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Abstract

BACKGROUND:

Isoform-specific function of doublecortin-like kinase 1 (DCLK1) has highlighted the key role of the DCLK1-S (short isoform) in the maintenance, progression, and invasion of the tumor.

OBJECTIVE:

This study was designed to produce an anti-DCLK1-S polyclonal antibody to evaluate DCLK1-S in human colorectal cancer (CRC) specifically.

METHODS:

The expression pattern and clinical significance of DCLK1-S were assessed in a well-defined tissue microarray (TMA) series of 348 CRC and 51 adjacent normal tissues during a follow-up period of 108 months.

RESULTS:

Expression of DCLK1-S was significantly higher in CRC samples compared to adjacent normal samples ( $P<$ 0.001). Cytoplasmic expression of DCLK1-S was significantly higher in the tumors at the advanced stage of cancer and with poorer differentiation ( $P<$ 0.001, $P=$ 0.02). The patients with CRC whose tumors showed higher cytoplasmic expression of DCLK1-S had worse disease-specific survival (DSS) (log-rank test, $P=$ 0.03) and 5-year DSS rates ( $P=$ 0.01). Additionally, an improved prognostic value was observed in the patients with CRC with high DCLK1-S expression vs. its moderate expression (HR: 2.70, 95% CI: 0.98–7.38; $p=$ 0.04) by multivariate analysis.

CONCLUSIONS:

Our findings strongly supported that high cytoplasmic expression of DCLK1-S compared to its moderate expression could be considered an independent prognostic factor influencing DSS.

Keywords

Colorectal cancer DCLK1-S polyclonal antibody immunohistochemistry tissue microarray

1. Introduction

Colorectal cancer (CRC) is the fourth most frequently diagnosed cancer and the leading cause of global death [1]. CRC also makes up the third most frequent cancer in males and second in females in Iran, with the increase of almost twice the mortality rate from 2.87 in 1990 to 6.8 in 2017 in 100,000 in both sexes [2, 3]. Despite developments in anticancer treatments, most CRC cases are resistant to conventional cancer therapies. Surgery, radiotherapy, chemotherapy, and targeted therapy are used as the current treatment for patients with CRC. Combining these traditional methods with new targeted therapy strategies could increase the survival rate of patients with CRC [4]. Therefore, identifying biomarkers can be considered an essential step for improving targeted molecular therapies (immunotherapy) and increasing the survival of patients with cancer. Considering the crucial role of cancer stem cells (CSCs) in tumorigenesis, tumor invasion, and drug-resistance, many efforts have been made to discover specific markers to target CSCs in various cancers, including CRC [5, 6, 7, 8]. Among various markers used for identification of CRCCSCs, doublecortin-like kinase 1 (DCLK1), as a putative CSC marker has been shown to have a crucial oncogenic role in various in-vivo and in-vitro experiments, particularly in pancreatic and colon cancers [7, 9, 10, 11, 12] so that, DCLK1 is called as a well-known CRCCSC marker distinguished cancer cells from normal cells in Apc Min/ $+$ mice [9].

In the recent decades, many experiments have focused on the biological function of two different transcripts of DCLK1; long isoform (DCLK1-L, NM_004734.4) and short isoform (DCLK1-S, NM_ 001195415.1) with a molecular weight of $\sim$ 80–82 and $\sim$ 45–50 KDa, respectively [11, 13, 14]. Evidence has suggested more than 98% of similarity between DCLK1-L and DCLK1-S isoforms at the C-terminal end of both proteins. At the same time, the only difference in the sequence of six amino acids was found at the N-terminal end of the DCLK1-S protein [11, 12]. Further molecular evidence regarding the isoform-specific function of DCLK1 in tumor initiation, proliferation, and aggressiveness has shown the oncogenic role of DCLK1-L at the early stages and DCLK1-S in progression and invasion of CRC and pancreatic cancer [10, 15, 16]. Epigenetically, due to the silencing of the 5’ ( $\alpha$ )-promoter of the DCLK1 gene as an early event during CRC carcinogenesis, overexpression of DCLK1 in CRC experiments was a vague issue [11]. This controversy was addressed when the oncogenic biological role of an alternative transcriptional $\beta$ -promoter of DCLK1 was clarified [11]. O’Connell et al. suggested that DCLK1-S isoform originated from the $\beta$ -promoter mainly related to aggressive behavior and CSC characteristics, however, DCLK1-L isoform was basically reported in normal or low stage CRC cells [11, 17]. They showed higher expression of DCLK1-S in the patients with CRC with shorter overall survival by quantitative real-time polymerase chain reaction (qRT-PCR) technique and ultimately they suggested DCLK1-S as a specific CRC biomarker for characterization of high-risk patients after colonoscopy [11, 12]. Other molecular experiments have reported overexpression of DCLK1-S vs. DCLK1-L in tumor cells compared to normal cells influenced by forkhead-box-D3 (FOXD3) gene in the regulation of DCLK1-S in colorectal carcinomas [18]. However, more investigations are required to define the specific biological difference between these two isoforms yet.

In our previous research, expression pattern and prognostic significance of DCLK1 have been studied in colorectal, gastric, and bladder carcinomas [19, 20, 21, 22, 23] using available commercial anti-DCLK1 antibodies that can detect C-terminal domain of both DCLK1-L/S isoforms or only N-terminal end of DCLK1-L isoform. In contrast, the role of the main isoform to overexpress DCLK1 has remained unknown. To the best of our knowledge, there is no commercial anti-DCLK1-S antibody. Also, no study has been previously performed to investigate the prognostic significance of DCLK1-S on tumor tissues, particularly in CRC by immunohistochemistry (IHC). Therefore, for addressing these gaps, the present study was conducted to generate an anti-DCLK1-S-specific polyclonal antibody elicited by six amino acids synthetic peptide that has been reported as the only different form in the sequence homology epitopes of both DCLK1 isoforms (DCLK1-L and DCLK1-S) [12].

