FLIM of NAD(P)H in monolayer cell cultures

(A) Representative FLIM images of colorectal cancer cell lines. Scale bar = 50 μm. For FLIM: ex. 750 nm, reg. 450–490 nm. (B) The relative contribution of free NAD(P)H (a1, %) in cell cultures. Box shows the median and the quartiles Q1 and Q3, whiskers show minimum and maximum. Dots indicate individual cells (n=280 for HT29 cells, n=185 for HCT116 cells, n=146 for CaCo2 cells, n=138 for CT26 cells). p-values are shown in Table S4. (C) The distribution of the NAD(P)H-a1 for the cell lines. The bimodality index (BI-a1) is shown on each diagram.

The Bimodality Index BI-a1 and Dispersity D-a1 of NAD(P)H in cultured cells, mouse tumors and patients’ tumor samples

FLIM of NAD(P)H in mouse tumors in vivo

(A) FLIM images of NAD(P)H of tumor cells in mouse models in vivo. Scale bar = 50 μm. For FLIM: ex. 750 nm, reg. 450– 490 nm. (B) Representative histological slices of tumors, hematoxylin/eosin (HE) staining, initial magnification 20×. Scale bar = 50 μm. (C) The relative contribution of free NAD(P)H (a1, %) in tumors (numbered 1–3) obtained from different cell lines. Box shows the median and the quartiles Q1 and Q3, whiskers show minimum and maximum. Dots indicate individual cells (n=280 for HT29, n=340 for HCT116, n=160 for CaCo2, n=350 for CT26). p-values are shown in Table S4. (D) Representative distributions of the NAD(P)H-a1 for each type of tumor. The bimodality index (BI-a1) is shown on the diagrams.

FLIM of NAD(P)H in patients’ tumor samples ex vivo

(A) Representative FLIM images of patient tumors. Scale bar = 50 μm. For FLIM: ex. 750 nm, reg. 450–490 nm. (B) Histopathology of tumors, hematoxylin/eosin (HE) staining, initial magnification 20×. Scale bar = 50 μm. (C) The relative contribution of free NAD(P)H (a1, %) in patients’ tumors (numbered 1–29). Box shows the median and the quartiles Q1 and Q3, whiskers show minimum and maximum. Dots are the measurements from the individual cells. (D) Representative distributions of the NAD(P)H-a1 for patients’ tumors. The bimodality index (BI-a1) is shown on the diagrams.

Information about patients and their colorectal tumors

The relationships between metabolic heterogeneity and clinicopathological characteristics of patients’ tumors

(A) Plots of SHAP analysis for the built decision tree models to determine the importance of dispersion (D) and bimodality index (BI) of the fluorescence decay parameters of NAD(P)H. The higher the value of the variable, the more red the dot is. (B) Box-plots of D-a1 with highest significance, * p-val < 0.05.

FLIM of NAD(P)H in monolayer cell cultures

The distribution of the NAD(P)H-τm for the cell lines. The bimodality index (BI-τm) is shown on each diagram.

The relationships between parameters BI-τm (A) and BI-a1 (B) and clinicopathological characteristics of patients’ tumors

No significant differences were found between the groups.

Immunohistochemical analysis of the expression of EpCAM (green, epithelial cells marker) and vimentin (red, mesenchymal cells marker) in tumors

(A) Immunofluorescence images of HT29, HCT116, CaCo2 and CT26 mouse tumors. Scale bar = 100 μm. (B) Immunofluorescence images of patients’ tumors (numbered from 6 to 21). Scale bar = 100 μm. (С) The ratio of EpCAM-positive to vimentin-positive areas in tumor sections. (D) The bimodality index (BI-a1) and dispersion (D-a1) plotted against the EpCAM/vimentin ratio in patient tumor samples. Pearson correlation r is shown on the plots.

Immunohistochemical analysis of the expression of GLUT3 and LDHA in patients’ tumors

(A) Representative immunohistochemical images of GLUT3 expression. Scale bar = 50 μm (magnification x200) and 20 μm (magnification x630). (B) Representative immunohistochemical images of LDHA expression. Scale bar = 50 μm (magnification x200) and 20 μm (magnification x630). (С) Semi-quantitative evaluation of the expression level by staining intensity.

Autofluorescence of cofactors FAD and NAD(P)H

Representative fluorescence intensity images of FAD (ex. 900 nm, em. 500–550 nm) and NAD(P)H (ex. 750 nm, em. 450–490 nm) in cultured cells (A), tumor xenografts in vivo (B) and patient tumor ex vivo (№ 11) (C). Scale bar = 50 μm. D – The number of photons per pixel recorded by FLIM for flavins in ex vivo patient samples. Mean±SD, n=7 patients.

NAD(P)H fluorescence decay parameters of colorectal cancer cells in monolayer cultures in vitro and in mouse tumors in vivo

τm – mean lifetime, τ1 – short lifetime component, τ2 – long lifetime component, a1 – relative contribution of the short lifetime component, BI-τm – bimodality index of the mean lifetime.

Clinicopathological characteristics of patients’ tumors

NAD(P)H fluorescence decay parameters of patients’ tumors ex vivo

Statistical significance of the differences of NAD(P)H a1-% between different cell lines and tumors (p-values)