|Year : 2016 | Volume
| Issue : 3 | Page : 79-84
Hyaluronic Acid in Normal and Neoplastic Colorectal Tissue: Electrospray Ionization Mass Spectrometric and Fluor Metric Analysis
Ana Paula Cleto Marolla1, Jaques Waisberg2, Gabriela Tognini Saba2, Demétrius Eduardo Germini2, Maria Aparecida da Silva Pinhal1
1 Department of Biochemistry, Federal University of São Paulo, São Paulo, SP, Brazil
2 Department of Surgery, State Public Servant Hospital, São Paulo, SP, Brazil
|Date of Submission||23-Mar-2016|
|Date of Acceptance||27-May-2016|
|Date of Web Publication||21-Jun-2016|
Dr. Jaques Waisberg
Department of Surgery, State Public Servant Hospital, Rua Pedro de Toledo, 1800 - Vila Clementino, São Paulo 04029-000, SP
Source of Support: None, Conflict of Interest: None
Aim: The aim of the study was to analyze the expression of hyaluronic acid (HA) and its type in human colorectal cancer (CRC) and non-neoplastic mucosa tissues.
Methods : The study included 64 adult CRC patients, who met all the inclusion and exclusion criteria. Two tissue samples (neoplastic and non-neoplastic mucosa) from each patient's large intestine were collected and were subjected to electrospray ionization mass spectrometry (ESI-MS) and fluorimetric assays.
Results: The analysis of colorectal neoplastic tissue and non-neoplastic mucosa by ESI-MS allowed the identification of HA and its oligosaccharide fragments. Low molecular weight (LMW), nonbiotinylated isoform of HA, and its fragments were identified in both neoplastic and non-neoplastic mucosa. The expression of HA was found to be slightly lower in tumor tissue (0.561 mg HA/g tissue) than in colorectal non-neoplastic mucosa (0.579 mg HA/g tissue), although the difference was not statistically significant (P = 0.87). This result was probably influenced by the nonbiotinylated LMW-HA. For the specific group of patients that did not present lymph node metastasis, the average HA levels were higher in tumor tissue (0.674 mg HA/g tissue) than in non-neoplastic tissue (0.529 mg HA/g tissue), which managed to reach statistical significance (P = 0.04). For the group of patients with lymph node involvement, no difference between tumor and nontumor tissue was observed. The HA expression among the tumor tissues within the variables of each clinicopathological parameters assessed failed to elicit any significant difference (location, P = 0.62; size, P = 0.13; lymph node invasion, P = 0.57; degree of cellular differentiation, P = 0.46; venous infiltration, P = 0.73; lymphatic infiltration, P = 0.36; neural infiltration, P = 0.28; tumor node metastasis classification, P = 0.15; and presence of synchronous metastases, P = 0.35; initial versus advanced stage, P = 0.37).
Conclusions: The expression of HA was found to be slightly lower in tumor tissue than in colorectal non-neoplastic mucosa, although this difference was not statistically significant. This finding probably influenced the lower expression of HA in tumor tissue than in colorectal non-neoplastic mucosa. Compared to normal tissues, HA levels are significantly increased in the tumor tissues unless they exhibit lymph node metastasis. Otherwise, the expression of HA in tumor tissue did not correlated with the other clinicopathological parameters.
Keywords: Colorectal neoplasms, extracellular matrix, hyaluronic acid, mass electrospray ionization, spectrometry
|How to cite this article:|
Marolla AP, Waisberg J, Saba GT, Germini DE, Pinhal MA. Hyaluronic Acid in Normal and Neoplastic Colorectal Tissue: Electrospray Ionization Mass Spectrometric and Fluor Metric Analysis. Cancer Transl Med 2016;2:79-84
|How to cite this URL:|
Marolla AP, Waisberg J, Saba GT, Germini DE, Pinhal MA. Hyaluronic Acid in Normal and Neoplastic Colorectal Tissue: Electrospray Ionization Mass Spectrometric and Fluor Metric Analysis. Cancer Transl Med [serial online] 2016 [cited 2018 Oct 23];2:79-84. Available from: http://www.cancertm.com/text.asp?2016/2/3/79/184319
| Introduction|| |
Colorectal cancer (CRC) is one of the most common causes of cancer-related deaths worldwide, a considerable mortality of which is due to metastasis to other organs, mainly the liver. 
The cancer microenvironment and the interactions between cancer cells and surrounding tissue play a pivotal role in tumor development and progression. , Cancer invasion and progression involves the motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. 
