# Strong Expression of Chemokine ReceptorCXCR4 by Renal Cell Carcinoma Correlates with Advanced Disease

Authors: Thomas C. Wehler; Claudine Graf; Stefan Biesterfeld; Walburgis Brenner; Jörg Schadt; Ines Gockel; Martin R. Berger; Joachim W. Thüroff; Peter R. Galle; Markus Moehler; Carl C. Schimanski
Journal: Journal of Oncology
 (2008)

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## Abstract

Diverse chemokines and their receptors have been associated with tumor growth, tumor dissemination, and local immune escape. In different tumor entities, the level of chemokine receptorCXCR4 expression has been linked with tumor progression and decreased survival. The aim of this study was to evaluate the influence of CXCR4 expression on the progression of human renal cell carcinoma. CXCR4 expression of renal cell carcinoma was assessed by immunohistochemistry in 113 patients. Intensity of CXCR4 expression was correlated with both tumor and patient characteristics. Human renal cell carcinoma revealed variable intensities of CXCR4 expression. Strong CXCR4 expression of renal cell carcinoma was significantly associated with advanced T-status (P=.039), tumor dedifferentiation (P = .0005), and low hemoglobin (P = .039). In summary, strong CXCR4 expression was significantly associated with advanced dedifferentiated renal cell carcinoma.


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## Body

## 1. Introduction

Renal  cell  carcinoma  (RCC) is the  sixth leading cause
of cancer-related deaths in the Western world and comprises 2-3% of all newly
diagnosed malignancies in adults. Among the different kidney neoplasms, it
represents with 85% the largest fraction [1]. The age-adjusted incidence of RCC
in Western nations is 5–12/100 000 in
women or men, respectively, with a peak incidence in the 6th decade [2]. In
practice, the only curable treatment is nephrectomy performed in early stages of
the disease. However, about 30–50% of patients
have already metastases at presentation, and approximately one third of the nephrectomized
patients relapse and progress with metastatic disease. The preferential sites
of metastasis are the regional lymph nodes, the lung, the liver, and the bones.
Survival strongly depends on the tumor stage at presentation. The 5-year
survival rate is approximately 50%, whereas the median survival in case of
metastasis is less than one year [3–5]. The current
standard treatment for metastasized RCC consists of the application of IFN-α
and IL-2 [6]. Recently, phase II clinical trials using receptor-tyrosine
kinase (RTK) inhibitors have shown more promising results and lead to
approval by the Food and Drug Administration (FDA) and European Medicines
Agency (EMEA) [2].In vivo and in vitro results from different tumor
entities suggest that organ-specific metastasis is partially governed by
interactions of chemokine receptors on cancer cells and their corresponding
chemokines expressed in  target organs and the tumor bed. This process is
considered to direct lymphatic and hematogenous spread and furthermore influences the
sites of metastatic growth [7]. Chemokines and their respective
G-protein-coupled receptors were initially described to mediate different pro-
and anti-inflammatory responses [8]. In particular, the high expression of
stromal cell derived factor 1α (SDF-1α), also known as CXCL12, by endothelial
cells, biliary epithelial cells, bone marrow stromal cells, and lymph nodes
results in a chemotactic gradient attracting CXCR4 expressing
lymphocytes into those organs [9–15]. Most recently, CXCR4 has shifted into focus as it
is the most common chemokine receptor expressed on cancer cells [16]. It was
suggested to play an important role in tumor spread of colorectal, breast, and oral
squamous cell carcinoma as all of them commonly metastasize to SDF-1α expressing organs [17–20]. Data
obtained from in vitro as well as from murine in vivo models, analyzing the
metastatic ability of CXCR4 in expressing cancer cells, underlined the
key role of CXCR4 for tumor cell malignancy, as activation of CXCR4 by SDF-1α induced migration, invasion, and angiogenesis of cancer
cells [21–23].Therefore, we evaluated the
expression ofCXCR4 in renal cancer cell lines and specimens and
correlated these results with the patients’ clinicopathological parameters and
survival.


## 2. Materials and Methods

### 2.1. Tissue Samples

Renal cell
carcinoma samples were
intraoperatively obtained from 113 patients with renal clear cell carcinoma who
underwent surgery at the Department of Urology of the University of Mainz. The morphological classification of the carcinomas was conducted according to
World Health Organization (WHO) specifications. Patients were followed up on a
regular basis depending on the procedure performed.


