1.

Introduction

The epithelial–mesenchymal transition (EMT) is a process in which epithelial cells dedifferentiate to become mesenchymal cells.¹ It is an essential step in embryonic development, wound healing, and cancer metastasis. Regarding the role of EMT in cancer, metastasis is the primary cause of death of cancer patients.² To metastasize, stationary epithelial cancer cells switch off expression of epithelial markers such as E-cadherin and lose epithelial characteristics such as cell–cell adhesion and polarity. Meanwhile, cells turn on expression of mesenchymal markers such as vimentin and acquire a mesenchymal morphology and ability to migrate and invade.^3,4

Cancer cells in general have elevated anabolic metabolism,^3,5 in order to synthesis cellular components for aberrant proliferation. While protein synthesis is tightly controlled in normal cells, this process is dysregulated in cancer cells, leading to fast and uncontrolled protein synthesis.⁶ Glucose uptake is also generally elevated, in the presence or absence of oxygen (i.e., the Warburg effect).⁷ The uptaken glucose is believed to serve a variety of functions, including providing building blocks for cell mass synthesis, energy production, redox balance, and lactate secretion.⁸ Lipogenesis is also upregulated in cancer cells. This includes both de novo lipogenesis from glucose and direct scavenging of exogenous free fatty acids from surroundings.^9–11

Although elevated anabolism in cancer cells is generally accepted, metabolic changes during cancer EMT is not well studied. A few recent works have shed light on this topic, mainly through analysis of gene expression. During EMT, cancer cell protein synthesis is quickly inhibited on loss of cell–cell adhesion to preserve ATP, because protein synthesis consumes a large amount of it.¹² In both pancreatic cancer and breast cancer cells, increased expressions are observed for glucose transporters, lactate dehydrogenase A, and lactate exporter MCT4.^13,14 Pyruvate entry into the TCA cycle is increased due to decreased activity of pyruvate dehydrogenase kinase 4.¹⁵ Oxidative phosphorylation is increased to consume more glucose for ATP production to support mesenchymal cell motility.¹⁶ Glucose anabolism in the pentose phosphate pathway, serine biosynthetic pathway, gluconeogenesis, and de novo lipogenesis is all decreased.^14,17 Synthesis of phosphatidylcholine, an important component of membrane lipids, seems slower in mesenchymal cells.¹⁸ Expression of fatty acid translocase is increased during EMT, suggesting direct scavenging activity.^18,19 Together, these results suggest that mesenchymal cells might exhibit faster catabolism and slower anabolism than the epithelial counterpart.

Although the previous work on gene expression provides important insight into metabolic changes during cancer EMT, a direct visualization of the relevant metabolites is lacking, especially at the single-cell level. We here directly compared various metabolisms in the epithelial and mesenchymal cells of the breast cancer cell MCF-7 through stimulated Raman scattering (SRS) microscopy, using deuterium or alkyne tag-labeled amino acids, glucose, choline, and fatty acids. Vibrational imaging by SRS is a rapidly growing field.^20–24 It is becoming the most powerful vibrational microscopy for biology, owing to its superb sensitivity, imaging speed, three-dimensional optical sectioning, Raman spectral fidelity, a strict linear concentration dependence, straightforward image interpretation, and quantification.^20–28 Recently, a new bioorthogonal imaging strategy is emerging, by introducing vibrational tags such as deuterium or alkyne labels to small biomolecules.^29–32 Through deuterium labeling in amino acids, glucose, cholesterol, and fatty acids, SRS microscopy was applied to visualize protein synthesis,³² de novo lipogenesis,³³ intracellular cholesterol storage,³⁴ and metabolic activity in live tissues.^35,36 Through alkyne labeling of glucose, choline, nucleic acids, and fatty acids, SRS microscopy was applied to study the metabolism of glucose uptake,³⁷ choline metabolism,³¹ cell proliferation,^31,35 and membrane synthesis.³¹

In this work, we employed deuterium-labeled glucose (D7-Glc), deuterium-labeled amino acids (CD-AA), deuterium-labeled palmitate acid (d31-PA), and alkyne-labeled choline (propargylcholine) to directly study glucose metabolism, protein synthesis, fatty acid metabolism, and choline metabolism, respectively, during EMT of the MCF-7 cell model. Slower protein synthesis and membrane synthesis are observed after EMT. Interestingly, new information regarding the metabolism of lipid droplets has been revealed in mesenchymal cells.

