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Antiangiogenic therapies are considered a promising strategy against solid tumors. Their aim is to inhibit the formation of new blood vasculature, thereby reducing the oxygen and nutrient supply to prevent further tumor growth and spreading. However, the strategy has seen limitations, as survival benefits are modest and often accompanied with increased tumor aggressiveness in form of invasion and metastasis. Antiangiogenic induced changes in the tumor microenvironment, such as hypoxia, mechanical stress or extracellular acidification can activate different receptors of tumoral and stromal cells and induce an extensive remodeling of the entire tumor microenvironment, with the overall goal to invade nearby tissues and regain access to the vasculature. In this regard, receptor tyrosine kinases have been studied intensively and especially the inhibition of c-Met has given promising results, characterized by a reduction in invasiveness and prolonged survival. Receptors that sense changes in the extracellular matrix like integrins or proteoglycans can also induce downstream signaling that stimulates the expression of remodeling factors such as new matrix components, enzymes or chemoattractants. Targeting multiple receptors and sensors of cancer cells simultaneously might represent an effective second line treatment that prevents the formation of malignant side effects.

Supplementary Figure 1. Expression of CXCR4 by immunofluorescence and of mouse Cxcr4 and human CXCR7 by RT-PCR; Supplementary Figure 2. Decrease in number of CXCR4-positive cells arising from AMD3100 treatment; Supplementary Figure 3. Absence of effect of AMD3100 on fibrosis, necrosis and proliferation; Supplementary Figure 4. Inhibition of angiogenesis by AMD3100; Supplementary Figure 5. Cytometric analysis of CTCs in blood of OVA17 implanted animals; Supplementary Figure 6. Cytokeratin 19 mRNA analysis in blood from ovarian carcinoma patients; Supplementary Figure 7. CXCR4 immunostaining in SKOV-3 tumors; Supplementary Figure 8. Absence of effect of shCXCR4 expression in SKOV-3 cells proliferation and viability, but decrease of angiogenesis in tumors; Supplementary Figure 9. Cytometric analysis of CTCs in blood of SKOV-3-sh control and SKOV-3-shCXCR4 863 inoculated animals; Supplementary Figure 10. CXCR4 inhibition by shRNA expression blocks Src and ERKs activation; Supplementary Figure 11. Cytometric analysis of GFP and CXCR4 expression in cells isolated from SKOV-3 xenografts; Supplementary Figure 12. CXCR4 and E-cadherin expression in consecutive sections of OVA17 tumors

Purpose:FGFR1 amplification (FGFR1amp) is recurrent in metastatic breast cancer (MBC) and is associated with resistance to endocrine therapy and CDK4/6 inhibitors (CDK4/6is). Multi-tyrosine kinase inhibitors (MTKIs) and selective pan-FGFR inhibitors (FGFRis) are being developed for FGFR1amp breast cancer. High-level FGFR amplification and protein expression by IHC have identified breast cancer responders to FGFRis or MTKIs, respectively.Experimental Design:Here, we used preclinical models and patient samples to identify predictive biomarkers to these drugs. We evaluated the antitumor activity of an FGFRi and an MTKI in a collection of 17 breast cancer patient–derived xenografts (PDXs) harboring amplification in FGFR1/2/3/4 and in 10 patients receiving either an FGFRi/MTKI. mRNA levels were measured on FFPE tumor samples using two commercial strategies. Proliferation and angiogenesis were evaluated by detecting Ki-67 and CD31 in viable areas by immunofluorescence.Results:High FGFR1–4 mRNA levels but not copy-number alteration (CNA) is associated with FGFRi response. Treatment with MTKIs showed higher response rates than with FGFRis (86% vs. 53%), regardless of the FGFR1–4 mRNA levels. FGFR-addicted PDXs exhibited an antiproliferative response to either FGFRis or MTKIs, and PDXs exclusively sensitive to MTKI exhibited an additional antiangiogenic response. Consistently, the clinical benefit of MTKIs was not associated with high FGFR1–4 mRNA levels and was observed in patients previously treated with antiangiogenic drugs.Conclusions:Tailored therapy with FGFRis in molecularly selected MBC based on high FGFR1–4 mRNA levels warrants prospective validation in patients with CDK4/6i-resistant luminal breast cancer and in patients with TNBC without targeted therapeutic options.

