Hypoxic Tumor Microenvironments Reduce Collagen I Fiber Density

Abstract

Although the mechanisms through which hypoxia influences several phenotypic characteristics such as angiogenesis, selection for resistance to apoptosis, resistance to radiation and chemotherapy, and increased invasion and metastasis are well characterized, the relationship between tumor hypoxia and components of the extracellular matrix (ECM) is relatively unexplored. The collagen I (Col1) fiber matrix of solid tumors is the major structural part of the ECM. Col1 fiber density can increase tumor initiation, progression, and metastasis, with cancer cell invasion occurring along radially aligned Col1 fibers. Here we have investigated the influence of hypoxia on Col1 fiber density in solid breast and prostate tumor models. Second harmonic generation (SHG) microscopy was used to detect differences in Col1 fiber density and volume between hypoxic and normoxic tumor regions. Hypoxic regions were detected by fluorescence microscopy, using tumors derived from human breast and prostate cancer cell lines stably expressing enhanced green fluorescent protein (EGFP) under transcriptional control of the hypoxia response element. In-house fiber analysis software was used to quantitatively analyze Col1 fiber density and volume from the SHG microscopy images. Normoxic tumor regions exhibited a dense mesh of Col1 fibers. In contrast, fewer and structurally altered Col1 fibers were detected in hypoxic EGFP-expressing tumor regions. Microarray gene expression analyses identified increased expression of lysyl oxidase and reduced expression of some matrix metalloproteases in hypoxic compared with normoxic cancer cells. These results suggest that hypoxia mediates Col1 fiber restructuring in tumors, which may impact delivery of macromolecular agents as well as dissemination of cells.

Extracellular matrix and cell signalling: the dynamic cooperation of integrin, proteoglycan and growth factor receptor

“…Collagen is the major structural component in the formation of solid tumours and collagen fibre density can contribute to tumour initiation, invasion and metastasis (Gao et al 2010, Kakkad et al 2010. It has been shown that the increased expression of lysyl oxidase (LOX), an enzyme that covalently crosslinks collagen and elastin, is responsible for excess collagen deposition and hypoxiainduced metastasis of lung cancer through induction of b1 integrin signalling (Gao et al 2010).…”

“…It has been shown that the increased expression of lysyl oxidase (LOX), an enzyme that covalently crosslinks collagen and elastin, is responsible for excess collagen deposition and hypoxiainduced metastasis of lung cancer through induction of b1 integrin signalling (Gao et al 2010). Kakkad et al (2010) also report that hypoxia causes reduction of collagen I fibre density and restructuring of the ECM, thus contributing to the tumour cell dissemination.…”

Collagen I fiber density increases in lymph node positive breast cancers: pilot study

“…Snapfrozen primary breast tumor specimens were placed in Tissue Tek OCT freezing compound (Sakura Finetek USA), cryosectioned with a microcryotome (Microm International) at 100-μm thickness for whole-mount sections and 5-μm thickness for adjacent sections, fixed with 4% paraformaldehyde (Sigma-Aldrich) solution, stained for nuclei with Hoechst 33342 (Invitrogen, Carlsbad, CA), and mounted with Faramount aqueous mounting medium (DakoCytomation) as previously described. 19 For testing the within-tumor consistency of SHG-detected Col1 interfiber distances and Col1 fiber volume, we obtained FFPE samples from four different biopsy passes of the same breast cancer for four human specimens, which were grade 3,…”

“…3,4,18 We have performed a small retrospective SHG microscopy study to determine if the Col1 fiber signature in primary breast tumors is altered in patients presenting with lymph node metastasis (LN þ ) compared to patients with uninvolved lymph nodes (LN − ). We used our in-house automated fiber analysis software as previously described, 19 which is free of user variability, and observed that primary breast cancers in LN þ patients have significantly denser Col1 fiber signatures than primary breast cancer in LN − patients. To validate our in-house software results, we additionally used two commonly used texture analysis techniques to analyze Col1 images: [20][21][22] the gray level co-occurrence matrix (GLCM) and the Fourier transform (FT).…”

MiRNA and Proline Metabolism in Cancer

“…Although proline can be obtained from the dietary proteins, an important source of proline is from the degradation of collagen in the ECM by sequential enzymatic catalysis of matrix metalloproteinases (MMPs) and prolidase [9,23]. The upregulation of MMPs in tumors has been considered a critical step for tumor progression and invasion [24][25][26]. A number of reports have shown that proline concentration is increased in various tumors, which may result from the upregulated MMPs degrading collagen.…”