2.

Materials and Methods

3.

Results

Spontaneous Raman spectra of MCF-7 cells grown in unlabeled or labeled mediums are shown in Fig. 1. Without any labels (bottom gray), the spectrum has a silent region in 1800 to $2400{\mathrm{cm}}^{-1}$ where no Raman peaks from other biological molecules exist. After culturing in mediums supplemented with labeled metabolites, Raman peaks from incorporated metabolic labels appear in the silent region. Metabolites from deuterium-labeled glucose, amino acids, and palmitate acids all have broad Raman peaks, ranging from 2050 to $2300{\mathrm{cm}}^{-1}$, which are from the C-D bonds vibration. For SRS imaging, we pick the highest peaks for each label, i.e., $2133{\mathrm{cm}}^{-1}$ for D7-Glc and CD-AA, and $2109{\mathrm{cm}}^{-1}$ for d31-PA. In contrast, propargylcholine has a signature sharp Raman peak at $2142{\mathrm{cm}}^{-1}$ from the $\mathrm{C}\equiv \mathrm{C}$ alkyne tag.

Fig. 1

(a) Spontaneous Raman spectra of MCF-7 cells cultured with metabolic labels. MCF-7 cells cultured in regular medium do not have any Raman peak in 1800 to $2400{\mathrm{cm}}^{-1}$ (gray curve on bottom). Cells cultured in deuterium- or alkyne-labeled metabolites show Raman peaks that are signature of the label (cyan shade). (b) Representative SRS images of MCF-7 cells cultured in mediums with metabolic labels. Left, incorporated metabolites. Middle, cell silent region that is far away from Raman peak of metabolic labels. Right, SRS images at $1655{\mathrm{cm}}^{-1}$ from amide vibration that represent intrinsic protein pool. Scale bars, $50\mu \mathrm{m}$.

Representative SRS images of MCF-7 cultured in the labeled mediums are also shown in Fig. 1(b). Left panels are SRS images targeted at the signature frequencies of the C-D or $\mathrm{C}\equiv \mathrm{C}$ vibrations. When the frequency is moved away to $2000{\mathrm{cm}}^{-1}$, where the metabolic labels do not show any Raman signatures, no SRS signals can be detected (middle panels). SRS images of the same cells at a frequency of $1655{\mathrm{cm}}^{-1}$ (amide vibration attributed mainly to proteins) show a strong signal from the total protein pool illustrating the cell morphology (right panels).

We next aim to study metabolic changes during EMT of MCF-7 cells. We induced EMT using a standard EMT inducer from R&D systems. We first validated the method by immunofluorescence staining of the cells before and after EMT, using antibodies for E-cadherin and vimentin, which are well-established markers for epithelial and mesenchymal cells, respectively. The images are shown in Fig. 2. As expected, MCF-7 loses E-cadherin and acquires vimentin after EMT. We then followed this validated protocol to induce EMT of MCF-7 for the study of metabolism here. Epithelial and mesenchymal MCF-7 cells are cultured in mediums with d31-PA, D7-Glc, CD-AA, or propargylcholine for 1 to 2 days. SRS images were acquired for both types of cells, and they are shown in Figs. 3Fig. 4Fig. 5–6.

Fig. 2

Immunofluorescence of MCF-7 shows cells undergo epithelial–mesenchymal transition. Before EMT, cells have bright E-cadherin (red) staining but weak vimentin (green) staining. After EMT, E-cadherin expression decreased while vimentin expression increased. Scale bars, $50\mu \mathrm{m}$.

Fig. 3

CD-AA incorporation in MCF-7 during EMT. C-D SRS images of mesenchymal cells are darker than epithelial cells, meanwhile mesenchymal cells have lots of lipid droplets, which appear as bright puncta in both C-D and ${\mathrm{CH}}_2$ SRS images. Analysis of image reveals that C-D incorporation decreased by $\sim 30\%$ during EMT, reflecting slower protein synthesis. Number of intracellular lipid droplets increased from less than 10 to around 50. Scale bar, $50\mu \mathrm{m}$.

