Compact Spheroids Research Articles

Gene expression analysis of tumor spheroids reveals a role for suppressed DNA mismatch repair in multicellular resistance to alkylating agents.

Drug resistance is a major obstacle in the successful treatment of cancer. Thus, elucidation of the mechanisms responsible is a critical first step in trying to prevent or delay such manifestations of resistance. In this regard, three-dimensional multicellular tumor cell spheroids are intrinsically more resistant to virtually all anticancer cytotoxic drugs than conventional monolayer cultures. We have employed the EMT-6 subline PC5T, which forms highly compact spheroids, and differential display to identify candidate genes whose expression differs between monolayer and spheroids. Approximately 5,000 bands were analyzed, revealing 26 to be differentially expressed. Analysis of EMT-6 tumor variants selected in vivo for acquired resistance to alkylating agents identified eight genes whose expression correlated with drug resistance in tumor spheroids. Four genes (encoding Nop56, the NADH SDAP subunit, and two novel sequences) were found to be down-regulated in EMT-6 spheroids and four (encoding 2-oxoglutarate carrier protein, JTV-1, and two novel sequences) were up-regulated. Analysis of the DNA mismatch repair-associated PMS2 gene, which overlaps at the genomic level with the JTV-1 gene, revealed PMS2 mRNA to be down-regulated in tumor spheroids, which was confirmed at the protein level. Analysis of PMS2(-/-) mouse embryo fibroblasts confirmed a role for PMS2 in sensitivity to cisplatin, and DNA mismatch repair activity was found to be reduced in EMT-6 spheroids compared to monolayers. Dominant negative PMS2 transfection caused increased resistance to cisplatin in EMT-6 and CHO cells. Our results implicate reduced DNA mismatch repair as a determinant factor of reversible multicellular resistance of tumor cells to alkylating agents.

Formation of melanocyte spheroids on the chitosan-coated surface

The search for biocompatible materials that can maintain function of melanocytes as the cellular patch is a feasible alternative for use in the autologous melanocyte transplantation for vitiligo. In this study, we demonstrated that the surface of chitosan-coated polystyrene wells supported the growth and phenotype expression of melanocytes. Depending on the seeding density and culture time, melanocytes were monolayered or spheroidal in morphology. At seeding densities above 10×10 3 cells/cm 2, human melanocytes started to aggregate on the surface of chitosan after 2 days in culture. These aggregates grew into compact melanocyte spheroids on day 3 and more melanocyte spheroids were observed when a higher seeding density was used. Cells remained viable in the spheroids and grew into dendritic melanocytes when they were reinoculated on polystyrene wells. Conversely, the time for the formation of melanocyte spheroids needed a longer period at lower seeding density. For example, melanocytes at as low as 1.25×10 3 cells/cm 2 did not aggregate until the 20th day of culture. In order to interpret the phenomenon further, we proposed the formation of melanocyte spheroids on the chitosan is mediated by a balance between two competing forces: the interactions of cell–chitosan and cell–cell.

Multicellular gastric cancer spheroids recapitulate growth pattern and differentiation phenotype of human gastric carcinomas

Background & Aims: Advanced gastric cancer has a poor prognosis and is largely unresponsive to currently available chemotherapeutic drugs. The development of more effective therapies would be aided by better preclinical models. Methods: An in vitro multicellular gastric cancer spheroid model was established using the liquid overlay technique and compared with the corresponding xenografts in immunodeficient mice. Results: Twelve of 17 (71%) gastric cancer cell lines reflected growth characteristics of their parental gastric carcinomas in three-dimensional culture. Thus, cell lines derived from peritoneal and pleural carcinomatosis grew as single cells (HSC-39, KATO-II, KATO-III) and cell aggregates (SNU-5, SNU-16). Cell lines representing adenosquamous (MKN-1) and tubular differentiation (MKN-28, MKN-74, N87) formed partly compact multicellular spheroids recapitulating the tumor architecture of the respective original tumor. The differentiated phenotype was lost after subcutaneous implantation of the in vitro spheroids in mice. The degree of morphologic differentiation was reflected by the levels of mucin and constitutive E-cadherin expression. Heterogeneous changes of other adhesion molecules (EpCAM, α2β1, CD44s, Lex, sLex) were observed. In contrast, cell lines derived from poorly differentiated gastric carcinomas (Hs-746T, RF-1, RF-48) formed fully compact spheroids mimicking the poorly differentiated phenotype, were E-cadherin negative, and showed only CD44s up-regulation. Conclusions: Recapitulating some complexity of their in vivo counterparts, multicellular gastric cancer spheroids may represent a physiologically valid model for studying the biology of this cancer, and testing new therapeutic strategies.GASTROENTEROLOGY 2001;121:839-852

