Doxorubicin-Mediated Bone Loss in Breast Cancer Bone Metastases Is Driven by an Interplay between Oxidative Stress and Induction of TGFβ

<div><p>Breast cancer patients, who are already at increased risk of developing bone metastases and osteolytic bone damage, are often treated with doxorubicin. Unfortunately, doxorubicin has been reported to induce damage to bone. Moreover, we have previously reported that doxorubicin treatment increases circulating levels of TGFβ in murine pre-clinical models. TGFβ has been implicated in promoting osteolytic bone damage, a consequence of increased osteoclast-mediated resorption and suppression of osteoblast differentiation. Therefore, we hypothesized that in a preclinical breast cancer bone metastasis model, administration of doxorubicin would accelerate bone loss in a TGFβ-mediated manner. Administration of doxorubicin to 4T1 tumor-bearing mice produced an eightfold increase in osteolytic lesion areas compared untreated tumor-bearing mice (<i>P</i> = 0.002) and an almost 50% decrease in trabecular bone volume expressed in BV/TV (<i>P</i> = 0.0005), both of which were rescued by anti-TGFβ antibody (1D11). Doxorubicin, which is a known inducer of oxidative stress, decreased osteoblast survival and differentiation, which was rescued by N-acetyl cysteine (NAC). Furthermore, doxorubicin treatment decreased Cu-ZnSOD (SOD1) expression and enzyme activity <i>in vitro</i>, and treatment with anti-TGFβ antibody was able to rescue both. In conclusion, a combination therapy using doxorubicin and anti-TGFβ antibody might be beneficial for preventing therapy-related bone loss in cancer patients.</p></div

Doxorubicin mediates bone loss by elevating oxidative stress.

<p>(A) C400 oxidation show increase in reactive oxygen species (ROS) upon <i>in vitro</i> doxorubicin (0.01 ug/ml, 20 hours) treatment increases oxidative stress in the mouse bone marrow stromal cells, which was decreased by concomitant treatment with 1D11(25 µg/ml). Data represents average percentage of C400 positive cells from triplicate samples. (B) RT-PCR showing a decrease in SOD1 (copper zinc superoxide dismutase 1) and GPx expression was noted in MC3T3 mouse osteoblast cells upon treatment with doxorubicin (0.01 µg/ml, 20 hours), which was returned to normal level by co-treatment with anti-TGFβ antibody 1D11(25 ug/ml). (C) SOD1 expression normalized against GAPDH expression, quantified by Image J. (D) GPx expression normalized against GAPDH expression, quantified by Image J. (E) SOD1 activity was performed using MC3T3 cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078043#s4" target="_blank">Materials and Methods</a> section. The inhibition of SOD1 activity was measured by formation of NBT-diformazan from NBT following 20 hours treatment in either serum free alpha-MEM media alone, or supplemented with 0.01 ug/ml doxorubicin, 25 µg/ml anti-TGFβ antibody and a combination of both doxorubicin and anti-TGFβ antibody. A drastic inhibition of SOD1 activity was noted following doxorubicin treatment which was restored by anti-TGFβ antibody. (F) Calvarial osteoblasts from wild type mice (3–4 days old pups) were cultured until confluent and treated with osteoblast differentiation media supplemented with doxorubicin (0.01 µg/ml), N-acetyl cysteine (NAC, 20 mM) treatment, or a combination of both, or media alone until mineralized matrix was formed. Quantification of Von Kossa staining images from at least 3 different fields were done using Metamorph software.</p

Doxorubicin treatment accelerates bone loss in preclinical breast cancer bone metastasis model.