Fig. 4

Propargylcholine incorporation in MCF-7 during EMT. $\mathrm{C}\equiv \mathrm{C}$ SRS images of mesenchymal cells are darker than epithelial cells. Meanwhile mesenchymal cells have lots of lipid droplets, which appear as bright puncta in both $\mathrm{C}\equiv \mathrm{C}$ and ${\mathrm{CH}}_2$ SRS images. Analysis of image reveals that C-D incorporation decreased by $\sim 80\%$ during EMT, reflecting slower membrane synthesis. Number of intracellular lipid droplets increased from less than 10 to around 50. Scale bar, $50\mu \mathrm{m}$.

Fig. 5

D7-Glc incorporation in MCF-7 during EMT. C-D SRS images of mesenchymal cells are darker than epithelial cells. Meanwhile mesenchymal cells have lots of lipid droplets, which appear as bright puncta in both C-D and ${\mathrm{CH}}_2$ SRS images. Analysis of image reveals that C-D incorporation decreased by $\sim 50\%$ during EMT, reflecting slower biomass synthesis. Number of intracellular lipid droplets increased from less than 10 to around 50. Scale bar, $50\mu \mathrm{m}$.

Fig. 6

d31-PA incorporation in MCF-7 during EMT. In epithelial cells, d31-PA are mainly incorporated into membrane lipids, as evidenced by the resemblance to ${\mathrm{CH}}_2$ of lipids. In mesenchymal cells, d31-PA is also largely incorporated into lipid droplets, which also appear in ${\mathrm{CH}}_2$ lipids channel. Analysis of C-D SRS signal of individual cells reveals that without considering lipid droplets, d31-PA incorporation into membrane lipids is less after EMT. When all lipid droplets are included, overall C-D SRS is higher after EMT. Number of intracellular lipid droplets increased from less than 10 to around 50. Scale bar, $50\mu \mathrm{m}$.

In Fig. 3, before EMT, CD-AA showed bright C-D SRS intensity, reflecting rapid protein synthesis in epithelial cells. The bright puncta are from nucleoli with fast protein turnover.³² After EMT, C-D SRS intensity in mesenchymal cells is visually darker than epithelial cells, indicating reduced protein synthesis after EMT. Analysis of the images revealed a reduction of around 30% in CD-AA incorporation. This is consistent with reports from other groups that protein synthesis¹² is slowed down during cancer EMT. Meanwhile, cells also acquire a large amount of lipid droplets, which appear as bright puncta in the ${\mathrm{CH}}_2$ lipid channel at $2845{\mathrm{cm}}^{-1}$. The average number of lipid droplets increased from less than 10 per cell in epithelial cells to around 50 per cell in mesenchymal cells.

Figure 4 shows choline metabolism during EMT of MCF-7 cells. The signal of propargylcholine reflects the synthesis activity of choline phospholipids which is the main component of biological membrane.^31,38 Before EMT, the alkyne tag from propargylcholine shows bright SRS images at its peak Raman frequency $2142{\mathrm{cm}}^{-1}$, across the whole cell. This reflects active membrane synthesis in rapidly proliferating epithelial cells. After EMT, however, SRS images of the alkyne tag become much dimmer, and the majority of propargylcholine concentrates in the endoplasmic reticulum (ER) region. This reflects that there is less membrane synthesis in mesenchymal cells, which does not proliferate as fast as epithelial cells, and choline is synthesized only into regions where lipids turnover is fast, such as ER. Analysis of image intensity reveals the incorporation of choline reduced by around 80% after EMT. Our imaging result is consistent with reports that choline anabolic metabolism¹⁸ is slowed down during cancer EMT.

Figure 5 shows D7-Glc metabolism during MCF-7 EMT. Glucose can be synthesized into proteins, DNA, and lipids inside cells. In epithelial cells, it appears that glucose is incorporated into both proteins and membrane lipids, reflecting fast protein and membrane synthesis in rapidly proliferating epithelial cells. In mesenchymal cells, however, the overall C-D signal is weaker across cytoplasm than epithelial cells, and it is concentrated in bright droplets (indicated by green arrows). These bright droplets also appear on the ${\mathrm{CH}}_2$ SRS channel at a frequency of $2845{\mathrm{cm}}^{-1}$ (indicated by green arrows). Therefore, in mesenchymal cells, apart from being synthesized into membrane lipids, glucose is also metabolized and stored into lipid droplets as energy storage. SRS intensity analysis revealed that total incorporation of the C-D from D7-Glc is reduced by around 50% during MCF-7 EMT, and the average number of lipid droplets increased from less than 10 per cell in epithelial cells to around 50 per cell in mesenchymal cells.

