Introduction
CRC is the 3rd leading cause of cancer-related mortalities worldwide (Ferlay et al.
2015; Siegel et al.
2016). CRC is a highly malignant disease with tumor cells having a tremendous ability of metastasizing to distant organs, including liver, lungs, bones and brain. Among these potential organs, liver is the pre-dominant site for CRC metastasis, as it offers both, a suited soil and the first vascular bed for the circulating tumor cells (Sheth and Clary
2005; Valderrama-Trevino et al.
2017; Zarour et al.
2017). At the first round of medical check-up/ surgery for primary CRC, almost 15–20% of the patients are diagnosed with liver metastasis (synchronous metastasis). Furthermore, a significant proportion of CRC patients (> 50%) develop liver metastasis over the course of the disease (metachronous metastasis) and account for a major fraction of CRC associated mortality (Helling and Martin
2014; Jegatheeswaran et al.
2013; Valderrama-Trevino et al.
2017). Hepatic resection, along with systemic adjuvant regimens, is the present day therapeutic option for CRC liver metastasis, but it cures only a limited proportion of the patients (< 20%) and often cannot inhibit disease recurrence (House et al.
2011; Konopke et al.
2012; Tol and Punt
2006; Tomlinson et al.
2007). To summarize, after liver metastases have been established, available surgical and combinational therapies are of minor assistance to cure the disease, leading to a significantly reduced 5-year survival rate (10–15%) (Adam
2007; Adam et al.
2015; Alberts
2012; Riihimaki et al.
2016). In this scenario, it is of paramount importance to identify new therapeutic targets and means for improving the treatment options to possibly cure CRC liver metastasis.
In recent years, the chemokine network has been exploited extensively in search of new prognostic markers and therapeutic targets for treating cancers. Chemokines are basically a class of secretory chemo-attractant cytokines (8–14 kDa), which mediate a variety of physiological functions including cellular migration, development, survival, inflammatory responses and angiogenesis (Hughes and Nibbs
2018; Raman et al.
2011). In addition to their homeostatic and inflammatory functions, chemokines are being investigated for their potential role in cancer progression. Multiple aspects of tumor biology like angiogenesis, leukocyte infiltration and metastasis are affected via the chemokine network in an auto- and/or paracrine manner (Lacalle et al.
2017; Liu et al.
2017; Lopez-Cotarelo et al.
2017; Massara et al.
2016). In view of their significant pro- or anti-cancer effects, strategies are being developed to exploit the chemokine network for therapeutic purposes. Along these lines, the developed entities for targeting the chemokine network including antibodies, antagonists or small molecules are being investigated in pre-clinical settings or even clinical trials (Mollica Poeta et al.
2019; Mukaida et al.
2014).
Alterations in chemokine expression levels have been witnessed during CRC development, invasion and metastasis. Subsequent effects of these modulations mainly depend upon the type of chemokine, the corresponding concentration and on source/target cells (Emmanouil et al.
2018; Itatani et al.
2016; Ryu et al.
2018). CCR5 (CD195) along with its three known ligands (CCL3, CCL4, CCL5) comprises an important axis of the chemokine network and mediates multiple physiological functions as well as others related to malignancies including CRC (Aldinucci and Casagrande
2018; Fuente et al.
2018; Oliveira et al.
2014; Singh et al.
2018; Walens et al.
2019). These multifunctional properties of CCR5 are largely attributed to the expression of this receptor on a variety of cells including leukocytes, stromal and cancer cells. A differential expression profile of the CCR5 axis has been reported in CRC and its liver metastasis. As far as the functional importance of CCR5 is considered, the majority of the reports have supported a pro-tumor role of the CCR5 axis in CRC progression (Chang et al.
2012; Pervaiz et al.
2015; Sasaki et al.
2014; Schimanski et al.
2011). Owing to its vital role in CRC progression, the CCR5 axis is currently in the spotlight of consideration as a therapeutic target. Numerous strategies including development of specific antagonists and antibodies are being deployed to block the CCR5 axis of CRC cells for therapeutic purposes. In addition, CCR5 blockage on other cells including cells of the immune system has been proposed to be very effective in reducing CRC burden and its liver metastasis (Halama et al.
2016; Tanabe et al.
