Effects Of Cell-cell Interactions Research Articles

Integrating mechanism-based T cell phenotypes into a model of tumor-immune cell interactions.

Interactions between cancer cells and immune cells in the tumor microenvironment influence tumor growth and can contribute to the response to cancer immunotherapies. It is difficult to gain mechanistic insights into the effects of cell-cell interactions in tumors using a purely experimental approach. However, computational modeling enables quantitative investigation of the tumor microenvironment, and agent-based modeling, in particular, provides relevant biological insights into the spatial and temporal evolution of tumors. Here, we develop a novel agent-based model (ABM) to predict the consequences of intercellular interactions. Furthermore, we leverage our prior work that predicts the transitions of CD8+ T cells from a naïve state to a terminally differentiated state using Boolean modeling. Given the details incorporated to predict T cell state, we apply the integrated Boolean-ABM framework to study how the properties of CD8+ T cells influence the composition and spatial organization of tumors and the efficacy of an immune checkpoint blockade. Overall, we present a mechanistic understanding of tumor evolution that can be leveraged to study targeted immunotherapeutic strategies.

Effects of cell-cell interactions between A. oryzae and Z. rouxii on morphology and secondary metabolites

Aspergillus oryzae and Zygosaccharomyces rouxii are important microorganisms in the food brewing process, and interactions between fungi can produce various secondary metabolites. Herein, metabolic profiling was used to investigate the differential metabolites and metabolic pathways of A. oryzae and Z. rouxii co-culture in solid medium. The results demonstrated that the fungal breakage rate increased significantly, and the enzyme activity decreased in the co-culture. Furthermore, A. oryzae mycelium grew above the Z. rouxii. By detecting the metabolites of the fungus cell, an increase in the co-culture was detected in the contents of l-Arginine and l-Glutamate. On the other hand, the contents of l-Leucine and l-Isoleucine decreased and affected various metabolic pathways (Arginine biosynthesis, Isoleucine degradation and Valine biosynthesis). In summary, the results of this study support essential information regarding the interaction and cell metabolisms between food-brewing fungi.

Effects of Cell-Cell Interactions between a. Oryzae and Z. Rouxii on Morphology and Secondary Metabolites

Effects of Cell-Cell Interactions between a. Oryzae and Z. Rouxii on Morphology and Secondary Metabolites

4168 Understanding ECM-Based Drug Resistivity in Breast Cancer

OBJECTIVES/GOALS: Cell-cell (CC) and cell-matrix interactions (CM) are known to affect drug sensitivity of cancer cells, but are not effectively recapitulated using 2D platforms. This research aims to determine how cell and matrix interactions confer drug resistivity in 3 distinct culturing models: 2D (no CM/limited CC), 3D spheroids (CC) and 3D fibronectin (both). METHODS/STUDY POPULATION: We examined four breast cancer cell types. The cells were derived from a nonmetastatic primary tumor (HMLE-E2) or overt bone-metastasis (BM). Transglutaminase 2 (TGM2), a matrix crosslinking protein, is overexpressed in metastatic bone tumors and may play a key role in matrix-conferred drug resistivity. In a gain-of-function model, TGM2 was upregulated in HMLE-E2 cells and compared to shTGM2 knockdown BM cells. Growth rates were analyzed using metabolic activity over 8 days, and drug sensitivity to Neratinib (0-1000 nM) was analyzed via cell titer. To account for the different transport properties of the 3 distinct culture environments, we developed a mathematical model for each condition, allowing us to normalize the drug sensitivity results across models to effectively compare true biological resistivity. RESULTS/ANTICIPATED RESULTS: We observed that increased cellular levels of TGM2 significantly increase the growth rate and drug resistivity of cells on fibronectin matrices. Interestingly, in 2D cultures, TGM2 expression was correlated with higher Neratinib resistivity but did not affect growth rates. In spheroid models without a significant matrix component, that rely solely on cell-cell junctions, high levels of TGM2 were correlated with lower survival rates. Lower levels of TGM2 are correlated with a more epithelial phenotype, and using our mathematical model we have identified significant transport differences between high and low TGM2 spheroids. We theorize that the low TGM2 spheroids have denser packing, which lowers the rate of diffusion and, thus reduces the effective concentration of the drug to the majority of the cells. DISCUSSION/SIGNIFICANCE OF IMPACT: Our studies indicate that the cellular response to drugs can be altered by changes in both transport properties of the tissue and the CM interactions. By systematically investigating the effects of CC interactions and CM interactions, we can use mathematical models to delineate physical means of drug resistivity from a biologically driven resistance.

