Overexpression of ClC-3 Chloride Channel in Chondrosarcoma: An In Vivo Immunohistochemical Study with Tissue Microarray.

Immunohistochemical analysis demonstrated that ClC-3 chloride channel expression in normal cartilage tissue was significantly thinner in normal chondrocyte and well-differentiated chondrosarcoma (WDC) ( Figure 1A, 1B ) in comparison to moderately- and poorly-differentiated chondrosarcoma that presented high expression of ClC-3 chloride channel. As shown in Table 2 , among the 44 cases of WDC, only 29.55% exhibited low expression levels of ClC-3 chloride channel ( Figure 1A, 1B ). However, among the 18 cases of MDC and PDC, 94.44% exhibited high levels of ClC-3 chloride channel expression. Besides, the rate of grade >3 (high expression of ClC-3) was 0%, 16.66%, and 41.66% in well-, moderately- and poorly-differentiated chondrosarcoma, respectively ( Figure 1C–1F ). The statistical analysis demonstrated significant difference among the 3 groups ( Table 2 , χ 2 =33.53, P <0.05). The investigation of ClC-3 chloride channel subcellular localization in the chondrosarcoma tissues showed that among the 44 cases of WDC, which exhibited low expression levels of ClC-3 chloride channel expression, ClC-3 was noticed in the nucleus, ( Figure 2A, 2B ). In the 6 cases of MDC and 12 cases of PDC, ClC-3 chloride channel subcellular localization presented overexpression 83.33% (5 out of 6 cases) and 91.66% (11 out of 12 cases). It mainly localized in the cytoplasm and in both nucleus and cytoplasm, respectively ( Figure 2C, 2D ). Additionally, the immunohistochemical staining of mesenchymal chondrosarcoma (MC) tissues and dedifferentiation chondrosarcoma (DDC) tissues showed that subcellular localization of ClC-3 chloride channel also in the cytoplasm and in both nucleus and cytoplasm among the 6 and 4 cases, respectively ( Figure 2E, 2F ). We also noticed that ClC-3 chloride channel localization changes with the change of malignant degree, and showed significant difference among the groups ( Table 3 , χ 2 =48.51, P <0.05). The present study also investigated the effect of age on ClC-3 immunoreactivity in varying degrees of differentiated chondrosarcoma. The occurrence of chondrosarcoma diseases exhibits a certain degree of age bias, with a higher percentage detected in young patients. As shown in Figure 3 , the mean age of the 75 patients was 38.8 years old. The ratio of age in the 75 chondrosarcoma tissues significantly indicated that 21.33%, (16 out of 75 samples) were from patients aged <30 years, 73.33% (55 out of 75 samples) were from patients 30≤ age 2) was 18.75% (3 out of 16 samples) from patients aged <30 years, 21.81% (12 out of 55 samples) in those 30≤ age <60 years, and 25.00% (1 out of 4 samples) in those age ≥60 years. However, chi-square statistical analysis for the 3 age groups demonstrated no significant difference among the 3 age groups ( Table 4 , χ 2 =0.271, P >0.05). There were sex-associated differences in ClC-3 chloride channel in chondrosarcoma tissue. The occurrence of chondrosarcoma also exhibits a certain degree of sex bias, with a higher percentage detected in males. Thus, the present study compared the expression of ClC-3 chloride channel in various degrees of chondrosarcoma tissues from male and female. Among the 75 cases of chondrosarcoma tissue, the percentage of samples with high ClC-3 expression level (>2) was 9.09% (2 out of 22 cases) in females and 24.52% (13 out of 53 cases) in males. However, the chi-square statistical analysis of gender groups demonstrated no significant differences ( Table 4 , χ 2 =2.32, P >0.05).

