Tasquinimod Inhibits Prostate Cancer Growth in Bone Through Alterations in the Bone Microenvironment

Lisa U. Magnusson,1 Malin Hagberg Thulin,1 Pascale Plas,2 Anders Olsson,3 Jan-Erik Damber,1 and Karin Wel´en1*
1Sahlgrenska Cancer Center, Department of Urology, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden
2IPSEN Oncology and Biomarkers, Les Ulis, France
3Active Biotech AB, Lund, Sweden

BACKGROUND. Tasquinimod (ABR-215050) is an orally active quinoline-3-carboxamide analog that inhibits occurrence of experimental metastasis and delays disease progression of castration resistant prostate cancer in humans. Its mechanism of action is not fully elucidated, but previous studies show immunomodulatory and anti-angiogenic effects. The aim of the present study was to investigate the tumor inhibiting effect of tasquinimod in bone of castrated mice as well as to elucidate its working mechanism related to bone microenvironment.

METHODS. Effects of tasquinimod on prostate cancer metastasis to bone was studied in an intratibial xenograft model. Animals were treated with tasquinimod and tumor establishment and growth, immunological status, as well as markers for bone remodeling were analyzed. Direct effects of tasquinimod on osteoblasts were studied in vitro.

RESULTS. Establishment and growth of tumors in the bone after intratibial implantation in castrated mice was suppressed by tasquinimod treatment. The treatment effect was linked to decreased potential for immunosuppression in the pre-metastatic niche in bone (lower levels of CD206 and Arg1 expression in combination with increased iNOS expression) as well as in the tumor microenvironment (less Gr1 and CD206 staining). The shift to a pro-inflammatory, anti-tumorigenic milieu was also reflected in serum by increased levels of IFN-g, CCL4, IL-5, LIX, IP-10, and MCP-1 as well as decreased TGF-b.

Tasquinimod treatment also affected expression of factors involved in the pre-metastatic niche in the bone microenvironment (Lox, Cdh2, Cdh11, and Cxcl12). In addition, tasquinimod treatment caused a decreased osteogenic response indicated by decreased expression of Ocn, Runx2, and Col1a2 and increased expression of osteoclast stimulating CSF2. In vitro studies on mouse osteoblasts showed impaired osteoblast mineralization upon tasquinimod treatment.

CONCLUSIONS. The present study shows that tasquinimod reduces establishment and progression of tumor growth in bone likely through a combination of effects on the pre- metastatic niche, homing, immunological status, and osteogenesis. It was concluded that tasquinimod interferes with the metastatic process, presumably by inhibition of tumor

Grant sponsor: Swedish Cancer Society; Grant number: CAN2011/534; Grant sponsor: ALF/Vastra Gotalandsregionen; Grant number: ALFGBG-138351; Grant sponsor: Hillevi Fries Research Foundation; Grant sponsor: Percy Falks Foundation for Prostate and Breast Cancer Research; Grant sponsor: Assar Gabrielssons Foundations for Clinical Research; Grant sponsor: Wilhelm and Martina Lundgrens Research Foundation; Grant sponsor: Active Biotech AB.
Disclosure: AO is employed by Active Biotech AB, and PP is employed by IPSEN, both companies involved in the development of tasquinimod for commercial purposes.

KEY WORDS: tasquinimod; prostate cancer; metastasis; pre-metastatic niche; bone microenvironment; immunosuppression

INTRODUCTION

Prostate cancer (PC) is the most commonly diag- nosed cancer and the sixth leading cause of cancer- related death among men worldwide [1]. Localized disease is treated with radiation or surgical removal of the prostate gland with curative intention. Patients with distant metastases, mainly in lymph nodes and skeleton, are treated with androgen deprivation ther- apy, inhibiting the growth of androgen-dependent cancer cells. However, the majority of these cases relapse into a castration resistant stage for which currently several new drugs are used (e.g., abirater- one, enzalutamide, and radium-223) with shown survival benefits [2–4].

