Spontaneous cell–cell fusion is essential in many important biological processes, such as fertilization, placenta development, bone and muscle formation, tissue regeneration, immune response, tissue repair and stem cell differentiation [
1‐
4].
In the field of cancer research, Otto Aichel first hypothesized that leucocyte–tumour cell fusion or hybridization could lead to the emergence of malignant cells and neoplasms in 1911 [
5]. Cell–cell fusion has been recognized as an important biological process in cancer evolution since Pawelek et al. discovered that normal cell–tumour cell fusion is involved in tumour initiation and metastasis [
6,
7] and Miller FR et al. demonstrated that in vitro tumour–tumour cell hybrids were more malignant and chemoresistant [
8,
9]. In addition, spontaneous cell–cell fusion may occur during chronic inflammation, which is related to neoplasia [
10]. Thus, spontaneous cell–cell fusion could play important roles in many aspects of tumour progression, such as the generation of cancer stem cells and the acquisition of metastatic potential or multidrug resistance [
11,
12]. Chemotherapy has been utilized to treat cancer for many years, and this approach kills most of the tumour cells, but patients still have a poor prognosis because of drug resistance caused by intratumoural heterogeneity and other factors [
13,
14]. Multiple ways can lead to this heterogeneity. Stochastic genetic or epigenetic changes are well-established mechanisms involving intrinsic differences among cancer cells [
15,
16]. Genetic and phenotypic heterogeneity are major causes of variations in chemotherapeutic responses [
17]. Cell–cell fusion can lead to heterogeneity [
18]. Cells with different genetic backgrounds that hybridize in vivo may enrich for multiple important mutations and then develop drug-resistant subclones, as has been reported based on in vitro experiments [
8,
19]. Moreover, except mutations occurred in stem cells, epithelial–mesenchymal transition (EMT) and de-differentiation of differentiated cells or cells in the late progenitor stage, spontaneous cell–cell fusion can produce recurrence cancer stem cells (rCSCs) after prognosis as well, which could increase the patient’s risk of experiencing recurrent or metastatic disease [
12,
20].
Therefore, spontaneous cell–cell fusion has a considerable impact on chemoresistance, metastasis and prognosis in cancer treatment. In vitro experiments can provide preliminary information on cell fusion during tumour evolution, but these experiments do not recapitulate in vivo tumour evolution under selective pressures, such as chemotherapy. Furthermore, there have been only a few reports on in vivo tumour cell fusion in human cancer and the corresponding chemotherapeutic response, which is the key evidence that illustrates the important roles of cell fusion in tumour evolution [
6,
19,
21,
22] and its critical impact on cancer treatment.
Here, we introduced enhanced green fluorescent protein (EGFP) and red fluorescent protein (mCherry) transgenes into the SKBR3 cell line to perform xenograft experiments in Balb/c-nu mice and treated these xenograft mice with epirubicin chemotherapy to construct an in vivo chemotherapy tumour evolution model that better mimics cancer evolution in patients. Cell hybrids, which express dual fluorescence in a single cell, were detected by fluorescence-activated cell sorting (FACS). Furthermore, chemotherapy was found to promote tumour cell hybridization in vivo by generating more hybrids in the outer section of the tumour. These results provide evidence to support previous studies on cell–cell fusion in vitro [
6,
8,
19] and provoke safety concerns regarding chemotherapy.