IL-1 belongs to a family of 11 members and is one of the seven receptor-agonists with pro-inflammatory activity. Beyond its biological role as a regulator of the innate immune response, IL-1 is involved in stress and chronic inflammation, therefore it is responsible for several pathological conditions. In particular, IL-1 is known to exert a critical function in malignancies, influencing the tumor microenvironment and promoting cancer initiation and progression. Thus, it orchestrates immunosuppression recruiting pro-tumor immune cells of myeloid origin. Furthermore, new recent findings showed that this cytokine can be directly produced by tumor cells in a positive feedback loop and contributes to the failure of targeted therapy. Activation of anti-apoptotic signaling pathways and senescence are some of the mechanisms recently proposed, but the role of IL-1 in tumor cells refractory to standard therapies needs to be further investigated.

Cetuximab (CTX) is a monoclonal antibody targeting the epidermal growth factor receptor (EGFR), commonly used to treat patients with metastatic colorectal cancer (mCRC). Unfortunately, objective remissions occur only in a minority of patients and are of short duration, with a population of cells surviving the treatment and eventually enabling CTX resistance. Our previous study on CRC xenopatients associated poor response to CTX with increased abundance of a set of pro-inflammatory cytokines, including the interleukins IL-1A, IL-1B and IL-8. Stemming from these observations, our current work aimed to assess the role of IL-1 pathway activity in CTX resistance. We employed a recombinant decoy TRAP IL-1, a soluble protein combining the human immunoglobulin Fc portion linked to the extracellular region of the IL-1-receptor (IL-1R1), able to sequester IL-1 directly from the medium. We generated stable clones expressing and secreting a functional TRAP IL-1 into the culture medium. Our results show that IL-1R1 inhibition leads to a decreased cell proliferation and a dampened MAPK and AKT axes. Moreover, CRC patients not responding to CTX blockage displayed higher levels of IL-1R1 than responsive subjects, and abundant IL-1R1 is predictive of survival in patient datasets specifically for the consensus molecular subtype 1 (CMS1). We conclude that IL-1R1 abundance may represent a therapeutic marker for patients who become refractory to monoclonal antibody therapy, while inhibition of IL-1R1 by TRAP IL-1 may offer a novel therapeutic strategy.

Extracellular vesicles (EVs) are of great interest to study the cellular mechanisms of cancer development and to diagnose and monitor cancer progression. EVs are a highly heterogeneous population of cell derived particles, which include microvesicles (MVs) and exosomes (EXOs). EVs deliver intercellular messages transferring proteins, lipids, nucleic acids, and metabolites with implications for tumour progression, invasiveness, and metastasis. Epidermal Growth Factor Receptor (EGFR) is a major driver of cancer. Tumour cells with activated EGFR could produce EVs disseminating EGFR itself or its ligands. This review provides an overview of EVs (mainly EXOs and MVs) and their cargo, with a subsequent focus on their production and effects related to EGFR activation. In particular, in vitro studies performed in EGFR-dependent solid tumours and/or cell cultures will be explored, thus shedding light on the interplay between EGFR and EVs production in promoting cancer progression, metastases, and resistance to therapies. Finally, an overview of liquid biopsy approaches involving EGFR and EVs in the blood/plasma of EGFR-dependent tumour patients will also be discussed to evaluate their possible application as candidate biomarkers.

