Presence of crown-like structures in breast adipose tissue; differences between healthy controls, BRCA1/2 gene mutation carriers and breast cancer patients.

PURPOSE: Crown-like structures (CLS) in breast adipose tissue are associated with inflammation and a potential factor in breast cancer behaviour. Whether this effect varies between breast cancer subtypes and is influenced by BMI and BRCA mutation status is presently unknown. Therefore, we compared CLS presence between adipose tissue of healthy controls, BRCA1/2 gene mutation carriers and breast cancer patients, and assessed the relation of CLS with clinical outcome in breast cancer patients. METHODS: Immunohistochemical staining for CD68 was performed on breast adipose tissue sections of 48 healthy controls, 78 BRCA1/2 gene mutation carriers and 259 breast cancer patients. CLS presence and index (CLS/cm2) were correlated with BMI, BRCA status, tumour presence, intrinsic tumour subtype and tumour characteristics. Associations with clinical outcome were assessed. RESULTS: CLS were more often present in breast cancer patients compared to BRCA carriers and healthy controls. CLS presence was associated with the presence of breast cancer and high BMI. CLS were more often present in Luminal-B-like tumours compared to the other subtypes. No correlations between CLS and BRCA status or age was found. In TNBC, CLS were related to lymphovascular invasion. No association with survival was found. CONCLUSION: In conclusion, CLS were more frequently present in breast adipose tissue of breast cancer patients compared to BRCA1/2 gene mutation carriers and healthy controls. Furthermore, our study provides evidence of the association between obesity and presence of CLS. The prognostic significance and impact on clinical outcome of differences in CLS numbers should be further assessed in prospective studies.

Assessing the role of tumour-associated macrophage subsets in breast cancer subtypes using digital image analysis.

PURPOSE: The number of M1-like and M2-like tumour-associated macrophages (TAMs) and their ratio can play a role in breast cancer development and progression. Early clinical trials using macrophage targeting compounds are currently ongoing. However, the most optimal detection method of M1-like and M2-like macrophage subsets and their clinical relevance in breast cancer is still unclear. We aimed to optimize the assessment of TAM subsets in different breast cancer subtypes, and therefore related TAM subset numbers and ratio to clinicopathological characteristics and clinical outcome. METHODS: Tissue microarrays of 347 consecutive primary Luminal-A, Luminal-B, HER2-positive and triple-negative tumours of patients with early-stage breast cancer were serially sectioned and immunohistochemically stained for the pan-macrophage marker CD68 and the M2-like macrophage markers CD163, CSF-1R and CD206. TAM numbers were quantified using a digital image analysis algorithm. M1-like macrophage numbers were calculated by subtracting M2-like TAM numbers from the total TAM number. RESULTS: M2-like markers CD163 and CSF-1R showed a moderate positive association with each other and with CD68 (r ≥ 0.47), but only weakly with CD206 (r ≤ 0.06). CD68 + , CD163 + and CSF-1R + macrophages correlated with tumour grade in Luminal-B tumours (P < 0.001). Total or subset TAM numbers did not correlate with disease outcome in any breast cancer subtype. CONCLUSION: In conclusion, macrophages and their subsets can be detected by means of a panel of TAM markers and are related to unfavourable clinicopathological characteristics in Luminal-B breast cancer. However, their impact on outcome remains unclear. Preferably, this should be determined in prospective series.

Comprehensive characterization of the prostate tumor microenvironment identifies CXCR4/CXCL12 crosstalk as a novel antiangiogenic therapeutic target in prostate cancer.

