Figure 1.
Doxorubicin treatment accelerates bone loss in preclinical breast cancer bone metastasis model.
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 (P = 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 (P<0.05) in bone resorption upon doxorubicin treatment in non-tumor bearing mice. Statistical analysis was performed using student’s T–test. P<0.05 was considered significant. At least 5 mice were used in each group for these experiments.
Figure 2.
Anti-TGFβ antibody treatment improves doxorubicin-mediated inhibition of osteoblast differentiation and increases the frequency of osteoblast colony forming units.
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 vivo 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 Materials and Methods 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.
Figure 3.
Anti-TGFβ antibody inhibits doxorubicin-mediated increase in osteoclast formation.
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 Materials and Methods. (C) Osteoblast-mediated osteoclast formation was also done using a co-culture system as described in Materials and Methods. Student T-test was performed and P<0.05 was considered significant.
Figure 4.
Anti-TGFβ antibody rescues doxorubicin mediated bone loss in breast cancer bone metastasis.
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.
Figure 5.
Doxorubicin mediates bone loss by elevating oxidative stress.
(A) C400 oxidation show increase in reactive oxygen species (ROS) upon in vitro 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 Materials and Methods 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.