2016). Nevertheless, development of more specific and clinically relevant CCR5 inhibitors to target this chemokine axis in cancers is a continuing process.
In this study, following the CCR5 inhibition using gene specific siRNAs or an FDA approved antagonist (maraviroc), we investigated the role of CCR5 receptor in CRC progression and metastasis in pre-clinical settings. For this purpose, human (SW480, SW620) and rat (CC531) CRC cell lines were used in a series of in vitro assays. Furthermore, time-dependent modulations in expressional profiling of the CCR5 axis during CRC liver metastasis were determined using a related animal model. In addition, the potential of CCR5 inhibition by maraviroc was assessed regarding its capability to restrict CRC liver metastasis progression in vivo. Furthermore, we measured the circulatory and tumor associated levels of CCR5 and its ligands (CCL3, CCL4, CCL5) in serum (ELISA), primary tumors (qRT-PCR; IHC) and matched liver metastases (IHC) from CRC patients to assess potential morbidity-related changes.
Materials and methods
Cell lines and chemicals
Human (SW480 and SW620) and rat (CC531) colon adenocarcinoma cell lines were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and Cell Line Service (CLS, Eppelheim, Germany), respectively. The cells were cultured and maintained under standard incubation conditions (5% CO2, 37 ˚C, humidified atmosphere) in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 2 mM L-glutamine, 10% fetal bovine serum (FBS, Gibco: 10,270–106), 100 µg/ml streptomycin and 100 IU/ml penicillin. Cell lines were routinely tested (every 3 months) for mycoplasma contamination using the VenorGem PCR kit (Minerva Biolabs, Berlin, Germany) and passaged two to three times per week to maintain logarithmically growing cell populations. For propagation, the cells were washed with PBS, trypsinized (0.05% trypsin) and cell pellets were collected by centrifugation at 1500–2000 rpm for 5 min. The cells were counted by a Neubauer chamber and re-suspended at desired cell densities according to the experimental needs. Purified compound and commercially available tablets of maraviroc were purchased from Selleck Chemical Co. China (UK-427857) and Viiv Healthcare GmbH, Germany, respectively.
CCR5 expression and knockdown
CCR5 expression was assessed in untreated human CRC cell lines (SW480 and SW620) using quantitative real-time PCR (qRT-PCR) and western blot methodologies as described in below sections. Afterwards, small interfering RNA (siRNA) duplexes were designed against the human CCR5 gene (Sequence 1: 5′-AUUGAUACUGACUGUAUGG-3′, Sequence 2: 5′-AGAUGAACACCAGUGAGUAGAGCGG-3′, Invitrogen), while nonspecific siRNA (mock) was purchased from Ambion, Berlin, Germany (cat#AM4615). Following the manufacturer’s instructions of the transfecting reagent (X-tremeGENE 9, Roche, Mannheim, Germany), the cells were cultured to 50–60% confluence prior to transfection with siRNAs (200 nM, 24–72 h) in 96, 24, 12, 6-well plates or 25 cm2 cell culture flasks as per demand of the experiments.
Quantitative polymerase chain reaction (qRT-PCR)
CCR5 knockdown efficacy was evaluated by qRT-PCR, where total RNA was extracted from the cell pellets by using RNeasy Mini kit (Qiagen, Hilden, Germany) followed by synthesis of complementary DNA (cDNA) by Maxima reverse transcriptase (Thermo Scientific, Schwerte, Germany). The CCR5 transcript was detected using a mixture of gene specific primers (Table
1), 2X LC480 Master Mix (Roche, Mannheim, Germany) and an appropriate probe from the Human Universal Probe Library (Roche, Mannheim, Germany), which was amplified in a LightCycler 480 Real-Time PCR system. The samples were processed in triplicate and the expression level of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used as reference to normalize the data.