The channel proteins of the ClC family are widely distributed in prokaryotes and eukaryotes. ClC family members are expressed in tissues and cells. It plays various and important roles in physiological functions, ranging from the control of cell volume to the regulation of information transfer between cells [ 1 – 3 ]. Loss of function of the ClC gene by gene mutation or other means can lead to a several congenital diseases [ 4 ]. Channel dysfunctions have been associated with Dent disease [ 5 ], osteosclerosis [ 4 ], Bartter syndrome [ 6 ], congenital epilepsy [ 7 ]. There is increasing evidence that ion channel dysfunction is also involved in cancers targeting ion channels, which is a promising strategy for the treatment of cancer [ 3 , 8 , 9 ]. ClC-3 is located on chromosome 4, 4q33. The ClC-3 chloride channel realizes anionic transport across the membrane in the form of dimers. The activation and inactivation of ClC-3 channels are regulated by various factors, such as anion concentration level, H + concentration level, calcium ion presence, osmotic pressure, energy, or anti-tumor drugs [ 8 , 10 – 12 ]. It is widely involved in the basic life processes [ 13 – 15 ]. Moreover, ClC-3 is a member of ClC voltage gated Cl − channel gene super family [ 10 ]. However, the role of ClC-3 chloride channels as a component of volume-regulated anion channels (VRAC) are a controversial issue. A large amount of accumulated data indicates that ClC-3 is more highly expressed in cancer tissues such as glioma, nasopharyngeal carcinoma, breast cancer, and human cervical squamous cell carcinoma. It plays an important role in regulating cell migration and erosion, cell cycle and drug-induced apoptosis [ 2 , 8 , 14 , 16 , 17 ]. Chondrosarcoma is a chondrogenic bone tumor that can be classified into several types based on the presence of pathological lesions, preexisting lesions, and histological grading [ 18 ]. Most chondrosarcomas are primary, whereas secondary chondrosarcomas are caused by benign progenitor cell lesions such as osteochondromas [ 19 ]. Chondrosarcomas are usually a group of slow-growing, heterogeneous, primary bone malignancies characterized by the formation of hyaline cartilage tumor tissue. The prognosis of chondrosarcoma is related to the histologic grade and whether the tumor margin is removed completely. Most chondrosarcomas have a good prognosis, but once distant metastases, such as lung metastasis, occur, this is a red flag [ 19 – 21 ]. However, the expression of the ClC-3 chloride channel in chondrosarcoma has not been reported. To address this issue, we evaluated the immunoreactivity of the ClC-3 chloride channel in chondrosarcoma and explored its clinical significance.

Primary chondrosarcoma is a malignant chondrogenic tumor characterized by the formation of cartilage matrix by tumor cells and the direct formation of nidus. Primary chondrosarcomas are classified into 7 histological subtypes, including conventional intramedullary chondrosarcoma, clear cell chondrosarcoma, juxtacortical chondrosarcoma, myxoid chondrosarcoma, and dedifferentiated chondrosarcoma [ 25 , 26 ]. Chondrosarcoma is a common malignant bone tumor in the skeletal system, and its treatment is challenging for orthopedists. Since it is not sensitive to radiotherapy and chemotherapy, the only effective treatment at present is surgical excision. However, incomplete surgical resection or metastasis is associated with poor survival. Therefore, it is of great significance to study the risk factors of chondrosarcoma and more effective treatment methods for improving the survival rate of patients [ 26 , 27 ]. TMA studies of histopathological materials are often used to study malignant diseases. In this study, we reported for the first time the immunoreactivity of ClC-3 chloride channel in TMA-based chondrosarcoma. Among the 18 cases of moderately- and poorly-differentiated chondrosarcoma (MDC and PDC), 94.44% exhibited high levels of ClC-3 chloride channel expression. With the increase of tumor malignancy (from high differentiation to low differentiation), the expression level of ClC-3 chloride channel was significantly increased. The size of samples for MDC and PDC was small in this study. However, we have used these differences just as indication for further