MSCs as Tumor-Specific Vectors for the Delivery of Anticancer Agents-A Potential Therapeutic Strategy in Cancer Diseases: Perspectives for Quinazoline Derivatives.

Mesenchymal stem cells (MSCs) are considered to be a powerful tool in the treatment of various diseases. Scientists are particularly interested in the possibility of using MSCs in cancer therapy. The research carried out so far has shown that MSCs possess both potential pro-oncogenic and anti-oncogenic properties. It has been confirmed that MSCs can regulate tumor cell growth through a paracrine mechanism, and molecules secreted by MSCs can promote or block a variety of signaling pathways. These findings may be crucial in the development of new MSC-based cell therapeutic strategies. The abilities of MSCs such as tumor tropism, deep migration and immune evasion have evoked considerable interest in their use as tumor-specific vectors for small-molecule anticancer agents. Studies have shown that MSCs can be successfully loaded with chemotherapeutic drugs such as gemcitabine and paclitaxel, and can release them at the site of primary and metastatic neoplasms. The inhibitory effect of MSCs loaded with anti-cancer agents on the proliferation of cancer cells has also been observed. However, not all known chemotherapeutic agents can be used in this approach, mainly due to their cytotoxicity towards MSCs and insufficient loading and release capacity. Quinazoline derivatives appear to be an attractive choice for this therapeutic solution due to their biological and pharmacological properties. There are several quinazolines that have been approved for clinical use as anticancer drugs by the US Food and Drug Administration (FDA). It gives hope that the synthesis of new quinazoline derivatives and the development of methods of their application may contribute to the establishment of highly effective therapies for oncological patients. However, a deeper understanding of interactions between MSCs and tumor cells, and the exploration of the possibilities of using quinazoline derivatives in MSC-based therapy is necessary to achieve this goal. The aim of this review is to discuss the prospects for using MSC-based cell therapy in cancer treatment and the potential use of quinazolines in this procedure.

Quinazoline Derivatives as Potential Therapeutic Agents in Urinary Bladder Cancer Therapy.

Cancer diseases remain major health problems in the world despite significant developments in diagnostic methods and medications. Many of the conventional therapies, however, have limitations due to multidrug resistance or severe side effects. Bladder cancer is a complex disorder, and can be classified according to its diverse genetic backgrounds and clinical features. A very promising direction in bladder cancer treatment is targeted therapy directed at specific molecular pathways. Derivatives of quinazolines constitute a large group of chemicals with a wide range of biological properties, and many quinazoline derivatives are approved for antitumor clinical use, e.g.,: erlotinib, gefitinib, afatinib, lapatinib, and vandetanib. The character of these depends mostly on the properties of the substituents and their presence and position on one of the cyclic compounds. Today, new quinazoline-based compounds are being designed and synthesized as potential drugs of anticancer potency against bladder cancers.

The Impact of SARS-CoV-2 Infection on Fertility and Female and Male Reproductive Systems.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains a huge challenge for contemporary healthcare systems. Apart from widely reported acute respiratory distress syndrome (ARDS), the virus affects many other systems inducing a vast number of symptoms such as gastrointestinal, neurological, dermatological, cardiovascular, and many more. Currently it has also been hypothesized that the virus might affect female and male reproductive systems; SARS-CoV-2 infection could also have a role in potential disturbances to human fertility. In this article, we aimed to review the latest literature regarding the potential effects of SARS-CoV-2 infection on female and male reproductive systems as well as fertility, in general.

Endosialin (TEM1) as a Diagnostic, Progression, and Prognostic Serum Marker for Patients With Colorectal Cancer-A Preliminary Study.

