Effect of immune modulation on the skeletal muscle mitochondrial exercise response: An exploratory study in mice with cancer.

Cancer causes mitochondrial alterations in skeletal muscle, which may progress to muscle wasting and, ultimately, to cancer cachexia. Understanding how exercise adaptations are altered by cancer and cancer treatment is important for the effective design of exercise interventions aimed at improving cancer outcomes. We conducted an exploratory study to investigate how tumor burden and cancer immunotherapy treatment (anti-PD-1) modify the skeletal muscle mitochondrial response to exercise training in mice with transplantable tumors (B16-F10 melanoma and EO771 breast cancer). Mice remained sedentary or were provided with running wheels for ~19 days immediately following tumor implant while receiving no treatment (Untreated), isotype control antibody (IgG2a) or anti-PD-1. Exercise and anti-PD-1 did not alter the growth rate of either tumor type, either alone or in combination therapy. Untreated mice with B16-F10 tumors showed increases in most measured markers of skeletal muscle mitochondrial content following exercise training, as did anti-PD-1-treated mice, suggesting increased mitochondrial content following exercise training in these groups. However, mice with B16-F10 tumors receiving the isotype control antibody did not exhibit increased skeletal muscle mitochondrial content following exercise. In untreated mice with EO771 tumors, only citrate synthase activity and complex IV activity were increased following exercise. In contrast, IgG2a and anti-PD-1-treated groups both showed robust increases in most measured markers following exercise. These results indicate that in mice with B16-F10 tumors, IgG2a administration prevents exercise adaptation of skeletal muscle mitochondria, but adaptation remains intact in mice receiving anti-PD-1. In mice with EO771 tumors, both IgG2a and anti-PD-1-treated mice show robust skeletal muscle mitochondrial exercise responses, while untreated mice do not. Taken together, we postulate that immune modulation may enhance skeletal muscle mitochondrial response to exercise in tumor-bearing mice, and suggest this as an exciting new avenue for future research in exercise oncology.

Effects of exercise and anti-PD-1 on the tumour microenvironment.

Immune checkpoint inhibition is highly effective in treating a subset of patients with certain cancers, such as malignant melanoma. However, a large proportion of patients will experience treatment resistance, and other tumour types, such as breast cancer, have thus far proven largely refractory to immune checkpoint inhibitors as single agents. Exercise has been associated with improved cancer patient survival, has known immune-modulatory effects, may improve anti-tumour immunity and may normalise tumour blood vessels. Therefore, we hypothesised that post-implant exercise would boost the effect of concurrent immunotherapy by enhancing anti-tumour immune responses and improving tumour blood flow. To investigate this, mice with EO771 breast tumours or B16-F10 melanomas received anti-PD-1, an isotype control antibody or no treatment. Mice were randomised to exercise (voluntary wheel running) or no exercise at tumour implant. Exercise reduced the number of CD8+T cells in EO771 (p = 0.0011) but not B16-F10 tumours (p = 0.312), and reduced the percentage of CD8+T cells within the total T cell population in both tumour types (B16-F10: p = 0.0389; EO771: p = 0.0015). In contrast, the combination of exercise and anti-PD-1 increased the percentage of CD8+T cells in EO771 (p = 0.0339) but not B16-F10 tumours. Taken together, our results show that exercise and anti-PD-1 induce changes in the tumour immune microenvironment which are dependant on tumour type.

Gene and Protein Expression Is Altered by Ascorbate Availability in Murine Macrophages Cultured under Tumour-Like Conditions.

