Rheumatoid Synovial Fluid and Acidic Extracellular pH Modulate the Immunomodulatory Activity of Urine-Derived Stem Cells.

Urine-derived stem cells (UdSCs) possess a remarkable anti-inflammatory and immune-modulating activity. However, the clinical significance of UdSCs in autoimmune inflammatory diseases such as rheumatoid arthritis (RA) is yet to be explored. Hence, we tested the UdSCs response to an articular RA microenvironment. To simulate the inflamed RA joint more authentically in vitro, we treated cells with rheumatoid synovial fluids (RASFs) collected from RA patients, serum deprivation, acidosis (pH 7.0 and 6.5), and their combinations. Firstly, the RASFs pro-inflammatory status was assessed by cytokine quantification. Then, UdSCs were exposed to the RA environmental factors for 48 h and cell proliferation, gene expression and secretion of immunomodulatory factors were evaluated. The immunosuppressive potential of pre-conditioned UdSCs was also assessed via co-cultivation with activated peripheral blood mononuclear cells (PBMCs). In all experimental conditions, UdSCs' proliferation was not affected. Conversely, extracellular acidosis considerably impaired the viability/proliferation of adipose tissue-derived stem cells (ATSCs). In the majority of cases, exposure to RA components led to the upregulated expression of IL-6, TSG6, ICAM-1, VCAM-1, and PD-L1, all involved in immunomodulation. Upon RASFs and acidic stimulation, UdSCs secreted higher levels of immunomodulatory cytokines: IL-6, IL-8, MCP-1, RANTES, GM-CSF, and IL-4. Furthermore, RASFs and combined pretreatment with RASFs and acidosis promoted the UdSCs-mediated immunosuppression and the proliferation of activated PBMCs was significantly inhibited. Altogether, our data indicate that the RA microenvironment certainly has the capacity to enhance UdSCs' immunomodulatory function. For potential preclinical/clinical applications, the intra-articular injection might be a reasonable approach to maximize UdSCs' therapeutic efficiency in the RA treatment.

Novel model of triple-negative breast cancer produces viable circulating tumor cells and rapid lung metastasis for functional testing in vivo.

Breast cancer metastases are the main reason for women´s highest cancer mortality. Even though tumor cell dissemination via circulating tumor cells (CTC) released from the primary site is a very ineffective process, distant metastases appear in 46% of triple-negative breast cancer (TNBC) patients corresponding to the disease aggressiveness. Laboratory models for functional testing which mimic the spread of metastatic cells are needed for efficient investigation of the underlying mechanisms and therapeutic intervention. Here, we describe novel isogenic variants LMC3 and CTC3 of human TNBC cell line MDA-MB-231 that were derived by repeated injection of tumor cells into the tail vein of immunodeficient mice and subsequent selection of metastatic cells from lung metastases. These variants have increased migration potential, altered expression profiles, and elevated tumorigenic potential. Moreover, cell line CTC3 readily produces metastases in the lungs and bone marrow and detectable viable circulating tumor cells in the blood. This model enables rapid and cost-efficient strategies for biomarker exploration and novel intervention approaches to limit the CTC presence in the blood and hence tumor dissemination.

The third dimension of tumor microenvironment-The importance of tumor stroma in 3D cancer models.

Recent advances in the three-dimensional (3D) cancer models give rise to a plethora of new possibilities in the development of anti-cancer drug therapies and bring us closer to personalized medicine. Three-dimensional models are undoubtedly more authentic than traditional two-dimensional (2D) cell cultures. Nowadays, they are becoming preferentially used in most cancer research fields due to their more accurate biomimetic characteristics. On the contrary, they still lack the cellular and matrix complexity of the native tumor microenvironment (TME). This review focuses on the description of existing 3D models, the incorporation of TME and fluidics into these models, and their perspective in the future research. It is clear that such an improvement would need not only biological but also technical progress. Therefore, the modern approach to anti-cancer drug discovery should involve various fields.

Chemotherapy-triggered changes in stromal compartment drive tumor invasiveness and progression of breast cancer.