In the current study, for the first time, membranous, cytoplasmic, and nuclear localization of DCLK1-S elicited by polyclonal anti-DCLK1-S antibody, generated in our laboratory was evaluated in a large number of formalin-fixed, paraffin-embedded (FFPE) series of CRC samples by tissue microarray (TMA)-based IHC analysis. The prognostic significance of short isoform of DCLK1 (DCLK1-S) was also investigated in this series of patients with CRC.

2. Materials and methods

2.1 Production of anti-DCLK1-S-specific polyclonal antibody

Polyclonal antibody to DCLK1-S was generated based on the protocols were described previously [24, 25]. DCLK1-S specific antibody was produced and the reactivity of rabbits sera as well as the purified antibodies was evaluated by ELISA. A synthetic peptide corresponding to the published amino acid sequence of DCLK1-S isoform (MLELIE) was selected as immunogen [12]. A cysteine residue was added to C-terminal of peptide (NH2-Met-Leu-Glu-Leu-Ile-Glu-Cys-COOH) for conjugation with Maleimide-activated keyhole limpet hemocyanin (mcKLH) (Thermo Scientific, Rockford, IL, USA) and used for immunization of two female New Zealand White Rabbits purchased from Pasteur Institute of Iran. Two rabbits were immunized with 250 $\mu$ g of KLH-peptide mixed with complete Freund’s adjuvant. After three weeks, four boosters of 125 $\mu$ g KLH-peptide mixed with incomplete Freund’s adjuvant were injected on four occasions at 14-day intervals. Test bleeds were taken before each injection, and serum was obtained and tested by ELISA against DCLK1-S peptide. The serum was compared with prebleeds taken from the same rabbit before immunization and stored in $-$ 20 ${}^{\circ}$ C [25]. The main isotype of polyclonal antibody against DCLK1-S was IgG.

To assess conjugation efficacy, the peptide was also conjugated with Imject Maleimide-activated bovine serum albumin (BSA) (Thermo Scientific, Rockford, IL, USA). Conjugation efficacy was investigated by analysis of the electrophoretic pattern of the BSA-conjugate [24]. Production of DCLK1-S specific antibodies was evaluated by enzyme-linked immunosorbent assay (ELISA) Anti-DCLK1-S specific antibodies were purified by the peptide coupled to CNBr-activated Sepharose-4B. The purity and reactivity of the purified antibodies, were assessed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and ELISA techniques, respectively. The immunoreactivity pattern of the generated antibody was compared with available commercial anti-DCLK1 antibody; ab31704 (which is commonly used to detect long and short DCLK1 isoforms; Abcam, UK) in CRC tissues by IHC method.

2.2 Indirect ELISA

Reactivity of rabbits sera, as well as the purified anti DCLK1-S antibodies, was tested by ELISA Briefly, the coating was performed using 10 $\mu$ g/ml of the peptide diluted in phosphate-buffered saline (PBS, 0.15 M, pH 7.2), which was incubated at 37 ${}^{\circ}$ C for 1 h followed by overnight incubation at 4 ${}^{\circ}$ C. After several washes, plates were blocked using 2.5% skimmed milk at 37 ${}^{\circ}$ C for 1 h and then, serial dilutions of rabbits sera or the purified antibodies were added. Following serum incubation (37 ${}^{\circ}$ C for 1.5 h) and washing, a horseradish peroxidase (HRP) conjugate of sheep anti-rabbit immunoglobulin (Ig) antibody (Avicenna Research Institute, Tehran, Iran) diluted 1:1500 in phosphatebuffered salineTween 20 (PBS-T) was added to the wells (37 ${}^{\circ}$ C, 1.5 h). Following washing, substrate, 3, 3’, 5, 5’-tetramethylbenzidine (TMB) (Pishtaz Teb Zaman, Iran) was added and optical density (OD) values were measured at the wavelength of 450 nm on a microplate reader (Synergy HTXMulti-Mode Reader, BioTek, USA).

2.3 Sample collection and patients characteristics

FFPE series of 385 CRC samples and 70 matched adjacent normal tissues were collected from university hospitals including Hasheminejad, Rasool Akram, and Firoozgar during 2010–2019 in Tehran, Iran. Clinicopathological information including age, gender, tumor size, tumor differentiation, vascular and neural invasion, lymph node involvement, tumor, nodes, and metastases (TNM) staging, distant metastasis, and tumor recurrence was collected from the patients’ documents. None of the patients with CRC in this study had received neoadjuvant treatment before surgery. Size cutoff and TNM stage of the tumors were determined according to the American joint committee on cancer/union for international cancer control (AJCC/UICC) and the TNM staging system, respectively [26]. The patients’ data were kept anonymous in all steps of the study. The interval from the date of surgery to the date of death by CRC was used for disease-specific survival (DSS) analysis.

Moreover, progression-free survival (PFS) was defined as the interval between the primary surgery and the last follow-up visit if the patient showed no evidence of disease, tumor recurrence, or distant CRC metastasis. The current study was approved by the Human Research Ethics Committee of the Iran University of Medical Sciences, Tehran, Iran (Ref No: IR.IUMS.REC 1396.32186). All the procedures performed in this study were following the 1964 Declaration of Helsinki and its later amendments. Informed consent was obtained from all participants, parents or legally authorized representatives of participants at the time of sample collection with routine consent forms.

