Hypoxic 3D in vitro culture models reveal distinct resistance processes to TKIs in renal cancer cells

The aim of this study is to determine the effect of hypoxia on axitinib and sorafenib-treated renal cell carcinoma (RCC) cells. Hypoxia is a crucial factor influencing transcription process via protein modulation, which was shown i.e. in pancreatic cancer. Until now, hypoxia has been defined as associated with poorer outcome and inducing chemotherapy resistance in solid tumors. The unique phenomenon of pseudo-hypoxia connected with vhl mutation was observed in clear-cell, but not in papillary RCC, and the treatment of this subtype of cancer is still challenging. Despite the introduction of new antiangiogenic targeted therapies (inter alia tyrosine kinase inhibitors, TKIs), patients still develop both primary and acquired resistance. Overcoming resistance to TKIs, also in papillary RCC, may be possible by finding significantly modified protein expression. To do this, hypoxic 3D in vitro models must be developed to mimic both molecular pathways typical for low oxygen tension and cell–cell dynamics in tumor-like spatial structures

Hypoxic 3D in vitro culture models reveal distinct resistance processes to TKIs in renal cancer cells

The aim of this study is to determine the effect of hypoxia on axitinib and sorafenib-treated renal cell carcinoma (RCC) cells. Hypoxia is a crucial factor influencing transcription process via protein modulation, which was shown i.e. in pancreatic cancer. Until now, hypoxia has been defined as associated with poorer outcome and inducing chemotherapy resistance in solid tumors. The unique phenomenon of pseudo-hypoxia connected with vhl mutation was observed in clear-cell, but not in papillary RCC, and the treatment of this subtype of cancer is still challenging. Despite the introduction of new antiangiogenic targeted therapies (inter alia tyrosine kinase inhibitors, TKIs), patients still develop both primary and acquired resistance. Overcoming resistance to TKIs, also in papillary RCC, may be possible by finding significantly modified protein expression. To do this, hypoxic 3D in vitro models must be developed to mimic both molecular pathways typical for low oxygen tension and cell–cell dynamics in tumor-like spatial structures

The role of chitosan in the induction of immune response in mice infected with nematodes

Odpowiedź obronna przeciwko nicieniom pasożytniczym zależna jest zwykle od mechanizmów regulowanych przez limfocyty Th2. Wzbudzenie tej populacji następuje w wyniku aktywacji komórek odpowiedzi wrodzonej, jednak wiele pasożytów moduluje reakcje układu odpornościowego wywołując immunosupresję, aby utrzymać się w żywicielu. Prowadzi to do chronicznego zarażenia i znacznie utrudnia kontrolę inwazji, zarówno u ludzi, jak i zwierząt hodowlanych. Poza podawaniem leków przeciwpasożytniczych, strategią zwalczania/ kontrolowania nicieni pasożytniczych może być wzmacnianie naturalnych mechanizmów obronnych żywiciela. Istnieje wiele związków, które mogą polaryzować odpowiedź immunologiczną w kierunku Th2 zależnej i jednym z nich jest chitozan. Jest to naturalny polimer, deacetylowana pochodna chityny, która występuje m. in. w strukturach nicieni. Uważa się, że związek ten może być rozpoznawany przez układ odpornościowy żywiciela i aktywować go, wpływając na równowagę odpowiedzi immunologicznej. Celem pracy było określenie w jaki sposób chitozan, polimer N-acetyloglukozaminy i glukozaminy, wpływa na odpowiedź immunologiczną oraz czy może on zwiększać skuteczność odpowiedzi przeciwpasożytniczej. W badaniach in vitro i in vivo oceniano jak chitozan aktywuje komórki odpowiedzi immunologicznej oraz czy wpływa na przebieg zarażenia nicieniami: Heligmosomoides polygyrus i Trichinella spiralis. Stwierdzono, że chitozan, w zależności od masy cząsteczkowej, wykazuje odmienne właściwości aktywowania in vitro komórek odpowiedzi wrodzonej. Podany in vivo dootrzewnowo, chitozan o dużej masie cząsteczkowej wywołał początkowo silny stan zapalny charakteryzujący się napływem neutrofilów i niedojrzałych monocytów oraz produkcją cytokin prozapalnych, jak i regulatorowych. Po zaprzestaniu podawania chitozanu obserwowano pojawianie się mechanizmów związanych z gojeniem tkanek; alternatywną aktywację makrofagów oraz wydzielanie TGF-β i IL-10. Zarażenie H. polygyrus myszy, którym podawano chitozan, skutkowało zahamowaniem objawów stanu zapalnego, bez znacznego wpływu na przebieg zarażenia. Z kolei u myszy, którym podawano chitozan, w czasie inwazji T. spiralis występował wyższy poziom zarażenia, a odpowiedź na polimer zdominowała odpowiedź przeciwpasożytniczą i obniżyła efektywność mechanizmów obronnych. Stan zapalny wywołany przez chitozan wpływał także na odpowiedź nabytą; zahamował proliferację limfocytów T CD4+, ale wzbudził produkcję przeciwciał IgA krzyżowo rozpoznających antygeny nicieni oraz sam polimer. Uzyskane wyniki wskazują, że chitozan nie jest specyficznym adiuwantem odpowiedzi Th2 zależnej; przetwarzany przez komórki może wywoływać stan zapalny, który ma długotrwały wpływ na mechanizmy odpornościowe żywiciela.Protective immune response against parasitic nematodes is usually mediated by Th2 dependant mechanisms. Induction of those is initiated by activated cells of innate immunity, however many parasites modify immune response leading to immunosuppression and maintain within the host. This leads to chronicity and hampers control of infections both in humans and livestock. Apart from drug therapy, amplification of natural immunity may be a strategy to limit parasitic infections. Among compounds that polarise immune response towards Th2 mechanisms, chitosan may be listed. It is a naturally occurring polymer derived from chitin, that is present also in nematode structures. It is suggested that it can be recognised by the immune system and activate immune response causing its polarisation. Aim of this thesis was to establish how chitosan, polymer of N-acetyloglucosamine and glucosamine, influences immune system and antiparasitic responses. In in vitro and in vivo models it was evaluated how chitosan activates immune cells and affects the course of infection with nematodes: Heligmosomoides polygyrus or Trichinella spiralis. It was observed that chitosan activates innate immunity and this effect depends on molecular weight of the polymer. After intraperitoneal injections, high molecular weight chitosan induced inflammation characterized by influx of neutrophils and immature monocytes and production of proinflammatory and regulatory cytokines. After the treatment was terminated, tissue healing processes were observed; alternative activation of macrophages and secretion of TGF-β and IL-10. Infection of mice that obtained chitosan with H. polygyrus alleviated inflammation but the invasion was unchanged. However when chitosan treated mice were infected with T. spiralis, increased level of infection was observed as response to the polymer dominated and decreased protection. Chitosan induced inflammation altered also adaptive responses; proliferation of CD4+ T lymphocytes was reduces but secretion of IgA occurred that were cross-reactive to nematode antigens and polymer itself. Obtained results suggest that chitosan is not a specific adjuvant of Th2 responses; processed by cells it induces inflammation that alters host immunity in long-term perspective

