**3. Results**

## *3.1. IHC Scoring*

Staining intensities were rated using a scoring system ranging from 0 to 3, with 0 indicating no staining, 1 being mild, 2 moderate, and 3 strong staining. Score values were obtained by multiplying the staining intensity by the percentage of positive cells. The percentage of positive cells was analyzed with 10% increments. For 10–20% of positive cells a median value of 15% was used for statistics. Examples for immunohistochemical scorings are shown in Figure 1.

**Figure 1.** Immunohistochemical staining of VDAC1 and SDHA in the tumor center and periphery and normal adjacent tissue. (**A**–**C**) VDAC1 (case 28); (**D**–**F**) SDHA (case 4); (**A**,**D**) normal tissue; (**B**,**E**) tumor center; (**C**,**F**) tumor periphery. The following score values were evaluated for the images: Score value = intensity × percent positive cells; (**A**) 150 = 2 × 75; (**B**) 295 = 3 × 85; (**C**) 97.5 = 1.5 × 65; (**D**) 255 = 3 × 85; (**E**) 187.5 = 2.5 × 75; (**F**) 130 = 2 × 65. Magnification = 400×. Scale bar = 100 μm.

#### *3.2. Expression of VDAC1 and Subunits of the Five OXPHOS Complexes*

VDAC1 was used as a marker for the mitochondrial mass. It is highly expressed in the outer mitochondrial membrane which is otherwise relatively sparse of proteins. Therefore it represents the gold standard for determination of the mitochondrial amount. Protein complexes of the OXPHOS are localized in the inner mitochondrial membrane where they transport electrons to generate a proton gradient used by the ATP synthase (complex V) to make ATP. Subunits for each of the five OXPHOS complexes were analyzed in CCCs. Complex I (NADH coenzyme Q oxidoreductase) is the largest multisubunit complex of the OXPHOS system with a molecular mass of 970 kDa consisting of 45 subunits [21,22]. NDUFS4 is an iron–sulfur cluster-containing subunit incorporated during a very late stage of complex I assembly essential for complex I function. Complex II (succinate dehydrogenase) is the smallest complex consisting of four subunits and the only complex exclusively encoded by the nuclear DNA. Complex III (coenzyme Q: cytochrome c-oxidoreductase) consists of 22 subunits. Cytochrome b is the only mtDNA-encoded subunit of complex III [23]. Complex IV (cytochrome c oxidase) represents the last complex of the respiratory chain catalyzing the terminal step in reduction O**<sup>2</sup>**. Three complex IV subunits are encoded by mtDNA. Complex V (ATP synthase) uses the protons translocated by the respiratory chain enzymes for production of ATP [23,24]. Complex I, complex III, and complex IV are furthermore organized in even bigger protein complexes, of which the most abundant one is termed respirasome [25].

Significantly higher VDAC1 expression was observed in the tumor center compared to adjacent normal tissue (*p* < 0.0001) (Figure 1A–C, Figure 2A,C and Figure 3A).

**Figure 2.** Immunohistochemical staining of a CCC and adjacent control tissue. (**A**,**D**,**G**,**J**,**M**,**P**) liver control tissue; (**B**,**E**,**H**,**K**,**N**,**O**) control bile duct tumor; (**C**,**F**,**I**,**L**,**O**,**R**) tumor center; (**A**–**C**) VDAC1; (**D**–**F**) NDUFS4; (**G**–**I**) SDHA; (**J**–**L**) UQCRC2; (**M**–**O**) MT-CO1; (**P**–**R**) ATP5F1A. Images of the liver and tumor center were taken at 100× magnification. For bile ducts a 400× magnification is shown. Scale bars = 100 μm. Red arrows highlight bile ducts. Case 11, a 59-year-old man, is shown.

**Figure 3.** Score values of staining of the OXPHOS complexes and VDAC1 in CCCs. (**A**) VDAC1, (**B**) NDUFS4, (**C**) SDHA, (**D**) UQCRC2, (**E**) MT-CO1, (**F**) ATP5F1A. The mean score values ± SD of the staining of control tissues, tumor center, tumor periphery, and the average of center and periphery are given. \*\*\*\* *p* < 0.0001, \*\*\* *p* < 0.001, \*\* *p* < 0.01, \* *p* < 0.05.

