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Dysbiotic stress increases the sensitivity of the tumor vasculature to radiotherapy and c-Met inhibitors

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Abstract

Antibiotic-induced microbial imbalance, or dysbiosis, has systemic and long-lasting effects on the host and response to cancer therapies. However, the effects on tumor endothelial cells are largely unknown. Therefore, the goal of the current study was to generate matched B16-F10 melanoma associated endothelial cell lines isolated from mice with and without antibiotic-induced dysbiosis. After validating endothelial cell markers on a genomic and proteomic level, functional angiogenesis assays (i.e., migration and tube formation) also confirmed their vasculature origin. Subsequently, we found that tumor endothelial cells derived from dysbiotic mice (TEC-Dys) were more sensitive to ionizing radiotherapy in the range of clinically-relevant hypofractionated doses, as compared to tumor endothelial cells derived from orthobiotic mice (TEC-Ortho). In order to identify tumor vasculature-associated drug targets during dysbiosis, we used tandem mass tag mass spectroscopy and focused on the statistically significant cellular membrane proteins overexpressed in TEC-Dys. By these criteria c-Met was the most differentially expressed protein, which was validated histologically by comparing tumors with or without dysbiosis. Moreover, in vitro, c-Met inhibitors Foretinib, Crizotinib and Cabozantinib were significantly more effective against TEC-Dys than TEC-Ortho. In vivo, Foretinib inhibited tumor growth to a greater extent during dysbiosis as compared to orthobiotic conditions. Thus, we surmise that tumor response in dysbiotic patients may be greatly improved by targeting dysbiosis-induced pathways, such as c-Met, distinct from the many targets suppressed due to dysbiosis.

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Acknowledgements

The study was supported by P20GM103625: the Center for Microbial Pathogenesis and Host Inflammatory Responses grant through the NIH National Institute of General Medical Sciences Centers of Biomedical Research Excellence, as well as in part by a seed grant from the Vice Chancellor of Research & Innovation, and the Arkansas Biosciences Institute and the Winthrop P. Rockefeller Cancer Institute to R.P.M. Dings. The study was also supported in part by the Translational Research Institute (TRI), grant TL1 TR003109 through the National Center for Advancing Translational Sciences of the National Institutes of Health (NIH) to S.V. Jenkins. K.B. Vang and R.J. Griffin received support from NSF OIA-1457888 through the Center for Advanced Surface Engineering. J.W. Leung was supported by NIH (K22CA204354 and R35GM137798) and Arkansas Breast Cancer Research Program (AWD00054499 and AWD00053730).

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Authors and Affiliations

  1. Department of Radiation Oncology, University of Arkansas for Medical Sciences, Little Rock, AR, USA

    Samir V. Jenkins, Mohammad Alimohammadi, Alexia S. Terry, Robert J. Griffin, Justin W. Leung & Ruud P. M. Dings

  2. Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA

    Alan J. Tackett & Stephanie D. Byrum

  3. Arkansas Children’s Research Institute, 13 Children’s Way, Little Rock, AR, 72202, USA

    Alan J. Tackett & Stephanie D. Byrum

  4. Center for Integrative Nanotechnology Sciences, University of Arkansas at Little Rock, Little Rock, AR, USA

    Kieng B. Vang

  5. Department of Radiation Oncology, University of Arkansas for Medical Sciences, 4301 W. Markham Street, Mail Slot #771, Little Rock, AR, 72205, USA

    Ruud P. M. Dings

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  1. Samir V. Jenkins
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Contributions

All authors contributed in part to elements of the study’s conception, design or execution. Material preparation, data collection and analysis were performed by SVJ, MA, AST, KBV, SDB, and RPMD. The first draft of the manuscript was written by RPMD and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Ruud P. M. Dings.

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Supplementary information

Below is the link to the electronic supplementary material.

Supplementary material 1 (PS 6469 kb)

Supplemental Fig. 1. Molecular marker authentication of TEC-Ortho and TEC-Dys validating endothelial origin. Representative composite RT-PCR blot of TEC-Ortho and TEC-Dys expressing endothelial cell markers Tie-2, TNFR1, NRP1, Galectin-1 (Gal-1), and Galectin-3 (Gal-3).

