Combined thermal ablation and liposomal granulocyte-macrophage colony stimulation factor increases immune cell trafficking in a small animal tumor model.

PURPOSE: To characterize intratumoral immune cell trafficking in ablated and synchronous tumors following combined radiofrequency ablation (RFA) and systemic liposomal granulocyte-macrophage colony stimulation factor (lip-GM-CSF). METHODS: Phase I, 72 rats with single subcutaneous R3230 adenocarcinoma were randomized to 6 groups: a) sham; b&c) free or liposomal GM-CSF alone; d) RFA alone; or e&f) combined with blank liposomes or lip-GM-CSF. Animals were sacrificed 3 and 7 days post-RFA. Outcomes included immunohistochemistry of dendritic cells (DCs), M1 and M2 macrophages, T-helper cells (Th1) (CD4+), cytotoxic T- lymphocytes (CTL) (CD8+), T-regulator cells (T-reg) (FoxP3+) and Fas Ligand activated CTLs (Fas-L+) in the periablational rim and untreated index tumor. M1/M2, CD4+/CD8+ and CD8+/FoxP3+ ratios were calculated. Phase II, 40 rats with double tumors were randomized to 4 groups: a) sham, b) RFA, c) RFA-BL and d) RFA-lip-GM-CSF. Synchronous untreated tumors collected at 7d were analyzed similarly. RESULTS: RFA-lip-GMCSF increased periablational M1, CTL and CD8+/FoxP3+ ratio at 3 and 7d, and activated CTLs 7d post-RFA (p<0.05). RFA-lip-GMSCF also increased M2, T-reg, and reduced CD4+/CD8+ 3 and 7d post-RFA respectively (p<0.05). In untreated index tumor, RFA-lip-GMCSF improved DCs, M1, CTLs and activated CTL 7d post-RFA (p<0.05). Furthermore, RFA-lip-GMSCF increased M2 at 3 and 7d, and T-reg 7d post-RFA (p<0.05). In synchronous tumors, RFA-BL and RFA-lip-GM-CSF improved DC, Th1 and CTL infiltration 7d post-RFA. CONCLUSION: Systemic liposomal GM-CSF combined with RFA improves intratumoral immune cell trafficking, specifically populations initiating (DC, M1) and executing (CTL, FasL+) anti-tumor immunity. Moreover, liposomes influence synchronous untreated metastases increasing Th1, CTL and DCs infiltration.

PFKFB3-mediated Pro-glycolytic Shift in Hepatocellular Carcinoma Proliferation.

BACKGROUND & AIMS: Metabolic reprogramming, in particular, glycolytic regulation, supports abnormal survival and growth of hepatocellular carcinoma (HCC) and could serve as a therapeutic target. In this study, we sought to identify glycolytic regulators in HCC that could be inhibited to prevent tumor progression and could also be monitored in vivo, with the goal of providing a theragnostic alternative to existing therapies. METHODS: An orthotopic HCC rat model was used. Tumors were stimulated into a high-proliferation state by use of off-target liver ablation and were compared with lower-proliferating controls. We measured in vivo metabolic alteration in tumors before and after stimulation, and between stimulated tumors and control tumors using hyperpolarized 13C magnetic resonance imaging (MRI) (h13C MRI). We compared metabolic alterations detected by h13C MRI to metabolite levels from ex vivo mass spectrometry, mRNA levels of key glycolytic regulators, and histopathology. RESULTS: Glycolytic lactate flux increased within HCC tumors 3 days after tumor stimulation, correlating positively with tumor proliferation as measured with Ki67. This was associated with a shift towards aerobic glycolysis and downregulation of the pentose phosphate pathway detected by mass spectrometry. MRI-measured lactate flux was most closely coupled with PFKFB3 expression and was suppressed with direct inhibition using PFK15. CONCLUSIONS: Inhibition of PFKFB3 prevents glycolytic-mediated HCC proliferation, trackable by in vivo h13C MRI.

Myofibroblasts: A key promoter of tumorigenesis following radiofrequency tumor ablation.

