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Osteoblasts suppress high bone turnover caused by osteolytic breast cancer in-vitro
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y e x c r
Research Article
Osteoblasts suppress high bone turnover caused by osteolytic breast cancer in-vitro Roman Krawetz a,⁎, Yiru Elizabeth Wu a , Derrick E. Rancourt a , John Matyas b a
Department of Oncology, Faculty of Medicine, University of Calgary, Canada Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Canada
b
A R T I C L E I N F O R M AT I O N
AB ST R AC T
Article Chronology:
The skeleton is the most common site of breast cancer metastasis, which can occur in up to 85% of
Received 25 June 2008
patients during their lifetime. The morbidity associated with bone metastases in patients with
Revised version received
breast cancer includes pathological fractures, bone pain, hypercalcaemia, and spinal cord
27 April 2009
compression. When breast cancer metastasizes to bone, the balance of bone resorption
Accepted 28 April 2009
(mediated by osteoclasts) and bone formation (mediated by osteoblasts) favors bone resorption,
Available online 9 May 2009
which leads to net bone destruction (i.e., osteolysis). Anti-resorptive agents such as bisphosphonates are commonly used to treat bone resorption in osteoporosis or osteolytic cancer patients. However,
Keywords:
bisphosphonates by themselves are unable to rebuild lost bone tissue, and can cause severe side
Bone organ culture
effects. In this study, we developed a bovine bone explant culture system and have observed that
Osteotropic breast cancer
murine osteoblasts can modulate the activity of osteotropic human breast cancer cells on this
Osteoblasts
substrate. Using markers of bone metabolism, we observe diminished bone turnover in organ
Bone turnover
culture following the addition of exogenous osteoblasts. The data presented in this study supports further investigation into the use of cytotherapies to limit breast cancer mediated osteolysis. Crown Copyright © 2009 Published by Elsevier Inc. All rights reserved.
Introduction The skeleton undergoes continuous remodeling: Bone is resorbed by osteoclasts and is deposited by osteoblasts in a coordinated fashion. The tight balance between osteoclasts and osteoblasts is deregulated in diseases such as osteoporosis, rheumatoid arthritis, and osteolytic metastasis. When breast cancer metastasizes to bone, it activates osteoclasts, creating osteolytic lesions [1]. Tumorinduced osteolysis is mediated by bone-resorbing osteoclasts, whose number and activity are influenced by a variety of osteoclast-stimulating factors that are produced in the bone marrow microenvironment and act in a paracrine fashion [2]. Osteoblasts, for instance, regulate osteoclast activity through expression of the cytokine RANKL (receptor activator of nuclear factor-κB ligand), which promotes osteoclast differentiation.
Osteoblasts also express the decoy receptor of RANKL; osteoprotegerin (OPG), which acts to inhibit RANKL activity [3,4]. In cancer, as in osteoporosis, bisphosphonates act as specific inhibitors of osteoclastic activity, and thereby have special utility in treating hypercalcaemia of malignancy [5]. Although targeted inhibition of osteoclasts by bisphosphonates can slow osteolysis, inhibition is typically incomplete, and bone lesions continue to progress without healing. Unfortunately, few therapies are currently available to promote sustained regeneration of damaged bone. In order to investigate new therapies against metastasisinduced osteolysis, various in-vivo and in-vitro models have been developed. The mouse has proven to be a very powerful animal model of human breast cancer metastasis, as xenografts of the human breast cancer cell line MDA-MB-231 can be transplanted
⁎ Corresponding author. University of Calgary, 331-3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1. E-mail address: [email protected] (R. Krawetz). 0014-4827/$ – see front matter. Crown Copyright © 2009 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2009.04.026
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into immunocompromised animals. For example, when injected systemically into BalbCnu/nu (nude) mice or rats, these cells metastasize to a variety of locations, including the skeleton [6,7]. The murine model has been used extensively to develop therapies aimed at mitigating the morbidity of breast cancer metastasis [8– 11]. A common approach is the competitive inhibition of cell activities using antibodies and peptides directed against bonetrophic integrin proteins that appear on both metastatic cancer cells and osteoblasts [12,13]. Clinically, metabolic bone diseases are diagnosed by measuring bone mineral density (BMD). However, BMD is a static measure of bone structure and a baseline BMD is of little value in predicting the rate of osteolytic progression. However, when BMD is combined with markers of bone metabolism, more reliable predictions may be made of whole-body bone turnover. Metabolic markers of bone metabolism include bone-specific alkaline phosphatase (BAP) for osteoblast activity, and tartrate-resistant acid phosphatase (TRAP) for osteoclastic activity. BAP is synthesized by osteoblasts and is involved in the calcification of bone matrix. BAP is one of a number of different tissue isoenzymes of alkaline phosphatase, and is considered to be a highly specific marker of the bone-forming activity of osteoblasts. Moreover, BAP can be used to differentiate between serum sampled from tumor patients with or without bone metastases. In patients with bone metastases, elevated BAP levels strongly predict the presence of metastases; patients without bone metastases have BAP levels similar to non-cancer reference groups [14]. Serum TRAP is a marker of osteoclast activity and bone resorption [15–18]. As the normal concentration of serum TRAP levels is very low, elevated serum TRAP is a sensitive marker of bone metastasis in breast cancer patients. Indeed, TRAP and BAP levels in breast cancer patients with bone metastases are significantly higher than in normal subjects [19–21]. In this study we have developed and validated an in-vitro assay of overall bone turnover in explants co-cultured with human breast cancer cells. In this experimental system, the osteolytic and osteoblastic activity of osteotropic human breast cancer cells on native bone substrate can be influenced by the addition of exogenous osteoblasts. We therefore submit that this in-vitro system can be used to screen new treatments of osteolysis by providing a more relevant in-vivo cellular environment compared to traditional static cell culture.
Materials and methods Bovine bone discs Fresh bovine distal femurs were obtained from a local meat distributor. Eight millimeter-diameter bovine trabecular bone cores were harvested from the femoral condyles using a custom coring bit. The cylindrical cores were then cut into discs approximate 1 mm in height. The discs were rinsed with PBS containing 50 U/ml penicillin and 50 μg/ml streptomycin (Invitrogen), and weighed on an analytical balance accurate to 0.001 g. Bone discs ranged from 0.960 g to 0.169 g with a median weight of 0.139 g. The discs were then transferred to 24 well Primaria™ culture dishes (BD) and cultured in media containing DMEM without phenol red (Invitrogen), 10% FBS (Invitrogen),
50 U/ml penicillin and 50 μg/ml streptomycin, 1% non-essential amino acids NEAA (Invitrogen), 50 μg/ml ascorbic acid (Sigma), 5 × 10− 8 M vitamin D3 (Sigma), 10 mM beta-glycerol phosphate (Sigma), and 0.1 mM 2-mercaptoethanol (Invitrogen) for 24 h.
Cell culture After the discs were cultured for 24 h, murine osteoblasts (7F2 ATCC# CRL-12557), human breast cancer cells (MDA-MB-231 ATCC# HTB-26), or combinations of the two cell types were added together to the bone disc cultures. Either 1000 or 2000 cells of each type were seeded on the bone discs. MDA-MB-231 cells were stably transfected with a CMV-GFP-Luc2 reporter vector (gift from Dr. Frank Jirik, University of Calgary).