Supplementary material 2 (PS 515 kb)

Supplemental Fig. 2. TEC-Dys and TEC-Ortho do not express melanoma markers. (a) B16-F10 express GP100, whereas TEC-Dys do not. (b) Quantification of GP100 and (c) MART-1 on B16-F10, TEC-Ortho, TEC-Dys and 2H11. Data presented as mean ± SEM (n = 5 per melanoma marker pooled from 2 representative experiments). **P < 0.01, **P < 0.001 two-sided t-test. FMO = fluorescence minus one.

Supplementary material 3 (PS 119 kb)

Supplemental Fig. 3. Ampicillin, neomycin, metronidazole, and vancomycin do not directly influence TEC-Ortho, TEC-Dys or 2H11. The cell viability of -TEC-Ortho, TEC-Dys and 2H11 after 72 h being exposed to various concentrations of (a) Ampicillin, (b) Neomycin, (c) Metronidazole, and (d) Vancomycin. Data presented as means ± SD pooled from 2 experiments.

Supplementary material 4 (PS 140 kb)

Supplemental Fig. 4. No differential in intracellular ROS production by TEC-Ortho and TEC-Dys during radiation as measured by dichlorofluorescein diacetate. Cells were seeded in 96-well plates (Corning Costar #3603) at 5000 cells per well and allowed to adhere overnight. Medium was removed and cells were incubated for 1 hour with 1 μM dichlorofluorescein diacetate in PBS. Cells were then irradiated and the fluorescence (492 nm excitation, 515 nm emission) was measured. Background ROS was measured with the same incubation time and 0 Gy radiation and subtracted from radiation values.

Supplementary material 5 (PS 204 kb)

Supplemental Fig. 5. Mean dispersion plot showing all proteins identified by TMT-MS3 between TEC-Ortho and TEC-Dys. X-axis is the log2 intensity for each protein across all samples. Y-axis is log2 fold change of FDR-adjusted P-value. FDR-adjusted P-values < 0.05 and fold change > 2 are highlighted in red and fold change < -2 are highlighted in blue.

Supplementary material 6 (PS 129 kb)

Supplemental Fig. 6. C-Met inhibitor JNJ-38877605 has no effect on TEC-Ortho and TEC-Dys. The cell viability of Tec-Ortho and TEC-Dys after 72 hr being exposed to various concentrations of JNJ-38877605. Data presented as means ± SEM, pooled from 3 experiments with triplicates.

Supplementary material 7 (PS 429 kb)

Supplemental Fig. 7. Heat shock protein 27 (HSP27), part of the VEGF signaling pathway, is differentially expressed in TEC-Dys as compared to TEC-Ortho. Based on TMT-MS3 analysis, proteins deemed significantly overexpressed in TEC-Dys were superimposed in blue on the Kyoto encyclopedia of genes and genomes (KEGG) VEGF pathway. HSP27 was identified within this pathway.

Supplementary material 8 (PS 955 kb)

Supplemental Fig. 8. Heat shock protein 27 (HSP27), part of the receptor tyrosine kinase (RTK), and mitogen activated protein kinase (MAPK) signaling pathway is differentially expressed in TEC-Dys as compared to TEC-Ortho. Based on TMT-MS3 analysis, proteins deemed significantly overexpressed in TEC-Dys were superimposed in blue on the Kyoto encyclopedia of genes and genomes (KEGG) MAPK pathway. HSP27 was identified within this pathway.

Supplementary material 9 (PS 688 kb)

Supplemental Fig. 9. No significant proteomic changes within the phosphatidylinositol 3-kinase (PI3K-AKT) signaling pathway were detected in TEC-Dys as compared to TEC-Ortho. Based on TMT-MS3 analysis, no proteins within the PI3K-AKT pathway were deemed significantly overexpressed in TEC-Dys. The PI3K-AKT signaling pathway is depicted based on the Kyoto encyclopedia of genes and genomes (KEGG).

Supplementary material 10 (PS 174 kb)

Supplemental Table 1. Primer sequences of endothelial cell markers.

Supplementary material 11 (PS 223 kb)

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Jenkins, S.V., Alimohammadi, M., Terry, A.S. et al. Dysbiotic stress increases the sensitivity of the tumor vasculature to radiotherapy and c-Met inhibitors. Angiogenesis 24, 597–611 (2021). https://doi.org/10.1007/s10456-021-09771-z

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  • DOI: https://doi.org/10.1007/s10456-021-09771-z

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