Differential effects of low and high molecular weight chitosan administered intraperitoneally to mice infected with heligmosomoides polygyrus

Aim of the study was to compare the effect of different molecular weight chitosan on activity of peritoneal cells of mice during immunosupression caused by adult stages of Heligmosomoides polygyrus. We observed that intraperitoneal injections of chitosan induce cell infiltration, but the activity of recruited cells differed depending on the type of polysaccharide used. Low molecular weight chitosan activated cells with inflammatory characteristics, while high molecular weight polysaccharide reduced cell responsiveness to stimulation. Although IgA titers in the peritoneal fluid were elevated, chitosan treatments had no effect on the level of infection

In vivo stimulation of peritoneal cells by chitosan administered in drinking water to mice

The aim of the study was to determine immunostimulant properties of chitosan administered alimentary to BALB/c mice. We observed that chitosan feeding effected in activation of cells from the peritoneal cavity. The cells produced less nitric oxide with simultaneous enhanced activity of arginase and higher expression of receptor for IL-4. What is more, chitosan caused increased number of cells expressing MHC class II. The study confirms that chitosan can stimulate immune system what potentially makes it useful candidate for adjuvant

Distinctive Properties of Endothelial Cells from Tumor and Normal Tissue in Human Breast Cancer

Tumor microenvironments shape aggressiveness and are largely maintained by the conditions of angiogenesis formation. Thus, endothelial cells’ (ECs) biological reactions are crucial to understand and control the design of efficient therapies. In this work, we used models of ECs to represent a breast cancer tumor site as well as the same, healthy tissue. Cells characterization was performed at the transcriptome and protein expression levels, and the cells functional biological responses (angiogenesis and permeability) were assessed. We showed that the expression of proteins specific to ECs (ACE+, VWF+), their differentiation (CD31+, CD 133+, CD105+, CD34-), their adhesion properties (ICAM-1+, VCAM-1+, CD62-L+), and their barrier formation (ZO-1+) were all downregulated in tumor-derived ECs. NGS-based differential transcriptome analysis confirmed CD31-lowered expression and pointed to the increase of Ephrin-B2 and SNCAIP, indicative of dedifferentiation. Functional assays confirmed these differences; angiogenesis was impaired while permeability increased in tumor-derived ECs, as further validated by the distinctly enhanced VEGF production in response to hypoxia, reflecting the tumor conditions. This work showed that endothelial cells differed highly significantly, both phenotypically and functionally, in the tumor site as compared to the normal corresponding tissue, thus influencing the tumor microenvironment

Endothelial Cells as Tools to Model Tissue Microenvironment in Hypoxia-Dependent Pathologies

