CXCR4 Expression in Prostate Cancer Progenitor Cells

Tumor progenitor cells represent a population of drug-resistant cells that can survive conventional chemotherapy and lead to tumor relapse. However, little is known of the role of tumor progenitors in prostate cancer metastasis. The studies reported herein show that the CXCR4/CXCL12 axis, a key regulator of tumor dissemination, plays a role in the maintenance of prostate cancer stem-like cells. The CXCL4/CXCR12 pathway is activated in the CD44+/CD133+ prostate progenitor population and affects differentiation potential, cell adhesion, clonal growth and tumorigenicity. Furthermore, prostate tumor xenograft studies in mice showed that a combination of the CXCR4 receptor antagonist AMD3100, which targets prostate cancer stem-like cells, and the conventional chemotherapeutic drug Taxotere, which targets the bulk tumor, is significantly more effective in eradicating tumors as compared to monotherapy

Hypoxia-Activated Small Molecule-Induced Gene Expression

Hypoxia, conditions of reduced oxygen, occur in a wide variety of biological contexts, including solid tumours and bacterial biofilms, which are relevant to human health. Consequently, the development of chemical tools to study hypoxia is vital. Here we report a hypoxia-activated small molecule-mediated gene expression system using a bioreductive prodrug of the inducer isopropyl 1-thio-β-D-galactopyranoside (IPTG). As a proof-of-concept we have placed the production of a green fluorescent protein under the control of hypoxia. Our system has the potential to be extended to regulate the production any given protein of choice.</div

Hypoxia-Activated Small Molecule-Induced Gene Expression

<div><div><div><p>Hypoxia, conditions of reduced oxygen, occur in a wide variety of biological contexts, including solid tumours and bacterial biofilms, which are relevant to human health. Consequently, the development of chemical tools to study hypoxia is vital. Here we report a hypoxia-activated small molecule-mediated gene expression system using a bioreductive prodrug of the inducer isopropyl 1-thio-β-D-galactopyranoside (IPTG). As a proof-of-concept we have placed the production of a green fluorescent protein under the control of hypoxia. Our system has the potential to be extended to regulate the production any given protein of choice.</p></div></div></div

Verteporfin-induced lysosomal compartment dysregulation potentiates the effect of sorafenib in hepatocellular carcinoma.

Lysosomal sequestration of anti-cancer compounds reduces drug availability at intracellular target sites, thereby limiting drug-sensitivity and inducing chemoresistance. For hepatocellular carcinoma (HCC), sorafenib (SF) is the first line systemic treatment, as well as a simultaneous activator of autophagy-induced drug resistance. The purpose of this study is to elucidate how combination therapy with the FDA-approved photosensitizer verteporfin (VP) can potentiate the antitumor effect of SF, overcoming its acquired resistance mechanisms. HCC cell lines and patient-derived in vitro and in vivo preclinical models were used to identify the molecular mechanism of action of VP alone and in combination with SF. We demonstrate that SF is lysosomotropic and increases the total number of lysosomes in HCC cells and patient-derived xenograft model. Contrary to the effect on lysosomal stability by SF, VP is not only sequestered in lysosomes, but induces lysosomal pH alkalinization, lysosomal membrane permeabilization (LMP) and tumor-selective proteotoxicity. In combination, VP-induced LMP potentiates the antitumor effect of SF, further decreasing tumor proliferation and progression in HCC cell lines and patient-derived samples in vitro and in vivo. Our data suggest that combination of lysosome-targeting compounds, such as VP, in combination with already approved chemotherapeutic agents could open a new avenue to overcome chemo-insensitivity caused by passive lysosomal sequestration of anti-cancer drugs in the context of HCC

CXCR4⁺ populations in DU145 and PC3 cell lines are multipotent.

<p>FACS purified CXCR4⁺ and CXCR4⁻ DU145 and PC3 cells were cultured under differentiation conditions in the presence of 10% FBS.CXCR4⁺ cells differentiate toCK5⁺ basal epithelial cells (10.7%), CK5⁺/CK18⁺ intermediate cells (32.2%), and CK18⁺ luminal epithelial cells (57.1%). In contrast, CXCR4⁻ cells differentiate to CK18⁺cells (91.9% of total cell population) and CK5⁺/CK18⁺ cells (8% of total cell population) but not basal epithelial cells. Scale bars indicate 15 µm.</p

Overexpression of CXCR4 inprostate cancer progenitors.

