Extracellular vesicles in glioblastoma: Biomarkers and therapeutic tools.

Glioblastoma (GBM) is the most aggressive tumor among the gliomas and intracranial tumors and to date prognosis for GBM patients remains poor, with a median survival typically measured in months to a few years depending on various factors. Although standardized therapies are routinely employed, it is clear that these strategies are unable to cope with heterogeneity and invasiveness of GBM. Furthermore, diagnosis and monitoring of responses to therapies are directly dependent on tissue biopsies or magnetic resonance imaging (MRI) techniques. From this point of view, liquid biopsies are arising as key sources of a variety of biomarkers with the advantage of being easily accessible and monitorable. In this context, extracellular vesicles (EVs), physiologically shed into body fluids by virtually all cells, are gaining increasing interest both as natural carriers of biomarkers and as specific signatures even for GBM. What makes these vesicles particularly attractive is they are also emerging as therapeutical vehicles to treat GBM given their native ability to cross the blood-brain barrier (BBB). Here, we reviewed recent advances on the use of EVs as biomarker for liquid biopsy and nanocarriers for targeted delivery of anticancer drugs in glioblastoma.

Lysosomal lipid switch sensitises to nutrient deprivation and mTOR targeting in pancreatic cancer.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with limited therapeutic options. However, metabolic adaptation to the harsh PDAC environment can expose liabilities useful for therapy. Targeting the key metabolic regulator mechanistic target of rapamycin complex 1 (mTORC1) and its downstream pathway shows efficacy only in subsets of patients but gene modifiers maximising response remain to be identified. DESIGN: Three independent cohorts of PDAC patients were studied to correlate PI3K-C2γ protein abundance with disease outcome. Mechanisms were then studied in mouse (KPC mice) and cellular models of PDAC, in presence or absence of PI3K-C2γ (WT or KO). PI3K-C2γ-dependent metabolic rewiring and its impact on mTORC1 regulation were assessed in conditions of limiting glutamine availability. Finally, effects of a combination therapy targeting mTORC1 and glutamine metabolism were studied in WT and KO PDAC cells and preclinical models. RESULTS: PI3K-C2γ expression was reduced in about 30% of PDAC cases and was associated with an aggressive phenotype. Similarly, loss of PI3K-C2γ in KPC mice enhanced tumour development and progression. The increased aggressiveness of tumours lacking PI3K-C2γ correlated with hyperactivation of mTORC1 pathway and glutamine metabolism rewiring to support lipid synthesis. PI3K-C2γ-KO tumours failed to adapt to metabolic stress induced by glutamine depletion, resulting in cell death. CONCLUSION: Loss of PI3K-C2γ prevents mTOR inactivation and triggers tumour vulnerability to RAD001 (mTOR inhibitor) and BPTES/CB-839 (glutaminase inhibitors). Therefore, these results might open the way to personalised treatments in PDAC with PI3K-C2γ loss.

Visualizing Galectin-3 Binding Protein Expression with ImmunoPET.