Proteoglycans and glycosaminoglycans (GAGs), which are the major constituents of the ECM, impact various cellular functions.  They have unique structural characteristics that enable specific interactions with the matrix proteins and cell surface receptors to regulate key cell functions and affect cancer growth and progression. ,
Hyaluronic acid (HA) or hyaluronan is a widespread polysaccharide that is found on the cell surface and in the ECM of almost every human tissue.  HA is an anionic, nonsulfated GAG composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. ,
HA is synthesized in the plasma membrane by the enzyme HA synthase and extruded into the ECM.  Normally, elevated concentrations of HA are associated with the physiological tissue remodeling process characterized by rapid cellular proliferation, as in healing wounds and fetal development. ,
HA is a major component of the pericellular matrix surrounding tumor cells, including colorectalcarcinomas.  HA is known to facilitate tumor progression by enhancing cell adhesion, growth, invasion, angiogenesis, and metastasis. ,,,, HA synthesis is regulated by the surrounding stromal fibroblasts producing openings through connective tissue and cancer cells, thus facilitating tumor dissemination. , However, whether HA oligosaccharides have a role in tumor progression currently remains uncertain due to an inability to analyze their concentration in tumors. , Furthermore, there is an imperative goal to highlight potential therapeutic targets in the rapidly developing field of glycan-based therapy. ,
Mass spectrometry (MS) is an analytical technique that is used to identify unknown compounds, quantify known materials, and elucidate the chemical and structural properties of molecules, based on the generation of ions that are subsequently detected.  This approach facilitates an appreciation of how changes in the GAG structure can regulate physiological as well as pathological processes. , Further, MS and gel electrophoresis procedures can be used to establish the length of an oligosaccharide chain and the presence of specific functional groups.  Sample preparation for these sensitive techniques often requires enzymatic treatments to produce oligosaccharide sequences for subsequent analysis. 
There are two techniques for ionizing large biological molecules. First, matrix-assisted laser desorption ionization, a MS technique that is based on desorption and the laser ionization of proteins aided by the matrix, which analyzes the mass through the flight time of ion analysis of the tube (time of flying [ToF]). The other technique is electrospray ionization (ESI)-MS, a technique for ionizing large biological molecules that use MS based on ionization by electric pulses in a liquid medium. ,,
The fluorimetric technique is an analytical process for identifying and characterizing minute amounts of the substance by the excitation with a beam of ultraviolet light while detecting and measuring the characteristic wavelength of the fluorescent light that is being emitted.  A fluorimeter is a device that is used to measure the parameters of fluorescence; its intensity and wavelength distribution of its emission spectrum after excitation by a certain spectrum of light. These parameters are used to identify the presence and the amount of specific molecules in a medium. ,
The HA is excessively synthesized in cancer; increased HA serum levels and deposition in tumor tissue are often associated with the malignant progression of CRC. ,,, Constitutive interactions between HA and CD44 on tumor cells induce various anti-apoptotic cell survival pathways through the formation of a signaling complex that contains activated receptor tyrosine kinases. ,,
The aim of the present study is to analyze the expression level of HA in surgically excised human CRC and non-neoplastic mucosa tissues. ESI-MS and fluorimetric technique was used as the tool to achieve this aim.
| Methods|| |
All the procedures, involving human participants, performed in the study were in accordance with the ethical standards of the Institutional and/or National Research Committee, the Declaration of Helsinki, and its later amendments or comparable ethical standards.
This work is an observational, longitudinal, prospective study on patients who underwent resection of a sporadic colorectal carcinoma.
The inclusion criteria were adult patients of both genders, with sporadic CRC confirmed by histopathological examination of the resected tumor. Exclusion criteria were the presence of other concurrent or previous malignancy in any other site, neoadjuvant treatment, presence of concomitant inflammatory bowel disease, and those patients presenting hereditary CRC syndromes.
The following clinical, anatomical, and pathological data were collected from the medical records of the patients: clinical characteristics (age, gender, and ethnicity), macroscopic tumor characteristics (location and size), microscopic characteristics (lymph node invasion, degree of cellular differentiation and venous and lymphatic and neural infiltration), tumor node metastasis (TNM) classification, and presence of synchronous metastases. The preoperative tumor staging was based on the results of colonoscopy, rigid proctosigmoidoscopy, carcinoembryonic antigen (CEA) plasma levels, magnetic resonance imaging of the pelvis, and helical computed tomography of the abdomen and chest. The follow-up procedures performed after the diagnosis were clinical history and pertinent physical examination for every 3-6 months for the first 3 years and annually thereafter; serum CEA testing for every 2-3 months in patients with Stage II or III disease for ≥ 2 years; and colonoscopy for every 3-5 years. The average follow-up time was 39.1 months (range: 9-72 months).