Cell counting by Flow Cytometry Bone discs were seeded with cells as described earlier and then treated with Accutase™ (Millipore) for 3 treatments of 5 min at each time point to release the cells for counting. The supernatant was collected and the cells were fixed in 4% paraformaldehyde. Cells obtained from the bone discs were stained using the following antibodies: anti-ERα to detect 7F2 osteoblasts (Santa Cruz; sc-7207), anti-GFP to detect MDA-MB-231 breast cancer cells (Santa Cruz; sc-8334), anti-TRAP to detect bovine osteoclasts (Santa Cruz; sc-28204) or bone sialoprotein (BSP) to detect bovine osteoblasts (Iowa Developmental Studies Hybridoma Bank; WVIDI9C5). Primary antibodies were directly conjugated to R-Phycoerythrin (R-PE) for anti-ERα, Alexa Fluor 488 for anti-GFP or antiBSP, and Alexa Fluor 647 for TRAP all using Zenon conjugation kits (Invitrogen) following the manufacturer's recommended method. The single cell suspension was subjected to fluorescence-activated cell sorting on days 15, 30 and 45 using a FACS Calibur instrument and the CellQuest software from Becton Dickinson (Germany). Anti-mouse IgG R-PE, 488 or 647 were used as isotype controls depending on the primary conjugation molecule used. Each count was performed using triplicate cultures. To obtain the cell count from each sample CountBright™ absolute counting beads (Invitrogen) were pre-mixed with each sample as per the manufacturer's instructions and were gated separately.
Immunofluorescence To assess short term viability and attachment behavior, osteoblasts and breast cancer cells were labeled with the vital cell-tracking dyes Oregon Green and Fura Red respectively (Molecular Probes: O-6807, Molecular Probes: F-3021), and incubated with the bone cores for 48 h. The discs were subsequently rinsed with PBS and visualized using a fluorescence microscope (Olympus 1X70 microscope equipped with a SPOT RT Color camera and SPOT software [v4.0.9]). To assess whether the transplanted cells retained their phenotypes up to 45 days in bone explant coculture, breast cancer cells and osteoblasts were stained with antibodies to GFP, TRAP and bone sialoprotein (BSP), then visualized by confocal microscopy. Briefly, bone cores were fixed in 4% paraformaldehyde overnight at 4 °C, washed in PBS, and permeabilized in 1% saponin. The cores were then blocked using 3% BSA in PBS and incubated with primary antibody (anti-GFP, anti-TRAP, or anti-BSP) at a 1:50 concentration in 3% BSA in PBS overnight at 4 °C. The samples were then washed in PBS and
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incubated with an appropriate secondary antibody (Alexa Fluor 488 or 568: Invitrogen) and incubated at 4 °C overnight, samples were then washed and mounted. A Zeiss 510 confocal microscope with 488 nm and 568 nm filters was used to capture images, and the images were prepared with the Zeiss LSM image browsing software (v.4.0.0.157).
Alkaline and acid phosphatase detection To asses bone turnover in long term culture, the culture was maintained for 45 days and 100 µl of culture fluid was analyzed on days 15, 30 and 45 using acid (Sigma CS-0740) and alkaline (BioRad 172-1063) phosphatase detection kits. The medium was changed as necessary, however, 24 h before each time point (15, 30 and 45 days) every well was replaced with 1000 µl of fresh medium. Alkaline and acid phosphatase detection was carried out as follows: 100 μl of medium was incubated with acid or alkaline phosphatase reaction buffer at 37 °C for 30 min. The reaction was stopped with the addition of 50 μl of 1 M NaOH. The absorbance at 420 nm was quantified using a Bio-Rad Benchmark Plus plate reader. The alkaline and acid phosphatase absorbance values were normalized by subtracting background absorbance values from control treatments (medium alone), the culture vessel (∼0.045 at 420 nm), each cell type cultured alone, the bone disc alone (the medium from an average bone disc ∼ 0.139 g produced an absorbance value of 0.100 at 420 nm). Each treatment and assay was performed in triplicate. Statistical analysis (repeated measures ANOVA) was performed using GraphPad Prism4 (GraphPad Software) and significance was set at p < 0.05.
Devitalization of bone discs To study the influence of endogenous bone cells on bone disc metabolism, bone discs were prepared as above and devitalized by placing them into dishes containing 100% ethanol, repeatedly freezing to −80 °C, and thawing at room temperature. After the 3rd freeze-thaw cycle, devitalized bone discs were placed in 24well Primaria™ culture dishes (BD) and cultured in a medium as described above.
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visible within the bone discs. Moreover, when osteoblasts and breast cancer cells were co-cultured on the bone discs both cell types were observed in close proximity to each other (the colocalization of GFP and BSP, yellow, represents the human breast cancer cells whereas green staining represents osteoblasts). TRAP positive cells were also observed at 45 days in organ culture, signifying that bovine osteoclasts or differentiating progenitors were still viable after long term culture periods.
Cell counting To determine the proliferation of cells seeded on the bone discs over the 45 day culture period we utilized Flow Cytometry (FACS). When 1000 7F2 cells were seeded on a bone disc and then harvested for FACS analysis on days 15, 30 or 45 of culture we found an increase in cell number from day 0 to day 15 with a plateau by day 30 (Fig. 2A). We did not observe a decrease in 7F2 cell number by day 45 with approximately 35,000 total cells. When 1000 MDA-MB-231 cells were seeded on a devitalized bone disc, an increase in cell number was observed by day 15, with a plateau by day 30 (Fig. 2B) at approximately 60,000 total cells. When 1000 7F2 cells and 1000 MDA-MB-231 cells were co-cultured together on a devitalized bone disc we observed similar cell growth as before for 7F2 cells (approximately 45,000 total cells on day 45), however, we only observed 45,000 MDA-MB-231 cells on day 30 with a decrease of cells observed by day 45 (Fig. 2C), compared to 60,000 total MDA-MB-231 cells when cultured alone. We then cocultured 1000 7F2 and 1000 MDA-MB-231 cells together on a vital bone disc and found that MDA-MB-231 cells displayed a similar growth profile to MDA-MB-231 cells cultured on devitalized bone discs, however 7F2 cells reached a plateau on day 30 with 35,000 total cells and this decreased to under 20,000 by day 45 (Fig. 2D). We also observed endogenous TRAP positive cells at all time points with a peak at day 30 of approximately 7500 cells (Fig. 2D). When unseeded devitalized bone discs were cultured for 45 days we found no cells present as expected (Fig. 2E), however, in untreated discs approximately 15,000 endogenous bovine cells were present by day 45. Within this population we observed 1500 TRAP positive cells (Fig. 2E), and approximately 900 BSP positive cells by day 45 in culture (Fig. 2E).
Results
Analysis of bone turnover
Immunofluorescence of cells seeded onto bone discs
Bone turnover was assessed once it was established that mouse and human cells adhered to and survived for extended periods in culture. Bovine bone discs were cultured alone, with osteoblasts, with breast cancer cells, or with osteoblasts and breast cancer cells together. When cultured with bone discs, both murine osteoblasts and human breast cancer cells alone increased alkaline phosphatase activity in the medium at all time points measured (15, 30 and 45 days) (Fig. 3A). Human breast cancer cells induced significantly more alkaline phosphatase activity in culture medium than osteoblasts at all time points measured. When equal numbers of osteoblasts and breast cancer cells were co-cultured, the amount of alkaline phosphatase activity in the medium resembled that of osteoblasts alone. When the number of breast cancer cells was doubled and co-cultured with 1000 osteoblasts, there was an increase in alkaline phosphatase levels compared to the co-culture of equal numbers of osteoblasts and breast cancer cells. In contrast, alkaline phosphatase levels were lower when osteoblast numbers
Cells labeled with vital tracking dyes and seeded on bovine bone discs were visualized using fluorescence microscopy (Fig. 1). Fluorescence microscopy of bone discs seeded with murine osteoblasts (Fig. 1A) and human breast cancer cells (Fig. 1B) demonstrated that these cell types attach to bovine bone cores invitro after 48 h of culture. The longevity of murine and human cells in co-culture with bovine femur explants was observed by either seeding bone discs with both osteoblasts and breast cancer cells (Figs. 1C–E), or leaving them unseeded (Fig. 1F) and then cultured for 45 days. Bone discs were fixed and stained with anti-GFP (Fig. 1D) to recognize human breast cancer cells, bone sialoprotein (Fig. 1C) to recognize murine osteoblasts and human breast cancer cells, or TRAP (Fig. 1F) to recognize bovine osteoclasts. After 45 days in culture, both osteoblasts and breast cancer cells were clearly
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Fig. 1 – Bone explant culture and vital staining. Cells were added to organ cultures at 24 h. (A, B) Vital staining of cells after 48 h in culture with bone discs. (A) Osteoblasts labeled with Oregon Green; (B) breast cancer cells labeled with Fura Red. Note that both murine osteoblasts and human cancer cells adhere firmly to the surface of bone discs after rinsing in PBS. (The bar equals 100 μm for A–B.) Immunofluorescence of cells after 45 days in culture (C–F) with transmitted light channel showing the outline of bone trabeculae (white). (C) Murine osteoblasts stained green with anti-BSP, (D) human breast cancer cells stained red with anti-GFP, (E) overlay of green osteoblasts and yellow breast cancer cells, (F) unseeded bone discs stained green with anti-TRAP antibody to visual native osteoclasts. Images C–E were taken from the same field of view on an identical bone disc, while image F was produced from a separate bone disc. Secondary control for TRAP antibody (inset F).
