Mimicking the Tumor Niche: Methods for Isolation, Culture, and Characterization of Cancer Stem Cells and Multicellular Spheroids.

Cancer stem cells (CSCs) play a significant role in driving several tumor hallmarks. Their behavior and tumor progression are strictly related to the tumor microenvironment (TME). The dynamic interplay between CSCs and TME drives metastasis, chemoresistance, and disease relapse. In this chapter, we describe different techniques and protocols for isolating, culturing, and characterizing CSCs and we explain the methodology for the culture of multicellular spheroids comprising CSCs.

Unlocking the effective alliance of ß-lapachone and hydroxytyrosol against triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is characterised by its aggressiveness and resistance to chemotherapy, demanding the development of effective strategies against its unique characteristics. Derived from lapacho tree bark, ß-lapachone (ß-LP) selectively targets cancer cells with elevated levels of the detoxifying enzyme NQO1. Hydroxytyrosol (HT) is a phenolic compound derived from olive trees with important anticancer properties that include the inhibition of cancer stem cells (CSCs) and metastatic features in TNBC, as well as relevant antioxidant activities by mechanisms such as the induction of NQO1. We aimed to study whether these compounds could have synergistic anticancer activity in TNBC cells and the possible role of NQO1. For this pourpose, we assessed the impact of ß-LP (0.5 or 1.5 µM) and HT (50 and 100 µM) on five TNBC cell lines. We demonstrated that the combination of ß-LP and HT exhibits anti-proliferative, pro-apoptotic, and cell cycle arrest effects in several TNBC cells, including docetaxel-resistant TNBC cells. Additionally, it effectively inhibits the self-renewal and clonogenicity of CSCs, modifying their aggressive phenotype. However, the notable impact of the ß-LP-HT combination does not appear to be solely associated with the levels of the NQO1 protein and ROS. RNA-Seq analysis revealed that the combination's anticancer activity is linked to a strong induction of endoplasmic reticulum stress and apoptosis through the unfolded protein response. In conclusion, in this study, we demonstrated how the combination of ß-LP and HT could offer an affordable, safe, and effective approach against TNBC.

Unraveling the Potential of miRNAs from CSCs as an Emerging Clinical Tool for Breast Cancer Diagnosis and Prognosis.

Breast cancer (BC) is the most diagnosed cancer in women and the second most common cancer globally. Significant advances in BC research have led to improved early detection and effective therapies. One of the key challenges in BC is the presence of BC stem cells (BCSCs). This small subpopulation within the tumor possesses unique characteristics, including tumor-initiating capabilities, contributes to treatment resistance, and plays a role in cancer recurrence and metastasis. In recent years, microRNAs (miRNAs) have emerged as potential regulators of BCSCs, which can modulate gene expression and influence cellular processes like BCSCs' self-renewal, differentiation, and tumor-promoting pathways. Understanding the miRNA signatures of BCSCs holds great promise for improving BC diagnosis and prognosis. By targeting BCSCs and their associated miRNAs, researchers aim to develop more effective and personalized treatment strategies that may offer better outcomes for BC patients, minimizing tumor recurrence and metastasis. In conclusion, the investigation of miRNAs as regulators of BCSCs opens new directions for advancing BC research through the use of bioinformatics and the development of innovative therapeutic approaches. This review summarizes the most recent and innovative studies and clinical trials on the role of BCSCs miRNAs as potential tools for early diagnosis, prognosis, and resistance.

Interferon-Alpha Decreases Cancer Stem Cell Properties and Modulates Exosomes in Malignant Melanoma.

Malignant melanoma (MM) can spread to other organs and is resistant in part due to the presence of cancer stem cell subpopulations (CSCs). While a controversial high dose of interferon-alpha (IFN-α) has been used to treat non-metastatic high-risk melanoma, it comes with undesirable side effects. In this study, we evaluated the effect of low and high doses of IFN-α on CSCs by analyzing ALDH activity, side population and specific surface markers in established and patient-derived primary cell lines. We also assessed the clonogenicity, migration and tumor initiation capacities of IFN-α treated CSCs. Additionally, we investigated genomic modulations related to stemness properties using microRNA sequencing and microarrays. The effect of IFN-α on CSCs-derived exosomes was also analyzed using NanoSight and liquid chromatography (LC-HRMS)-based metabolomic analysis, among others. Our results showed that even low doses of IFN-α reduced CSC formation and stemness properties, and led to a significant decrease in the ability to form tumors in mice xenotransplants. IFN-α also modulated the expression of genes and microRNAs involved in several cancer processes and metabolomics of released exosomes. Our work suggests the utility of low doses of interferon, combined with the analysis of metabolic biomarkers, as a potential clinical approach against the aggressiveness of CSCs in melanoma.