Galectin-3 binding protein (Gal-3BP) is a glycoprotein that is overexpressed and secreted by several cancers and has been implicated as a marker of both tumor progression and poor prognosis in melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, and breast cancer. The expression of Gal-3BP by a variety of neoplasms makes it an enticing target for both diagnostics and therapeutics, including immuno-positron emission tomography (immunoPET) probes and antibody-drug conjugates (ADCs). Herein, we report the development, in vitro characterization, and in vivo evaluation of a pair of Gal-3BP-targeting radioimmunoconjugates for 89Zr-immunoPET. A humanized anti-Gal-3BP antibody, 1959, and its corresponding ADC, 1959-sss/DM4 (DM4 = ravtansine), were modified with desferrioxamine (DFO) to yield DFO-1959 and DFO-1959-sss/DM4 immunoconjugates bearing 1-2 DFO/monoclonal antibody. Both DFO-modified immunoconjugates retained their affinity for Gal-3BP in enzyme-linked immunosorbent assay experiments. The chelator-bearing antibodies were radiolabeled with zirconium-89 (t1/2 ≈ 3.3 d) to produce radioimmunoconjugates ─ [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 ─ with high specific activity (>444 MBq/mg, >12 mCi/mg) and stability (>80% intact after 168 h in human serum at 37 °C). In mice bearing subcutaneous Gal-3BP-secreting A375-MA1 xenografts, [89Zr]Zr-DFO-1959 clearly delineated tumor tissue, reaching a maximum tumoral activity concentration (54.8 ± 15.8%ID/g) and tumor-to-background contrast (tumor-to-blood = 8.0 ± 4.6) at 120 h post-injection. The administration of [89Zr]Zr-DFO-1959 to mice bearing subcutaneous Gal-3BP-expressing melanoma patient-derived xenografts produced similarly promising results. [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 exhibited nearly identical pharmacokinetic profiles in the mice bearing A375-MA1 tumors, though the latter produced higher uptake in the spleen and kidneys. Both [89Zr]Zr-DFO-1959 and [89Zr]Zr-DFO-1959-sss/DM4 effectively visualized Gal-3BP-secreting tumors in murine models of melanoma. These results suggest that both probes could play a role in the clinical imaging of Gal-3BP-expressing malignancies, particularly as companion theranostics for the identification of patients likely to respond to Gal-3BP-targeted therapeutics such as 1959-sss/DM4.

LGALS3BP is a potential target of antibody-drug conjugates in oral squamous cell carcinoma.

OBJECTIVE: The aim of the present study was to evaluate the expression of intracellular and vesicular LGALS3BP in oral squamous cell carcinoma (OSCC) patients and available cell lines to explore its potential as a target for antibody-drug conjugate (ADC) therapy. METHODS: Free and vesicular LGALS3BP expression levels were evaluated in cancer tissues from a cohort of OSCC patients as well as in a panel of OSCC cell lines through immunohistochemistry, qRT-PCR, Western Blot analysis, and ELISA. RESULTS: LGALS3BP resulted in being highly expressed in the cytoplasm of tumour cells in OSCC patient tissues. A strong correlation was found between high LGALS3BP expression levels and aggressive histological features of OSCC. Biochemistry analysis performed on OSCC cell lines showed that LGALS3BP is expressed in all the tested cell lines and highly enriched in cancer-derived extracellular vesicles. Moreover, LGALS3BP high-expressing HOC621 and CAL27 OSCC cell lines showed high sensitivity to the ADC-payload DM4, with an IC50 around 0.3 nM. CONCLUSIONS: The present study highlights that LGALS3BP is highly expressed in OSCC suggesting a role as a potential diagnostic biomarker and therapeutic target for ADC-based therapy.

BAG3 induces fibroblasts to release key cytokines involved in pancreatic cell migration.

Pancreatic ductal adenoma carcinoma (PDAC) is considered one of the deadliest solid cancers as it is usually diagnosed in advanced stages and has a poor response to treatment. The enormous effort made in the last 2 decades in the oncology field has not led to significant progress in improving early diagnosis or therapy for PDAC. The stroma of PDAC plays an active role in tumour initiation and progression and includes immune cells and stromal cells. We previously reported that Bcl2-associated athanogene (BAG3) secreted by PDAC cells activates tumour-associated macrophages to promote tumour growth. The disruption of this tumour-stroma axis by the anti-BAG3 H2L4 therapeutic antibody is sufficient to delay tumour growth and limit metastatic spreading in different PDAC preclinical models. In the present study, we examined the role of BAG3 to activate human fibroblasts (HF) in releasing cytokines capable of supporting tumour progression. Treatment of fibroblasts with recombinant BAG3 induced important changes in the organisation of the cytoskeleton of these cells and stimulated the production of interleukin-6, monocyte chemoattractant protein-1/C-C motif chemokine ligand 2, and hepatocyte growth factor. Specifically, we observed that BAG3 triggered a depolymerisation of microtubules at the periphery of the cell while they were conserved in the perinuclear area. Conversely, the vimentin-based intermediate filaments increased and spread to the edges of the cells. Finally, the conditioned medium (CM) collected from BAG3-treated HF promoted the survival, proliferation, and migration of the PDAC cells. Blocking of the PDAC-fibroblast axis by the H2L4 therapeutic anti-BAG3 antibody, resulted in inhibition of cytokine release and, consequently, the inhibition of the migratory phenotype conferred by the CM to PDAC cells.