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Fig. 2 – Cell counts from the bone discs. (A) One-thousand 7F2 cells seeded on a vital bone disc and counted using FACS analysis. (B) One-thousand MDA-MB-231 cells seeded on a vital bone disc. (C) One-thousand 7F2 and MDA-MB-231 cells co-cultured on a devitalized bone disc. (D) One-thousand 7F2 and MDA-MB-231 cells co-cultured on a vital bone disc with TRAP positive cells labeled. (E) Unseeded vital or devitalized bone discs with labeled TRAP and BSP positive cells and unlabeled endogenous cells. All cell counts were gated to exclude debris.
were doubled relative to breast cancer cells compared to the coculture of equal numbers of osteoblasts and breast cancer cells. Human breast cancer cells, when added alone to bovine cores, induced an increase in acid phosphatase activity at all time points. In contrast, osteoblasts alone added to bone discs did not induce detectable amounts of acid phosphatase activity during the 45 day culture period (Fig. 3B). Similar to alkaline phosphatase, equal numbers of osteoblasts and breast cancer cells cultured together resulted in a decrease in acid phosphatase activity (approximately 50% from levels observed in breast cancer alone). When the number of breast cancer cells was doubled, the effect of the
osteoblasts was muted, but not lost completely. Correspondingly, when the number of osteoblasts was doubled relative to breast cancer cells, no difference was observed in acid phosphatase activity in comparison to equal numbers of osteoblasts and human breast cancer cells.
Devitalized bone discs do not respond to breast cancer cells Devitalized bone discs seeded with osteoblasts, breast cancer cells, or osteoblasts and breast cancer cells were assayed for alkaline (Fig. 4A) or acid (Fig. 4B) phosphatase activity on days 15, 30 and 45 of culture.
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Fig. 3 – Osteoblasts abrogate bone turnover in the presence of osteolytic breast cancer cells. Osteoblasts (7F2) and/or human breast cancer (MDA-MB-231) cells were cultured with bovine core discs and the levels of alkaline phosphate (A) or acid phosphatase (B) in the media were assayed every 15 days. (A) ⁎ = 7F2 vs. MDA-MB-231, + = 7F2 vs. 7F2 & 2X MDA-MB-231, @ = MDA-MB-231 vs. 2X 7F2 & MDA-MB-231, # = MDA-MB-231 vs. 7F2 & MDA-MB-231. (B) ⁎ = 7F2 vs. MDA-MB-231, + = 7F2 vs. 7F2 & 2X MDA-MB-231, @ = 7F2 vs. 7F2 & MDA-MB-231, # = MDA-MB-231 vs. 2X 7F2 & MDA-MB-231. (⁎, +, @, # P < 0.05).
Even without endogenous bovine cells within the bone disc, murine osteoblasts were still able to secrete alkaline phosphatase into the medium. In the absence of endogenous bovine cells, human breast cancer cells did not significantly induce the release of alkaline or acid phosphatase into the medium over the 45 day culture period. To test if osteoblasts and breast cancer cells survived 45 days in culture when cultured on devitalized bovine bone discs, we performed immunofluorescence of osteoblast and breast cancer cells (Figs. 5A–C) or on discs left unseeded (Fig. 5D). These devitalized discs were stained with anti-bone sialoprotein to identify osteoblasts and cancer cells (Fig. 5A), anti-GFP to identify cancer cells (Fig. 5B), or TRAP to identify endogenous bovine osteoclasts (Fig. 5D). Similar to non-devitalized bone, osteoblasts and breast cancer cells persist throughout the 45 days when cultured on devitalized bone discs. However, after the devitalization procedure no bovine osteoclasts were detectable.
Delayed introduction of osteoblasts As osteoblasts had a dramatic effect on bone alkaline and acid phosphatase activity in the presence of breast cancer cells, we next
tested whether murine osteoblasts could compete with previously seeded breast cancer cells. Murine osteoblasts were added 10 days after bovine bone core cultures were established and seeded immediately with breast cancer cells; results from these cultures were compared to control experiments in which breast cancer cells and osteoblasts were both seeded on day 1 (24 h after the initial harvest of bone discs). Alkaline and acid phosphatase levels in the medium were assayed every 15 days as before. Here, alkaline (Fig. 6A) and acid (Fig. 6B) phosphatase activity were higher in cultures where osteoblasts were introduced on day 10 of the culture period.
Discussion The newly understood biological interplay between osteoblasts and osteoclasts suggests that alterations to one cell population can be used to manipulate the biological activity of the other population. As the overall turnover of bone represents the collective activities of osteoblasts and osteoclasts, the present studies included the development of an assay of bone turnover in-
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Fig. 4 – Devitalized bone discs do not show an osteolytic response to MDA-MB-231 treatment. Endogenous bovine cells within the bone disc were killed off using ethanol washes coupled with repeated freezing events at − 80 °C. Discs were subjected to the same analysis as demonstrated in Fig. 2. Without endogenous osteoblasts present for MDA-MB-231 cells to interact with, no differences between controls and MDA-MB-231 cells are observed when discs are assayed for alkaline (A) or acid phosphatase (B) activity. ⁎ = 7F2 vs. MDA-231-MB. (⁎P < 0.05).
vitro using clinical markers of bone turnover (i.e., alkaline and acid phosphatase). Here we demonstrated that bone turnover in our explant culture system is influenced by osteolytic breast cancer cells and murine osteoblasts. In this study, murine osteoblasts and human osteolytic cancer cells clearly influenced one another when co-cultured on a bovine bone substrate. We observed that cancer cells were able to induce rapid bone turnover, and that this effect can be mitigated in part through the administration of osteoblasts. We have also demonstrated that within this bone explant culture system murine osteoblasts and human breast cancer cells are viable for at least 45 days in culture. It is interesting to note that the expansion rate of both these cell types in this culture system was not exponential and that each cell type reached a plateau in expansion around day 30 in culture. However, in experiments where osteoblasts and cancer cells were co-cultured together on the vital bone discs we observed a decrease in osteoblast numbers by day 45 of culture. Since we did not observe the same effect when the two cell lines were cultured on devitalized bone discs, there must be some signaling events between the osteoblasts, breast cancer cells and native bovine cells that limit the expansion or survival of the
osteoblasts. We observed a change in proliferation rates of both 7F2 and MDA-MB-231 cells when they were cultured on vital vs. devitalized bone discs. Furthermore, when these cell types were cultured on vital bone discs we observed the appearance of a TRAP positive cell population native to the bone disc. We also observed the appearance of an endogenous; albeit, rare BSP positive cell population over the duration of culture. It is possible that our bone disc extraction protocol killed off the majority of mature bovine osteoblasts, which BSP is a marker of, and that the surviving precursors only matured towards the end of our experiment. Therefore, if we cultured the bone discs for longer periods of time, the number of bovine osteoblasts may increase. However, since these cells were observed at very low levels (less than 1000 cells) at each of our experimental time points, (day 15, 30 or 45) we do not believe that this population of cells significantly affected our outcome measures. However, these cells would account for the background levels of alkaline phosphatase we detected in unseeded bone discs, which was subtracted at each time point as described in the methods. We also demonstrated that although the total number of 7F2 cells is reduced when co-cultured with MDA-MB-231 cells on vital
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Fig. 5 – Immunofluorescence of devitalized bone discs. (A) Murine osteoblasts stained green with anti-BSP, (B) human breast cancer cells stained red with anti-GFP, (C) combination of green osteoblasts and yellow breast cancer cells, (D) unseeded bone discs stained green with anti-TRAP antibody to visualize native osteoclasts, which are not abundant within the devitalized bone discs. The overlaid brightfield (white) channel (A–D) shows the exogenous cells in relation to the trabecular structure of the bone disc. Images A–C were taken from the same field of view on an identical bone disc, and image D was produced from a separate bone disc.
