Invasive phenotype induced by low extracellular pH requires mitochondria dependent metabolic flexibility.

Metabolic reprogramming is required for tumors to meet the bioenergetic and biosynthetic demands of malignant progression. Numerous studies have established a causal relationship between oncogenic drivers and altered metabolism, most prominently aerobic glycolysis, which supports rapid growth and affects the tumor microenvironment. Less is known about how the microenvironment modulates cancer metabolism. In the present study, we found that low extracellular pH, a common feature of solid tumors, provoked PDAC cells to decrease glycolysis and become resistant to glucose starvation. This was accompanied by increased dependency on mitochondrial metabolism, in which long-chain fatty acids became a primary fuel source. Consistent with previous reports, low pH enhanced tumor cell invasiveness. A novel finding was that limiting PDAC metabolic flexibility by either suppression of oxidative phosphorylation capacity or the pharmacological inhibition of fatty-acid oxidation prevented invasion induced by low extracellular pH. Altogether, our results suggest for the first time that targeting fatty-acid oxidation may be a viable adjunct strategy for preventing metastatic progression of pancreatic cancer mediated by the acidic tumor compartment.

Strategies to target long-lived plasma cells for treating hemophilia A inhibitors.

Long-lived plasma cells (LLPCs) can persistently produce anti-factor VIII (FVIII) antibodies which disrupt therapeutic effect of FVIII in hemophilia A patients with inhibitors. The migration of plasma cells to BM where they become LLPCs is largely controlled by an interaction between the chemokine ligand CXCL12 and its receptor CXCR4. AMD3100 combined with G-CSF inhibit their interactions, thus facilitating the mobilization of CD34(+) cells and blocking the homing of LLPCs. These reagents were combined with anti-CD20 to reduce B-cells and the specific IL-2/IL-2mAb (JES6-1) complexes to induce Treg expansion for targeting anti-FVIII immune responses. Groups of mice primed with FVIII plasmid and protein respectively were treated with the combined regimen for six weeks, and a significant reduction of anti-FVIII inhibitor titers was observed, associated with the dramatic decrease of circulating and bone marrow CXCR4(+) plasma cells. The combination regimens are highly promising in modulating pre-existing anti-FVIII antibodies in FVIII primed subjects.

Intraosseous delivery of lentiviral vectors targeting factor VIII expression in platelets corrects murine hemophilia A.

Intraosseous (IO) infusion of lentiviral vectors (LVs) for in situ gene transfer into bone marrow may avoid specific challenges posed by ex vivo gene delivery, including, in particular, the requirement of preconditioning. We utilized IO delivery of LVs encoding a GFP or factor VIII (FVIII) transgene directed by ubiquitous promoters (a MND or EF-1α-short element; M-GFP-LV, E-F8-LV) or a platelet-specific, glycoprotein-1bα promoter (G-GFP-LV, G-F8-LV). A single IO infusion of M-GFP-LV or G-GFP-LV achieved long-term and efficient GFP expression in Lineage(-)Sca1(+)c-Kit(+) hematopoietic stem cells and platelets, respectively. While E-F8-LV produced initially high-level FVIII expression, robust anti-FVIII immune responses eliminated functional FVIII in circulation. In contrast, IO delivery of G-F8-LV achieved long-term platelet-specific expression of FVIII, resulting in partial correction of hemophilia A. Furthermore, similar clinical benefit with G-F8-LV was achieved in animals with pre-existing anti-FVIII inhibitors. These findings further support platelets as an ideal FVIII delivery vehicle, as FVIII, stored in α-granules, is protected from neutralizing antibodies and, during bleeding, activated platelets locally excrete FVIII to promote clot formation. Overall, a single IO infusion of G-F8-LV was sufficient to correct hemophilia phenotype for long term, indicating that this approach may provide an effective means to permanently treat FVIII deficiency.

The dataset from administration of single or combined immunomodulation agents to modulate anti-FVIII antibody responses in FVIII plasmid or protein primed hemophilia A mice.

Hemophilia A mice with pre-existing inhibitory antibodies against factor VIII (FVIII) were treated with single agents, AMD3100 and GCS-F, respectively. Inhibitor titers in treated mice and control HemA inhibitors mice were followed over time. Total B cells and plasma cells (PCs) were characterized by flow cytometry. HemA inhibitor mice were then treated with a combination regimen of IL-2/IL-2mAb complexes plus rapamycin and AMD3100. Finally, HemA inhibitor mice were treated with a new combination therapy using include IL-2/IL-2mAb complexes + Anti-CD20+AMD3100+G-CSF. The timeline of combination therapy was illustrated. Inhibitor titers following treatment in FVIII plasmid or protein induced inhibitor mice were evaluated overtime. A representative figure and gating strategies to characterize the subsets of Treg cells and B cells are presented. Please see http://dx.doi.org/10.1016/j.cellimm.2016.01.005 [1] for interpretation and discussion of these data and results.