Journal: Advanced Science

Article Title: The Osteoblastic Microenvironment Determines the Fate of Breast Cancer Cells Disseminated in the Bone Marrow

doi: 10.1002/advs.202509980

Figure Lengend Snippet: RUNX2 increases the accumulation of MDA231 cells as micrometastases in bone marrow. (A) Experimental schedule for the bone colonization of MDA231‐derived cells in SCID mice. An osteogenic premetastatic niche (PMN) was established by injecting MDA231 CDH11 high /ITGA5 high extracellular vesicles into SCID mice via the tail vein for 3 weeks (2 doses/week). MDA231 RUNX2‐OE cells and control cells were injected into the mice via the left ventricle. (B) H&E staining images demonstrating osteoblasts (indicated by red triangles) in homeostatic bone (HB) and the PMN. (C) X‐ray images showing the bone mass in the HB and the PMN microenvironments. (D) Bar charts quantifying the osteoblast number as the ratio of osteoblast counts to bone perimeter in /mm (N.Ob/B.Pm) and bone mass as the bone volume fraction (BV/TV). (E) Western blot analysis demonstrating increased RUNX2 protein levels in MDA231 RUNX2‐OE cells compared with those in control cells. (F) Representative X‐ray images, pan‐cytokeratin (pan‐CK) immunohistochemical staining images, and TRAP staining images showing osteolytic lesions, tumor cell distribution, and activated osteoclasts, respectively. (G) Pie charts depicting the incidence of DTCs, micrometastases (micromets), and osteolytic lesions formed by control cells and RUNX2‐OE cells within HB and the PMN. The scale bars in the inset images indicate 20 µm. (H) Bar charts quantifying the tumor surface, erosion surface, and the number of TRAP + osteoclasts normalized to the total bone surface (N.Oc/BS in mm 2 ). (I) Bar charts illustrating the abundance and size of micrometastases in the bone marrow of mice without detectable bone lesions. (J) Representative KI67 fluorescence immunohistochemical staining images. Pan‐CK was used to label the tumor cells, while DAPI was used to stain the nuclei. (K) Bar chart illustrating the reduced numbers of KI67 + tumor cells in micrometastases compared with tumor cells within bone colonization. The data are displayed as the means ± SDs. n.s., not significant; * p <0.05, ** p <0.01, *** p <0.001 compared with the corresponding controls, as determined by Student's t‐test.

Article Snippet: To evaluate the effect of osteoblast differentiation status on osteoclast activity, MC3T3‐E1 cells in the upper chamber were induced with osteogenic media or 0.5 ng/mL PTH(1‐34) (MCE, HY‐P0059).

Techniques: Derivative Assay, Control, Injection, Staining, Western Blot, Immunohistochemical staining, Fluorescence

Journal: Advanced Science

Article Title: The Osteoblastic Microenvironment Determines the Fate of Breast Cancer Cells Disseminated in the Bone Marrow