Free fatty acids are important building blocks for lipid synthesis for proliferating cells.³⁹ Figure 6 shows the incorporation result of d31-PA. In epithelial cells, the distribution of the C-D SRS signal from d31-PA resembles the SRS signal at $2845{\mathrm{cm}}^{-1}$ of ${\mathrm{CH}}_2$ vibration mainly from lipids. Hence, in epithelial cells, d31-PA is actively taken up by cells and incorporated into membrane lipids, reflecting fast membrane synthesis in rapidly proliferating epithelial cells from the scavenging pathway. In mesenchymal cells, membrane C-D SRS is weaker compared with epithelial cells. Moreover, C-D appeared in the bright lipid droplets (indicated by green arrows). These droplets colocalize with lipid droplets in the ${\mathrm{CH}}_2$ channel (indicated by green arrows). SRS intensity analysis of individual cells reveals that, when not considering lipid droplets, d31-PA incorporation into membrane lipids is decreased by around 20% during EMT. However, when lipid droplets are included, the overall intracellular C-D derived from d31-PA increased by around 20% during EMT. Therefore, our data revealed a previously unknown phenomenon: after EMT, membrane synthesis from scavenged free fatty acid is decreased likely due to the decreased cell proliferation, but the overall fatty acids uptake is increased, and the majority of it is converted to triglyceride (which is the major component of lipid droplets) and stored as energy in the form of lipid droplets.

4.

Discussion and Conclusion

Epithelial–mesenchymal transition is a critical step in cancer progression and metastasis. While the metabolism change during EMT has been studied through analysis of mRNA and protein levels of key metabolic enzymes, direct microscopic imaging of metabolism at the single-cell level is challenging, primarily due to lack of imaging probes. By using metabolites that are labeled with distinct vibration tags, here we are able to visualize the metabolism of various small metabolites such as fatty acid, amino acids, glucose, and choline. We found that the incorporation rates of amino acids, choline, and glucose are all decreased by various amounts after EMT. These results indicate the need of mesenchymal cells to restrict biosynthesis of proteins and lipids (which consumes energy) and to preserve energy for its migration and invasion.

Our chemical imaging approach also revealed previous unknown information. The change of glucose metabolism during EMT is not only in its overall incorporation into biomass but also where it is incorporated into (Fig. 5). In epithelial cells, C-D from D7-Glc shows homogeneous distribution across cytoplasm, indicating its incorporation into proteins and membrane lipids. In mesenchymal cells, de novo synthesis from glucose is severely reduced, reflecting the stalled proliferation of mesenchymal cells. However, synthesis of triglycerides in lipid droplets from glucose is obviously increased relative to epithelial cells. Lipid droplet is a form of energy storage, and our result indicates that mesenchymal cells store more energy, and one way to do it is through de novo lipogenesis from glucose. Along a similar line, d31-PA metabolism changes not only quantitatively but also qualitatively (Fig. 6). Epithelial cells mainly build free palmitic acids into membrane lipids, reflecting its needs for fatty acid to sustain fast proliferation. Mesenchymal cells uptake even more fatty acid, although it does not need fatty acid for lipid synthesis. The uptaken fatty acids are not built into membrane but into lipid droplets. Together, the direct imaging approach taken in this study is indispensable in unraveling this microscopic information at subcellular level.

Regarding lipid droplets, here we observed their accumulation in mesenchymal cells, both from the de novo lipogenesis pathway (Fig. 5) and from the fatty acid scavenging pathway (Fig. 6). Recently, the lipid droplet has emerged as an important player in cancer biology.^40–42 Accumulation of lipid droplets was also observed in prostate cancer cell EMT.⁴³ More malignant tumor tissues tend to accumulate more lipid droplets.^40,44 Inhibition of fatty acid synthase reverses the EMT and malignancy of breast cancer and glioblastoma cancer.^45,46 Together, it seems that lipid droplets might play important functions in maintaining malignancy and mesenchymal phenotype of cancer cells.