Increased microvascular blood content is an early event in colon carcinogenesis

Abstract

Background: Increased premalignant epithelial microvascular blood content is a common theme in neoplastic transformation; however, demonstration of this phenomenon in colon carcinogenesis has been stymied by methodological limitations. Our group has recently developed a novel optics technology, four dimensional elastic light scattering fingerprinting (4D-ELF), which allows examination of the colonic mucosal architecture with unprecedented accuracy. In this study, we utilised 4D-ELF to probe the preneoplastic colonic microvasculature. Methods: Colonic mucosal blood content was assessed by 4D-ELF at serial preneoplastic time points from azoxymethane (AOM) treated Fisher 344 rats and age matched control animals. We also examined the pretumorigenic intestinal mucosa of the MIN mouse, and compared with wild-type mice. Finally, in a pilot study, we examined superficial blood content from the endoscopically normal mid transverse colon in 37 patients undergoing screening colonoscopy. Results: In the AOM treated rat model, augmentation of superficial mucosal and total mucosal/superficial submucosal blood supply preceded the appearance of aberrant crypt foci (ACF) and temporally and spatially correlated with future ACF occurrence. These findings were replicated in MIN mice. The 4D-ELF based results were corroborated with immunoblot analysis for haemoglobin on mucosal scrapings from AOM treated rats. Moreover, 4D-ELF analysis of normal human colonic mucosa indicated that there was a threefold increase in superficial blood in patients who harboured advanced adenomas. Conclusion: We report, for the first time, that blood content is increased in the colonic microvasculature at the earliest stages of colon carcinogenesis. These findings may provide novel insights into early biological events in colorectal carcinogenesis and have potential applicability for screening.

Spectroscopic Microvascular Blood Detection From the Endoscopically Normal Colonic Mucosa: Biomarker for Neoplasia Risk

“…Importantly, this early increase in blood supply (EIBS) was confined to the regions where future neoplasia would develop. This was confirmed using conventional techniques (Western blot analysis for hemoglobin from colonic mucosal scrapings), although latter was markedly less sensitive than 4D-ELF 11 . While the biological mechanisms remain incompletely elucidated, studies in the AOM-treated rat implicate the role of nitric oxide synthase (iNOS) 12 .…”

“…Indeed, the only statistically significant difference for either OHb or DHb readings between controls (no neoplasia) and adenoma-harboring patients was at 100 µm. This tissue depth corresponded to the mucosal pericryptal capillary network obtained in the MIN mouse and AOM-treated rat using our bench-top instrument 11 . This supports the concept that only a fraction of the total colonic blood supply (the most superficial) is involved in the phenomena of EIBS thus underscoring the importance of polarization gating to selectively probe this area and avoid being overshadowed by blood from deeper tissues.…”

“…11,14 Briefly, we assumed minimal path length variability due to differences in optical properties. Therefore, the attenuation due to absorption has an inverse exponential relationship with the absorber concentration: (1) where I(λ) is one of the polarization components of the collected light (ΔI, I ‖ , or I ⊥ ), I scattering (λ) represents the scattering signal if the sample were devoid of absorbers.…”

“…For penetration depth, aqueous suspensions of microspheres with various scattering properties in the range of colon tissue (anisotropy coefficient g = 0.73-0.93, mean free path length ls = 90-289 µm) were used. [11][12][13][14] The polarization signals were collected from tissue phantoms of various thickness (~ 50 µm to 4 mm). Then, we estimated each representative penetration depth from ΔI, I ‖ , and I ⊥ as the average of optical thickness (optical thickness τ = ls*L; τ =1 represents one scattering event on average) by the rate of the increase of the polarization signals with tissue phantom thickness.…”

“…We have recently developed four dimensional elastic light scattering fingerprinting (4D-ELF) 10 which allows heretofore unprecedented quantification of the colonic mucosal microvasculature. Using this approach, we observed that mucosal microvascular blood content was increased in the histologically normal mucosa of two experimental models of colonic neoplasia, the azoxymethane (AOM)-treated rat and the MIN mouse 11 . These occurred prior to development of adenomas or even ACF.…”