Tumour-associated macrophages (TAMs) are ubiquitously present in tumours and commonly associated with poor prognosis. In immune cells, ascorbate affects epigenetic regulation, differentiation and phenotype via its co-factor activity for the 2-oxoglutarate dependent dioxygenase enzymes. Here, we determined the effect of ascorbate on TAM development in response to tumour microenvironmental cues. Naïve murine bone marrow monocytes were cultured with Lewis Lung Carcinoma conditioned media (LLCM) or macrophage colony-stimulating factor (MCSF) to encourage the development into tumour-associated macrophages. Cells were stimulated with hypoxia (1% O2), with or without ascorbate (500 µM) supplementation. Cells and media were harvested for gene, cell surface marker and protein analyses. LLCM supported bone marrow monocyte growth with >90% of cells staining CD11b+F4/80+, indicative of monocytes/macrophages. LLCM-grown cells showed increased expression of M2-like and TAM genes compared to MCSF-grown cells, which further increased with hypoxia. In LLCM-grown cells, ascorbate supplementation was associated with increased F4/80 cell surface expression, and altered gene expression and protein secretion. Our study shows that ascorbate modifies monocyte phenotype when grown under tumour microenvironmental conditions, but this was not clearly associated with either a pro- or anti-tumour phenotype, and reflects a complex and nuanced response of macrophages to ascorbate. Overall, ascorbate supplementation clearly has molecular consequences for TAMs, but functional and clinical consequences remain unknown.

Effect of post-implant exercise on tumour growth rate, perfusion and hypoxia in mice.

Preclinical studies have shown a larger inhibition of tumour growth when exercise begins prior to tumour implant (preventative setting) than when training begins after tumour implant (therapeutic setting). However, post-implantation exercise may alter the tumour microenvironment to make it more vulnerable to treatment by increasing tumour perfusion while reducing hypoxia. This has been shown most convincingly in breast and prostate cancer models to date and it is unclear whether other tumour types respond in a similar way. We aimed to determine whether tumour perfusion and hypoxia are altered with exercise in a melanoma model, and compared this with a breast cancer model. We hypothesised that post-implantation exercise would reduce tumour hypoxia and increase perfusion in these two models. Female, 6-10 week old C57BL/6 mice were inoculated with EO771 breast or B16-F10 melanoma tumour cells before randomisation to either exercise or non-exercising control. Exercising mice received a running wheel with a revolution counter. Mice were euthanised when tumours reached maximum ethical size and the tumours assessed for perfusion, hypoxia, blood vessel density and proliferation. We saw an increase in heart to body weight ratio in exercising compared with non-exercising mice (p = 0.0008), indicating that physiological changes occurred with this form of physical activity. However, exercise did not affect vascularity, perfusion, hypoxia or tumour growth rate in either tumour type. In addition, EO771 tumours had a more aggressive phenotype than B16-F10 tumours, as inferred from a higher rate of proliferation (p<0.0001), a higher level of tumour hypoxia (p = 0.0063) and a higher number of CD31+ vessels (p = 0.0005). Our results show that although a physiological training effect was seen with exercise, it did not affect tumour hypoxia, perfusion or growth rate. We suggest that exercise monotherapy is minimally effective and that future preclinical work should focus on the combination of exercise with standard cancer therapies.

The effect of CSE gene deletion in caerulein-induced acute pancreatitis in the mouse.

Hydrogen sulfide (H2S) has been reported to be involved in the signaling of the inflammatory response; however, there are differing views as to whether it is pro- or anti-inflammatory. In this study, we sought to determine whether endogenously synthesized H2S via cystathionine-γ-lyase (CSE) plays a pro- or anti-inflammatory role in caerulein-induced pancreatitis. To investigate this, we used mice genetically deficient in CSE to elucidate the function of CSE in caerulein-induced acute pancreatitis. We compared the inflammatory response and tissue damage of wild-type (WT) and CSE knockout (KO) mice following 10 hourly administrations of 50 µg/kg caerulein or saline control. From this, we found that the CSE KO mice showed significantly less local pancreatic damage as well as acute pancreatitis-associated lung injury compared with the WT mice. There were also lower levels of pancreatic eicosanoid and cytokines, as well as reduced acinar cell NF-κB activation in the CSE KO mice compared with WT mice. Additionally, in WT mice, there was a greater level of pancreatic CSE expression and sulfide-synthesizing activity in caerulein-induced pancreatitis compared with the saline control. When comparing the two saline-treated control groups, we noted that the CSE KO mice showed significantly less pancreatic H2S-synthesizing activity relative to the WT mice. These results indicate that endogenous H2S generated by CSE plays a key proinflammatory role via NF-κB activation in caerulein-induced pancreatitis, and its genetic deletion affords significant protection against acute pancreatitis and associated lung injury.