BACKGROUND: Crosstalk between neoplastic and stromal cells fosters prostate cancer (PCa) progression and dissemination. Insight in cell-to-cell communication networks provides new therapeutic avenues to mold processes that contribute to PCa tumor microenvironment (TME) alterations. Here we performed a detailed characterization of PCa tumor endothelial cells (TEC) to delineate intercellular crosstalk between TEC and the PCa TME. METHODS: TEC isolated from 67 fresh radical prostatectomy (RP) specimens underwent multi-omic ex vivo characterization as well as orthogonal validation of both TEC functions and key markers by immunohistochemistry (IHC) and immunofluorescence (IF). To identify cell-cell interaction targets in TEC, we performed single-cell RNA sequencing (scRNA-seq) in four PCa patients who underwent a RP to catalogue cellular TME composition. Targets were cross-validated using IHC, publicly available datasets, cell culture expriments as well as a PCa xenograft mouse model. RESULTS: Compared to adjacent normal endothelial cells (NEC) bulk RNA-seq analysis revealed upregulation of genes associated with tumor vasculature, collagen modification and extracellular matrix remodeling in TEC. PTGIR, PLAC9, CXCL12 and VDR were identified as TEC markers and confirmed by IF and IHC in an independent patient cohort. By scRNA-seq we identified 27 cell (sub)types, including endothelial cells (EC) with arterial, venous and immature signatures, as well as angiogenic tip EC. A focused molecular analysis revealed that arterial TEC displayed highest CXCL12 mRNA expression levels when compared to all other TME cell (sub)populations and showed a negative prognostic role. Receptor-ligand interaction analysis predicted interactions between arterial TEC derived CXCL12 and its cognate receptor CXCR4 on angiogenic tip EC. CXCL12 was in vitro and in vivo validated as actionable TEC target by highlighting the vessel number- and density- reducing activity of the CXCR4-inhibitor AMD3100 in murine PCa as well as by inhibition of TEC proliferation and migration in vitro. CONCLUSIONS: Overall, our comprehensive analysis identified novel PCa TEC targets and highlights CXCR4/CXCL12 interaction as a potential novel target to interfere with tumor angiogenesis in PCa.

High hepatocyte growth factor expression in primary tumor predicts better overall survival in male breast cancer.

BACKGROUND: Breast cancer is rare in men, but management is focused on tumor characteristics commonly found in female breast cancer. The tumor microenvironment of male breast cancer is less well understood, and insight may improve male breast cancer management. The hepatocyte growth factor (HGF)/c-MET axis and the stromal cell-derived factor-1 (CXCL12)/C-X-C chemokine receptor type 4 (CXCR4) axis are prognostic in women with breast cancer. We aimed to investigate these factors in male breast cancer and correlate them with patient survival. METHODS: From 841 Dutch males with breast cancer who were enrolled in the EORTC 10085/TBCRC/BIG/NABCG International Male Breast Cancer Program (NCT01101425) and diagnosed between 1990 and 2010, archival primary tumor samples were collected. Tissue microarrays were constructed with 3 cores per sample and used for immunohistochemical analysis of HGF, c-MET, CXCL12, and CXCR4. Overall survival (OS) of the patients without metastases (M0) was analyzed using the Kaplan-Meier method. The value of the markers regarding OS was determined using univariable and multivariable Cox regression analyses, providing hazard ratios (HRs) and 95% confidence intervals (95% CIs). RESULTS: Of 720 out of 841 patients, sufficient tissue was available for analysis; 487 out of 720 patients had M0 disease. Patients with high HGF expression and high CXCL12 expression had a superior OS (low vs high expression of both markers, 7.5 vs 13.0 years, hazard ratio [HR] 0.64, 95% CI 0.49-0.84, P = 0.001 [HGF]; 9.1 vs 15.3 years, HR 0.63, 95% CI 0.45-0.87, P = 0.005 [CXCL12]). Multivariate analysis identified HGF as an independent predictor for OS (HR 0.64, 95% CI 0.47-0.88, P = 0.001). CONCLUSIONS: HGF and CXCL12 tumor expression appear to identify male breast cancer patients with a relatively good prognosis. Possibly, this could support male breast cancer-specific management strategies in the future.

Hormone receptors as a marker of poor survival in epithelial ovarian cancer.