Role of galectin 3 binding protein in cancer progression: a potential novel therapeutic target.

The lectin galactoside-binding soluble 3 binding protein (LGALS3BP) is a secreted, hyperglycosylated protein expressed by the majority of human cells. It was first identified as cancer and metastasis associated protein, while its role in innate immune response upon viral infection remains still to be clarified. Since its discovery dated in early 90 s, a large body of literature has been accumulating highlighting both a prognostic and functional role for LGALS3BP in cancer. Moreover, data from our group and other have strongly suggested that this protein is enriched in cancer-associated extracellular vesicles and may be considered a promising candidate for a targeted therapy in LGALS3BP positive cancers. Here, we extensively reviewed the literature relative to LGALS3BP role in cancer and its potential value as a therapeutic target.

Role of BAG3 in cancer progression: A therapeutic opportunity.

BAG3 is a multifunctional protein that can bind to heat shock proteins (Hsp) 70 through its BAG domain and to other partners through its WW domain, proline-rich (PXXP) repeat and IPV (Ile-Pro-Val) motifs. Its intracellular expression can be induced by stressful stimuli, while is constitutive in skeletal muscle, cardiac myocytes and several tumour types. BAG3 can modulate the levels, localisation or activity of its partner proteins, thereby regulating major cell pathways and functions, including apoptosis, autophagy, mechanotransduction, cytoskeleton organisation, motility. A secreted form of BAG3 has been identified in studies on pancreatic ductal adenocarcinoma (PDAC). Secreted BAG3 can bind to a specific receptor, IFITM2, expressed on macrophages, and induce the release of factors that sustain tumour growth and the metastatic process. BAG3 neutralisation therefore appears to constitute a novel potential strategy in the therapy of PDAC and, possibly, other tumours.

miR-574-5p as RNA decoy for CUGBP1 stimulates human lung tumor growth by mPGES-1 induction.

MicroRNAs (miRs) are important posttranscriptional regulators of gene expression. Besides their well-characterized inhibitory effects on mRNA stability and translation, miRs can also activate gene expression. In this study, we identified a novel noncanonical function of miR-574-5p. We found that miR-574-5p acts as an RNA decoy to CUG RNA-binding protein 1 (CUGBP1) and antagonizes its function. MiR-574-5p induces microsomal prostaglandin E synthase-1 (mPGES-1) expression by preventing CUGBP1 binding to its 3'UTR, leading to an enhanced alternative splicing and generation of an mPGES-1 3'UTR isoform, increased mPGES-1 protein expression, PGE2 formation, and tumor growth in vivo. miR-574-5p-induced tumor growth in mice could be completely inhibited with the mPGES-1 inhibitor CIII. Moreover, miR-574-5p is induced by IL-1ß and is strongly overexpressed in human nonsmall cell lung cancer where high mPGES-1 expression correlates with a low survival rate. The discovered function of miR-574-5p as a CUGBP1 decoy opens up new therapeutic opportunities. It might serve as a stratification marker to select lung tumor patients who respond to the pharmacological inhibition of PGE2 formation.-Saul, M. J., Baumann, I., Bruno, A., Emmerich, A. C., Wellstein, J., Ottinger, S. M., Contursi, A., Dovizio, M., Donnini, S., Tacconelli, S., Raouf, J., Idborg, H., Stein, S., Korotkova, M., Savai, R., Terzuoli, E., Sala, G., Seeger, W., Jakobsson, P.-J., Patrignani, P., Suess, B., Steinhilber, D. miR-574-5p as RNA decoy for CUGBP1 stimulates human lung tumor growth by mPGES-1 induction.

Antibody-Drug Conjugates: The New Frontier of Chemotherapy.