Colorectal cancer (CRC) is one of the most common cancers worldwide usually diagnosed in the advanced stage. In this study, the serum concentration of tumor endothelial marker 1 (TEM1) was measured and correlated with clinicopathological features to evaluate whether TEM1 might serve as a biomarker for early CRC diagnosis, progression, and prognosis. The concentration of TEM1 was measured in the serum samples of 45 patients with CRC and 35 healthy individuals using enzyme-linked immunosorbent assay test. The mean serum concentration of TEM1 was significantly higher in the patients with CRC compared to the healthy individuals (1.31 ± 0.16 vs 0.92 ± 0.90 ng/mL; P < .001). The mean concentration of TEM1 significantly increased in the patients having CRC with early stage (stage I + II) compared to noncancer control individuals (stage I + II vs control 1.21 ± 0.13 ng/mL: 0.92 ± 0.90 ng/mL; P < .001). The TEM1 concentration in blood serum also showed a significant association with the development of T stages (P < .001), N stages (P < .001), and M stages (P = .006). The TEM1 sensitivity and specificity in CRC detection are higher than routinely used blood markers (carcinoembryonic antigen [CEA] and carbohydrate antigen [Ca 19-9]). Patients with high TEM1 concentration (≥1.055 ng/mL) had a worse overall survival rate compared to the patients having CRC with low TEM1 concentration (<1.055 ng/mL). In conclusion, TEM1 can act as a potential diagnostic, progression, and prognostic serum biomarker for patients with CRC; TEM1 might be a good supplement for commonly used markers CEA and Ca 19-9.

Serum TEM5 and TEM7 concentrations correlate with clinicopathologic features and poor prognosis of colorectal cancer patients.

PURPOSE: Colorectal cancer (CRC) is a serious threat worldwide; therefore, discovering sensitive and specific serum biomarkers for early stages of CRC is a great challenge. In this study, we evaluated whether tumour endothelial marker 5 (TEM5) and 7 (TEM7) circulating in blood serum can be useful as blood-based markers for detection, progression, and prognosis in CRC patients. Moreover, their specificity and sensitivity in the early diagnosis of CRC were compared with common carcinoma diagnostic markers, i.e. CEA and Ca 19-9. MATERIALS AND METHODS: The study included 45 CRC patients and 35 healthy individuals. The serum concentration of TEM5 and TEM7 were quantified using sandwich ELISA. RESULTS: The mean TEM5 and TEM7 serum concentrations were statistically significantly higher in the CRC patients than in the healthy controls. Moreover, the mean TEM5 and TEM7 concentrations were statistically significantly higher in the serum of patients with late stage (III/IV) compared to early stage (I/II) cancer. The TEM5 and TEM7 values increased along the development of the T, N, and M stages. The TEM5 and TEM7 sensitivity and specificity in CRC detection were higher than routinely used blood markers (CEA, Ca19-9). The high TEM5 and TEM7 concentrations were associated with worse overall survival compared to CRC patients of low TEM5 and TEM7 concentrations. CONCLUSIONS: Taken together, these findings suggest that TEM5 and TEM7 serum concentrations can be considered as useful biomarkers for the detection of CRC patients and for monitoring cancer progression and identifying patients with a high possibility of poor survival.

Interactions between microRNA-200 family and Sestrin proteins in endometrial cancer cell lines and their significance to anoikis.

In the present study, we intend to determine whether Sestrin proteins 1, 2, and 3 (SESN1-3) are targets of microRNA-200 family (miR-200) in endometrial cancer (EC) Ishikawa, AN3CA, KLE, and RL 95-2 cell lines and to investigate how these potential interactions influence anoikis resistance of EC cell lines. The luciferase reporter assay, qRT-PCR, and western blotting assays were used to verify whether SESN1-3 are direct targets of miR-200. Moreover, the anoikis assay and transient transfections of miR-200 mimics or inhibitors into EC cell lines were performed to evaluate the modulatory role of miR-200 and SESN proteins on anoikis resistance. We demonstrated that SESN2 protein is a direct target of mir-141 in KLE and RL-95-2 EC cell lines and the functional interaction of miR-141 and SESN2 protein has a downstream effect on anoikis resistance and SESN2 expression level in Ishikawa and AN3CA cell lines. Moreover, we have shown that SESN3 protein is a direct target of miR-200b, miR-200c, and miR-429 in Ishikawa, AN3CA, and KLE cell lines. Our results show that manipulation of miR-200b, miR-200c, and miR-429 expression patterns also has an influence on anoikis resistance in EC cell lines. In conclusion, we identified new interactions between miR-200 and the oxidative stress response SESN proteins that affect anoikis resistance in human EC cells.

