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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 15  |  Issue : 1  |  Page : 75-81

Detection of cancer stem cell-related markers in different stages of colorectal carcinoma patients of Indian origin by immunohistochemistry


1 Department of Biochemistry, Center of Excellence in Molecular Biology and Regenerative Medicine, JSS Medical College and Hospital, JSS Academy of Higher Education and Research, Mysore, Karnataka, India
2 Department of Surgery, JSS Medical College and Hospital, JSS University, Mysore, Karnataka, India
3 Department of Pathology, JSS Medical College and Hospital, JSS University, Mysore, Karnataka, India

Date of Web Publication13-Mar-2019

Correspondence Address:
Dr. Akila Prashant
Department of Biochemistry, Centre of Excellence in Molecular Biology and Regenerative Medicine, JSS Medical College, JSS Academy of Higher Education and Research, Mysore - 570 015, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_991_16

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 > Abstract 


Context: Although the incidence rate of colorectal cancer (CRC) in all Indian cancer registries is very close to the lowest rate in the world, westernization has shown an increasing trend in the recent years. Recurrence is reported in CRC because the slowly proliferating stem cells escape the chemotherapeutic regimen.
Aim: To detect the presence of CD133 and CD44 in human CRC specimens and to correlate the level of marker expression with tumor staging.
Materials and Methods: We included 26 colorectal carcinoma patients between 20 and 70 years of age. Histological and immunohistochemical analysis of CD133 and CD44 was done in sections of 5 μm prepared from paraffin-embedded blocks with most representative areas.
Statistical Analysis: All analyses were performed using Microsoft Excel 2010 and SPSS version 22.
Results: CD133 expression was seen exclusively on the cell membrane at the glandular luminal surface with dot-like cytoplasmic staining. In the normal mucosa, CD44 expression was seen in the superficial region of the cell, whereas in most of the carcinomas, the staining was localized in the basolateral region of the cell. Both CD133 and CD44 showed significant correlation with tumor stage.
Conclusions: In the present study, CD133 and CD44 show significant correlation with tumor staging. Cancer stem cell markers have shown similar pattern of expression in the patients of Indian origin. Using combination of markers for staging is preferred as it increases the sensitivity and specificity.

Keywords: CD133, CD44, colorectal cancer, immunohistochemistry, Indian patients


How to cite this article:
Chaitra L P, Prashant A, Gowthami C S, Hajira B, Suma M N, Mahesh S S, Manjunath G V, Sheeladevi C S. Detection of cancer stem cell-related markers in different stages of colorectal carcinoma patients of Indian origin by immunohistochemistry. J Can Res Ther 2019;15:75-81

How to cite this URL:
Chaitra L P, Prashant A, Gowthami C S, Hajira B, Suma M N, Mahesh S S, Manjunath G V, Sheeladevi C S. Detection of cancer stem cell-related markers in different stages of colorectal carcinoma patients of Indian origin by immunohistochemistry. J Can Res Ther [serial online] 2019 [cited 2019 Aug 22];15:75-81. Available from: http://www.cancerjournal.net/text.asp?2019/15/1/75/243509




 > Introduction Top


Colorectal cancer (CRC) is the tumor developing in epithelial cells lining gastrointestinal tract[1] due to abnormal division of cells lining large intestine.[2] CRC is the most frequent neoplasm and most common type of digestive tract malignancy.[3] Usually, CRC develops in adults aged more than 50 years and is uncommon before 40 years of age.[4]

Globalization of human culture has the hidden impact over one's health that seeks medical attention. CRC being the more prevalent has potential to attack due to changing lifestyle ubiquitously. It is the third most commonly diagnosed cancer in the world and is more common in developed countries. India is a country in transition from a developing to a developed nation. The reflection of westernized trends became common among Indians in such a way that it has modified the Indian lifestyle with a changing profile of burden at different cancer sites. Although the incidence rate of CRC in all Indian cancer registries is very close to the lowest rate in the world, changes in lifestyle and dietary habits have shown an increasing trend in the recent years. Furthermore, Indian immigrants to the United Kingdom and the United States have a higher incident rate of CRC clearly, suggesting the role of lifestyle and dietary habits in the causation of CRC.[5] This suggests that with economic transition, there will be a big increase in the burden of CRC in India.