studies. The survival rate of chondrosarcoma patients was closely related to the grade of metastasis and malignancy. Nie et al. analysis of 3737 patients and found that the 5-year overall survival rate of chondrosarcoma was 73.9%. However, if there was remote metastasis, non-differentiation or single radiation, the 5-year survival rate was greatly reduced, below 30%. The characteristic of “distant” stage, “undifferentiated” grade, and single radiation had very low 5-year survival rates [ 28 ]. Wang et al. considered that high histological grade was an independent risk factor for chondrosarcoma [ 29 ]. Fiorenza et al. also considered that high histological grade was an independent risk factor for survival and prognosis evaluation, but the location and surgical type was not, after they analyzed 153 patients with non-metastatic chondrosarcoma [ 30 ]. Our results also demonstrated that localization of ClC-3 chloride channel changed with the change of malignant degree. Among the 44 cases of WDC, ClC-3 level was low expression and present in the nucleus. In the 6 cases of MDC and 12 cases of PDC, the subcellular localization of ClC-3 chloride channel was overexpression and mainly localized in the cytoplasm and in both of nucleus and cytoplasm. The significance of the different localization in different malignant degrees remains unclear. It has been suggested that ClC-3 chloride channels may perform different functions with difference distributions. Some of the data have showed that ClC-3 chloride channels were distributed on the cell membrane and were related to cell cycle and apoptosis [ 31 , 32 ]. ClC-3 chloride channels have also been located in the intracellular compartment and associated with changes in the intracellular acidic environment, thereby increasing the isolation of anti-tumor drugs and leading to resistance to chemotherapy drugs [ 33 , 34 ]. It has been shown that ClC-3 induces tumor mediating multidrug resistance by activating the nuclear factor kappa B signaling pathway [ 17 ]. Chondrosarcoma, as a common malignant bone tumor, mostly occurs in patients younger than 30 years of age, and the incidence of chondrosarcoma increases gradually over the age of 35 years. Our study found that the grading of chondrosarcoma increased with age. But the ratio of age in chondrosarcoma significantly showed that the incidence of chondrosarcoma increased gradually in the age range of 30≤ age <60 years. Interestingly, marital status was identified as an independent prognostic factor for chondrosarcoma. Widowed patients have been reported to show the worst chondrosarcoma cancer-specific survival performance compared with married, divorced, and single controls [ 35 ]. Several studies have confirmed that marital status is an independent prognostic factor for cancer patients [ 36 – 38 ]. We also looked at gender differences in the incidence of chondrosarcoma in this study. The occurrence of chondrosarcoma also exhibits a certain degree of sex bias, with a high percentage detected in males. We found a 2.4: 1 prevalence predilection in males versus females. Among the 75 cases of chondrosarcoma tissue, the percentage of samples with high ClC-3 expression level (>2) was also significantly higher in males. Gender differences in chondrosarcoma disease have been well established. Arshi et al. analyzed 973 cases of chondrosarcoma and found a 3: 2 prevalence predilection in males versus females [ 39 ]. Shao et al. analyzed 40 Chinese cases of extra skeletal myxoid chondrosarcoma and found a gender ratio of 1.7: 1 [ 40 ]. Moreover, the etiology of osteosarcoma has not been fully illuminated. Tumor characteristics understanding may play an important role in guiding development of targeted therapy for these diseases. In this study, we found that the ClC-3 chloride channels were significantly increased in chondrosarcoma in highly malignant tissue. The high expression of ClC-3 chloride channels may be related to the malignant proliferation and erosion in chondrosarcoma diseases. We and others have found that high expression of ClC-3 chloride channels are associated with endometriosis, nasopharyngeal cancer, breast cancer, and peritoneal adhesion. However, the mechanism of ClC-3 chloride channels in regulating the pathogenesis of chondrosarcoma needs further study.