Abstract 2834: Metastatic colorectal cancer: characterization of distinct histological growth patterns which demonstrate different response to current treatment regimes

Abstract Colorectal cancer (CRC) is the third most common cancer in North America, and the third leading cause of cancer-related death. About 50% of patients will be diagnosed with CRC liver metastasis (CRCLM) during the course of their disease. We have identified two major histological growth patterns (HGP) in CRCLM resected from patients. In the desmoplastic HGP (DHGP), the tumor cells are enclosed within a desmoplastic stromal ring, physically separating them from the normal liver tissue. In the replacement HGP (RHGP), the tumor cells replace normal hepatocytes without disrupting adjacent cells or structures within the liver parenchyma. We have recently shown that patients with RHGP lesions who received neoadjuvant bevacizumab plus chemotherapy had a worse pathological response and five-year overall survival. Our group has also shown that RHGP CRCLM is resistant to anti-angiogenic therapy due to it promoting vessel co-option as a means of tumor vascularization, rather than VEGF-dependent angiogenesis as seen in DHGP. However, the molecular mechanisms that drive the CRCLM HGPs remain unknown. This project aims to shed light on the biological processes that underlie the diversity of HGPs, with an emphasis on the key genes and pathways that support the RHGP. To test this, we have: Aim 1) performed RNAseq on chemonaïve RHGP and DHGP CRCLM lesions to identify pathways that are significantly upregulated in the RHGP. We have validated these genes via real-time PCR (RT-PCR), and a subset by immunohistochemistry (IHC). Aim 2) To further refine the RHGP gene signature and to examine the effects of cell-cell interactions on hepatocyte and tumor cell gene expression, individual cells have been isolated from distinct regions within RHGP CRCLM tissues using laser capture microdissection (LCM), followed by single-cell transcriptomics analyses. Our preliminary data have identified three major pathways with differentially expressed genes: i. cell motility, ii. cell junctions, and iii. ECM-receptor interactions, which are upregulated in RHGP relative to the DHGP and normal liver. A panel of RHGP-specific genes has been identified and validated via RT-PCR. We selected a subset of these genes and are performing IHC to further validate and locate their expression. We have also performed LCM and are in the process of performing transcriptomics analyses – the data will be presented. We expect to gain some insight into the mechanisms responsible for driving and/or maintaining the RHGP lesions. In the longer term, this work will result in identification of targets in the HGPs that will help stratify patients in terms of treatment. It would appear that we have fewer treatment options for the RHGP patients and it would be important to utilize the data to develop new treatment strategies for those patients. These new therapies would lead to further clinical trials. Citation Format: Vincent Palmieri, Anthoula Lazaris, Hussam Alamri, Abdellatif Amri, Nisreen Ibrahim, Pablo Zoroquiain, Peter Vermeulen, Peter Metrakos. Metastatic colorectal cancer: characterization of distinct histological growth patterns which demonstrate different response to current treatment regimes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2834. doi:10.1158/1538-7445.AM2017-2834

Interleukin‐6 induces the lineage commitment of bone marrow‐derived mesenchymal multipotent cells through down‐regulation of Sox2 by osteogenic transcription factors

