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Abstract Background Physical activity and metformin pharmacotherapy are associated with improved clinical outcomes in breast and colorectal cancer survivors. Myokines are cytokines secreted from skeletal muscle that may mediate these associations. Methods This hypothesis‐generating analysis used biospecimens collected from a multi‐centre 2 × 2 factorial randomized design of 116 patients with stage I–III breast and colorectal cancer who were randomized to 12 weeks of (1) aerobic exercise (moderate intensity titrated to 220 min/week); (2) metformin (850 mg daily for 2 weeks and then titrated to 850 mg twice per day); (3) aerobic exercise and metformin; or (4) control. Fourteen myokines were quantified using a multiplex panel. Myokine concentrations were log‐transformed, and main effects analyses were conducted using linear mixed‐effects regression models. The type I error rate was controlled with the Holm sequential testing procedure. Results Randomization to exercise increased leukaemia inhibitory factor (1.26 pg/mL, 95% confidence interval [CI]: 0.69, 1.84; adjusted P = 0.001) and interleukin‐15 (2.23 pg/mL, 95% CI: 0.87, 3.60; adjusted P = 0.013) compared with randomization to no exercise. Randomization to metformin decreased apelin (−2.69 pg/mL, 95% CI: −4.31, −1.07; adjusted P = 0.014) and interleukin‐15 (−1.74 pg/mL, 95% CI: −2.79, −0.69; adjusted P = 0.013) compared with randomization to no metformin. Metformin decreased myostatin, irisin, oncostatin M, fibroblast growth factor 21 and osteocrin; however, these changes were not statistically significant after correction for multiple comparisons. Conclusions This pilot study demonstrates that randomization to exercise and metformin elicit unique effects on myokine concentrations in cancer patients. This hypothesis‐generating observation warrants further basic, translational and clinical investigation and replication.

The aggregation of cancer cells provides a survival signal for disseminating cancer cells; however, the underlying molecular mechanisms have yet to be elucidated. Using qPCR gene arrays, this study investigated the changes in cancer-specific genes as well as genes regulating mitochondrial quality control, metabolism, and oxidative stress in response to aggregation and hypoxia in our progressive ovarian cancer models representing slow- and fast-developing ovarian cancer. Aggregation increased the expression of anti-apoptotic, stemness, epithelial-mesenchymal transition (EMT), angiogenic, mitophagic, and reactive oxygen species (ROS) scavenging genes and functions, and decreased proliferation, apoptosis, metabolism, and mitochondrial content genes and functions. The incorporation of stromal vascular cells (SVF) from obese mice into the spheroids increased DNA repair and telomere regulatory genes that may represent a link between obesity and ovarian cancer risk. While glucose had no effect, glutamine was essential for aggregation and supported proliferation of the spheroid. In contrast, low glucose and hypoxic culture conditions delayed adhesion and outgrowth capacity of the spheroids independent of their phenotype, decreased mitochondrial mass and polarity, and induced a shift of mitochondrial dynamics towards mitophagy. However, these conditions did not reduce the appearance of polarized mitochondria at adhesion sites, suggesting that adhesion signals that either reversed mitochondrial fragmentation or induced mitobiogenesis can override the impact of low glucose and oxygen levels. Thus, the plasticity of the spheroids’ phenotype supports viability during dissemination, allows for the adaptation to changing conditions such as oxygen and nutrient availability. This may be critical for the development of an aggressive cancer phenotype and, therefore, could represent druggable targets for clinical interventions.

BackgroundOvarian cancer cells aggregate during or after exfoliation from the primary tumor to form threedimensional spheroids. Spheroid formation provides a survival advantage during peritoneal dissemination in nutrient and oxygen-depleted conditions which is accompanied by a suppressed metabolic phenotype and fragmented mitochondria. Upon arrival to their metastatic sites, spheroids adhere to peritoneal organs and transition to a more epithelial phenotype to support outgrowth and invasion. In this study, we investigated the plasticity of mitochondrial morphology, dynamics, and function upon adhesion.MethodsUsing our slow-developing (MOSE-L) and fast-developing (MOSE-LTICv) ovarian cancer models, we mimicked adhesion and reoxygenation conditions by plating the spheroids onto tissue culture dishes and changing culture conditions from hypoxia and low glucose to normoxia with high glucose levels after adhesion. We used Western Blot, microscopy and Seahorse analyses to determine the plasticity of mitochondrial morphology and functions upon adhesion, and the impact on proliferation and invasion capacities.ResultsIndependent of culture conditions, all spheroids adhered to and began to grow onto the culture plates. While the bulk of the spheroid was unresponsive, the mitochondrial morphology in the outgrowing cells was indistinguishable from cells growing in monolayers, indicating that mitochondrial fragmentation in spheroids was indeed reversible. This was accompanied by an increase in regulators of mitobiogenesis, PGC1a, mitochondrial mass, and respiration. Reoxygenation increased migration and invasion in both cell types but only the MOSE-L responded with increased proliferation to reoxygenation. The highly aggressive phenotype of the MOSE-LTICv was characterized by a relative independence of oxygen and the preservation of higher levels of proliferation, migration and invasion even in limiting culture conditions but a higher reliance on mitophagy. Further, the outgrowth in these aggressive cells relies mostly on proliferation while the MOSE-L cells both utilize proliferation and migration to achieve outgrowth. Suppression of proliferation with cycloheximide impeded aggregation, reduced outgrowth and invasion via repression of MMP2 expression and the flattening of the spheroids.DiscussionOur studies indicate that the fragmentation of the mitochondria is reversible upon adhesion. The identification of regulatory signaling molecules and pathways of these key phenotypic alterations that occur during primary adhesion and invasion is critical for the identification of druggable targets for therapeutic intervention to prevent aggressive metastatic disease.

Ovarian cancer is the deadliest gynecological cancer in women, with a survival rate of less than 30% when the cancer has spread throughout the peritoneal cavity. Aggregation of cancer cells increases their viability and metastatic potential; however, there are limited studies that correlate these functional changes to specific phenotypic alterations. In this study, we investigated changes in mitochondrial morphology and dynamics during malignant transition using our MOSE cell model for progressive serous ovarian cancer. Mitochondrial morphology was changed with increasing malignancy from a filamentous network to single, enlarged organelles due to an imbalance of mitochondrial dynamic proteins (fusion: MFN1/OPA1, fission: DRP1/FIS1). These phenotypic alterations aided the adaptation to hypoxia through the promotion of autophagy and were accompanied by changes in the mitochondrial ultrastructure, mitochondrial membrane potential, and the regulation of reactive oxygen species (ROS) levels. The tumor-initiating cells increased mitochondrial fragmentation after aggregation and exposure to hypoxia that correlated well with our previously observed reduced growth and respiration in spheroids, suggesting that these alterations promote viability in non-permissive conditions. Our identification of such mitochondrial phenotypic changes in malignancy provides a model in which to identify targets for interventions aimed at suppressing metastases.