bone discs this does not account fully for the changes seen in the alkaline or acid phosphatase levels. One possible explanation is that by day 30 or 45 the 7F2 cells have mineralized sufficiently into the bone disc and therefore were more difficult to extract than the other cell populations. More importantly we did see changes in cell proliferation of the osteoblasts and cancer cells when in co-culture compared to single culture demonstrating that there is an interaction between these cell types. Although normalizing the enzymatic data to total cell number could be used to represent the changes between treatments, we have to remember that in any coculture system each cell type is not necessarily having an equal effect on each outcome measure, such as we demonstrate here. Therefore, normalizing to total cell number in this co-culture system would incorrectly average the outcome measures (in this case alkaline and acid phosphatase), and we would not be able to describe the interaction observed when these distinct cell populations interact.
A cascade of events is required for breast cancer cells to metastasize to bone. Breast cancer cells must first localize, or ‘home’, to the bone. Once seeded, cancer cell proliferation proceeds. Although the exact mechanisms of osteotropism are unclear, it is known that metastatic breast cancer cells can express integrins ordinarily peculiar to bone cells (osteopontin, bone sialoprotein, and osteocalcin), which likely promote the adherence of metastatic cells to bone [22]. Once cancer cells have metastasized to bone they may recruit and influence bone cells that can mediate resorption. Osteolysis is mediated directly by osteoclasts, and, in this culture system, resident osteoclast activity was observed in bone discs as evident by TRAP positive cells. In suppressing osteolysis, we speculate that exogenous osteoblasts added to bone cultures compete for sites on the bone surface, which might otherwise be occupied by cancer cells. Our experiments in which cells were added at later times also suggest that osteoblasts are unable to displace previously established cancer cells, which remained on
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Fig. 6 – Alkaline/acid phosphatase levels increase when osteoblasts are added to bone discs 10 days after breast cancer cell are introduced. (A) Alkaline phosphatase levels are lower when bone discs cultured with breast cancer cells and osteoblasts were added on day 1 vs. day 10. (B) With the exception of the acid phosphatase levels on day 15, acid phosphatase is lower in bone disc cultures where osteoblasts and breast cancer cells are plated with the bone discs on day 1. ⁎ = Day 10 lane vs. day 1 lane at same time point. (⁎P < 0.001).
bone discs at the end of the time course. We speculate that osteoblasts are less effective at reducing resident cancer cell activity and bone turnover after 10 days of cancer cell growth because of fewer occupiable sites on the bone surface. This hypothesis can also explain the results obtained when osteoblast numbers were doubled. Here we observe an effect close to that of osteoblasts co-cultured with breast cancer cells in a 1:1 ratio; however, bone turnover is not reduced by half. It may be that there is a finite number of occupiable sites on the bone discs, and if breast cancer cells get a clear foothold, it is improbable that osteoblasts can displace adherent cancer cells. This reinforces the need for the development of therapeutic strategies, which can help dislodge osteotropic cancer from the bone [12,13]. The system that we have developed is compatible with a chemical biological approach and may be used to identify such factors. Previous studies of metastasis to bone have demonstrated that breast cancer cells can alter osteoblast adhesion and prevent differentiation [23]. Moreover, osteoblast-conditioned medium reportedly promotes breast cancer cell proliferation [23]. These studies typically employ cell culture systems in which breast cancer cells are treated with osteoblastic factors, or are co-cultured with
osteoblasts. To more closely mimic the in situ environment of bone, we chose to develop an organ culture environment that supports the attachment and growth of exogenous cells, including osteoblasts and breast cancer cells. There have been many studies that demonstrate changes in cellular behavior when cells are cultured in different environments (normally 2-dimensional vs. 3-dimensional culture systems) [24,25]. In the case of osteoblasts and cancer cells we have taken the culture system to the next level in which we employ a 3D culture system that also presents the correct in-vivo tissue/cellular environment. Using this organ culture system we were able to determine that the exogenous addition of osteoblasts reduces bone turnover stimulated by metastatic breast cancer cells. Whereas cytotherapies are not a current treatment of breast cancer metastasis, such an approach may have advantages when combined with current treatments (such as bisphosphonates alone). For example, whereas bisphosphonates have a clear role in treating an osteolytic disease by their action on osteoclasts [26], bisphosphonates alone do not promote the regeneration of lost bone material. Our data suggest that osteoblasts might stimulate the repair of bone material and therefore might augment bisphosphonate therapy by promoting bone regeneration.
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This study has demonstrated that exogenous cells can be cultured/co-cultured in a viable bone environment for long periods of time in-vitro, and that within this culture environment we can get a more accurate picture of what is taking place in-vivo when osteoblasts and/or breast cancer cells are cultured together. This culture system also allows for the environment (bone disc) to interact with the cells through signaling pathways by introducing new cell types (osteoclast) that would normally be present in-vivo. By utilizing this new approach, we have not only described a culture system that more appropriately describes an in-vivo environment, but we have also demonstrated that breast cancer induced osteolysis of bone can be mitigated with exogenous osteoblasts in bone disc organ culture. Hence, the in-vitro system developed in this report can be used to test other cytotherapies alone or in combination with pharmacological approaches.
[10]
[11]
[12]
[13]
Acknowledgments [14]
This work was supported by an operating grant from the Alberta Breast Cancer Research Initiative. EW was a recipient of summer studentships from the Canadian Institutes of Health Research, Musculoskeletal Health & Arthritis and the Alberta Cancer Board. DER is a Senior Scholar of the Alberta Heritage Foundation for Medical Research. JRM is an Investigator of the Arthritis Society.