A small subset of cancer cells has been identified which initiate new tumor growth and is more resistant to therapy than differentiated tumor cells. Isolation and characterization of these slowly proliferating cancer stem cells escaping chemotherapeutic regimen play a major role in the development of new diagnostic and therapeutic procedures.[6] Several surface biomarkers have been used in the identification and isolation of these stem cells.

CD133 was discovered in 1997 on normal hematopoietic stem cells.[7] It is seen on normal stem cells, cancer stem cells, embryonic stem cells, stem cell niche, and circulatory endothelial progenitor.[8] CD133 serves as a marker of cancer stem cell isolation in various tumors including colon, brain, prostate, liver, pancreas, lung,[3] so it considered as the molecule of the moment.[7] In colon cancer, CD133+ subpopulation of colon cancer cells has been demonstrated to be highly enriched in tumor-initiating colon cancer stem cells that have the ability to self-renew and to recapitulate the bulk tumor population.[9],[10] CD133 is a five-transmembrane domain glycoprotein with molecular weight 120 KDa containing 865 amino acids.[11] CD133 gene is located on chromosome 4p15.32.[12] CD133 exists in cholesterol-rich domain lipid rafts[13] and is localized to membrane protrusions and microvilli.[14] Although the physiological function of CD133 remains unknown, it is a marker of asymmetric division, lineage plasticity, tumor cell dormancy, and inherent embryonic gene expression.[8]

Another potential colon CSC marker is CD44 which is a multifunctional class I transmembrane glycoprotein,[15] with a molecular weight of 85–90 kDa. It acts as a specific receptor for hyaluronic acid, promoting migration in normal cells and highly expressed in almost every cancer cell in its standard or variant form.[16] It is mainly associated with proteins that monitor the extracellular changes and critical in regulating cell adhesion, proliferation, growth, survival, motility, migration, angiogenesis, and differentiation.[15]

Since immunohistochemistry (IHC) is a relatively simple, cheap, and easily applicable procedure, we evaluated the expression of the marker proteins CD133 and CD44 in colorectal carcinoma patients of Indian origin and associated the level of marker expression with classical clinicopathological variables.


 > Materials and Methods Top


Inclusion criteria

The patients included in the study have been described earlier.[17] The study was conducted for 1 year. Briefly, 26 patients between 20 and 70 years of age who gave informed consent were included in the study. Eleven out of them had carcinoma of colon, 12 had carcinoma of rectum, and 3 had carcinoma of rectosigmoid site. Institutional ethical committee clearance was taken from JSS Medical College, Mysore, before the commencement of the study.

Exclusion criteria

Patients on preoperative chemotherapy or radiotherapy and mortality in the postoperative period were not included in the study. Furthermore, cases with inflammatory bowel disease, noninherited polyposis CRC, and familial adenomatous polyposis were excluded from the study.

Immunohistochemical staining

Paraffin-embedded sections were prepared for surgically resected tumor specimens and adjacent normal tissue. Histological and IHC analysis of CD133 and CD44 was done in sections of 5 μm prepared from paraffin-embedded sections with most representative areas. Omission of the primary antibody served as negative control. Glioblastoma was used as positive control for CD133 and tonsil was used as positive control for CD44.

