Modeling of response to endocrine therapy in a panel of human luminal breast cancer xenografts.

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.

Establishment and characterisation of a new breast cancer xenograft obtained from a woman carrying a germline BRCA2 mutation.

BACKGROUND: The BRCA2 gene is responsible for a high number of hereditary breast and ovarian cancers, and studies of the BRCA2 biological functions are limited by the lack of models that resemble the patient's tumour features. The aim of this study was to establish and characterise a new human breast carcinoma xenograft obtained from a woman carrying a germline BRCA2 mutation. METHODS: A transplantable xenograft was obtained by grafting a breast cancer sample into nude mice. The biological and genetic profiles of the xenograft were compared with that of the patient's tumour using histology, immunohistochemistry (IHC), BRCA2 sequencing, comparative genomic hybridisation (CGH), and qRT-PCR. Tumour response to standard chemotherapies was evaluated. RESULTS: Histological profile identified the tumour as a basal-like triple-negative breast cancer. Targeted BRCA2 DNA sequencing of the xenograft showed the presence of the mutation previously identified in the carrier. Comparative genomic hybridisation array profiles of the primary tumour and the xenograft revealed a high number of similar genetic alterations. The therapeutic assessment of the xenograft showed sensitivity to anthracyclin-based chemotherapy and resistance to docetaxel. The xenograft was also highly sensitive to radiotherapy and cisplatin-based treatments. CONCLUSIONS: This study describes a new human breast cancer xenograft obtained from a BRCA2-mutated patient. This xenograft provides a new model for the pre-clinical drug development and for the exploration of the drug response biological basis.

Newly characterised ex vivo colospheres as a three-dimensional colon cancer cell model of tumour aggressiveness.

BACKGROUND: New models continue to be required to improve our understanding of colorectal cancer progression. To this aim, we characterised in this study a three-dimensional multicellular tumour model that we named colospheres, directly obtained from mechanically dissociated colonic primary tumours and correlated with metastatic potential. METHODS: Colorectal primary tumours (n=203) and 120 paired non-tumoral colon mucosa were mechanically disaggregated into small fragments for short-term cultures. Features of tumours producing colospheres were analysed. Further characterisation was performed using colospheres, generated from a human colon cancer xenograft, and spheroids, formed on agarose by the paired cancer cell lines. RESULTS: Colospheres, exclusively formed by viable cancer cells, were obtained in only 1 day from 98 tumours (47%). Inversely, non-tumoral colonic mucosa never generated colospheres. Colosphere-forming capacity was statistically significantly associated with tumour aggressiveness, according to AJCC stage analysis. Despite a close morphology, colospheres displayed higher invasivity than did spheroids. Spheroids and colospheres migrated into Matrigel but matrix metalloproteinase (MMP)-2 and MMP-9 activity was detected only in colospheres. Mouse subrenal capsule assay revealed the unique tumorigenic and metastatic phenotype of colospheres. Moreover, colospheres and parental xenograft reproduced similar CD44 and CD133 expressions in which CD44+ cells represented a minority subset of the CD133+ population. CONCLUSION: The present colospheres provide an ex vivo three-dimensional model, potentially useful for studying metastatic process.

CD44 targeting reduces tumour growth and prevents post-chemotherapy relapse of human breast cancers xenografts.

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.

Changes in the status of p53 affect drug sensitivity to thymidylate synthase (TS) inhibitors by altering TS levels.

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.

Potentiation of antitumour activity of docetaxel by combination with trastuzumab in a human prostate cancer xenograft model and underlying mechanisms.

Antitumour activity of docetaxel (Taxotere) in hormone-dependent (HD) and hormone-independent (HID) prostate cancer PAC120 xenograft model was previously reported, and its level was associated with HER2 protein expression. In the present study, we evaluate the antitumour effects of docetaxel combined with trastuzumab (Herceptin), an anti-HER2 antibody. Although trastuzumab alone had no effect on tumour growth, it potentiated the antitumour activity of docetaxel in HD tumours and more strongly in HID variants. Using the HID28 variant, we show that docetaxel treatment of tumour-bearing mice induces an increased HER2 mRNA expression of the tyrosine kinase receptor of 25-fold 24 h after docetaxel treatment, while HER2 protein and p-AKT decreased. This was followed by an increase of HER2 protein 3 days (two-fold) after docetaxel treatment and by a strong HER2 release in the serum of treated mice; expression of phospho-ERK, p27, BCL2 and HSP70 concomitantly increased. Similar molecular alterations were induced by docetaxel plus trastuzumab combination, except for that there was a transient and complete disappearance of AR and HSP90 proteins 24 h after treatment. We show that in addition to its known effects on tubulin and mitotic spindles, docetaxel induces complex signalisation pathway mechanisms in surviving cells, including HER2, which can be pharmacologically targeted. This study suggests that the docetaxel/trastuzumab combination may prove an effective therapeutic approach for HER2-expressing hormone-refractory prostate cancer.