[15]
[16] REFERENCES [17] [1] C.S. Galasko, Mechanisms of lytic and blastic metastatic disease of bone, Clin. Orthop. 169 (1982) 20–27. [2] O.S. Nielsen, A.J. Munro, I.F. Tannock, Bone metastases: pathophysiology and management policy, J. Clin. Oncol. 9 (1991) 509–524. [3] G.P. Thomas, S.U. Baker, J.A. Eisman, E.M. Gardiner, Changing RANKL/OPG mRNA expression in differentiating murine primary osteoblasts, J. Endocrinol. 170 (2001) 451–460. [4] F. Gori, L.C. Hofbauer, C.R. Dunstan, T.C. Spelsberg, S. Khosla, B.L. Riggs, The expression of osteoprotegerin and RANK ligand and the support of osteoclast formation by stromal-osteoblast lineage cells is developmentally regulated, Endocrinology 141 (2000) 4768–4776. [5] J.J. Body, A. Borkowski, A. Cleeren, O.L. Bijvoet, Treatment of malignancy associated hypercalcemia with intravenous aminohydroxypropylidene diphosphonate, J. Clin. Oncol. 4 (1986) 1177–1183. [6] B.F. Boyce, T. Yoneda, T.A. Guise, Factors regulating the growth of metastatic cancer in bone, Endocr. Relat. Cancer 6 (1999) 3333–3347. [7] M. Neudert, C. Fischer, B. Krempien, F. Bauss, M.J. Seibel, Site-specific human breast cancer (MDA-MB-231) metastases in nude rats: model characterisation and in vivo effects of ibandronate on tumour growth, Int. J. Cancer 107 (2003) 468–477. [8] L.J. Murray, T.J. Abrams, K.R. Long, T.J. Ngai, L.M. Olson, W. Hong, P. K. Keast, J.A. Brassard, A.M. O'Farrell, J.M. Cherrington, N.K. Pryer, SU11248 inhibits tumor growth and CSF-1R-dependent osteolysis in an experimental breast cancer bone metastasis model, Clin. Exp. Metastasis 20 (2003) 757–766. [9] S. Hiscox, L. Morgan, D. Barrow, C. Dutkowskil, A. Wakeling, R.I. Nicholson, Tamoxifen resistance in breast cancer cells is
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / y e x c r
Research Article
Osteoblasts suppress high bone turnover caused by osteolytic breast cancer in-vitro Roman Krawetz a,⁎, Yiru Elizabeth Wu a , Derrick E. Rancourt a , John Matyas b a
Department of Oncology, Faculty of Medicine, University of Calgary, Canada Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Canada
b
A R T I C L E I N F O R M AT I O N
AB ST R AC T
Article Chronology:
The skeleton is the most common site of breast cancer metastasis, which can occur in up to 85% of
Received 25 June 2008
patients during their lifetime. The morbidity associated with bone metastases in patients with
Revised version received
breast cancer includes pathological fractures, bone pain, hypercalcaemia, and spinal cord
27 April 2009
compression. When breast cancer metastasizes to bone, the balance of bone resorption
Accepted 28 April 2009
(mediated by osteoclasts) and bone formation (mediated by osteoblasts) favors bone resorption,
Available online 9 May 2009
which leads to net bone destruction (i.e., osteolysis). Anti-resorptive agents such as bisphosphonates are commonly used to treat bone resorption in osteoporosis or osteolytic cancer patients. However,
Keywords:
bisphosphonates by themselves are unable to rebuild lost bone tissue, and can cause severe side
Bone organ culture
effects. In this study, we developed a bovine bone explant culture system and have observed that
Osteotropic breast cancer
murine osteoblasts can modulate the activity of osteotropic human breast cancer cells on this
Osteoblasts
substrate. Using markers of bone metabolism, we observe diminished bone turnover in organ
Bone turnover
culture following the addition of exogenous osteoblasts. The data presented in this study supports further investigation into the use of cytotherapies to limit breast cancer mediated osteolysis. Crown Copyright © 2009 Published by Elsevier Inc. All rights reserved.
Introduction The skeleton undergoes continuous remodeling: Bone is resorbed by osteoclasts and is deposited by osteoblasts in a coordinated fashion. The tight balance between osteoclasts and osteoblasts is deregulated in diseases such as osteoporosis, rheumatoid arthritis, and osteolytic metastasis. When breast cancer metastasizes to bone, it activates osteoclasts, creating osteolytic lesions [1]. Tumorinduced osteolysis is mediated by bone-resorbing osteoclasts, whose number and activity are influenced by a variety of osteoclast-stimulating factors that are produced in the bone marrow microenvironment and act in a paracrine fashion [2]. Osteoblasts, for instance, regulate osteoclast activity through expression of the cytokine RANKL (receptor activator of nuclear factor-κB ligand), which promotes osteoclast differentiation.
Osteoblasts also express the decoy receptor of RANKL; osteoprotegerin (OPG), which acts to inhibit RANKL activity [3,4]. In cancer, as in osteoporosis, bisphosphonates act as specific inhibitors of osteoclastic activity, and thereby have special utility in treating hypercalcaemia of malignancy [5]. Although targeted inhibition of osteoclasts by bisphosphonates can slow osteolysis, inhibition is typically incomplete, and bone lesions continue to progress without healing. Unfortunately, few therapies are currently available to promote sustained regeneration of damaged bone. In order to investigate new therapies against metastasisinduced osteolysis, various in-vivo and in-vitro models have been developed. The mouse has proven to be a very powerful animal model of human breast cancer metastasis, as xenografts of the human breast cancer cell line MDA-MB-231 can be transplanted
⁎ Corresponding author. University of Calgary, 331-3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1. E-mail address: [email protected] (R. Krawetz). 0014-4827/$ – see front matter. Crown Copyright © 2009 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2009.04.026
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into immunocompromised animals. For example, when injected systemically into BalbCnu/nu (nude) mice or rats, these cells metastasize to a variety of locations, including the skeleton [6,7]. The murine model has been used extensively to develop therapies aimed at mitigating the morbidity of breast cancer metastasis [8– 11]. A common approach is the competitive inhibition of cell activities using antibodies and peptides directed against bonetrophic integrin proteins that appear on both metastatic cancer cells and osteoblasts [12,13]. Clinically, metabolic bone diseases are diagnosed by measuring bone mineral density (BMD). However, BMD is a static measure of bone structure and a baseline BMD is of little value in predicting the rate of osteolytic progression. However, when BMD is combined with markers of bone metabolism, more reliable predictions may be made of whole-body bone turnover. Metabolic markers of bone metabolism include bone-specific alkaline phosphatase (BAP) for osteoblast activity, and tartrate-resistant acid phosphatase (TRAP) for osteoclastic activity. BAP is synthesized by osteoblasts and is involved in the calcification of bone matrix. BAP is one of a number of different tissue isoenzymes of alkaline phosphatase, and is considered to be a highly specific marker of the bone-forming activity of osteoblasts. Moreover, BAP can be used to differentiate between serum sampled from tumor patients with or without bone metastases. In patients with bone metastases, elevated BAP levels strongly predict the presence of metastases; patients without bone metastases have BAP levels similar to non-cancer reference groups [14]. Serum TRAP is a marker of osteoclast activity and bone resorption [15–18]. As the normal concentration of serum TRAP levels is very low, elevated serum TRAP is a sensitive marker of bone metastasis in breast cancer patients. Indeed, TRAP and BAP levels in breast cancer patients with bone metastases are significantly higher than in normal subjects [19–21]. In this study we have developed and validated an in-vitro assay of overall bone turnover in explants co-cultured with human breast cancer cells. In this experimental system, the osteolytic and osteoblastic activity of osteotropic human breast cancer cells on native bone substrate can be influenced by the addition of exogenous osteoblasts. We therefore submit that this in-vitro system can be used to screen new treatments of osteolysis by providing a more relevant in-vivo cellular environment compared to traditional static cell culture.
Materials and methods Bovine bone discs Fresh bovine distal femurs were obtained from a local meat distributor. Eight millimeter-diameter bovine trabecular bone cores were harvested from the femoral condyles using a custom coring bit. The cylindrical cores were then cut into discs approximate 1 mm in height. The discs were rinsed with PBS containing 50 U/ml penicillin and 50 μg/ml streptomycin (Invitrogen), and weighed on an analytical balance accurate to 0.001 g. Bone discs ranged from 0.960 g to 0.169 g with a median weight of 0.139 g. The discs were then transferred to 24 well Primaria™ culture dishes (BD) and cultured in media containing DMEM without phenol red (Invitrogen), 10% FBS (Invitrogen),
50 U/ml penicillin and 50 μg/ml streptomycin, 1% non-essential amino acids NEAA (Invitrogen), 50 μg/ml ascorbic acid (Sigma), 5 × 10− 8 M vitamin D3 (Sigma), 10 mM beta-glycerol phosphate (Sigma), and 0.1 mM 2-mercaptoethanol (Invitrogen) for 24 h.