2.4 TMA construction

As described previously, colorectal tissue arrays were prepared and constructed by a TMA instrument (Minicore; ALPHELYS, Plaisir, France), as described previously [6, 27, 28]. Pathologist pointed three representative areas of the tumors on hematoxylin & eosin (H & E) slides of CRC samples. Different regions of each tumor were punched into TMA recipient blocks in three copies. Based on TMA studies, each core would denote the whole tissue’s staining pattern with 90% accuracy, while using two cores or more could increase this accuracy by 95–99%, respectively [29]. In this study, three cores were evaluated from each tissue sample to overcome heterogeneity in antigen expression and increase the accuracy and validity of the analysis. The mean score of the three cores was calculated as the final score for IHC analysis of DCLK1-S.

2.5 Immunohistochemistry

Immunostaining of CRC slides (whole tissue and TMA slides) was performed by standard chain polymer-conjugated antibody (EnVision; standard EnVision-HRP kit, Bio pharmadx), as described previously [6, 21, 30]. Briefly, following de-waxing at 60 ${}^{\circ}$ C for 30 min and rehydration steps, heat-activated antigen was retrieved by autoclaving the sections in citrate buffer (10 mM, pH 6) at 95 ${}^{\circ}$ C for 11 min. Endogenous peroxidase activity and non-specific binding sites were blocked by H2O2 (3 %) and 5 % normal sheep serum diluted in protein block (Dako, CA, USA). The sections were incubated overnight using a polyclonal anti-DCLK1-S antibody generated in our laboratory (0.75 $\mu$ g/ml) at 4 ${}^{\circ}$ C. Then, the sections were treated with the secondary antibody, ${}^{\text{TM}}$ Mouse/Rabbit PolyVue HRP/DAB Detection kit (standard EnVision-HRP kit, Bio pharmadx), at room temperature (RT). The 3; 3’-diaminobenzidine (DAB, Dako, Denmark) substrate as a chromogen was used to visualize immune signals. The sections were counterstained with hematoxylin (Dako, Denmark) for 5 min, were dehydrated and finally, were mounted. Humans CRC whole tissue was selected as positive control for DCLK1-S. In negative reagent controls, the primary antibody was replaced by non-immune rabbit IgG (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) as isotype control to confirm specific bindings of anti-DCLK1-S antibody. In addition, in all the experiments, the specificity of the produced antibodies was evaluated by blocking antibodies with saturating concentration (1:100 molar ratio) of immunizing peptide before incubating the slides with the primary antibody. The primary antibody was also replaced by TBS as a negative control to confirm the absence of non-specific bindings of the secondary antibody. Normal and cancerous tissues of the ovary [31], testis, and skin were also used as negative control tissues based on protein atlas databases for further confirmation of the specificity of our polyclonal antibody. Digital images were captured by an H550S microscope (Nikon, Japan) and a Digital sight DS-LS camera (Nikon, Japan).

2.6 Evaluation of immunostaining

The scoring step was performed semi-quantitatively in a coded manner by two pathologists (MA and MR) who were blinded to clinical and pathological parameters (as described previously) [32], and a consensus agreement was achieved. Given a deal to the final score of DCLK1-S expression, the general distribution of the tumor cells was assessed at 10 $\times$ magnification. Then, positive cells were evaluated semi-quantitatively at higher magnifications, and final scores were given, respectively. Percentage of positive cells, the intensity of the staining, and histochemical score ( $H$ -score) were three scoring methods by which the expression pattern of DCLK1-S was evaluated. The intensity of immunostaining was divided into four groups; 0 (negative), 1 (weak), 2 (moderate), and 3 (strong) staining. The percentage of positive cells was valued semi-quantitatively and was scored as 0–100%. The $H$ -score was obtained by multiplying intensity (0–3) by percentage scores (0–100%), which led to the generation of the final scores of 0–300. In the present study, $H$ -score was divided into three groups: 0–100 (low expression), 101–200 (moderate), and 201–300 (high expression) [33].

Figure 1.

Different localization pattern of DCLK1-S expression in CRC tissues by IHC staining: considering our findings A) cytoplasmic expression, B) membranous, and C) nucleus expression of DCLK1-S were observed in CRC samples (All images were taken at 400 $\times$ magnification).

Figure 2.

Immunohistochemical staining of DCLK1-S in CRC tissues and negative controles. (A) Negative, (B) low, (C) moderate, and (D) high expression of DCLK1-S in CRC tissue. Replacement of the primary antibodies by (E) pre-immune purified rabbit IgG (isotype control) and (F) Tris Buffer Saline, and (G) primary antibody saturated with immunizing peptide, as the negative reagent controls. (H) Negative expression of DCLK1-S in adjacent normal tissue. The results showed that increased cytoplasmic expression of DCLK1-S in CRC samples in comparison to adjacent normal samples (All images were taken at 200 $\times$ magnification).

2.7 Statistical analysis

SPSS software version 22 (IBM Corp, USA) was used for statistical analysis. Pearson’s $\chi^{2}$ and Spearman’s correlation tests were performed to analyze the significance of association and correlation between DCLK1-S protein expression and clinicopathological features. Moreover, Kruskal-Wallis and Mann-Whitney U tests were carried out to compare the groups. DSS and PFS analysis were used to adopting the Kaplan-Meier method, with along the log-rank test to estimate curves between the groups with 95% confidence interval (CI). To determine the variables were influenced by DSS or PFS, Cox (Proportional Hazards) Regression was performed. All the categorical data were described by N (%), a valid percentage and quantitative data were exhibited as mean, standard deviation (SD), median, and quartile (Q1, Q3). We reported our results by a two-sided $P$ -value and a $P$ -value $<$ 0.05 was considered statistically significant.