CCR5 | AACCAGGCGAGAGACTTGTG | GATCCAACTCAAATTCCTTCTCA |
CCL3 | CAGAATCATGCAGGTCTCCAC | GCGTGTCAGCAGCAAGTG |
CCL4 | CTTCCTCGCAACTTTGTGGT | CAGCACAGACTTGCTTGCTT |
CCL5 | TGCCCACATCAAGGAGTATTT | TTTCGGGTGACAAAGACGA |
GAPDH | AGCCACATCGCTCAGACAC | GCCCAATACGACCAAATCC |
Immunoblotting
Western blot analysis was used to assess knockdown of CCR5 at protein levels. To that purpose, the experimental cells were harvested, washed with PBS and stored in liquid nitrogen. Subsequently, to extract the protein content, the pellets were lysed with RIPA buffer (150 mM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, pH 8.0) supplemented with complete protease inhibitor cocktail tablets (Roche, Mannheim, Germany). Afterwards, the supernatant was collected by centrifugation (14,000 rpm/4 °C, 20 min) and quantified for protein concentration using the Pierce Protein Assay. The total protein lysates (30–50 µg) were subjected to electrophoresis on 4–12% gradient polyacrylamide SDS gels followed by transfer onto PVDF membranes and probing for CCR5 protein using specific primary antibody as per manufacturer’s instructions (Cell Signaling Technologies, Frankfurt, Germany). Immunoblots were developed using a HRP-conjugated anti-mouse (Cell Signaling Technologies, Frankfurt, Germany) and ECL-System (Amersham Pharmacia Biotech, Munich, Germany). Levels of β-actin (Santa Cruz Biotechnology) were used to normalize the data, and relative concentrations were measured by densitometric analysis of digitized autographic images using the ImageJ Program.
Cell proliferation assay
Cell proliferation was assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) dye reduction assay. In brief, the cells were counted in a Neubauer’s chamber, suspended in RPMI-1640 complete medium and seeded in 96-well plates at pre-optimized cell density (5 × 103 cells/100 μl medium/well). After an incubation period of 24 h, CCR5 was knocked-down by siRNA or blocked by increasing concentrations of purified maraviroc (1.5–750 µM) dissolved in ethanol (100 mM stock). Correspondingly, highest ethanol (the vehicle) concentrations used were ≤ 0.75% (by volume) in any of the samples exposed to maraviroc. Following treatment, the cells were incubated at standard conditions for 24, 48 or 72 h. Thereafter, MTT solution (10 mg/ml in PBS) was added (10 μl/well) and plates were incubated for another 3 h in the incubator. Afterwards, the old medium was discarded and formed crystals of formazan were dissolved by adding 100 μl/well of acidified solvent (0.04 N HCl in 2-propanol). Optical densities were measured by an ELISA plate reader at 540 nm absorbance wavelength and 690 nm reference filters. Cell survival rates were shown as percentage of controls transfected with mock siRNAs, or treated with equal concentrations of the vehicle (ethanol), while the inhibitory concentrations (IC) were calculated by GraphPad Prism 6 software.
The effects of targeting CCR5 on clonogenic ability of CRC cells were assessed by colony formation assay. Following siRNA-mediated knockdown of CCR5 or blockage by maraviroc IC20 (SW480: 213 µM, SW620: 148 µM, CC531: 432 µM) for 48 h, 5 × 102 cells/1.5 ml semiliquid medium (0.4% methylcellulose and 30% FBS in RPMI-1640 medium) were transferred to six-well plates. After an incubation period of 6–8 days under standard culture conditions, clusters of cells were counted by an inverted microscope (Leitz Fluovert FU Microscope, Wetzlar, Germany). Clusters with more than 10 cells were recorded as colony-forming units and categorized as small (< 30 cells) or large (≥ 30 cells) colonies. Data sets were represented as percentage of the controls (mock transfected siRNA or treated with the vehicle only).
Migration assay
To study the effects of targeting CCR5 (siRNA/maraviroc) on directional migration of the cells, we used a two compartment model separated by an 8 μm polycarbonate membrane (Millicell, Millipore, Germany) with a chemo-attractant (FBS) in the lower compartment. Briefly, the bottom of 24-well plates was covered with 250 μl of FBS, then gently over layered by 650 μl semi-liquid medium (0.4% methylcellulose and 20% FBS in RPMI-1640 medium) and incubated under standard conditions for 24 h to build a chemotaxis gradient. Following the transfection with CCR5 or mock siRNAs for 48 h, the cells were counted and equal numbers were seeded (5 × 104 cells/200 ul Optimem media) into hanging Millicell inserts with polycarbonate membrane. For the maraviroc group, respective cell numbers were transferred to Millicell inserts and allowed to migrate in the presence of compound (IC20) or vehicle only. Migrating cells were counted under an inverted microscope (Leitz Fluovert FU Microscope, Wetzlar, Germany) for 24, 48, and 72 h time intervals, while the filters with non-migrated cells were placed each day onto wells of a new plate with fresh chemotaxis gradient.