A guide for endometrial cancer cell lines functional assays using the measurements of electronic impedance.

Endometrial cancer cell lines are critical tools to investigate the molecular mechanism of tumorigenesis using the end point cell-based assay such as proliferation, cytotoxicity, apoptosis, anoikis or migration and invasion. The proper assay optimization and performance is essential for physiologically relevant results interpretation. In this study we use label-free real-time cell analysis platform (xCELLigence) to optimize growing conditions for proliferation and migration experiments of two types of endometrial cancer cell lines HEC-1-B, HEC-1-A, KLE, and Ishikawa. Profiling of cell lines by cell index measurement in proliferation and migration experiments was performed. Our experimental approach allowed us to monitor particular stage of the cell growth, to see the relation between seeding density and dynamic cell growth as well as to choose the optimal serum concentration as chemoattractant in migration experiment. The highest rate of proliferation was shown for Ishikawa cells. The rapid pace of cellular migration was observed in case of KLE and HEC-1-B cells as compared to weak migratory activity of Ishikawa cells. The cell index that reflects the cell status characterized real-time cytological profile of each analyzed cell line. These cell profiles were crucial for better planning the classical end-point assays used in further research.

In vitro and in vivo activity of miR-92a-Locked Nucleic Acid (LNA)-Inhibitor against endometrial cancer.

BACKGROUND: Endometrial cancer is the most common cancer of the female reproductive tract. Based on our previous studies we speculated that miR-92a exhibited pro-oncogenic properties in endometrial cancer, and therefore its inhibition could be used as a therapeutic measure in this disease. Therefore in the present study we aimed to investigate both in vitro and in vivo if inhibition of miR-92a in endometrial cancer would limit cancer cells proliferation. METHODS: miR-92a expression was evaluated in four endometrial cancer cell lines using qPCR. Inhibition of miR-92a activity was obtained in endometrial cancer cell lines by a transient transfection of a custom designed Locked Nucleic Acid (LNA)-Inhibitor, developed to work both in vitro and in vivo. In vitro proliferation studies were performed using xCELLigence RTCA DP system. In vivo experiment was performed in Cby.Cg-Foxn1 < nu>/cmdb mice bearing endometrial cancer xenografts, which were intraperitoneally injected with nine dosages of 25 mg/kg of miR-205-LNA-inhibitor. RESULTS: qPCR revealed increased expression of miR-92a in HEC-1-B, Ishikawa and AN3CA cells. LNA-i-miR-92a inhibited endometrial cancer growth in vitro. It was also demonstrated that systemic administration of LNA-i-miR-92a was feasible and exerted inhibitory effect on endometrial cancer xenograft growth in vivo with only mild toxic effects in treated animals, however the effect was observed until 12th experimental day and the last three dosages did not maintain the attenuating effect with the acceleration of tumor growth observed at the end and after cessation of the intraperitoneal therapy. CONCLUSIONS: Taken together, these results indicate that intraperitoneal delivery of miR-92a-LNA-modified-inhibitor is feasible, devoid of significant toxicity and moderately inhibits endometrial cancer growth in vivo, and therefore warrants further studies investigating other routes of inhibitor delivery possibly in other animal models.

Locked nucleic acid-inhibitor of miR-205 decreases endometrial cancer cells proliferation in vitro and in vivo.