Mucinous differentiation features associated with hormonal escape in a human prostate cancer xenograft.

Many theories mention hypersensitive, promiscuous, outlaw or bypass signalling pathways to explain the acquisition of hormone independence in prostate cancer. Hormonal escape of prostate tumours is marked by many biological changes, including mucinous and neuroendocrine differentiation. Since expression of several mucins has been linked to carcinoma tumour progression, we have characterised the expression of mucins at both RNA and protein levels in an in vivo model of prostate cancer in hormonal escape. Using PAC120, a xenograft of a human hormone-dependent prostate tumour, and its hormone-independent variants, we analysed the expression of mucins (MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC6) by immunohistochemistry or reverse transcriptase (RT)-PCR. While the parental PAC120 tumour was a compact poorly-differentiated tumour of Gleason score 9 (5+4), hormone-independent variants displayed mucinous, neuroendocrine-like or mixed histological changes; these changes were stable through serial transplantations or after testosterone supply. MUC1 mRNA was expressed in both PAC120 and the hormone-independent variants, although at variable levels. All tumours displayed a high and constant expression of MUC2 and no expression of MUC4 mRNA. While MUC1 was expressed in all xenografts whatever their hormone dependence status, MUC2, MUC5B and MUC6 were preferentially expressed in hormone-independent variants. The loss of hormone dependence in this prostate cancer xenograft model is therefore marked by irreversible histological alterations, mucinous or neuro-endocrine, associated with an expression of secretory MUC2, MUC5B and MUC6, independent of the histological differentiation subtype. These data point to mucinous differentiation as an important step in the acquisition of hormone independence in this cancer, and suggest that secretory mucins might participate in an unknown pathway of hormonal escape in prostate cancer.

Correlation between genetic alterations and growth of human malignant glioma xenografted in nude mice.

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.

[A new model of human prostate cancer, the PAC120 xenograft]. / Un nouveau modèle de cancer humain de prostate, PAC120.

Prostate cancer is the second cause of cancer death in men. Often, initialy hormono-independent, escape from anti-androgen therapy is a key event of tumoral progression showing an hormone-independent phenotype. To study morphological, genetic and molecular bases associated with the hormono-dependence escape, a new model of human adenocarcinoma prostate xenograft, PAC120, was established with its hormono-dependent and independent variants. Its growth was strongly inhibited by surgical castration or by administration of the new gonadotrophin-releasing hormone antagonist, FE 200486 (Ferring, San Diego, CA). Evolution to hormono-independence was frequently associated with a mucoid differentiation or a neuroendocrine-like pattern, with the apparition of new chromosomic alterations and variations of human gene expressions. PAC120 xenograft is a new model of hormone-dependent prostate cancer, opening the opportunity to study the hormone dependence escape mechanism and to evaluate the efficacity of new therapeutics.

Influence of epidermal growth factor receptor (EGFR), p53 and intrinsic MAP kinase pathway status of tumour cells on the antiproliferative effect of ZD1839 ("Iressa").

of 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 microM (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 IC(50) values for cell sensitivity to ZD1839 ranged from 6 to 31 microM and a significant inverse correlation (P=0.022, r=0.82) between IC(50) 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 IC(50) 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.

Bone sialoprotein, bone morphogenetic protein 6 and thymidine phosphorylase expression in localized human prostatic adenocarcinoma as predictors of clinical outcome: a clinicopathological and immunohistochemical study of 43 cases.

