ObjectiveAlthough counting of circulating tumour cells (CTC) has attracted a broad interest as potential markers of tumour progression and treatment response, the lack of functional characterisation of these cells had become a bottleneck in taking these observations to the clinic. Our objective was to culture these cells in order to understand them and exploit their therapeutic potential to the full.DesignHere, hypothesising that some CTC potentially have cancer stem cell (CSC) phenotype, we generated several CTC lines from the blood of patients with advanced metastatic colorectal cancer (CRC) based on their self-renewal abilities. Multiple standard tests were then employed to characterise these cells.ResultsOur CTC lines self-renew, express CSC markers and have multilineage differentiation ability, both in vitro and in vivo. Patient-derived CTC lines are tumorigenic in subcutaneous xenografts and are also able to colonise the liver after intrasplenic injection. RNA sequencing analyses strikingly demonstrate that drug metabolising pathways represent the most upregulated feature among CTC lines in comparison with primary CRC cells grown under similar conditions. This result is corroborated by the high resistance of the CTC lines to conventional cytotoxic compounds.ConclusionsTaken together, our results directly demonstrate the existence of patient-derived colorectal CTCs that bear all the functional attributes of CSCs. The CTC culture model described here is simple and takes <1 month from blood collection to drug testing, therefore, routine clinical application could facilitate access to personalised medicine.Clinical Trial RegistrationClinicalTrial.gov NCT01577511.

The immune system plays a major role in the surveillance and control of malignant cells, with the presence of tumor infiltrating lymphocytes (TILs) correlating with better patient prognosis in multiple tumor types. The development of ‘checkpoint blockade’ and adoptive cellular therapy has revolutionized the landscape of cancer treatment and highlights the potential of utilizing the patient’s own immune system to eradicate cancer. One mechanism of tumor-mediated immunosuppression that has gained attention as a potential therapeutic target is the purinergic signaling axis, whereby the production of the purine nucleoside adenosine in the tumor microenvironment can potently suppress T and NK cell function. The production of extracellular adenosine is mediated by the cell surface ectoenzymes CD73, CD39, and CD38 and therapeutic agents have been developed to target these as well as the downstream adenosine receptors (A1R, A2AR, A2BR, A3R) to enhance anti-tumor immune responses. This review will discuss the role of adenosine and adenosine receptor signaling in tumor and immune cells with a focus on their cell-specific function and their potential as targets in cancer immunotherapy.

Adenosine is an immunosuppressive factor that limits anti-tumor immunity through the suppression of multiple immune subsets including T cells via activation of the adenosine A2A receptor (A2AR). Using both murine and human chimeric antigen receptor (CAR) T cells, here we show that targeting A2AR with a clinically relevant CRISPR/Cas9 strategy significantly enhances their in vivo efficacy, leading to improved survival of mice. Effects evoked by CRISPR/Cas9 mediated gene deletion of A2AR are superior to shRNA mediated knockdown or pharmacological blockade of A2AR. Mechanistically, human A2AR-edited CAR T cells are significantly resistant to adenosine-mediated transcriptional changes, resulting in enhanced production of cytokines including IFNγ and TNF, and increased expression of JAK-STAT signaling pathway associated genes. A2AR deficient CAR T cells are well tolerated and do not induce overt pathologies in mice, supporting the use of CRISPR/Cas9 to target A2AR for the improvement of CAR T cell function in the clinic.

Metabotropic glutamate receptor subtype 5 (mGluR5) is a potential drug target in neurological and psychiatric disorders, and subtype-selective allosteric modulators have attracted much attention as potential drug candidates. In this study, the binding sites of three novel 2-methyl-6-(phenylethynyl)pyridine (MPEP)-derived negative allosteric modulators, 2-, 3-, and 4-BisPEB, have been characterized. 2-, 3-, and 4-BisPEB are 1,3-bis(pyridinylethynyl)-benzenes and differ only by the position of the nitrogen atoms in the pyridine rings. Despite their high structural similarity, 2-BisPEB [1,3-bis(pyridin-2-ylethynyl)-benzene, nitrogen atoms in ortho positions], with an IC 50 value in the nanomolar range, is significantly more potent than the 3-and 4-pyridyl analogs. Mutational analysis, directed by a previously published mGluR5 homology model, was used to determine key residues for the ligand-receptor interactions that may explain the potency differences of 2-, 3-, and 4-BisPEB. Residues Ile651, Pro655, Tyr659, Asn747, Trp785, Phe788, Tyr792, Ser809, and Ala810 were found to have critical roles for the activity of one or more of the three BisPEBs and the reference compound MPEP. The mutagenesis data suggest that the higher potency of 2-BisPEB is due to hydrogen bonding to Ser809 because the S809A mutation made 2-BisPEB equipotent to 3-and 4-BisPEB (IC 50 , 1-2.5 M). The potency of MPEP was also greatly affected by S809A (52-fold), suggesting that a Ser809-mediated hydrogen bond is also a key interaction between MPEP and mGluR5. Potential binding modes of 2-, 3-, and 4-BisPEB obtained by molecular docking to the mGluR5 homology model provide a structural context for the reported major mutational effects.