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are a heterogeneous population of cells that differ in size and morphology. BM-MSCs become committed to the osteogenic lineage as senescence approaches and lose multipotency. Nevertheless, little is known about the effects of cell-cell interaction between different populations on stemness loss and lineage commitment. The current study aimed to identify mechanisms by which cell-cell interactions between heterogeneous BM-MSCs affect stemness and lineage commitment of multipotent subpopulation. The lineage commitment of primitive multipotent cells was strongly induced in the presence of cytokines secreted by senescent-like cells in a cell culture insert system. Senescent-like cells secreted higher levels of interleukin-6 (IL-6) than primitive multipotent cells in a human cytokine array. IL-6 induced the lineage commitment and stemness loss in multipotent cells by decreasing Sox2 expression. Furthermore, we confirmed that IL-6 decreased the transcriptional activity of Sox2 through up-regulation of Runx2 and Dlx5. We suggest a mechanism by which IL-6 modulates the expression of Sox2, resulting in decreased multipotency and causing primitive multipotent cells to undergo osteogenic lineage commitment. This is the first study to identify mechanisms in which the cell-cell interactions between the different populations play important roles in the stemness loss and lineage commitment of multipotent populations.-Yoon, D. S., Kim, Y. H., Lee, S., Lee, K.-M., Park, K. H., Jang, Y., Lee, J. W. Interleukin-6 induces the lineage commitment of bone marrow-derived mesenchymal multipotent cells through down-regulation of Sox2 by osteogenic transcription factors.

Adhesive Dynamics

Adhesive dynamics (AD) is a method for simulating the dynamic response of biological systems in response to force. Biological bonds are mechanical entities that exert force under strain, and applying forces to biological bonds modulates their rate of dissociation. Since small numbers of events usually control biological interactions, we developed a simple method for sampling probability distributions for the formation or failure of individual bonds. This method allows a simple coupling between force and strain and kinetics, while capturing the stochastic response of biological systems. Biological bonds are dynamically reconfigured in response to applied mechanical stresses, and a detailed spatio-temporal map of molecules and the forces they exert emerges from AD. The shape or motion of materials bearing the molecules is easily calculated from a mechanical energy balance provided the rheology of the material is known. AD was originally used to simulate the dynamics of adhesion of leukocytes under flow, but new advances have allowed the method to be extended to many other applications, including but not limited to the binding of viruses to surface, the clustering of adhesion molecules driven by stiff substrates, and the effect of cell-cell interaction on cell capture and rolling dynamics. The technique has also been applied to applications outside of biology. A particular exciting recent development is the combination of signaling with AD (so-called integrated signaling adhesive dynamics, or ISAD), which allows facile integration of signaling networks with mechanical models of cell adhesion and motility. Potential opportunities in applying AD are summarized.

Effects of Morphology vs. Cell–Cell Interactions on Endothelial Cell Stiffness

Biological processes such as atherogenesis, wound healing, cancer cell metastasis, and immune cell transmigration rely on a delicate balance between Cell-Cell and cell-substrate adhesion. Cell mechanics have been shown to depend on substrate factors such as stiffness and ligand presentation, while the effects of Cell-Cell interactions on the mechanical properties of cells has received little attention. Here, we use atomic force microscopy to measure the Young's modulus of live human umbilical vein endothelial cells (HUVECs). In varying the degree of Cell-Cell contact in HUVECs (single cells, groups, and monolayers), we observe that increased cell stiffness correlates with an increase in cell area. Further, we observe that HUVECs stiffen as they spread onto a glass substrate. When we weaken Cell-Cell junctions (i.e., through a low dose of cytochalasin B or treatment with a VE-cadherin antibody), we observe that cell-substrate adhesion increases, as measured by focal adhesion size and density, and the stiffness of cells within the monolayer approaches that of single cells. Our results suggest that while morphology can roughly be used to predict cell stiffness, Cell-Cell interactions may play a significant role in determining the mechanical properties of individual cells in tissues by careful maintenance of cell tension homeostasis.