Pathogenesis of endometrial cancer has been connected with alterations of microRNA expression and in particular miR-205 up-regulation was consistently reported in this carcinoma. Presented study aimed to investigate if inhibition of miR-205 expression using LNA-modified-nucleotide would attenuate endometrial cancer cells proliferation in vitro and in vivo.In the course of the study we found that the proliferation of endometrial cancer cells (HEC-1-B, RL-95, KLE, Ishikawa) transfected with LNA-miR-205-inhibitor and evaluated using real time cell monitoring as well as standard cell proliferation assay, was significantly decreased. Next, LNA-miR-205-inhibitor was used to assess the in vivo effects of miR-205 inhibition of endometrial cancer growth. Cby.Cg-Foxn1/cmdb mice bearing endometrial cancer xenografts were intraperitoneally injected with nine dosages of 25mg/kg of miR-205-LNA-inhibitor or scramble control or phosphatase buffered saline and were observed for 32 days. We found that systemic administration of miR-205-LNA-inhibitor was technically possible, and exerted inhibitory effect on endometrial cancer xenograft growth in vivo with only mild toxic effects in treated animals.In conclusion our results suggest that systemic delivery of miR-205-LNA-inhibitor is feasible, devoid of significant toxicity, and could be a promising treatment strategy for endometrial cancer. Therefore it warrants further studies in other animal models.

Selection and validation of endogenous controls for microRNA expression studies in endometrioid endometrial cancer tissues.

OBJECTIVES: microRNAs comprise a family of small, non-coding RNAs, which regulate gene expression at the posttranscriptional level. Multiple studies implicated important roles of microRNAs in various malignancies including endometrioid endometrial carcinoma (EEC). qPCR is widely used in the studies investigating microRNA expression. Relative quantification of microRNA expression requires proper normalization methods and endogenous controls are widely used for this purpose. The aim of this study was experimental identification of stable endogenous controls for normalization of microRNA qPCR expression studies in EEC. METHODS: Expression of twelve candidate endogenous controls (miR-16, miR-26b, miR-92a, RNU44, RNU48, U75, U54, U6, U49, RNU6B, RNU38B, U18A) was investigated in tissue samples obtained from 45 patients (30 EEC, 15 normal endometrium) using qPCR. Stability of candidate endogenous controls was evaluated using NormFinder, geNorm, BestKeeper and equivalency test. The results were then validated using larger group of samples. RESULTS: RNU48, U75 and RNU44 were identified as stably and equivalently expressed between malignant and normal tissues. Both NormFinder and geNorm indicated that those three snRNAs were optimal for qPCR data normalization in EEC tissues. CONCLUSIONS: In conclusion, we suggest that average expression of those snoRNAs could be used as a reliable endogenous control in microRNA qPCR studies in endometrioid endometrial cancer. In addition to identifying suitable endogenous controls in EEC, our study presents an appropriate strategy for validation of candidate reference genes for any microRNA qPCR study.

[The assessment of selected molecules belonging to B7 family on the mature dendritic cells in laryngeal cancer patients]. / Ocena ekspresji wybranych molekul z rodziny B7 na powierzchni dojrzalych komórek dendrytycznych u chorych na raka krtani.

