title: Proliferating fibroblasts and HeLa cells co-cultured in vitro reciprocally influence growth patterns, protein expression, chromatin features and cell survival.
abstract: OBJECTIVE
  to identify biological interactions between proliferating fibroblasts and HeLa cells in vitro.
  
  
  METHODS
  Fibroblasts were isolated from both normal and tumour human tissues. Coverslip co-cultures of HeLa and fibroblasts in various ratios with medium replacement every 48 h were studied using fixed cell staining with dyes such as Giemsa and silver staining, with immunochemistry for Ki-67 and E-cadherin, with dihydrofolate reductase (DHFR) enzyme reaction, as well as live cell staining for non-specific esterases and lipids. Other techniques included carmine cell labeling, autoradiography and apoptosis assessment.
  
  
  RESULTS
  Under conditions of feeding and cell: cell ratios allowing parallel growth of human fibroblasts and HeLa cells, co-cultured for up to 20 days, a series of phenomena occur consecutively: profound affinity between the two cell types and exchange of small molecules; encircling of the HeLa colonies by the fibroblasts and enhanced growth of both cell types at their contact areas; expression of carbonic anhydrase in both cell types and high expression of non-specific esterases and cytoplasmic argyrophilia in the surrounding fibroblasts; intense production and secretion of lipid droplets by the surrounding fibroblasts; development of a complex net of argyrophilic projections of the fibroblasts; E-cadherin expression in the HeLa cells; from the 10th day onwards, an increasing detachment of batches of HeLa cells at the peripheries of colonies and appearance of areas with many multi-nucleated and apoptotic HeLa cells, and small HeLa fragments; from the 17th day, appearance of fibroblasts blocked at the G2-M phase. Co-cultures at approximately 17-20 days display a cell-cell fight with foci of (a) sparse growth of both cell types, (b) overgrowth of the fibroblasts and (c) regrowth of HeLa in small colonies. These results indicate that during their interaction with HeLa cells in vitro, proliferating fibroblasts can be activated against HeLa. This type of activation is not observed if fibroblast proliferation is blocked by contact inhibition of growth at confluency, or by omitting replacement of the nutrient medium.
  
  
  CONCLUSIONS
  The present observations show that: (a) interaction between proliferating fibroblasts and HeLa cells in vitro drastically influences each other's protein expression, growth pattern, chromatin features and survival; (b) these functions depend on the fibroblast/HeLa ratio, cell topology (cell-cell contact and the architectural pattern developed during co-culture) and frequent medium change, as prerequisites for fibroblast proliferation; (c) this co-culture model is useful in the study of the complex processes within the tumour microenvironment, as well as the in vitro reproduction and display of several phenomena conventionally seen in tumour cytological sections, such as desmoplasia, apoptosis, nuclear abnormalities; and (d) overgrown fibroblasts adhering to the boundaries of HeLa colonies produce and secrete lipid droplets.
authors: International Institute of Anticancer Research, Kapandriti, Attiki, Greece editor@iiar-anticancer.org.
authors: John G
authors: JG
authors: Delinasios
authors: International Institute of Anticancer Research, Kapandriti, Attiki, Greece.
authors: Flora
authors: F
authors: Angeli
authors: International Institute of Anticancer Research, Kapandriti, Attiki, Greece.
authors: George
authors: G
authors: Koumakis
authors: Department of Experimental Medicine, Medical School, University of Manchester, Manchester, U.K.
authors: Shant
authors: S
authors: Kumar
authors: Department of Experimental Medicine, Medical School, University of Manchester, Manchester, U.K.
authors: Wen-Hui
authors: WH
authors: Kang
authors: Institute of Histology and Embryology, "A. Gemelli" Faculty of Medicine, Catholic University of the Sacred Heart, Rome, Italy.
authors: Gigliola
authors: G
authors: Sica
authors: Institute of Histology and Embryology, "A. Gemelli" Faculty of Medicine, Catholic University of the Sacred Heart, Rome, Italy.
authors: Fortunata
authors: F
authors: Iacopino
authors: Institute of Histology and Embryology, "A. Gemelli" Faculty of Medicine, Catholic University of the Sacred Heart, Rome, Italy.
authors: Gina
authors: G
authors: Lama
authors: Department of Clinical Biochemistry and Pharmacology, Ben-Gurion University of the Negev, Beersheba, Israel.
authors: Sergio
authors: S
authors: Lamprecht
authors: Department of Clinical Biochemistry and Pharmacology, Ben-Gurion University of the Negev, Beersheba, Israel.
authors: Ina
authors: I
authors: Sigal-Batikoff
authors: Proteomics Research Unit, Biomedical Research Foundation of the Academy of Athens, Athens, Greece.
authors: George T
authors: GT
authors: Tsangaris
authors: Department of General Surgery, Metaxa Cancer Hospital, Piraeus, Greece.
authors: Christos D
authors: CD
authors: Farfarelos
authors: Department of Plastic Surgery, Metaxa Cancer Hospital, Piraeus, Greece.
authors: Maria C
authors: MC
authors: Farfarelos
authors: Department of Maxillofacial Surgery, Attikon University Hospital, Athens, Greece.
authors: Eleftherios
authors: E
authors: Vairaktaris
authors: Department of Maxillofacial Surgery, Attikon University Hospital, Athens, Greece.
authors: Stavros
authors: S
authors: Vassiliou
authors: International Institute of Anticancer Research, Kapandriti, Attiki, Greece.
authors: George J
authors: GJ
authors: Delinasios
mesh_terms: D002450:Cell Communication / Q000235:genetics; D049109:Cell Proliferation / Q000235:genetics*; D002470:Cell Survival / Q000235:genetics; D002843:Chromatin / Q000235:genetics; D018920:Coculture Techniques; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D017154:Stromal Cells / Q000473:pathology; D059016:Tumor Microenvironment / Q000235:genetics*
chemical_list: D002843:Chromatin
keywords: E-cadherin; Fibroblasts; G2-M phase; HeLa; Ki-67; apoptosis; carmine labeling; cell affinity; cell-cell interactions; co-culture; desmoplasia; dihydrofolate reductase (DHFR); lipids; multi-nucleated cells; non-specific esterases (NSE); nuclear abnormalities; proliferating fibroblasts; silver staining; stromal fibroblasts; tumour microenvironment