, to reinforce their chemoresistance. The efficacy of the CyCAV regimen, associated with or without verapamil (given 24 h before CyCAV on days 1–5), was tested on the growth of these SCLC. Verapamil (25 mg/kg) improved the antitumour effect of CyCAV in mice bearing SCLC-6T, SCLC-41T and SCLC-75 tumours, although toxicity was observed. Verapamil modestly delayed the plasma clearance of ADM. Two daily injections of 10 mg/kg of verapamil, administered at a 3 h interval, proved to be effective, whereas the same total dose administered as a bolus was not. These results indicate that the association of some reversers of MDR, including drugs possibly interacting with Pgp, might potentiate SCLC combined chemotherapy.

In vitro attachment assays were carried out to assess adhesion between two basement membrane proteins, type IV collagen and laminin, and rat rhabdomyosarcoma (RMS) cell lines with different metastatic potentials. Whereas cells did not adhere to type IV collagen, adhesion to laminin appeared to be very sensitive as maximal adhesion was achieved in dose-response assays with only nanograms of laminin. Adhesion was mediated by interactions between coated laminin and cell surface components, probably receptors, but not endogenous laminin. Laminin-mediated adhesion of RMS cell lines was compared with that of the MCF-7 (human mammary carcinoma) and the L6 (rat myoblast) cell lines. In dose-response assays, RMS cell lines required 10 times less laminin to reach half-maximal attachment rates than MCF-7 and L6 cell lines. Two laminin fragments, P1 and E8, which are structurally and immunologically distinct as shown by alpha-helix content, SDS-PAGE and monoclonal antibody mapping, supported adhesion by RMS cells and L6 myoblasts, but MCF-7 adhered only to P1. This fragment was 10 times less active than laminin in RMS cell lines. Attachment in dose-response assays and adhesion inhibition studies by antibodies revealed that E8 accounted for the activity of laminin in RMS cell adhesion. Adhesion in the RMS cell lines was dominated by interaction with E8 regardless of metastatic potential.

In vitro cloned lines derived from a primary nickel-induced rat rhabdomyosarcoma exhibited diverse levels of susceptibility to spontaneous NK activity. The presence of NK target structures was revealed by competition assays on all cloned cell lines, and the NK susceptibility of the tumour lines varied according to their osmotic fragility. Tumour cell lines derived from metastatic lung nodules presented similar NK susceptibilities to cells originating from the primary tumour. However, cloned cell lines differed in their capacity to form lung colonies after i.v. injection, and in their potential for invading lungs after s.c. primary tumour development. No correlation was found between lung colonization potential and NK resistance. Studies of the correlation between metastatic potential and NK sensitivity revealed that (1) all the NK resistant tumour cells were highly metastatic; (2) NK susceptible tumour cells could be either highly or weakly metastatic. Therefore, highly metastatic tumour cells could be either resistant or susceptible to NK lysis. We conclude that the property of resistance to NK contributes to a high metastatic potential. However, other properties could counterbalance and finally prevail over NK susceptibility thus enabling NK susceptible cell lines to be also highly metastatic.

Spleen cells of mice receiving an i.p. injection of a sublethal dose of cyclophosphamide (CY) have the ability to inhibit tumor growth in vitro. This effect is dose dependent and is maximal at the peak of the spleen regeneration which follows the phase of atrophy due to CY toxicity.This cytostasis is neither tumor-specific nor strain-restricted and the cells responsible for this inhibition of tumor cell multiplication have the characteristics of macrophages: they lack the Thy 1–2 antigenic marker, appear in CY treated nude mice, stick on plastic vessels, and are retained by adherence columns (nylon wool or Sepha-dex G10); their activity is greatly reduced when a specific macropha-ge toxic reagent such as carrageenan is added to the cultures.The effector cells are similar to those which are able to suppress the response of normal splenocytes to T and B mitogens, and which appear in the same conditions of induction, after CY injection.