Effects of S-propargyl-cysteine (SPRC) in caerulein-induced acute pancreatitis in mice.

Hydrogen sulfide (H(2)S), a novel gaseous messenger, is synthesized endogenously from L-cysteine by two pyridoxal-5'-phosphate-dependent enzymes, cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE). S-propargyl-cysteine (SPRC) is a slow H(2)S releasing drug that provides cysteine, a substrate of CSE. The present study was aimed to investigate the effects of SPRC in an in vivo model of acute pancreatitis (AP) in mice. AP was induced in mice by hourly caerulein injections (50 µg/kg) for 10 hours. Mice were treated with SPRC (10 mg/kg) or vehicle (distilled water). SPRC was administered either 12 h before or 3 h before the induction of pancreatitis. Mice were sacrificed 1 h after the last caerulein injection. Blood, pancreas and lung tissues were collected and processed to measure the plasma amylase, plasma H(2)S, myeloperoxidase (MPO) activities and cytokine levels in pancreas and lung. The results revealed that significant reduction of inflammation, both in pancreas and lung was associated with SPRC given 3 h prior to the induction of AP. Furthermore, the beneficial effects of SPRC were associated with reduction of pancreatic and pulmonary pro-inflammatory cytokines and increase of anti-inflammatory cytokine. SPRC administered 12 h before AP induction did not cause significant improvement in pancreatic and lung inflammation. Plasma H(2)S concentration showed significant difference in H(2)S levels between control, vehicle and SPRC (administered 3 h before AP) treatment groups. In conclusion, these data provide evidence for protective effects of SPRC in AP possibly by virtue of its slow release of endogenous H(2)S.

Bone marrow-derived mesenchymal stem cells influence early tendon-healing in a rabbit achilles tendon model.

BACKGROUND: A repaired tendon needs to be protected for weeks until it has accrued enough strength to handle physiological loads. Tissue-engineering techniques have shown promise in the treatment of tendon and ligament defects. The present study tested the hypothesis that bone marrow-derived mesenchymal stem cells can accelerate tendon-healing after primary repair of a tendon injury in a rabbit model. METHODS: Fifty-seven New Zealand White rabbits were used as the experimental animals, and seven others were used as the source of bone marrow-derived mesenchymal stem cells. The injury model was a sharp complete transection through the midsubstance of the Achilles tendon. The transected tendon was immediately repaired with use of a modified Kessler suture and a running epitendinous suture. Both limbs were used, and each side was randomized to receive either bone marrow-derived mesenchymal stem cells in a fibrin carrier or fibrin carrier alone (control). Postoperatively, the rabbits were not immobilized. Specimens were harvested at one, three, six, and twelve weeks for analysis, which included evaluation of gross morphology (sixty-two specimens), cell tracing (twelve specimens), histological assessment (forty specimens), immunohistochemistry studies (thirty specimens), morphometric analysis (forty specimens), and mechanical testing (sixty-two specimens). RESULTS: There were no differences between the two groups with regard to the gross morphology of the tendons. The fibrin had degraded by three weeks. Cell tracing showed that labeled bone marrow-derived mesenchymal stem cells remained viable and present in the intratendinous region for at least six weeks, becoming more diffuse at later time-periods. At three weeks, collagen fibers appeared more organized and there were better morphometric nuclear parameters in the treatment group (p < 0.05). At six and twelve weeks, there were no differences between the groups with regard to morphometric nuclear parameters. Biomechanical testing showed improved modulus in the treatment group as compared with the control group at three weeks (p < 0.05) but not at subsequent time-periods. CONCLUSIONS: Intratendinous cell therapy with bone marrow-derived mesenchymal stem cells following primary tendon repair can improve histological and biomechanical parameters in the early stages of tendon-healing.