Tasquinimod (ABR-215050; CAS number: 254964- 60-8) is an orally active quinoline-3-carboxamide that was previously shown to inhibit PC growth in experimental models [5]. In a phase II clinical trial for castration resistant prostate cancer (CRPC) tasquini- mod treatment resulted in an increase of the median progression-free survival [6]. In a randomized pla- cebo-controlled pivotal clinical study, tasquinimod reduced the risk of radiographic cancer progression or death compared to placebo (rPFS, HR ¼ 0.69, 95% CI: 0.60–0.80) in patients with metastatic CRPC who had not received chemotherapy, but tasquinimod did not extend overall survival (HR ¼ 1.097, 95%CI: 0.938– 1.282) [7]. In addition, it has been shown that the effect of tasquinimod is most pronounced in patients with bone metastases [6].

Tasquinimod inhibits establishment of experimen- tal PC in bone [8] and has shown beneficial clinical effects in patients with bone metastases [6]. The mechanisms behind its function in bone are largely unknown. Tasquinimod efficiently binds and blocks S100A9 [9] as well as HDAC4 [10] and modulates the tumor microenvironment, possibly through HIF-regu- lation, acting as an inhibitor of immunosuppres- sion [11], angiogenesis [12], and metastasis [8]. It interferes with tumor angiogenesis as shown by decreased micro vessel density and increased secre- tion of the anti-angiogenic protein thrombospondin-1 in experimental tumors [13]. This may be mediated by tasquinimod interference with HIF-1a dependent transcription [10] and its downstream targets in- volved in hypoxic responses, such as the major angiogenic factor VEGF [14]. Tasquinimod treatment also results in changes in the number of tumor- infiltrating regulatory myeloid cells, such as myeloid derived suppressor cells (MDSCs) [15] and tumor associated macrophages (TAMs) [16], and reduces the immunosuppressive potential in the tumor microen- vironment [11]. Tasquinimod directly binds the S100A9 protein, which has been shown to be an important factor in the creation of the pre-metastatic niche as well as in recruitment and differentiation of MDSCs [17–19]. S100A9 can also be expressed by osteoblasts and osteoclasts, as well as chondrocytes, and the expression patterns vary with maturation and location in the bone [15]. In addition, it has been shown that MDSCs, TAMs, and osteoblasts can all express receptors for S100A9 [20,21], making these cell types potential tasquinimod targets.

PC cells are prone to home to bone due to intrinsic properties [22]. Tumor cells preferentially home to the endosteal niche in the bone, in many ways mimicking the homing mechanisms of hematopoietic stem cells (HSC) [23]. In this pre-metastatic niche, the bone is lined with osteoblasts which secrete homing factors such as CXCL12 [24] and express osteoblast cadherin (CDH11) known to mediate tumor cell-osteoblast contact in bone metastases [25,26]. The endosteal HSC niche also comprises SNO cells, immature osteoblasts that express high level of N-cadherin (CDH2) [27]. Although the role of osteoblastic N-cadherin in homing of HSC and/or cancer cells is unclear [28] it has been shown that increased N-cadherin is associ- ated with increased metastasis in PC [29,30]. In addition, lysyl oxidase (LOX), an enzyme involved in crosslinking collagens, which is important for both the recruitment of MDSCs to the pre-metastatic niche and the regulation of bone turnover influencing tumor establishment [31–33], can be produced by osteoblasts and influence their differentiation [34]. Also the bone marrow microenvironment is important for the homing efficacy of tumor cells.

PC metastases in bone are primarily sclerotic (osteoblastic), that is, they build bone. Osteoblasts are the cells responsible for the formation and mineralization of new bone. We and others have previously shown that osteoblasts are stimulated by PC tumor cells [35–38]. In addition, we demon- strated that osteoblasts induce a more aggressive phenotype along with osteomimicry in osteoblastic CRPC cells [35]. Together with their role in the
pre-metastatic niche, this makes osteoblasts impor- tant players in both establishment and growth of sclerotic bone metastases.