3. Results

3.1 Production of antibody and characterization

3.1.1 Production of anti-DCLK1-S antibody

Efficacy of conjugation and immunization was evaluated by SDS-PAGE analysis of the BSA-conjugate and ELISA test, respectively. The smear pattern of SDS-PAGE and the absence of free peptide showed effective conjugation of BSA-peptide (supplementary Fig. 1A). The immunoreactivity of the antibodies was confirmed by the ELISA test. The results showed high immunoreactivity of the antibodies reaching to plateau as low as an antibody concentration of 1250 ng/ml (Supplementary Fig. 1B).

3.1.2 Validation of anti-DCLK1-S antibody

Immunoreactivity of anti-DCLK1-S was evaluated by the IHC method on CRC whole tissues as positive control samples and humans normal and cancerous tissues of testis, ovary, and skin as negative control samples. In addition, all the experiments used isotype control and primary antibody saturated with immunizing peptides as negative reagent controls. The results clearly showed a specific pattern of immunoreactivity in CRC tissues compared to negative control tissues (Fig. 2 and supplementary Fig. 2). The antibody could strongly recognize DCLK1-S in CRC samples, and localization was restricted to the cytoplasmic area and partially in the cell membrane and nucleus area of CRC samples (Fig. 1). No staining was observed in humans in normal and cancerous tissues of testis, ovary, and skin, demonstrating specific immunoreactivity of the produced anti-DCLK1-S antibody (supplementary Fig. 2).

The commercial anti-DCLK1 antibody, which can recognize the C-terminal end of both long and short isoforms of DCLK1 (anti-DCLK1-L/S antibody; ab31704) was used to compare the expression pattern of DCLK1-L/S vs. DCLK1-S. Results showed no difference in the expression pattern using the generated anti-DCLK1-S antibody and the commercial anti-DCLK1-L/S antibody, as shown in supplementary Fig. 3. No staining was also observed in negative control sections incubated by pre-adsorbed or substituted by pre-immune-purified rabbit IgG as primary antibody (Fig. 2).

3.2 Immunohistochemical evaluation of anti-DCLK1-S antibody in CRC tissues

3.2.1 Study population

Considering technical problems during TMA construction and IHC staining, among all 385 collected CRC samples and 70 adjacent normal tissues, 348 CRC and 51 adjacent normal tissue samples remained for statistics analysis of DCLK1-S expression. Patients had a mean age of 60 $\pm$ 14.4 years old, and males accounting for 51.8% of our study population. Mean tumor size was equal to 5 $\pm$ 2.2 cm (ranged from 2 to 14 cm), of which 236 (67.6%) were less than 5 cm. All the clinicopathological characteristics of the study population are summarized in Table 1.

3.2.2 Expression of DCLK1-S in the patients with CRC and adjacent normal samples

IHC analysis results showed that most CRC samples (293/348, 84.2%) had a positive DCLK1-S expression. All the positive samples represented cytoplasmic localization of DCLK1-S in tumor cells area (293/293, 100%), which 9.5% (28/293) of them showed both cytoplasmic and nuclear expressions and 4.7% (14/293) of them showed both cytoplasmic and membranous expression of DCLK1-S (Fig. 1). The findings of the intensity of staining, percentage of positive tumor cells, and $H$ -score in the cytoplasm of CRC and adjacent normal tissue samples were demonstrated in Table 2. Our findings indicated the high expression of DCLK1-S in CRC samples compared to adjacent normal tissue samples (Fig. 3).

Figure 3.

Box plot analysis of DCLK1-S expression levels in CRC specimens versus adjacent normal tissues. The results of Mann-Whitney U test showed that there is a statistically significant differences in the median expression levels of DCLK1-S among CRC and adjacent normal tissues ( $P<$ 0.001). Based on standard definitions, each box-plot demonstrated the median (bold line) and interquartile lines (box) outliers (circle), and extreme observations (star).

3.2.3 Cytoplasmic expression of DCLK1-S was associated with tumor aggressiveness in CRC tissues

The results of Pearson’s $\chi^{2}$ test revealed a statistically significant association between overexpression of DCLK1-S and advanced TNM stage ( $P<$ 0.001) as well as the increased tumor differentiation ( $P=$ 0.03). Furthermore, the results of Spearman’s correlation test exhibited a significant direct correlation between cytoplasmic DCLK1-S expression and TNM stage ( $P<$ 0.001) and also tumor differentiation ( $P=$ 0.02). Moreover, the Kruskal-Wallis test results indicated a statistically significant difference between the median cytoplasmic expression level of DCLK1-S and tumor differentiation ( $P=$ 0.04). Mann-Whitney $U$ test also showed a statistically significant difference in the median expression level of cytoplasmic DCLK1-S between poor and moderate tumor differentiation ( $P=$ 0.04). Results of statistical analysis did not show any significant association between DCLK1-S expression and

Table 1
The association between cytoplasmic DCLK1S expression and clinicopathological parameters of colorectal cancer (CRC) samples (intensity of staining and h-score)