VDAC1 levels in the tumor periphery were similar to those in normal tissue and significantly lower than in the tumor center (*p* < 0.001) (Figure 1A–C and Figure 3A). NDUFS4 (subunit of complex I) expression did not differ between normal tissue and the tumor center or the periphery (Figures 2 and 3B). SDHA (subunit of complex II) levels were significantly lower in the tumor periphery than in the tumor center (*p* < 0.05) and in adjacent normal tissue (*p* < 0.001) (Figure 1D–F and Figure 3C). No significant difference was detected between the tumor center and normal tissue (Figure 1D,E). UQCRC2 (subunit of complex III) expression was lower in the periphery compared to the control tissue (*p* < 0.01) (Figure 3D). MT-CO1 (subunit of complex IV) protein levels were significantly reduced in both the center (*p* < 0.0001) and periphery (*p* < 0.001) of the tumors compared to normal tissue (Figure 3E). Finally, ATP5F1A (subunit of complex V/ATP synthase) was significantly diminished in the tumor periphery (*p* < 0.001) and showed a trend to lower levels in the center, compared to controls (Figure 3F).

Areas with cells negative for one or more OXPHOS subunits were found in both tumors and normal tissue (Figure 2D–F,M–O): 3% (tumor center), 21% (tumor periphery), and 25% (normal tissue) of the cases showed a loss of VDAC1 in more than 30% of the analyzed cells; 43% (tumor center), 60% (tumor periphery), and 35% (normal tissue) of the cases showed a loss of NDUFS4 (Figure 2F); 30% (tumor center), 54% (tumor periphery), and 11% (normal tissue) showed a loss of SDHA; 34% (tumor center), 54% (tumor periphery), and 10% (normal tissue) showed a loss of UQCRC2; 45% (tumor center) (Figure 2O), 41% (tumor periphery), 0% (normal tissue) showed a loss of MT-CO1; and 23% (tumor center), 30% (tumor periphery), and 0% (normal tissue) showed a loss of ATP5F1A.

#### *3.3. Associations between OXPHOS Subunit and VDAC1 Expression and Clinical Outcome*

A significant inverse correlation between the percentage of VDAC1-positive cells and MT-CO1-negative cells was detected (*p* = 0.0093). A significant inverse correlation was found between VDAC1 score values and UICC stage (*p* = 0.0065; R = −0.4855) (Table 2).


**Table 2.** Correlations of OXPHOS subunits and VDAC1 with respect to clinical parameters.

VDAC1 levels in the tumor center were lower in cases with lymph node involvement (*p* = 0.0201). The tumor periphery showed a similar trend (mean score values N0 = 162 ± 31 versus N1 = 147 ± 46). The same trend was present when metastasis occurred in the tumor center (mean score value for M0 = 229 ± 54; mean score value for M1 = 186 ± 48) (Table 3). No di fferences were observed in the tumor periphery with respect to M stage. Significantly lower NDUFS4 levels were present in males compared to females (*p* = 0.0454).

**Table 3.** Mean staining scores of OXPHOS subunits and VDAC1 with respect to clinical parameters.


The non-parametric Mann–Whitney test was used for analysis.

We divided the cases into high/moderate and low expressors. High expressors were defined as having staining intensities above 2. Since, in general, the staining intensities were lower for NDUFS4 and MT-CO1, high expressors for these subunits were defined by staining intensities above 1.5. A significant di fference (*p* < 0.05) in survival was observed between these groups (high/moderate vs. low expressors) for VDAC1 expression (Figures 4 and 5).

**Figure 4.** High and low VDAC1 expressors with low and high survival times. (**A**) High VDAC1 expression in case 3 with a survival time of 0.49 months. (**B**) Low VDAC1 expression in case 5 with a survival time of 30.05 months. Magnification 100×. Scale bar = 100 μm.

**Figure 5.** Kaplan–Meier plot of patients with cholangiocellular carcinomas exhibiting low/moderate or high VDAC1 staining intensity. High expressors are shown in red and low expressors in green. (**A**) VDAC1; (**B**) NDUFS4; (**C**) SDHA; (**D**) UQCRC2; (**E**) MT-CO1; (**F**) ATP5F1A. Significantly shorter survival was present in low/moderate expressors (*p* < 0.05).

No correlations were found between survival and expression of any of the markers of differentiation (epithelial: CK7, CK19; mesenchymal: vimentin), cell cycle proteins (p16, p27, p53 and Ki67) and OXPHOS subunits. Kaplan–Meier analysis revealed that individuals with tumors with high VDAC1 expression (staining intensity > 2) had significantly reduced overall survival (*p* < 0.05) compared to low expressors (staining intensity ≤ 2). None of the OXPHOS complexes was significantly associated with survival. Score values, extensities (percent positive/negative cells) and the intensities were used for the overall survival analysis. However, only the intensity of VDAC1 staining was associated with survival. Therefore, we suppose that the staining intensity might be an independent prognostic factor for CCCs indicating more an on–off-phenomenon than a gradient mechanism.