Ratiometric Two-Photon Near-Infrared Probe to Detect DPP IV in Human Plasma, Living Cells, Human Tissues, and Whole Organisms Using Zebrafish.

DPP IV, otherwise known as CD26 lymphocyte T surface antigen, is a transmembrane glycoprotein also found in circulation in the blood. It plays an important role in several processes like glucose metabolism and T-cell stimulation. Moreover, it is overexpressed in renal, colon, prostate, and thyroid human carcinoma tissues. It can also serve as a diagnostic in patients with lysosomal storage diseases. The biological and clinical importance of having readouts for the activity of this enzyme, in physiological and disease conditions, has led us to design a near-infrared (NIR) fluorimetric probe that also has the characteristics of being ratiometric and excitable by two simultaneous NIR photons. The probe consists of assembling an enzyme recognition group (Gly-Pro) (Mentlein, 1999; Klemann et al., 2016) on the two-photon (TP) fluorophore (derivative of dicyanomethylene-4H-pyran, DCM-NH2) disturbing its NIR characteristic internal charge transfer (ICT) emission spectrum. When the dipeptide group is released by the DPP IV-specific enzymatic action, the donor-acceptor DCM-NH2 is restored, forming a system that shows high ratiometric fluorescence output. With this new probe, we have been able to detect, quickly and efficiently, the enzymatic activity of DPP IV in living cells, human tissues, and whole organisms, using zebrafish. In addition, due to the possibility of being excited by two photons, we can avoid the autofluorescence and subsequent photobleaching that the raw plasma has when it is excited by visible light, achieving detection of the activity of DPP IV in that medium without interference.

TGFß Governs the Pleiotropic Activity of NDRG1 in Triple-Negative Breast Cancer Progression.

In triple-negative breast cancer (TNBC), the pleiotropic NDRG1 (N-Myc downstream regulated gene 1) promotes progression and worse survival, yet contradictory results were documented, and the mechanisms remain unknown. Phosphorylation and localization could drive NDRG1 pleiotropy, nonetheless, their role in TNBC progression and clinical outcome was not investigated. We found enhanced p-NDRG1 (Thr346) by TGFß1 and explored whether it drives NDRG1 pleiotropy and TNBC progression. In tissue microarrays of 81 TNBC patients, we identified that staining and localization of NDRG1 and p-NDRG1 (Thr346) are biomarkers and risk factors associated with shorter overall survival. We found that TGFß1 leads NDRG1, downstream of GSK3ß, and upstream of NF-κB, to differentially regulate migration, invasion, epithelial-mesenchymal transition, tumor initiation, and maintenance of different populations of cancer stem cells (CSCs), depending on the progression stage of tumor cells, and the combination of TGFß and GSK3ß inhibitors impaired CSCs. The present study revealed the striking importance to assess both total NDRG1 and p-NDRG1 (Thr346) positiveness and subcellular localization to evaluate patient prognosis and their stratification. NDRG1 pleiotropy is driven by TGFß to differentially promote metastasis and/or maintenance of CSCs at different stages of tumor progression, which could be abrogated by the inhibition of TGFß and GSK3ß.

EZH2 endorses cell plasticity to non-small cell lung cancer cells facilitating mesenchymal to epithelial transition and tumour colonization.

Reversible transition between the epithelial and mesenchymal states are key aspects of carcinoma cell dissemination and the metastatic disease, and thus, characterizing the molecular basis of the epithelial to mesenchymal transition (EMT) is crucial to find druggable targets and more effective therapeutic approaches in cancer. Emerging studies suggest that epigenetic regulators might endorse cancer cells with the cell plasticity required to conduct dynamic changes in cell state during EMT. However, epigenetic mechanisms involved remain mostly unknown. Polycomb Repressive Complexes (PRCs) proteins are well-established epigenetic regulators of development and stem cell differentiation, but their role in different cancer systems is inconsistent and sometimes paradoxical. In this study, we have analysed the role of the PRC2 protein EZH2 in lung carcinoma cells. We found that besides its described role in CDKN2A-dependent cell proliferation, EZH2 upholds the epithelial state of cancer cells by repressing the transcription of hundreds of mesenchymal genes. Chemical inhibition or genetic removal of EZH2 promotes the residence of cancer cells in the mesenchymal state during reversible epithelial-mesenchymal transition. In fitting, analysis of human patient samples and tumour xenograft models indicate that EZH2 is required to efficiently repress mesenchymal genes and facilitate tumour colonization in vivo. Overall, this study discloses a novel role of PRC2 as a master regulator of EMT in carcinoma cells. This finding has important implications for the design of therapies based on EZH2 inhibitors in human cancer patients.