<p>Five-week old female Balb/C mice received either 5 mg/Kg doxorubicin (i.p. once weekly for three weeks) or PBS and microCT of tibiae were performed. For orthotopic breast cancer bone metastasis model, another set of four-week old female Balb/C mice were injected in the left #4 mammary fat pad using a bone-tropic 4T1 cell line and upon tumor development (palpable size after 1 week) mice were treated with either PBS or doxorubicin (5 mg/Kg doxorubicin, i.p., once weekly for three weeks) and microCT and histology of tibiae were performed. (A) Schematic representation of orthotopic injection of 4T1 cells in mammary fat pad. (B) Histology of mouse tibia showing tumor cells in the bone. (C) Representative microCT images (3D reconstruction) of tibiae collected from non-tumor bearing and tumor-bearing Balb/C mice after three weeks of treatment and (D) Quantification of average BV/TV showing further decrease in trabecular bone volume in tumor-bearing mice upon doxorubicin treatment, compared to non-tumor bearing mice. (E) Average BV/TV were assessed using microCT analysis of tibiae collected from 4 week old athymic nude mice mouse treated with either vehicle or doxorubicin showing loss of trabecular bone volume (<i>P</i> = 0.02). (F) Representative X-ray images showing bone loss upon doxorubicin treatment, compared to control. Quantification of (G) osteolytic lesion area and (H) osteolytic lesion numbers in tumor-bearing Balb/C mice receiving 4T1 cell via cardiac injection and treated with either PBS or doxorubicin (5 mg/Kg) once per week for three weeks. (I) Ratlaps ELISA showing significant increase (<i>P</i><0.05) in bone resorption upon doxorubicin treatment in non-tumor bearing mice. Statistical analysis was performed using student’s T–test. <i>P</i><0.05 was considered significant. At least 5 mice were used in each group for these experiments.</p

Anti-TGFβ antibody inhibits doxorubicin-mediated increase in osteoclast formation.

<p>Mononuclear cells from spleen (A) and bone marrow (B) from C57BL/6 were isolated and cultured for 15 days in the presence of MCSF and RANKL until mature osteoclasts are formed and scored using TRAP staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078043#s4" target="_blank">Materials and Methods</a>. (C) Osteoblast-mediated osteoclast formation was also done using a co-culture system as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078043#s4" target="_blank">Materials and Methods</a>. Student T-test was performed and P<0.05 was considered significant.</p

Anti-TGFβ antibody rescues doxorubicin mediated bone loss in breast cancer bone metastasis.

<p>MDA-MB-231 cells (1×10⁶) were inoculated via left cardiac ventricle of four-week old female athymic nude mice and treated with PBS, doxorubicin (5 mg/Kg, once weekly for 4 weeks, i.p.), 1D11 (10 mg/Kg, three times per weekly for 4 weeks) and a combination of doxorubicin and 1D11 for three weeks. (A) Representative microCT images of mice tibiae from each treatment group (B) Quantification of trabecular bone volume (BV/TV), (C) Histology of mice tibia from each group revealed trabecular bone loss upon doxorubicin treatment, both of which were rescued upon anti-TGFβ antibody treatment. Quantification of (D) average trabecular numbers and (E) average trabecular thickness. Representative microCT images from tumor-bearing mice in which (F) 4T1 cells (10⁵) were inoculated either via intracardiac route or (H) in the #4 mammary fat pad of four-week old female Balb/c mice which received either PBS, doxorubicin (5 mg/Kg, once weekly for 4 weeks, i.p.), 1D11 (10 mg/Kg, three times per weekly for 4 weeks) or a combination of doxorubicin and 1D11 for three weeks. Quantification of microCT images show significant loss of trabecular bone volume (BV/TV) in both intracardiac (G) and orthotopic (I) models. At least 5 mice were assessed in each group and P<0.05 was considered significant.</p

Anti-TGFβ antibody treatment improves doxorubicin-mediated inhibition of osteoblast differentiation and increases the frequency of osteoblast colony forming units.