The tissue sections were placed in an incubator at 80°C for 2 h, deparaffinized, and rehydrated by serial immersion in Hellendahl jars containing graded ethanol solution of 100%, 96%, 80%, and 70% followed by rinsing in deionized water. For antigen retrieval, plastic Hellendahl jar was filled with 1X target retrieval solution and submerged the tissue sections completely in the solution. Heat uncovered at 250–300 W in a microwave for 15 min. Any evaporated target retrieval solution was replaced and left to cool to room temperature. Sections were washed for 1 × 5 min with deionized water, followed by 1 × 5 min with tris-buffered saline (TBS), and endogenous peroxidase activity was blocked by immersing slide in 3% H2O2 solution for 30 min at room temperature followed by washing in TBS again three times. Nonspecific binding of CD133 and CD44 pure antibody was blocked by incubating slide in a humidified chamber at room temperature for 10 min in the blocking serum (HK083-5K; BioGenex, Fremont, CA, USA). Supernatant was drained off by tilting the slide and then blot dried. Each slide was then incubated for 2 h at 4°C with primary anti-CD133 antibody (AC133; Miltenyi Biotec, Auburn, CA, USA) at a dilution of 1:300 in the blocking buffer, anti-CD44 antibody (AM310-5M; BioGenex, Fremont, CA, USA) undiluted followed by washing the slides three times using TBS. Sections were further treated with Super Sensitive™* Polymer-HRP detection system and subsequently biotinylated secondary antibody for 1 h (HK519-06K; BioGenex, Fremont, CA, USA). Tissue sections were incubated in diaminobenzidine used as a chromogen for 10 min at 37°C. After washing in tap water three times, slight hematoxylin counterstaining was done. The slides were then dehydrated in graded ethanol solution and mounted with dibutyl phthalate xylene.

Semi-quantitative analysis of slides

The IHC staining results were evaluated and scored independently by two pathologists. To avoid scoring bias, we used four-tiered scoring method. For CD133, the IHC staining was scored as 0 when there was no expression at all, 1+ when the expression of CD133 was detected in 1%–10% of the whole tumor area, while 2+ and 3+ when it was expressed in 11%–50% and 51%–100% of the tumor area, respectively. Tumors with CD133 expression on over 10% of whole tumor area were considered as CD133 positive. For CD44, tumor cells showing cytoplasmic or membranous staining were regarded as positive. A cutoff <10% of positive neoplastic cells was used to define low expression, 10%–50% to define moderate expression, and >50% to define extensive expression.

Statistical analysis

All analyses were performed using Microsoft Excel 2010 and IBM SPSS Statistics for Windows, Version 22.0. Armonk, NY: IBM Corp. Descriptive statistical measures such as mean, standard deviation, and percentage were applied. Other inferential statistical tests such as unpaired t-test, Chi-square test, and Fisher's exact test for assessing relation between the expression of CD133 and CD44 with tumor staging. The difference are interpreted as statistically significant when P < 0.05. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated by cross-tabulation.


 > Results Top


A total of 26 cases were involved, and according to tumor location, 11 were from colon, 12 from rectum, and 3 from rectosigmoid site. The mean age of the patients were 52.88 ± 13.5 years between 20 and 70 years. The number of cases included in different stages of TNM and Duke's stage was similar, accordingly 6 cases were in Stage I, 10 in Stage II, and 10 in Stage III. Histologically tumors were adenocarcinomas, and most of the tumors were moderately differentiated.

CD133 expression

CD133 expression was detected in 21 of the 26 tumors (80.8%); the remaining tumors showed negative expression [Figure 1]a. Positive expression was seen exclusively on the cell membrane at the glandular luminal surface with dot-like cytoplasmic staining [Figure 1]b. There was no CD133 expression in the paired normal colon tissue. Of the 21 tumors, 46.2% showed low expression (<10% of neoplastic cells), 34.6% showed moderate expression (10%–50% of neoplastic cells), and no cases showed extensive expression (>50% of neoplastic cells).
Figure 1: Expression of CD133 in Stage I colon cancer. Stage I colorectal cancer showing negative for the expression of CD133 by immunohistochemistry (×20) (a). CD133 positive staining was localized to luminal membrane of tumor glands along with intraglandular luminal debris showing positivity. Cytoplasmic expression was not defined (×40) (b). Appreciable staining was taken by nearly 50% of tumor glands (2+)

