Abstract 2643: ROS dependent PGC1alpha driven mitochondrial biogenesis within the melanoma microenvironment is a pre-requisite for mitochondrial transfer from MSC to malignant cells
Authors: Kumar Prakrit , Mistry Jayna , Moore Jamie , Hellmich Charlotte , Jibril Aisha , Morris Christopher , Moncrieff Marc , Bowles Kristian , Rushworth Stuart 
Published: September 2020

Abstract
The tumor microenvironment is essential for the growth and proliferation of melanoma. Recently, reports have shown that mitochondria are transferred from mesenchymal stromal cells (MSCs) to melanoma tumor cells through tunneling nanotubes (TNTs), a process which supports their proliferation in-vitro and in-vivo. Moreover, we have shown in leukemia that tumor superoxide provides the stimulus to drive mitochondrial transfer in this way. In the present study we specifically examine the effects of melanoma cells on MSC mitochondrial biogenesis and subsequent mitochondrial transfer to the malignant cell. Primary melanoma cells, and MSCs derived from the bone marrow of patients without melanoma were obtained after informed consent and under approval from the UK Health Research Authority, and in accordance with the Declaration of Helsinki. Primary cells were characterized using flow cytometry. Genes involved in mitochondria biogenesis, fission/fusion and activity were analysed using Real-time PCR and Western blotting. Mitochondrial transfer was assessed and quantified in-vitro using a MitoTracker green FM staining and flow cytometry. Melanoma signals were quenched by various inhibitors including glutathione and diphenyleneiodonium (DPI). Melanoma proliferation and tumor volume, in an NSG xenograft subcutaneous model, were monitored through bioluminescent live animal imaging. First, we confirmed primary melanoma cell identity using CD45-, CD31-, CD166+ and CD146+ staining using flow cytometry. Next, we quantified levels of mitochondrial transfer from MSC to melanoma, with and without inhibition of superoxide activity. We found that the anti-oxidants glutathione and DPI significantly reduced levels of mitochondrial transfer from MSCs to melanoma. We next quantified mitochondrial biogenesis within MSCs, showing that MSCs had increased mitochondrial DNA and increased MitoTracker green FM staining in response to co-culture with melanoma. Next, we found that this increase in mitochondrial content also occurred in response to hydrogen peroxide, which mimics the effects of melanoma-derived ROS. In addition, we show that PGC1α (master regulator of mitochondrial biogenesis) levels in the nucleus of MSCs increased when cultured with melanoma. Moreover, knockdown of PGC1α inhibited mitochondrial biogenesis in MSCs when cultured with melanoma or activated with hydrogen peroxide. Furthermore, transfer of mitochondria from MSCs to melanoma was substantially reduced when MSC-derived PGC1α was silenced. Finally, we engrafted control or PGC1α knockdown MSCs together with melanoma cell lines into the sub-cutaneous tissue of NSG mice. We found significantly reduced tumor volume when the melanoma was transplanted with the PGC1α knockdown MSCs compared to transplant of melanoma with the control knockdown MSC. Here we report that the ROS generated by melanoma cells drives MSC mitochondrial biogenesis and subsequent mitochondrial transfer from MSCs to melanoma cells. Targeting this pathway may provide an effective novel therapeutic approach in melanoma.
Citation Format: Prakrit Kumar, Jayna Mistry, Jamie Moore, Charlotte Hellmich, Aisha Jibril, Christopher Morris, Marc Moncrieff, Kristian Bowles, Stuart Rushworth. ROS dependent PGC1alpha driven mitochondrial biogenesis within the melanoma microenvironment is a pre-requisite for mitochondrial transfer from MSC to malignant cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2643.