Role of CD44 in clear cell renal cell carcinoma invasiveness after antiangiogenic treatment

Treballs Finals de Grau de Farmàcia, Facultat de Farmàcia, Universitat de Barcelona, 2017. Tutor/a: Joan Carles Rodríguez Rubio.[eng] During last century, big effort to understand the biochemical basis of cancer was carried out. One of the principal branches of these cancer investigations used drugs to prevent the formation of new blood vessels –process called angiogenesis– responsible for the nutrients supply of the tumour. These drugs are generally called antiangiogenics. It was discovered that some types of tumour have or develop resistance to these drugs when treatment was long enough. For that reason, mechanisms of resistance, aggressiveness, invasion and/or metastasis after the treatment are nowadays relevant to study. Recently, a protein that could be involved in the increased invasiveness of tumour cells after the antiangiogenic treatment appeared. This project collects some evidence that indicates that this protein, called CD44, might play a role in the increased invasion after antiangiogenic treatment in mouse models of renal carcinoma.[cat] Durant l’últim segle, s’ha fet un gran esforç per aprofundir en la basant bioquímica de la investigació contra el càncer. Una de les branques principals d’aquesta investigació utilitza fàrmacs que prevenen la formació de nous vasos sanguinis –procés anomenat angiogènesis- encarregats de nodrir el tumor. Aquests fàrmacs es diuen generalment antiangiogènics. S’ha descobert que alguns tipus de tumor tenen o desenvolupen resistència a aquests fàrmacs quan el tractament és prou llarg. Per aquesta raó, actualment s’està investigant profundament quins són els mecanismes pels quals apareix aquesta resistència, així com també perquè els tumors es tornen més agressius, invasius i/o metastàtics després del tractament. Recentment s’ha descobert una proteïna que podria estar involucrada en l’augment de la invasivitat de les cèl·lules tumorals després del tractament antiangiogènic. Aquest treball recull algunes de les evidències que apunten cap al paper de la proteïna CD44 en l’increment de la invasió tumoral post-tractament amb fàrmacs antiangiogènics en models ratolins de càncer renal

CD46 costimulation and calcitriol modulate CD8+ T cells.

<p>(<b>A</b>) Purified CD8+ T cells from 3 healthy donors were left unstimulated or stimulated as indicated by immobilized anti-CD3 and anti-CD3/CD46 antibodies, in presence or absence of calcitriol. (<b>A</b>) The expression levels of surface CD46, CD28, CD25, OX40, PD-1 and 4-1BB were determined by flow cytometry after 3 days. (<b>B</b>) The levels of CTLA-4 and Foxp3 were determined after intracellular staining. (<b>C</b>) Proliferation was determined by ³H incorporation. The levels of IFNγ in the supernatants were determined by ELISA (mean ± SEM). The data shown for this donor are representative of the three donors.</p

Calcitriol modulates CD46 expression in CD4+ T cells.

<p>Purified CD4+ T cells from healthy donors were left unstimulated (US), or were stimulated by immobilized anti-CD3, anti-CD3/CD28, or anti-CD3/CD46 antibodies as indicated in presence of calcitriol (10⁻⁷M) or ethanol as vehicle control. CD46 expression was monitored by flow cytometry. The representative expression of CD46 after 5 days of culture is shown in (<b>A</b>). The average expression of CD46 detected after 2 or 5 days for the different donors analyzed (mean ± SEM; n = 15) is shown in (<b>B</b>). Samples were analyzed using the Wilcoxon test.</p

Calcitriol promotes CTLA-4, Foxp3 and CD25 expression on CD4+ T cells but not in MS.

<p>(<b>A</b>) Purified CD4+ T cells from MS patients or healthy controls (HC) were left unstimulated or stimulated by immobilized anti-CD3 or anti-CD3/CD46 antibodies in presence of calcitriol (10⁻⁷M) or ethanol, for 5 days. Expression of CTLA-4, Foxp3 and CD25 was then monitored by flow cytometry. (<b>B</b>) The representative data showing the concomitant increase in CD25 or CTLA-4 with the downregulation of CD46 upon CD3/CD46 costimulation for one healthy donor are shown. (<b>C</b>) The representative data showing changes in CD25 expression following addition of calcitriol in one healthy donor and one patient with MS are shown.</p

Calcitriol-treated cell supernatants decrease proliferation of bystander CD4+ T cells.

<p>(<b>A</b>) Purified CD4+ T cells were CFSE pre-labeled and then activated by anti-CD3 or anti-CD3/CD28 antibodies, in presence or absence of culture supernatants from CD46-costimulated T cells with or without calcitriol from either a healthy donor or a patient with MS, as indicated. Proliferation was assessed by flow cytometry after 3 days. (<b>B</b>) The data obtained with the supernatants from CD46-costimulated T cells from 3 healthy donors are represented. (<b>C</b>) CFSE-labeled naïve T cells were activated by anti-CD3 antibodies (1 µg/ml), in presence of cell supernatants from CD46-costimulated T cells with or without calcitriol (n = 3). A blocking anti-IL-10 antibody or control IgG1 was added to the culture. Proliferation was assessed after 3 days.</p

Calcitriol promotes concomitant CD46 downregulation and CD28 upregulation in CD46-costimulated CD4+ T cells.

<p>Purified CD4+ T cells from a cohort of RRMS patients (MS) (n = 11) or healthy controls (HC) (n = 15) were stimulated by immobilized anti-CD3 or anti-CD3/CD46 antibodies as indicated in presence of calcitriol (10⁻⁷M) or ethanol as vehicle control for 5 days. Expression of CD46 (<b>A</b>) or CD28 (<b>B</b>) was monitored by flow cytometry. Samples were analyzed using the Wilcoxon test. (<b>C</b>) The dot-plot showing the concomitant changes in expression of CD46 and CD28 following calcitriol treatment on CD46-costimulated T cells is represented for one healthy donor.</p

Calcitriol allows the switch from Th1 to Tr1 in CD4+ T cells.

<p>(<b>A</b>) Purified CD4+ T cells from one healthy donor were left unstimulated or stimulated as indicated by immobilized anti-CD3 and anti-CD3/CD46 antibodies, in presence or absence of IL-2 and calcitriol. After 2 days, the production of IL-10 and IFNγ was assessed using the secretion catch assay (Miltenyi). (<b>B</b>) The production of IL-10 and IFNγ in the supernatants from the same wells was also determined by ELISA. The IL-10:IFNγ ratio is also represented (mean ± SEM; samples were analyzed using Student's t-test).</p

Characteristics of the donors used in this study as to sex, age and disease status.

<p>Characteristics of the donors used in this study as to sex, age and disease status.</p

Calcitriol restores the IL-10:IFNγ ratio upon CD46 costimulation of CD4+ T cells in MS.

<p>Purified CD4+ T cells from healthy controls (HC) or RRMS patients (MS) were left unstimulated or were stimulated as indicated by immobilized antibodies for 4 days, in presence or absence of calcitriol (10⁻⁷M). (<b>A</b>) Proliferation was determined by ³H incorporation. The levels of IL-10 (<b>B</b>) and IFNγ (<b>C</b>) in the supernatants were determined by ELISA (mean ± SEM; samples were analyzed using the Wilcoxon test). (<b>D</b>) The IL-10:IFNγ ratio is also represented to bypass the change related to differences in proliferation (mean ± SEM; samples were compared using the Mann Whitney U-test).</p

Additional file 1 of The impact of professional midwives and mentoring on the quality and availability of maternity care in government sub-district hospitals in Bangladesh: a mixed-methods observational study

Additional file 1:Table S1. Quotations and codes contributing to the theme “resistance to change”