Numerical Simulation of Mass Transport in a Microchannel Bioreactor With Cell Micropatterning

Micropatterning of two different cell types based on surface modification allows spatial control over two distinct cell subpopulations. This study considers a micropatterned coculture system, which has release and absorption parts alternately arranged at the base, and each part has a single cell type. A micropattern unit was defined and within each unit, there are one release part and one absorption part. The cells in the absorption parts consume species, which are secreted by the cells in the release parts. The species concentrations at the micropatterned cell base were computed from a three-dimensional numerical flow model incorporating mass transport. Different combined parameters were developed for the release and absorption parts to make the data collapse in each part. Combination of the collapse data in the release and absorption parts can be used to predict the concentration distribution through the whole channel. The correlated results were applied to predict the critical length ratio of the release and absorption parts for an actual micropatterned system (Bhatia et al., 1999, "Effect of Cell-Cell Interactions in Preservation of Cellular Phenotype: Co-Cultivation of Hepatocytes and Nonparenchymal Cell," FASEB J. 13, pp. 1883-1900) to avoid species insufficiency based on basic fibroblast growth factor (bFGF). The mass transfer effectiveness was found to be higher with more numbers of micropattern units. The optimal condition for micropatterned coculture bioreactors is achieved by having the product of the length ratio and the reaction ratio equal to 1. This condition was used to optimize the mass transfer in the micropatterned system (Bhatia et al., 1999, "Effect of Cell-Cell Interactions in Preservation of Cellular henotype: Co-Cultivation of Hepatocytes and Nonparenchymal Cell," FASEB J. 13, pp. 1883-1900) based on bFGF.

Effects of tumor stroma interaction on global gene expression in breast cancer

10006 Background: Perturbations in cell-cell interaction are a key feature of cancer. However, the systematic effects of cell-cell interaction on global gene expression in cancer are largely unexplored. We hypothesized that gene expression signatures induced by cell-cell interaction might be of clinical relevance. Methods: We simulated tumor-stroma interaction in vitro by systematically co-cultivating each of 7 different breast cancer cell lines with stromal fibroblasts from 3 different sites, and determined associated gene expression changes with cDNA microarrays. A dataset of pretreatment gene expression profiles from 295 early stage breast cancers (stage 1 and 2) with a median follow up of 12.6 years allowed us to evaluate the prognostic significance of the gene expression signatures of specific cell-cell interactions derived from our ex vivo models. Results: The most prominent response to epithelial-mesenchymal interaction was an induction of interferon-response genes (IRG), observed in 4 of the 7 breast cancer cell lines in co-culture with fibroblasts, but not in normal mammary epithelial cells. In response to close contact with these breast cancer cells, the fibroblasts secreted type I interferons, which, in turn, induced expression of the IRG in the tumor cells. Immunohistochemical analysis of human breast cancer tissues showed that Stat1, the key transcriptional activator of the IRG, and itself an IRG, was expressed in a subset of the cancers, with a striking pattern of elevated expression in the cancer cells in contact with, or close proximity to, the tumor stroma - paralleling the response seen in our ex vivo model. In vivo, expression of the IRGs was remarkably coherent, providing a basis for segregation of the 295 early-stage breast cancers into two groups by unsupervised hierarchical clustering with the IRG. Tumors with high expression levels (n=161) of IRG were associated with significantly shorter overall survival; 59% at 10 years versus 80% at 10 years for tumors with low expression levels (n=134) (log-rank p=0.001). Conclusions: Our results suggest that an interaction between some breast cancer cells and stromal fibroblasts can induce an interferon response, and that this response may be associated with a greater propensity for tumor progression. No significant financial relationships to disclose.