Wound healing assay
The effect of targeting CCR5 on CRC cell mobility was assessed by using a wound healing assay. In brief, the cells were seeded in 12-well plates (1 × 105 cells/well) and allowed to grow as monolayer under standard incubation conditions. Next day, the cells were knocked-down with respective siRNAs (gene specific or mock) for 48 h, followed by the creation of a straight scratch using a 200 μl sterile pipette tip. Free-floating cells from the wells were removed carefully and optimum medium (500 µl/well) with reduced FBS (0.5%) was added. With regard to the CCR5 antagonist (maraviroc), the cells were exposed to the compound (IC20) or vehicle only after 48 h of seeding. The images were captured by Axio Observer Z1 microscope (Carl Zeiss, Oberkochen, Germany) in both cases for zero and 24 h to monitor the “scratch healing” process.
Cell cycle panel and signaling pathway
In a previous study, we reported that targeting CCR5 by maraviroc induces significant arrest in G0/G1 phase of cell cycle in CRC cells (Pervaiz et al.
2015). To figure out the mechanistic reasoning for these previously observed effects, we used a ready-made Human Cell Cycle Regulation Panel (Cat. 05,339,359,001, Roche) and qRT-PCR methodology. The panel contains probes/primers for 84 cell cycle relevant genes (Supplementary File 1), appropriate controls (genomic DNA, RT-negative and positive controls) and 7 reference genes to monitor overall amplification and normalization of the data. Briefly, metastatic CRC cells (SW620) were exposed to maraviroc (IC
75/48 h) followed by extraction of total RNA and cDNA synthesis as described above. qRT-PCR was performed using 50 µl cDNA (0.5 µl/well) prepared from 1000 ng extracted RNA along with 2X LC480 Master Mix (Roche, Mannheim, Germany) in a LightCycler 480 Real-Time PCR System. After normalization of the data sets, relative fold changes were calculated by the 2− △△Ct method. Based on the results from this panel, a signaling pathway was predicted with the help of
Ingenuity Pathway Analysis software (Redwood, USA) at the Proteomics and Genomics core facility of DKFZ, Heidelberg.
Microarray analysis
Microarray analysis was performed to highlight the expressional modification in CCR5 and its cognate ligands (CCL3, CCL4, and CCL5) during the process of CRC liver metastasis (Georges et al.
2012). In brief, RFP-labelled CRC cells (CC531) were transplanted to the rat liver via the hepatic portal vein, which creates an animal model mimicking liver metastasis. Transplanted cells were re-isolated by FACS after discrete time intervals (3, 6, 9, 14 and 21 days) followed by RNA extraction with the RNeasy Mini kit (Qiagen, Hilden, Germany). In addition to this, a fraction of re-isolated cells was cultured in vitro for 14 and 22 days to compare the results with those from tumor cells grown in vivo. Following the extraction procedure, quality of extracted RNA was determined by gel analysis while using the total RNA Nano chip assay on an Agilent 2100 Bioanalyzer (Agilent Technologies GmbH, Berlin, Germany). RNA samples with RNA integrity number (RIN) values ≥ 8.5 were selected for further expression profiling.
Discussion
Accumulating evidences have shown that CCR5 along with its ligands plays an important role in tumor progression and organ specific homing of cancer cells during metastasis. Based on these findings, strategies are being materialized for blocking the CCR5 axis to uncover resulting antineoplastic effects and therapeutic relevance in cancers (Aldinucci and Casagrande
2018; Casagrande et al.
2019; Mencarelli et al.
2013; Ochoa-Callejero et al.
2013; Suarez-Carmona et al.
2019; Tan et al.
2009; Velasco-Velazquez et al.
2012). Regarding CRC prognosis and its metastasis, the CCR5 axis has earned considerable attention over the last few years as a novel biomarker and therapeutic option (Cambien et al.