### 2.2. Immunohistochemical Staining

The  avidin-biotin-complex method
(LSAB+ System-HRP Kit,  Dako Cytomation, Hamburg, Germany) was used to
detect the proteinCXCR4 (anti-CXCR4, dilution 1 : 300; Capralogics
Inc., Mass, USA).
Formalin-fixed and paraffin-embedded tissues were deparaffinized and
subsequently microwaved (600 W, 15 minutes) in citrate buffer (ph 6.0). After
preincubation with hydrogen peroxide (LSAB+ System-HRP Kit, Dako Cytomation, Hamburg,
Germany) and human AB plasma (Department of Transfusion, University of Mainz,
Mainz, Germany), the primary antibodies were applied for one hour at room
temperature. After incubation with the secondary antibody (LSAB+ System-HRP
Kit, Dako Cytomation, Hamburg, Germany),
the avidin-biotin complex was added and the enzyme activity was visualized with
diaminobenzidine (LSAB+ System-HRP Kit, Dako Cytomation, Hamburg, Germany).
Counterstaining was performed with haematoxylin (Roth, Karlsruhe, Germany).
For negative controls only the secondary antibody was used. A negative control
was performed for each sample (N=113). For positive controls formalin-fixed and
paraffin-embedded tissue samples of the human spleen were applied.


### 2.3. Evaluation of Immunostaining

Immunostaining was evaluated by three authors
independently (T.C. Wehler, C. Graf, S. Biesterfeld), blinded to patient
outcome and all clinicopathologic findings. The immunohistochemical staining
was analyzed according to a scoring method as previously validated and
described [17]. The tumors were classified into four groups based on
the homogeneous staining intensity: 0, absent; 1, weak; 2, intermediate; 3,
strong staining.  In the case of heterogeneous staining within the same sample, the respective higher score was chosen, if more than 50% of cells revealed a higher staining intensity. If expression intensity was exactly in between two scores, the authors agreed on 0.5 point-steps. If evaluations
did not agree, specimens were re-evaluated and reclassified according to the
assessment given most frequently by the observers.


### 2.4. Statistics

The correlation ofCXCR4 staining
intensity with clinicopathological patterns was assessed with the χ2 test and with the unpaired Student t-test (one/two sided), when appropriate. Survival rates were visualized
applying Kaplan-Meier curves, and P-values were determined by log-rank
test. P<.05 was considered significant and P<.001
highly significant in all statistical analyses.


## 2.1. Tissue Samples

Renal cell
carcinoma samples were
intraoperatively obtained from 113 patients with renal clear cell carcinoma who
underwent surgery at the Department of Urology of the University of Mainz. The morphological classification of the carcinomas was conducted according to
World Health Organization (WHO) specifications. Patients were followed up on a
regular basis depending on the procedure performed.


## 2.2. Immunohistochemical Staining

The  avidin-biotin-complex method
(LSAB+ System-HRP Kit,  Dako Cytomation, Hamburg, Germany) was used to
detect the proteinCXCR4 (anti-CXCR4, dilution 1 : 300; Capralogics
Inc., Mass, USA).
Formalin-fixed and paraffin-embedded tissues were deparaffinized and
subsequently microwaved (600 W, 15 minutes) in citrate buffer (ph 6.0). After
preincubation with hydrogen peroxide (LSAB+ System-HRP Kit, Dako Cytomation, Hamburg,
Germany) and human AB plasma (Department of Transfusion, University of Mainz,
Mainz, Germany), the primary antibodies were applied for one hour at room
temperature. After incubation with the secondary antibody (LSAB+ System-HRP
Kit, Dako Cytomation, Hamburg, Germany),
the avidin-biotin complex was added and the enzyme activity was visualized with
diaminobenzidine (LSAB+ System-HRP Kit, Dako Cytomation, Hamburg, Germany).
Counterstaining was performed with haematoxylin (Roth, Karlsruhe, Germany).
For negative controls only the secondary antibody was used. A negative control
was performed for each sample (N=113). For positive controls formalin-fixed and
paraffin-embedded tissue samples of the human spleen were applied.


## 2.3. Evaluation of Immunostaining

Immunostaining was evaluated by three authors
independently (T.C. Wehler, C. Graf, S. Biesterfeld), blinded to patient
outcome and all clinicopathologic findings. The immunohistochemical staining
was analyzed according to a scoring method as previously validated and
described [17]. The tumors were classified into four groups based on
the homogeneous staining intensity: 0, absent; 1, weak; 2, intermediate; 3,
strong staining.  In the case of heterogeneous staining within the same sample, the respective higher score was chosen, if more than 50% of cells revealed a higher staining intensity. If expression intensity was exactly in between two scores, the authors agreed on 0.5 point-steps. If evaluations
did not agree, specimens were re-evaluated and reclassified according to the
assessment given most frequently by the observers.


## 2.4. Statistics

The correlation ofCXCR4 staining
intensity with clinicopathological patterns was assessed with the χ2 test and with the unpaired Student t-test (one/two sided), when appropriate. Survival rates were visualized
applying Kaplan-Meier curves, and P-values were determined by log-rank
test. P<.05 was considered significant and P<.001
highly significant in all statistical analyses.