BACKGROUND: Chemotherapy remains a standard treatment option for breast cancer despite its toxic effects to normal tissues. However, the long-lasting effects of chemotherapy on non-malignant cells may influence tumor cell behavior and response to treatment. Here, we have analyzed the effects of doxorubicin (DOX) and paclitaxel (PAC), commonly used chemotherapeutic agents, on the survival and cellular functions of mesenchymal stromal cells (MSC), which comprise an important part of breast tumor microenvironment. METHODS: Chemotherapy-exposed MSC (DOX-MSC, PAC-MSC) were co-cultured with three breast cancer cell (BCC) lines differing in molecular characteristics to study chemotherapy-triggered changes in stromal compartment of the breast tissue and its relevance to tumor progression in vitro and in vivo. Conditioned media from co-cultured cells were used to determine the cytokine content. Mixture of BCC and exposed or unexposed MSC were subcutaneously injected into the immunodeficient SCID/Beige mice to analyze invasion into the surrounding tissue and possible metastases. The same mixtures of cells were applied on the chorioallantoic membrane to study angiogenic potential. RESULTS: Therapy-educated MSC differed in cytokine production compared to un-exposed MSC and influenced proliferation and secretory phenotype of tumor cells in co-culture. Histochemical tumor xenograft analysis revealed increased invasive potential of tumor cells co-injected with DOX-MSC or PAC-MSC and also the presence of nerve fiber infiltration in tumors. Chemotherapy-exposed MSC have also influenced angiogenic potential in the model of chorioallantoic membrane. CONCLUSIONS: Data presented in this study suggest that neoadjuvant chemotherapy could possibly alter otherwise healthy stroma in breast tissue into a hostile tumor-promoting and metastasis favoring niche. Understanding of the tumor microenvironment and its complex net of signals brings us closer to the ability to recognize the mechanisms that prevent failure of standard therapy and accomplish the curative purpose.

Mapping and Analysis of Swi5 and Sfr1 Phosphorylation Sites.

The evolutionarily conserved Swi5-Sfr1 complex plays an important role in homologous recombination, a process crucial for the maintenance of genomic integrity. Here, we purified Schizosaccharomyces pombe Swi5-Sfr1 complex from meiotic cells and analyzed it by mass spectrometry. Our analysis revealed new phosphorylation sites on Swi5 and Sfr1. We found that mutations that prevent phosphorylation of Swi5 and Sfr1 do not impair their function but swi5 and sfr1 mutants encoding phosphomimetic aspartate at the identified phosphorylation sites are only partially functional. We concluded that during meiosis, Swi5 associates with Sfr1 and both Swi5 and Sfr1 proteins are phosphorylated. However, the functional relevance of Swi5 and Sfr1 phosphorylation remains to be determined.

Targeting of Deregulated Wnt/ß-Catenin Signaling by PRI-724 and LGK974 Inhibitors in Germ Cell Tumor Cell Lines.

The majority of patients with testicular germ cell tumors (GCTs) can be cured with cisplatin-based chemotherapy. However, for a subset of patients present with cisplatin-refractory disease, which confers a poor prognosis, the treatment options are limited. Novel therapies are therefore urgently needed to improve outcomes in this challenging patient population. It has previously been shown that Wnt/ß-catenin signaling is active in GCTs suggesting that its inhibitors LGK974 and PRI-724 may show promise in the management of cisplatin-refractory GCTs. We herein investigated whether LGK-974 and PRI-724 provide a treatment effect in cisplatin-resistant GCT cell lines. Taking a genoproteomic approach and utilizing xenograft models we found the increased level of ß-catenin in 2 of 4 cisplatin-resistant (CisR) cell lines (TCam-2 CisR and NCCIT CisR) and the decreased level of ß-catenin and cyclin D1 in cisplatin-resistant NTERA-2 CisR cell line. While the effect of treatment with LGK974 was limited or none, the NTERA-2 CisR exhibited the increased sensitivity to PRI-724 in comparison with parental cell line. Furthermore, the pro-apoptotic effect of PRI-724 was documented in all cell lines. Our data strongly suggests that a Wnt/ß-catenin signaling is altered in cisplatin-resistant GCT cell lines and the inhibition with PRI-724 is effective in NTERA-2 CisR cells. Further evaluation of Wnt/ß-catenin pathway inhibition in GCTs is therefore warranted.

The Role of BRCA1/2-Mutated Tumor Microenvironment in Breast Cancer.