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CD133 expression and clinicopathological features

The relationship between CD133 expression and a range of clinico-histopathological variables is summarized in [Table 1]. There was a significant correlation between CD133 staining and tumor stage (P = 0.002), with Stage I and II having mostly <10% staining and Stage III having 11%–50% staining. Lymph node involvement was more common in CD133-positive cases with 11%–50% staining (P = 0.001). Duke's Stage A and B were either negative or had <10% of the cells staining for CD133, whereas Duke's Stage C had 11%–50% of the cells staining for CD133 (P = 0.002). No significant relationship was observed between CD133 IHC expression and other clinicopathological variables studied such as sex (P = 0.053), age (P = 0.839), and tumor site (P = 0.868). Although there was a significant increase in CD133 expression in moderately differentiated adenocarcinoma, this cannot be considered because of less number of cases in the other tumor grades.
Table 1: CD133 expression according to clinicopathological parameters in colorectal cancer patients

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CD44 expression

CD44 expression was detected in 23 of the 26 tumors (86.4%). Of these, 19.2% showed low expression (<10% of neoplastic cells), 26.9% showed moderate expression (10%–50% of neoplastic cells), and 42.3% showed extensive expression (>50% of neoplastic cells).

In the normal colorectal mucosa the cell surface, CD44 immunoreactivity was confined to the basal part of the crypts [Figure 2]a. Different staining patterns of CD44 were observed. In the normal mucosa, CD44 expression was seen in the superficial region of the cell, whereas in most of the carcinomas, the staining was localized in the basolateral region of the cell [Figure 2]b and [Figure 2]c. CD44 expression was found in some lymphocytes and macrophages which also showed superficial and basal staining pattern and were used as internal controls. Nerve fibers in submucosa and muscularis mucosa also showed similar type of staining patterns where myelin sheath showed strong stain.
Figure 2: Expression of CD44 in normal colonic mucosa and colon cancer. In the normal colorectal mucosa the cell surface CD44 immunoreactivity was confi ned to the basal part of the crypts (×40) and CD44 expression was seen in the superficial region of the cell (a) whereas, in tumor glands, the staining was localized to basolateral region of the cell (b)

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CD44 expression and clinicopathological features

The relationship between CD44 expression and a range of clinico-histopathological variables is summarized in [Table 2]. There was a significant correlation between CD44 staining and tumor stage (P = 0.003). Most of the cases in Stage I had <10% of the cells staining for CD44, most of the cases in Stage II had 11%–50% of the cells staining for CD44, and most of the cases in Stage III had >50% of the cells staining for CD44. Lymph node involvement was more common in cases with >50% of staining for CD44 (P = 0.032). A similar pattern was observed in Duke's staging (P = 0.003). No significant relationship was observed between CD44 expression and other clinicopathological variables studied such as sex (P = 0.803), age (P = 0.875), tumor site (P = 0.397), and tumor grade (P = 0.101).
Table 2: CD44 expression according to clinicopathological parameters in colorectal cancer patients

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Sensitivity, specificity, PPV, and NPV of CD133 and CD44 in combination against the individual marker for staging of colorectal carcinoma are listed in [Table 3].
Table 3: Sensitivity, specificity, positive predictive value, negative predictive value of markers in combination

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 > Discussion Top


CRC is a global problem[18] affecting both men and women equally.[19] Incidence is rising annually in developing countries[20] and also in most Asian countries.[18] Incidence is also increasing in economically transitioning countries due to high-fat diet, smoking, and physical inactivity.[3] Various pathophysiological mechanisms are implicated in the pathogenesis of CRC. CRC normally originates from stem cells or progenitor cells located at the bottom of the crypt which undergoes clonal expansion.[21] Genetic alterations are involved in CRC pathogenesis. Dominant oncogenes and tumor suppressor gene both are affected with these genetic mutations. Ras gene activation and suppression of adenomatous polyposis coli gene and p53 gene have been implicated in the development of CRC.[22] Dysfunctional regulation of Wnt/beta-catenin pathway is responsible for the formation of CRC.[23]