The present study investigates the effect of tasqui- nimod on tumor growth in bone in a castrated setting, as well as its effects on the tumor-permissive proper- ties of the bone microenvironment in terms of homing factors, immunological status, and osteoblastic func- tion. The major findings are that tasquinimod reduces establishment and progression of tumor growth in bone likely through a combination of an increased inflammatory response and decreased immunosup- pression, altered pre-metastatic niche, homing, and osteogenesis resulting in impaired tumor growth. In conclusion, this study and clinical data show that tasquinimod interferes with the metastatic process, foremost by inhibition of tumor establishment.

MATERIALS AND METHODS

Cell Lines and Culture Conditions

The castration-resistant cell line LNCaP-19 has been established in our laboratory from LNCaP cells [30]. It has an increased angiogenic and invasive potential compared to its parental cell line [39]. In contrast to LNCaP, it also has metastatic poten- tial [8,29] and gives rise to osteoblastic tumors in bone [35]. Cells were maintained as previously de- scribed [31]. MC3T3-E1 cells, clone 4, were obtained from the ATCC (Rockville, MD). Osteoblasts were maintained in ascorbic acid free aMEM (Invitrogen, Carlsbad, CA) and 10% FBS. Conditioned media was obtained as previously described [35]. The cells were tested and found to be free from mycoplasma.

In Vivo Experiments on Intratibial Tumors

To evaluate the potential effects of tasquinimod on tumor growth in the bone microenvironment, athymic BALB/c Nude mice (age 8 weeks; Charles River Laboratories International, Inc.) were used. Castration was performed prior to tumor cell implantation via scrotal incision. Bone injections were performed un- der anesthesia with a similar procedure as previously described [8,35]. A total of 500,000 LNCaP-19 cells suspended in 7 ml matrigel (BD Bioscience) were injected directly into the left tibial bone marrow cavity. After the procedure, animals received analge- sics (carprofen; 5 mg/kg) for the following 3 days. Tasquinimod (10 mg/kg/day) was administered via drinking water from day 0 to the end of the experi- ment (the control group received normal tap water). Two independent experiments with six mice in each group (treated/untreated) were performed in parallel.

The two experiments were subsequently analyzed together. The experiment was discontinued after 9 weeks and tibia were collected and fixed in forma- lin, decalcified in 12.5% EDTA and embedded in paraffin. Tumor establishment and size was assessed in H&E stained sections of the tibiae. Tumor take was defined as percentage of animals with a detectable tumor (detectable by H&E staining of sections of the left tibiae) at termination of the experiment. Most tumors that grew in the bone marrow were large and filled the width of the bone marrow cavity, approxi- mately 1.0 mm3 in size. All animal experiments were conducted in accordance with animal ethical guide- lines and approved by the animal ethical committee in Gothenburg.

Real-Time Q-PCR

Total RNA was extracted using RNeasy Mini Plus kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. RNA concentration was measured on a NanoDrop (Thermo Fisher Scientific Inc.) and the RNA reversely transcribed into cDNA using the VILO superscript cDNA synthesis kit (Invitrogen). RT-qPCR was performed using an ABI Prism 7500 Fast Sequence Detector (Applied Biosystems, Foster City, CA) and specific TaqMan probes as follows: Cxcl12 (Mm00445553_m1), Ocn (Mm03413826_m1), Col1a2 (Mm00483888_m1), Runx2 (Mm00501584_m1), Csf2 (Mm01290062_m1), Csf3 (Mm00438335_g1), Lox (Mm00495386_m1), Cdh2 (Mm01162497_m1), Cdh11 (Hs00156438_m1), Mrc1/ Cd206 (Mm00485148_m1), Arg1 (Mm00475988_m1), Nos2 (Mm00440502_m1), S100a9 (Mm00656925_m1), 18S (Hs99999901_s1), and Gapdh (Mm99999915_g1), purchased as TaqMan Gene Expression Assays (Applied Biosystems). PCR parameters were in accor- dance with the manufacturer’s protocol and the ddCt method was used for relative mRNA quantification. The expression levels of each sample were normalized against 18S rRNA and PCR reactions for target genes, and controls were performed in duplicates for all samples.

