Marta Baiocchi

Heart infarct in NOD-SCID mice: Therapeutic vasculogenesis by transplantation of human CD34+ cells and low dose CD34+KDR+ cells

Hematopoietic (Hem) and endothelial (End) lineages derive from a common progenitor cell, the hemangioblast: specifically, the human cord blood (CB) CD34+KDR+ cell fraction comprises primitive Hem and End cells, as well as hemangioblasts. In humans, the potential therapeutic role of Hem and End progenitors in ischemic heart disease is subject to intense investigation. Particularly, the contribution of these cells to angiogenesis and cardiomyogenesis in myocardial ischemia is not well established. In our studies, we induced myocardial infarct (MI) in the immunocompromised NOD‐SCID mouse model, and monitored the effects of myocardial transplantation of human CB CD34+ cells on cardiac function. Specifically, we compared the therapeutic effect of unseparated CD34+ cells vs. PBS and mononuclear cells (MNCs); moreover, we compared the action of the CD34+KDR+ cell subfraction vs. the CD34+KDR– subset. CD34+ cells significantly improve cardiac function after MI, as compared with PBS/MNCs. Similar beneficial actions were obtained using a 2‐log lower number of CD34+KDR+ cells, while the same number of CD34+KDR– cells did not have any effects. The beneficial effect of CD34+KDR+ cells may mostly be ascribed to their notable resistance to apoptosis and to their angiogenic action, since cardiomyogenesis was limited. Altogether, our results indicate that, within the CD34+ cell population, the CD34+KDR+ fraction is responsible for the improvement in cardiac hemodynamics and hence represents the candidate active CD34+ cell subset.

New models for cancer research: human cancer stem cell xenografts.

Human cancer stem cells represent promising tools for new approaches to pathway-targeted drug discovery and preclinical screening. Because of their distinctive capability to recapitulate the development of the original tumors in vivo, the study of human cancer stem cells (CSCs) largely relies on models of xenograft transplantation into immunodeficient mice. In this frame, immunity and microenvironment-related issues need careful consideration. Here we shortly revise present knowledge on xenograft-related aspects of human CSCs studies.

Proliferation State and Polo‐Like Kinase1 Dependence of Tumorigenic Colon Cancer Cells

Tumor‐initiating cells are responsible for tumor maintenance and relapse in solid and hematologic cancers. Although tumor‐initiating cells were initially believed to be mainly quiescent, rapidly proliferating tumorigenic cells were found in breast cancer. In colon cancer, the proliferative activity of the tumorigenic population has not been defined, although it represents an essential parameter for the development of more effective therapeutic strategies. Here, we show that tumorigenic colon cancer cells can be found in a rapidly proliferating state in vitro and in vivo, both in human tumors and mouse xenografts. Inhibitors of polo‐like kinase1 (Plk1), a mitotic kinase essential for cell proliferation, demonstrated maximal efficiency over other targeted compounds and chemotherapeutic agents in inducing death of colon cancer‐initiating cells in vitro. In vivo, Plk1 inhibitors killed CD133+ colon cancer cells leading to complete growth arrest of colon cancer stem cell‐derived xenografts, whereas chemotherapeutic agents only slowed tumor progression. While chemotherapy treatment increased CD133+ cell proliferation, treatment with Plk1 inhibitors eliminated all proliferating tumor‐initiating cells. Quiescent CD133+ cells that survived the treatment with Plk1 inhibitors could be killed by subsequent Plk1 inhibition when they exited from quiescence. Altogether, these results provide a new insight into the proliferative status of colon tumor‐initiating cells both in basal conditions and in response to therapy and indicate Plk1 inhibitors as potentially useful in the treatment of colorectal cancer. Stem Cells2012;30:1819–1830

Bisperoxovanadium, a phospho-tyrosine phosphatase inhibitor, reprograms myogenic cells to acquire a pluripotent, circulating phenotype