The tissue samples were obtained from 64 adult patients who underwent a surgical resection of sporadic CRC between 2005 and 2006. Thirty-seven (57.8%) patients were female and 27 patients (42.2%) were male. The mean age was 68.5 ± 7.3 years (ranging from 44 to 81 years). All the patients were Caucasians. The CRC was situated in the rectum in 32 (50.0%) patients and in the colon in another 32 (50.0%) patients, 17 (53.1%) of them were located in the right colon and the remaining 15 (46.9%) in the left colon.
Regarding the CRC, macroscopic (location and size)/microscopic (degree of tumor differentiation, level of invasion, lymphatic/vascular invasion, perineural infiltration, and lymph node involvement) characteristics and TNM classification of UICC (2010) were obtained.
Two colorectal tissue samples were obtained from each patient for the ESI-MS and fluorimetric assays; one sample was obtained from the representative macroscopic area of the CRC and another from the non-neoplastic colorectal mucosa that was located 10 cm cranially from the tumor.
The neoplastic and non-neoplastic tissues were minced and homogenized in two volumes of acetone for 24 h, and the acetone was changed four times. These homogenates were kept at room temperature for 48 h. The material was centrifuged at 1300 g for 20 min, discarding the supernatant and drying the precipitate under vacuum. The dry powder obtained from the tissue was weighed and submitted to proteolysis in the presence of maxatase (Biocon, Rio de Janeiro, RJ, Brazil) (2 mg/100 mg of dry tissue) in 0.05 M Tris-HCl buffer, pH 8.0, containing 0.15 M NaCl at a ratio of 10 mL of enzyme solution per 1 g of dry tissue. Trichloroacetic acid (10% end concentration) was added to the mixture and maintained at 4°C for 15 min after incubation. After approximately 48 h at 60°C, the nucleic acids and proteins that remained in the solution were precipitated in an ice bath by adding TCA (10% end concentration). After 10 min, the solution was centrifuged (1300 g, 15 min) to remove the precipitate. Two volumes of methanol were added to the supernatant slowly and with constant stirring for the precipitation of GAGs. The amount of GAGs containing supernatant after centrifugation was obtained (10 min, 3500 g, 4°C). GAGs were precipitated by adding 2 volumes of methanol (24 h, −20°C). The precipitate was collected by centrifugation (20 min, 3500 g, 4°C), dried, and dissolved in distilled water (50 mL of water per g of acetone powder). An aliquot of this solution was separated, dried under vacuum, and suspended in deoxyribonuclease solution to inactivate the nucleic acids.
The studies of ESI-MS were conducted at the Brazilian Synchrotron Light Laboratory (Campinas, São Paulo, Brazil) using the Micromass Q-ToF instrument (Micromass Inc., Milford, MA, USA) to detect the presence of HA in the CRC and non-neoplastic tissue samples. The composition of the electrospray solvent was acetonitrile-phosphoric acid, and the corresponding flow rate was 5 μL/min.
The HA obtained from the tissues was degraded with specific enzymes and was diluted in acetonitrile phosphoric acid (1:1), centrifuged at 1400 g, injected into the ESI-MS apparatus with a mass-load ratio of 4 kDa, and exposed to nebulization. The device was programed to act in a negative way, that is, by selecting only negative ions with energy in the cone of 80 V and subjected to a flow rate of 5 μL/min.
HA and its degradation products were analyzed by electrophoresis on agarose gel in a 1,3-diaminopropane-acetate 0.05 M, pH 9.0 buffer. After electrophoresis (5 V/cm, for 1 h), the blade was immersed in 0.1% cetrimide solution for the precipitation of HA for a minimum of 2 h. The samples were applied to agarose gel blades, and then they were subjected to electrophoresis (5 V/cm for 1 h) in a box cooled to 5°C. Since these compounds have an anionic charge, the origin of the gel corresponded to the negative pole.
The gel was dried under hot air stream and artificial lighting. After 90 min, the compounds were stained with toluidine blue solution, and the excess was removed with a solution of 1% acetic acid and 50% ethanol.