Nanomedicine as a Promising Tool to Overcome Immune Escape in Breast Cancer.

Breast cancer is the most common type of malignancy and leading cause of cancer death among women worldwide. Despite the current revolutionary advances in the field of cancer immunotherapy, clinical response in breast cancer is frequently below expectations, in part due to various mechanisms of cancer immune escape that produce tumor variants that are resistant to treatment. Thus, a further understanding of the molecular events underlying immune evasion in breast cancer may guarantee a significant improvement in the clinical success of immunotherapy. Furthermore, nanomedicine provides a promising opportunity to enhance the efficacy of cancer immunotherapy by improving the delivery, retention and release of immunostimulatory agents in targeted cells and tumor tissues. Hence, it can be used to overcome tumor immune escape and increase tumor rejection in numerous malignancies, including breast cancer. In this review, we summarize the current status and emerging trends in nanomedicine-based strategies targeting cancer immune evasion and modulating the immunosuppressive tumor microenvironment, including the inhibition of immunosuppressive cells in the tumor area, the activation of dendritic cells and the stimulation of the specific antitumor T-cell response.

Anti-CD44-Conjugated Olive Oil Liquid Nanocapsules for Targeting Pancreatic Cancer Stem Cells.

The latest trends in cancer research and nanomedicine focus on using nanocarriers to target cancer stem cells (CSCs). Specifically, lipid liquid nanocapsules are usually developed as nanocarriers for lipophilic drug delivery. Here, we developed olive oil liquid NCs (O2LNCs) functionalized by covalent coupling of an anti-CD44-fluorescein isothiocyanate antibody (αCD44). First, O2LNCs are formed by a core of olive oil surrounded by a shell containing phospholipids, a nonionic surfactant, and deoxycholic acid molecules. Then, O2LNCs were coated with an αCD44 antibody (αCD44-O2LNC). The optimization of an αCD44 coating procedure, a complete physicochemical characterization, as well as clear evidence of their efficacy in vitro and in vivo were demonstrated. Our results indicate the high targeted uptake of these αCD44-O2LNCs, and the increased antitumor efficacy (up to four times) of paclitaxel-loaded-αCD44-O2LNC compared to free paclitaxel in pancreatic CSCs (PCSCs). Also, αCD44-O2LNCs were able to selectively target PCSCs in an orthotopic xenotransplant in vivo model.

Development of a Biomimetic Hydrogel Based on Predifferentiated Mesenchymal Stem-Cell-Derived ECM for Cartilage Tissue Engineering.

The use of decellularized extracellular matrix (dECM) as a biomaterial has been an important step forward for the development of functional tissue constructs. In addition to tissues and organs, cell cultures are gaining a lot of attention as an alternative source of dECM. In this work, a novel biomimetic hydrogel is developed based on dECM obtained from mesenchymal stem cells (mdECM) for cartilage tissue engineering. To this end, cells are seeded under specific culture conditions to generate an early chondrogenic extracellular matrix (ECM) providing cues and elements necessary for cartilage development. The composition is determined by quantitative, histological, and mass spectrometry techniques. Moreover, the decellularization process is evaluated by measuring the DNA content and compositional analyses, and the hydrogel is formulated at different concentrations (3% and 6% w/v). Results show that mdECM derived hydrogels possess excellent biocompatibility and suitable physicochemical and mechanical properties for their injectability. Furthermore, it is evidenced that this hydrogel is able to induce chondrogenesis of mesenchymal stem cells (MSCs) without supplemental factors and, furthermore, to form hyaline cartilage-like tissue after in vivo implantation. These findings demonstrate for the first time the potential of this hydrogel based on mdECM for applications in cartilage repair and regeneration.

Antioxidants for the Treatment of Breast Cancer: Are We There Yet?

