Fig 2: Endogenous tagging of TUBB3 does not compromise cell integrity. (A) Endogenous tagging of β3-tubulin was performed in rat-derived PC12 (left) and mouse-derived Neuro2A (right) cell lines. Both cell lines showed a consistent increase in expression of β3-tubulin-GFP after NGF stimulation (100 ng/mL) as analyzed by flow cytometry. (B) Endogenous labeling of β3-tubulin does not compromise cell proliferation of either PC12 (left) or Neuro-2A (right) neuronal precursors, as observed by a time course cell viability assay using direct counting of Hoechst/PI staining of the cell culture between 0 and 10 days of culture. We observe no statistically significant difference in the rate of cell proliferation between cells that have undergone endogenous labeling and control cells, providing evidence that the CRISPR-mediated labeling approach did not compromise cell viability. (C) WST-1 assay does not reveal any changes in cell metabolic activity after endogenous tagging of β3-tubulin in either PC12 (left) or Neuro-2A (right) cell lines. We observe no statistically significant difference in the cell metabolic activity between cells that have undergone endogenous labeling and control cells, providing evidence that the CRISPR-mediated labeling approach did not affect the cell’s overall differentiation or viability. Statistics: two-way ANOVA.

Fig 3: CRISPR/Cas9-based HITI approach for experimental labeling of endogenous β3-tubulin. (A) To generate a culture of PC12-TUBB3-GFP cells, neuronal PC12 precursors were transfected with the vector containing Cas9, sgRNA, and the GFP reporter donor sequence, which integrates into the β3-tubulin locus. We used a single vector that contains spCas9 controlled by a constitutive CMV promoter, the gRNA controlled by a U6 promoter, and the GFP donor DNA sequence flanked by target sequences that match the genomic TUBB3-targeted region. The gRNA creates a double-strand break and removes the GFP donor DNA from the plasmid for genomic integration of GFP at the β3-tubulin locus target sequence. The correct orientation of GFP is ensured by the reverse orientation of the target sequence sites and protospacer-adjacent motif (PAM) compared to the TUBB3 locus. This design allows Cas9 to self-correct GFP its orientation if integration occurs backward. (B) Strategy to isolate PC12 culture transfected with the knock-in vector. Cells were sorted using fluorescence-activated single-cell sorting (FACS) to select cells that acquired GFP fluorescence signal as presented in the dot plot (transfected). Wild-types (WT) cells served as control. GFP+ cells were collected and regrown in 96-well plates to generate isogenic cultures. Cells were then extracted from the well to regenerate the desired culture. (C) Confocal microscopy observation of PC12-TUBB3-GFP culture treated with NGF to induce differentiation and the development of extended neurites reveals the expression of a GFP network throughout the cytoplasm of the neuron precursor cells, with particular enrichment within the neurites where β3-tubulin acts as a scaffold (white arrows). The GFP signal reveals a typical and expected tubular network structure specific to tubulin networks such as the targeted β3-tubulin. (D,E) Fluorescence microscopy imaging confirms increased GFP signal in PC12-TUBB3-GFP neuronal precursors after their differentiation. The top pictures show brightfield imaging confirming the change in neuronal morphology between untreated cells (D) and cells treated with NGF at 50 ng/mL for 5 days to induce differentiation (E). The bottom picture highlights GFP expression in these cells and the significant increase in signal, validating the reporter’s ability to report neuronal differentiation. Blue: DAPI staining of the nucleus. Green: TUBB3-GFP. Scale bars: 50 µm.

Fig 4: Breast cancer plasticity promotes neuron precursor axonogenesis. (A) To test the model developed for studying cancer-induced neuronal differentiation, we developed a model of breast cancer plasticity and epithelial-mesenchymal transition (EMT). The NMuMG cell cultures from ATCC are mostly epithelial cells but are heterogenous cultures having mixed phenotypes, with a few cells exhibiting mesenchymal morphologies (red arrows). NMuMG clones with epithelial (NMuMG-E) and mesenchymal (NMuMG-M) morphologies were isolated by single-cell seeding and regrowing of isogenic culture having epithelial or mesenchymal characteristics. (B) Western blot analysis of the regenerated culture confirms the occurrence of an EMT, consisting of the expression of the epithelial marker E-cadherin in NMuMG-E cells that disappears in NMuMG-M cells and the acquisition of the expression of mesenchymal markers fibronectin, N-cadherin, and Slug in NMuMG-M cells (Original Western blot, see Supplementary File S1). GAPDH serves as a loading control. (C) Wound healing assay shows a greater velocity of migration in NMuMG-M cells compared to NMuMG-E cells, confirming the biological impact of the EMT on the cells’ aggressive behaviors. (D) To verify the relevance of NMuMG E/M to reflect the cell EMT-related plasticity functionally, we performed wound healing assays and quantified them by live-cell microscopy with the workflow published by Jonkman and colleagues [47]. (E) Fluorescence microscopy imaging of PC12-TUBB3-GFP cells in coculture with NMuMG-E or NMuMG-M constitutively expressing the mCherry fluorophore (red Channel), show increased GFP signal with the NMuMG-M cells, consistent with the development of extended neurites (White arrow). (F) PC12-TUBB-GFP cells were cultivated as unstimulated monoculture (Alone), incubated in direct coculture with either epithelial (+E cells) or mesenchymal (+M cells) cells, or treated with NGF (100 ng/mL) (+NGF) for 5 days as a positive control for differentiation. Flow cytometry revealed a moderate induction of PC12 neuronal precursor differentiation under NMuMG-E stimulation but a robust induction of differentiation under stimulation with NMuMG-M, where neuronal cells developed even more differentiation than those stimulated with NGF. (G) Cocultures of PC12-TUBB3-GFP-mCherry (constitutively expressing mCherry fluorophore) mixed with NMuMG-E or NMuMG-M cells were analyzed using an automated fluorescent plate reader. The mCherry expression allowed normalization of the assay by reflecting quantities of PC12 cells in the wells, and the mCherry/GFP ratio was used as an indicator of neuronal differentiation. Statistics: unpaired Student t-test ** p < 0.01; *** p < 0.001. Scale bar: 50 µm.