Peroxisome proliferator activated receptor γ (PPARγ) agonists for treatment of neoplastic diseases - in vitro data and early clinical observations in chronic myelomnocytic leukemia (CMML) and in liposarcoma

Peroxisome proliferator activated receptor 3' (PPAR3') agonists for treatment of neoplastic diseases in vitro data and early clinical observations in chronic myelomnocyfie leukemia (CMML) and in fiposareoma. C. Den71inser, A. Mdhle, F. Machicao, R. M61de, P. Brossart, H.-U. Hiring, L. Kanz, K. Dittmann. University Clinic Tfibingnn, D-72076 Tfibingen, Germany. PPAR 7 is a nuclear hormone receptor which modulates gene expression upon agonist binding and heterodimelzation with activated retinoid X receptor. PPAR 7 agonists comprise both natural (cyclopentenone prostanoids) and pharmaceutical compounds (e.g. thiamlidinediones) and have been shown to exert antiproliferative effects in several cell lines in vitro. We investigated whether the thiazolidinediones troglitazone (TgO) or rosiglitazone (ROS) have an effect on proliferation ofmononuclenr ceils from healthy individuals and form patients with CMML in vitro. At a concemration of 50pM, TRO reduced DH]thymidine incorporation in mononudear celh from healthy individuals (n=5) or from CMML patients (n=10) during 24 h of incubation to 57_+18% (mean_+gD) or to 9-+7% of control, g o s had similar but less potent antiproliferative effects in CMML ceils in vitro whereas ceils from healthy individuals were not significantly affected. Our in vitro data prompted us to offer TRO to 3 selected CMML patients. Another 4 CMML pationts were treated with ROS. Furthermore, 3 patients with metastasized liposarcoma were treated with TRO or ROS. Treatment with either drug was very well tolerated. Control of monocyte counts was transiently improved in 5 of the 7 CMML patients. In 2 of the 3 liposarcoma patients minor responses and tran~emt disease stabilizations were noted. Our results suggest that PPAR 7 agonists may have therapeutic potentiel in CMML and in liposarcoma justifying further study of this non-genotoxic approach. Multicellular gasta'ic eaneer spheroids represent a valid model for pre-dinical testing of new therapeutic strategies B, Mayer, G. Klement, M. Kuneko, S. Jothy and R.S. Kerbel Gastric carcinoma is largely unresponsive to current chemotherapy and better pro~linical models to study therapeutic targets are urgently needed. An in vitro multicellular gastric cancer spheroid model was established, and compared to the subcutaneous xenogra~s in nu/nu mice as to: i) morphological and functional differentiation and ii) expres~on of cell adhesion molecules. Twelve out of 17 (71%) gastric cancer cell lines grown in 3D reflected growth characteristics of then parental gastric carcinomas. Cell lines derived from peritoneal and pleural carcinomatous grew as single cells (HSC-39, KATO-II, KATO-III) or cell aggregates (SNU-5, SNU-16). Adenosquamous (MKN-1) and tubularly differentiated (MKN-28, MKN-74, N87) cell lines formed partly compact multicellular spheroids and also reflected their parental architecture. Most importantly, difl'ereatiation was lost afier s.c. implantation of these preformed spheroids. Levels ofnmcin and constitutive E-cadherin expression reflected the degree of morphological differentiation, but the changes ot other adhesion molecules (EpCAM, c~2131, CD44s, Le', sLe') were heterogeneous. In contrast, cell lines derived from poorly differanfiated gastric carcinomas (Hs-746T, RF-1, RF-48) formed fully compact spheroids mimicking the poorly differentiated phenotype, were E-cadherin negative, and showed only CD44s upregulation. In summary, rm~Mcellular gastric cancer spheroid recapitulate the complexity of their in wvo counterparts, and represent a superior model for studying the biology of this cancer than s.c. xenogrefls. They embody a physiologically relevant model to test new therapeutic strategies such as differentiating and antiadhesion agents.