doi: 10.1002/advs.202509980

Figure Lengend Snippet: Abnormal activation of the osteogenic microenvironment reactivates quiescent basal‐like cancer cells. (A) Experimental schedule for PTH‐reactivated bone colonization. MDA231 RUNX2‐OE cells were injected into SCID mice via the left ventricle. The mice were then treated with 100 µg/kg PTH for 10 consecutive days. Mice inoculated with PBS served as controls. (B) Representative BLI, X‐ray, H&E staining, immunohistochemical staining for pan‐CK, and TRAP staining images demonstrating the progression of bone lesions and the activity of osteoclasts. (C) Pie charts displaying the incidence of DTCs/micrometastases and osteolytic lesions in mice treated with PTH and in control mice. (D) Bar charts showing the incidence of osteolytic bone colonization, tumor surface, and erosion surface in PTH‐treated mice and control mice. (E) Experimental schedule involving the injection of MDA231 RUNX2‐OE cells through the left ventricle, followed by treatment with either a single dose of low‐dose estradiol cypionate (E2, 0.3 mg/kg) or weekly high‐dose E2 (2 mg/kg) for 3 weeks via subcutaneous injection, starting 7 days post‐inoculation of cancer cells. Mice injected with an equal volume of the solvent corn oil served as controls. (F) Representative BLI, X‐ray, H&E staining, immunohistochemical staining for pan‐CK, and TRAP staining images illustrating the progression of bone lesions and the activity of osteoclasts. (G) Pie charts depicting the incidence of DTCs/micrometastases and osteolytic lesions in mice treated with various doses of E2. (H) Bar charts illustrating the incidence of osteolytic bone colonization, tumor surface, and erosion surface in mice subjected to low‐dose E2, high‐dose E2, and control conditions. The scale bars in the inset images indicate 20 µm. The data are presented as the means ± SDs. * p <0.05 and ** p <0.01 compared with the control group, as determined by Fisher's exact probability method or Student's t test.

Article Snippet: To evaluate the effect of osteoblast differentiation status on osteoclast activity, MC3T3‐E1 cells in the upper chamber were induced with osteogenic media or 0.5 ng/mL PTH(1‐34) (MCE, HY‐P0059).

Techniques: Activation Assay, Injection, Staining, Immunohistochemical staining, Activity Assay, Control, Solvent

Journal: Advanced Science

Article Title: The Osteoblastic Microenvironment Determines the Fate of Breast Cancer Cells Disseminated in the Bone Marrow

doi: 10.1002/advs.202509980

Figure Lengend Snippet: RUNX2 promotes the colonization of luminal‐like MCF7 cells within a highly mineralized osteogenic microenvironment induced by E2. (A) Experimental schedule for bone colonization of MCF7‐derived cells in SCID mice. MCF7 RUNX2‐OE cells and control cells were injected into the mice via the left ventricle. E2 (2 mg/kg) was administered via subcutaneous injection weekly to support MCF7 tumor growth, starting 1 week before tumor cell injection. (B) Representative micro‐CT and H&E staining images showing increased bone mass and a reduced marrow cavity in SCID mice administered E2. Bar graph illustrating the bone volume fraction (BV/TV) in mice treated with E2 compared with in control mice. (C) Western blot analysis of RUNX2 protein levels in MCF7 RUNX2‐OE cells and control cells. (D) Representative BLI images showing the systemic distribution of tumor cells following left ventricular inoculation on day 0. X‐ray and H&E staining images displaying bone lesions in mice after sacrifice. TRAP staining images indicating osteoclast activity. (E) Representative micro‐CT images displaying bone lesions in SCID mice on day 38. The osteolytic lesions are marked by asterisks and arrows. (F) Pie charts illustrating the incidence of osteolytic bone colonization in mice injected with MCF7 RUNX2‐OE cells compared with those in mice injected with control cells. (G) Bar charts showing the tumor surface, erosion surface, and TRAP + surface normalized to the bone surface. The data are displayed as the means ± SDs. ** p <0.01 and *** p <0.001 compared to the control group, as determined by Student's t‐test.

Article Snippet: To evaluate the effect of osteoblast differentiation status on osteoclast activity, MC3T3‐E1 cells in the upper chamber were induced with osteogenic media or 0.5 ng/mL PTH(1‐34) (MCE, HY‐P0059).