Immunoassay

Cytokines, chemokines, and growth factors were measured directly in mice serum using Multiplex Immuno-assay kits (Magnetic beads Milliplex kits MCYTOMAG-70K, MSCRMAG-42K, MAGMAG-24K, and TGFBMAG-64K, Merck-Millipore) following the manufacturer’s instructions. Signal detection was performed on Luminex 200 (Luminex) and MFI (Median Fluorescence Intensity) was recorded. The quantification for each cytokine was determined using standard curve analyzed with a five Parameter Logistic model (XLfit software, version 5.3, IDBS). For each cytokine in each assay, Low Limit of Detection (LLOD) and Quantification (LLOQ) were calculated.

Immunohistochemistry

Intratibial tumor tissue from the intratibial experi- ment (EDTA decalcified and paraffin embedded) was sectioned, deparaffinized, and rehydrated. Antigen unmasking was performed using heat or proteinase K, and endogenous peroxidase was blocked by hydro- gen peroxide (3%) in tap water. Primary antibodies (Gr1; BD Pharmingen (Bedford, MA), 550291, clone RB6-8C5, CD206; Abcam, (Cambridge, UK), ab64693, CD11b; Abcam, ab75476) were incubated over night at 4°C. Signals were detected by a HRP labeled second- ary IgG (K4003, EnVision rabbit, Dako, Glostrup, Denmark) and developed using the chromogen 3.30- diaminobenzidine (DAB).

Quantification of Gr1 stained area and CD206 cell number as well as tumor area was defined using computerized color selection in BioPix iQ 2.2.3 (Goth- enburg, Sweden, see www.biopix.se for further infor- mation). For Gr1 and CD206 five consecutive sections were stained and quantified. For tumor quantification the tumor area on every 5th slide (containing five sections) was quantified using Biopix.

Detection of Mineralization With von Kossa and OsteoImage Staining

MC3T3-E1 cells were seeded in six well plates at a density of 1 × 105 or 2 × 105 cells per well in aMEM with 10% FBS or RPMI-1640 with 10% FBS or DCC, respectively. After 48 hr, medium was changed to conditioned media (CM) or promineralization media (aMEM supplemented with 10 mM b-glycerophos- phate (Sigma–Aldrich, St Louis, MO) and 50 mg/ml L-AA (Sigma)). A total of 10 mM tasquinimod was used in the treated group. Mineral deposition was determined by von Kossa staining after 21 days. Briefly cells were washed with PBS and fixed with 95% ice cold ethanol. For calcium retention, the fixed cells were exposed to 60 W UV light in a solution of 5% silver nitrate for 60 min for visualization. For the mineralization assays, medium was changed every 3 days. For quantification of formed hydroxyapatite, OsteoImage (Lonza) was used according to manufac- turer’s instructions.

Statistics

Statistical calculations were performed using Graph Pad Prism software (GraphPad Software Inc.,CA). Statistical differences between groups were performed using Student’s t-Test or Mann–Whitney U-Test where appropriate. All data are presented as mean SEM. A P-value less than 0.05 was considered significant (*P< 0.05; **P < 0.005; ***P < 0.001).

RESULTS

Tasquinimod Decreased Tumor Take and Tumor Growth in Bone Clinically, tasquinimod inhibits metastatic CRPC with the largest effects in patients with bone metastasis [3]. Also in pre-clinical settings tasquini- mod has previously been shown to inhibit tumor establishment in bone [8]. To investigate if, and to what extent, that inhibitory effect is valid in the more clinically relevant situation of androgen depri- vation, LNCaP-19 cells were injected in the tibial bone marrow of castrated nude mice. The tumor take in the untreated mice was 94%, whereas tumors could be detected in only 25% of the tasquinimod treated mice (P < 0.001, Fig. 1A). In addition to affecting tumor establishment, tasquini- mod also influenced the growth of the tumor. The mean volume of the untreated tumors was 1.36 mm3 (n = 12), whereas treated tumors were only 0.26 mm3 (n = 4) (P < 0.01, Fig. 1B). The un- treated tumors displayed an osteoblastic phenotype with newly formed trabecular bone, as previously described [35] (Fig. 1C). Phenotype evaluation was not possible of the treated tumors due to their small volume.