Endothelial cells (ECs) lining the blood vessels are important players in many biological phenomena but are crucial in hypoxia-dependent diseases where their deregulation contributes to pathology. On the other hand, processes mediated by ECs, such as angiogenesis, vessel permeability, interactions with cells and factors circulating in the blood, maintain homeostasis of the organism. Understanding the diversity and heterogeneity of ECs in different tissues and during various biological processes is crucial in biomedical research to properly develop our knowledge on many diseases, including cancer. Here, we review the most important aspects related to ECs’ heterogeneity and list the available in vitro tools to study different angiogenesis-related pathologies. We focus on the relationship between functions of ECs and their organo-specificity but also point to how the microenvironment, mainly hypoxia, shapes their activity. We believe that taking into account the specific features of ECs that are relevant to the object of the study (organ or disease state), especially in a simplified in vitro setting, is important to truly depict the biology of endothelium and its consequences. This is possible in many instances with the use of proper in vitro tools as alternative methods to animal testing

Spheroid Culture Differentially Affects Cancer Cell Sensitivity to Drugs in Melanoma and RCC Models

2D culture as a model for drug testing often turns to be clinically futile. Therefore, 3D cultures (3Ds) show potential to better model responses to drugs observed in vivo. In preliminary studies, using melanoma (B16F10) and renal (RenCa) cancer, we confirmed that 3Ds better mimics the tumor microenvironment. Here, we evaluated how the proposed 3D mode of culture affects tumor cell susceptibility to anti-cancer drugs, which have distinct mechanisms of action (everolimus, doxorubicin, cisplatin). Melanoma spheroids showed higher resistance to all used drugs, as compared to 2D. In an RCC model, such modulation was only observed for doxorubicin treatment. As drug distribution was not affected by the 3D shape, we assessed the expression of MDR1 and mTor. Upregulation of MDR1 in RCC spheroids was observed, in contrast to melanoma. In both models, mTor expression was not affected by the 3D cultures. By NGS, 10 genes related with metabolism of xenobiotics by cytochrome p450 were deregulated in renal cancer spheroids; 9 of them were later confirmed in the melanoma model. The differences between 3D models and classical 2D cultures point to the potential to uncover new non-canonical mechanisms to explain drug resistance set by the tumor in its microenvironment

Colitis promotes adaptation of an intestinal nematode: a Heligmosomoides polygyrus mouse model system.

The precise mechanism of the very effective therapeutic effect of gastrointestinal nematodes on some autoimmune diseases is not clearly understood and is currently being intensively investigated. Treatment with living helminths has been initiated to reverse intestinal immune-mediated diseases in humans. However, little attention has been paid to the phenotype of nematodes in the IBD-affected gut and the consequences of nematode adaptation. In the present study, exposure of Heligmosomoides polygyrus larvae to the changed cytokine milieu of the intestine during colitis reduced inflammation in an experimental model of dextran sulphate sodium (DSS)- induced colitis, but increased nematode establishment in the moderate-responder BALB/c mouse strain. We used mass spectrometry in combination with two-dimensional Western blotting to determine changes in protein expression and changes in nematode antigens recognized by IgG1 in mice with colitis. We show that nematode larvae immunogenicity is changed by colitis as soon as 6 days post-infection; IgG1 did not recognize highly conserved proteins Lev-11 (isoform 1 of tropomyosin α1 chain), actin-4 isoform or FTT-2 isoform a (14-3-3 family) protein. These results indicate that changes in the small intestine provoked by colitis directly influence the nematode proteome. The unrecognized proteins seem to be key antigenic epitopes able to induce protective immune responses. The proteome changes were associated with weak immune recognition and increased larval adaptation and worm growth, altered localization in the intestine and increased survival of males but reduced worm fecundity. In this report, the mechanisms influencing nematode survival and the consequences of changed immunogenicity that reflect the immune response at the site colonized by the parasite in mice with colitis are described. The results are relevant to the use of live parasites to ameliorate IBD

Biodegradable Chitosan Decreases the Immune Response to Trichinella spiralis in Mice

The purpose of this study was to evaluate the potential of chitosan units released during natural degradation of the polymer to activate the immune system against T. spiralis infection. High molecular weight chitosan was injected intraperitoneally into C57BL/6 mice. Flow cytometry and cytokine concentration, measured by ELISA, were used to characterize peritoneal cell populations during T. spiralis infection. The strong chemo-attractive properties of chitosan caused considerable infiltration into the peritoneal cavity of CD11b+ cells, with reduced expression of MHC class II, CD80, CD86, Dectin-1 or CD23 receptors in comparison to T. spiralis-infected mice. After prolonged chitosan biodegradation, cell populations expressing IL-4R, MR and Dectin-1 receptors were found to coexist with elevated IL-6, IL-10, TGF-β and IgA production. IgA cross-reacted with T. spiralis antigen and chitosan. It was found that chitosan treatment attracted immune cells with low activity, which resulted in the number of nematodes increasing. The glucosamine and N-acetyl-D-glucosamine residues were recognized by wheat germ agglutinin (WGA) lectin and therefore any biodegradable chitosan units may actively downregulate the immune response to the parasite. The findings are relevant for both people and animals treated with chitosan preparations