PURPOSE: Skeletal metastases are the hallmark of advanced prostate cancer and recurrence after local surgery is common. Currently to our knowledge no biological markers predict the risk of disease progression in individuals with localized prostate cancer. In a search for predictive markers we evaluated the expression of bone sialoprotein and bone morphogenetic protein 6, 2 bone related proteins, and the angiogenic factor thymidine phosphorylase. MATERIALS AND METHODS: The study population included 43 men who presented with localized prostate cancer treated with radical prostatectomy. Bone sialoprotein, bone morphogenetic protein 6 and thymidine phosphorylase expression was assessed by immunohistochemical testing. Results were analyzed in relation to pathological disease stage, Gleason score and clinical outcome. Clinical followup was 4.3 to 11.4 years after surgery (median 7.9). RESULTS: Disease did not progress in 17 of the 43 cases, while recurrence and/or metastasis developed in the other 26 at a median of 6.5 and 6.9 years, respectively. Bone sialoprotein and bone morphogenetic protein 6 expression detected in 28 (65%) and 29 (67%) of the 43 samples, respectively, was significantly associated (p = 0.0001). Thymidine phosphorylase detected in 26 samples (60%) was not related to bone sialoprotein and/or bone morphogenetic protein 6 positivity. Bone sialoprotein and/or bone morphogenetic protein 6 expression correlated with bone metastasis, while thymidine phosphorylase expression was related to local recurrence (p = 0.002 and/or 0.007, and 0.00007, respectively). On multivariate analysis only the correlation of thymidine phosphorylase expression with recurrence remained statistically significant (p = 0.002). Co-expression of the 3 markers was observed in the samples of 10 of the 11 patients (90%) with bone metastases and only in 5 of the 17 (29%) who were disease-free. CONCLUSIONS: This study indicates that the expression of bone sialoprotein, bone morphogenetic protein 6 and thymidine phosphorylase determined at a clinically early stage of disease by a simple immunohistochemical technique would enable subgroups of patients to be identified that are at different risks of bone metastasis or recurrence. Detection of such markers would provide additional prognostic information that would be useful for patients with intermediate or low Gleason score or stage disease. These patients would benefit from a more adapted clinical follow-up.

Single alteration of p53 or E-cadherin genes can alter the surgical resection benefit in an experimental model of colon cancer.

PURPOSE: p53 and E-cadherin mutations are associated with a high risk of metastatic potential and local recurrence after colorectal surgery. LoVo, a human colon cancer cell line expressing a wild-type p53 and a normal E-cadherin, was studied. Clone LoVo-XC17 was obtained from LoVo cells transfected with a vector bearing a p53 273his mutation. Clone LoVo-92R4 was obtained from LoVo by culture cells with an E-cadherin down-regulation. LoVo, LoVo-XC17, and LoVo-92R4 were studied for in vivo behavior in a surgical intracolonic graft model. METHODS: Ten nude mice were used per cell line. A colonic tumor was obtained by tumor implantation into the cecal wall. The cecal tumor was resected at Day 15; at this time the volumes of the different tumors were similar. RESULTS: Surgical resection of the LoVo tumor led to 100 percent disease-free animals at one month. Surgical resection of mice grafted with the LoVo-XC17 line did not cure any mice (0/10; P = 0.001). Mice had local recurrences (10/10), mesenteric lymph node metastases (9/10), liver metastases (2/10), and peritoneal carcinomatosis (8/10). Surgical resection of LoVo-92R4 tumors led to cures in 30 percent (3/10), whereas 70 percent had isolated mesenteric lymph node metastases (7/10; P = 0.003). CONCLUSION: In this model surgical tumor resection was consistently effective for colonic tumors with functional p53 and E-cadherin, it was consistently ineffective with tumors displaying a mutated p53, and it was partially effective with E-cadherin-deficient tumors. This study shows that the alteration of a single gene can be associated with a profound alteration of surgical resection benefit.

Distinct experimental efficacy of anti-Fas/APO-1/CD95 receptor antibody in human tumors.

Ligation of the Fas receptor (FasR) is a key step in apoptosis induction. Using a series of human tumor cells (SNB19, SNB79, 143N2, and SHEP), we observed a distinct efficacy of human anti-FasR antibody with an apparent correlation with Fas cell surface antigen expression. In contrast, all cells studied expressed detectable FasR mRNA transcripts. For all anti-FasR antibody-sensitive tumor cells, we showed a similar efficacy of Mab according to dose fractionation and injection site. We showed that, when injected into nude mice bearing human osteosarcoma 143N2, neuroblastoma SHEP, prostatic cancer PAC120, and the two glioblastomas SNB19 and SNB79, anti-FasR Mab induces significant inhibition of the growth rate of 143N2, SHEP, and PAC120 tumors, but has no efficacy on SNB19 and SNB79 tumors, with a relationship between in vitro and in vivo sensitivity to anti-FasR antibody. Altogether, these results suggest the antitumor potential of anti-FasR antibody in human neoplasms.

Clinical and experimental progression of a new model of human prostate cancer and therapeutic approach.