OBJECTIVE: Androgen receptor (AR), estrogen receptor α and ß (ERα, ERß), and progesterone receptor (PR) are potential therapeutic targets in epithelial ovarian cancer. In this study we evaluate the prognostic value of these hormone receptors in ovarian cancer patients. METHODS: In a prospective multicenter randomized controlled phase II trial 196 ovarian cancer patients were randomized to carboplatin/docetaxel±celecoxib. Of 121 patients sufficient tumor tissue was available for hormone receptor analysis. Tissue micro-arrays were stained for AR, ERα, ERß, and PR. Cluster analysis was performed to identify subgroups based on hormone receptor expression profile. Receptor expression was correlated to progression-free survival (PFS) and overall survival (OS) in uni- and multivariate analysis. RESULTS: AR, ERα, ERß, and PR were expressed in respectively 10%, 31%, 73%, and 19%. In patients with synchronous metastasis tissue available (n=69 patients), discordant receptor expression was observed in 9-32%. ERß-expression was associated with poor PFS and OS (hazard ratios 1.88 and 1.92). Clustering analysis revealed a subgroup with hormone receptor negative disease that had a favorable PFS and OS. CONCLUSION: Hormone receptors are expressed in the majority of ovarian cancer tumors and may serve as therapeutic targets. Clustering analysis can reveal subgroups with different outcome, which may prove valuable in selecting patients for endocrine therapy.

Lapatinib and 17AAG reduce 89Zr-trastuzumab-F(ab')2 uptake in SKBR3 tumor xenografts.

Human epidermal growth factor receptor-2 (HER2) directed therapy potentially can be improved by insight in drug effects on HER2 expression. This study evaluates the effects of the EGFR/HER2 tyrosine kinase inhibitor lapatinib, the heat shock protein-90 inhibitor 17AAG, and their combination, on HER2 expression with in vivo HER2-PET imaging. Lapatinib and 17AAG effects on EGFR and HER2 membrane expression were determined in vitro using flow cytometry of human SKBR3 tumor cells. Effect of lapatinib on HER2 internalization was studied in vitro by (89)Zr-trastuzumab-F(ab')(2) internalization. For in vivo evaluation, (89)Zr-trastuzumab-F(ab')(2) µPET imaging was performed two times with a 7 day interval. Lapatinib was administered for 6 days, starting 1 day after the baseline scan. 17AAG was given 1 day before the second (89)Zr-trastuzumab-F(ab')(2) injection. Imaging data were compared with ex vivo biodistribution analysis and HER2 immunohistochemical staining. 17AAG treatment lowered EGFR expression by 41% (P = 0.016) and HER2 by 76% (P = 0.022). EGFR/HER2 downregulation by 17AAG was inhibited by lapatinib pretreatment. Lapatinib reduced internalization of (89)Zr-trastuzumab-F(ab')(2) with 25% (P = 0.0022). (89)Zr-trastuzumab-F(ab')(2) tumor to blood ratio was lowered 32% by lapatinib (P = 0.00004), 34% by 17AAG (P = 0.0022) and even 53% by the combination (P = 0.011). Lapatinib inhibits HER2 internalization and 17AAG lowers HER2 membrane expression. Both drugs reduce (89)Zr-trastuzumab-F(ab')(2) tumor uptake. Based on our findings, supported by previous preclinical data indicating the antitumor potency of lapatinib in combination with HSP90 inhibition, combination of these drugs deserves further investigation.

Fluorescent image-guided surgery in breast cancer by intravenous application of a quenched fluorescence activity-based probe for cysteine cathepsins in a syngeneic mouse model.

PURPOSE: The reoperation rate for breast-conserving surgery is as high as 15-30% due to residual tumor in the surgical cavity after surgery. In vivo tumor-targeted optical molecular imaging may serve as a red-flag technique to improve intraoperative surgical margin assessment and to reduce reoperation rates. Cysteine cathepsins are overexpressed in most solid tumor types, including breast cancer. We developed a cathepsin-targeted, quenched fluorescent activity-based probe, VGT-309, and evaluated whether it could be used for tumor detection and image-guided surgery in syngeneic tumor-bearing mice. METHODS: Binding specificity of the developed probe was evaluated in vitro. Next, fluorescent imaging in BALB/c mice bearing a murine breast tumor was performed at different time points after VGT-309 administration. Biodistribution of VGT-309 after 24 h in tumor-bearing mice was compared to control mice. Image-guided surgery was performed at multiple time points tumors with different clinical fluorescent camera systems and followed by ex vivo analysis. RESULTS: The probe was specifically activated by cathepsins X, B/L, and S. Fluorescent imaging revealed an increased tumor-to-background contrast over time up to 15.1 24 h post probe injection. In addition, VGT-309 delineated tumor tissue during image-guided surgery with different optical fluorescent imaging camera systems. CONCLUSION: These results indicate that optical fluorescent molecular imaging using the cathepsin-targeted probe, VGT-309, may improve intraoperative tumor detection, which could translate to more complete tumor resection when coupled with commercially available surgical tools and techniques.