<p>(A) Cells grown under sphere forming conditions showed an increased expression level of CXCR4 as analyzed by RT-PCR and Western blot analysis. For sphere formation, single cells were plated at 500 cells/mL in 10-cm dishes with an ultralow attachment surface and grown in serum-free epithelial basal medium for 7 days. (B) Flow cytometry analysis showed significant enrichment of the CXCR4⁺ population within CD44⁺/CD133⁺ cells compared to the total cell population for DU145 and PC3 cells (p<0.001). The cells were triple stained and analyzed with a BD LSR II flow cytometer. (C) Representative fluorescent images of CD133 and CD44 co-immunostaining showing that CXCR4⁺ DU145 and PC3 cells have a higher proportion of CD44⁺/CD133⁺ cells compared to CXCR4⁻ cells. Cells expressing high of low levels of CXCR4 were FACS-purified, plated in 384 well black clear bottom plates at a density of 100 cells/well in serum-free epithelial basal medium. After 18 hours, the cells were fixed with 3.7<b>%</b> formaldehyde in PBS and stained with anti-CD133 and anti-CD44 antibodies. Cells in at least five randomly selected fields of view were counted for each condition. Arrows show the triple positive cells. Scale bars indicate 15 µm. *- p value<0.05. (D) Immunostaining of paraffin-embedded sections of xenograft tumors formed by FACS purified CD44⁺/CD133⁺ and CD44⁻/CD133⁻ cells showed more than 13% CXCR4⁺ cells in tumors derived from CD44⁺/CD133⁺ cells compared to 2.2% CXCR4⁺ cells in xenograft tumors derived from CD44⁻/CD133⁻ cells. A total of 10³ FACS-sorted DU145 CD133⁺/CD44⁺ or CD133⁻/CD44⁻ cells embedded in BD matrigel were injected s.c. into NOD/SCID mice. Tumors were allowed to grow for 42 days until the tumors produced by DU145 CD133⁺/CD44⁺ reached a size of 400 mm³ and the tumors produced by DU145 CD133⁻/CD44⁻ reached a size of 125 mm³. Cells in at least five randomly selected fields of view were counted for each condition. Scale bars indicate 30 µm. (E) CXCR4 immunostaining on paraffin-embedded sections of xenograft tumors made by the cells grown under sphere forming and monolayer conditions showed more than 6% CXCR4⁺ cells in sphere-derived tumors as compared to 1.4% of CXCR4⁺ cells in monolayer-derived xenograft tumors. **- p value<0.01.Scale bars indicate 30 µm.</p

CXCR4 neutralization leads to attenuation of the CD44⁺/CD133⁺ prostate progenitor population.

<p>(A) DU145 cells were plated in 96-well low-attachment plates at 100 cells per well (5 replicates) and the spheres were grown in serum-free, EBM medium with supplements. The antibodies were replenished daily. Cells were imaged with an Acumen eX3 microplate cytometer and spheres were detected using image analysis software. The sphere size was measured by GFP intensity. The spheres were discriminated from cell debris using a Gaussian filter. The spheres included in the analysis are outlined in red and indicated by arrows. Representative data from one of two independent experiments is shown; *- p value<0.05. (B) Flow cytometry analysis revealed attenuation of CD44⁺/CD133⁺ population in DU145 cells treated with 10 µg/ml neutralizing anti-CXCR4 (mouse monoclonal IgG, clone 44716, R&D Systems) or 10 µg/ml control antibody (mouse IgG isotype control, Lifespan Bioscience Inc.) for 5 days. The cell were grown in medium supplemented with 2% FBS. Culture medium was refreshed every second day; *- p value<0.05. (C) Western blot analysis of DU145 cells treated with 10 µg/ml neutralizing anti-CXCR4 antibody for 5 days demonstrated downregulation of the PI3K/AKT pathway activation compared to the cells treated with 10 µg/ml control antibody. The cell were grown in medium supplemented with 2% FBS. Culture medium was refreshed every second day. (D) Preincubation of prostate cancer cells with neutralizing anti-CXCR4 antibody significantly delays tumor growth. 5×10⁵ DU145 cells pretreated with neutralizing anti-CXCR4 or control antibody for 5 days were embedded in BD matrigel and injected s.c. into NOD/SCID mice.*- p value<0.01.</p