Techniques: Derivative Assay, Control, Injection, Micro-CT, Staining, Western Blot, Activity Assay

Journal: Advanced Science

Article Title: The Osteoblastic Microenvironment Determines the Fate of Breast Cancer Cells Disseminated in the Bone Marrow

doi: 10.1002/advs.202509980

Figure Lengend Snippet: An unmineralized osteogenic microenvironment exacerbates osteolytic lesions caused by luminal‐like MCF7 cells. (A) Experimental diagram showing the bone colonization of MCF7‐derived cells within different osteogenic microenvironments established by the administration of E2. SCID mice were administered E2 (2 mg/kg) weekly, either alone or in combination with dexamethasone (DEX, 0.5 µg/mL in drinking water) or a low‐calcium diet. (B) Representative BLI images showing the systemic distribution of tumor cells following left ventricular inoculation on day 0. X‐ray and H&E staining images depict bone lesions in mice after sacrifice, whereas the TRAP staining images indicate osteoclast activity. (C) Representative micro‐CT images showing bone lesions in SCID mice on day 38. The osteolytic lesions are marked by asterisks and arrows. (D,E) Bar charts presenting the incidence of osteolytic bone colonization (D), tumor surface, erosion surface, and TRAP + osteoclast surface (E). The scale bars in the inset images indicate 20 µm. The data are displayed as the means ± SDs. * p <0.05, ** p <0.01 and *** p <0.001 compared with the respective E2‐treated mice, as determined by Student's t test.

Article Snippet: To evaluate the effect of osteoblast differentiation status on osteoclast activity, MC3T3‐E1 cells in the upper chamber were induced with osteogenic media or 0.5 ng/mL PTH(1‐34) (MCE, HY‐P0059).

Techniques: Derivative Assay, Staining, Activity Assay, Micro-CT

Journal: Advanced Science

Article Title: The Osteoblastic Microenvironment Determines the Fate of Breast Cancer Cells Disseminated in the Bone Marrow

doi: 10.1002/advs.202509980

Figure Lengend Snippet: Osteoblasts and the bone matrix exhibit distinct effects on RUNX2‐overexpressing breast cancer cells in different osteoblastic microenvironments in vitro. Mouse primary osteoblasts (mOBs) were induced in osteogenic media supplemented with 50 µg/mL L‐ascorbic acid and 10 m m β‐glycerophosphate disodium for 0, 2, 4, 6, 8, 10, or 12 days. mOBs were isolated by digesting the cells with 0.25% trypsin‐EDTA, while the bone matrix (BM) was obtained by removing the cell components by treatment with 20 m m NH 4 OH and 0.5% Triton X‐100 for 5 min. (A) Representative ALP staining and alizarin S staining images after different durations of osteogenic induction. (B) Western blot analysis of osteogenesis‐related protein levels. (C) Staging of the osteoblastic microenvironment in vitro on the basis of various induction durations. (D,E) Effects of mOBs and BM at different stages of differentiation on the chemotactic (D) and proliferative (E) capacities of GFP‐labeled MDA231 RUNX2‐OE cells. Calcium nodules are circled with red dashed lines. (F,G) Effects of mOBs and BM at different stages of differentiation on the chemotactic (F) and proliferative (G) capacities of GFP‐labeled MCF7 RUNX2‐OE cells. Calcium nodules are circled with red dashed lines. The data are presented as the means ± SDs. *** p <0.001 compared with the respective control group (0 days), as determined by Student's t‐test.

Article Snippet: To evaluate the effect of osteoblast differentiation status on osteoclast activity, MC3T3‐E1 cells in the upper chamber were induced with osteogenic media or 0.5 ng/mL PTH(1‐34) (MCE, HY‐P0059).

Techniques: In Vitro, Isolation, Staining, Western Blot, Labeling, Control

Journal: Frontiers in Physiology

Article Title: Thiamet-G facilitates reparative dentin formation via modulating O-GlcNAcylation and inflammation

doi: 10.3389/fphys.2025.1739168

Figure Lengend Snippet: Effect of Thiamet-G on human dental pulp stem cells (hDPSCs). (A) Experimental design for MTS assay to examine cell viability and proliferation. (B) MTS assay shows that Thiamet-G treatment facilitates dose-dependent hDPSC proliferation. (C) Experimental design for ALP activity assay. (D) Thiamet-G treatment significantly induces ALP activity in hDPSCs compared to the control, particularly after 14 days (E) Thiamet-G treatment increases the expression of reparative dentin-related markers, including Alp, Bmp2, Bsp, Dspp, Opn, Gsk3β, and RUNX2, compared to the control. Ns non-significant, * p < 0.03, ** p < 0.002, *** p < 0.0002, and **** p < 0.0001. NC negative control, GM growth medium, OM osteogenic medium.