2011; Chen et al.
2019; Nishikawa et al.
2019; Zimmermann et al.
2010). We have also contributed to this notion recently and showed that targeting CCR5 by an FDA-approved antagonist (maraviroc) induces anti-cancer effects and inhibits the tumor growth in vivo (Huang et al.
2020; Pervaiz et al.
2015,
2019). In the present study, we validated that targeting the CCR5 receptor via RNAi or an antagonist induces significant antineoplastic effects, including inhibition of proliferation, migration, colony formation and interference with cell cycle-related signaling cascades. Furthermore, implantation of CRC cells in rat liver (mimicking a CRC liver metastasis model) revealed a course-dependent induction of the CCR5 axis during liver colonization. Targeting the CRC cells via maraviroc in this liver metastasis model led to complete remission of growing liver metastasis. Lastly, circulatory- and tumor-associated expression changes of genes related to CCR5 axis were assessed in primary and metastatic clinical CRC samples.
Maraviroc, a competitive (non-allosteric) antagonist of the CCR5 receptor, was originally designed as an entry inhibitor for R5-HIV infections. Owing to mounting importance of the CCR5 axis in cancer, maraviroc turned out to be an immediately available drug for therapeutic purposes (Blanco and Ochoa-Callejero
2016). Characterized by a favorable pharmacological profile and minimal liver toxicity, the compound has been used recently in a phase I clinical trial (NCT01736813) to treat patients with CRC liver metastasis (Halama et al.
2016). In this particular study, when patients with metastatic CRC were given maraviroc (300 mg twice per day), Halama et al. highlighted the pro-tumor effects of infiltrating immune cells via the CCR5 axis. Interestingly, blockage of CCR5 by maraviroc re-polarized the immune cells to cause anti-tumor effects and reduced the subsequent disease burden. Profound success of this first clinical trial has attracted considerable attention of the scientific and medical community to further explore the CCR5 axis for treatment of advanced stage CRC. Currently, another phase I clinical trial (NCT03274804) is going on, where patients with refractory microsatellite stable metastatic CRC are being treated with a combination of pembrolizumab (anti-PD-1 antibody) and maraviroc. This trial possibly will reveal a new horizon in using a CCR5 antagonist like maraviroc in combination with other targeted agents. As these clinical studies have highlighted the concept of immune remodeling via the CCR5 axis, our pre-clinical data point to a direct anticancer effect of maraviroc in breast, pancreatic and CRC cells (Huang et al.
2020; Pervaiz et al.
2015,
2019). Furthermore, the concentrations used in our in vivo studies (25 mg/kg) are in the pharmacological range as shown by a calculated human equivalent dose of 242 mg/day (Nair and Jacob
2016). Based on these studies, it can be hypothesized that targeting the CCR5 axis implies using a double edged sword with direct antineoplastic effects against tumor cells and remodeling the immune system for ensuing anti-tumor effects.
In the present study, we identified significant anti-proliferative effects by targeting CCR5 via either gene specific siRNAs or maraviroc. Interestingly, siRNA-mediated knockdown led to a pronounced inhibition of CCR5 at mRNA (60–80% after 48–72 h) but not at protein levels (< 40%). The reduced inhibition at protein level could be due to the long half-life of the CCR5 protein present in membrane structures or to epigenetic cellular feedback loop(s) to maintain certain CCR5 protein levels. In spite of the poorly affected protein levels, targeting CCR5 inhibited the survival of selected human (SW480: primary, SW620: metastatic) and rat (CC531) CRC cell lines in vitro. A possible explanation is that CCR5 interacts with multiple ligands and various signalling cascades to play a pivotal role in metabolic and proliferative events (Gao et al.
2017; Oppermann
2004). Thus, it is not too surprising to witness a substantial inhibition of cell survival even after a small change in protein levels following siRNA knockdown. As far as maraviroc is concerned, relatively high concentrations (1.5–750 µM) were used in the in vitro part of this study. However, when tested clinically, the test compound was well tolerated in healthy persons (up to 1200 mg/day) and patients with viral infections and cancers (up to 300 mg/day twice daily) with no clear adverse effects on haematology and hepatobiology (Emmelkamp and Rockstroh
2007; Halama et al.