We report the clinical evolution of a prostate cancer, metastasizing to lungs and bones, recurring locally, and escaping from anti-androgen therapy. Key event of biological progression of the patient's tumor was the coincidence of allelic imbalance accumulation and of bone metastases occurrence. The recurrent tumor was established as the transplantable xenograft PAC120 in nude mice, where it grew locally. PAC120 displayed the same immunophenotype of the original tumor (positive for keratin, vimentin, prostatic acid phosphatase, and Leu-7) and expressed human HOXB9, HOXA4, HER-2/neu, and prostate-specific antigen genes, as detected by reverse transcriptase-polymerase chain reaction. It formed lung micrometastases detected by mRNA expression of human genes. Cytogenetic analysis demonstrated numerous alterations reflecting the tumor evolution. PAC120 was still hormone-dependent; its growth was strongly inhibited by the new gonadotropin-releasing hormone antagonist FE 200486 but weakly by gonadotropin-releasing hormone superagonist D-Trp(6)-luteinizing-hormone releasing hormone (decapeptyl). Tumor growth inhibition induced by anti-hormone therapy was linked to the hormone deprivation degree, more important and more stable with FE 200486 than with D-Trp(6)-luteinizing-hormone releasing hormone. Surgical castration of mice led to tumor regressions but did not prevent late recurrences. Transition to hormone-independent tumors was frequently associated with a mucoid differentiation or with a neuroendocrine-like pattern. Independent variations of mRNA expression of HER-2/neu and prostate-specific antigen were observed in hormone-independent tumors whereas HOXB9 gene expression was constant. In conclusion, PAC120 xenograft, a new model of hormone-dependent prostate cancer retained the progression potential of the original tumor, opening the opportunity to study the hormone dependence escape mechanism.

Topoisomerase I-DNA covalent complexes in human colorectal cancer xenografts with different p53 and microsatellite instability status: relation with their sensitivity to CTP-11.

The topoisomerase I poison CPT-11 has proved efficient for the treatment of untreated metastatic colorectal cancers (CRC) and those refractory to fluoropyrimidines. However, the interpatient variability is important. A previous in vitro study suggested that measurements of the level of topoisomerase I-DNA intermediates trapped by camptothecin may be useful to estimate the chemosensitivity of colon carcinoma cell lines. To verify this hypothesis, we developed an immuno-assay to detect covalent topoisomerase I-DNA complexes in a series of human colorectal cancers xenografted in nude mice. Six human CRCs were selected for their distinctive p53 and microsatellite instability (MSI) status. Tumour lysates, prepared from mice untreated or treated with CPT-11, were fractionated onto CsCl gradients to separate free and DNA-bound topoisomerase I by centrifugation. Interestingly, significant levels of DNA-topoisomerase I complexes were detected in the tumours most responsive to the treatment with CPT-11, irrespective of their MSI and p53 phenotypes. Our in vivo study fully agrees with the predictions from the in vitro data indicating that evaluation of topoisomerase I-DNA complexes would be useful to predict the response of CRC to a treatment with CPT-11.

Synergistic efficacy of 3n-butyrate and 5-fluorouracil in human colorectal cancer xenografts via modulation of DNA synthesis.

BACKGROUND & AIMS: Butyrate, produced in the colon lumen, maintains mucosal cell homeostasis. Poorly diffusible, its access is compromised in growing colon cancers and absent in distant metastases. Butyrate regulates DNA synthesis. We postulated that systemic administration of butyrate should reduce colon cancer growth and enhance 5-fluorouracil (5-FU) efficacy. METHODS: A stable derivative of butyrate (3n-But) was used. The antitumoral efficacy of 5-FU and 3n-But, alone or combined, was evaluated in human colorectal cancers (hCRCs) subcutaneously, orthotopically, or intrasplenically grafted into nude mice. Thymidylate synthase (TS) and thymidine kinase (TK) mRNA expression, proliferation, apoptosis, and cell cycle alterations were studied. RESULTS: In vivo, 5-FU alone inhibited growth of only 3 of the 12 hCRCs tested and 3n-But alone had no effect; the 5-FU/3n-But combination inhibited growth of all 16 hCRCs tested. The hCRCs differed in their p53 and microsatellite instability status. 5-FU/3n-But decreased TK and TS mRNA expression by 20- and 40-fold, respectively, and TS activity by 75%, stopped cell proliferation without affecting cell differentiation, and significantly enhanced apoptosis. 3n-But potentiated the efficacy of Tomudex and methotrexate, 2 TS inhibitors, but not that of oxaliplatin. In vitro, 5-FU/3n-But inhibited [3H]thymidine but not bromodeoxyuridine incorporation and induced apoptosis in hCRC cell lines. Cells treated with 5-FU/3n-But did not accumulate in G1 nor in S phase of the cell cycle, while 5-FU and 3n-But arrested the cycle in S and in G1 phase, respectively. 3n-But prevented the cell rescue from 5-FU-induced cytotoxicity by uridine or thymidine. CONCLUSIONS: 3n-But and TS inhibitors acted synergistically against colorectal cancers, independently of the genetic alterations of the hCRCs. The mechanism of action of 5-FU/3n-But could be enhanced reduction of TS and prevention of thymidine salvage in DNA synthesis.

