Chapter III - Additional Results
3.2 Arg2 overexpression in melanoma dampens adaptive anti-tumor immune
increased cancer aggressiveness. Despite these correlative observations, to date, a formal
assessment of the pro-tumoral role of Arginase 2 had not been reported in the literature. In general, tumor masses are highly heterogenous and frequently infiltrated by various cell types, thus comprising differential potential sources of arginase activity, as demonstrated for MDSCs.
The results described below allowed us to gain better understanding of the role of Arg2 expressed by mouse melanoma tumor cells and can be regarded as an extension of the data included in the publication above (Figure II-6).
To start addressing the hypothesis that Arg2 might play a pro-tumoral role in cancer cells, we forced the overexpression of Arg2 by transduction of the B16-OVA cell line using the same lentiviral vectors used for DC2114 (Figure III-1a). Analogously to MC38-OVA, the B16-OVA cell line also expresses the chicken ovalbumin protein as a surrogate tumor antigen. After transduction, the Arg2-transduced tumor cells showed a 2-log increased in Arg2 mRNA levels relative to empty-vector controls (Figure III-2a), and a massive increase in Arg2 protein levels (Figure III-2b).
The overexpression of Arg2 did not alter the cell-intrinsic capacity of these tumor cells to proliferate in vitro (Figure III-2c) and did not alter either their tumorigenic potential or tumor growth rate when implanted in immunocompromised Rag2-/- mice (Figure III-2d). However, in WT mice possessing a fully-functional adaptive immune system, Arg2-overexpressing B16-OVA tumors grew significantly faster and more aggressively than the empty-vector control tumors. Taken together, these results suggested that, like in colorectal carcinoma MC38-OVA cells, Arg2 overexpression promotes tumor growth because it dampens the anti-tumor immune response.
Because B16-OVA is a highly immunogenic tumor, we sought to determine whether pre-existing antigen-specific anti-tumor T cell responses would be potent enough to cancel the pro-tumoral effects of Arg2 overexpression. To address this question, WT mice were pre-immunised with MHC class I or MHC class II OVA peptides and CpG-B seven days prior to tumor
challenge. Two weeks after tumor implantation, Arg2-overexpressing tumors showed faster and more aggressive growth in mice immunised with both the OVA257-264 peptide (Figure III-2f) and OVA323-339 peptide (Figure III-2g). These results suggested that Arg2 overexpression by tumor cells can suppress anti-tumor immune responses despite the prior elicitation of CD8+ or CD4+ T cell driven antigen-specific responses.
Taken together, these results support the view that Arginase 2 can act as a pro-tumoral enzyme due to its ability to dampen anti-tumor immune responses. The enzymatic activity of arginases also promotes the biosynthesis of polyamines, which are known to be critical molecules for tumor cell proliferation. Moreover, several basic studies and clinical trials have demonstrated that arginine-auxotrophic tumors are highly sensitive to systemic arginine
depletion (induced by bloodstream infusion of pegylated-Arg1), suggesting that arginine metabolism is essential for tumor cell metabolism and survival. Thus, we sought to determine whether endogenous Arginase 2 expression is essential for B16 melanoma cell growth or immune escape.
Figure III-2. Arginase 2 overexpression in tumor cells promotes tumor growth by dampening adaptive anti-tumor immunity. (a) mRNA and (b) protein levels of Arg2 in parental, pWPI-transduced and pWPI-Arg2 B16-OVA cells. (c) In vitro tumor cell proliferation of pWPI and Arg2 B16-OVA cells. Indicated below, the doubling times calculated (in hours) for each condition. (d,e) Tumor growth rate of pWPI-transduced and pWPI-Arg2 B16-OVA cells in (d) Rag2-/- hosts and (e) WT hosts. (f,g) Tumor growth rate of pWPI-transduced and pWPI-Arg2 B16-OVA cells in (f) CpG-B + OVA257-264 or (e) CpG-B + OVA323-339 pre-immunised WT hosts.
To address this question, we generated Arg2-deficient B16-OVA cells by CRISPR/Cas9-mediated gene editing, thus ablating basal Arg2 expression observed in these cells (Figure III-2a.b). With the aim of targeting the first coding exon of Arg2 at three different loci, we generated three distinct OFP-coding GeneArt® CRISPR Nuclease vectors by cloning an
Arg2-targeting crRNA into each vector (Figure III-3a). This vector-based approach delivers the Cas9 protein and the gRNA in a non-integrative DNA format thus allowing transient action of the CRISPR/Cas9 machinery. Moreover, the encoded OFP is separated from the Cas9 protein by the self-cleaving 2A sequence, allowing fluorescent-based sorting of the vector-containing cells.