2016). Given the aggressive and invasive nature of CRC cells, we evaluated the importance of the CCR5 axis for cellular invasion and metastasis. Inhibition of CCR5 led to a decline in cell movement, invasiveness and colony formation ability (Mencarelli et al.
2013; Pervaiz et al.
2019; Singh et al.
2018; Velasco-Velazquez et al.
2012). Keeping in mind the primary chemo-attractant property of any chemokine axis, the possible inhibition of migratory activities of cancer cells can be foreseen after inhibiting a vital axis like that of CCR5. In addition to migration, chemokines have been shown to affect important functional aspects like cellular proliferation, apoptosis and cell cycle (Legler and Thelen
2018). In a previous study, we observed a significant cell cycle arrest in G1 phase of the cell cycle in CRC cells after blocking CCR5 by maraviroc (Pervaiz et al.
2015). In the present study, we explored potential signaling cascades underlying the previously observed cytostatic effects. Expressional profiling of 84 cell cycle-related genes followed by
Ingenuity Pathway analysis (IPA) revealed that CCR5 primarily interferes with the “G1/S checkpoint regulation” in CRC cells. In the light of available reports and our data (Fig.
3), we envision that CCR5 blockage leads to the alteration of multiple genes and related pathways of the cell cycle. Nevertheless, investigations that are more detailed are required to understand the CCR5-mediated effects on cell cycle-related signaling cascades in depth.
As we know, liver metastasis is a lethal condition and accounts for almost more than 50% of CRC-related deaths. Cellular processes and complex underlying molecular events, responsible for CRC liver metastasis, are poorly understood. Thus, there is a pressing need to identify metastasis-related changes in the tumor cells. More importantly, it is required to relate molecular changes accurately to their time of occurrence, so that target genes and pathways could be manipulated at the right time for therapeutic purposes. To understand time-dependent metastasis-related genetic changes, CC531 cells were implanted in rat livers and re-isolated for expressional profiling by cDNA microarray. The analysis revealed significant induction of the CCR5 axis in CC531 cells during the initial phase (3 days) of liver colonization. Remarkably, at later stages this increase was less impressive and almost normalized at the final stage (21 days) of liver colonization (Fig.
4). Which factors imposed these dynamic alterations on the CCR5 axis is an open question that deserves more attention. Here, we can speculate about the influence of the tumor microenvironment playing a pivotal role during the progression of cancers. Specifically, interactions of the implanted CRC cells with liver cells and/or immunological effector cells could be driving forces in the transient changes of the CCR5 axis. Furthermore, the possibility of epigenetic modifications within the tumour cells cannot be ruled out, which may lead to marked induction of the CCR5 axis. From a clinical perspective, the transient early up-regulation of the CCR5 axis should be investigated following resection of a primary CRC for improving the treatment options by e.g. reducing the rise of CCR5 ligands in the liver environment. Present data show a significant role of the CCR5 axis during early liver metastasis and indicate a period during which the respective CRC cells are sensitive towards CCR5 blockade. Keeping in mind the multiple functions of the CCR5 axis, including cellular adhesions, proliferation, survival and immune modulation to support tumor growth in a secondary organ (liver), targeting the CCR5 axis at this period should have profound effects against metastasis development.
To validate our above-mentioned hypothesis, CC531 cells were implanted in rat livers followed by treatment with daily intra-peritoneal administration of maraviroc (25 mg/Kg/day). To assess the sensitivity of the tumor cells to a chemotherapeutic agent, a second animal group was treated with gemcitabine in parallel (50 mg/Kg/week). In untreated animals, a continuous growth of tumor cells was observed. Animals treated with gemcitabine showed a moderate reduction in tumor burden. Likely reasons for these marginal effects can be explained from the fact that gemcitabine is used clinically in combination with other drugs for maximum anticancer effects, while we used it as a single agent. Outstandingly, complete tumor remission (undetectable signals during BLI) was observed in animals treated with maraviroc during the in vivo experiments. Almost similar, but less impressive anticancer effects have been reported by others where significant reduction of growing tumor mass has been observed when using maraviroc in other malignancies (Casagrande et al.
2019; Mencarelli et al.