Immunosuppressive Microenvironment in Tumor Bearing Mice

Tasquinimod targets the tumor microenvironment by enhancing the host immune response [11], and the action of different myeloid immune cells of the bone marrow influences the formation of metastases [16]. In femoral bone marrow (from intratibially implanted mice), the mRNA levels of Mrc1 (Cd206, Fig. 2A, P < 0.01), a marker for immunosuppressive M2 macrophages as well as the number of CD206 positive cells in the tumors (visualized with IHC staining), was decreased by tasquinimod treatment (Fig. 2B and C) suggesting a less immunosuppressive macrophage profile in treated bone marrow. In line with this, the expression level of Arg1 was decreased (P < 0.001) whereas Nos2 was increased (P < 0.05, Fig. 2A), also skewing the Arg1/Nos2 ratio towards a less immunosuppressive milieu.

In the intratibial tumors the number of Gr1-positive cells that co-localized with CD11b staining per tumor area was much smaller in the tumors from treated mice (n = 4), indicating a decrease in MDSC in the treated tumors (Fig. 2D and E).

Fig. 1. Effects of tasquinimod on LNCaP-19 tumors in bone. (A) Frequency of intratibial tumors in control and treated castrated mice.(B) Volume of intratibial LNCaP-19 tumors in control versus tasquinimod treated animals. (C) Representative pictures of tibiae with tumors from control (left) and tasquinimod treated (right) mice. Tumor areas are marked with yellow using the digital tool Biopix. TASQ = tasquinimod. ** = P < 0.01, *** = P < 0.001.

Pro-Inflammatory Cytokines are Increased in Serum by Tasquinimod

The inflammatory status was also investigated at the systemic level by measuring pro- and anti- inflammatory factors in serum from treated and control mice. The shift from an immunosuppressive towards a more pro-inflammatory status by tasqui- nimod treatment seen in the bone was supported by increased systemic levels of IFNg (P < 0.001), CCL4 (P < 0.05), IL-5 (P < 0.05), MCP-1 (P < 0.05), and IP-10 (P < 0.05) along with decreased levels of LIX (CXCL5, P < 0.05) and TGFb (P < 0.01), which together would indicate a more pro-inflammatory situation that locally promote M1 polarization of macrophages in the treated mice (Fig. 3A–G). Opposing this however, the level of the anti- inflammatory, M2-inducing IL-4 was increased by tasquinimod treatment (Fig. 3H, P < 0.001). The levels of all analytes measured in serum are listed in Supplementary Table S1.

Fig. 2. Tasquinimod modulates the immunosuppressive environment in vivo. (A) Gene expression of markers related to immunosup- pression in bone marrow cells from femur. (B) Number of CD206 positive cells (IHC)/tumor area (mm2), P = 0.105. (C) IHC staining of CD206 in tumor. (D) Gr1/tumor area (%) in tumors quantified with IHC, P = 0.33. (E) Co-localization of Gr1 (left) and CD11b (right). TASQ = tasquinimod. ** = P < 0.01, *** = P < 0.001.

Fig. 3. Effects of tasquinimod treatment on immunoactive protein levels in serum. The serum levels of (A) IFN-g (pg/ml), (B) CCL4 (pg/ml), (C) IL-5 (pg/ml), (D) MCP-1 (pg/ml), (E) IP-10 (pg/ml), (F) LIX (ng/ml), (G) TGF-b (ng/ml), and (H) IL-4 (pg/ml) in control and tasquinimod treated animals. TASQ = tasquinimod. * = P< 0.05, ** = P < 0.01, *** = P < 0.001.

Interference With the Pre-Metastatic Niche in the Bone Microenvironment

The presence of a tumor in the mouse enables the bone marrow to react upon signals from tumor cells in order to start forming the pre-metastatic niche and recruit tumor cells. The effect of tasquini- mod on these processes was evaluated in prepara- tions of femoral bone of intratibial implanted mice. The expression of Lox, encoding an enzyme in- volved in ECM remodeling during niche forma- tion [40], was decreased by tasquinimod in femoral bone together with the cadherins N-cadherin (Cdh2) and osteoblast-cadherin (Cdh11). Measuring Cxcl12 mRNA expression levels in femoral bone revealed a not statistically significant decrease (P = 0.0517) in expression of this homing factor by tasquinimod treatment (Fig. 4).