Adult colonic epithelium shows an organized structure with crypts and dynamic structures which are self-renewing. Multipotent stem cells are localized at the bottom of the crypt which generates mature cells on transient amplification. Colonocytes, mucus-secreting goblet cells, and enteroendocrine cells are involved in colonic epithelial function. Colonic epithelium is replaced every 5 days.[24] CRC is considered to be a stem cell disease.[25]

We are in the process of identifying the presence of cancer stem cells in colorectal carcinoma of Indian origin. After the identification of cancer stem cells through detection of specific markers along with the signaling pathways that govern self-renewal and differentiation ability of cancer stem cells, there is surely a way for the development of cancer stem cell-specific therapy.[26] Identification of specific markers and signaling pathways could help reduce the public health issue due to cancer by the development of cancer stem cell-directed therapy. This remains as an important step to design anticancer drugs. With the available literature, less toxic anticancer drugs could be developed and this requires multidisciplinary approach with the cooperation between researchers and practitioners. Research is on for the detection of signaling pathways which are involved in the function of cancer stem cells (Wnt, Notch, and Hedgehog) so that medicament can be developed to destroy cancer stem cells by targeting critical step in the path.[27]

Since CRC is rising annually in India due to change in lifestyle and adoption to western behavior, this study was taken up to investigate the expression of the well-known stem cell makers CD133 and CD44 in colorectal carcinoma of Indian origin. Furthermore, studies on individual markers have shown inconclusive results which probed us to study combination of markers to identify the cancer stem cell.

CD133 was used as a first marker for the identification of CRC stem cells.[9],[10] It is a five-transmembrane glycoprotein which is made up of an extracellular tail at N-terminal end and short intracellular tail at C-terminal end that can be alternatively spliced or phosphorylated.[14] CD133 has five promoters, three out of five are involved in CD133 transcription.[12] There are three isoforms of CD133. The physiological function of CD133 is yet to be clarified. However, it is said that it establishes and maintains plasma membrane protrusions, cell–cell interaction, and cell–matrix interactions, thereby maintaining cell polarity and migration.[28] Binding domain present at N-terminus of CD133 helps in cell–cell contact.[29] CD133 is also involved in signal transduction.[30]

The study by Horst et al. showed that CD133 was expressed on the cell membrane at the glandular luminal surface of colorectal tumor cells with shedding into the lumina. The tumor cells in direct contact with luminal cell surface were positive for CD133. The intraglandular cellular debris also was positive for the expression of CD133.[31] Similar pattern of staining was observed in our study which confirms that the antibody was specific to the protein. Some studies have reported different patterns and distributions of CD133 in colorectal carcinomas. This could arise from using different antibodies or using a different method for scoring the positivity of CD133 expression. To avoid scoring bias, we used four-tiered scoring method which was recently reported to quantitatively grade CD133 positivity.[12],[32] Moderately differentiated tumors showed higher level of CD133 expression which is in line with the findings of the previous studies on colon cancer.[31],[33]

Culturing of colon cancer stem cells in serum-free medium developed tumor spheres from fresh CRC specimens, whereas there was no tumorsphere formation in cases belonging to Stage I or IIA, indicating less cancer stem cells and smaller in size in those stages.[34] Moderately differentiated tumors and rectal tumors show enhanced expression of CD133 than poorly differentiated mucinous adenocarcinoma. High CD133 expression was seen in advanced T-stage. T0 and T1 stages show low expression of CD133.[35] The present study also demonstrated significant correlation of CD133 and TNM staging with P = 0.002 and is statistically significant.