Breast cancer is the most frequent cancer and the leading cause of cancer death in women. Oxidative stress and the generation of reactive oxygen species (ROS) have been related to cancer progression. Compared to their normal counterparts, tumor cells show higher ROS levels and tight regulation of REDOX homeostasis to maintain a low degree of oxidative stress. Traditionally antioxidants have been extensively investigated to counteract breast carcinogenesis and tumor progression as chemopreventive agents; however, there is growing evidence indicating their potential as adjuvants for the treatment of breast cancer. Aimed to elucidate whether antioxidants could be a reality in the management of breast cancer patients, this review focuses on the latest investigations regarding the ambivalent role of antioxidants in the development of breast cancer, with special attention to the results derived from clinical trials, as well as their potential use as plausible agents in combination therapy and their power to ameliorate the side effects attributed to standard therapeutics. Data retrieved herein suggest that antioxidants play an important role in breast cancer prevention and the improvement of therapeutic efficacy; nevertheless, appropriate patient stratification based on "redoxidomics" or tumor subtype is mandatory in order to define the dosage for future standardized and personalized treatments of patients.

The Inhibitory Role of miR-486-5p on CSC Phenotype Has Diagnostic and Prognostic Potential in Colorectal Cancer.

Colorectal cancer (CRC) is the third most frequent cancer worldwide and the second cause of cancer deaths. Increasing evidences supports the idea that the poor prognosis of patients is related to the presence of cancer stem cells (CSCs), a cell population able to drive cancer recurrence and metastasis. The deregulation of microRNAs (miRNAs) plays a role in the formation of CSC. We investigated the role of hsa-miR-486-5p (miR-486-5p) in CRC, CSCs, and metastasis, in order to reach a better understanding of the biomolecular and epigenetic mechanisms mir-486-5p-related. The expression of miR-486-5p was investigated in three different matrices from CRC patients and controls and in CSCs obtained from the CRC cell lines HCT-116, HT-29, and T-84. In the human study, miR-486-5p was up-regulated in serum and stool of CRC patients in comparison with healthy controls but down-regulated in tumor tissue when compared with normal mucosa. miR-486-5p was also down-regulated in the sera of metastatic patients. In vitro, miR-486-5p was down-regulated in CSC models and it induced an inhibitory effect on stem factors and oncogenes in the main pathways of CSCs. Our results provide a step forward in understanding the role of mir-486-5p in CRC and CSC, and suggest that further studies are needed to investigate its diagnostic and prognostic power, possibly in combination with other biomarkers.

Cancer stem cell secretome in the tumor microenvironment: a key point for an effective personalized cancer treatment.

Cancer stem cells (CSCs) represent a tumor subpopulation responsible for tumor metastasis and resistance to chemo- and radiotherapy, ultimately leading to tumor relapse. As a consequence, the detection and eradication of this cell subpopulation represent a current challenge in oncology medicine. CSC phenotype is dependent on the tumor microenvironment (TME), which involves stem and differentiated tumor cells, as well as different cell types, such as mesenchymal stem cells, endothelial cells, fibroblasts and cells of the immune system, in addition to the extracellular matrix (ECM), different in composition to the ECM in healthy tissues. CSCs regulate multiple cancer hallmarks through the interaction with cells and ECM in their environment by secreting extracellular vesicles including exosomes, and soluble factors such as interleukins, cytokines, growth factors and other metabolites to the TME. Through these factors, CSCs generate and activate their own tumor niche by recruiting stromal cells and modulate angiogenesis, metastasis, resistance to antitumor treatments and their own maintenance by the secretion of different factors such as IL-6, VEGF and TGF-ß. Due to the strong influence of the CSC secretome on disease development, the new antitumor therapies focus on targeting these communication networks to eradicate the tumor and prevent metastasis, tumor relapse and drug resistance. This review summarizes for the first time the main components of the CSC secretome and how they mediate different tumor processes. Lastly, the relevance of the CSC secretome in the development of more precise and personalized antitumor therapies is discussed.

CSC Radioresistance: A Therapeutic Challenge to Improve Radiotherapy Effectiveness in Cancer.