Tasquinimod Decreases Osteoblast Markers In Vivo

Prostate cancer bone metastases are predomi- nantly osteoblastic and a bi-directional stimulatory interaction between cancer cells and osteoblasts has been described [35]. To evaluate the effect of tasquinimod directly on osteogenesis and osteoblast differentiation, expression levels of osteogenic fac- tors were investigated in tumor-free femoral bone from intratibially implanted mice. The osteoblast marker osteocalcin (Ocn, P < 0.05), the bone matrix collagen Col1a2 (P = 0.141), and the osteogenic tran- scription factor Runx2 (P < 0.05) were decreased by tasquinimod treatment, whereas the osteoclast marker GM-CSF (Csf-2) was increased (P < 0.001, Fig. 5A). In addition the osteoblast inhibitory cytokine G-CSF (CSF-3) was found to be increased in serum (P < 0.001, Fig. 5B) although Csf-3 mRNA- expression was unaffected in femoral bone (data not shown). Together these results show that tasqui- nimod has an inhibitory effect on osteoblast differentiation and function in vivo.

Fig. 4. Altered expression of factors related to the pre- metastatic niche by tasquinimod treatment. Relative expression of Lox, Cdh2 (N-cadherin), Cdh11 (osteoblast-cadherin), and Cxcl12, P = 0.0517) in femoral bone of control and tasquinimod treated animals. TASQ = tasquinimod. * = P < 0.05, ** = P < 0.01.

Fig. 5. Inhibitory effects of tasquinimod on osteogenesis. (A) Decreased expression of osteoblast markers Ocn (Bglap), Col1A2 (ns) and Runx2, and increased expression of osteoclast marker GM-CSF (Csf2) in femoral bone after tasquinimod treatment. (B) Increased serum concentration of G-CSF. TASQ =
tasquinimod. * = P < 0.05, *** = P < 0.001.

Tasquinimod Inhibits Tumor Cell-Induced Osteoblast Function In Vitro

To verify the indicated effect of tasquinimod on osteoblasts found in vivo, and also to see if tasquini- mod interferes with tumor cell-osteoblast interaction, in vitro experiments were performed. Previously, we have shown that CM from cultured LNCaP-19 cells induced osteoblast differentiation and mineraliza- tion [35]. In a similar setting tasquinimod altered the osteoblast phenotype towards a more elongated fibro- blast-like appearance, which could indicate decreased osteoblast differentiation (Fig. 6A). Moreover, func- tional assays detecting formation of hydroxyapatite and calcification, that is, mineralization, also showed that tasquinimod reduced these activities to the basal levels without the presence of CM from LNCaP-19 (aMEM controls) (Fig. 6B). Von Kossa staining further revealed an inhibitory effect of tasquinimod on CM- induced mineralization compared to untreated con- trol (data not shown).

