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The BAG3- and SIRPα- mediated pathways trigger distinct cellular targets and signaling mechanisms in pancreatic cancer microenvironment. To explore their functional connection, we investigated the effects of their combined blockade on cancer growth in orthotopic allografts of pancreatic cancer mt4-2D cells in immunocompetent mice. The anti-BAG3 + anti-SIRPα mAbs treatment inhibited (p = 0.007) tumor growth by about the 70%; also the number of metastatic lesions was decreased, mostly by the effect of the anti-BAG3 mAb. Fibrosis and the expression of the CAF activation marker α-SMA were reduced by about the 30% in animals treated with anti-BAG3 mAb compared to untreated animals, and appeared unaffected by treatment with the anti-SIRPα mAb alone; however, the addition of anti-SIRPα to anti-BAG3 mAb in the combined treatment resulted in a > 60% (p < 0.0001) reduction of the fibrotic area and a 70% (p < 0.0001) inhibition of CAF α-SMA positivity. Dendritic cells (DCs) and CD8+ lymphocytes, hardly detectable in the tumors of untreated animals, were modestly increased by single treatments, while were much more clearly observable (p < 0.0001) in the tumors of the animals subjected to the combined treatment. The effects of BAG3 and SIRPα blockade do not simply reflect the sum of the effects of the single blockades, indicating that the two pathways are connected by regulatory interactions and suggesting, as a proof of principle, the potential therapeutic efficacy of a combined BAG3 and SIRPα blockade in pancreatic cancer., (© 2022. The Author(s).)

Delivery of recombinant adeno-associated virus (rAAV) vectors to the newborn liver is followed by a rapid loss of episomal vector copies because of hepatocyte proliferation. In selected hepatocytes, integration of rAAV genomes can lead to a sustained expression of the transgene. The safety of in vivo gene therapy with single-stranded AAV vectors has been questioned in a study reporting a high incidence of hepatocellular carcinoma, associated with provirus integration events in mice that receive an single-stranded AAV injection at birth. To investigate the tumour-initiating potential of the newly established self-complementary AAV (scAAV) vectors in the liver, groups of newborn rats received intravenous injection of a scAAV vector encoding the green fluorescent protein (GFP), or were injected with phosphate-buffered saline (PBS) or diethylnitrosamine (DEN), a well-known liver tumour initiator. The rats were fed on a diet containing 2-acetylaminofluorene, a potent liver tumour-promoting agent to accelerate the carcinogenic process. After 2 months, the animals were killed and their livers analysed. Preneoplastic nodules were identified by glutathion S-transferase-p (GSTp) staining, and GFP expression was detected by immunohistochemistry. Vector genome integration events were analysed. The numbers of GSTp-positive foci were comparable in the PBS and the scAAV-GFP groups and significantly higher in the DEN group. The proportion of GSTp-positive foci that also expressed GFP was low and in the range expected for random occurrence. No specific integration hot spots were detected by linear amplification-mediated-PCR in transduced liver. In conclusion, scAAV transduction of newborn rat liver does not trigger preneoplastic lesions suggesting an absence of liver tumourigenesis.