Androgen receptor expression inversely correlates with immune cell infiltration in human epidermal growth factor receptor 2-positive breast cancer.

INTRODUCTION: Although targeting human epidermal growth factor receptor 2 (HER2) is a meaningful treatment in HER2-positive breast cancer, ultimately resistance develops. Androgen receptor (AR) expression and immune cell infiltration are thought to be involved in trastuzumab response and may, therefore, be of interest as additional targets for therapy in HER2-positive breast cancer. AIM: To improve insights into the presence among AR expression, immune cell infiltration and HER2, we analysed HER2-positive breast tumours. METHODS: Primary tumours of 221 patients treated with trastuzumab for metastatic disease were selected. HER2 status was centrally confirmed. AR, T-cells (CD3 and CD8), programmed cell death protein 1 (PD-1) and PD-1 ligand 1 immunohistochemical staining and M2 tumour-associated macrophages (TAMs; CD68 and CD163) immunofluorescence were performed. Tumour-infiltrating lymphocytes were evaluated by haematoxylin and eosin staining. RESULTS: Sufficient tumour material was available for 150 patients. Oestrogen receptor was expressed in 51.3% of the tumours and AR in 81.3% of the tumours. AR expression was inversely correlated with M2 TAM (Pearson's r = -0.361, P < 0.001), CD3+ (r = -0.199, P < 0.030) and CD8+ (r = -0.212, P < 0.021) T-cell infiltration. Clustering analysis showed high immune cell infiltration in AR low-expressing tumours, and low immune cell infiltration in AR-high expressing tumours. CONCLUSION: AR expression inversely correlates with immune cell infiltration in HER2-positive breast cancer.

Reduced inflammatory response in cigarette smoke exposed Mrp1/Mdr1a/1b deficient mice.

BACKGROUND: Tobacco smoke is the principal risk factor for chronic obstructive pulmonary disease (COPD), though the mechanisms of its toxicity are still unclear. The ABC transporters multidrug resistance-associated protein 1 (MRP1) and P-glycoprotein (P-gp/MDR1) extrude a wide variety of toxic substances across cellular membranes and are highly expressed in bronchial epithelium. Their impaired function may contribute to COPD development by diminished detoxification of noxious compounds in cigarette smoke. METHODS: We examined whether triple knock-out (TKO) mice lacking the genes for Mrp1 and Mdr1a/1b are more susceptible to develop COPD features than their wild-type (WT) littermates. TKO and WT mice (six per group) were exposed to 2 cigarettes twice daily by nose-only exposure or room air for 6 months. Inflammatory infiltrates were analyzed in lung sections, cytokines and chemokines in whole lung homogenates, emphysema by mean linear intercept. Multiple linear regression analysis with an interaction term was used to establish the statistical significances of differences. RESULTS: TKO mice had lower levels of interleukin (IL)-7, KC (mouse IL-8), IL-12p70, IL-17, TNF-alpha, G-CSF, GM-CSF and MIP-1-alpha than WT mice independent of smoke exposure (P < 0.05). IL-1-alpha, IL-6, IL-8, IL-13, IL-17, TNF-alpha, G-CSF, GM-CSF and MCP-1 increased after smoke exposure in both groups, but the increase in IL-8 was lower in TKO than WT mice (P < 0.05) with a same trend for G-CSF (P < 0.10). Smoke-induced increase in pulmonary inflammatory cells in WT mice was almost absent in TKO mice. The mean linear intercept was not different between groups. CONCLUSION: Mrp1/Mdr1a/1b knock-out mice have a reduced inflammatory response to cigarette smoke. In addition, the expression levels of several cytokines and chemokines were also lower in lungs of Mrp1/Mdr1a/1b knock-out mice independent of smoke exposure. Further studies are required to determine whether dysfunction of MRP1 and/or P-gp contribute to the pathogenesis of COPD.