DISCUSSION

Metastasis to bone is associated with higher mor- tality in patients with PC [41] and prevention of bone metastasis would likely increase both survival and quality of life in these patients. In the present study, we investigate the potential of tasquinimod to inhibit PC growth in bone and to alter the metastatic niche to diminish further tumor establishment.Although its working mechanism is largely un- known, tasquinimod has a documented clinical effect on disease progression in patients with bone metasta- ses [6], and the results from the present study show that it inhibits both tumor establishment and growth in castrated mice. The decreased intratibial tumor take upon tasquinimod treatment in the present study indicates that it affects early stages of tumor establish- ment in bone. In addition to decreased tumor estab- lishment, tasquinimod also affected the tumor growth in bone, once the tumors were established. This is in line with several studies demonstrating inhibition of tumor growth of subcutaneous xenograft tumors by tasquinimod treatment [10,12,42,43] although a previ- ous study failed to show a growth effect of tasquini- mod on intratibial tumors in intact mice [8]. Direct effects of tasquinimod on tumor cell proliferation have previously not been suggested as a major mechanism behind its anti-tumor activity. Instead, the immune modulatory function of the drug has gained interest in this aspect. Tumor cells directly affect the immune system and induce an immuno-suppressive environment, for example by recruiting and activating MDSCs and shifting the phenotype of tumor infiltrat- ing macrophages, which increases the possibility for tumor cells to grow. One of the target molecules for tasquinimod, S100A9, is involved in MDSC recruit- ment and stimulation [19]. It has previously been shown that the growth potential of a tumor is markedly affected by the immunological status of the individual, both systemically and locally in the tumor microenvironment [16,44]. The pro-inflammatory con- tingent of the immune system is generally said to be anti-tumorigenic, stimulating the immune system to sequester growth of and kill the tumor cells, whereas immunosuppressive functions help the tumor to evade the immune response and more readily grow and metastasize. In line with what earlier has been demonstrated in syngeneic tumors [11], tasquinimod treatment caused a marked decrease in Cd206 and Arg1 expression and increase in Nos2 expression in femoral bone marrow giving a decreased Arg1/Nos2 ratio. This likely reflects a shift from M2 towards M1 polarization of macrophages and a less immunosup- pressive microenvironment leading to reduced possi- bilities for tumor establishment. The tumors in the treated animals showed fewer Gr1-positive possible MDSCs indicating a diminished MDSC recruitment and maturation. Many of the immunosuppressive actions of MDSCs are mediated through the T-cell axis effects that cannot be fully investigated in a xenograft model system lacking T-cells. However, it has been shown that MDSCs can also suppress NK cell activation [45,46] and potentiate cancer growth in T-cell deficient BALB/c Nude mice. In line with the effects on gene expression and cell populations in the present study the systemic inflammatory status also indicates a more pro-inflammatory response in the treated group, as has been reported previously [6], with increased serum levels of IFN-g, CCL4, IL-5, IP- 10, and MCP-1 along with decreased levels of LIX (CXCL5) and TGF-b. In contrast, IL-4 levels are also increased. Although IL-4 is generally considered to be a tumor-promoting molecule, tumor suppressing effects have also been detected [47]. The indicated decrease in immunosuppressive phenotype in the pre-metastatic niche in response to tasquinimod treat- ment will likely contribute to decreased potential for tumor growth in this niche.

Fig. 6. Tasquinimod inhibits the stimulation of osteoblasts by cancer cells in vitro. (A) Reduced osteoblast differentiation of MC3T3 cells in conditioned media from LNCaP-19 cancer cells after tasquinimod treatment. Scale bar = 200 mm. (B) Inhibited stimulation of osteoblast mineralization by LNCaP-19 conditioned media after tasquinimod treatment measured with OsteoImage at day 21. TASQ = tasquinimod. * = P < 0.05.

Even if tumor cells are injected directly into tibia in the present model and no actual homing to bone from the circulation takes place, the mechanism of homing may still be involved in localizing the cells in the bone marrow to the pre-metastatic endosteal niche where tumor establishment is most effi- cient [48]. The fact that not all implantations give rise to tumors indicate that simply ending up in the bone marrow cavity is not enough to initiate tumor growth in bone, and that we actually can modify this capacity by tasquinimod treatment. The hypoxia-induced homing factor CXCL12 has previ- ously been shown to be decreased by tasquinimod treatment in the pre-metastatic niche of putative metastatic organs [8]. In the present study, the Cxcl12 inhibition was not as pronounced as in previous studies but nevertheless a trend of re- duced expression of homing factor Cxcl12 in the femoral bone suggests that tasquinimod could be an effective inhibitor of the very early stages of tumor homing to bone thereby preventing further metastatic progression. The expression of another HIF-dependent gene, Lox, was also decreased by tasquinimod treatment which likely contributes to impaired preparations of the pre-metastatic niche. LOX has been shown to play an important role in the establishment of osteolytic tumors in bone but its role in sclerotic tumors is less defined [32]. Decreased stimulation of bone degradation by decreased LOX-levels can contribute to diminished establishment of osteoblastic tumors as it has been shown that the osteolytic response is involved also in establishment of osteoblastic tumors in bone [49]. Actual adhesion of tumor cells to the endosteal niche has not been extensively studied, but HSC requires adhesion to osteoblasts for their mainte- nance [50]. In the present study, tasquinimod inhibited expression of both N-cadherin and osteo- blast-cadherin in tumor free femoral bone. The importance of these changes is difficult to interpret, since the role of cadherins in the endosteal niche is not clarified [28,51]. However, N-cadherin and osteoblast-cadherin have been demonstrated to be of importance for PC metastasis and adhesion to osteoblasts [25,26,29,30] suggesting that decreased levels in bone would reduce the potential for tumor cells to use these mechanisms for establishment.

