Modulation of stress and immune response by Amblyomin-X results in tumor cell death in a horse melanoma model

We have investigated Amblyomin-X-treated horse melanomas to better understand its mode of action through transcriptome analysis and the in vivo model. Amblyomin-X is a Kunitz-type homologous protein that selectively leads to the death of tumor cells via ER stress and apoptosis, currently under investigation as a new drug candidate for cancer treatment. Melanomas are immunogenic tumors, and a better understanding of the immune responses is warranted. Equine melanomas are spontaneous and not so aggressive as human melanomas are, as this study shows that the in vivo treatment of encapsulated horse melanoma tumors led to a significant reduction in the tumor size or even the complete disappearance of the tumor mass through intratumoral injections of Amblyomin-X. Transcriptome analysis identified ER- and mitochondria-stress, modulation of the innate immune system, apoptosis, and possibly immunogenic cell death activation. Interactome analysis showed that Amblyomin-X potentially interacts with key elements found in transcriptomics. Taken together, Amblyomin-X modulated the tumor immune microenvironment in different ways, at least contributing to induce tumor cell death.

melanoma cells consist of many different cell types, and many of these cells can respond to external perturbations via inflammatory cytokines and transcribe other genes through autocrine and paracrine stimuli. Virus and bacteria infections trigger "Pattern-Recognition Receptors" (PRR), like TLRs but also RLR after the cell invasion. In the cytoplasm these pathogens are recognized by dead-box RLR helicases like DDX58 (RIG-I), IFIH1  and DHX58 (LGP2) having MAVS as a target. Also by non-RLR helicases like DDX60 (a DEG), DHX3, DDX9 and DHX33 comprising NLRP3 inflammasome as a target. Other midstream genes like IFIT, NOD2, LRRFIP1 and PKR, having as target NF-κBB and CTNNB1 (β-catenin) 1 , were observed. Furthermore, double-stranded RNA activates OAS genes (OAS1, OAS2, OAS3) that lead to the synthesis of 2′-5′ oligoA and RNase L activation 2 .

The effect of Amblyomin-X on Inflammation
As previously mentioned, there must be an initial response increasing the expression level of many cytokines, most of them related to inflammatory pathways, like IL1β (produced by activated macrophages and proteolytically processed to the active form by CASP1), IL-6 (a critical protein acting in acute and chronic inflammation), CXCL8 (IL-8, secreted primarily by neutrophils), and CCL2 (MCP-1, involved in immunoregulatory and inflammatory processes, acting as an antitumoral gene). By analyzing different enriched pathways, we could observe distinct inflammatory responses causing increased expression of the previously mentioned cytokines, besides genes like DEFB4A (Beta-Defensin 2, a microbicidal and cytotoxic peptide page 2 / 22 secreted by neutrophils and regulated by inflammation). Another inflammatory pathway is TREM1 response; "Immune response: TREM1 signaling pathway"), increasing the expression levels of IL-6, CCL2 and IL8. Finally, TNF pathway was also enriched, although TNF-α was moderately regulated, being a proinflammatory cytokine secreted by macrophages and also produced downstream of the RLR pathway. Furthermore, TNF receptor TNF-R1 was a DEG.
Downstream of TNF-R1 we observed two critical paths, one via JUN (AP-1), reaching two DEGs, MMP3 and IL-6, and another via NF-κBB complex and possibly related to important DEGs such as IL-6, IL1β, SFK (SRC family kinase, a proto-oncogene related to development and growth), and IL1RN (IL1 receptor agonist that inhibits IL1α and IL1β, modulating the inflammatory response of the IL1 gene family). Summarizing, inflammatory responses could be achieved, via TREM1, IL1β, IL-6, and possibly TNF and HMGB1-RAGE pathways. IL-8 must also be part of the inflammatory response, but it is the biggest confounding factor in our analysis. Moreover, IL-6 is a central hub for 6hx0h and also for 12hx0h. A part of these results was supported by interactomics profile of Amblyomin-X, where immunogenic protein such as TLR2 and CD2 were identified as binding partners.

ER-stress and Proteasome Inhibition & production
Many proteins that make up the main proteasome complex (the 20S and 26S units) started the transcription late after 12 h, enriching the proteasome pathway, through a set of none DEGs found in module M9 of coexpression analysis. This means that some cell types may have need to build new proteasome complexes. However, the pathway "Proteasome Inhibition" (PI) could not be enriched. It is worth noting that PI has already been evaluated and confirmed by multiple biochemical and cellular biological techniques [4][5][6][7]. One possible hypothesis of proteasome activity inhibition is HTT binding to 20S/26S [8][9]; and possible page 3 / 22 consequences are: a) oxidative stress, mitochondria-stress, CASP3 (and CASP9), and also release of cytochrome-c, all very well established in our transcriptomics results.

Pathways not found in transcriptomics
In addition, we were expecting certain pathways to be enriched, like endocytosis and vesicle transport pathways, but nothing could be seen. A possible hypothesis are post-translational modifications not detected by RNA-Seq, or the transcriptional machinery had no need to produce the related proteins.

RNA extraction and library preparation
Total RNA was isolated from cultured cells with Trizol (Ambion, Life Technologies) and

Quality and filtering fastq Reads
Raw sequencing reads contaminants were removed with Bowtie version 2 2.2.5 [13].
Trimmomatic [14] was used to trim and remove reads with low-complexity and homopolymer enriched regions, poly-A/T/N tails, adapter sequences and low-quality bases, while fastq-mcf version 1.04.662 40 was used for sequencing quality control [15]. The reads were filtered out if more than 90% of reads corresponding to a homopolymer or low-complexity regions, and if the mean quality score was lower than 25 in a 15 window size. After trimming, all reads smaller than 40 bp were discarded. A quality check was performed using FastQC. Hisat2 [16] was used to align reads against horse (Equus caballus) reference genome (annotation version 89) [17]. assays, transcript levels were normalized to RPL18 and represented as relative abundance using the delta Ct method [18]. Two controls for the RT step, one without primer (-primer) and the other without reverse transcriptase (-RT) were performed, followed by qRT-PCR with the pair of primers, to confirm the absence of RNA self-priming and of genomic DNA contamination in the RT, respectively. Conditions for qRT-PCR reactions were: 40 cycles of 95°C/15 sec, 60°C/1 min, using the specific primers listed in (Table S14).