Studying cancer metastasis: Existing models, challenges and future perspectives.

Cancer metastasis causes most cancer-related deaths. Several model systems to study the complex and multi step process of metastasis exist, including in vitro systems, ex-vivo organ slices, Drosophila Melanogaster and zebrafish models and the use of the chorio allantoic membrane (CAM) of fertilized chicken eggs. These models are relatively easy and cheap but often lack the opportunity to study the complete metastasis cascade. More complex but also more expensive is the use of animal models including the more recently developed patient derived tumor xenografts (PDTX). In this review, we give an overview of the existing metastatic models, discuss the challenges of improving current models to enhance translation from the preclinical to the clinical setting and consider future perspectives.

Difference in CXCR4 expression between sporadic and VHL-related hemangioblastoma.

Central nervous system hemangioblastomas occur sporadically and in patients with von Hippel-Lindau (VHL) disease due to a VHL germline mutation. This mutation leads to enhanced transcription of chemokine receptor 4 (CXCR4), its ligand (CXCL12) and vascular endothelial growth factor A (VEGFA). We aimed to determine in VHL-related and sporadic hemangioblastomas CXCR4, CXCL12, and VEGFA protein expression and to correlate this to hemangioblastoma size and expression in normal surrounding tissue. 27 patients with a hemangioblastoma were included for analysis of immunohistochemistry of tissue, MRI and DNA. Hemangioblastomas overexpress CXCR4, CXCL12, and VEGFA compared to normal surrounding tissue. In sporadic hemangioblastomas the mean percentage of CXCR4 positive hemangioblastoma cells was 16 %, SD 8.4, in VHL-related hemangioblastomas 8 %, SD 4.4 (P = 0.002). There was no relation between preoperative tumor size and CXCR4 or CXCL12 expression. Compared to normal surrounding tissue CXCR4, CXCL12, and VEGFA were overexpressed in hemangioblastomas. Most interestingly, sporadic hemangioblastomas overexpress CXCR4 compared to VHL-related hemangioblastoma.

Molecular Fluorescence Endoscopy Targeting Vascular Endothelial Growth Factor A for Improved Colorectal Polyp Detection.

UNLABELLED: Small and flat adenomas are known to carry a high miss-rate during standard white-light endoscopy. Increased detection rate may be achieved by molecular fluorescence endoscopy with targeted near-infrared (NIR) fluorescent tracers. The aim of this study was to validate vascular endothelial growth factor A (VEGF-A) and epidermal growth factor receptor (EGFR)-targeted fluorescent tracers during ex vivo colonoscopy with an NIR endoscopy platform. METHODS: VEGF-A and EGFR expression was determined by immunohistochemistry on a large subset of human colorectal tissue samples--48 sessile serrated adenomas/polyps, 70 sporadic high-grade dysplastic adenomas, and 19 hyperplastic polyps--and tissue derived from patients with Lynch syndrome--78 low-grade dysplastic adenomas, 57 high-grade dysplastic adenomas, and 31 colon cancer samples. To perform an ex vivo colonoscopy procedure, 14 mice with small intraperitoneal EGFR-positive HCT116(luc) tumors received intravenous bevacizumab-800CW (anti-VEGF-A), cetuximab-800CW (anti-EGFR), control tracer IgG-800CW, or sodium chloride. Three days later, 8 resected HCT116(luc) tumors (2-5 mm) were stitched into 1 freshly resected human colon specimen and followed by an ex vivo molecular fluorescence colonoscopy procedure. RESULTS: Immunohistochemistry showed high VEGF-A expression in 79%-96% and high EGFR expression in 51%-69% of the colorectal lesions. Both targets were significantly overexpressed in the colorectal lesions, compared with the adjacent normal colon crypts. During ex vivo molecular fluorescence endoscopy, all tumors could clearly be delineated for both bevacizumab-800CW and cetuximab-800CW tracers. Specific tumor uptake was confirmed with fluorescent microscopy showing, respectively, stromal and cell membrane fluorescence. CONCLUSION: VEGF-A is a promising target for molecular fluorescence endoscopy because it showed a high protein expression, especially in sessile serrated adenomas/polyps and Lynch syndrome. We demonstrated the feasibility to visualize small tumors in real time during colonoscopy using a NIR fluorescence endoscopy platform, providing the endoscopist a wide-field red-flag technique for adenoma detection. Clinical studies are currently being performed in order to provide in-human evaluation of our approach.

