Search

Precise diagnosis and prognosis are key in prevention and reduction of morbidity and mortality in all types of cancers. Here we show that changes in the collagen fibres in the main histological subtypes of canine mammary gland carcinomas are directly associated with the tumour behaviour and the animal survival time and could become a useful tool in helping with diagnosis. Imaging by second harmonic generation and multiphoton excited fluorescence microscopy were performed to evaluate the collagen and cellular segment parameters in cancer biopsies. We present a retrospective study of 45 cases of canine mammary cancer analysing 836 biopsies regions including normal mammary gland tissue, benign mixed tumours, carcinoma in mixed tumour, carcinosarcoma, micropapillary carcinoma and solid carcinoma. The image analyses and the comparison between the tumour types allowed to assess the collagen fibre changes during tumour progression. We demonstrate that the collagen parameters correlate with the clinical and pathological data, the results show that in neoplastic tissues, the collagen fibres are more aligned and shorter as compared to the normal tissues. There is a clear association of the mean fibre length with the dogs survival times, the carcinomas presenting shorter collagen fibres indicate a worse survival rate.

Many cancers adeptly modulate metabolism to thrive in fluctuating oxygen conditions; however, current tools fail to image metabolic and vascular endpoints at spatial resolutions needed to visualize these adaptations in vivo. We demonstrate a high-resolution intravital microscopy technique to quantify glucose uptake, mitochondrial membrane potential (MMP), and SO 2 to characterize the in vivo phentoypes of three distinct murine breast cancer lines. Tetramethyl rhodamine, ethyl ester (TMRE) was thoroughly validated to report on MMP in normal and tumor-bearing mice. Imaging MMP or glucose uptake together with vascular endpoints revealed that metastatic 4T1 tumors maintained increased glucose uptake across all SO 2 ("Warburg effect"), and also showed increased MMP relative to normal tissue. Non-metastatic 67NR and 4T07 tumor lines both displayed increased MMP, but comparable glucose uptake, relative to normal tissue. The 4T1 peritumoral areas also showed a significant glycolytic shift relative to the tumor regions. During a hypoxic stress test, 4T1 tumors showed significant increases in MMP with corresponding significant drops in SO 2 , indicative of intensified mitochondrial metabolism. Conversely, 4T07 and 67NR tumors shifted toward glycolysis during hypoxia. Our findings underscore the importance of imaging metabolic endpoints within the context of a living microenvironment to gain insight into a tumor's adaptive behavior.

Recent developments in optical tissue clearing and microscopic imaging have advanced three-dimensional (3D) visualization of intact tissues and organs at high resolution. However, to expand applications to oncology, critical limitations of current methods must be addressed. Here we describe transparent tissue tomography (T3) as a tool for rapid, three-dimensional, multiplexed immunofluorescent tumor imaging. Cutting tumors into sub-millimeter macrosections enables simple and rapid immunofluorescence staining, optical clearing, and confocal microscope imaging. Registering and fusing macrosection images yields high resolution 3D maps of multiple tumor microenvironment components and biomarkers throughout a tumor. The 3D maps can be quantitatively evaluated by automated image analysis. As an application of T3, 3D mapping and analysis revealed a heterogeneous distribution of programmed death-ligand 1 (PD-L1) in Her2 transgenic mouse mammary tumors, with high expression limited to tumor cells at the periphery and to CD31 + vascular endothelium in the core. Also, strong spatial correlation between CD45 + immune cell distribution and PD-L1 expression was revealed by T3 analysis of the whole tumors. Our results demonstrate that a tomographic approach offers simple and rapid access to high-resolution three-dimensional maps of the tumor immune microenvironment, offering a new tool to examine tumor heterogeneity.