Rats bearing a transplanted nickel-induced rhabdomyosarcoma (RMS 9-4/0), treated with chlorozotocin (CZT), an alkylating agent, showed an amplified metastatic invasion of the lung (median of 165 lung tumour nodules, compared to 3 for untreated controls). A higher level of metastatic invasion (200 nodules) was reached spontaneously after the grafting of the S4T line, which was obtained by successive in vivo passages of RMS 9-4/0 cells in CZT treated rats. S4T tumour cells also invaded the liver and a considerable proportion of the lymph nodes. The NT4T line, obtained by successive in vivo passages in untreated rats, showed a lesser degree of enhancement of metastatic capacity (57 nodules). Both derived lines proved to be more aggressive than the parental, proliferated more rapidly, and were resistant to CZT toxicity. Only the non-treated lineage became more resistant to NK lysis. The S4T line lost its myogenic differentiation and was best described as a fibrohistiosarcoma, whereas NT4T did not. Chromosome analysis demonstrated a reduced range of chromosome number per cell in both lines. We conclude that both S4T and NT4T tumours became more metastatic than RMS 9-4/0 as the result of tumour progression through in vivo passages, and that in addition S4T acquired a spontaneously higher metastatic potential, similar to that which occurred in rats grafted with RMS 9-4/0 or NT4T tumours and treated by CZT. This suggests an inheritable mutation in the S4T line. Images Figure 3

Mouse amniotic fluids (MAF) were tested for their inhibitory effect on the transformation of lymphocytes by Concanavalin A (Con A), Phytohemagglutinin (PHA) or Lipopolysaccharide of Escherichia coli (LPS), and on mixed lymphocyte cultures (MLC). Eight out of the thirteen samples tested contained inhibitory activity equal or superior to 50% of the controls. Pure preparations of mouse alpha-fetoprotein (AFP) were obtained from these or other MAF pools by immunoadsorption and/or by affinity chromatography on oestradiol-Sepharose. None of the isolated samples of AFP, whether originating from immunosuppressive MAF pools or from pools lacking activity, exhibited a significant inhibitory effect on the in vitro transformation of lymphocytes. The suppressive properties of the MAF samples and the non suppressive activity of samples of pure AFP were not affected by the addition of oestradiol-17β or after removal of endogenous oestrogens by exhaustive dialysis against 15% dioxan solutions.

The effects of carcinogenic nickel [(Ni) CAS: 7440-02-0] and Ni compounds on the natural killer (NK) cell activity of rat peripheral blood mononuclear cells (PBMCs) were studied. Rhabdomyosarcomas were locally induced by one im injection of Ni or Ni subsulfide [(Ni3S2) CAS: 12035-72-2] dust in the hind leg of WAG rats. A weakly tumorigenic dose of 5 mg Ni3S2 (tumor incidence, 2%) induced a transient decrease of PBMC NK activity against YAC-1 cells in vitro (from the 17th to the 23d wk after Ni3S2 inoculation), which could be restored by in vivo injections of partially purified rat fibroblastic interferon (IFN). Injection of 20 mg Ni (tumor incidence, 47.5%) produced a long-lasting depression of NK cell activity (from the 8th to the 23d wk). In vivo chronic IFN treatment of the Ni-injected rats neither restored NK cell activity nor affected the tumor incidence. However, NK cells of Ni-treated animals responded normally to IFN in vitro. Prospective analysis of individual NK cell responses showed that a persistent depression of basal NK cell activity was restricted to rats that subsequently developed a tumor. In these animals the time between carcinogen treatment and clinical detection of the primary tumor was positively correlated with the mean level of NK cell activity (3-4 determinations/rat). Admixture of manganese to Ni inhibited the development of tumors and also prevented the depression of NK cell activity produced by Ni alone. Noncarcinogenic Ni oxide stimulated NK cell activity. These results point out the possible involvement of NK cells in resistance to Ni-induced carcinogenesis.

Two microanalytical methods, electron probe X ray analysis (E P M A) and ion mass analysis (I A M) were used to study gallium incorporation in normal tissues (kidney, liver, mammary gland, bone marrow, bone tissue) and in experimental tumors. The very high sensitivity of I M A makes possible the detection of very low concentration of gallium (1 ppm) with a spatial resolution of 0.5 micron, on the other hand, E P M A of lower sensitivity (100 ppm) makes possible the relation between the gallium concentration and the ultrastructure of the cell. It was shown that gallium is concentrated in the lysosomes of both types of tissues, where it is precipitated in an insoluble form. In addition, gallium is systematically combined with phosphorus in these precipitates. These observations suggest an active intralysosomal concentrating mechanism related to the presence of local phosphatase activity.