Supplementary Materials and Methods

Analysis of PTEN expression in PanNETs ; Supplemental Figure 1 Legend

Supplementary Figure 1. FGFR1-4 DNA CN, mRNA levels and ROC curves analysis. A) Waterfall plot representing the best antitumor response of FGFRi in 24 patients from 8 different cancer types treated at VHIO. FGFR1/2 amplification status and high FGFR1-4 mRNA levels are indicated in the underneath panel. B) Copy number (CN) values of FGFR1-4 by MSK-IMPACT in the FGFR/FGFamp PDX collection and categorized analysis in PDXs harboring CN amplification (CN>4), according to the response to rogaratinib (responders (SD and PR) and non-responders (PD)). C) Evaluation of FGFR1/4 CN ratio by FISH ratio (the dashed line indicates the CN ratio>2.2 that is considered amplified) and categorized analysis according to the response to rogaratinib. D) Correlation analysis between the CN values (X-axis) and mRNA levels (Y-axis) of FGFR1-4 obtained from MSK-IMPACT or nCounter, respectively. Pearson correlation coefficient (r2) is shown. E) Categorized analysis of FGFR1-4 mRNA composite biomarker according to the response to rogaratinib, obtained from HTG or qPCR data. Statistical test: Mann-Whitney U test. ROC curve analysis the FGFR1-4 mRNA composite biomarker, obtained from HTG or qPCR (AUC=0.89, both).

Analysis of GDC-0941 effects on cell proliferation ; Supplemental Figure 2 Legend

Purpose:FGFR1 amplification (FGFR1amp) is recurrent in metastatic breast cancer (MBC) and is associated with resistance to endocrine therapy and CDK4/6 inhibitors (CDK4/6is). Multi-tyrosine kinase inhibitors (MTKIs) and selective pan-FGFR inhibitors (FGFRis) are being developed for FGFR1amp breast cancer. High-level FGFR amplification and protein expression by IHC have identified breast cancer responders to FGFRis or MTKIs, respectively.Experimental Design:Here, we used preclinical models and patient samples to identify predictive biomarkers to these drugs. We evaluated the antitumor activity of an FGFRi and an MTKI in a collection of 17 breast cancer patient–derived xenografts (PDXs) harboring amplification in FGFR1/2/3/4 and in 10 patients receiving either an FGFRi/MTKI. mRNA levels were measured on FFPE tumor samples using two commercial strategies. Proliferation and angiogenesis were evaluated by detecting Ki-67 and CD31 in viable areas by immunofluorescence.Results:High FGFR1–4 mRNA levels but not copy-number alteration (CNA) is associated with FGFRi response. Treatment with MTKIs showed higher response rates than with FGFRis (86% vs. 53%), regardless of the FGFR1–4 mRNA levels. FGFR-addicted PDXs exhibited an antiproliferative response to either FGFRis or MTKIs, and PDXs exclusively sensitive to MTKI exhibited an additional antiangiogenic response. Consistently, the clinical benefit of MTKIs was not associated with high FGFR1–4 mRNA levels and was observed in patients previously treated with antiangiogenic drugs.Conclusions:Tailored therapy with FGFRis in molecularly selected MBC based on high FGFR1–4 mRNA levels warrants prospective validation in patients with CDK4/6i-resistant luminal breast cancer and in patients with TNBC without targeted therapeutic options.