Interactions between osteoblasts and cancer cells are believed to be important for establishment and development of PC bone metastases [35]. Osteo- blasts have been shown to induce a more aggres- sive and osteoblast-like phenotype in prostate cancer cells through soluble factors and cancer cells, in turn, influence osteoblast proliferation and differ- entiation [35]. In the present study, tasquinimod decreases femoral expression of Runx2 together with genes encoding two major matrix building proteins, osteocalcin and collagen 1A2, both under the regulation of RUNX2, in tumor-bearing mice. RUNX2 is the major transcription factor for osteo- blasts and also crucial for bone development in mice, indicating a prominent downregulation of osteoblast differentiation and activity in the femoral bone. This is supported by the increased serum levels of the osteoblast inhibitory cytokine G-CSF (CSF3). Increased stimulation of osteoclasts (by higher expression levels of GM-CSF (Csf2)) can also contribute to counteracting not only the osteoblastic activity in these tumors, but also the vicious cycle of bone metastasis and, hence, decrease CRPC growth in bone.The effect of tasquinimod on osteoblast activity was further supported in the in vitro set up where treatment of osteoblasts with tasquinimod in cell culture caused impaired osteoblast differentiation.

The cells stay in a more fibroblast like stage (Fig. 6A), both visibly and in respect to mineralization. In a previous control experiment, NIH3T3-E1 fibroblasts did not induce any of the osteoblast differentiation markers in response to conditioned medium from LNCaP-19 and did not increase aggressiveness of cancer cells [35]. These results indicate that tasquini- mod can directly target osteoblasts, inducing the observed in vivo effects.
Effects of tasquinimod on the bone milieu may be acting through the S100A9–TLR4 axis as Tlr4 is expressed in femoral bone (data not shown). How- ever, secretion of S100A9 by osteoblasts could not be demonstrated using mass spectometry of conditioned media from undifferentiated/unstimulated osteo- blasts or cancer cells (data not shown). This does not rule out that S100A9 is expressed and produced at some point during osteoblast differentiation or cancer cell development, since S100A8 and A9 previously have been suggested to be involved in processes of osteoblast maturation and extra cellular matric calcifi- cation [52]. Besides possible S100A9-coupled actions, the effects of tasquinimod on HIF-dependent tran- scription has been shown to work through interac- tions between tasquinimod and HDAC4 [10] and it is possible that the direct effects of tasquinimod on osteoblasts work through the HDAC4-HIF1a pathway since HDAC4 has been shown to be important for osteoblast function [53–55].

Our data show that tasquinimod in this in vivo model impairs establishment and progression of osteoblastic CRPC in bone likely through a combina- tion of alterations in the pre-metastatic niche, de- creased homing, increased inflammation, decreased immunosuppression, and decreased osteogenesis. Im- portantly, this study supports previous pre-clinical and clinical data showing that tasquinimod interferes with the metastatic process, foremost by inhibition of tumor establishment. From a clinical perspective, this suggests that a major role of tasquinimod would be to inhibit metastatic spread, and thereby be most effi- cient in earlier stages of disease.

ACKNOWLEDGMENTS

We thank Karin Larsson and Anita Fae for excellent technical assistance.

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