			Intensity of staining $N$ (%)									H-score $N$ (%)
Patients and tumor characteristics	Total samples $N$ (%)		Negative (0)		Weak (1 $+$ )		Moderate (2 $+$ )		Strong (3 $+$ )		$P$ -value	Low (0–100)		Moderate (100–200)		High (200–300)		$P$ -value
Colorectal cancer
Mean age, years (range)
$\leqslant$ 60	176	(52.9)	27	(15.3)	85	(48.3)	46	(26.2)	18	(10.2)		120	(68.2)	50	(28.4)	6	(3.4)
$>$ 60	157	(47.1)	28	(17.8)	85	(54.1)	26	(16.6)	18	(11.5)	0.21	125	(79.6)	28	(17.9)	4	(2.5)	0.11
Not identified
Gender
Male	177	(51.8)	29	(16.4)	91	(51.4)	35	(19.8)	22	(12.4)	0.59	130	(73.5)	42	(23.7)	5	(2.8)	0.98
Female	165	(48.2)	26	(15.8)	86	(52.1)	39	(23.6)	14	(8.5)		122	(73.9)	38	(23)	5	(3.1)
Mean tumor size (mm)
$\leqslant$ 5	236	(67.6)	31	(13.2)	125	(53.5)	56	(23.5)	24	(9.8)		169	(71.6)	59	(25)	8	(3.4)
$>$ 5	112	(32.4)	24	(21.4)	54	(48.2)	21	(18.8)	13	(11.6)	0.20	85	(75.9)	24	(21.4)	3	(2.7)	0.76
Not identified
Differentiation
Well	121	(35)	18	(14.9)	64	(52.9)	26	(21.5)	13	(10.7)		95	(78.5)	20	(16.5)	6	(5)
Moderate	192	(55.5)	32	(16.7)	100	(52.1)	40	(20.8)	20	(10.4)	0.82	139	(72.4)	50	(26)	3	(1.6)	0.02
Poor	33	(9.5)	5	(15.2)	14	(42.4)	11	(33.3)	3	(9.1)		19	(57.6)	13	(39.4)	1	(3)
Not identified
Primary tumor (PT) stage
pT1	52	(17.6)	9	(17.3)	31	(59.7)	10	(19.2)	2	(3.8)		43	(82.7)	8	(15.4)	1	(1.9)
pT2	133	(44.9)	21	(15.8)	73	(54.9)	26	(19.5)	13	(9.8)	0.29	104	(78.2)	27	(20.3)	2	(1.5)	$<$ 0.001
pT3	100	(33.8)	21	(21)	46	(46)	17	(17)	16	(16)		73	(73)	23	(23)	4	(4)
PT4	11	(3.7)	1	(9.1)	6	(54.5)	1	(9.1)	3	(27.3)		8	(72.7)	0	(0)	3	(27.3)
Not identified
Vascular invasion (VI)
Present	59	(17.2)	14	(23.7)	18	(30.5)	18	(30.5)	9	(15.3)		36	(61)	21	(35.6)	2	(3.4)
Absent	284	(82.8)	40	(14.1)	160	(56.3)	57	(20.1)	27	(9.5)	0.13	215	(75.7)	61	(21.5)	8	(2.8)	0.078
Not identified
Lymph node invasion (LNI)
Involved	118	(34.3)	25	(21.2)	48	(40.7)	27	(22.8)	18	(15.3)	0.009	79	(66.9)	33	(28)	6	(5.1)	0.007
None	226	(65.7)	29	(12.8)	130	(57.5)	49	(21.7)	18	(8)		173	(76.5)	49	(21.7)	4	(1.8)
Not identified
Neural invasion (NI)
Involved	67	(19.5)	12	(17.9)	28	(41.8)	19	(28.4)	8	(11.9)	0.30	42	(62.7)	24	(35.8)	1	(1.5)	0.41
None	276	(80.5)	42	(15.3)	150	(54.3)	56	(20.3)	28	(10.1)		209	(75.7)	58	(21)	9	(3.3)
Not identified
Distant metastasis
Present	67	(28.5)	9	(13.4)	30	(44.8)	20	(29.9)	8	(11.9)	0.51	45	(67.2)	18	(26.8)	4	(6)	0.46
Absent	168	(71.5)	19	(11.3)	91	(54.2)	36	(21.4)	22	(13.1)		118	(70.2)	44	(26.2)	6	(3.6)
Not identified
Tumor recurrence
Yes	72	(30.6)	9	(12.5)	33	(45.9)	22	(30.5)	8	(11.1)	0.33	46	(64)	22	(30.5)	4	(5.5)	0.48
No	163	(69.4)	19	(11.7)	88	(54)	34	(20.8)	22	(13.5)		117	(71.8)	40	(24.5)	6	(3.7)
Not identified

$P$ value; Pearson’s $\chi^{2}$ test. H-score histological score. Values in bold are statistically significant.

other clinicopathological characteristics. All findings are available in Table 1.

Table 2

Cytoplasmic expression of DCLK1s in colorectal cancer (CRC) and adjacent normal tissue samples

Scoring system	CRC samples $N$ (%)		Adjacent normal tissue samples $N$ (%)	$P$ -value
Intensity of staining
Negative (0)	55	(15.8)	28 (54.9)
Weak ( $+$ 1)	180	(51.7)	20 (39.2)	$<$ 0.001
Moderate ( $+$ 2)	77	(22.2)	3 (5.9)
Strong ( $+$ 3)	36	(10.3)	0 (0)
Percentage of positive tumor cells
$<$ 25%	83	(23.9)	33 (64.7)
25–50%	29	(8.3)	2 (3.9)	$<$ 0.001
51–75%	56	(16.1)	4 (7.8)
$>$ 75%	180	(51.7)	12 (23.6)
H-score (3 groups)
Low (0–100)	255	(73.3)	48 (94.1)
Moderate (101–200)	83	(23.8)	3 (5.9)	$<$ 0.001
High (201–300)	10	(2.9)	0 (0)
Total	348		51

H-score histological score. $P$ value is based on Mann-Whitney $U$ test. Values in bold are statistically significant.