The dosage of HA was performed by the fluorimetric method. The HA samples from proteolysis of the colorectal tissue were diluted in Tris-HCl 0.05M buffer, pH 7.75, containing 0.15M NaCl and 1% bovine serum albumin. The solutions were applied in triplicates to an ELISA plate reader (Model ELX 800, Universal Microplate Reader, BioTek Instruments Inc., Highland Park, WI, USA) that was previously treated with a HA probe represented by the globular portion together with the binding protein, named link protein, obtained from bovine nasal cartilage. After incubation for 12 h at 40°C, the plate was washed six times with wash buffer (Tris-HCl) and incubated with a biotinylated HA probe. The plates were shaken for 2 h at room temperature, followed by washing with Tris-HCl. The revelation was performed by incubating with streptavidin (PerkinElmer, Wallac Oy, Turku, Finland) labeled with europium for 30 min under agitation. Again, the plate was washed with Tris-HCl and then incubated with enriching solution (PerkinElmer, Wallac Oy, Turku, Finland) to release the europium retained on the plate. Europium fluorescence was measured by fluorimeter, and the concentration of HA was determined by using the fluorimetric-specific program for that system.
The values obtained in the study of each continuous variable were organized and expressed as mean and standard deviations. Absolute and relative frequencies were used for categories. Statistical associations were determined by using the Student's t-test. The statistical significance level adopted was 5% (P < 0.05), and the results were analyzed using SPSS™ software (Statistical Package for Social Sciences, SPSS Inc., Chicago, IL, USA), version 15.0.
| Results|| |
The diameter of the tumor was ≥ 5 cm in 32 (50%) patients and < 5 cm in the remaining 32 (50%), 32 (50%) patients had lymph node metastases, 20 (31.2%) had venous invasion, 20 (31.2%) had lymphatic vessel invasion, and 10 (17.1%) had perineural infiltration. Regarding the degree of tumor differentiation, 52 (81.2%) neoplasms were classified as moderately differentiated, 11 (17.2%) as well differentiated, and 1 (1.6%) as poorly differentiated. The depth of tumor invasion was T3 in 44 (68.7%), T2 in 18 (28.1%), and T4 in 2 (3.1%) tumors. Recurrence of the CRC occurred in 15 (23.4%) patients, and 8 of them (12.5%) passed away due to the neoplasia (an average follow-up of 39.1 months).
The analysis of colorectal neoplastic tissue and non-neoplastic mucosa by ESI-MS allowed the identification of HA and its oligosaccharide fragments [Figure 1].
|Figure 1. Electrospray ionization mass spectrometry of colorectal tissue showing the structures of hyaluronic acid and its degradation into oligosaccharide fragments|
Click here to view
In general, the expression of HA was found to be slightly lower in tumor tissue (0.561 mg HA/g tissue) than in colorectal non-neoplastic mucosa (0.579 mg HA/g tissue), although this difference was not statistically significant (P = 0.87). However, for the specific group of patients that did not present lymph node metastasis, the average HA levels were higher in tumor tissue (0.674 mg HA/g tissue) than in nonneoplastic tissue (0.529 mg HA/g tissue), which managed to reach statistical significance (P = 0.04). For the group of patients with lymph node involvement, no difference between tumor and nontumor tissue was observed [Table 1].
|Table 1: Concentration of hyaluronic acid in the non-neoplastic and tumor tissues in the presence or absence of lymph node metastasis|
Click here to view
The HA expression among the tumor tissues within the variables of each clinicopathological parameters assessed failed to elicit any significant difference (location, P = 0.62; size, P = 0.13; lymph node invasion, P = 0.57; degree of cellular differentiation, P = 0.46; venous infiltration, P = 0.73; lymphatic infiltration, P = 0.36; neural infiltration, P = 0.28; TNM classification, P = 0.15; and presence of synchronous metastases, P = 0.35).
Forty-two (65.6%) patients belonged to the initial stage of cancer and the remaining 22 (34.4%) to the advanced stage. There was no statistically significant (P = 0.37) correlation between the stage of cancer and the expression of HA.
Electrophoresis in Tris-acetate gel was used to verify the HA profile which confirmed the presence of low molecular weight HA (LMW-HA) in both colorectal neoplastic tissue and non-neoplastic mucosa.
| Discussion|| |
Especially in human cancers, HA concentrations are usually higher in malignant than in normal tissues. CRC is considered to be enriched with HA and it may support tumor growth by stimulating anchorage-independent growth and proliferation of tumor cells. , Moreover, HA may also actively promote tumor cell adhesion, migration, and metastasis and may also protect against immune surveillance. , Furthermore, tumor cells may take an advantage of HA-rich ECMs to invade more easily into the surrounding tissues. ,
In the present study, the analysis of colorectal neoplastic tissue and non-neoplastic mucosa by ESI-MS allowed the identification of HA and its oligosaccharide fragments. Samples were subjected to ESI-MS to verify the presence of HA and to characterize its degradation into oligosaccharide fragments. The fragmentation obtained with the ionization of samples corresponds to those described in the literature.  However, whether these small HA oligosaccharides have a role in tumor progression currently remains uncertain.  On the other hand, it was verified that cancer cells covered by HA coat may be protected from cytotoxic cells  or chemotherapeutic agents.  Thus, the measurement of HA tumors' content may be useful for the identification of a subcategory of patients whose tumors may be chemoresistant. 