## 3. Results

### 3.1. Tumor Characteristics and Patient Profiles

The selected group of patients represents the
typical characteristics of renal cell carcinoma in industrialized countries.


### 3.2. Immunohistochemical Staining ofCXCR4 in Renal Cell Carcinoma

The  staining of normal human kidney tissue forCXCR4 revealed a cytoplasmatic expression and in only few specimens an additional weak membranous location of CXCR4 (see Figure 1). A nuclear staining of CXCR4 was not observed. In renal
cell carcinoma, the respective expression rate for CXCR4 was 100%
(113/113) and varied from weak (34%), intermediate (42%), to strong (24%).
Negative controls of human renal cancer remained negative for all tissue
samples (N=113, not shown). Glomeruli did not reveal any CXCR4 expression and thus served as internal negative control. As internal positive control, splenic lymphocytes
(strong CXCR4 expression) and tubuli cells (intermediate CXCR4 expression) were used.
Similarly, inflammatory infiltrates in kidney tissue (data not shown) depicted
a strong CXCR4 expression.Figure 1
The
figure depictsCXCR4 expression in healthy kidney and cancer samples. While
glomeruli did not depict any CXCR4 expression, tubuli did reveal a
medium-strong predominantly cytoplasmic CXCR4 expression. All cancer
samples did reveal a cytoplasmatic expression of CXCR4 ranging from weak
(34%) to medium (42%) and strong (24%).


### 3.3. Relevance ofCXCR4 Expression in Renal Cell Carcinoma

StrongCXCR4 expression significantly correlated with
dedifferentiated (P=.0005) and progressed renal cell carcinoma, indicated by T-status (P=.039;
see Table 1). Furthermore, strong CXCR4 expression revealed a
significant association with low hemoglobin values (P=.039) and a nonsignificant trend towards
increased thrombocytes (P=.089/P=.18, resp.). No
correlation was seen for age, size, survival, or creatinine values.Table 1
Patient and tumor characteristics dependent on intensity ofCXCR4 expression.
CXCR4 expressionStatisticsWeakMediumStrongTotal number39 (34%)47 (42%)27 (24%)Average age (years)63.866.3n.s.GenderFemale36 (42%)8 (30%)n.s.Male50 (58%)19 (70%)Grading1/265 (78%)11 (41%)P=.00053/418 (22%)15 (59%)T-status1/264 (76%)15 (56%)P=.0393/420 (24%)12 (44%)Average size (cm)5.76.0n.s.Survival (months)29.736.8n.s.Average creatinin (mg/dl)1.111.07n.s.Average hemoglobin (g/dl)14.3213.25P=.019(1-sided)P=.039(2-sided)Average thrombocytes (/nl)271313P=.089(1-sided)P=.18(2-sided)


## 3.1. Tumor Characteristics and Patient Profiles

The selected group of patients represents the
typical characteristics of renal cell carcinoma in industrialized countries.


## 3.2. Immunohistochemical Staining ofCXCR4 in Renal Cell Carcinoma

The  staining of normal human kidney tissue forCXCR4 revealed a cytoplasmatic expression and in only few specimens an additional weak membranous location of CXCR4 (see Figure 1). A nuclear staining of CXCR4 was not observed. In renal
cell carcinoma, the respective expression rate for CXCR4 was 100%
(113/113) and varied from weak (34%), intermediate (42%), to strong (24%).
Negative controls of human renal cancer remained negative for all tissue
samples (N=113, not shown). Glomeruli did not reveal any CXCR4 expression and thus served as internal negative control. As internal positive control, splenic lymphocytes
(strong CXCR4 expression) and tubuli cells (intermediate CXCR4 expression) were used.
Similarly, inflammatory infiltrates in kidney tissue (data not shown) depicted
a strong CXCR4 expression.Figure 1
The
figure depictsCXCR4 expression in healthy kidney and cancer samples. While
glomeruli did not depict any CXCR4 expression, tubuli did reveal a
medium-strong predominantly cytoplasmic CXCR4 expression. All cancer
samples did reveal a cytoplasmatic expression of CXCR4 ranging from weak
(34%) to medium (42%) and strong (24%).