3.2.4 Cytoplasmic expression of DCLK1-S was a poor prognostic factor of disease-specific survival (DSS) in CRC tissues

Among 348 CRC samples included in this study, 235 (67.5%) samples had a history of tumor recurrence, distant metastasis, or cancer-related death events. Distant metastasis and tumor recurrence occurred in 67 (28.5%) and 72 (30.6%) of patients, respectively, while in 80 (34%) of patients, metastasis and recurrence were not observed. During the follow-up period, cancer-related death and the other causes of death were documented in 67 (90.5%) and 7 patients (9.5%), respectively. Mean and median follow-up times were equal to 43.5 (SD $=$ 29.7) and 38 (21, 38) months, with a range of 1–108 months.

The results of Kaplan-Meier survival analysis represented that the CRC patients with high cytoplasmic expression of DCLK1-S had shorter DSS compared to those with moderate and low cytoplasmic DCLK1-S expression (log-rank test, $P=$ 0.03) (Fig. 4a). Mean $\pm$ SD of DSS time was equal to 48 $\pm$ 14, 69 $\pm$ 6, and 81 $\pm$ 3.5, for the patients with high, moderate, and low cytoplasmic DCLK1-S expression, respectively. There was no significant difference between PFS and the patients with high, moderate, and low cytoplasmic expression of DCLK1-S (log-rank test, $P=$ 0.17) (Fig. 4b). Mean $\pm$ SD of PFS time was equal to 48 $\pm$ 14, 67.7 $\pm$ 6, and 74 $\pm$ 3.6, for the patients with high, moderate, and low cytoplasmic DCLK1-S expression, respectively. Moreover, 5-year DSS was obtained as 50, 62, and 74%, for the patients with high, moderate, and low levels of cytoplasmic expression of DCLK1-S, respectively (mean $\pm$ SD of DSS time was equal to 48 $\pm$ 14, 69 $\pm$ 6, and 81 $\pm$ 3.5, for the patients with high, moderate, and low cytoplasmic expression of DCLK1-S, respectively $P=$ 0.01).

Figure 4.

Kaplan-Meier curves for disease-specific survival (DSS) and progression-free survival (PFS) according to the expression levels of DCLK1-S in CRC. (A, B). The mean time was (48 $\pm$ 14, 69 $\pm$ 6, and 81 $\pm$ 3.5) for DSS and (48 $\pm$ 14, 67.7 $\pm$ 6, and 74 $\pm$ 3.6) for PFS in the patients with high, moderate, and low cytoplasmic DCLK1-S expression, respectively. Moreover, Kaplan-Meier survival analysis showed that increased cytoplasmic expression of DCLK1-S are significantly related to a DSS ( $P=$ 0.034) and are not significantly related to PFS ( $P=$ 0.175).

Table 3

Univariate and multivariate Cox regression analyses of potential prognostic factor for disease-specific survival (DSS) in patients with colorectal cancer (CRC)

Covariate	Univariate analysis		Multivariate analysis
	HR (95% CI)	$P$ -value	HR (95% CI)	$P$ -value
Cytoplasmic DCLK1s expression		0.04		0.14
High versus Low	1.61 (0.97–2.68)	0.06	1.22 (0.67–2.22)	0.51
High versus Moderate	2.58 (1.023–6.52)	0.04	2.70 (0.98–7.38)	0.04
Age	1.65 (0.79–2.34)	0.04	1.73 (1.007–1.99)	0.02
Tumor differentiation		0.01		0.94
Moderate versus well	1.46 (0.86–2.49)	0.15	1.34 (0.75–2.38)	0.23
Poor versus well	2.99 (1.47–6.11)	0.003	2.31 (0.85–6.31)	0.31
Primary tumor (PT) stage		0.02		0.22
pT2 versus pT1	1.15 (0.49–2.67)	0.74	1.09 (0.46–2.57)	0.83
pT3 versus pT1	2.38 (1.04–5.45)	0.04	1.85 (0.78–4.38)	0.15
pT4 versus pT1	1.08 (0.22–5.24)	0.91	0.94 (0.17–4.98)	0.94
Vascular invasion (VI)	1.84 (1.08–3.11)	0.02	–	–
Neural invasion (NI)	0.57 (0.34–0.94)	0.03	–	–
Lymph node invasion (LNI)	0.45 (0.28–0.71)	0.001	–	–
Distant metastasis	0.17 (0.10–0.28)	$<$ 0.001	–	–
Tumor recurrence	0.18 (0.11–0.30)	$<$ 0.001	–	–

HR: hazard ratio, CI: confidence interval. The variables with $P$ value less than 0.05 and HR more than 1.0 were included in multivariable analyses. Values in bold are statistically significant.

As demonstrated in Table 3, the results of the univariate analysis showed that cytoplasmic expression of DCLK1-S, age, tumor differentiation, and TNM stage were significant risk factors influencing DSS. Moreover, the prognostic value was increased in the high cytoplasmic expression of DCLK1-S vs. moderate expression of DCLK1-S (HR: 2.70, 95% CI: 0.98–7.38; $p=$ 0.04) in the patients with CRC in multivariate analysis. Therefore, high cytoplasmic expression of DCLK1-S compared to moderate expression could be considered as an independent prognostic factor of DSS in multivariate analysis.