The stroma of the majority of malignant tumors, including CRC, has been shown to contain more than the standard level of HA content in analogous to non-neoplastic tissues. , In advanced malignancies, it has been suggested that raised levels of HA may be the consequence of blocked lymphatic drainage, which is the route of clearance of the tissue's HA. , In a cohort of 72 CRC patients, Schmaus et al. found that increased HA and its oligosaccharide fragments concentrations are associated with lymphatic vessel invasion by tumor cells and the formation of lymph node metastasis. This was the opposite of the results found in the present study, where, in patients without lymph node involvement, the average HA expression was significantly higher in tumor tissue than in non-neoplastic tissue, whereas in patients with lymph node involvement, no difference between tumor and nontumor tissue was observed.
In general, HA is increased in tumors. , However, in the present study, there was no statistically significant difference in the expression of HA between colorectal carcinoma tissue and non-neoplastic mucosa. There are two kinds of HA: LMW-HA and high molecular weight HA (HMW-HA). The result of the expression of HA in the colorectal carcinoma tissue and non-neoplastic mucosa obtained in the present study may be due to the fact that the peaks found in the mass spectrum and gel electrophoresis refer, at least in part, to the LMW-HA. This finding supports the assertion that the quantification of HA was influenced by the nonbiotinylated LMW-HA as the biotinylated binding protein that was used in the process is not fully connected to the LMW-HA present in tumor tissues. In the present study, the expression of HA was found to be lower in tumor tissue than in colorectal non-neoplastic mucosa, although this difference was not statistically significant. This result was probably influenced by the nonbiotinylated LMW-HA.
HMW-HA is indicative of healthy tissues, while LMW-HA seems to promote angiogenesis and activate signaling pathways that are critical for cancer progression. , The LMW-HA fragments could be truncated products of the synthetic reaction but could also be the result of hyaluronidase activity,  which could contribute to the lower, although not significant, HA expression in tumor tissue than in colorectal non-neoplastic mucosa found in this series.
Oligosaccharides of degraded HA have been credited with a number of biological functions that are not exerted by HMW-HA.  Electrophoresis in Tris-acetate gel was used to verify the HA profile found in the presence of LMW-HA in the colorectal neoplastic tissue and non-neoplastic mucosa. Alaniz et al. have shown that LMW-HA, but not HMW-HA, was found to significantly reduce the CRC growth in vitro and in vivo. The elicited response is partially mediated by activated dendritic cells. , These findings suggest that LMW-HA in the model of CRC triggers an immune system activation that is likely involved in the inhibition of tumor growth. Thus, LMW-HA was proposed as a candidate molecule for therapeutic adjuvant treatments in CRC immunotherapy. ,
Physiologically, HA is turned over continuously in a process, in which initial intermediate-sized fragments produced extracellularly are internalized and further degraded.  Extracellular HA degradation is affected by secreted or membrane-bound hyaluronidases.  Although HA fragments are normally rapidly cleared, increased synthesis and breakdown of HA in the tumor context could potentially lead to the accumulation of small HA oligosaccharides in the interstitial fluid.  In experimental systems, HA oligosaccharides have a wide range of biological activities that are not exerted by HMW-HA. 
The technique of identification of HA performed in our assays found the presence of LMW-HA that this method depends on the interaction of HA with the binding protein, and such interaction is dependent on the size of HA. In addition, peaks found in the MS also referred to LMW-HA. Thus, the measurement of HA may not have been efficient for its expression in the analysis because the biotinylated binding protein used in the process may not be linked to its maximum efficiency when LMW-HA is present in the tissue tumor.
This study also suggests the potential of ESI-MS as an analytical tool to study the interactions of highly heterogeneous GAGs such as HA. The broad applicability of this powerful platform offers an insight into how changes in the cell surface and extracellular GAG composition and sequence influence the ability of cells and tissues to dynamically alter the responses to signaling molecules. ECM-based scaffolds may also be beneficial for future studies seeking prognostic and diagnostic stromal markers and targets for antineoplastic drugs. Thus, this approach provides a window into understanding how changes at the molecular level manifest with respect to cellular phenotype.
Financial support and sponsorship
This study was partially supported and financed by the Coordination for the Improvement of Education Personnel (CAPES, Brazil) (Project No. Unifesp2006pgcm).
Conflicts of interest
There are no conflicts of interest.
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