c

c. (scale club?=?200?m) (d). Significant differences Eprosartan mesylate were decided using ANOVA (Tukeys multiple comparisons test) Eprosartan mesylate (b) and Tukeys unpaired t-test with Welchs correction (c). Asterisks indicate significant differences when the complementation of growth Eprosartan mesylate medium with medium or exosomes from parental or clonal cell lines increased the growth rate of the other clones. Complementation assays, co-growth and co-injection of mKO E10 and GFP C3 clonal cell lines increased the efficiency of invasion and migration. Conclusions These findings support a model where interplay between clones confers aggressiveness, and which may allow identification of the factors involved in cellular communication that could play a role in clonal cooperation and thus represent new targets for preventing tumor progression. Electronic supplementary material The online version of this article (10.1186/s12885-019-5883-y) contains supplementary material, which is available to authorized users. is the largest diameter of the tumor and the smallest one. All animals were euthanized 34?days after inoculation to compare the primary tumor size and composition and the number and extent of lung metastases between groups. The tumors and lungs were weighed, fixed with paraformaldehyde 4%, and later processed for histopathological analyses (hematoxylin and eosin staining). The metastasis growth rate of the MDA-MB-231 and clonal cell lines was evaluated by intravenous (IV) injection of 2.5??106 cells into the caudal tail vein of 10 animals per group (five groups). All animals were euthanized 36?days after inoculation. Animals underwent gross necropsy consisting of a macroscopic evaluation. Lungs were excised, weighed, fixed and processed for histopathological analysis. Immediately following dissection, the tumors and lungs were fixed for 24?h by immersion in paraformaldehyde 4%. After fixation, the tissue was dehydrated to enable embedding with paraffin. Five-micron-thick sections were cut from fixed, paraffin-embedded tissues and mounted on poly-L-lysine-coated glass slides. Sections were deparaffinized in xylene and rehydrated in graded alcohol. The presence of clonal cells in the primary tumor and metastases were detected by fluorescence on confocal microscopy (Spectral Confocal Microscope FV1000 (Olympus)). To avoid spectral overlapping of the different fluorescent proteins, Eprosartan mesylate a Lambda scan was performed from 470 to 635?nm followed by spectral deconvolution using FV10-ASW 4.2 software. Images were quantified using the program ImageJ. In vivo zebrafish tumor xenograft assays Zebrafish (and describe a model where the interplay of clones confers aggressiveness, and which may allow the identification of factors involved in cellular communication and metastasis. Thus, clonal heterogeneity allows the malignant cell line to acquire the greatest malignant potential. Conventional models propose that each metastasis originates from a single tumor cell [55C57]. However, recent studies using mouse models of cancer have exhibited that multiple RAD26 subclones undergo polyclonal seeding and demonstrate interclonal cooperation between multiple subclones [7, 58]. Our results confirm (Fig. ?(Fig.3d)3d) that metastasis could be formed either by a single (Fig. ?(Fig.3e)3e) or several clonal cell lines (Fig. ?(Fig.3d).3d). However, even in cases of metastasis formed by several clonal cell lines, each metastasis contained one predominant clone (Fig. ?(Fig.3d).3d). Comparative studies indicate monoclonal patterns of seeding, suggesting that clones compete to metastasize. However, polyclonal seeding, in which multiple clones from the primary tumor seed the same metastasis, is also observed, indicating subclones might cooperate as well as compete to metastasize [7, 59]. Eprosartan mesylate In our model the cells were injected as a mix of single cells, therefore the metastasis formed by more than one cell line originated from several cells that reached the lung together, demonstrating that this cells actually interact to form the metastasis. Several studies call into question the theory of clonal progression by the progressive accumulation of genetic alterations and selection of more aggressive clones, supporting instead the proposed theory of clonal cooperation between tumor clones [20C23, 25C27]. Tumor multiclonality is also supported by the field cancerization theory [60, 61], which says that there are many genetic alterations in the normal tissue surrounding tumors that can give rise to independent clones. Similarly, supporting interpretations can be drawn from the stem cell hypothesis, as diverse clones can derive from more than one pluripotent stem cell [62, 63], and the Big Bang model of colorectal tumor growth where the tumor grows predominantly as a single expansion populated by numerous intermixed subclones [64]. Clonal cooperation has recently been suggested in studies of single cell sequencing [62, 65, 66]. The present study further supports the idea that there are several clones that together confer the properties of malignancy, thus strengthening the concept of clonal cooperation, whereby clones synergistically provide certain selective advantages for proliferation, resistance to apoptosis, induction of angiogenesis, and conversation with environmental factors and inflammatory cells [20, 21,.