4. Discussion

DCLK1, a putative CSC marker is a member of microtubule-associated proteins whose biological function has been identified in different human cancers, particularly in colorectal and pancreatic carcinomas [10, 12, 34]. The previous studies have also reported that suppressing DCLK1 had a significant direct correlation with tumor invasiveness and drug resistance in renal clear cell cancer and pancreatic cancer [35, 36]. In CRC, upregulation of DCLK1 is significantly associated with tumor progression, aggressiveness, metastasis, and poor prognosis [37]. Therefore, the oncogenic role of DCLK1 in a wide range of biological studies has suggested the prominence of the DCLK1 marker as a promising molecular target in the treatment of CRC. From this point, finding distinguished CSC markers which have not been identified on normal stem cells play a fundamental role in targeted therapy strategies and tumor eradication. Nakanishi et al. revealed CSC characteristics of DCLK1 in intestinal tumor cells but not in normal stem cells in-vivo [9]. Vreugdenhil et al. cloned different transcripts of DCLK1 and discovered two distinguishing alternative splicing variants of DCLK1 transcripts; DCLK-short and DCLK-long [38]. The previous studies have also highlighted these transcripts’ isoform-specific function and localization profile in Wistar rats’ brains [14]. Interestingly, epigenetic differences have discovered that DCLK1-S isoform ( $\sim$ 47 KDa), originated from $\beta$ -promoter plays a significant role during colon carcinogenesis by the response to oncogenic pathways. In comparison, DCLK1-L isoform ( $\sim$ 82 KDa) could be detected in the early stage of CRC and normal colonic mucosal samples vs. high-stage tumors [11, 17]. These investigations have strongly supported that DCLK1-S as an isoform of DCLK1 is identified as a requisite factor in tumor invasion, proliferation, and metastasis potential of CRC cells [17].

Although many preliminary studies have confirmed the clinical value of DCLK1 expression in tumor progression and aggressiveness, the available commercial anti-DCLK1 antibodies widely used in the previous studies could detect sequence homology epitopes of both isoforms, DCLK1-L and DCLK1-S, or only DCLK1-L isoform; hence, the specific implication of DCLK1-S has remained largely unknown.

In our previous works, the oncopathological value of DCLK1 expression was evaluated in various cancers, including colorectal, bladder, and gastric carcinomas; results were in line with other studies [19, 21, 35, 39, 40, 41, 42]. In a comprehensive study on 472 bladder cancer cases, an increase was found in the expression of DCLK1 (DCLK1-L/S) that associated with more aggressive tumors, advanced stages, and poorer DSS in the patients with bladder cancer [21]. Additionally, results of our previous study on local and circulating DCLK1 (DCLK1-L) in 58 fresh CRC tissues and their correspondent normal margins demonstrated significantly higher expression of DCLK1 in the patients with CRC having advanced-stage and higher tumor grade in both protein and mRNA expression levels [40]. We have recently reported a highly significant expression of DCLK1 (DCLK1-L/S) in CRC cases compared to adenomatous and non-adenomatous colonic polyps and a positive significant correlation between DCLK1 expression and tumor size, tumor differentiation, and lymph node involvement [22]. Other clinical investigations have also represented that higher expression of DCLK1 (DCLK1-L/S) had a significant positive correlation with tumor invasion, metastasis, and shorter survival in patients with renal clear cell carcinoma and bladder cancer [35, 42]. However, it is important to state that the commercial anti-DCLK1 antibodies used in these studies can only recognize the C-terminal end of DCLK1-L/S protein or only the N-terminal end of DCLK1-L isoform; thus, further studies are required to investigate the biological role of DCLK1-S in tumor progression and invasiveness.

Since the absence of the commercial anti-DCLK1-S antibody was the primary obstacle for assessing this molecule at the protein level, in the present study a polyclonal specific antibody against DCLK1-S was successfully developed in our laboratory. This will provide further opportunities to investigators to obtain more accurate and valuable findings related to DCLK1-S localization and its clinical significance as the unique DCLK1 isoform responsible for invasiveness and aggressive behavior in various tumors by the IHC method.

Our findings demonstrated that most CRC samples with positive expression of DCLK1-S had pronounced cytoplasmic localization, besides partially nuclear and very low membranous expression patterns of S-isoform. In parallel, the results of immune electron microscopy (IEM) have confirmed the presence of S-isoform in the cytosol, mitochondrial, and cell membrane fractions of HCT-116 and COLO205-S-GFP cell lines, unlike L-isoform that was present mainly in cell membrane fractions of HEK293 cells only expressing L-isoform [12]. Other clinical studies that used anti-DCLK1-L/S or anti-DCLK1-L antibodies have also reported mainly cytoplasmic and membranous localization of DCLK1 in gastric, colorectal, and bladder carcinoma tissues [20, 21, 32, 42]. Meanwhile in the current study, the nucleus localization of DCLK1-S was shown in tumor cells area.

Besides, O’Connell et al. suggested that DCLK1-S was the main DCLK1 isoform expressing in humans CRC cell lines vs. DCLK1-L markedly expressing in normal colorectal cell lines; thus, DCLK1-S can be used as a promising prognostic and targeted-therapeutic marker in the treatment of CRC [11]. Other studies have demonstrated higher expression of DCLK1-S in polyps from high-risk vs. low-risk patients and downregulation of DCLK1-L/FOXD3 in polyps from high-risk compared to low-risk patients, indicating the potential suppressor function of FOXD3 in the regulation of DCLK1-S [18]. The existence of FOXD3 in normal or low-risk polyps inhibited expression of DCLK1-S, while methylation of FOXD3 in cancerous cells can cause overexpression of DCLK1-S in high-risk cases.