Radiofrequency ablation (RFA) of intrahepatic tumors induces distant tumor growth through activation of interleukin 6/signal transducer and activator of transcription 3 (STAT3)/hepatocyte growth factor (HGF)/tyrosine-protein kinase Met (c-MET) pathway. Yet, the predominant cellular source still needs to be identified as specific roles of the many types of periablational infiltrating immune cells requires further clarification. Here we report the key role of activated myofibroblasts in RFA-induced tumorigenesis and successful pharmacologic blockade. Murine models simulating RF tumorigenic effects on a macrometastatic tumor and intrahepatic micrometastatic deposits after liver ablation and a macrometastatic tumor after kidney ablation were used. Immune assays of ablated normal parenchyma demonstrated significantly increased numbers of activated myofibroblasts in the periablational rim, as well as increased HGF levels, recruitment other cellular infiltrates; macrophages, dendritic cells and natural killer cells, HGF dependent growth factors; fibroblast growth factor-19 (FGF-19) and receptor of Vascular Endothelial Growth Factor-1 (VEGFR-1), and proliferative indices; Ki-67 and CD34 for microvascular density. Furthermore, macrometastatic models demonstrated accelerated distant tumor growth at 7d post-RFA while micrometastatic models demonstrated increased intrahepatic deposit size and number at 14 and 21 days post-RFA. Multi-day atorvastatin, a selective fibroblast inhibitor, inhibited RFA-induced HGF and downstream growth factors, cellular markers and proliferative indices. Specifically, atorvastatin treatment reduced cellular and proliferative indices to baseline levels in the micrometastatic models, however only partially in macrometastatic models. Furthermore, adjuvant atorvastatin completely inhibited accelerated growth of macrometastasis and negated increased micrometastatic intrahepatic burden. Thus, activated myofibroblasts drive RF-induced tumorigenesis at a cellular level via induction of the HGF/c-MET/STAT3 axis, and can be successfully pharmacologically suppressed.

In vivo detection of distal tumor glycolytic flux stimulated by hepatic ablation in a breast cancer model using hyperpolarized 13C MRI.

PURPOSE: Hepatic thermal ablation therapy can result in c-Met-mediated off-target stimulation of distal tumor growth. The purpose of this study was to determine if a similar effect on tumor metabolism could be detected in vivo with hyperpolarized 13C MRI. MATERIALS AND METHODS: In this prospective study, female Fisher rats (n = 28, 120-150 g) were implanted with R3230 rat breast adenocarcinoma cells and assigned to either: sham surgery, hepatic radiofrequency ablation (RFA), or hepatic RFA + adjuvant c-Met inhibition with PHA-665752 (RFA + PHA). PHA-665752 was administered at 0.83 mg/kg at 24 h post-RFA. Tumor growth was measured daily. MRI was performed 24 h before and 72 h after treatment on 14 rats, and the conversion of 13C-pyruvate into 13C-lactate within each tumor was quantified as lactate:pyruvate ratio (LPR). Comparisons of tumor growth and LPR were performed using paired and unpaired t-tests. RESULTS: Hepatic RFA alone resulted in increased growth of the distant tumor compared to sham treatment (0.50 ± 0.13 mm/day versus 0.11 ± 0.07 mm/day; p < 0.001), whereas RFA + PHA (0.06 ± 0.13 mm/day) resulted in no significant change from sham treatment (p = 0.28). A significant increase in LPR was seen following hepatic RFA (+0.016 ± 0.010, p = 0.02), while LPR was unchanged for sham treatment (-0.048 ± 0.051, p = 0.10) or RFA + PHA (0.003 ± 0.041, p = 0.90). CONCLUSION: In vivo hyperpolarized 13C MRI can detect hepatic RFA-induced increase in lactate flux within a distant R3230 tumor, which correlates with increased tumor growth. Adjuvant inhibition of c-Met suppresses these off-target effects, supporting a role for the HGF/c-Met signaling axis in these tumorigenic responses.