Taking into account the factors of high incidence rate, prevalence and mortality, breast cancer represents a crucial social and economic burden. Most cases of breast cancer develop as a consequence of somatic mutations accumulating in mammary epithelial cells throughout lifetime and approximately 5-10% can be ascribed to monogenic predispositions. Even though the role of genetic predispositions in breast cancer is well described in the context of genetics, very little is known about the role of the microenvironment carrying the same aberrant cells impaired by the germline mutation in the breast cancer development and progression. Based on the clinical observations, carcinomas carrying mutations in hereditary tumor-suppressor genes involved in maintaining genome integrity such as BRCA1/2 have worse prognosis and aggressive behavior. One of the mechanisms clarifying the aggressive nature of BRCA-associated tumors implies alterations within the surrounding adipose tissue itself. The objective of this review is to look at the role of BRCA1/2 mutations in the context of breast tumor microenvironment and plausible mechanisms by which it contributes to the aggressive behavior of the tumor cells.

Decreased levels of circulating cytokines VEGF, TNF-ß and IL-15 indicate PD-L1 overexpression in tumours of primary breast cancer patients.

Programmed death ligand 1 (PD-L1) overexpression has been associated with poor clinical outcomes in several human cancers whose increased malignant behaviour might be related to PD-L1 mediated systemic immunological tolerance. This study aims to verify if circulating cytokines may serve as a proxy for non-invasive identification of sensitive prognostic biomarkers reflecting tumour and its microenvironment. Immunohistochemistry was used to measure PD-L1 expression in tumour tissue sections of 148 chemonaïve breast cancer (BC) patients. The panel of 51 cytokines was analysed using multiplex bead arrays. High PD-L1 expression in tumours was associated with shorter progression-free survival (HR 3.25; 95% CI 1.39-7.61; P = 0.006) and low circulating levels of three multifunctional molecules; VEGF, TNF-ß and IL-15 (P = 0.001). In multivariate analysis, patients with low VEGF had 4.6-fold increased risk of PD-L1 overexpression (P = 0.008), present in 76.5% of patients with all these three cytokines below the median (vs. 35.6% among the others; P = 0.002). The area under the curve value of 0.722 (95% CI 0.59-0.85; P = 0.004) shows that this combination of cytokines has a moderate ability to discriminate between PD-L1 high vs. PD-L1 low patients. Plasma cytokines, therefore, could serve as potential non-invasive biomarkers for the identification of high-risk BC cases.

Increased Stromal Infiltrating Lymphocytes Are Associated with the Risk of Disease Progression in Mesenchymal Circulating Tumor Cell-Positive Primary Breast Cancer Patients.

Circulating tumor cells (CTCs) and the immune infiltration of tumors are closely related to clinical outcomes. This study aimed to verify the influence of stromal lymphocyte infiltration and the immune context of tumor microenvironment on the hematogenous spread and prognosis of 282 chemotherapy naïve primary BC patients. To detect the presence of mesenchymal CTCs, RNA extracted from CD45-depleted peripheral blood was interrogated for the expression of mesenchymal gene transcripts. Tumor-infiltrating lymphocytes (TILs) were detected in the stromal areas by immunohistochemistry, using CD3, CD8, and CD45RO antibodies. The concentrations of 51 plasma cytokines were measured by multiplex bead arrays. TILs infiltration in mesenchymal CTC-positive patients significantly decreased their progression-free survival (HR = 4.88, 95% CI 2.30-10.37, p < 0.001 for CD3high; HR = 6.17, 95% CI 2.75-13.80, p < 0.001 for CD8high; HR = 6.93, 95% CI 2.86-16.81, p < 0.001 for CD45ROhigh). Moreover, the combination of elevated plasma concentrations of transforming growth factor beta-3 (cut-off 662 pg/mL), decreased monocyte chemotactic protein-3 (cut-off 52.5 pg/mL) and interleukin-15 (cut-off 17.1 pg/mL) significantly increased the risk of disease recurrence (HR = 4.838, 95% CI 2.048-11.427, p < 0.001). Our results suggest a strong impact of the immune tumor microenvironment on BC progression, especially through influencing the dissemination and survival of more aggressive, mesenchymal CTC subtypes.

Inflammation-Based Scores Increase the Prognostic Value of Circulating Tumor Cells in Primary Breast Cancer.