Cell culture After the discs were cultured for 24 h, murine osteoblasts (7F2 ATCC# CRL-12557), human breast cancer cells (MDA-MB-231 ATCC# HTB-26), or combinations of the two cell types were added together to the bone disc cultures. Either 1000 or 2000 cells of each type were seeded on the bone discs. MDA-MB-231 cells were stably transfected with a CMV-GFP-Luc2 reporter vector (gift from Dr. Frank Jirik, University of Calgary).
Cell counting by Flow Cytometry Bone discs were seeded with cells as described earlier and then treated with Accutase™ (Millipore) for 3 treatments of 5 min at each time point to release the cells for counting. The supernatant was collected and the cells were fixed in 4% paraformaldehyde. Cells obtained from the bone discs were stained using the following antibodies: anti-ERα to detect 7F2 osteoblasts (Santa Cruz; sc-7207), anti-GFP to detect MDA-MB-231 breast cancer cells (Santa Cruz; sc-8334), anti-TRAP to detect bovine osteoclasts (Santa Cruz; sc-28204) or bone sialoprotein (BSP) to detect bovine osteoblasts (Iowa Developmental Studies Hybridoma Bank; WVIDI9C5). Primary antibodies were directly conjugated to R-Phycoerythrin (R-PE) for anti-ERα, Alexa Fluor 488 for anti-GFP or antiBSP, and Alexa Fluor 647 for TRAP all using Zenon conjugation kits (Invitrogen) following the manufacturer's recommended method. The single cell suspension was subjected to fluorescence-activated cell sorting on days 15, 30 and 45 using a FACS Calibur instrument and the CellQuest software from Becton Dickinson (Germany). Anti-mouse IgG R-PE, 488 or 647 were used as isotype controls depending on the primary conjugation molecule used. Each count was performed using triplicate cultures. To obtain the cell count from each sample CountBright™ absolute counting beads (Invitrogen) were pre-mixed with each sample as per the manufacturer's instructions and were gated separately.
Immunofluorescence To assess short term viability and attachment behavior, osteoblasts and breast cancer cells were labeled with the vital cell-tracking dyes Oregon Green and Fura Red respectively (Molecular Probes: O-6807, Molecular Probes: F-3021), and incubated with the bone cores for 48 h. The discs were subsequently rinsed with PBS and visualized using a fluorescence microscope (Olympus 1X70 microscope equipped with a SPOT RT Color camera and SPOT software [v4.0.9]). To assess whether the transplanted cells retained their phenotypes up to 45 days in bone explant coculture, breast cancer cells and osteoblasts were stained with antibodies to GFP, TRAP and bone sialoprotein (BSP), then visualized by confocal microscopy. Briefly, bone cores were fixed in 4% paraformaldehyde overnight at 4 °C, washed in PBS, and permeabilized in 1% saponin. The cores were then blocked using 3% BSA in PBS and incubated with primary antibody (anti-GFP, anti-TRAP, or anti-BSP) at a 1:50 concentration in 3% BSA in PBS overnight at 4 °C. The samples were then washed in PBS and
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incubated with an appropriate secondary antibody (Alexa Fluor 488 or 568: Invitrogen) and incubated at 4 °C overnight, samples were then washed and mounted. A Zeiss 510 confocal microscope with 488 nm and 568 nm filters was used to capture images, and the images were prepared with the Zeiss LSM image browsing software (v.4.0.0.157).
Alkaline and acid phosphatase detection To asses bone turnover in long term culture, the culture was maintained for 45 days and 100 µl of culture fluid was analyzed on days 15, 30 and 45 using acid (Sigma CS-0740) and alkaline (BioRad 172-1063) phosphatase detection kits. The medium was changed as necessary, however, 24 h before each time point (15, 30 and 45 days) every well was replaced with 1000 µl of fresh medium. Alkaline and acid phosphatase detection was carried out as follows: 100 μl of medium was incubated with acid or alkaline phosphatase reaction buffer at 37 °C for 30 min. The reaction was stopped with the addition of 50 μl of 1 M NaOH. The absorbance at 420 nm was quantified using a Bio-Rad Benchmark Plus plate reader. The alkaline and acid phosphatase absorbance values were normalized by subtracting background absorbance values from control treatments (medium alone), the culture vessel (∼0.045 at 420 nm), each cell type cultured alone, the bone disc alone (the medium from an average bone disc ∼ 0.139 g produced an absorbance value of 0.100 at 420 nm). Each treatment and assay was performed in triplicate. Statistical analysis (repeated measures ANOVA) was performed using GraphPad Prism4 (GraphPad Software) and significance was set at p < 0.05.
Devitalization of bone discs To study the influence of endogenous bone cells on bone disc metabolism, bone discs were prepared as above and devitalized by placing them into dishes containing 100% ethanol, repeatedly freezing to −80 °C, and thawing at room temperature. After the 3rd freeze-thaw cycle, devitalized bone discs were placed in 24well Primaria™ culture dishes (BD) and cultured in a medium as described above.
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visible within the bone discs. Moreover, when osteoblasts and breast cancer cells were co-cultured on the bone discs both cell types were observed in close proximity to each other (the colocalization of GFP and BSP, yellow, represents the human breast cancer cells whereas green staining represents osteoblasts). TRAP positive cells were also observed at 45 days in organ culture, signifying that bovine osteoclasts or differentiating progenitors were still viable after long term culture periods.
Cell counting To determine the proliferation of cells seeded on the bone discs over the 45 day culture period we utilized Flow Cytometry (FACS). When 1000 7F2 cells were seeded on a bone disc and then harvested for FACS analysis on days 15, 30 or 45 of culture we found an increase in cell number from day 0 to day 15 with a plateau by day 30 (Fig. 2A). We did not observe a decrease in 7F2 cell number by day 45 with approximately 35,000 total cells. When 1000 MDA-MB-231 cells were seeded on a devitalized bone disc, an increase in cell number was observed by day 15, with a plateau by day 30 (Fig. 2B) at approximately 60,000 total cells. When 1000 7F2 cells and 1000 MDA-MB-231 cells were co-cultured together on a devitalized bone disc we observed similar cell growth as before for 7F2 cells (approximately 45,000 total cells on day 45), however, we only observed 45,000 MDA-MB-231 cells on day 30 with a decrease of cells observed by day 45 (Fig. 2C), compared to 60,000 total MDA-MB-231 cells when cultured alone. We then cocultured 1000 7F2 and 1000 MDA-MB-231 cells together on a vital bone disc and found that MDA-MB-231 cells displayed a similar growth profile to MDA-MB-231 cells cultured on devitalized bone discs, however 7F2 cells reached a plateau on day 30 with 35,000 total cells and this decreased to under 20,000 by day 45 (Fig. 2D). We also observed endogenous TRAP positive cells at all time points with a peak at day 30 of approximately 7500 cells (Fig. 2D). When unseeded devitalized bone discs were cultured for 45 days we found no cells present as expected (Fig. 2E), however, in untreated discs approximately 15,000 endogenous bovine cells were present by day 45. Within this population we observed 1500 TRAP positive cells (Fig. 2E), and approximately 900 BSP positive cells by day 45 in culture (Fig. 2E).