<p>Mouse bone marrow cells were flushed and allowed to attach for two days. Bone marrow stromal cells were trypsinized and replated as 1×10⁶ cells per well in six well plates for fibroblast colony forming units (CFU-F) and 2×10⁶ cells for osteoblast colony forming units (CFU-OB). Cells were cultured either using DMEM-F12 media (10% FBS) alone or supplemented with either doxorubicin (0.01 ug/ml), 1D11(25 µg/ml) or a combination of both until fibroblast colonies were formed. CFU-OB were cultured using osteoblast differentiation media (alpha-MEM+10% FBS) containing ascorbic acid and β glycerophosphate with similar concentration of doxorubicin (0.01 µg/ml) and/or 1D11(25 µg/ml). Upon microscopic colony formation, media were aspirated, plates were washed in PBS, fixed with 10% neutral buffered formalin and stained to score (A) Average number of fibroblast colony forming units (CFU-F) per 1×10⁶ bone marrow cells. (B) Average number of osteoblast colony forming units (CFU-OB) per 2×10⁶ bone marrow cells. (C) Ex <i>vivo</i> osteoblast mineralization assay was performed using mouse calverial osteoblasts isolated from 3 days old pups and plated in triplicate. Upon confluence, cells were grown in osteoblast differentiation media containing ascorbic acid and β glycerophosphate, in presence of doxorubicin (0.01 µg/ml), 1D11(25 µg/ml) or a combination until mineralized matrix were formed. Von Kossa staining was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078043#s4" target="_blank">Materials and Methods</a> and mineralization was scored using Metamorph software in each set and compared with media only group. Student T-test was performed to calculate p-values. P>0.05 was considered significant. N = 6 for each group was used in this experiment. (D) RT-PCR for expression of RANKL, OPG, OCN and OPN from MC3T3 cells treated with media alone, doxorubicin (0.01 µg/ml, 20 hours), anti-TGFβ antibody (25 ug/ml) and a combination of doxorubicin and 1D11.</p

Anti-TGFβ antibody decreased osteoclast numbers and <i>in vitro</i> osteoclastogenesis.

<p><b>Panel a:</b> Boxplot of number of TRAP positive osteoclasts per millimeter of bone surface in MBA-MB-231 tumor-bearing tibiae, showing significantly decreased osteoclasts upon 1D11 treatment, compared to 13C4. Wilcoxon rank-sum p-value = 0.027. Mean ± standard deviation = 13C4: 0.7733±0.3002, 1D11: 0.4539±0.1141. N = at least 6). <b>Panel b:</b> To assess the effect of anti-TGFβ treatment directly on osteoclast population, an <i>ex vivo</i> osteoclastogenesis assay was performed using bone marrow mononuclear cells. Bone marrow mononuclear cells were isolated and cultured in presence of 13C4 (control antibody), 1D11(anti-TGFβ antibody), TGFβ+13C4 or TGFβ+1D11 in presence of both RANKL and MCSF. Both 13C4 and 1D11 was used at a concentration of 25 µg/ml. TGFβ as used at a concentration of 5 ng/ml. Osteoclasts were stained using using a Leucocyte acid phosphatase (TRAP) kit as per manufacturer's instruction (Sigma-Aldrich) and TRAP positive cells (reddish brown) were counted under microscope. Boxplots of number of osteoclasts by group for osteoclastogenesis assay show that treatment with 1D11 significantly reduced TGFβ-mediated osteoclast formation. Wilcoxon rank-sum p-value for TGFβ and TGFβ + 1D11 groups is 0.01. Mean ± standard deviation = TGFβ: 26.5±3.83, TGFβ + 1D11: 17.83±4.17. Data presented here is representative of two independent experiments. <b>Panel c:</b> To assess the effect of anti-TGFβ antibody on osteoblast-mediated osteoclastogenesis, bone marrow mononuclear cells were cultured on a layer of primary mouse calverial osteoblasts in the presence of either control antibody (13C4) or the anti-TGFβ antibody (1D11). After 7–10 days, TRAP staining was performed to identify mature osteoclasts (indicated by arrow). <b>Panel d:</b> Osteoblast mediated osteoclastogenesis increases significantly upon 1D11 treatment compared to control, Wilcoxon rank-sum p-value = 0.006. Mean ± standard deviation = 13C4: 26.5±11.31, 1D11: 4.83±2.48. Data presented here is representative of two independent experiments.</p

Anti-TGFβ antibody improves trabecular architecture in tumor bearing mice tibia and femur.

