Comparison of standard and HD FT-IR with multimodal CARS/TPEF/SHG/FLIMS imaging in the detection of the early stage of pulmonary metastasis of murine breast cancer.

Lungs, due to their high oxygen availability and vascularization, are an ideal environment for cancer cell migration, metastasis and tumour formation. These processes are directly connected with extracellular matrix (ECM) remodelling, resulting from cancer cell infiltration and preparation of the environment suitable for tumour growth. Herein, we compare the potential of fast, label-free and non-destructive methods of Fourier-transform infrared spectroscopy (FT-IR) in standard and high definition (HD) modes with nonlinear coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), two-photon excited fluorescence (TPEF) and a fluorescence lifetime imaging (FLIM) technique for lung metastasis detection. We show their potential in the detection of lung macrometastasis, in which we already observed the ECM remodelling. The CARS image revealed a dense cell fraction typical of ECM remodeling and reduction of the TPEF signal together with an increase of fluorescence lifetime predominantly due to NAD(P)H suggesting metabolic changes in the metastatic foci. FT-IR spectroscopy allowed not only for macrometastasis detection but also their stage definition based mainly on the analysis of proteins, RNA and glycogen fractions. The multimodal approach additionally suggested partial enzymatic degradation of elastin in ECM and collagen remodelling.