UNLABELLED: B7-H1, B7-H2 and B7-H4 molecules play various roles in the adaptive immune system and are broadly expressed on many cells, especially those localized in cancer tissue. The aim of the study was to assess the surface expression of B7-H1, B7-H2 and B7-H4 molecules on the mature dendritic cells (DC) generated from peripheral blood monocytes of laryngeal cancer patients and healthy donors. MATERIAL AND METHODS: Forty-four male patients treated surgically for squamous cell carcinoma of the larynx were included in the study. Peripheral blood from twelve healthy male donors was used as a control. Mononuclear cells were separated from all individuals by density gradient centrifugation, incubated with anti-CD14 microbeads, and passed through MACS separation columns. The CD14 positive cell population was used to prepare monocyte derived DC. Laryngeal cancer tissue was obtained from patients during surgical treatment and homogenized to prepare tumor cell lysates for further stimulation. We evaluated with the use of flow cytometry method the percentage of cells with an expression and mean fluorescent intensity (MFI) of surface markers, such as: CD83, B7-H1, B7-H2, B7-H4. RESULTS: Our study revealed that the percentage of mature dendritic cells, stimulated with autologous tumor cell lysates, with the expression of B7-H1 molecule in patients was lower than in healthy donors (61.81 ± 25.58% vs 93.02 ± 4.63%, p=0.007). In laryngeal cancer patients, the percentage of CD83+/B7-H2+ cells was higher than in healthy individuals (18.32 ± 10.74% vs 2.89 ± 0.43%, p=0.019). CONCLUSIONS: There is a relation between the presence of laryngeal cancer and the expression of B7 family molecules.

[The evaluation of apoptosis in peripheral blood lymphocytes in laryngeal cancer patients]. / Ocena apoptozy limfocytów krwi obwodowej u chorych leczonych z powodu raka krtani.

UNLABELLED: The laryngeal cancer is the most often cancer among others in head and neck region. It occurs mostly among 55 and 69. Its development depends on immunological state of the body. Vitality of the immunological system cells was considered due to growth, treatment sensitivity and prognosis of some neoplasms. The aim of this work were estimation and comparison the phenomenon of lymphocytes T and B apoptosis in laryngeal cancer patients treated with surgery and radiotherapy. MATERIAL AND METHODS: The material were 30 patients hospitalized in The Department of Otolaryngology Medical University of Lublin. They all were treated with surgery or surgery and radiotherapy. Apoptosis was estimated on different stages of the treatment process. All samples were examined with the flow cytometry method. The control group were 21 patients hospitalized because of the suspicion of the apnea syndrome, which wasn't confirmed with polysomnographic examination. RESULTS: Results of this study show significantly increasing percentage of peripheral blood apoptotic B (CD19+) cells caused by surgical treatment. The results considering radiotherapy showed different influence on the phenomenon of immunological cells apoptosis, still those results weren't significant. CONCLUSIONS: The surgical treatment causes increased amount of apoptotic peripheral blood lymphocytes.

Evaluation of monocyte-derived dendritic cells, T regulatory and Th17 cells in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors.

Immunotherapy with dendritic cells (DC) may constitute a new and advantageous option for patients with chronic myeloid leukemia (CML) who respond to therapy with tyrosine kinase inhibitors (TKI), but do not reach complete cytogenetic or molecular remission. In this study, we evaluated the immunophenotype of DC generated from monocytes (Mo-DC) of patients with CML and the influence of TKI therapy on the results of CML-DC generation. We also measured the percentages of T regulatory cells (Tregs) as well as Th17 cells in 19 untreated patients suffering from CML, and in 28 CML patients treated with TKI. We found that DC can be reliably generated from the peripheral blood CD14+ cells of untreated CML patients. But we observed a persistent expression of CD14 monocyte marker on DC from CML patients, together with lower percentages of Mo-DC with expression of CD1a (p = 0.002), CD80 (p = 0.0005), CD83 (p = 0.0004), and CD209 (p = 0.02) compared to healthy donors. There was an adverse correlation between WBC count and the percentage of Mo-DC with co-expression of CD80 and CD86 (R = -0.63; p = 0.03). In patients treated with TKI, we observed higher efficacy of DC generation in seven-day cultures, compared to untreated patients. Expression of CD209 on DC was higher in patients treated with TKI (0.02). The duration of TKI therapy correlated adversely with MFI for CD1a (R = -0.49; p = 0.006) and positively with MFI for CD83 (R = 0.63; p = 0.01). Percentages of CD4+CD25highFoxP3+ cells (p = 0.0002) and Th17 cells (p = 0.02) were significantly higher in untreated CML patients compared to healthy controls. There was a significant correlation between the percentage of Treg cells and the percentage of peripheral blood basophiles (R = 0.821; p = 0.02). There were no changes in Tregs or Th17 cell percentages in CML patients after six months of TKI therapy. However, the expression of intracellular IL-17 in Th17 cells correlated negatively with the time of TKI therapy in the whole group of treated patients (R = -0.516; p = 0.04). We noted a correlation between IL-6 serum level and peripheral blood WBC count (R = 0.492; p = 0.04). There was also an inverse correlation between the serum level of IL-6 and the duration of TKI therapy (R = -0.66; p = 0.03). Taken together, our data shows that mature DC can be generated from CML patients treated with TKI, and that the yield of Mo-DC is higher in patients treated with TKI than in patients with active disease. This should encourage further trials with DC immunotherapy in patients with cytogenetic response after TKI therapy. We also found increased frequencies of T regulatory and Th17 cells in CML patients, which might suggest their potential role in immunity against this disease. Further studies are needed to determine if manipulation of these cell populations might improve the results of DC immunotherapy.