Our comparative immunostaining findings showed a similar pattern of immunoreactivity by anti-DCLK1-S antibody and antiDCLK1-L/S antibody, which both represented strong staining of DCLK1 in CRC tissues. A similar staining pattern in both antibodies may be due to the fact that DCLK1-S is the predominant isoform in CRC tissues. Although, further studies are required to show different expressions of these two isoforms.

Evaluating the circulatory DCLK1-S could lead to a better understanding of its biology and function in the tumor. However, this retrospective study aimed to investigate the DCLK1-S local protein expression as a novel isoform in the colorectalcancer tissues and its association with CRC patients’ outcome therefore, FFPE series of CRC samples were collected. The current study provided novel and interesting insights into the expression pattern and prognostic significance of DCLK1-S in a well-defined series of CRC tissues. Results of IHC analysis revealed a significant upregulation of cytoplasmic DCLK1-S expression in CRC samples compared to adjacent normal tissues. Furthermore, a significant association was found between high expression of DCLK1-S and advanced TNM stage, the increased tumor differentiation, and poor DSS in these cases. Consistent with our study, only one study pointed to a significant clinical value of DCLK1-S expression associated with tumor aggressiveness at mRNA level in the patients with CRC [11]. Additionally, our results showed that 5-year DSS was equal to 50, 62, and 74% for the patients with high, moderate, and low cytoplasmic expression of DCLK1-S, respectively.

5. Conclusions

Taken together, to better understand the specific role of the main DCLK1 isoform in the aggressiveness of CRC tumors, a specific polyclonal anti-DCLK1-S antibody was produced identified only by six particular amino acids related to short isoform and its specificity was validated by IHC method in CRC samples. Our findings strongly supported the critical role of DCLK1-S as the DCLK1 isoform, which led to tumor aggressiveness and worsened DSS in CRC samples. Significantly, high cytoplasmic expression of DCLK1-S protein compared to moderate expression was considered an independent poor prognostic factor of DSS in these series of CRC cases. Hence, additional investigations and larger trials on the potential therapeutic role of DCLK1-S as a novel CRC agent can shed new light on the treatment of patients with CRC.

Authors’ contributions

Z.M. and R.Gh. designed and supervised the work; E.K. wrote the manuscript, did the literature search, data acquisition, and statistical analysis constructed TMA blocks, and performed all experiments. L.S. interpreted the data, helped to prepare the Tables, and contributed to edit the manuscript. M.R. scored TMAs slides after immunohistochemically staining and helped to prepare the Fig. 2. L.E. helped to prepare the blood sampling of the rabbits in the animal experiment section. M.R. helped with the literature search and prepared Fig. 1. M.A. contributed to manuscript edit and data analysis. All authors read and approved the final manuscript.

Funding

Research reported in this publication was supported by Elite Researcher Grant Committee under award number [982625] from the National Institutes for Medical Research Development (NIMAD), Tehran, Iran. The current study was part of a PhD thesis with Grant Number: 96-03-126-32186, which was carried out at the Oncopathology research center at the Iran University of Medical Sciences.

Ethics approval and consent to participate

The current study was approved by the Human Research Ethics Committee of the Iran University of Medical Sciences, Tehran, Iran (Ref No: IR.IUMS.REC 1396.32186). All the procedures performed in this study were under the 1964 Declaration of Helsinki and its later amendments. Informed consent was obtained from all participants, parents or legally authorized representatives of participants at the time of sample collection with routine consent forms.

Supplementary data

The supplementary files are available to download from http://dx.doi.org/10.3233/CBM-210330.

sj-pdf-1-cbm-10.3233_CBM-210330.pdf - Supplemental material

Supplemental material, sj-pdf-1-cbm-10.3233_CBM-210330.pdf

Footnotes

Acknowledgments

We are grateful to our colleague at Iran University of Medical Sciences who provided insight and expertise that greatly assisted the research. Apart from this, it must be declared that the authors received no specific funding for this work.

Conflict of interest

The authors declare no competing interests.

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Cytoplasmic expression of DCLK1-S,a novel DCLK1 isoform,is associated with tumor aggressiveness and worse disease-specific survival in colorectal cancer

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

1. Introduction

2. Materials and methods

2.1 Production of anti-DCLK1-S-specific polyclonal antibody

2.2 Indirect ELISA

2.3 Sample collection and patients characteristics

2.4 TMA construction

2.5 Immunohistochemistry

2.6 Evaluation of immunostaining

3. Results

3.1 Production of antibody and characterization

3.1.1 Production of anti-DCLK1-S antibody

3.1.2 Validation of anti-DCLK1-S antibody

3.2 Immunohistochemical evaluation of anti-DCLK1-S antibody in CRC tissues

3.2.1 Study population

3.2.2 Expression of DCLK1-S in the patients with CRC and adjacent normal samples

Table 1 The association between cytoplasmic DCLK1S expression and clinicopathological parameters of colorectal cancer (CRC) samples (intensity of staining and h-score)

5. Conclusions

Authors’ contributions

Funding

Ethics approval and consent to participate

Supplementary data

sj-pdf-1-cbm-10.3233_CBM-210330.pdf - Supplemental material

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
The association between cytoplasmic DCLK1S expression and clinicopathological parameters of colorectal cancer (CRC) samples (intensity of staining and h-score)