Supplementary Table S2 HTG OBP panel raw data

Purpose: Germ cell tumors (GCT) of the testis are highly curable, but those patients who are refractory to cisplatin (CDDP)-based combination chemotherapy have a poor prognosis. Therefore, identifying new alternatives for treatment remains a priority. Several studies support an important role for angiogenesis in GCTs, suggesting that antiangiogenic treatment might be a good alternative. Sunitinib is an oral multitarget tyrosine kinase receptor inhibitor with antiangiogenic and antitumor activities. In the present study, we evaluated the effect of sunitinib, CDDP, or the combination of both drugs using an orthotopic model of human testicular GCT.Experimental Design: Mice were implanted with four different testicular tumors: a yolk sac, two choriocarcinomas, and a CDDP-resistant choriocarcinoma variant induced in mice by continuous exposure to CDDP. Mice were treated with vehicle, CDDP, sunitinib, or the combination of both drugs and their effects on tumors were analyzed.Results: We observed a significant inhibition in tumor growth accompanied by longer survival after sunitinib treatment. Combination therapy with CDDP significantly enhanced these effects. Sunitinib induced apoptosis, reduced tumor cell proliferation and tumor vasculature, and inhibited vascular endothelial growth factor receptor 1, 2, and 3 and platelet-derived growth factor receptor α phosphorylation without affecting phosphorylation of other tyrosine kinase receptors. More importantly, tumor growth inhibition induced by sunitinib was also observed in the induced CDDP-resistant choriocarcinoma model.Conclusions: Taken together, these results suggest that sunitinib might be a new alternative for treatment of CDDP-refractory patients.

Assessment of cell proliferation upon inhibition of p110α ; Supplemental Figure 5 Legend

Supplementary Figure Legends

Survival curves upon genetic inactivation of individual p110s ; Supplemental Figure 3 Legend

Index of all Supplementary Data

This file contains information about protein immunoblotting, histopathological analysis, and cell biological assays in cultured cells.

Supplementary figures on vessel phenotype, tumor effects, combo treatment, macrophage phenotype and effects, and human validation.

Expression of some angiogenesis related genes in response to anti-Sema4D and anti-VEGFR2 treatment

One of the main consequences of inhibition of neovessel growth and vessel pruning produced by angiogenesis inhibitors is increased intratumor hypoxia. Growing evidence indicates that tumor cells escape from this hypoxic environment to better nourished locations, presenting hypoxia as a positive stimulus for invasion. In particular, anti-VEGF/R therapies produce hypoxia-induced invasion and metastasis in a spontaneous mouse model of pancreatic neuroendocrine cancer (PanNET), RIP1-Tag2. Here, a novel vascular-targeting agent targeting semaphorin 4D (Sema4D) demonstrated impaired tumor growth and extended survival in the RIP1-Tag2 model. Surprisingly, although there was no induction of intratumor hypoxia by anti-Sema4D therapy, the increase in local invasion and distant metastases was comparable with the one produced by VEGFR inhibition. Mechanistically, the antitumor effect was due to an alteration in vascular function by modification of pericyte coverage involving platelet-derived growth factor B. On the other hand, the aggressive phenotype involved a macrophage-derived Sema4D signaling engagement, which induced their recruitment to the tumor invasive fronts and secretion of stromal cell–derived factor 1 (SDF1) that triggered tumor cell invasive behavior via CXCR4. A comprehensive clinical validation of the targets in different stages of PanNETs demonstrated the implication of both Sema4D and CXCR4 in tumor progression. Taken together, we demonstrate beneficial antitumor and prosurvival effects of anti-Sema4D antibody but also unravel a novel mechanism of tumor aggressivity. This mechanism implicates recruitment of Sema4D-positive macrophages to invasive fronts and their secretion of proinvasive molecules that ultimately induce local tumor invasion and distant metastasis in PanNETs.Significance:An anti-semaphorin-4D vascular targeting agent demonstrates antitumor and prosurvival effects but also unravels a novel promalignant effect involving macrophage-derived SDF1 that promotes tumor invasion and metastasis, both in animal models and patients.See related commentary by Tamagnone and Franzolin, p. 5146

Analysis of angiogenic islet upon genetic inactivation of p110α, p110β and p110Î' ; Supplemental Figure 4 Legend

Ranked list of proteins present in anti-Sema4D treated macrophage conditioned media obtained in the GSEA analysis of the proteomic data