CD44 is an adhesion molecule which is involved in cell–cell interaction and cell–matrix interactions and also plays an important role in lymphopoiesis, myelopoiesis, lymphocyte homing, macrophage activation, tumor progression, and metastasis. It was first identified in 1982 in lymphocytes. It is a transmembrane glycoprotein which is expressed as a standard form (CD44H) and as numerous splice variant forms, that is CD44V, which is seen in tumor invasion and it is linked to the development and spread of malignancies. Some of the studies also show the downregulation or CD44 aggressiveness or it can be said that expression of the CD44 variant epitopes does not correlate with either tumor progression or metastasis to the liver from CRC. Sometimes, problem may occur or may cause complications by the existence of numerous CD44 isoforms which may have remarkable homology in their antigenic properties, increasing the possibilities of cross-reactivity between the antibodies. Its expression is normally weak in the colon and mainly seen in bases of crypts. CD44 and its isoforms are mostly found in the basal layer of esophagus, the myoepithelial cells of the breast, the pneumocytes and bronchial epithelium, of the crypts of small and large intestine, and also several human cancers. Recent studies have found that CD44 glycoprotein was associated with tumor progression and metastasis cascade. Metastatic tumor cells and activated lymphocytes have some similar characteristics such as invasive behavior and motility, expansion, and accumulation on draining lymph nodes. This will prove that malignant cells might use CD44 protein for metastasizing. Therefore, it is associated with development and spread of malignancies. It also plays an important role in complicated processes which is proved by most of the studies. In lymphomas, in cervical and gastric cancer, high expression of CD44 was seen in advanced tumor stage in poor prognosis. In human epithelial tumors, CD44 not only localized in neoplastic cells but also weakly expressed in normal adjacent tissues. CD44 isoforms localization on cancer cells has been correlated to tumor stage and prognosis.[36]

We studied the expression of CD44 in different stages of CRC. High expression of CD44 was found in Duke's Stage B and C with P = 0.003. In our data, we also identified difference in the quantity of CD44 marker in tumor grading. Although it was not statistically significant, we found that CD44 extensively expressed in moderately differentiated adenocarcinomas with P = 0.101. There was significant correlation between CD44 staining and tumor stage, with a P = 0.003. Pitule et al. had shown that when the patients were stratified according to tumor lymph node invasion, there was a decrease in CD44 expression in sequence from N0 to N2.[37] However, we have found that lymph node involvement was more common in cases, with >50% of staining for CD44 (P = 0.032). Discrepancy in the results may be due to several reasons such as use of different antibodies, existence of numerous isoforms of CD44, and mode of reporting the IHC staining.

In Stage I, Stage II, and Stage III, combination of CD133+CD44 expression was detected in 2, 7, and 9 colorectal carcinoma cases, indicating that the level of marker expression increases with advanced lesions. The combination of CD133+CD44 had better sensitivity, specificity, PPV, and NPV against the individual marker for staging of colon cancer. Small sample size is the limitation of the study, and hence, large multicenter studies may be carried out for confirmation of the study results.


 > Conclusions Top


Cancer stem cell markers have shown similar pattern of expression in the patients of Indian origin. Using combination of markers is better as it increases the sensitivity and specificity. Further studies can be carried out using this combination either to isolate the CRC stem cells or to use this marker combination as therapeutic target in CRC stem cell-directed therapy.

Acknowledgment

We would like to acknowledge Vision Group on Science and Technology (VGST), Government of Karnataka, for funding this project under their Establishment of Centres of Excellence in Science, Engineering, and Medicine Program in 2012–2013.

We would also like to acknowledge Department of Science and Technology, Government of India, for funding the CEMR Laboratory under their Funds for Improvement of S&T Infrastructure (DST-FIST) Program in 2012.

We also acknowledge the financial assistance rendered by Indian Council of Medical Research (ICMR) for the postgraduate student towards her dissertation.

Financial support and sponsorship

The study was financially supported by VGST, ICMR, DST-FIST 2012, and JSSU.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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