Rapid acquisition of multicellular drug resistance after a single exposure of mammary tumor cells to antitumor alkylating agents.

Clinical drug resistance is either intrinsic (de novo) or often acquired rapidly in conjunction with chemotherapy. By contrast, the selection of drug-resistant mutant cell lines in monolayer culture systems is usually a more protracted process. Sublines of mouse EMT-6 mammary tumor cells selected for resistance to various alkylating agents in vivo after serial passage into syngeneic mice manifest their resistance in vitro only when cultured as three-dimensional multicellular aggregates or spheroids. We examined whether a single exposure of mouse EMT-6 or human MDA-MB-231 breast cancer cells to alkylating agents in vitro is sufficient for the induction of a resistance phenotype, which may be detected by re-applying the drugs to cells grown as three-dimensional aggregates. Mouse EMT-6 and human MDA-MB-231 breast cancer cells cultured as three-dimensional aggregates were exposed to a single dose of alkylating agent for 1-5 days. Aggregates were dispersed, and cells were plated as monolayer cultures for up to 8 weeks to allow for recovery. Colony-forming ability was assessed after a subsequent alkylating-agent exposure of cells cultured as monolayers or three-dimensional aggregates. A single in vitro exposure to 12.5-microM cisplatin (CDDP) for 5 days or 25 microM 4-hydroperoxycyclophosphamide (4-O2H CTX) for 1 or 3 days without changing the medium was sufficient to induce transient but substantial resistance in EMT-6 cells as determined by clonogenic assays. Such resistance was not detected when monolayer cell cultures were used. The concentration of 4-O2H-CTX and the length of time the cells remained in three-dimensional culture after initial exposure to this drug was associated with the degree of subsequent drug resistance of cells grown as three-dimensional cultures. Furthermore, this acquired resistance after a single drug exposure was accompanied by changes in the three-dimensional architecture of the cell aggregates, which now formed much more compact multicellular spheroids. Similarly, a single exposure to 4-O2H-CTX was enough to bring about resistance in MDA-MB-231 cells detectable only in three-dimensional cultures, as well as the change in three-dimensional architecture. Rapid acquisition of resistance likely represents a physiologic mechanism of adaptation operative at the multicellular level rather than a stable genetic change and may be one of the reasons for the rapid development of drug resistance acquired by tumors in vivo. In vivo drug exposure may result in transient and low levels of drug resistance that may nevertheless be clinically relevant.

Growth advantage ("clonal dominance") of metastatically competent tumor cell variants expressed under selective two- or three-dimensional tissue culture conditions.

In previous experiments it was shown that injection into syngeneic CBA/J mice of cell mixtures containing an excess of non-metastatic SP1 mouse mammary carcinoma cells with a ras transfected metastatic variant of SP1 called C1, always resulted in the eventual dominance of the C1 subpopulation at the site of inoculation. This occurred despite the growth rates of the two cell populations being identical in vivo when grown separately. The means by which the C1 subpopulation achieved "clonal dominance" is thought to involve its responsiveness to stimulatory paracrine growth factors liberated by the non-metastatic SP1 population. The clonal dominance process, however, could not be recapitulated in conventional monolayer tissue culture conditions in which SP1 and C1 cells were grown together in high concentrations of serum, i.e. under non-limiting culture conditions. We now show that clonal dominance of C1 cells can be observed when the cell mixture is maintained in tissue culture for extended periods, or when the cells are grown under selective, limiting conditions, some of which may mimic growth conditions in vivo more accurately. These conditions were a) growth in low (limiting) serum concentrations; and b) growth as three-dimensional multicellular aggregates, i.e. as "tumor spheroids". Under all of these conditions dominance of the C1 subpopulation always took place, but with an efficiency 6- to 40-fold less than generally observed in vivo. C1 cells were also able to form more stable (compact) spheroids compared to SP1 cells. Entrapment of the latter in mixed C1/SP1 spheroids increased the recovery of the SP1 cells suggesting some kind of "rescue" mechanism in which cells are protected from physical forces by three-dimensional structure. The relevance of these in vitro interactions for clonal dominance in primary tumors and metastasis in vivo are discussed.