ATP-binding cassette (ABC) transporters in normal and pathological lung.

ATP-binding cassette (ABC) transporters are a family of transmembrane proteins that can transport a wide variety of substrates across biological membranes in an energy-dependent manner. Many ABC transporters such as P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) are highly expressed in bronchial epithelium. This review aims to give new insights in the possible functions of ABC molecules in the lung in view of their expression in different cell types. Furthermore, their role in protection against noxious compounds, e.g. air pollutants and cigarette smoke components, will be discussed as well as the (mal)function in normal and pathological lung. Several pulmonary drugs are substrates for ABC transporters and therefore, the delivery of these drugs to the site of action may be highly dependent on the presence and activity of many ABC transporters in several cell types. Three ABC transporters are known to play an important role in lung functioning. Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene can cause cystic fibrosis, and mutations in ABCA1 and ABCA3 are responsible for respectively Tangier disease and fatal surfactant deficiency. The role of altered function of ABC transporters in highly prevalent pulmonary diseases such as asthma or chronic obstructive pulmonary disease (COPD) have hardly been investigated so far. We especially focused on polymorphisms, knock-out mice models and in vitro results of pulmonary research. Insight in the function of ABC transporters in the lung may open new ways to facilitate treatment of lung diseases.

Human stromal cells are required for an anti-breast cancer effect of zoledronic acid.

Previous studies suggested that bisphosphonate zoledronic acid exerts an anti-tumor effect by interacting with the microenvironment. In this study, we aimed to elucidate the mechanism behind the anti-breast cancer effect of zoledronic acid.Here we showed that zoledronic acid did not influence in vitro human breast cancer cell survival, but did affect human stromal cell survival. Breast cancer cell death in co-culture with stromal cells was analyzed in vitro by fluorescent microscopy and flowcytometry analysis. In co-culture, the addition of stromal cells to breast cancer cells induced tumor cell death by zoledronic acid, which was abolished by transforming growth factor (TGF)-ß. In the in vivo chicken chorioallantoic membrane model, zoledronic acid reduced the breast cancer cells fraction per tumor only in the presence of human stromal cells. Zoledronic acid decreased TGF-ß excretion by stromal cells and co-cultures. Moreover, supernatant of zoledronic acid treated stromal cells reduced phospho-Smad2 protein levels in breast cancer cells. Thus, zoledronic acid exerts an anti-breast cancer effect via stromal cells, accompanied by decreased stromal TGF-ß excretion and reduced TGF-ß signaling in cancer cells.

CXCR4 and CXCL12 Expression in Rectal Tumors of Stage IV Patients Before and After Local Radiotherapy and Systemic Neoadjuvant Treatment.

