We propose 293T is a valuable tool to study cancer stem/progenitor features in that it rapidly generates both spheres and tumors that are readily serially passaged, and maintains significant populations of the tumor-initiating phenotypes described in the cancer stem cell literature. In this study, we observed that transplanting 293T cells into the cleared mammary fat pads of immunocompromised mice resulted in serially transplantable rapidly growing tumors. Propagation of spheres from tumor tissue or directly from the cell line enriched for ALDH activity and CD44+/CD24- markers and expression of EMT markers as well as genes that promote stem cell survival. Papers characterizing similar features in existing cancer cell lines have demonstrated a wide range in each of the purported cancer stem cell populations in these lines making the lines available to screen simultaneously against each surrogate limited.
The 293T cell line was developed through transformation of human embryonic kidney cells using the sheared Adenovirus 5 (Ad5) DNA . It expresses genes permitting the replication of E1-deficient, non-replicating adeno and adeno-associated viral vectors leading to their common use of generating viral vectors for laboratory and gene therapy use. From the initial report in 1977, the transformation was surprisingly inefficient. Only two of eight transformations were successful with only one of twenty cultures forming a single colony in each case. Attempts to isolate these were not successful, however and a single transformed colony was observed in one dish not discarded 75 days after the original experiment. Partial serum starvation (2%) was used to select for the transformed phenotype and eventually the isolated culture was expanded. The culture was reported to contain cells of several morphologies of generally epitheliod appearing cells. Injection of 2 × 107 cells subcutaneously into nude mice yielded no tumors after 6-8 weeks. Re-injection at that time resulted in tumors 15-20 weeks after the first injection in 3/20 animals. In stark contrast, Shen et al  report that in cells passaged greater than 65 times, the tumorgenicity increases to 100% with tumors formed in two weeks. We have reproduced these findings with 100% tumor formation when injected into the cleared mammary fat pads of immunocompromised mice and further report that these tumors are serially transplantable with similarly aggressive biology and resembled poorly differentiated, high grade malignant primary tumor on histology. It's noteworthy that there is controversy on the origins of HEK 293T cells as it has been assumed that they were generated by transformation of either a fibroblastic, endothelial or epithelial cell all of which are abundant in kidney. However, the fact that the cells originated from cultured kidney cells does not say much about the exact cellular origin of the HEK 293T, as embryonic kidney cultures may contain small numbers of almost all cell types of the body. In fact, a study by Graham and colleagues provided evidence that these cells and several other human cell lines generated by adenovirus transformation of human embryonic kidney cells have many neuronal progenitor properties, suggesting that the adenovirus was taken up and transformed a neuronal lineage cell in the original kidney culture . Thus, the spheres generated from 293T cells under stem-cell promoting culture condition may have features observed in neurospheres.
Given the in vivo and histological findings, we explored known cancer stem cell surrogates and signaling pathways described in various cancer studies and confirm this cell line faithfully recapitulates the described biology of purported cancer stem cells. Previous studies showed that nuclear and cytoplasmic extracts from undifferentiated cells can reprogram 293T cells into a stem cell-like phenotype with activation of embryonic stem cell genes and outgrowth of spherical structures. As a result of treatment of 293T monolayer cells with embryonic carcinoma cell extract, spherical colonies of 293T cells develop and induce Oct4 gene expression which in turn induces the expression several Oct4-responsive genes like Sox2, Nanog and Rex1 [42–44]. Consistent with these findings, our study has found activation of the pluripotency genes as well as formation of spheres in suspension culture suggesting that the stem cell promoting culture condition promotes dedifferentiation. The notion that cells with properties of cancer stem cells can be generated from differentiated somatic cells has been proposed in a recent study by Dean and colleagues . These authors demonstrated that culturing retinoblastoma-deficient mouse embryonic fibroblasts in suspension culture form spherical structures, re-express embryonic stem cell markers and at least a subset of this population adopts characteristics of cancer stem cells. Likewise, recent accounts from Weinberg's group  and others  suggest the possibility of reversion or reprogramming of differentiated cancer cells into cancer stem cells.
Several studies from our lab have shown that breast cancer stem/progenitor cells are resistant to radiation compared to bulk, differentiated cells [9, 16, 48, 49]. Phillips and colleagues also demonstrated the radioresistance of putative breast cancer stem/progenitor cells by comparing the radiosensitivity of cells derived from the CD44+/CD24-subpopulation of MCF-7 cell line grown as spheres vs. monolayers . The resistance of cancer stem cells to radiation has been shown to be mediated by the β-catenin [9, 49] or Notch signaling pathway . Similar findings were reported in brain tumors demonstrating that CD133-expressing glioma stem cells are relatively resistant to radiation in vivo and in culture compared to non-stem cells . Likewise, in our study the stem cell-enriched population from the 293T cells is much more resistant and with increased expression of β-catenin and Notch1 compared to the adherent population. Moreover, a study by Li et al demonstrated that the percentage of cancer stem cells, assayed by mammosphere forming efficiency, was significantly increased after chemotherapy highlighting the chemoresistant nature of cancer stem cells .
The molecular basis underlying the EMT process involves multiple changes in expression and function of proteins that include vimentin and cadherins [51–53]. Acquired expression of vimentin by carcinoma cells often symbolizes mesenchymal-like cell transformation  while loss of E-cadherin or gain of N-cadherin on tumor cell surface is frequently observed in malignant carcinomas and also correlated with enhanced aggressiveness and dedifferentiation . Recent studies have demonstrated that induction of EMT using Twist and Snail in transformed mammary epithelial cells creates populations that are highly enriched for cancer stem cells as evidenced by increased CD44+/CD24- expression, mammosphere formation and tumor seeding ability . These cells showed attributes of mesenchymal phenotype including expression of vimentin, fibronectin and n-cadherin. Similarly, in a recent study in pancreatic cell lines the knockdown Zeb1, another EMT-inducer, affected not only the initial formation of spheres but also led to reduction of sphere numbers in subsequent generations indicating its role in self-renewal and maintenance of the stem cell phenotype. In our current study, we found higher expression of these EMT transcription factors in spheres from 293T cells vs. monolayer cells in line with the stem cell properties of 293T spheres.
MicroRNAs are emerging as important regulators of cellular differentiation and EMT. Recent findings have associated let-7 microRNAs with stem cell maintenance and loss of let-7 in cancer has been reported to cause dedifferentiation and self-renewal of breast cancer cells . Consistent with our finding that the 293T spheres have markedly reduced Let-7 expression and increased H-ras and HMGA2, two known let-7 targets these authors showed that cancer stem cell population have markedly reduced let-7 and increased H-ras and HMGA2 expression. By expressing let-7 in cancer stem cells, the authors also found that let-7 regulates self renewal, multipotent differentiation, and the ability to form tumors . Several recent studies have identified the miR-200 family and miR-205 as key regulators of EMT and enforcers of the epithelial phenotype of cancer cells [38, 40]. MiR-200 was found to directly target the mRNA of the E-cadherin transcriptional repressors Zeb1 and Zeb2. While ectopic expression of miR-200 caused up-regulation of E-cadherin in cancer cell lines and reduced their motility, its inhibition reduced E-cadherin expression, increased expression of vimentin and induced EMT . Another microRNA, miR-335, has been identified as metastasis suppressor microRNA in human breast cancer . It inhibits metastatic cell invasion and migration through targeting of the progenitor cell transcription factor Sox4 and extracellular matrix component tenascin C. Moreover, the loss of expression of this microRNA is associated with poor distal metastasis-free survival .