In recent years, antibody-drug conjugates (ADCs) have become promising antitumor agents to be used as one of the tools in personalized cancer medicine. ADCs are comprised of a drug with cytotoxic activity cross-linked to a monoclonal antibody, targeting antigens expressed at higher levels on tumor cells than on normal cells. By providing a selective targeting mechanism for cytotoxic drugs, ADCs improve the therapeutic index in clinical practice. In this review, the chemistry of ADC linker conjugation together with strategies adopted to improve antibody tolerability (by reducing antigenicity) are examined, with particular attention to ADCs approved by the regulatory agencies (the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA)) for treating cancer patients. Recent developments in engineering Immunoglobulin (Ig) genes and antibody humanization have greatly reduced some of the problems of the first generation of ADCs, beset by problems, such as random coupling of the payload and immunogenicity of the antibody. ADC development and clinical use is a fast, evolving area, and will likely prove an important modality for the treatment of cancer in the near future.

PLC-gamma-1 phosphorylation status is prognostic of metastatic risk in patients with early-stage Luminal-A and -B breast cancer subtypes.

BACKGROUND: Phospholipase Cγ1 (PLCγ1) is highly expressed in human tumours. Our previous studies reported that both stable and inducible PLCγ1 down-regulation can inhibit formation of breast-cancer-derived experimental lung metastasis. Further, high expression of PLCγ1 and its constitutively activated forms (i.e., PLCγ1-pY1253, PLCγ1-pY783) is associated with worse clinical outcome in terms of incidence of distant metastases, but not of local relapse in T1-T2, N0 breast cancer patients. METHODS: In the present retrospective study, we analysed the prognostic role of PLCγ1 in early breast cancer patients stratified according to the St. Gallen criteria and to their menopausal status. PLCγ1-pY1253 and PLCγ1-pY783 protein expression levels were determined by immunohistochemistry on tissue microarrays, and were correlated with patients' clinical data, using univariate and multivariate statistical analyses. RESULTS: In our series, the prognostic value of PLCγ1 overexpression was restricted to Luminal type tumours. From multivariate analyses, pY1253-PLCγ1High was an independent prognostic factor only in postmenopausal patients with Luminal-B tumours (hazard ratio [HR], 2.4; 95% confidence interval [CI], 1.1-5.3; P = 0.034). Conversely, PLCγ1-pY783High was a remarkably strong risk factor (HR, 20.1; 95% CI, 2.2-178.4; P = 0.003) for pre/perimenopausal patients with Luminal-A tumours. CONCLUSIONS: PLCγ1 overexpression is a strong predictive surrogate marker of development of metastases in early Luminal-A and -B breast cancer patients, being able to discriminate patients with high and low risk of metastases. Therefore, targeting the PLCγ1 pathway can be considered of potential benefit for prevention of metastatic disease.

Functional and prognostic significance of the genomic amplification of frizzled 6 (FZD6) in breast cancer.

Frizzled receptors mediate Wnt ligand signalling, which is crucially involved in regulating tissue development and differentiation, and is often deregulated in cancer. In this study, we found that the gene encoding the Wnt receptor frizzled 6 (FZD6) is frequently amplified in breast cancer, with an increased incidence in the triple-negative breast cancer (TNBC) subtype. Ablation of FZD6 expression in mammary cancer cell lines: (1) inhibited motility and invasion; (2) induced a more symmetrical shape of organoid three-dimensional cultures; and (3) inhibited bone and liver metastasis in vivo. Mechanistically, FZD6 signalling is required for the assembly of the fibronectin matrix, interfering with the organization of the actin cytoskeleton. Ectopic delivery of fibronectin in FZD6-depleted, triple-negative MDA-MB-231 cells rearranged the actin cytoskeleton and restored epidermal growth factor-mediated invasion. In patients with localized, lymph node-negative (early) breast cancer, positivity of tumour cells for FZD6 protein identified patients with reduced distant relapse-free survival. Multivariate analysis indicated an independent prognostic significance of FZD6 expression in TNBC tumours, predicting distant, but not local, relapse. We conclude that the FZD6-fibronectin actin axis identified in our study could be exploited for drug development in highly metastatic forms of breast cancer, such as TNBC. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

The role of phospholipase Cγ1 in breast cancer and its clinical significance.

