Gemcitabine-induced Activation of Type III Interferons and Chemokine Cxcl10 in Hepatoma Cells

Question:

Hepatocellular carcinoma features poor response to systemic chemotherapy and high lethality. Marked chemoresistance and tumorigenicity might result from genetic heterogeneity and from escaping host immune surveillance, respectively. In preclinical models, the efficacy of chemotherapeutics was found to rely on a molecular pattern-driven activation of tumor-derived type I interferons (IFNs) boosting an antitumor response by involving the C-X-C motif chemokine 10 (Cxcl10). Considering known complementary interactions between type I and type III IFNs, this investigation elucidates the capability of transplantable murine hepatoma cells to activate type III IFNs in the absence and presence of chemotherapeutics.

Methods:

Isogenic Ifnl2/3-deficient clones were generated from murine Hepa 1 – 6 hepatoma cells (DMSZ Braunschweig) using CRISPR/Cas9 genome engineering. Knockout was validated by conventional PCR assay on gDNA and mRNA level as well as by sequencing. Parental and deficient lines were stimulated with the RNA analogue poly(I:C) and treated with gemcitabine, doxorubicin or oxaliplatin. The expression of selected IFNs and IFN effectors was quantified via qRT-PCR. Cell viability was assessed by MTS reduction assay.

Results:

Hepa 1 – 6 cells were shown to respond to various stimulatory regimens based on the pattern recognition receptor agonist poly(I:C) with both type I IFN and type III IFN (Ifnl2/3) gene expression. Beyond that, type III IFN gene activation could also be achieved by a short exposure to gemcitabine if combined with poly(I:C) priming in a synergistic manner. This induction was diminished by timed culture medium exchange or addition of RNA targeting nucleases, suggesting release of a mediating compound. Moreover, gemcitabine, rather than doxorubicin or oxaliplatin, was found to be a potent inducer of the IFN effector Cxcl10. However, chemokine inducibility by and chemosensitivity towards gemcitabine appeared to be independent of Ifnl2/3 gene expression as these biological responses were comparable in extent in CRISPR/Cas9 engineered Ifnl2/3-deficient isogenic clones.

Conclusions:

The nucleoside analogue gemcitabine was demonstrated to independently promote Ifnl2/3 and Cxcl10 activation in transplantable hepatoma cells. Both by-effect properties are expected to impact interactive immunoediting in hepatocellular carcinoma in vivo.