Radiotherapy (RT) is a modality of oncologic treatment that can be used to treat approximately 50% of all cancer patients either alone or in combination with other treatment modalities such as surgery, chemotherapy, immunotherapy, and therapeutic targeting. Despite the technological advances in RT, which allow a more precise delivery of radiation while progressively minimizing the impact on normal tissues, issues like radioresistance and tumor recurrence remain important challenges. Tumor heterogeneity is responsible for the variation in the radiation response of the different tumor subpopulations. A main factor related to radioresistance is the presence of cancer stem cells (CSC) inside tumors, which are responsible for metastases, relapses, RT failure, and a poor prognosis in cancer patients. The plasticity of CSCs, a process highly dependent on the epithelial-mesenchymal transition (EMT) and associated to cell dedifferentiation, complicates the identification and eradication of CSCs and it might be involved in disease relapse and progression after irradiation. The tumor microenvironment and the interactions of CSCs with their niches also play an important role in the response to RT. This review provides a deep insight into the characteristics and radioresistance mechanisms of CSCs and into the role of CSCs and tumor microenvironment in both the primary tumor and metastasis in response to radiation, and the radiobiological principles related to the CSC response to RT. Finally, we summarize the major advances and clinical trials on the development of CSC-based therapies combined with RT to overcome radioresistance. A better understanding of the potential therapeutic targets for CSC radiosensitization will provide safer and more efficient combination strategies, which in turn will improve the live expectancy and curability of cancer patients.

Deregulation of cancer-stem-cell-associated miRNAs in tissues and sera of colorectal cancer patients.

Colorectal cancer (CRC) is a deadly tumour in Western countries characterized by high cellular/molecular heterogeneity. Cancer stem cells (CSC) act in cancer recurrence, drug-resistance and in metastatic epithelial-to-mesenchymal transition. microRNAs (miRNAs) contribute to cancer is increasing, and miRNA roles in CSC phenotype and fate and their utility as CRC biomarkers have also been reported. Here, we investigated miR-21, miR-221, miR-18a, miR-210, miR-31, miR-34a, miR-10b and miR-16 expression in experimental ALDH+ and CD44+/CD326+ colorectal CSCs obtained from the human CRC cell lines HCT-116, HT-29 and T-84. Then, we moved our analysis in cancer tissue (CT), healthy tissue (HT) and serum (S) of adult CRC patients (n=12), determining relationships with clinical parameters (age, sex, metastasis, biochemical serum markers). Specific miRNA patterns were evident in vitro (normal, monolayers and CSCs) and in patients' samples stratified by TNM stage (LOW vs HIGH) or metastasis (Met vs no-Met). miR-21, miR-210, miR-34a upregulation ad miR-16 dowregulation associated with the CSCs phenotype. miR-31b robustly overexpressed in monolayers and CSCs, and in CT ad S of HIGH grade and Met patients, suggesting a role as marker of CRC progression and metastasis. miR-18a upregulated in all cancer models and associated to CSC phenotype, and to metastasis and age in patients. miR-10b downregulated in CT and S of LOW/HIGH grade and no-Met patients. Our results identify miRNAs useful as colorectal CSC biomarker and that miR-21, miR-210, miR-10b and miR-31b are promising markers of CRC. A specific role of miR-18a as metastatic CRC serum biomarker in adult patients was also highlighted.

miRNAs as radio-response biomarkers for breast cancer stem cells.

In breast cancer (BC), the presence of cancer stem cells (CSCs) has been related to relapse, metastasis, and radioresistance. Radiotherapy (RT) is an extended BC treatment, but is not always effective. CSCs have several mechanisms of radioresistance in place, and some miRNAs are involved in the cellular response to ionizing radiation (IR). Here, we studied how IR affects the expression of miRNAs related to stemness in different molecular BC subtypes. Exposition of BC cells to radiation doses of 2, 4, or 6 Gy affected their phenotype, functional characteristics, pluripotency gene expression, and in vivo tumorigenic capacity. This held true for various molecular subtypes of BC cells (classified by ER, PR and HER-2 status), and for BC cells either plated in monolayer, or being in suspension as mammospheres. However, the effect of IR on the expression of eight stemness- and radioresistance-related miRNAs (miR-210, miR-10b, miR-182, miR-142, miR-221, miR-21, miR-93, miR-15b) varied, depending on cell line subpopulation and clinicopathological features of BC patients. Therefore, clinicopathological features and, potentially also, chemotherapy regimen should be both taken into consideration, for determining a potential miRNA signature by liquid biopsy in BC patients treated with RT. Personalized and precision RT dosage regimes could improve the prognosis, treatment, and survival of BC patients.

Matrix metalloproteases and TIMPs as prognostic biomarkers in breast cancer patients treated with radiotherapy: A pilot study.