Satellite cells are the main source of myogenic progenitors in postnatal skeletal muscle, but their use in cell therapy for muscle disorders is limited because these cells cannot be delivered through circulation and they are rapidly exhausted in severe myopa‐thies. The search for alternative donor cells is ongoing, but none of the candidates so far show all the features required for successful colonization and repair of diseased muscle. In this study, we show that bisperox‐ovanadium, a phospho‐tyrosine phosphatase inhibitor, induces myogenic cells to acquire a gene expression profile and a differentiation potential consistent with the phenotype of a circulating precursors, while maintaining their myogenic potential. These effects are mediated, at least in part, by NF‐κB activation through the Tyr42‐IκB‐α phosphorylation, as shown by the expression of the dominant negative mutant form of the p50 NF‐κB subunit. Moreover, when bisperoxova‐nadium‐treated cells are injected into the femoral artery of α‐sarcoglican null dystrophic mice, they are able to circulate and to reach muscle tissue;importantly, they contribute to muscle regeneration, as shown by the expression of α‐sarcoglican in some fibers. Our observations indicate that bisperoxovanadium, or similar compounds, may prove very valuable to obtain and to expand, from committed cells, multipotent cell populations suitable for gene‐cell therapy applications and may help to understand the molecular basis of genome reprogramming and “stem‐ness.”—Castaldi, L., Serra, C., Moretti, F., Messina, G., Paoletti, R., Sampaolesi, M., Torgovnick, A., Baiocchi, M., Padula, F., Pisaniello, A., Molinaro, M., Cossu, G., Levrero, M., Bouché, M. Bisperoxovanadium, a phospho‐tyrosine phosphatase inhibitor, reprograms myogenic cells to acquire a plu‐ripotent, circulating phenotype. FASEB J. 21, 3573–3583 (2007)

Salinomycin Potentiates the Cytotoxic Effects of TRAIL on Glioblastoma Cell Lines

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to exhibit therapeutic activity in cancer. However, many tumors remain resistant to treatment with TRAIL. Therefore, small molecules that potentiate the cytotoxic effects of TRAIL could be used for combinatorial therapy. Here we found that the ionophore antibiotic salinomycin acts in synergism with TRAIL, enhancing TRAIL-induced apoptosis in glioma cells. Treatment with low doses of salinomycin in combination with TRAIL augmented the activation of caspase-3 and increased TRAIL-R2 cell surface expression. TRAIL-R2 upmodulation was required for mediating the stimulatory effect of salinomycin on TRAIL-mediated apoptosis, since it was abrogated by siRNA-mediated TRAIL-R2 knockdown. Salinomycin in synergism with TRAIL exerts a marked anti-tumor effect in nude mice xenografted with human glioblastoma cells. Our results suggest that the combination of TRAIL and salinomycin may be a useful tool to overcome TRAIL resistance in glioma cells and may represent a potential drug for treatment of these tumors. Importantly, salinomycin+TRAIL were able to induce cell death of well-defined glioblastoma stem-like lines.

Cancer Stem Cell-Based Models of Colorectal Cancer Reveal Molecular Determinants of Therapy Resistance

Colorectal cancer (CRC) therapy mainly relies on the use of conventional chemotherapeutic drugs combined, in a subset of patients, with epidermal growth factor receptor [EGFR]‐targeting agents. Although CRC is considered a prototype of a cancer stem cell (CSC)‐driven tumor, the effects of both conventional and targeted therapies on the CSC compartment are largely unknown. We have optimized a protocol for colorectal CSC isolation that allowed us to obtain CSC‐enriched cultures from primary tumor specimens, with high efficiency. CSC isolation was followed by in vitro and in vivo validation, genetic characterization, and drug sensitivity analysis, thus generating panels of CSC lines with defined patterns of genetic mutations and therapy sensitivity. Colorectal CSC lines were polyclonal and maintained intratumor heterogeneity in terms of somatically acquired mutations and differentiation state. Such CSC‐enriched cultures were used to investigate the effects of both conventional and targeted therapies on the CSC compartment in vivo and to generate a proteomic picture of signaling pathways implicated in sensitivity/resistance to anti‐EGFR agents. We propose CSC lines as a sound preclinical framework to test the effects of therapies in vitro and in vivo and to identify novel determinants of therapy resistance.