Article Snippet: After 24 h, the media was changed with osteogenic media (Alfa-MEM with 1% penicillin-streptomycin, 5% fetal bovine serum, 50 μM L-ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone) in presence or absence of Thiamet-G (S7213, Selleckchem.com ) with various concentrations.

Techniques: MTS Assay, ALP Activity Assay, Activity Assay, Control, Expressing, Negative Control

Journal: International Dental Journal

Article Title: Demineralized Dentin Matrix Promotes Bone Regeneration Through IDO1-Mediated Th17/Treg Cell Balance Modulation

doi: 10.1016/j.identj.2025.103853

Figure Lengend Snippet: The DDM extract promotes Treg cells differentiation via IDO1 to enhance BMSCs viability and osteogenic differentiation in the co-culture system of naïve CD4 + T cells and BMSCs. A, CCK-8 results showed that the DDM extract significantly increased BMSCs viability, which decreased with the IDO1 inhibitor. No significant difference was observed between the Control and DDM+Inhibitor groups. B, Azide-Azide coupling method for ALP staining, with the red arrow indicating the blue precipitate after staining. ALP content significantly increased with the DDM extract but decreased with the DDM+inhibitor treatment compared to the Control. 100X, 1cm:100µm. C, ARS staining, with the white arrow indicating the red calcium nodules after staining. Calcium nodules significantly increased with the DDM extract but decreased with the DDM+inhibitor treatment compared to the Control. 100X, 1cm:100µm. WB (D) and RT-qPCR (E) results indicated that the DDM extract significantly upregulated RUNX2, BSP, Osx, COL I, OPN, OCN, OPG and RANKL. With the DDM+IDO1 inhibitor, RUNX2, BSP, Osx, COL I, OPN, OCN and OPG decreased significantly while RANKL remained unchanged. Neither DDM nor DDM+inhibitor affected RANK expression. CD4+BMSC, co-culture of naïve CD4 + T cells and BMSCs; DDM+BMSC, culture of BMSCs with the DDM extract; CD4+DDM+BMSC, co-culture of naïve CD4 + T cells and BMSCs with the DDM extract; CD4+DDM+Inh+BMSC, co-culture of naïve CD4 + T cells and BMSCs with the DDM extract and IDO1 inhibitor; DDM+Inh+BMSC, culture of BMSCs with the DDM extract and IDO1 inhibitor.

Article Snippet: BMSCs were cultured in osteogenic induction media (Procell), and ALP staining was performed after 14 days.

Techniques: Co-Culture Assay, CCK-8 Assay, Control, Staining, Quantitative RT-PCR, Expressing

Journal: Clinical and Experimental Dental Research

Article Title: Gingival Stem Cell‐Conditioned Media and Low‐Level Laser Therapy Enhance Periodontal Ligament Stem Cells Function by Upregulating Wnt and TGF‐β Pathway Components: An In Vitro Study

doi: 10.1002/cre2.70151

Figure Lengend Snippet: Matrix mineralization as determined by AR staining after 2 weeks of osteogenic induction in the (A) control group, (B) osteogenic group, (C) GMS‐CM group, (D) LLLT group, and (E) GMS‐CM + LLLT group (×100 original magnification). (E) Bar chart of the mean and standard deviation of the AR staining absorbance. Bars labeled with different lowercase letters indicate statistically significant differences ( p < 0.05) between groups based on post hoc multiple comparisons. Bars sharing the same letter are not significantly different.

Article Snippet: Afterward, the media was replaced with osteogenic media (Millipore Sigma).

Techniques: Staining, Control, Standard Deviation, Labeling