Breast cancer, the most common malignancy among women, is usually detected at an early stage and has a low risk of relapse. Nevertheless, a significant number of patients cannot be cured solely by local treatment. Distinguishing between patients who are of low risk of relapse from those who are of high risk may have important implications to improve treatment outcomes. The PLC-γ1 signaling pathway promotes many physiological processes, including cell migration and invasion. Increasing evidence shows aberrant PLC-γ1 signaling implication in carcinogenesis including breast cancer. In this review, the role of PLC-γ1 in breast cancer and its clinical implications will be discussed, as well as its potential as a prognostic factor and a therapeutic target.

Therapeutic Efficacy of the Novel Stimuli-Sensitive Nano-Ferritins Containing Doxorubicin in a Head and Neck Cancer Model.

Doxorubicin is employed alone or in combination for the treatment of several hematological and solid malignancies; despite its efficacy, there are associated cardiotoxicity limits both in its application in patients with heart disease risk factors and also in its long-term use. HFt-MP-PAS40 is a genetically engineered human ferritin heavy chain (HFt)-based construct able to efficiently entrap and deliver doxorubicin to cancer cells. HF-MP-PAS contains a short motif sequence (defined as MP) responsive to proteolytic cleavage by tumor matrix metalloproteases (MMPs), located between each HFt subunit and a masking polypeptide sequence rich in proline (P), alanine (A), and serine (S) residues (PAS). This carrier displayed excellent therapeutic efficacy in a xenogenic pancreatic cancer model in vivo, leading to a significant increase in overall animal survival in treated mice. Herein, we describe the HFt-MP-PAS40-Dox efficacy against squamous cell carcinomas of the head and neck (HNSCC) with the goal of validating the application of our nano-drug for the treatment of different solid tumors. In addition, a tolerability study in healthy mice was also performed. The results indicate that HFt-MP-PAS40-Dox produced increased anti-tumor effects both in vitro and in vivo in comparison to the free drug in several HNSCC cell lines. In the acute toxicity studies, the maximum tolerated dose (MTD) of HFt-MP-PAS40-Dox was about 3.5 higher than the free drug: 25 mg/kg versus 7 mg/kg doxorubicin equivalents. Importantly, evaluation of heart tissues provided evidence that doxorubicin is less cardio-toxic when encapsulated inside the ferritin carrier. In conclusion, HFt-MP-PAS40-Dox may be administered safely at higher doses compared with the free drug, resulting in superior efficacy to control HNSCC malignancies.

The mitotic checkpoint kinase BUB1 is a direct and actionable target of MYB in adenoid cystic carcinoma.

Adenoid cystic carcinoma (ACC) is a head and neck cancer that frequently originates in salivary glands, but can also strike other exocrine glands such as the breast. A key molecular alteration found in the majority of ACC cases is MYB gene rearrangements, leading to activation of the oncogenic transcription factor MYB. In this study, we used immortalised breast epithelial cells and an inducible MYB transgene as a model of ACC. Molecular profiling confirmed that MYB-driven gene expression causes a transition into an ACC-like state. Using this new cell model, we identified BUB1 as a targetable kinase directly controlled by MYB, whose pharmacological inhibition caused MYB-dependent synthetic lethality, growth arrest and apoptosis of patient-derived cells and organoids.

Anti-LGALS3BP antibody-drug conjugate treatment induces durable and potent antitumor response in a preclinical model of adenoid cystic carcinoma.

