Despite their lethality and ensuing clinical and therapeutic relevance, circulating tumor cells (CTCs) from colorectal carcinoma (CRC) remain elusive, poorly characterized biological entities. We perfected a cell system of stable, primary lines from human CRC showing that they possess the full complement of ex- and in-vivo, in xenogeneic models, characteristics of CRC stem cells (CCSCs). Here we show how tumor-initiating, CCSCs cells can establish faithful orthotopic phenocopies of the original disease, which contain cells that spread into the circulatory system. While in the vascular bed, these cells retain stemness, thus qualifying as circulating CCSCs (cCCSCs). This is followed by the establishment of lesions in distant organs, which also contain resident metastatic CCSCs (mCCSCs). Our results support the concept that throughout all the stages of CRC, stemness is retained as a continuous property by some of their tumor cells. Importantly, we describe a useful standardized model that can enable isolation and stable perpetuation of human CRC's CCSCs, cCCSCs and mCCSCs, providing a useful platform for studies of CRC initiation and progression that is suitable for the discovery of reliable stage-specific biomarkers and the refinement of new patient-tailored therapies. FUND: This work was financially supported by grants from "Ministero della Salute Italiano"(GR-2011-02351534, RC1703IC36 and RC1803IC35) to Elena Binda and from "Associazione Italiana Cancro" (IG-14368) Angelo L. Vescovi. None of the above funders have any role in study design, data collection, data analysis, interpretation, writing the project.

G-quadruplex (G4) ligands are investigated to discover new anticancer drugs with increased cell-killing potency. These ligands can induce genome instability and activate innate immune genes at non-cytotoxic doses, opening the discovery of cytostatic immune-stimulating ligands. However, the interplay of G4 affinity/selectivity with cytotoxicity and immune gene activation is not well-understood. We investigated a series of closely related hydrazone derivatives to define the molecular bases of immune-stimulation activity. Although they are closely related to each other, such derivatives differ in G4 affinity, cytotoxicity, genome instability, and immune gene activation. Our findings show that G4 affinity of ligands is a critical feature for immune gene activation, whereas a high cytotoxic potency interferes with it. The balance of G4 stabilization versus cytotoxicity can determine the level of immune gene activation in cancer cells. Thus, we propose a new rationale based on low cell-killing potency and high immune stimulation to discover effective anticancer G4 ligands.

Immunotherapy has shown significant improvement in the survival of patients with hepatocellular carcinoma (HCC) compared to TKIs as first-line treatment. Unfortunately, approximately 30% of HCC exhibits intrinsic resistance to ICIs, making new therapeutic combinations urgently needed. The dysregulation of the Notch signaling pathway observed in HCC can affect immune cell response, reducing the efficacy of cancer immunotherapy. Here, we provide an overview of how Notch signaling regulates immune responses and present the therapeutic rationale for combining Notch signaling inhibition with ICIs to improve HCC treatment. Moreover, we propose using exosomes as non-invasive tools to assess Notch signaling activation in hepatic cancer cells, enabling accurate stratification of patients who can benefit from combined strategies.

Advanced hepatocellular carcinoma (HCC) has a dismal prognosis; however, the introduction of atezolizumab-bevacizumab combination has improved overall survival and novel immune checkpoint inhibitors are entering the clinics. Despite more therapeutic options being available, no biomarker guides treatment choice. Indeed, tissue-based analyses and complex analytical procedures hinder clinical translation. We explored the informativeness of a simple, non-invasive, repeatable cytofluorimetric assay on peripheral blood to predict response and survival in HCC patients treated with atezolizumab-bevacizumab. Twenty-five cirrhotic patients, 50 HCC patients undergoing atezolizumab-bevacizumab and an independent validation cohort of 25 HCC patients were subjected to a cytofluorimetric study of peripheral white blood cells (WBCs) to assess baseline programmed death-ligand 1-positive (PD-L1+) and cytotoxic T-lymphocyte antigen 4-positive (CTLA4+) cell percentage in the different populations and their early on-treatment variations. Immunophenotypes were evaluated against treatment response. RNA sequencing followed by RT-PCR validation were used to elucidate the molecular correlates of immunophenotypic observations. PD-L1+ cell percentage did not predict response either at baseline or when evaluating treatment-induced early changes. Conversely, the percentage of CTLA4+ lymphocytes at baseline showed a predictive significance (35.37 in responders versus 31.5 in non-responders, P = 0.03). More interestingly, the early CTLA4+ changes during treatment in lymphocytes (responders 0.95 versus non-responders 1.08, P = 0.05), monocytes (responders 0.95 versus non-responders 1.04, P = 0.03), granulocytes (responders 0.94 versus non-responders 1.14, P = 0.001) and, even stronger, the early CTLA4+ percentage change in the whole WBCs displayed a predictive significance in terms of time to progression (TTP) (P < 0.0001) and overall survival (OS) (P = 0.005). The immunophenotypic findings correlated with transcriptional modulation of CTLA4 target genes and genes involved in immune response. A repeatable, easy, non-invasive blood test predicts response to immunotherapy in patients with HCC, both in terms of TTP and OS. CTLA4+ cell percentage increase in non-responders suggests a possible resistance mechanism which deserves attention as a druggable target.