Cell-Cell Interaction-dependent Regulation of N-Acetylglucosaminyltransferase III and the Bisected N-Glycans in GE11 Epithelial Cells: INVOLVEMENT OF E-CADHERIN-MEDIATED CELL ADHESION

Changes in oligosaccharide structures are associated with numerous physiological and pathological events. In this study, the effects of cell-cell interactions on N-linked oligosaccharides (N-glycans) were investigated in GE11 epithelial cells. N-glycans were purified from whole cell lysates by hydrazinolysis and then detected by high performance liquid chromatography and mass spectrometry. Interestingly, the population of the bisecting GlcNAc-containing N-glycans, the formation of which is catalyzed by N-acetylglucosaminyltransferase III (GnT-III), was substantially increased in cells cultured under dense conditions compared with those cultured under sparse conditions. The expression levels and activities of GnT-III but not other glycosyltransferases, such as GnT-V and alpha1,6-fucosyltransferase, were also consistently increased in these cells. However, this was not observed in mouse embryonic fibroblasts or MDA-MB231 cells, in which E-cadherin is deficient. In contrast, perturbation of E-cadherin-mediated adhesion by treatment with EDTA or a neutralizing anti-E-cadherin antibody abolished the up-regulation of expression of GnT-III. Furthermore, we observed the significant increase in GnT-III activity under dense growth conditions after restoration of the expression of E-cadherin in MDA-MB231 cells. Our data together indicate that a E-cadherin-dependent pathway plays a critical role in regulation of GnT-III expression. Given the importance of GnT-III and the dynamic regulation of cell-cell interaction during tissue development and homeostasis, the changes in GnT-III expression presumably contribute to intracellular signaling transduction during such processes.

In vitro expression of proalpha1(I) collagen mRNA by human pre-osteoclastic cells.

In vitro studies have demonstrated that the extracellular matrix modulates the cell phenotype. In the present study we have investigated in vitro proalpha1(I) collagen mRNA expression in a human pre-osteoclastic cell line (FLG 29.1 cells) in basal condition and after various stimuli. In addition, in order to evaluate the effect of cell-cell interactions on collagen type I mRNA expression, we have cultured the human pre-osteoclastic cells FLG 29.1 with either the human osteoblast-like cell line Saos-2 or the bovine bone endothelial cell line BBE. We showed that the FLG 29.1 cells express proal (I) collagen mRNA, whose expression is modulated by phorbol esters (TPA). Co-culturing FLG 29.1 cells with either Saos-2 or BBE cells induced decrease of proalpha1(I) collagen mRNA expression.

CDO, a robo-related cell surface protein that mediates myogenic differentiation.

CDO, a member of the Ig/fibronectin type III repeat subfamily of transmembrane proteins that includes the axon guidance receptor Robo, was identified by virtue of its down-regulation by the ras oncogene. We report here that one prominent site of cdo mRNA expression during murine embryogenesis is the early myogenic compartment (newly formed somites, dermomyotome and myotome). CDO is expressed in proliferating and differentiating C2C12 myoblasts and in myoblast lines derived by treating 10T1/2 fibroblasts with 5-azacytidine, but not in parental 10T1/2 cells. Overexpression of CDO in C2C12 cells accelerates differentiation, while expression of secreted soluble extracellular regions of CDO inhibits this process. Oncogenic Ras is known to block differentiation of C2C12 cells via downregulation of MyoD. Reexpression of CDO in C2C12/Ras cells induces MyoD; conversely, MyoD induces CDO. Reexpression of either CDO or MyoD rescues differentiation of C2C12/Ras cells without altering anchorage-independent growth or morphological transformation. CDO and MyoD are therefore involved in a positive feedback loop that is central to the inverse relationship between cell differentiation and transformation. It is proposed that CDO mediates, at least in part, the effects of cell-cell interactions between muscle precursors that are critical in myogenesis.

Biofilm formation by Porphyromonas gingivalis and Streptococcus gordonii.