## 3.3. Relevance ofCXCR4 Expression in Renal Cell Carcinoma

StrongCXCR4 expression significantly correlated with
dedifferentiated (P=.0005) and progressed renal cell carcinoma, indicated by T-status (P=.039;
see Table 1). Furthermore, strong CXCR4 expression revealed a
significant association with low hemoglobin values (P=.039) and a nonsignificant trend towards
increased thrombocytes (P=.089/P=.18, resp.). No
correlation was seen for age, size, survival, or creatinine values.Table 1
Patient and tumor characteristics dependent on intensity ofCXCR4 expression.
CXCR4 expressionStatisticsWeakMediumStrongTotal number39 (34%)47 (42%)27 (24%)Average age (years)63.866.3n.s.GenderFemale36 (42%)8 (30%)n.s.Male50 (58%)19 (70%)Grading1/265 (78%)11 (41%)P=.00053/418 (22%)15 (59%)T-status1/264 (76%)15 (56%)P=.0393/420 (24%)12 (44%)Average size (cm)5.76.0n.s.Survival (months)29.736.8n.s.Average creatinin (mg/dl)1.111.07n.s.Average hemoglobin (g/dl)14.3213.25P=.019(1-sided)P=.039(2-sided)Average thrombocytes (/nl)271313P=.089(1-sided)P=.18(2-sided)


## 4. Discussion

The
expression of the chemokine receptorCXCR4 has been reported in various epithelial, mesenchymal, and hematopoietic tumors.
In several entities, its expression was linked to tumor dissemination and poor
prognosis [20, 24, 25]. CXCR4 expression can be increased as a result of intracellular second messengers such
as calcium [26] and cyclic AMP [27, 28] by the inactivation of the tumor
suppressor gene p53 and
overexpression of NFκB [29–31], by cytokines like IL-2, IL-10, or TGF-1β [26, 32] and by growth factors such as VEGF and EGF [33, 34]. In addition, Staller and colleagues could
demonstrate that CXCR4 is a hypoxia
inducible gene with a HIF-1α binding domain, and that its overexpression
in clear-cell renal cell carcinoma is due to a loss-of-function of the von
Hippel-Lindau (VHL) tumor suppressor
protein, which under normoxic conditions directs HIF-1α to ubiquitin-mediated degradation [35]. Loss of VHL stabilizes HIF-1α leading to
increased expression of hypoxia-response genes including VEGFA, CXCR4, its ligand SDF1α, and HIF-1α itself [36, 37]. They also
reported a positive correlation between strong CXCR4 expression and poor
tumor-specific survival independent of tumor stage and differentiation grade.
The latter is in contrast to the results obtained in our study.We analyzed
the expression profile ofCXCR4 in a
series of human renal cell carcinoma cell lines and 113 patients’ samples for
which exact tumor staging and followup data were available and correlated the
expression profile with clinicopathological data. The human renal cell
carcinoma tumor samples that are analyzed revealed varying intensities of CXCR4 expression ranging from weak to
strong, as previously described for pancreatic and colorectal cancer [38].
Interestingly, CXCR4 expression was downregulated in 34% and upregulated
in 24% of renal cell carcinoma as compared to original tubuli cells. 42% of
cancers revealed the identical expression intensity of CXCR4 as tubuli
cells. A cytoplasmatic staining of CXCR4 was observed in all cancers, whereas fewer cases depicted an additional
membranous  localization of CXCR4.
These observations are in line with a recently published study by
Zagzag and coworkers [44]. Furthermore, it was reported that CXCR4 surface expression
was higher in permanent cell lines than in primary tumor samples [39]. Noteworthy, an inducible translocation of CXCR4 from the cytoplasm to the membrane
has been reported previously in [29]. In addition, at least in breast cancer
cells, inhibited CXCR4 ubiquitination
was described as another mechanism contributing to increased CXCR4 surface levels [40].In our
renal cell carcinoma patients, a strongCXCR4 expression was
significantly associated as well with progressed cancer as indicated by the
T-status as with dedifferentiation. Our results are furthermore in line with
recent reports from our group and others, describing a similar effect of CXCR4 on disease progression in other
tumor entities [17, 41]. Hence, our data suggest a relevant influence of CXCR4 on proliferation and
differentiation of renal cell carcinoma with regard to the in vivo situation.
This hypothesis is strengthened by observations in a murine model, where the
metastatic capability of CXCR4-expressing
RCC cells strongly correlated with CXCR4 protein level on cancer cells and the SDF-1α expression in the target organs [23]. Therefore, CXCR4-expressing cancer cells are
certainly attracted to the typical “homing organs” such as lungs, bone marrow,
liver, and lymph-nodes showing a high SDF-1α expression [13, 42]. A pathophysiological
relevant fact worthwhile to be mentioned is that endothelial cells coexpress SDF-1α and VCAM-1,
thus mediating tumor-cell/endothelial cell attachment. CXCR4 activation by SDF-1α induces β-integrin expression, binding VCAM-1 on endothelial cell
[43, 44]. Similar pathophysiological processes must be proposed for renal cell
carcinoma dissemination.Therefore,CXCR4 might be an interesting
therapeutic target in a multimodal therapy of renal clear cell carcinoma.



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