2013; Ochoa-Callejero et al.
2013; Pervaiz et al.
2019; Velasco-Velazquez et al.
2012). At the end of experiments, the pathology of rats, especially the liver weights were in line with the BLI data. Needless to say, that our in vivo experiments indicate that targeting the CCR5 axis using maraviroc is a highly promising therapeutic option for CRC liver metastasis.
Cancer-related activation or inhibition of a chemokine network is a well-known phenomenon. It allows the tumor cells to cross-talk with surrounding stromal/immune cells for dictating the further progression. Considering this, it is worth to investigate alterations in chemokine expression during various stages of a cancer (Bian et al.
2019; Borsig et al.
2014; Huang et al.
2018). A number of studies have shown differential expression of CCR5-related ligands (CCL3, CCL4, CCL5) in peripheral blood and tumor samples of CRC. Furthermore, these variations were associated with varied prognosis and treatment outcomes (Fuente et al.
2018; Halama et al.
2016; Nishikawa et al.
2019; Yamaguchi et al.
2019). In this study, we analyzed the circulatory and tumor-associated levels of CCR5 and/or its ligands (CCL3, CCl4, CCL5) via ELISA and qRT-PCR/IHC, respectively. The results supported our working hypothesis; circulatory levels of the CCR5 cognate ligands (CCL3, CCL4, and CCL5) differ in CRC patients, when compared to healthy controls. Differential expression of these ligands could play a vital role in overall CRC prognosis as they can mediate a crosstalk between tumor cells and surrounding microenvironment to promote further tumor growth at primary locations and/or metastatic niches. Additionally, varied expression of these chemokine ligands can be exploited as biomarkers to detect CRC. However, careful consideration should be given to the fact that circulating levels of the ligands may not represent the actual levels at the tumor sites. Therefore, our results related to circulatory levels of the CCR5 ligands in CRC patients should be validated on larger sample pools and other populations as well. As far as the tumor-associated expressional profile of the CCR5 axis is concerned, the majority of available data indicate induction of this chemokine network with a pro-tumor role and shorter overall survival rate in CRC (Cambien et al.
2011; Erreni et al.
2009; Nishikawa et al.
2019; Zhang et al.
2018; Zimmermann et al.
2010). Furthermore, a distinct pattern of CCR5 expression has been reported recently in metastatic CRC liver specimens. The authors showed that intensity of CCR5 expression increases with primary tumor size, while a “patchy” pattern of the receptor (at least 10% of tumor cells negative for the CCR5 in a patchwork-like configuration) was observed in liver metastases (Suarez-Carmona et al.
2019). In our selected patient cohort, differential expression of CCR5 and its ligands (CCL3, CCL4, and CCL5) was observed in primary CRC tumors (Fig.
6b). To be precise, we identified a reduced average expression of CCR5 with an increasing primary tumor mass when compared with the healthy mucosa. Likewise, we observed variations in CCR5 stains during immunohistochemistry of the primary CRC tissues and matched metastatic lesions (Fig.
6c, d). These observations, at least in part, can be explained from our in vivo microarray data, which clearly shows temporal induction of the CCR5 axis during early tumor growth in the liver. This phenomenon can be exploited from the therapeutic perspective as well, where CCR5 blockage can lead to abrogation of vital signaling cascades required for tumor growth during metastasis. However, further studies will be required to dissect and understand the precise contribution of the CCR5 axis in CRC progression, especially during metastasis.
To conclude, inhibition of CCR5 induces cytotoxic and cytostatic effects in CRC cells. In vivo data demonstrated significant induction of the CCR5 axis in CRC cells especially during the early phase of liver colonization. Likely, in a similar fashion of time-dependent expressional modifications, varied levels of CCR5 were observed in the clinical samples collected at various phases of patients with liver metastasis. Blocking the CCR5 receptor via maraviroc led to complete remission of the tumor in an animal model mimicking CRC liver metastasis. The findings highlight CCR5 as an attractive therapeutic target, where CRC patients with early-stage liver metastasis could be more responsive towards this treatment approach. In this context, maraviroc is an already available FDA approved CCR5 antagonist and can be used in clinical settings as a monotherapy or in combination with other agents to possibly cure patients having CRC liver metastasis.
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