Confocal scanning laser microscopy (CSLM) was used to visualize and quantify biofilm formation by the oral bacteria Streptococcus gordonii and Porphyromonas gingivalis. A saliva-coated glass coverslip under continuous bacterial challenge and conditions of low shear force was used to investigate attachment to the salivary pellicle and also the effect of cell-cell interactions on the extent of colonization and biofilm development. S. gordonii bound to the salivary pellicle and outcompeted P. gingivalis for attachment sites. Both P. gingivalis and S. gordonii failed to establish substantial biofilm formation independently. However, biofilm formation did occur subsequent to initial adherence of P. gingivalis to S. gordonii cells deposited on the salivary pellicle. The commensal species S. gordonii may, therefore, provide an attachment substrate for colonization and biofilm accretion by the potential pathogen, P. gingivalis.

Biofilm formation by Porphyromonas gingivalis and Streptococcus gordonii

Confocal scanning laser microscopy (CSLM) was used to visualize and quantify biofilm formation by the oral bacteria Streptococcus gordonii and Porphyromonas gingivalis. A saliva‐coated glass coverslip under continuous bacterial challenge and conditions of low shear force was used to investigate attachment to the salivary pellicle and also the effect of cell‐cell interactions on the extent of colonization and biofilm development. S. gordonii bound to the salivary pellicle and outcompeted P. gingivalis for attachment sites. Both P. gingivalis and S. gordonii failed to establish substantial biofilm formation independently. However, biofilm formation did occur subsequent to initial adherence of P. gingivalis to S. gordonii cells deposited on the salivary pellicle. The commensal species S. gordonii may, therefore, provide an attachment substrate for colonization and biofilm accretion by the potential pathogen, P. gingivalis.

Cell‐cell interactions that modulate neuronal development in the leech

Mitotic lineage has been found to determine the cellular identity of leech neurons (reviewed in Stent et al., 1992), Int. Rev. Neurobiol. 33:109-133. However, the details of the adult phenotype of many neurons in the central nervous system of the leech have been shown to be shaped by interactions either with other neurons or with non-neuronal tissues in the environment. Four effects of cell-cell interactions will be considered in this article: stimulation of mitosis that generates new neurons, modulation of cell death or axonal retraction, modification of neurotransmitter metabolism, and modification of other physiological properties. In all cases, the interactions that modify development are thought to occur at a location distant from the soma, requiring that signals be transmitted a significant distance from the site of interaction to the metabolic machinery in the soma.

Effects of cell-cell interaction on the mRNA level of glyoxalase I gene in Saccharomyces cerevisiae

When cells of Saccharomyces cerevisiae were exposed to high cell-density environments, the glyoxalase I activity increased, and the amount of mRNA of the glyoxalase I gene in the yeast cells increased in accord with the enzyme activity. The mRNA level of the glyoxalase I gene in flocculent mutant cells of S. cerevisiae was also higher than that in the corresponding parental cells; therefore, cell-cell interaction may affect the mRNA level of the glyoxalase I gene in S. cerevisiae.

Cellular interactions and tubulin detyrosination in fibroblastic and epithelial cells

In mammalian cells most microtubules are enriched in tyrosinated alpha-tubulin (tyr-tubulin). Other subclasses of microtubules are present in variable amounts and some are enriched in detyrosinated alpha-tubulin (glu-tubulin). We examined the effect of cell-cell interactions on the level of glu-tubulin in microtubules. This was studied by quantitative immunofluorescence using antibodies against tyr- and glu-tubulin. We found that in cells which have established cell-cell contacts, the ratio of glu-/tyr-tubulin is higher than in isolated cells. We also examined the effect of cell-cell interactions on the glu-/tyr-tubulin ratio by using the antibody blocking method of Schulze and Kirschner [42]. Microtubules containing mainly tyr-tubulin had been blocked first by a polyclonal antibody against tyr-tubulin and several layers of secondary antibodies. The unblocked microtubules were then labeled by a monoclonal antibody against alpha-tubulin. Since the coating efficiency of microtubules by the anti-tyr tubulin depends on the amount of tyr-tubulin in each microtubule, this procedure allows the visualization of microtubules enriched or depleted in tyr-tubulin in specific domains of each cell. Microtubules were more extensively blocked in subconfluent than in confluent cells and preferentially at the periphery of the cytoplasm. In cells present at the margin of an artificial wound produced in a confluent monolayer, the amount of blocked microtubules increased slowly with time (between 2 and 4 h). These results are consistent with the hypothesis that cell-cell contacts lead to increased tubulin dytyrosination both in fibroblastic and epithelial cells.