OBJECTIVES: Adenoid cystic carcinoma (ACC) is a rare type of cancer that typically arises from glandular tissues, most commonly in the salivary glands. Although relatively rare, it represents a serious clinical issue as the management of the disease is highly complex being the only therapeutic options represented by invasive surgery and/or radiotherapy. In the present study, we have explored the potential of galectin-3 binding protein (LGALS3BP) as a novel target for antibody-drug conjugate (ADC) therapy in ACC. MATERIALS AND METHODS: RNAseq was conducted on a panel of 10 ACC patient-derived xenografts (PDX)s tissues and 6 normal salivary glands to analyze LGALS3BP gene expression. Protein expression was assessed in ACC PDX and primary tumor tissues using immunohistochemistry. Anti-LGALS3BP ADC named 1959-sss/DM4, was tested in high LGALS3BP expressing ACC PDX model ST1502B. RESULTS: RNAseq analysis revealed that LGALS3BP expression was highly expressed in ACC PDX tissues compared to normal salivary gland tissues. As evaluated by immunohistochemical analysis, LGALS3BP protein was found to be heterogeneously expressed in 10 ACC PDX and in tumor tissues derived from a cohort of 37 ACC patients. Further, treatment with 1959-sss/DM4 ADC led to durable tumor growth inhibition (TGI) in 100% of animals without observed toxicity. CONCLUSIONS: Our study provides strong evidence that LGALS3BP is a promising therapeutic target for ACC, warranting further expedited preclinical and clinical investigation.

HPLC-DAD validated method for DM4 and its metabolite S-Me-DM4 quantification in biological matrix for clinical and pharmaceutical applications.

The present study focuses on the development and validation of an HPLC-DAD methodology for the detection of a potent chemotherapeutic agent, Maytansinoid Ravtansine (DM4), and its metabolite, S-methyl-DM4 (S-Me-DM4), in plasma samples. Methodologically, after a simple protein precipitation with acetonitrile and after drying 1 mL of supernatant, the sample (suspended with N,N-Dimethylacetamide, DMA) was directly analyzed by HPLC under isocratic elution using a mobile phase comprising milliQ water and methanol (25:75, v:v), both acidified with 0.1 % v:v formic acid. Employing a flow rate of 1.0 mL/min and a reversed-phase GraceSmart RP18 column thermostated at 40 °C, we achieved complete resolution and separation of DM4 and S-Me-DM4 within 13 min. The optimized injection volume of 20 µL and the wavelength set at 254 nm were utilized for quantitative analyses. Rigorous validation has not only ensured its reliability and reproducibility but has also addressed potential limitations associated with methodological inconsistency. The limit of detection and quantification of the method were 0.025 and 0.06 µg/mL for both the analytes, respectively. The calibration curve showed a good linearity in the range 0.06-20 µg/mL. For both analytes, the intraday precision and trueness were 2.3-8.2 % and -1.1 to 3.1 %, respectively, while the interday values were 0.7-10.1 % and -10.4 to 7.5 %, respectively. The developed methodology enables the concurrent determination and quantification of free DM4 and its metabolite, free S-Me-DM4, making it a valuable tool for assessing the pharmacokinetics and pharmacodynamics of DM4-based therapies. In addition, the procedure was successfully applied to analyse the presence of free DM4 or its metabolite, free S-Me-DM4, in human plasma samples spiked with the 1959-sss/DM4 antibody-drug conjugate (ADC). The utilization of the herein validated methodology allowed to confirm the presence of these analytes, thereby providing insights into their potential release from the ADC structure.

Extracellular LGALS3BP: a potential disease marker and actionable target for antibody-drug conjugate therapy in glioblastoma.

Glioblastoma multiforme (GBM) is a lethal disease characterized by an overall survival of about 1 year, making it one of the most aggressive tumours, with very limited therapeutic possibilities. Specific biomarkers for early diagnosis as well as innovative therapeutic strategies are urgently needed to improve the management of this deadly disease. In this work, we demonstrated that vesicular galectin-3-binding protein (LGALS3BP), a glycosylated protein overexpressed in a variety of human malignancies, is a potential GBM disease marker and can be efficiently targeted by a specific antibody-drug conjugate (ADC). Immunohistochemical analysis on patient tissues showed that LGALS3BP is highly expressed in GBM and, compared with healthy donors, the amount of vesicular but not total circulating protein is increased. Moreover, analysis of plasma-derived extracellular vesicles from mice harbouring human GBM revealed that LGALS3BP can be used for liquid biopsy as a marker of disease. Finally, an ADC targeting LGALS3BP, named 1959-sss/DM4, specifically accumulates in tumour tissue, producing a potent and dose-dependent antitumor activity. In conclusion, our work provides evidence that vesicular LGALS3BP is a potential novel GBM diagnostic biomarker and therapeutic target deserving further preclinical and clinical validation.