Background: In the last years, several efforts have been made to classify colorectal cancer (CRC) into well-defined molecular subgroups, representing the intrinsic inter-patient heterogeneity, known as Consensus Molecular Subtypes (CMSs). Methods: In this work, we performed a meta-analysis of CRC patients stratified into four CMSs. We identified a negative correlation between a high level of anaplastic lymphoma kinase (ALK) expression and relapse-free survival, exclusively in CMS1 subtype. Stemming from this observation, we tested cell lines, patient-derived organoids and mice with potent ALK inhibitors, already approved for clinical use. Results: ALK interception strongly inhibits cell proliferation already at nanomolar doses, specifically in CMS1 cell lines, while no effect was found in CMS2/3/4 groups. Furthermore, in vivo imaging identified a role for ALK in the dynamic formation of 3D tumor spheroids. Consistently, ALK appeares constitutively phosphorylated in CMS1, and it signals mainly through the AKT axis. Mechanistically, we found that CMS1 cells display several copies of ALKAL2 ligand and ALK-mRNAs, suggesting an autocrine loop mediated by ALKAL2 in the activation of ALK pathway, responsible for the invasive phenotype. Consequently, disruption of ALK axis mediates the pro-apoptotic action of CMS1 cell lines, both in 2D and 3D and enhanced cell-cell adhesion and e-cadherin organization. In agreement with all these findings, the ALK signature encompassing 65 genes statistically associated with worse relapse-free survival in CMS1 subtype. Finally, as a proof of concept, the efficacy of ALK inhibition was demonstrated in both patient-derived organoids and in tumor xenografts in vivo. Conclusions: Collectively, these findings suggest that ALK targeting may represent an attractive therapy for CRC, and CMS classification may provide a useful tool to identify patients who could benefit from this treatment. These findings offer rationale and pharmacological strategies for the treatment of CMS1 CRC.

Three-dimensional (3D) culture systems like tumor spheroids represent useful in vitro models for drug screening and more broadly for cancer biology research, but the generation of uniform populations of spheroids remains challenging. The possibility to properly characterize spheroid properties would increase the reliability of these models. To address this issue different analysis were combined: i) a new device and relative analytical method for the accurate, simultaneous, and rapid measurement of mass density, weight, and size of spheroids, ii) confocal imaging, and iii) protein quantification, in a clinically relevant 3D model. The LoVo colon cancer cell line forming spheroids, treated with crizotinib (CZB) an ATP-competitive small-molecule inhibitor of the receptor tyrosine kinases, was employed to study and assess the correlation between biophysical and morphological parameters in both live and fixed cells. The new fluidic-based measurements allowed a robust phenotypical characterization of the spheroids structure, offering insights on the spheroids bulk and an accurate measurement of the tumor density. This analysis helps overcome the technical limits of the imaging that hardly penetrates the thickness of 3D structures. Accordingly, we were able to document that CZB treatment has an impact on mass density, which represents a key marker characterizing cancer cell treatment. Spheroid culture is the ultimate technology in drug discovery and the adoption of such precise measurement of the tumor characteristics can represent a key step forward for the accurate testing of treatment’s potential in 3D in vitro models.

Evidences of a crosstalk between Epidermal Growth Factor Receptor (EGFR) and Glucocorticoid Receptor (GR) has been reported, ranging from the modulation of receptor levels or GR mediated transcriptional repression of EGFR target genes, with modifications of epigenetic markers. The present study focuses on the involvement of EGFR positive and negative feedback genes in the establishment of cetuximab (CTX) resistance in metastatic Colorectal Cancer (CRC) patients. We evaluated the expression profile of the EGFR ligands TGFA and HBEGF, along with the pro-inflammatory cytokines IL-1B and IL-8, which were previously reported to be negatively associated with monoclonal antibody response, both in mice and patient specimens. Among EGFR negative feedback loops, we focused on ERRFI1, DUSP1, LRIG3, and LRIG1. We observed that EGFR positive feedback genes are increased in CTX-resistant cells, whereas negative feedback genes are reduced. Next, we tested the expression of these genes in CTX-resistant cells upon GR modulation. We unveiled that GR activation leads to an increase in ERRFI1, DUSP1, and LRIG1, which were shown to restrict EGFR activity, along with a decrease in the EGFR activators (TGFA and IL-8). Finally, in a cohort of xenopatients, stratified for response to cetuximab, we observed an inverse association between the expression level of LRIG1 and CRC progression upon CTX treatment. Our model implies that combining GR modulation to EGFR inhibition may yield an effective treatment strategy in halting cancer progression.