Lipid droplets: a new player in colorectal cancer stem cells unveiled by spectroscopic imaging

Abstract

The cancer stem cell (CSC) model is describing tumors as a hierarchical organized system and CSCs are suggested to be responsible for cancer recurrence after therapy. The identification of specific markers of CSCs is therefore of paramount importance. Here, we show that high levels of lipid droplets (LDs) are a distinctive mark of CSCs in colorectal (CR) cancer. This increased lipid content was clearly revealed by label-free Raman spectroscopy and it directly correlates with well-accepted CR-CSC markers as CD133 and Wnt pathway activity. By xenotransplantation experiments, we have finally demonstrated that CR-CSCs overexpressing LDs retain most tumorigenic potential. A relevant conceptual advance in this work is the demonstration that a cellular organelle, the LD, is a signature of CSCs, in addition to molecular markers. A further functional characterization of LDs could lead soon to design new target therapies against CR-CSCs.

Keywords: Colorectal cancer stem cells; Lipid droplets; Raman spectroscopy; Wnt/β-catenin pathway.

Figure 1

Raman characterization and mapping of colon cell samples. (A): Two different cell regions, indicated as * and **, respectively, can be clearly identified in the cell according to their Raman spectra. The Raman differences are due to four main peaks located at 1,300, 1,440, 1,740, and 2,850 cm⁻¹. Typical spectra from region * (top curve) and region ** (bottom curve) show that region * has higher expression of all these aforementioned peaks compared to region **, as clearly highlighted by the Raman difference spectrum ***; brightfield image of a CR-CSC and Raman imaging at 2,850 cm⁻¹ of the same cell are reported, highlighting the two different regions. (B): From the top row to the bottom one: NECCs, CR-CSCs, SDACs, and CCCs. Brightfield images are reported on the first column, while Raman images calculated at 1,300 and 2,850 cm⁻¹ are reported in the second and third column, respectively. The fourth column shows spectra averaged over the whole cell area, for each cell line. Raman images in the second column are similar to the corresponding ones of the third column, thus revealing that the two Raman modes at 1,300 and 2,850 cm⁻¹ are space-correlated. Peaks related to lipidic vibrations are more pronounced in the CR-CSCs (second row) compared to SDACs and CCCs, as it is evident both from Raman images and spectra. NECCs reported in the first row express the lowest Raman intensities of lipidic vibrations. Abbreviations: CCC, colon carcinoma cell; CR-CSC, colorectal cancer stem cell; NECC, normal epithelial colon cell; SDAC, sphere-derived adherent cell.

Figure 2

Lipid droplet quantification. (A): Comparison of typical z-projected confocal fluorescence images of the investigated cell lines stained with BODIPY 493/503. The lipid droplet content on CR-CSCs is higher compared to all the other cell lines. (B): Histograms overlay for flow-cytometry BODIPY fluorescence measurements regarding three of the CR-CSC (red) lines and their SDACs (black). Abbreviations: CCC, colon carcinoma cell; CR-CSC, colorectal cancer stem cell; NECC, normal epithelial colon cell; SDAC, sphere-derived adherent cell.

Figure 3

Transmission electron microscopy (TEM) to reveal lipid droplets on colorectal cells. (A–F): TEM images of the CR-CSCs, their differentiated forms (SDACs), CCCs, and NECCs. (A and B), parasagittal sections of (A) CR-CSCs and (B) SDACs belonging to human patient 1; (C and D), parasagittal sections of (C) CR-CSCs and (D) SDACs belonging to human patient 2; (E), parasagittal section of a CCC; (F), parasagittal section of a NECC. White arrowheads point to mitochondria; black arrowheads point to multivesicular bodies and late-endosome/lysosome hybrids. LDs are colored in red. n, nucleus. (G): TEM image of a LD in cross-section. The asterisk points to the endoplasmic reticulum. Inset: detail of the LD single membrane leaflet. (H): Volume fraction of LDs in the cytoplasm and in the whole cell. Error bars, SEM. Statistical significance is denoted by * (p ≤ .01, Student's t test). Scale bars are 4 µm for A–F and 100 nm for G. Abbreviations: CCC, colon carcinoma cell; CR-CSC, colorectal cancer stem cell; NECC, normal epithelial colon cell; SDAC, sphere-derived adherent cell.

Figure 4

Correlation of the expression levels of the Lipid Droplets with CD133 and Wnt/β-catenin. (A, B): The expression of the LDs in CD133^High and CD133^Low cells were analyzed by flow cytometry. Cells were stained with an anti-CD133 APC-conjugated and then with BODIPY 493/503. Both CD133^High samples (A and B red lines) have a higher expression of LDs compared to the CD133^Low (black lines). (C): Schematic representation of the TOP-GFP Wnt construct. (D, E): Cells were sorted for GFP expression (GFP^High and GFP^Low) and then stained for LDs with LD540 dye; both TOP-GFP samples have the same behavior showing as Wnt/β-catenin pathway expression clearly correlates with LDs quantity. Abbreviations: APC, allophycocyanin; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; TOP, TCF Optimal Promoter (TOP).

Figure 5

Clonogenic assay of CR-CSC LDs^High/Low subpopulations in vitro. Three different CR-CSC samples were tested for their clonogenic potential. (A): CR-CSCs were sorted for LDs^High and LDs^Low by fluorescence activated cell sorting for LDs using LD540 dye and then deposited 1, 2, 4, 8, 16, 32, 64, and 128 cells per well. (B): The estimated sphere-forming cells were analyzed using the extreme limiting dilution analysis as reported in the graph. All the three LDs^High cell samples have a significantly increased clonogenic potential compared to the LDs^Low cell samples. Error bars representing the SD of the mean of three independent experiments are shown. Significance is indicated (*, p < .05 and **, p < .01). Abbreviations: CR-CSC, colorectal cancer stem cell; LD, lipid droplet.

Figure 6

Cells with high lipid droplet content show cancer stem cell (CSC) tumorigenic features in vivo. (A): In vivo transfer of LD540 stained cultures. Different cell numbers from the indicated populations were injected into NOD/SCID mice after fluorescence-activated cell sorting. The percentage of successful tumor initiations after 17 weeks out of four injections for each condition is shown. The LDs^High fraction showed the highest tumor-initiating capacity (refer Fig. 6B for the kinetic of tumor appearance). (B): Time course of colon tumor growth after injection of 8,000 LDs^High or 8,000 LDs^Low CR-CSCs (tumor volumes were determined as described in Materials and Methods). (C): Representative picture of the xenotransplanted tumors. (D): Hematoxylin/eosin staining of xenografts shows clear evidence of colon carcinoma morphology, resembling the human parental phenotype. Abbreviation: LD, lipid droplet.