The Effects of Conventional Chemotherapeutics on the Activation of Type I Interferons (IFN-α/β) in Hepatic Cancer Cells in vitro: The Significance of Endogenous Type III Interferons (IFN-λ)

by Luca Maria Grothe
Doctoral thesis
Date of Examination:2023-05-02
Date of issue:2023-04-24
Advisor:Prof. Dr. Sabine Mihm
Referee:Prof. Dr. Sabine Mihm
Referee:Dr. Dieter Prof Kube
Persistent Address: http://dx.doi.org/10.53846/goediss-9849

 

 

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Abstract

English

HCC is characterized by limited therapy options, poor response rates to systemic chemotherapy, and high lethality. Preclinical cancer models have revealed the efficacy of genotoxic drugs to rely on the release of type I IFNs by neoplastic cells boosting an antitumor response. This thesis elucidates the capacity of the transplantable hepatoma cancer cell line Hepa 1-6 to activate type I IFNs (IFN-α/β), type III IFNs (IFN-λ) and IFN effectors, and compares the parental Hepa 1-6 cell line to CRISPR/Cas9-engineered Ifnl2/3-deficient isogenic clones in view of preclinical models. 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: Chemotherapy was demonstrated to be able to modulate poly(I:C)-primed Ifnl2/3 activation and subsequently IFN effector induction. The nucleoside analogue gemcitabine was demonstrated to independently promote Ifnl2/3 and Cxcl10 activation in transplantable hepatoma cells. Ifnl2/3-deficiency was shown to not influence in vitro chemosensitivity of murine Hepa 1-6 hepatoma cells. Nevertheless, both by-effect properties are expected to impact interactive immunoediting in hepatocellular carcinoma in vivo and thus the establishment of an antitumoral immune response.
Keywords: hepatocellular carcinoma; interferons; immunogenic cell death; CRISPR/Cas9; IFNL; Hepa 1-6; type III interferons; gemcitabine; doxorubicin; oxaliplatin; poly(I:C); antitumoral immune response; DAMPs; CXCL10; immunoediting
 
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