Acquired multicellular-mediated resistance to alkylating agents in cancer.

EMT-6 murine mammary tumor sublines highly resistant to cyclophosphamide, cis-diamminedichloro-platinum(II), or N,N',N"-triethylenethiophosphoramide were generated in vivo by sequential treatment of tumor-bearing mice with the respective drugs. Previous studies demonstrated the drug-resistant phenotypes of the sublines were not expressed in vitro when the cells were grown as monolayer cultures. We now show that expression of drug resistance--including patterns of cross-drug resistance observed in vivo--can be fully recapitulated in vitro when the cells are grown under in vivo-like, three-dimensional conditions--namely, as multicellular tumor spheroids. Moreover, the spheroids generated from all of the drug-resistant sublines manifested a much more compact structure. Immediate drug-sensitivity testing of single cells released by trypsin treatment from compact drug-resistant spheroids revealed that such cells lost much of their drug-resistant properties. The results suggest a possible mechanism of acquired drug resistance in tumors based on the response of a cell population (i.e., multicellular or tissue resistance) as opposed to classic (uni)cellular resistance mechanisms.

Physical characterization of histidine-rich protein from Plasmodium lophurae

The size and shape of Plasmodium lophurae histidine-rich protein have been determined by analytical centrifugation and electron microscopy. From the partial specific volume of 0.72 cc/g, the molecular weight was determined to be 43000. The sedimentation velocity studies indicated a coefficient of 1.32 S in 0.9 M acetic acid (pH 3.5), monodispersity and significant asymmetry. Darkfield electron microscopy revealed the major species to be compact oblate spheroids 12 nm in width and extended filamentous particles of average length 35 nm by 1.5 nm. Analysis of the sequence of the protein by the method of Garnier et al. (J. Mol. Biol. (1978) 120, 97–120) predicted that 82% of its residues would be found in three long α-helices. The protein's CD spectrum has a strong resemblance to that of poly( l-histidine) at pH 4–5, where the homopolymer is thought to be in a right-handed α-helical form. A single helix containing 300 residues would be 45 nm long, the largest length found by electron microscopy. From the electron-microscopic data, sedimentation coefficients of 1.6 and 1.95 S, respectively, were calculated for flexible-coil and extended-rod models, in closer agreement with the measured value of 1.3 S than the value calculated for a spherical model. Thus, the major species in acetic acid is probably an incompletely extended rod which, as the pH is increased to neutrality, condenses to form spherical molecular aggregates seen in the malaria parasite.

Isolation and analysis of nuclear bodies from estrogen-stimulated chick liver

The target cells of most steroid hormones contain prominent structures termed nuclear bodies (NB). Estrogen (ES) responsive tissues have very distinctive NB, whose development has been associated with both the level of ES receptors and their retention times in nuclei. It has been suggested that these NB may be large-scale ES binding centers at transcriptionally hyperstimulated sites. We examined the formation of NB in ES-stimulated rooster liver, after primary induction of vitellogenesis and during subsequent decay of this response. Distinct NB appear 6 h postinduction, initially as compact spheroids with ring-like profiles and later on as more complex morphotypes. Nuclei average 15–17 NB each at peak induction (24–48 h), but this declines again gradually as the hormonal response abates. This transient pattern is paralleled closely by the [ 3H]ES binding capacity of nuclei in vitro and the calculated number of specific hormone receptors present. There is no direct numerical correspondence, however, between the NB and the ES receptors, either on a one-to-one basis or as some simple ratio. Hence any links between ES binding activity in nuclei and the NB must be both complex and indirect. Nuclear bodies were isolated following lysis of nuclei by sonication and separation of subfractions on discontinuous sucrose gradients. The resultant NB-enriched fraction consisted primarily of protein, with traces of DNA and RNA. Analysis of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a complex pattern of polypeptides, some apparently unique and probably NB-derived.