<p>MicroCT analysis of the tibiae from MDA-MB-231tumor-bearing mice treated for 4 weeks, starting one day after tumor cell inoculation, revealed that suppression of TGFβ by the antibody 1D11 increased trabecular bone volume through increases in trabecular number, and this improved the connectivity of the trabeculae (lack of fenestrations), compared to isotype control. Wilcoxon rank-sum test was used for this analysis. Means and standard deviations by group for the MDA-MB-231 four week data with p-values from Wilcoxon rank-sum tests. Quantitative analysis of microCT data from MDA-MB-231 tumor-bearing mice treated with 13C4 or 1D11 for weeks. Trabecular bone volume (BV/TV), trabecular thickness (Tb.Th*), trabecular number (Tb.N*), and connectivity density (Conn.D), and mean volumetric density of the mineralized tissue (Tb.TMD) were calculated using the Scanco evaluation software.</p

Suppression of TGFβ by anti-TGFβ antibody 1D11 increased the mineral-to-collagen ratio.

<p>Confocal raman spectroscopy was performed on mice bearing MDA-MB-231 tumors in bone treated for 4 weeks with 1D11 or 13C4 antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027090#s2" target="_blank">materials and methods</a> section. At least nine spectra were analyzed per specimen and the mean mineral-to-collagen ratio, Type-B carbonate substitution, and crystallinity were scored. Both mineral-to-collagen ratio and carbonate substitution increased significantly upon 1D11 treatments compared to control. Mean ± standard deviation is shown, p value was determined using Wilcoxon test.</p

Anti-TGFβ antibody increases bone volume in tumor bearing mice.

<p>MDA-MB-231 cells were injected via intra-cardiac route in 4 week old female nude mice and 4T1 cells were injected in 4–5 week old female Balb/C mice. Mice were treated either with control antibody (13C4, 10 mg/kg) or anti-TGFβ antibody (1D11, 10 mg/kg) for 4 weeks, starting 1 day after tumor cell inoculation. Trabecular bone volume in the tibial metaphysis of tumor-bearing mice was analyzed by microCT. <b>Panel a:</b> Representative three dimensional reconstitutions of microCT images from both 13C4 and 1D11 treated groups from mice injected with MDA-MB-231 cells. <b>Panel b:</b> Boxplots of average BV/TV (bone volume/total volume) by group for the MDA-M<b>B</b>-231 tumor- bearing mice show significant increase in bone mass after treatment with anti-TGFβ antibody. Wilcoxon rank-sum p-value = <0.001. Mean ± standard deviation = 13C4: 0.06±0.04, 1D11: 0.32±0.09. N = at least 10. <b>Panel c.</b> Boxplots of average BV/TV (bone volume/total volume) by group for the 4T1 tumor- bearing mice show a significant increase in bone mass as a result of treatment with 1D11 as measured by BV/TV. Wilcoxon rank-sum p-value = 0.036. Mean ± standard deviation = 13C4: 0.09±0.01, 1D11: 0.11±0.01, N = at least 5. <b>Panel d:</b> Mouse calverial osteoblasts were isolated and cultured for 7–10 days as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027090#s2" target="_blank">Materials and Methods</a>, either in presence of anti-TGFβ antibody (1D11) or isotype control (13C4) and mineralized matrix formation was measured using Von Kossa staining as a surrogate for osteoblast differentiation. <b>Panel e:</b> Boxplot analysis reveals treatment with anti-TGFβ antibody (1D11) significantly increased percent areas of mineralized matrix. Images were taken from representative fields and quantified using Metamorph software. Wilcoxon rank-sum p-value = 0.005. Mean ± standard deviation = 13C4: 16±3.7, 1D11: 32.3±1. Data presented here is representative of two independent experiments.</p