Results
Analysis of bone turnover
Immunofluorescence of cells seeded onto bone discs
Bone turnover was assessed once it was established that mouse and human cells adhered to and survived for extended periods in culture. Bovine bone discs were cultured alone, with osteoblasts, with breast cancer cells, or with osteoblasts and breast cancer cells together. When cultured with bone discs, both murine osteoblasts and human breast cancer cells alone increased alkaline phosphatase activity in the medium at all time points measured (15, 30 and 45 days) (Fig. 3A). Human breast cancer cells induced significantly more alkaline phosphatase activity in culture medium than osteoblasts at all time points measured. When equal numbers of osteoblasts and breast cancer cells were co-cultured, the amount of alkaline phosphatase activity in the medium resembled that of osteoblasts alone. When the number of breast cancer cells was doubled and co-cultured with 1000 osteoblasts, there was an increase in alkaline phosphatase levels compared to the co-culture of equal numbers of osteoblasts and breast cancer cells. In contrast, alkaline phosphatase levels were lower when osteoblast numbers
Cells labeled with vital tracking dyes and seeded on bovine bone discs were visualized using fluorescence microscopy (Fig. 1). Fluorescence microscopy of bone discs seeded with murine osteoblasts (Fig. 1A) and human breast cancer cells (Fig. 1B) demonstrated that these cell types attach to bovine bone cores invitro after 48 h of culture. The longevity of murine and human cells in co-culture with bovine femur explants was observed by either seeding bone discs with both osteoblasts and breast cancer cells (Figs. 1C–E), or leaving them unseeded (Fig. 1F) and then cultured for 45 days. Bone discs were fixed and stained with anti-GFP (Fig. 1D) to recognize human breast cancer cells, bone sialoprotein (Fig. 1C) to recognize murine osteoblasts and human breast cancer cells, or TRAP (Fig. 1F) to recognize bovine osteoclasts. After 45 days in culture, both osteoblasts and breast cancer cells were clearly
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Fig. 1 – Bone explant culture and vital staining. Cells were added to organ cultures at 24 h. (A, B) Vital staining of cells after 48 h in culture with bone discs. (A) Osteoblasts labeled with Oregon Green; (B) breast cancer cells labeled with Fura Red. Note that both murine osteoblasts and human cancer cells adhere firmly to the surface of bone discs after rinsing in PBS. (The bar equals 100 μm for A–B.) Immunofluorescence of cells after 45 days in culture (C–F) with transmitted light channel showing the outline of bone trabeculae (white). (C) Murine osteoblasts stained green with anti-BSP, (D) human breast cancer cells stained red with anti-GFP, (E) overlay of green osteoblasts and yellow breast cancer cells, (F) unseeded bone discs stained green with anti-TRAP antibody to visual native osteoclasts. Images C–E were taken from the same field of view on an identical bone disc, while image F was produced from a separate bone disc. Secondary control for TRAP antibody (inset F).
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Fig. 2 – Cell counts from the bone discs. (A) One-thousand 7F2 cells seeded on a vital bone disc and counted using FACS analysis. (B) One-thousand MDA-MB-231 cells seeded on a vital bone disc. (C) One-thousand 7F2 and MDA-MB-231 cells co-cultured on a devitalized bone disc. (D) One-thousand 7F2 and MDA-MB-231 cells co-cultured on a vital bone disc with TRAP positive cells labeled. (E) Unseeded vital or devitalized bone discs with labeled TRAP and BSP positive cells and unlabeled endogenous cells. All cell counts were gated to exclude debris.
were doubled relative to breast cancer cells compared to the coculture of equal numbers of osteoblasts and breast cancer cells. Human breast cancer cells, when added alone to bovine cores, induced an increase in acid phosphatase activity at all time points. In contrast, osteoblasts alone added to bone discs did not induce detectable amounts of acid phosphatase activity during the 45 day culture period (Fig. 3B). Similar to alkaline phosphatase, equal numbers of osteoblasts and breast cancer cells cultured together resulted in a decrease in acid phosphatase activity (approximately 50% from levels observed in breast cancer alone). When the number of breast cancer cells was doubled, the effect of the
osteoblasts was muted, but not lost completely. Correspondingly, when the number of osteoblasts was doubled relative to breast cancer cells, no difference was observed in acid phosphatase activity in comparison to equal numbers of osteoblasts and human breast cancer cells.
Devitalized bone discs do not respond to breast cancer cells Devitalized bone discs seeded with osteoblasts, breast cancer cells, or osteoblasts and breast cancer cells were assayed for alkaline (Fig. 4A) or acid (Fig. 4B) phosphatase activity on days 15, 30 and 45 of culture.
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Fig. 3 – Osteoblasts abrogate bone turnover in the presence of osteolytic breast cancer cells. Osteoblasts (7F2) and/or human breast cancer (MDA-MB-231) cells were cultured with bovine core discs and the levels of alkaline phosphate (A) or acid phosphatase (B) in the media were assayed every 15 days. (A) ⁎ = 7F2 vs. MDA-MB-231, + = 7F2 vs. 7F2 & 2X MDA-MB-231, @ = MDA-MB-231 vs. 2X 7F2 & MDA-MB-231, # = MDA-MB-231 vs. 7F2 & MDA-MB-231. (B) ⁎ = 7F2 vs. MDA-MB-231, + = 7F2 vs. 7F2 & 2X MDA-MB-231, @ = 7F2 vs. 7F2 & MDA-MB-231, # = MDA-MB-231 vs. 2X 7F2 & MDA-MB-231. (⁎, +, @, # P < 0.05).
Even without endogenous bovine cells within the bone disc, murine osteoblasts were still able to secrete alkaline phosphatase into the medium. In the absence of endogenous bovine cells, human breast cancer cells did not significantly induce the release of alkaline or acid phosphatase into the medium over the 45 day culture period. To test if osteoblasts and breast cancer cells survived 45 days in culture when cultured on devitalized bovine bone discs, we performed immunofluorescence of osteoblast and breast cancer cells (Figs. 5A–C) or on discs left unseeded (Fig. 5D). These devitalized discs were stained with anti-bone sialoprotein to identify osteoblasts and cancer cells (Fig. 5A), anti-GFP to identify cancer cells (Fig. 5B), or TRAP to identify endogenous bovine osteoclasts (Fig. 5D). Similar to non-devitalized bone, osteoblasts and breast cancer cells persist throughout the 45 days when cultured on devitalized bone discs. However, after the devitalization procedure no bovine osteoclasts were detectable.
Delayed introduction of osteoblasts As osteoblasts had a dramatic effect on bone alkaline and acid phosphatase activity in the presence of breast cancer cells, we next
tested whether murine osteoblasts could compete with previously seeded breast cancer cells. Murine osteoblasts were added 10 days after bovine bone core cultures were established and seeded immediately with breast cancer cells; results from these cultures were compared to control experiments in which breast cancer cells and osteoblasts were both seeded on day 1 (24 h after the initial harvest of bone discs). Alkaline and acid phosphatase levels in the medium were assayed every 15 days as before. Here, alkaline (Fig. 6A) and acid (Fig. 6B) phosphatase activity were higher in cultures where osteoblasts were introduced on day 10 of the culture period.
Discussion The newly understood biological interplay between osteoblasts and osteoclasts suggests that alterations to one cell population can be used to manipulate the biological activity of the other population. As the overall turnover of bone represents the collective activities of osteoblasts and osteoclasts, the present studies included the development of an assay of bone turnover in-
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Fig. 4 – Devitalized bone discs do not show an osteolytic response to MDA-MB-231 treatment. Endogenous bovine cells within the bone disc were killed off using ethanol washes coupled with repeated freezing events at − 80 °C. Discs were subjected to the same analysis as demonstrated in Fig. 2. Without endogenous osteoblasts present for MDA-MB-231 cells to interact with, no differences between controls and MDA-MB-231 cells are observed when discs are assayed for alkaline (A) or acid phosphatase (B) activity. ⁎ = 7F2 vs. MDA-231-MB. (⁎P < 0.05).