Widespread in vivo efficacy of The-0504: A conditionally-activatable nanoferritin for tumor-agnostic targeting of CD71-expressing cancers.

Background: Cancer is still among the leading causes of death all over the world. Improving chemotherapy and minimizing associated toxicities are major unmet medical needs. Recently, we provided a preliminary preclinical evaluation of a human ferritin (HFt)-based drug carrier (The-0504) that selectively delivers the wide-spectrum topoisomerase I inhibitor Genz-644282 to CD71-expressing tumors. The-0504 has so far been evaluated on four different human tumor xenotransplant models (breast, colorectal, pancreatic and liver cancers). Methods: Herein, we extend our studies, by: (a) testing DNA damage in vitro, (b) treating eight additional tumor xenograft models in vivo with The-0504; (c) performing pharmacokinetic (PK) studies in rats; and (d) evaluating The-0504 anti-tumor xenotransplant efficacy by optimizing its administration schedule based on PK considerations. Results: Immunofluorescence demonstrated that The-0504 induces foci expressing the DNA double-strand break marker γH2AX. Expression increases up to 4-fold and is more persistent as compared to free Genz-644282. In vivo studies confirmed a remarkable anti-tumor activity of The-0504, resulting in tumor eradication in most murine xenograft models, regardless of embryological origin (e.g. epithelial, mesenchymal or neuroendocrine), and molecular subtypes. PK studies demonstrated a long persistence of The-0504 in rat serum (half-life of about 40 h as compared to 15 h of the free drug), with a 400-fold increase in peak concentrations as compared to the free drug. On this basis, we reduced The-0504 administration frequency from twice to once per week, with no appreciable loss in therapeutic efficacy in mice. Conclusion: The results presented here confirm that The-0504 is highly active against several human tumor xenotransplants, even when administered less frequently than previously reported. The-0504 may be a good candidate for further clinical development in a tumor histotype-agnostic setting.

HER-3 surface expression increases in advanced colorectal cancer representing a potential therapeutic target.

HER-3 (also known as ErbB-3) is a human epidermal growth factor receptor tyrosine kinases family member, and its expression in CRC (colorectal cancer) tissues was previously associated with poor prognosis. In this study, HER-3 expression was analyzed by immunohistochemistry in two cohorts of early and advanced metastatic CRC patients. The first cohort included 180 patients diagnosed with CRC in absence of lymph nodes or distant metastases (Stage I and Stage II), while the second was obtained from 53 advanced metastatic CRC patients who developed synchronous (SM) and metachronous (MM) liver metastases. In the first early-stage CRC cohort, 86 out of 180 (47.8%) tumors showed membranous expression of HER-3, with a mean percentage of positive tumor cells of 25.7%; conversely, in advanced metastatic CRC primary tumors, HER-3 was detected in all specimens, with a mean percentage of positive tumor cells of 76.1%. Kaplan-Meier curves showed that in the advanced metastatic CRC group, patients with HER-3high tumors had a significantly lower Cancer-Specific Survival (CSS) rate compared to patients with HER-3low tumors (p = 0.021). Importantly, this worse CSS rate was observed only in the MM subgroup of patients with HER-3high tumors (p = 0.002). Multivariate analysis confirmed that high HER-3 expression represents a significant and strong risk factor for death in patients developing MM liver metastases (Hazard Ratio = 64.9; 95% Confidence Interval, 4.7-886.6; p = 0.002). In addition, using a specific anti-HER-3 antibody-drug conjugate, named EV20/MMAF, we showed that HER-3 + CRC cells can be efficiently targeted in vitro and in vivo. Overall, this study confirms that surface HER-3 is highly expressed in CRC and reveals that HER-3 expression increases in metastatic CRC patients compared to early stage. Importantly, the results suggest that HER-3 has a prognostic and therapeutic value in patients developing MM liver metastases.