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Up-regulation of pro-angiogenic molecules and events does not relate with an angiogenic switch in metastatic osteosarcoma cells but to cell survival features

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Abstract

Osteosarcoma (OS) is the most frequent malignant bone tumor, affecting predominantly children. Metastases represent a major clinical challenge and an estimated 80% would present undetectable micrometastases at diagnosis. The identification of metastatic traits and molecules would impact in micrometastasis management. We demonstrated that OS LM7 metastatic cells secretome was able to induce microvascular endothelium cell rearrangements, an angiogenic-related trait. A proteomic analysis indicated a gain in angiogenic-related pathways in these cells, as compared to their parental-non-metastatic OS SAOS2 cells counterpart. Further, factors with proangiogenic functions like VEGF and PDGF were upregulated in LM7 cells. However, no differential angiogenic response was induced by LM7 cells in vivo. Regulation of the Fas–FasL axis is key for OS cells to colonize the lungs in this model. Analysis of the proteomic data with emphasis in apoptosis pathways and related processes revealed that the percentage of genes associated with those, presented similar levels in SAOS2 and LM7 cells. Further, the balance of expression levels of proteins with pro- and antiapoptotic functions in both cell types was subtle. Interestingly and of relevance to the model, Fas associated Factor 1 (FAF1), which participates in Fas signaling, was present in LM7 cells and was not detected in SAOS2 cells. The subtle differences in apoptosis-related events and molecules, together with the reported cell-survival functions of the identified angiogenic factors and the increased survival features that we observed in LM7 cells, suggest that the gain in angiogenesis-related pathways in metastatic OS cells would relate to a prosurvival switch rather to an angiogenic switch as an advantage feature to colonize the lungs. OS metastatic cells also displayed higher adhesion towards microvascular endothelium cells suggesting an advantage for tissue colonization. A gain in angiogenesis pathways and molecules does not result in major angiogenic potential. Together, our results suggest that metastatic OS cells would elicit signaling associated to a prosurvival phenotype, allowing homing into the hostile site for metastasis. During the gain of metastatic traits process, cell populations displaying higher adhesive ability to microvascular endothelium, negative regulation of the Fas–FasL axis in the lung parenchyma and a prosurvival switch, would be selected. This opens a new scenario where antiangiogenic treatments would affect cell survival rather than angiogenesis, and provides a molecular panel of expression that may help in distinguishing OS cells with different metastatic potential.

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Acknowledgements

We thank the Program for Technological Development in Tools for Health-PDTIS-FIOCRUZ for the use of the mass spectrometry facility.

Funding

This work was supported by a grant from the Agencia Nacional de Promoción Científica y tecnológica (ANPCyT) PICT N°1974.

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Author notes

  1. Luciana M. Gutiérrez and Matías Valenzuela Alvarez have contributed equally to this work.

Authors and Affiliations

  1. Remodeling Processes and Cellular Niches Laboratory, Instituto de Medicina Traslacional e Ingeniería Biomédica (IMTIB) - CONICET - Hospital Italiano Buenos Aires (HIBA), Instituto Universitario del Hospital Italiano (IUHI), Potosí 4240, C1199ACL, CABA, Argentina

    Luciana M. Gutiérrez, Matías Valenzuela Alvarez & Marcela F. Bolontrade

  2. Division of Pediatrics and Department of Cancer Biology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Unit #853, Houston, TX, 77030, USA

    Yuanzheng Yang & Eugenie S. Kleinerman

  3. CITNOBA CONICET-UNNOBA, Jorge Newbery 261, B6000, Junín, Argentina

    Fiorella Spinelli & Laura Alaniz

  4. Facultad de Ciencias Biomédicas, Instituto de Investigaciones en Medicina Traslacional (IIMT), CONICET, Universidad Austral, Pilar, Buenos Aires, Argentina

    María José Cantero & Mariana G. García

  5. Instituto Carlos Chagas Fiocruz/PR, Curitiba, 81350-010, Brazil

    Alejandro Correa

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  1. Luciana M. Gutiérrez
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Contributions

LMG and MVA performed experiments, analyzed data and designed figures; FS, MJC, MGG and YY performed experiments; AC conducted proteomic analysis and analyzed data; ESK, MGG and LA contributed with essential reagents and analyzed data; ESK provided OS cells. ESK and AC contributed with paper revision; MB conceived research and experiments and analyzed data. Authors discussed and commented the results on the manuscript.

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Correspondence to Marcela F. Bolontrade.

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10495_2021_1677_MOESM1_ESM.jpg

Supplementary file1 (JPG 62 kb) Supplementary Figure 1. Kaplan–Meier metastasis-free survival curves using the GSE42352 dataset. Curves represent high (blue) and low (red) gene expression in pretreatment high grade OS biopsy samples. A) PDGFA Kaplan-Meier metastasis-free survival, higher expression is worse (p 0.085). B) PDGFB Kaplan–Meier metastasis-free survival, higher expression is worse (p 0.046). C) PDGFC Kaplan–Meier metastasis-free survival, higher expression is worse (p 0.061). D) PDGFD Kaplan–Meier metastasis-free survival, higher expression is worse (p =0.009). E) FAF1Kaplan–Meier metastasis-free survival, higher expression is worse (p =0.056). F) VEGF Kaplan–Meier metastasis-free survival, higher expression is worse (p 0.028).

10495_2021_1677_MOESM2_ESM.docx

Supplementary file2 (DOCX 16 kb) Supplementary Table 1. LFQ intensity fold change for GO for biological pathway and biological processes terms in CM and cells. Analyses considering the relative abundance of the proteins (LFQ normalized intensities) were carried out using Funrich software. LFQ: label free quantification; GO: gene ontology; CM: conditioned medium; CM-LM7: conditioned medium from LM7 OS cells; CM-SAOS: conditioned medium from SAOS2 OS cells.

10495_2021_1677_MOESM3_ESM.docx

Supplementary file3 (DOCX 23 kb) Supplementary Table 2. OS cells apoptosis- related proteins at the intracellular compartment, classified by their pro- or antiapoptotic effect. Proteins are expressed as relative intensity values (normalized LFQ). FAF: Fas associated factor; BAG: BCL-2 associated athanogene; BAX: BCL-2 associated X protein; BCL2L13: BCL-2 like 13; BCLAF1: BCL-2 associated transcription factor 1; API5: Apoptosis inhibitor 5; AATF: Apoptosis antagonizing transcription factor; AIFM1: Apoptosis inducing factor mitochondrial 1; CASP3: caspase 3

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Gutiérrez, L.M., Valenzuela Alvarez, M., Yang, Y. et al. Up-regulation of pro-angiogenic molecules and events does not relate with an angiogenic switch in metastatic osteosarcoma cells but to cell survival features. Apoptosis 26, 447–459 (2021). https://doi.org/10.1007/s10495-021-01677-x

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