[Molecular and cell aspects of the cancer metastasis]. / Aspectos moleculares y celulares de la metastásis cancerosa.

The metastasis dissemination is a process which leads to a primary tumor cells to migrate, infiltrate host tissues and form a secondary tumor focus at distance. This clinic evolution is a consequence of the cancer natural history and is due to the appearance of new potentialities in tumoral cells, providing to a small quantity of them an invasive and metastatic capability. This dissemination is possible by many factors; among them the lack of cohesion in tumor tissue cells, forming new blood vessels, resistance to anoikis and posterior implantation of tumoral cells in a heterotypic site. The change of cohesion in cell systems, synthesis of proteolytic enzymes, cell motility, modification of signals induced by growth factors, or the substratum for environment adhesion, and lack immune recognition by the immunologic system, are the main contributors to the appearance of metastasis. A permissive ecosystem is necessary to implant tumoral cells in target organs such as: liver, lung and bone marrow. This special environment, which is more favorable to metastasis, supplies an stroma, adhesive systems, growth factors and neo-vascularization. It is important to point out that the development of a metastasis is preceded by some genetic changes which make possible the adaptation of malign cells to a new microenvironment.

Induction of invasion in vivo of alpha-catenin-positive HCT-8 human colon-cancer cells.

Variants from the HCT-8 colon-cancer cell line were implanted s.c. and orthotopically into nude mice. Well-differentiated HCT-8/E11 and HCT-8/E41 cells have a functional E-cadherin-catenin complex and are non-invasive into pre-cultured chick heart fragments in vitro, whereas poorly differentiated HCT-8/E11R1 cells are deficient in alpha-catenin protein and invasive in heart fragments. We investigated whether these differences were maintained in vivo. In contrast with in vitro observations, in vivo the 3 HCT-8 variants behaved very similarly and all formed undifferentiated tumors. The in vivo invasive behavior of HCT-8 cells was site-dependently modulated: HCT-8 cells invaded when injected into the cecum but not when injected s.c. Metastases to the liver or lungs were not observed. The composition and expression of the E-cadherin-catenin complex in nude mouse HCT-8 tumors was the same as in HCT-8 cells in culture on solid substrate. We conclude that the in vivo invasive behavior of HCT-8 cells is not determined by whether alpha-catenin is expressed or not but by as yet unidentified host factors.

Nuclear DNA content and chromatin pattern of rat rhabdomyosarcoma cell sublines with different metastatic potentials.

There is a constant need of features able to characterize potentially metastatic cells among the heterogeneous cell subpopulations which constitute a tumor. Image cytometry of metastatic tumor cells give rise to variable results, partly because of a heterogeneous origin of cells, or potential drug effects. The aim of this work was to characterize nuclear changes observed in metastatic cell clones issued in vitro from the same parental cell population The nuclear phenotypes of 6 cell sublines isolated from a rat rhabdomyosarcoma cell line and differing in their metastatic ability were evaluated by image cytometry on Feulgen-stained preparations. Densitometric [5], geometric [3] and textural [9] features were computed from each nuclear image. For each cell subline, a metastatic score, ranging from 0 to 10, was calculated on the basis of in vivo invasivity data, by measuring the number of pulmonary metastases observed after s.c. graft of tumor cells in rats. Data obtained were compared to karyotype, growth characteristics, and oncogene expressions of cell lines. The nuclear DNA content, the chromosome numbers, the cell sublines doubling times, and the distribution of cells within the cell cycle appear unrelated with this score. On the contrary, increase in metastatic ability is accompanied by changes in chromatin pattern as assessed by textural features. Progressive increase in chromatin condensation can be observed in cell sublines with increasing metastatic score. These results were confirmed by an unsupervised multivariate partitioning of rhabdomyosarcoma cells which identified two separate subsets whose distributions within the analyzed cell lines correlate with their metastatic ability. These data suggest that, in rat rhabdomyosarcoma cell sublines, metastatic ability could be associated with nuclear morphological changes at the level of chromatin texture.