We examined the expression of ClC-3 chloride channel in different types of chondrosarcoma and normal cartilage tissues. Our data provided the first evidence that the ClC-3 chloride channels were significantly increased in chondrosarcoma in highly malignant tissue. Besides, the subcellular localization of ClC-3 chloride channel was changed in association with malignant degree changes. Our results revealed the potential of ClC-3 chloride channel in the treatment of chondrosarcoma.

Paraffin-embedded tissue microarrays (TMAs) used in the present study were purchased from Alenabio (Alenabio, Xian, China). The chondrosarcoma and normal cartilage tissue TMAs (cat. no. OS805, Table 1 ) contained 80 carcinoma samples, including 44 well-differentiated chondrosarcoma (WDC) cases, 6 moderately-differentiated chondrosarcoma (MDC) cases, 12 poorly-differentiated chondrosarcoma (PDC) cases, 4 dedifferentiation chondrosarcoma cases, 8 mesenchymal chondrosarcoma (MC) cases, 1 chondrosarcoma of degeneration (D-C) case and 5 normal cartilage tissue samples (C). One case of moderately-to-poorly differentiated osteosarcoma was classified as poorly-differentiated osteosarcoma for statistics conveniently. On the basis of morphology, the chondrosarcoma carcinoma samples were graded, according to the tumor-node-metastasis grading system [ 22 ] by the supplier, indicating well-, moderately- or poorly-differentiated tissue, respectively. In total, there were 80 tissue samples in the microarray, with 1 sample from each patient. According to the previously described scheme, TMA was dewaxed with xylene, rehydrated with a graded alcohol series and antigen retrieval with 0.01 M sodium citrate buffer, in pH 6.0 at steady temperature. TMAs were then washed with phosphate-buffered saline (PBS, pH 7.4) 3 times and blocked with 3% H 2 O 2 (v/v in PBS) for 15 minutes at room temperature. Glass slides were then incubated for 20 minutes with 2% normal goat serum (v/v in PBS) at room temperature to block the non-specific binding. Where after, the TMAs were incubated overnight at 4°C with a rabbit polyclonal antibody against to ClC-3 antibody 1: 250 dilution (Abcam, Cambridge, UK). Following several washes with PBS, the sections were incubated with secondary antibody (biotinylated goat anti-mouse/rabbit IgG, Ultra-Sensitive™ SP kit (CHEMBIO, China) for 10 minutes at room temperature. The sections were then washed with PBS and incubated with streptavidin-peroxidase for 10 minutes. Finally, DAB/ACE (DAB color development kit, CHEMBIO, China) developed color for 10 minutes and then we added distilled water to stop the color development. The equivalent procedure was conducted for the blank controls, with the primary antibody replaced by antibody diluent. After immunohistochemical staining, images were captured with a DS-Ri2 digital camera (Nikon Corporation, Tokyo, Japan) mounted to a CX41 Nikon microscope (Nikon Corporation, Tokyo, Japan). The staining was scored according to the previously described 4-point system (score 0–3) [ 23 , 24 ] by a pathologist (double-blinded) as follows: score 3, dark staining that is easily visible and present in >50% of cells; score 2, focal areas of dark staining (50% of cells; score 1, focal moderate staining in <50% of cells or pale staining in any proportion of cells not easily observable at low power; and score 0, none of the aforementioned. A high level of expression was defined as a score of 2–3 and low level of expression was defined as a score of 0–1, as described previously. Considering the comparatively small sample size, an early tumor stage was defined as stages I and II, and an advanced stage was defined as stages III and IIIb. Well-differentiated carcinoma (WDC) was defined as grade 1, moderately-differentiated carcinoma (MDC) as grade 2 and poorly-differentiated carcinoma (PDC) was defined as grade 3 [ 24 ]. All data are expressed as n (%) and were compared using a χ 2 test. Fisher’s exact test was used for correction. Statistical analysis was performed using SPSS software (version 13.0; SPSS, Inc., Chicago, IL, USA). P <0.05 was considered to indicate a statistically significant difference.