Surgery remains the curative treatment modality for colorectal cancer in all stages, including stage IV with resectable liver metastasis. There is emerging evidence that the stress response caused by surgery as well as other perioperative therapies such as anesthesia and analgesia may promote growth of pre-existing micro-metastasis or potentially initiate tumor dissemination. Therapeutically targeting the perioperative period may therefore reduce the effect that surgical treatments have in promoting metastases, for example by combining β-adrenergic receptor antagonists and cyclooxygenase-2 (COX-2) inhibitors in the perioperative setting. In this paper, we highlight some of the mechanisms that may underlie surgery-related metastatic development in colorectal cancer. These include direct tumor spillage at the time of surgery, suppression of the anti-tumor immune response, direct stimulatory effects on tumor cells, and activation of the coagulation system. We summarize in more detail results that support a role for catecholamines as major drivers of the pro-metastatic effect induced by the surgical stress response, predominantly through activation of β-adrenergic signaling. Additionally, we argue that an improved understanding of surgical stress-induced dissemination, and more specifically whether it impacts on the level and nature of heterogeneity within residual tumor cells, would contribute to the successful clinical targeting of this process. Finally, we provide a proof-of-concept demonstration that ex-vivo analyses of colorectal cancer patient-derived samples using RGB-labeling technology can provide important insights into the heterogeneous sensitivity of tumor cells to stress signals. This suggests that intra-tumor heterogeneity is likely to influence the efficacy of perioperative β-adrenergic receptor and COX-2 inhibition, and that ex-vivo characterization of heterogeneous stress response in tumor samples can synergize with other models to optimize perioperative treatments and further improve outcome in colorectal and other solid cancers.

Posttreatment recurrence of colorectal cancer, the third most lethal cancer worldwide, is often driven by a subpopulation of cancer stem cells (CSC). The tight junction (TJ) protein claudin-2 is overexpressed in human colorectal cancer, where it enhances cell proliferation, colony formation, and chemoresistance While several of these biological processes are features of the CSC phenotype, a role for claudin-2 in the regulation of these has not been identified. Here, we report that elevated claudin-2 expression in stage II/III colorectal tumors is associated with poor recurrence-free survival following 5-fluorouracil-based chemotherapy, an outcome in which CSCs play an instrumental role. In patient-derived organoids, primary cells, and cell lines, claudin-2 promoted colorectal cancer self-renewal and in multiple mouse xenograft models. Claudin-2 enhanced self-renewal of ALDH CSCs and increased their proportion in colorectal cancer cell populations, limiting their differentiation and promoting the phenotypic transition of non-CSCs toward the ALDH phenotype. Next-generation sequencing in ALDH cells revealed that claudin-2 regulated expression of nine miRNAs known to control stem cell signaling. Among these, miR-222-3p was instrumental for the regulation of self-renewal by claudin-2, and enhancement of this self-renewal required activation of YAP, most likely upstream from miR-222-3p. Taken together, our results indicate that overexpression of claudin-2 promotes self-renewal within colorectal cancer stem-like cells, suggesting a potential role for this protein as a therapeutic target in colorectal cancer. Claudin-2-mediated regulation of YAP activity and miR-222-3p expression drives CSC renewal in colorectal cancer, making it a potential target for therapy. .

Since its discovery in 1992, mGluR5 has attracted significant attention and been linked to several neurological and psychiatric diseases. Ligand development was initially focused on the orthosteric binding pocket, but lack of subtype selective ligands changed the focus to the transmembrane allosteric binding pocket. This strategy has resulted in several drug candidates in clinical testing. In the present article we explore the orthosteric and allosteric binding pockets in terms of structure and ligand recognition across the mGluR subtypes and groups, and discuss the clinical potential of ligands targeting these pockets. We have performed binding mode analyses of non- and group-selective orthosteric ligands based on molecular docking in mGluR crystal structures and models. For the analysis of the allosteric binding pocket we have combined data from all mGluR5-mutagenesis studies, collectively reporting five negative allosteric modulators and 47 unique mutations, and compared it to the closest related homolog, mGluR1.

The immune system plays a major role in the surveillance and control of malignant cells, with the presence of tumor infiltrating lymphocytes (TILs) correlating with better patient prognosis in multiple tumor types. The development of &lsquo;checkpoint blockade&rsquo; and adoptive cellular therapy has revolutionized the landscape of cancer treatment and highlights the potential of utilizing the patient&rsquo;s own immune system to eradicate cancer. One mechanism of tumor-mediated immunosuppression that has gained attention as a potential therapeutic target is the purinergic signaling axis, whereby the production of the purine nucleoside adenosine in the tumor microenvironment can potently suppress T and NK cell function. The production of extracellular adenosine is mediated by the cell surface ectoenzymes CD73, CD39 and CD38 and therapeutic agents have been developed to target these as well as the downstream adenosine receptors (A1R, A2AR, A2BR, A3R) to enhance anti-tumor immune responses. This review will discuss the role of adenosine and adenosine receptor signaling in tumor and immune cells with a focus on their cell-specific function and their potential as targets in cancer immunotherapy.