A correlation between circulating tumor cells (CTCs) and monocytes in metastatic breast cancer (BC), where CTCs and monocyte-to-lymphocyte ratio (MLR) were predictors of overall survival (OS), was recently shown. Herein, we aimed to assess the association between CTCs and the complete blood count (CBC)-derived inflammation-based scores in 284 primary BC patients. CTCs were determined in CD45-depleted peripheral blood mononuclear cells by real time-PCR. This method allowed us to detect a subset of CTCs with an epithelial-to-mesenchymal transition phenotype (CTC EMT), previously associated with inferior outcomes in primary BC. In the present study, CTC EMT positivity (hazard ratio (HR) = 2.4; 95% CI 1.20-4.66, p = 0.013) and elevated neutrophil-to-lymphocyte ratio (NLR) (HR = 2.20; 95% CI 1.07-4.55; p = 0.033) were associated with shorter progression-free survival (PFS) in primary BC patients. Multivariate analysis showed that CTC EMT-positive patients with NLR ≥ 3 had 8.6 times increased risk of disease recurrence (95% CI 2.35-31.48, p = 0.001) compared with CTC EMT-negative patients with NLR < 3. Similarly, disease recurrence was 13.14 times more likely in CTC EMT-positive patients with MLR ≥ 0.34 (95% CI 4.35-39.67, p < 0.001). Given its low methodological and financial demands, the CBC-derived inflammation-based score determination could, after broader validation, significantly improve the prognostication of BC patients.

Permanent Pro-Tumorigenic Shift in Adipose Tissue-Derived Mesenchymal Stromal Cells Induced by Breast Malignancy.

During cancer progression, breast tumor cells interact with adjacent adipose tissue, which has been shown to be engaged in cancer aggressiveness. However, the tumor-directed changes in adipose tissue-resident stromal cells affected by the tumor-stroma communication are still poorly understood. The acquired changes might remain in the tissue even after tumor removal and may contribute to tumor relapse. We investigated functional properties (migratory capacity, expression and secretion profile) of mesenchymal stromal cells isolated from healthy (n = 9) and tumor-distant breast adipose tissue (n = 32). Cancer patient-derived mesenchymal stromal cells (MSCs) (MSC-CA) exhibited a significantly disarranged secretion profile and proliferation potential. Co-culture with MDA-MB-231, T47D and JIMT-1, representing different subtypes of breast cancer, was used to analyze the effect of MSCs on proliferation, invasion and tumorigenicity. The MSC-CA enhanced tumorigenicity and altered xenograft composition in immunodeficient mice. Histological analysis revealed collective cell invasion with a specific invasive front of EMT-positive tumor cells as well as invasion of cancer cells to the nerve-surrounding space. This study identifies that adipose tissue-derived mesenchymal stromal cells are primed and permanently altered by tumor presence in breast tissue and have the potential to increase tumor cell invasive ability through the activation of epithelial-to-mesenchymal transition in tumor cells.

Recent advances in understanding tumor stroma-mediated chemoresistance in breast cancer.

Although solid tumors comprise malignant cells, they also contain many different non-malignant cell types in their micro-environment. The cellular components of the tumor stroma consist of immune and endothelial cells combined with a heterogeneous population of stromal cells which include cancer-associated fibroblasts. The bi-directional interactions between tumor and stromal cells therefore substantially affect tumor cell biology.Herein, we discuss current available information on these interactions in breast cancer chemo-resistance. It is acknowledged that stromal cells extrinsically alter tumor cell drug responses with profound consequences for therapy efficiency, and it is therefore essential to understand the molecular mechanisms which contribute to these substantial alterations because they provide potential targets for improved cancer therapy. Although breast cancer patient survival has improved over the last decades, chemo-resistance still remains a significant obstacle to successful treatment.Appreciating the important experimental evidence of mesenchymal stromal cells and cancer-associated fibroblast involvement in breast cancer clinical practice can therefore have important therapeutic implications.

ALDH1A3 upregulation and spontaneous metastasis formation is associated with acquired chemoresistance in colorectal cancer cells.