CD3+/CD16+CD56+ cell numbers in peripheral blood are correlated with higher tumor burden in patients with diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma is the commonest histological type of malignant lymphoma, and remains incurable in many cases. Developing more efficient immunotherapy strategies will require better understanding of the disorders of immune responses in cancer patients. NKT (natural killer-like T) cells were originally described as a unique population of T cells with the co-expression of NK cell markers. Apart from their role in protecting against microbial pathogens and controlling autoimmune diseases, NKT cells have been recently revealed as one of the key players in the immune responses against tumors. The objective of this study was to evaluate the frequency of CD3(+)/CD16(+)CD56(+) cells in the peripheral blood of 28 diffuse large B-cell lymphoma (DLBCL) patients in correlation with clinical and laboratory parameters. Median percentages of CD3(+)/CD16(+)CD56(+) were significantly lower in patients with DLBCL compared to healthy donors (7.37% vs. 9.01%, p = 0.01; 4.60% vs. 5.81%, p = 0.03), although there were no differences in absolute counts. The frequency and the absolute numbers of CD3(+)/CD16(+)CD56(+) cells were lower in advanced clinical stages than in earlier ones. The median percentage of CD3(+)/CD16(+)CD56(+) cells in patients in Ann Arbor stages 1-2 was 5.55% vs. 3.15% in stages 3-4 (p = 0.02), with median absolute counts respectively 0.26 G/L vs. 0.41 G/L (p = = 0.02). The percentage and absolute numbers of CD3(+)/CD16(+)CD56(+) cells were significantly higher in DL -BCL patients without B-symptoms compared to the patients with B-symptoms, (5.51% vs. 2.46%, p = 0.04; 0.21 G/L vs. 0.44 G/L, p = 0.04). The percentage of CD3(+)/CD16(+)CD56(+) cells correlated adversely with serum lactate dehydrogenase (R= -445; p 〈 0.05) which might influence NKT count. These figures suggest a relationship between higher tumor burden and more aggressive disease and decreased NKT numbers. But it remains to be explained whether low NKT cell counts in the peripheral blood of patients with DLBCL are the result of their suppression by the tumor cells, or their migration to affected lymph nodes or organs.

Dendritic cells based immunotherapy of patient with chondrosarcoma--case report.

We present a case report of patient with intracranial chondrosarcoma and attempt to use vaccination of dendritic cells as the salvage therapy. To our knowledge, this is the first case report of DCs vaccination in the head and neck chondrosarcoma. Immunotherapy with allogeneic DCs stimulated with tumor cell lysates in this case was demonstrated to be feasible, safe and well tolerated. Unfortunately we did not observe any clinical or immune response during vaccination. CD4+ and CD8+ regulatory cells could be responsible for ineffectiveness of immunotherapy.