Metastatic rectal cancer patients could benefit from novel therapeutic approaches. The signaling network formed by chemokines and their receptors can promote metastasis and resistance to current anticancer treatments. This study assessed the expression of chemokine receptor 4 (CXCR4) and its ligand CXCL12 immuhistochemically in stage IV rectal tumors. Paraffin-embedded primary tumor collected before and after local radiotherapy and systemic treatment with bevacizumab, oxaliplatin and capecitabine was analyzed. Receptor and ligand expression was assessed in the cytoplasm and nucleus of tumor, stromal and normal rectal crypt cells. Baseline expression of CXCR4 and CXCL12 was correlated with patients' pathologic response to treatment. At diagnosis (n=46), 89% of the rectal tumors expressed cytoplasmic CXCR4 and 81% CXCL12. Nuclear CXCR4 expression in tumor cells was present in 30% and nuclear CXCL12 expression in 35% of the tumors. After radiochemotherapy and administration of bevacizumab, nuclear CXCL12 expression was observed in 79% of residual tumors, as compared to 31% of the paired tumor samples expressing nuclear CXCL12 before treatment (P=0.001). There were no differences in CXCR4 or CXCL12 expression at baseline between the patients who had (n=9) and did not have (n=30) a pathologic complete response. Our results show that CXCR4 and CXCL12 are extensively expressed in primary rectal tumors of patients presenting with metastatic disease, while radiochemotherapy and bevacizumab further upregulate CXCL12 expression. These data indicate the importance of the CXCR4/CXCL12 axis in rectal tumor biology, and may suggest the CXCR4/CXCL12 receptor-ligand pair as a potential therapeutic target in metastatic rectal cancer.

HER3, serious partner in crime: therapeutic approaches and potential biomarkers for effect of HER3-targeting.

The human epidermal growth factor receptor (HER) family members are targeted by a growing numbers of small molecules and monoclonal antibodies. Resistance against the epidermal growth factor receptor (EGFR) and HER2-targeting agents is a clinically relevant problem forcing research on optimizing targeting of the HER family. In view of its overexpression in tumors, and compensatory role in HER signaling, HER3 has gained much interest as a potential additional target within the HER family. It is the only member of the HER family lacking intrinsic tyrosine kinase activity and therefore its role in cancer has long been underestimated. Drugs that block HER3 or interfere with HER3 dimer signaling, including fully human anti-HER3 antibodies, bispecific antibodies and tyrosine kinase inhibitors (TKIs), are currently becoming available. Several compounds have already entered clinical trial. In the meantime potential biomarkers are tested such as tumor analysis of HER3 expression, functional assays for downstream effector molecules and molecular imaging techniques. This review describes the biology and relevance of HER3 in cancer, agents targeting HER3 and potential biomarkers for effect of HER3-targeting.

CXCR4 inhibition enhances radiosensitivity, while inducing cancer cell mobilization in a prostate cancer mouse model.

Preclinical studies show that stroma affects sensitivity of prostate cancer cells via activation of the CXCR4/CXCL12 pathway. Here we studied the effect of CXCR4 inhibition combined with irradiation in prostate cancer cells. In an in vitro co-culture with stromal cells, the CXCR4 inhibitor AMD3100 sensitized prostate cancer cell lines PC3-Luc and LNCaP to irradiation (P = 0.04). Tumor growth and metastasis were evaluated in mice xenografted with luciferase-expressing PC3 cells that received 5 Gy irradiation weekly ± 3.5 mg/kg AMD3100 daily intraperitoneally. The irradiated xenografts showed higher CXCR4 (P = 0.006) and CXCL12 (P = 0.01) expression, compared to controls. AMD3100 sensitized the xenografts to irradiation at the fourth week of treatment (P = 0.02). However AMD3100 also mobilized tumor cells at days 14 and 21 (P < 0.0001), as shown by bioluminescent imaging. In conclusion, AMD3100 transiently enhances prostate cancer radiosensitivity, but induces cancer cell mobilization.

Visualising dual downregulation of insulin-like growth factor receptor-1 and vascular endothelial growth factor-A by heat shock protein 90 inhibition effect in triple negative breast cancer.