An increasing number of reports highlight the fact that tumour cells are able to give rise in vitro to immunogenic variants, which are defined in vivo as being non tumorigenic, tum-. We have observed the emergence of immunogenic variants, derived from a primary nickel-induced rat rhabdomyosarcoma established in culture (RMS 9-4/0), resistant to treatment with the chloronitrosourea, chlorozotocin (CZT) (R-lines). They were separated from the whole population of cells by a cloning procedure. Furthermore, we demonstrate that the cloning procedure by itself allows the isolation of tum- variant designated as C-lines. In both cases, the tum- phenotype was observed after s.c. injection of cells into syngeneic rats with a broad range of R9 or C8 cells (10(4) to 10(7). This characteristic was inherited in a stable manner. Athymic mice developed tumours of rat rhabdomyosarcoma origin when grafted with 10(5) cells. Immunization of rats with one R variant (R9) tum- protected the rats grafted with the parental RMS 9-4/0 cells against metastatic invasion of the lungs, but not against local tumour growth, and rats grafted with a CZT-resistant tum+ cell variant S4T (in vivo-derived) against its hepatic and pulmonary metastases, while the local tumour progressed as usual. Immunization of rats with one C variant (C8) tum- cells did not protect them against either metastases or local growth of the implanted tumours. Both R and C lines cells became progressively resistant to NK- and macrophage-induced cytotoxicity. Splenic lymphocyte transfer from immune rats into nude mice, i.e., the Winn test, showed a complete degree of protection against C8 or R9 tumour growth. We conclude that two different antigenicities were revealed, one common to R9 and C8 cells in relation with their selection procedure by repeated cloning. Another antigenicity appeared in the R9 line, selected by CZT-resistance. The anti R9 cell immunization against CZT-resistant tum+ S4T could argue in favour of CZT action in the acquisition of R9 cell antigenicity. More likely, an amplification of antigens rather than induction of a new antigen could explain the protection of anti R9 immunized rats against parental tumour metastases.

The peripheral blood lymphocytes from 43 patients with sarcoidosis were cultured in the presence of various stimuli. Uptake of thymidine-3H was employed as a measure of in vitro DNA synthesis. The spontaneous DNA synthesis of lymphocytes from cases of sarcoidosis was slightly increased when compared with that of control cells. On the other hand, the response to phytohemagglutinin from control cells and that from cells of patients with sarcoidosis were not significantly different. Lymphocytes from patients with sarcoidosis exhibiting negative skin tests to tuberculin (PPD) failed to respond to that antigen in vitro whereas those from patients with a positive skin test showed an uptake of thymidine-3H which was similar to that of PPD positive controls. Lymphocytes from patients with sarcoidosis previously vaccinated with polio antigen, were shown to be stimulated in vitro by this specific antigen. A mitogenic factor (MF), prepared by incubating lymphocytes from patients highly sensitive to PPD antigen, was shown to induce DNA synthesis in vitro in lymphocytes from healthy donors, whether or not they were tuberculin sensitive, as well as in lymphocytes from patients with sarcoidosis. On these 3 populations of cells, a non-specific stimulatory effect was also demonstrated in vitro, with the use of pooled sera from patients with active pulmonary tuberculosis. Finally, testing the role of macrophages, it was shown that incubation of control sensitized macrophages with lymphocytes from patients with sarcoidosis, failed to reverse the unresponsiveness of these lymphocytes in the presence of tuberculin.

The effect of chronic uremia on the development of subcutaneously injected malignant tumoral cells was evaluated in 213 male Wistar AG rats made chronically uremic by simultaneous right nephrectomy and partial ligation of the left renal artery. The tumoral cells injected were stemming from a parental rhabdomyosarcoma (9-4/0) induced by intramuscular injection of 20 mg of colloidal nickel suspended in oil to a male Wistar rat. 54 sham-operated rats and 43 nonoperated animals served as control-groups. Renal function and tumoral growth were checked weekly up to the 60th postoperative day, at which time the surviving rats were sacrificed and submitted to autopsy. At day 15 after cell grafting, a tumoral lump could be felt by finger touch in 68% of the uremic rats, but in only 11% of the sham-operated and 14% of the nonoperated controls (p less than 0.0001). Throughout the study, the tumoral lumps which developed in the uremic animals were of significantly larger size than in the nonuremic controls. Pulmonary tumoral metastases were evidenced at autopsy in 95% of the uremic rats, but in only 50% of the sham-operated and in 54% of the nonoperated controls (p less than 0.005). These results indicate an apparently accelerating and amplifying effect of uremia on the development of a malignant tumor in the rat, for which a decrease in cell-mediated immunity associated with the uremic state still remains a questionable hypothesis.