Functional differentiation of alveolar type II epithelial cells in vitro: Effects of cell shape, cell-matrix interactions and cell-cell interactions

Alveolar type II epithelial cells rapidly lose characteristics of differentiated function when cultured on plastic dishes. We have attempted to circumvent this problem by culturing type II cells under conditions that might better reproduce their environment in vivo. Cell-matrix interactions were studied by culturing isolated adult rat type II cells on Engelbreth-Holm-Swarm (EHS) tumor basement membrane. Aggregates of type II cells formed on the surface of the matrix during 4 days in culture. Microscopic examination of these aggregates revealed cuboidal cells that retained more characteristics of differentiated type II cells than did cells cultured on plastic. Type II cells cultured on EHS matrix incorporated a higher percentage of acetate into phosphatidylcholine (PC) than did cells on plastic, and a higher percentage of this PC was saturated. Phosphatidylglycerol (PG) synthesis by these cells was no different from that seen in cells on plastic. The effects of cell-cell interactions and cell shape were evaluated by culturing type II cells on feeder layers that in turn were grown on collagen gels. The feeder layer cells included fetal rat lung fibroblasts, adult rat lung fibroblasts, fetal rat skin fibroblasts, bovine aortic endothelial cells, and rat mammary tumor epithelial cells. One-half of the gels remained attached to the culture dish and one-half of the gels were detached after 24 h and allowed to float free in the medium. Type II cells grown in association with any of the attached feeder layers became flattened and lost their differentiated phenotype. These cells incorporated no greater percentage of acetate into PC than did cells on plastic. Saturated PC synthesis was modestly increased. PG synthesis declined in parallel with that seen in cells cultured on plastic. Type II cells cultured on feeder layers that were detached assumed their native cuboidal shape and also exhibited many morphological characteristics of differentiated function. These cells incorporated a significantly greater percentage of acetate into PC compared to cells on either plastic or attached feeder layers. Saturated PC synthesis also increased markedly. These cells, however, incorporated no greater percentage of acetate into PG than did cells on plastic or attached feeder layers. These data suggest an important role for cell shape and cell-matrix interactions and maintenance of type II cell differentiation. The effects of cell-cell interactions, while beneficial, appear to be non-specific.

Effect of cell-cell interactions on drug sensitivity and growth of drug-sensitive and -resistant tumor cells in spheroids.

Multicellular spheroids were grown from mixtures of rat brain tumor cells sensitive (9L) and resistant (R3) to 1,3-bis(2-chloroethyl)-1-nitrosourea. Percentages of each cell subpopulation in these spheroids were estimated with the sister chromatid exchange assay and were found to be approximately the same as those used to initiate spheroids. Spheroids grown from 9L cells alone had a higher growth rate than spheroids grown from R3 cells alone. However, the growth rate of mixed-cell spheroids was essentially the same as that of pure 9L spheroids and was independent of the percentages of R3 cells in mixed-cell spheroids. The sensitivity of 9L cells in mixed-cell spheroids treated with 1,3-bis(2-chloroethyl)-1-nitrosourea, estimated by changes in the number of sister chromatid exchanges per metaphase induced by treatment, decreased as the percentage of R3 cells increased. These effects are probably the result of an interaction between the two cell subpopulations held in three-dimensional contact, a situation similar to that in tumors in situ. The results suggest why one cell subpopulation of tumors does not become dominant during growth and indicate that interactions between cell subpopulations can influence the sensitivity of one subpopulation to 1,3-bis(2-chloroethyl)-1-nitrosourea.