Figure III-3. Generation of Arg2-/- B16-OVA cell lines by CRISPR/Cas9 gene editing. (a) Vector system used in order to deliver all the molecular tools in DNA format. (b) Representative FACS plots of the transfection efficiency analysis based on percentage of OFP+ cells. (c) Lipofectamine-based transfection conditions tested. In the last column, percentage of OFP+ cells obtained 24 hours after transfection. (d) After transfection, the region surrounding the first exon of Arg2 was PCR-amplified in bulk-sorted OFP+ cells and misalignments caused by indel mutations introduced after Cas9 cleavage were analysed using a T7 endonuclease-based enzymatic digestion. Digested or undigested samples were run on a Bioanalyzer. (e) The band intensities obtained in (b) were used to calculate the cleavage efficiency of each gRNA construct used for Cas9 cleavage. (f) After transduction, cell clones were obtained by sorting one OFP+ cell in each well and, after clone expansion, frozen cells pellets were analysed by Western Blot for presence or absence of Arg2 protein. (g) Based on the presence or absence of Arg2 in Western Blots, the region surrounding the first exon of Arg2 was PCR-amplified and analysed by Sanger sequencing, primed by the previously used forward primer.
Three different transfection reagents aimed at nuclear delivery of the vector were tested (data not shown) and Lipofectamine 3000 showed the highest efficiency in terms of OFP+ cells.
Further optimisation of transfection conditions was performed for each vector (Figure III-3b,c), resulting in optimal transfection conditions (1.5 L of Lipofectamine and 2 g of DNA) across the three different gRNAs. Next, we sorted the OFP+ cells, PCR-amplified the 5’ coding region of Arg2, and interrogated the amplicons for insertion or deletion mutations using the T7
endonuclease-based GeneArt® Genomic Cleavage Detection kit (Figure III-3d,e). The digestion products were separated by electrophoresis and quantified using a Bioanalyzer (Figure III-3d).
Quantification of the digestion products showed that the CRISPR/Cas9 machinery efficiently induced indels in the interrogated region, in a range of 44% to 69% of the amplicons, although these numbers most likely represent an underestimation of all mutated alleles. Next, we derived 80 cell line clones by FACS sorting single-cells into 96-well plates with a MoFlow Astrios, expanded the clones and obtained cell pellets for Western blot detection of Arg2 (Figure III-3f).
We selected a subset of expressing clones as WT control clones plus a subset Arg2-deficient clones. Sanger sequencing chromatograms of the 5’ region of Arg2 allowed us to identify Arg2-expressing Arg2+/+ clones and Arg2-deficient Arg2-/- clones (Figure III-3g). The sequence information supported the observed defect in Arg2 protein synthesis, as the clones exhibited 7 nt frame-shift mutations (clones 4 and 6) or entire deletion of at least the first exon (clone 5). This analysis thus confirmed the existence of null mutations.
The ability to expand the Arg2-/- clones demonstrated that Arg2 is not essential for tumor cell growth in vitro. Further in vitro cell proliferation tests showed that, using several individual Arg2+/+ and Arg2-/- clones, both Arg2-expressing and Arg2-deficient clones grew at similar rates compared to the parental cell line (Figure III-4a,b), and demonstrated that the loss of Arg2 does not impact on their cell-intrinsic proliferative capacity.
Figure III-4. Constitutive Arg2 expression is not essential for B16 tumor cells and does not confer them any growth. (a,b) Tumor cell proliferation in vitro of (a) Arg2+/+ B16-OVA or (b) Arg2-/- B16-OVA cells, compared to B16-OVA parental cell line. (c,d) Three Arg2-/- B16-OVA clones and one Arg2+/+ B16-OVA clone were implanted into (c) Rag2-/- or (d) WT naïve hosts and clone tumor growth rates were compared to tumor growth of the B16-OVA parental cell line.
To address the requirement of Arg2 for the suppression of adaptive anti-tumor immunity, we transplanted one Arg2+/+ clone, three distinct Arg2-/- clones or the parental B16-OVA cell line into both immunocompromised Rag2-/- mice and immunocompetent WT mice (Figure III-4c,d).
In mice lacking control by T and B cells, all three types of B16-OVA tumors grew at similar
rates (Figure III-4c). In WT hosts, Arg2-deficient tumors also grew at similar rates compared to the Arg2-expressing clone and the parental cell line (Figure III-4d). These results demonstrated that the loss of Arg2 does not alter in vivo tumorigenic potential of this melanoma model and that Arg2 endogenous levels in B16 cells are not essential for their capacity to inhibit adaptive anti-tumor immune responses.
3.3 In vivo priming of Arg2-/- CD8+ T recapitulates their enhanced in vitro activation