PURPOSE: Triple negative breast cancer (TNBC) is biologically characterised by heterogeneous presence of molecular pathways underlying it. Insulin-like growth factor receptor-1 (IGF-1R) expression and vascular endothelial growth factor-A (VEGF-A) have been identified as key factors in these pathways in TNBC. In this study, we aimed at in vivo PET imaging the effect of heat shock protein (Hsp) 90 inhibition by means of NVP-AUY922 on these pathways, with zirconium-89 ((89)Zr) labelled antibodies targeting IGF-1R and VEGF-A. MATERIALS AND METHODS: In vitro NVP-AUY922 effects on cellular IGF-1R expression and VEGF-A secretion were determined in MCF-7 and MDA-MB-231 cell lines. Moreover human TNBC bearing MDA-MB-231 mice received 50mg/kg NVP-AUY922 or vehicle q3d intraperitoneally for 21days. PET scans with (89)Zr-MAB391 and (89)Zr-bevacizumab for visualisation of IGF-1R and VEGF-A were performed before and during treatment. Ex vivo biodistribution and correlative tissue analyses were performed. RESULTS: NVP-AUY922 treatment reduced IGF-1R expression and VEGF-A excretion in both cell lines. Hsp90 inhibition lowered tumour uptake on (89)Zr-MAB391-PET by 37.3% (P<0.01) and on (89)Zr-bevacizumab-PET by 44.4% (P<0.01). This was confirmed by ex vivo biodistribution with a reduction of 41.3% injected dose (ID)/g for (89)Zr-MAB391 and 37.8% ID/g for (89)Zr-bevacizumab, while no differences were observed for other tissues. This coincided with reduced IGF-1R expression and mean vessel density in the NVP-AUY922 treated tumours. CONCLUSION: (89)Zr-MAB391 and (89)Zr-bevacizumab PET reflect effect of Hsp90 inhibitors and can therefore potentially be used to monitor therapeutic effects of Hsp90 inhibitor therapy in TNBC.

A review on CXCR4/CXCL12 axis in oncology: no place to hide.

Classical chemotherapeutic anti-cancer treatments induce cell death through DNA damage by taking advantage of the proliferative behaviour of cancer cells. The more recent approach of targeted therapy (usually protein-targeted) has led to many treatments that are currently available or are under development, all of which are designed to strike at the critical driving forces of cancer cells. The interaction of the cancer cells with their microenvironment is one of these fundamental features of neoplasms that could be targeted in such cancer treatments. Haematological and solid tumour cells interact with their microenvironment through membrane chemokine receptors and their corresponding ligands, which are expressed in the tumour microenvironment. Important representatives of this system are the chemokine ligand CXCL12 and its receptor chemokine receptor 4 (CXCR4). This interaction can be disrupted by CXCR4 antagonists, and this concept is being used clinically to harvest haematopoietic stem/progenitor cells from bone marrow. CXCR4 and CXCL12 also have roles in tumour growth and metastasis, and more recently their roles in cancer cell-tumour microenvironment interaction and angiogenesis have been studied. Our review focuses on these roles and summarises strategies for treating cancer by disrupting this interaction with special emphasis on the CXCR4/CXCL12 axis. Finally, we discuss ongoing clinical trials with several classes of CXCR4 inhibitors, and their potential additive value for patients with a (therapy resistant) malignancy by sensitising cancer cells to conventional therapy.

Bevacizumab-induced normalization of blood vessels in tumors hampers antibody uptake.

In solid tumors, angiogenesis occurs in the setting of a defective vasculature and impaired lymphatic drainage that is associated with increased vascular permeability and enhanced tumor permeability. These universal aspects of the tumor microenvironment can have a marked influence on intratumoral drug delivery that may often be underappreciated. In this study, we investigated the effect of blood vessel normalization in tumors by the antiangiogenic drug bevacizumab on antibody uptake by tumors. In mouse xenograft models of human ovarian and esophageal cancer (SKOV-3 and OE19), we evaluated antibody uptake in tumors by positron emission tomographic imaging 24 and 144 hours after injection of (89)Zr-trastuzumab (SKOV-3 and OE19), (89)Zr-bevacizumab (SKOV-3), or (89)Zr-IgG (SKOV-3) before or after treatment with bevacizumab. Intratumor distribution was assessed by fluorescence microscopy along with mean vessel density (MVD) and vessel normalization. Notably, bevacizumab treatment decreased tumor uptake and intratumoral accumulation compared with baseline in the tumor models relative to controls. Bevacizumab treatment also reduced MVD in tumors and increased vessel pericyte coverage. These findings are clinically important, suggesting caution in designing combinatorial trials with therapeutic antibodies due to a possible reduction in tumoral accumulation that may be caused by bevacizumab cotreatment.