Breast cancer (BC) is the most common tumour in women and one of the most important causes of cancer death worldwide. Radiation therapy (RT) is widely used for BC treatment. Some proteins have been identified as prognostic factors for BC (Ki67, p53, E-cadherin, HER2). In the last years, it has been shown that variations in the expression of MMPs and TIMPs may contribute to the development of BC. The aim of this pilot work was to study the effects of RT on different MMPs (-1, -2, -3, -7, -8, -9, -10, -12 and -13) and TIMPs (-1 to -4), as well as their relationship with other variables related to patient characteristics and tumour biology. A group of 20 BC patients treated with RT were recruited. MMP and TIMP serum levels were analysed by immunoassay before, during and after RT. Our pilot study showed a slight increase in the levels of most MMP and TIMP with RT. However, RT produced a significantly decrease in TIMP-1 and TIMP-3 levels. Significant correlations were found between MMP-3 and TIMP-4 levels, and some of the variables studied related to patient characteristics and tumour biology. Moreover, MMP-9 and TIMP-3 levels could be predictive of RT toxicity. For this reason, MMP-3, MMP-9, TIMP-3 and TIMP-4 could be used as potential prognostic and predictive biomarkers for BC patients treated with RT.

GENYOi005-A: An induced pluripotent stem cells (iPSCs) line generated from a patient with Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) carrying a p.Thr196Ala variant.

Familial Platelet Disorder with associated Myeloid Malignancy (FPDMM) is a rare platelet disorder caused by mutations in RUNX1. We generated an iPSC line (GENYOi005-A) from a FPDMM patient with a non-previously reported variant p.Thr196Ala. Non-integrative Sendai viruses expressing the Yamanaka reprogramming factors were used to reprogram peripheral blood mononuclear cells from this FPDMM patient. Characterization of GENYOi005-A included genetic analysis of RUNX1 locus, Short Tandem Repeats profiling, alkaline phosphatase enzymatic activity, expression of pluripotency-associated factors and differentiation studies in vitro and in vivo. This iPSC line will provide a powerful tool to study developmental alterations of FPDMM patients.

Radiation and Stemness Phenotype May Influence Individual Breast Cancer Outcomes: The Crucial Role of MMPs and Microenvironment.

Breast cancer is the most common cancer in women. Radiotherapy (RT) is one of the mainstay treatments for cancer but in some cases is not effective. Cancer stem cells (CSCs) within the tumor can be responsible for recurrence and metastasis after RT. Matrix metalloproteases (MMPs), regulated mainly by tissue inhibitors of metalloproteinases (TIMPs) and histone deacetylases (HDACs), may also contribute to tumor development by modifying its activity after RT. The aim of this work was to study the effects of RT on the expression of MMPs, TIMPs and HDACs on different cell subpopulations in MCF-7, MDA-MB-231 and SK-BR-3 cell lines. We assessed the in vitro expression of these genes in different 3D culture models and induced tumors in female NSG mice by orthotopic xenotransplants. Our results showed that gene expression is related to the cell subpopulation studied, the culture model used and the single radiation dose administered. Moreover, the crucial role played by the microenvironment in terms of cell interactions and CSC plasticity in tumor growth and RT outcome is also shown, supporting the use of higher doses (6 Gy) to achieve better control of tumor development.

Pancreatic (pro)enzymes treatment suppresses BXPC-3 pancreatic Cancer Stem Cell subpopulation and impairs tumour engrafting.

Cancer stem cells (CSCs) subpopulation within the tumour is responsible for metastasis and cancer relapse. Here we investigate in vitro and in vivo the effects of a pancreatic (pro)enzyme mixture composed of Chymotrypsinogen and Trypsinogen (PRP) on CSCs derived from a human pancreatic cell line, BxPC3. Exposure of pancreatic CSCs spheres to PRP resulted in a significant decrease of ALDEFLUOR and specific pancreatic CSC markers (CD 326, CD 44 and CxCR4) signal tested by flow cytometry, further CSCs markers expression was also analyzed by western and immunofluorescence assays. PRP also inhibits primary and secondary sphere formation. Three RT2 Profiler PCR Arrays were used to study gene expression regulation after PRP treatment and resulted in, (i) epithelial-mesenchymal transition (EMT) inhibition; (ii) CSCs related genes suppression; (iii) enhanced expression of tumour suppressor genes; (iv) downregulation of migration and metastasis genes and (v) regulation of MAP Kinase Signalling Pathway. Finally, in vivo anti-tumor xenograft studies demonstrated high anti-tumour efficacy of PRP against tumours induced by BxPC3 human pancreatic CSCs. PRP impaired engrafting of pancreatic CSC's tumours in nude mice and displayed an antigrowth effect toward initiated xenografts. We concluded that (pro)enzymes treatment is a valuable strategy to suppress the CSC population in solid pancreatic tumours.