vitro using clinical markers of bone turnover (i.e., alkaline and acid phosphatase). Here we demonstrated that bone turnover in our explant culture system is influenced by osteolytic breast cancer cells and murine osteoblasts. In this study, murine osteoblasts and human osteolytic cancer cells clearly influenced one another when co-cultured on a bovine bone substrate. We observed that cancer cells were able to induce rapid bone turnover, and that this effect can be mitigated in part through the administration of osteoblasts. We have also demonstrated that within this bone explant culture system murine osteoblasts and human breast cancer cells are viable for at least 45 days in culture. It is interesting to note that the expansion rate of both these cell types in this culture system was not exponential and that each cell type reached a plateau in expansion around day 30 in culture. However, in experiments where osteoblasts and cancer cells were co-cultured together on the vital bone discs we observed a decrease in osteoblast numbers by day 45 of culture. Since we did not observe the same effect when the two cell lines were cultured on devitalized bone discs, there must be some signaling events between the osteoblasts, breast cancer cells and native bovine cells that limit the expansion or survival of the
osteoblasts. We observed a change in proliferation rates of both 7F2 and MDA-MB-231 cells when they were cultured on vital vs. devitalized bone discs. Furthermore, when these cell types were cultured on vital bone discs we observed the appearance of a TRAP positive cell population native to the bone disc. We also observed the appearance of an endogenous; albeit, rare BSP positive cell population over the duration of culture. It is possible that our bone disc extraction protocol killed off the majority of mature bovine osteoblasts, which BSP is a marker of, and that the surviving precursors only matured towards the end of our experiment. Therefore, if we cultured the bone discs for longer periods of time, the number of bovine osteoblasts may increase. However, since these cells were observed at very low levels (less than 1000 cells) at each of our experimental time points, (day 15, 30 or 45) we do not believe that this population of cells significantly affected our outcome measures. However, these cells would account for the background levels of alkaline phosphatase we detected in unseeded bone discs, which was subtracted at each time point as described in the methods. We also demonstrated that although the total number of 7F2 cells is reduced when co-cultured with MDA-MB-231 cells on vital
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Fig. 5 – Immunofluorescence of devitalized bone discs. (A) Murine osteoblasts stained green with anti-BSP, (B) human breast cancer cells stained red with anti-GFP, (C) combination of green osteoblasts and yellow breast cancer cells, (D) unseeded bone discs stained green with anti-TRAP antibody to visualize native osteoclasts, which are not abundant within the devitalized bone discs. The overlaid brightfield (white) channel (A–D) shows the exogenous cells in relation to the trabecular structure of the bone disc. Images A–C were taken from the same field of view on an identical bone disc, and image D was produced from a separate bone disc.
bone discs this does not account fully for the changes seen in the alkaline or acid phosphatase levels. One possible explanation is that by day 30 or 45 the 7F2 cells have mineralized sufficiently into the bone disc and therefore were more difficult to extract than the other cell populations. More importantly we did see changes in cell proliferation of the osteoblasts and cancer cells when in co-culture compared to single culture demonstrating that there is an interaction between these cell types. Although normalizing the enzymatic data to total cell number could be used to represent the changes between treatments, we have to remember that in any coculture system each cell type is not necessarily having an equal effect on each outcome measure, such as we demonstrate here. Therefore, normalizing to total cell number in this co-culture system would incorrectly average the outcome measures (in this case alkaline and acid phosphatase), and we would not be able to describe the interaction observed when these distinct cell populations interact.
A cascade of events is required for breast cancer cells to metastasize to bone. Breast cancer cells must first localize, or ‘home’, to the bone. Once seeded, cancer cell proliferation proceeds. Although the exact mechanisms of osteotropism are unclear, it is known that metastatic breast cancer cells can express integrins ordinarily peculiar to bone cells (osteopontin, bone sialoprotein, and osteocalcin), which likely promote the adherence of metastatic cells to bone [22]. Once cancer cells have metastasized to bone they may recruit and influence bone cells that can mediate resorption. Osteolysis is mediated directly by osteoclasts, and, in this culture system, resident osteoclast activity was observed in bone discs as evident by TRAP positive cells. In suppressing osteolysis, we speculate that exogenous osteoblasts added to bone cultures compete for sites on the bone surface, which might otherwise be occupied by cancer cells. Our experiments in which cells were added at later times also suggest that osteoblasts are unable to displace previously established cancer cells, which remained on
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Fig. 6 – Alkaline/acid phosphatase levels increase when osteoblasts are added to bone discs 10 days after breast cancer cell are introduced. (A) Alkaline phosphatase levels are lower when bone discs cultured with breast cancer cells and osteoblasts were added on day 1 vs. day 10. (B) With the exception of the acid phosphatase levels on day 15, acid phosphatase is lower in bone disc cultures where osteoblasts and breast cancer cells are plated with the bone discs on day 1. ⁎ = Day 10 lane vs. day 1 lane at same time point. (⁎P < 0.001).
bone discs at the end of the time course. We speculate that osteoblasts are less effective at reducing resident cancer cell activity and bone turnover after 10 days of cancer cell growth because of fewer occupiable sites on the bone surface. This hypothesis can also explain the results obtained when osteoblast numbers were doubled. Here we observe an effect close to that of osteoblasts co-cultured with breast cancer cells in a 1:1 ratio; however, bone turnover is not reduced by half. It may be that there is a finite number of occupiable sites on the bone discs, and if breast cancer cells get a clear foothold, it is improbable that osteoblasts can displace adherent cancer cells. This reinforces the need for the development of therapeutic strategies, which can help dislodge osteotropic cancer from the bone [12,13]. The system that we have developed is compatible with a chemical biological approach and may be used to identify such factors. Previous studies of metastasis to bone have demonstrated that breast cancer cells can alter osteoblast adhesion and prevent differentiation [23]. Moreover, osteoblast-conditioned medium reportedly promotes breast cancer cell proliferation [23]. These studies typically employ cell culture systems in which breast cancer cells are treated with osteoblastic factors, or are co-cultured with
osteoblasts. To more closely mimic the in situ environment of bone, we chose to develop an organ culture environment that supports the attachment and growth of exogenous cells, including osteoblasts and breast cancer cells. There have been many studies that demonstrate changes in cellular behavior when cells are cultured in different environments (normally 2-dimensional vs. 3-dimensional culture systems) [24,25]. In the case of osteoblasts and cancer cells we have taken the culture system to the next level in which we employ a 3D culture system that also presents the correct in-vivo tissue/cellular environment. Using this organ culture system we were able to determine that the exogenous addition of osteoblasts reduces bone turnover stimulated by metastatic breast cancer cells. Whereas cytotherapies are not a current treatment of breast cancer metastasis, such an approach may have advantages when combined with current treatments (such as bisphosphonates alone). For example, whereas bisphosphonates have a clear role in treating an osteolytic disease by their action on osteoclasts [26], bisphosphonates alone do not promote the regeneration of lost bone material. Our data suggest that osteoblasts might stimulate the repair of bone material and therefore might augment bisphosphonate therapy by promoting bone regeneration.
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This study has demonstrated that exogenous cells can be cultured/co-cultured in a viable bone environment for long periods of time in-vitro, and that within this culture environment we can get a more accurate picture of what is taking place in-vivo when osteoblasts and/or breast cancer cells are cultured together. This culture system also allows for the environment (bone disc) to interact with the cells through signaling pathways by introducing new cell types (osteoclast) that would normally be present in-vivo. By utilizing this new approach, we have not only described a culture system that more appropriately describes an in-vivo environment, but we have also demonstrated that breast cancer induced osteolysis of bone can be mitigated with exogenous osteoblasts in bone disc organ culture. Hence, the in-vitro system developed in this report can be used to test other cytotherapies alone or in combination with pharmacological approaches.
[10]
[11]
[12]
[13]
Acknowledgments [14]
This work was supported by an operating grant from the Alberta Breast Cancer Research Initiative. EW was a recipient of summer studentships from the Canadian Institutes of Health Research, Musculoskeletal Health & Arthritis and the Alberta Cancer Board. DER is a Senior Scholar of the Alberta Heritage Foundation for Medical Research. JRM is an Investigator of the Arthritis Society.
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