Dynamic regulation of the cancer stem cell compartment by Cripto-1 in colorectal cancer

Stemness was recently depicted as a dynamic condition in normal and tumor cells. We found that the embryonic protein Cripto-1 (CR1) was expressed by normal stem cells at the bottom of colonic crypts and by cancer stem cells (CSCs) in colorectal tumor tissues. CR1-positive populations isolated from patient-derived tumor spheroids exhibited increased clonogenic capacity and expression of stem-cell-related genes. CR1 expression in tumor spheroids was variable over time, being subject to a complex regulation of the intracellular, surface and secreted protein, which was related to changes of the clonogenic capacity at the population level. CR1 silencing induced CSC growth arrest in vitro with a concomitant decrease of Src/Akt signaling, while in vivo it inhibited the growth of CSC-derived tumor xenografts and reduced CSC numbers. Importantly, CR1 silencing in established xenografts through an inducible expression system decreased CSC growth in both primary and metastatic tumors, indicating an essential role of CR1 in the regulation the CSC compartment. These results point to CR1 as a novel and dynamically regulated effector of stem cell functions in colorectal cancer.

CHK1-targeted therapy to deplete DNA replication-stressed, p53-deficient, hyperdiploid colorectal cancer stem cells

Objective Cancer stem cells (CSCs) are responsible for tumour formation and spreading, and their targeting is required for tumour eradication. There are limited therapeutic options for advanced colorectal cancer (CRC), particularly for tumours carrying RAS-activating mutations. The aim of this study was to identify novel CSC-targeting strategies. Design To discover potential therapeutics to be clinically investigated as single agent, we performed a screening with a panel of FDA-approved or investigational drugs on primary CRC cells enriched for CSCs (CRC-SCs) isolated from 27 patients. Candidate predictive biomarkers of efficacy were identified by integrating genomic, reverse-phase protein microarray (RPPA) and cytogenetic analyses, and validated by immunostainings. DNA replication stress (RS) was increased by employing DNA replication-perturbing or polyploidising agents. Results The drug-library screening led to the identification of LY2606368 as a potent anti-CSC agent acting in vitro and in vivo in tumour cells from a considerable number of patients (∼36%). By inhibiting checkpoint kinase (CHK)1, LY2606368 affected DNA replication in most CRC-SCs, including RAS-mutated ones, forcing them into premature, lethal mitoses. Parallel genomic, RPPA and cytogenetic analyses indicated that CRC-SCs sensitive to LY2606368 displayed signs of ongoing RS response, including the phosphorylation of RPA32 and ataxia telangiectasia mutated serine/threonine kinase (ATM). This was associated with mutation(s) in TP53 and hyperdiploidy, and made these CRC-SCs exquisitely dependent on CHK1 function. Accordingly, experimental increase of RS sensitised resistant CRC-SCs to LY2606368. Conclusions LY2606368 selectively eliminates replication-stressed, p53-deficient and hyperdiploid CRC-SCs independently of RAS mutational status. These results provide a strong rationale for biomarker-driven clinical trials with LY2606368 in patients with CRC.

A Specific Mutational Signature Associated with DNA 8-Oxoguanine Persistence in MUTYH-defective Colorectal Cancer

8-Oxoguanine, a common mutagenic DNA lesion, generates G:C > T:A transversions via mispairing with adenine during DNA replication. When operating normally, the MUTYH DNA glycosylase prevents 8-oxoguanine-related mutagenesis by excising the incorporated adenine. Biallelic MUTYH mutations impair this enzymatic function and are associated with colorectal cancer (CRC) in MUTYH-Associated Polyposis (MAP) syndrome. Here, we perform whole-exome sequencing that reveals a modest mutator phenotype in MAP CRCs compared to sporadic CRC stem cell lines or bulk tumours. The excess G:C > T:A transversion mutations in MAP CRCs exhibits a novel mutational signature, termed Signature 36, with a strong sequence dependence. The MUTYH mutational signature reflecting persistent 8-oxoG:A mismatches occurs frequently in the APC, KRAS, PIK3CA, FAT4, TP53, FAT1, AMER1, KDM6A, SMAD4 and SMAD2 genes that are associated with CRC. The occurrence of Signature 36 in other types of human cancer indicates that DNA 8-oxoguanine-related mutations might contribute to the development of cancer in other organs.

How to Assess Drug Resistance in Cancer Stem Cells

Banks of genetically characterized cancer stem cells (CSCs) isolated from individual patients and grown as spheroids offer an invaluable approach to identify genetic determinants of drug resistance versus sensitivity, and to study new stem cell-directed therapies. Here, we describe our standardized procedure for in vitro drug screening on colorectal CSCs, taking irinotecan as an example.