BACKGROUND: Efficiency of colorectal carcinoma treatment by chemotherapy is diminished as the resistance develops over time in patients. The same holds true for 5-fluorouracil, the drug used in first line chemotherapy of colorectal carcinoma. METHODS: Chemoresistant derivative of HT-29 cells was prepared by long-term culturing in increasing concentration of 5-fluorouracil. Cells were characterized by viability assays, flow cytometry, gene expression arrays and kinetic imaging. Immunomagnetic separation was used for isolation of subpopulations positive for cancer stem cells-related surface markers. Aldehyde dehydrogenase expression was attenuated by siRNA. In vivo studies were performed on SCID/bg mice. RESULTS: The prepared chemoresistant cell line labeled as HT-29/EGFP/FUR is assigned with different morphology, decreased proliferation rate and 135-fold increased IC50 value for 5-fluorouracil in comparison to parental counterparts HT-29/EGFP. The capability of chemoresistant cells to form tumor xenografts, when injected subcutaneously into SCID/bg mice, was strongly compromised, however, they formed distant metastases in mouse lungs spontaneously. Derived cells preserved their resistance in vitro and in vivo even without the 5-fluorouracil selection pressure. More importantly, they were resistant to cisplatin, oxaliplatin and cyclophosphamide exhibiting high cross-resistance along with alterations in expression of cancer-stem cell markers such as CD133, CD166, CD24, CD26, CXCR4, CD271 and CD274. We also detected increased aldehyde dehydrogenase (ALDH) activity associated with overexpression of specific ALDH isoform 1A3. Its inhibition by siRNA approach partially sensitized cells to various agents, thus linking for the first time the ALDH1A3 and chemoresistance in colorectal cancer. CONCLUSION: Our study demonstrated that acquired chemoresistance goes along with metastatic and migratory phenotype and can be accompanied with increased activity of aldehyde dehydrogenase. We describe here the valuable model to study molecular link between resistance to chemotherapy and metastatic dissemination.

Cytotoxic response of 5-fluorouracil-resistant cells to gene- and cell-directed enzyme/prodrug treatment.

Gene-directed enzyme/prodrug therapy (GDEPT) mediated by mesenchymal stromal cells (MSC) was already approved for clinical study on a progressive disease refractory to standard therapy. In this work, we examined the effect of several GDEPT approaches on chemoresistant cells. First, we derived 5-fluorouracil (5-FU)-resistant variant of human colorectal adenocarcinoma cells HT-29 designated HT-29/EGFP/FUR. Our data show that the upregulation of thymidylate synthase (TS) and downregulation of thymidine phosphorylase (TP), orotate phosphoribosyl transferase (OPRT) and dihydropyrimidine dehydrogenase (DPD) contributed to the 5-FU resistance in cancer cells. Next, we combined the MSC expressing either yeast cytosine deaminase (CD-MSC) or fusion yeast CD::uracil phosphoribosyl transferase (CD::UPRT-MSC) and prodrug 5-fluorocytosine (5-FC) in a cell-mediated GDEPT approach. Bystander cytotoxic effect in the direct co-cultures of the tumor and therapeutic cells mixed in a 5:1 ratio resulted in 55% and 70% inhibition of proliferation, respectively. However, the acquired chemoresistance to 5-FU can be overcome by introducing the prodrug-converting transgene into the tumor cells. When the transgene CD::UPRT was expressed in the chemoresistant cells (CD::UPRT-FUR), substantial suicide effect and a 90% decrease in viability was observed using non-toxic concentration of 62.5 µg/ml 5-FC. In summary, we demonstrate here that the transgene introduction circumvented 5-FU resistance in the tumor cells.

Intentionally flawed manuscripts as means for teaching students to critically evaluate scientific papers.

As future scientists, university students need to learn how to avoid making errors in their own manuscripts, as well as how to identify flaws in papers published by their peers. Here we describe a novel approach on how to promote students' ability to critically evaluate scientific articles. The exercise is based on instructing teams of students to write intentionally flawed manuscripts describing the results of simple experiments. The teams are supervised by instructors advising the students during manuscript writing, choosing the 'appropriate' errors, monitoring the identification of errors made by the other team and evaluating the strength of their arguments in support of the identified errors. We have compared the effectiveness of the method with a journal club-type seminar. Based on the results of our assessment we propose that the described seminar may effectively complement the existing approaches to teach critical scientific thinking. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):22-30, 2018.