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Interstitial serum albumin empowers osteosarcoma cells with FAIM2 transcription to obtain viability via dedifferentiation

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Abstract

During hematogenous metastasis, cancer cells escape from primary lesions and enter into the circulatory system, and only a few can colonize distant organs. However, the mechanism of cell survival and metastasis in the hematopoietic environment remains unclear. Angiorrhea is the character of pathological neovascularization in malignant tumors and commonly detected in osteosarcoma (OS), a bone tumor that prefers circulatory metastasis. In the present study, we focused on the notable role of serum albumin, the highest content in blood plasma, on OS progression. Our results indicated that serum albumin might act as a barrier against exogenous cancer cells during hematogenous metastasis. OS cells with high metastatic potential could gradually obtain strong viability through dedifferentiation under the effect of serum albumin in the angiorrhea region. Further exploration showed that serum albumin could increase the intracellular calcium concentration by activating the voltage-dependent calcium channel Cav2.1 in OS cells to affect the cytoskeleton, sequentially leading to dedifferentiation. Dedifferentiated OS cells with increased FAS apoptosis inhibitory molecule 2 (FAIM2) expression would gradually acquire survival ability, whereas knockdown of FAIM2 caused apoptosis in serum albumin. Moreover, FAIM2 overexpression rescued the viability of OS cells with low metastatic potential in serum albumin. In clinical specimens, OS cells showed markedly stronger positive staining of FAIM2 in the angiorrhea area. Taken together, our findings indicate that serum albumin in the angiorrhea region is a critical substance during pulmonary metastasis of OS cells. Angiorrhea is a nonnegligible prognostic element and FAIM2 might serve as a promising therapeutic target.

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Funding

This work was supported by a grant from the National Natural Science Foundation of China (No. 31871413) and two Programs of Guangdong Science and Technology (2017B020230002 and 2016B030231001).

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Authors and Affiliations

  1. MOE Key Laboratory of Gene Function and Regulation, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006, People’s Republic of China

    Yubin Pan, Yu Zhang, Weifeng Tang & Yan Zhang

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  1. Yubin Pan
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  2. Yu Zhang
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  3. Weifeng Tang
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  4. Yan Zhang
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Corresponding author

Correspondence to Yan Zhang.

Ethics declarations

All of the animal experiments were conducted in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals after approval from the Animal Ethics Committee of Sun Yat-sen University.

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The authors declare that they have no conflicts of interest.

Additional information

Editor: Tetsuji Okamoto

Electronic supplementary material

Fig. S1.

SA-iOSCs representing characteristics of cancer stem cells. (a) IF of embryonic stem cells (ESCs) markers in adherent and SA-iOSCs MNNG/HOS cells. SSEA-1 is shown as red fluorescence and Sox2, CD133, and C-kit are shown as green fluorescence. Nuclei were stained with DAPI (Scale bar, 25 μm). (b) Micrographs of second and tenth passages of iOSCs induced by low-level serum albumin or TGF-β1 for 96 h (Scale bar, 100 μm). (c) Schematic diagram shows the differences in self-renewal ability between iOSCs induced by serum albumin or TGF-β1. (d) Detection of chemotherapeutic tolerance in adherent and SA-iOSCs MNNG/HOS cells (n = 5) under different concentrations of DOXO (0–400 ng/mL). P < 0.05=*. (JPG 804 kb)

Fig. S2.

Dedifferentiation process of MNNG/HOS cells was promoted by serum albumin. Representative microscopy images of MNNG/HOS cells cultured in serum-free medium supplemented with or without 0.5 mg/ml HSA or BSA (Scale bar, 100 μm). (JPG 979 kb)

Fig. S3.

Serum albumin did not affect OS cells through L-type VDCCs. (a) Cytotoxicity test of MNNG/HOS cells cultured with different concentrations of ω-conotoxin MVIIC, nitrendipine or nifedipine for 48 hours (n = 5). (b) Dedifferentiation of MNNG/HOS cells induced by serum albumin was not inhibited by 5 μM nitrendipine or 5 μM nifedipine treatment (Scale bar, 100 μm). (c) Cell death of U-2 OS cells induced by albumin was not inhibited by 5 μM nitrendipine or 5 μM nifedipine treatment (Scale bar, 100 μm). P < 0.05=*, P < 0.01=**, P < 0.001=***. (JPG 1324 kb)

Fig. S4.

The apoptosis induced by serum albumin in OS cells was mainly the extrinsic pathway of apoptosis. (a) qRT-PCR analysis of BCL2L1 mRNA expression level (n = 3) in MNNG/HOS cells and U-2 OS cells treated with gradient concentration of serum albumin for 96 h. (b) The expression levels of β-Actin, Bcl-xL, and cleaved caspase-8 in MNNG/HOS and U-2 OS cells were tested by WB after serum free, 5 mg/mL, and 50 mg/mL BSA treatment for 72 h. Representative images of WB are shown. P > 0.05= ns. (JPG 598 kb)

Fig. S5.

Knockdown or overexpression of FAIM2 in OS cells. mRNA levels (a, n = 3) and protein levels (b) of FAIM2 were downregulated by siRNA in MNNG/HOS cells. (c) TUNEL staining of MNNG/HOS-siRNA-NC and MNNG/HOS-siFAIM2 cells cultured in 50 mg/mL BSA for 48 h (Scale bar, 25 μm). The mRNA levels (d, n = 3) and protein levels (e) of FAIM2 were upregulated in U-2 OS cells. Representative images of WB are shown. (f) TUNEL staining of U2-pEGFP-N1 and U2-FAIM2 cells cultured in 50 mg/mL BSA for 48 h (Scale bar, 25 μm). (g) qRT-PCR analysis of FAIM2 expression in MNNG/HOS cells (n = 3). P < 0.01=**, P < 0.001=***. (JPG 1561 kb)

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Pan, Y., Zhang, Y., Tang, W. et al. Interstitial serum albumin empowers osteosarcoma cells with FAIM2 transcription to obtain viability via dedifferentiation. In Vitro Cell.Dev.Biol.-Animal 56, 129–144 (2020). https://doi.org/10.1007/s11626-019-00421-9

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