Purpose: Mutations in the PI3K pathway occur in 16% of patients with pancreatic neuroendocrine tumors (PanNETs), which suggests that these tumors are an exciting setting for PI3K/AKT/mTOR pharmacologic intervention. Everolimus, an mTOR inhibitor, is being used to treat patients with advanced PanNETs. However, resistance to mTOR-targeted therapy is emerging partially due to the loss of mTOR-dependent feedback inhibition of AKT. In contrast, the response to PI3K inhibitors in PanNETs is unknown.Experimental Design: In the current study, we assessed the frequency of PI3K pathway activation in human PanNETs and in RIP1-Tag2 mice, a preclinical tumor model of PanNETs, and we investigated the therapeutic efficacy of inhibiting PI3K in RIP1-Tag2 mice using a combination of pan (GDC-0941) and p110α-selective (GDC-0326) inhibitors and isoform-specific PI3K kinase-dead–mutant mice.Results: Human and mouse PanNETs showed enhanced pAKT, pPRAS40, and pS6 positivity compared with normal tissue. Although treatment of RIP1-Tag2 mice with GDC-0941 led to reduced tumor growth with no impact on tumor vessels, the selective inactivation of the p110α PI3K isoform, either genetically or pharmacologically, reduced tumor growth as well as vascular area. Furthermore, GDC-0326 reduced the incidence of liver and lymph node metastasis compared with vehicle-treated mice. We also demonstrated that tumor and stromal cells are implicated in the antitumor activity of GDC-0326 in RIP1-Tag2 tumors.Conclusions: Our data provide a rationale for p110α-selective intervention in PanNETs and unravel a new function of this kinase in cancer biology through its role in promoting metastasis. Clin Cancer Res; 22(23); 5805–17. ©2016 AACR.

List of cytokines modified in response to anti-Sema4D treatment (Cytokine array)

Supplementary Data from Sunitinib Inhibits Tumor Growth and Synergizes with Cisplatin in Orthotopic Models of Cisplatin-Sensitive and Cisplatin-Resistant Human Testicular Germ Cell Tumors

Supplementary Figure Legends 1-9 from Glycolytic Phenotype and AMP Kinase Modify the Pathologic Response of Tumor Xenografts to VEGF Neutralization

Supplementary Figures 1-9 from Glycolytic Phenotype and AMP Kinase Modify the Pathologic Response of Tumor Xenografts to VEGF Neutralization

Supplementary Methods from Glycolytic Phenotype and AMP Kinase Modify the Pathologic Response of Tumor Xenografts to VEGF Neutralization

Background More accurate predictive biomarkers in patients with gastroenteropancreatic neuroendocrine tumours (GEP-NETs) are needed. This study aims to investigate radiomics-based tumour phenotypes as a surrogate biomarker of the tumour vasculature and response prediction to antiangiogenic targeted agents in patients with GEP-NETs. Methods In this retrospective study, a radiomics signature was developed in patients with GEP-NETs and liver metastases receiving lenvatinib. Patients were selected from the multicentre phase II TALENT trial (NCT02678780) (development cohort). Radiomics variables were extracted from liver metastases in the pre-treatment CT-scans and selected using LASSO regression and minimum redundancy maximum relevance (mRMR). Logistic regression and Cox proportional-hazards models for radiomics and combined radiomics with clinical data were explored. The performance of the models was tested in an external cohort (test cohort). Associations between the radiomics score and vascularization factors in plasma were studied using hierarchical clustering and Mann-Whitney U test. Results A total of 89 patients were included in the study, 408 liver metastases where analysed. The CT-based radiomics signature was associated with clinical benefit in the development (training and validation sets) and test cohorts (AUC 0.75 [0.66-0.90], 0.67 [0.49-0.92] and 0.67 [0.43-0.91], respectively). The combined radiomics-clinical signature (including the radiomics score, Ki-67 index and primary tumour site) improved on radiomics-only signature performance (AUC 0.79 [95% CI 0.64-0.93]; PConclusions Radiomics-based phenotypes can provide valuable information about tumour characteristics, including vasculature, that are associated with response to antiangiogenics. Clinical Trial Registration This is a study of the Lenvatinib Efficacy in Metastatic Neuroendocrine Tumours (TALENT) phase II clinical trial (NCT02678780).

Background The metabolic and psychological management of pediatric type 1 diabetes mellitus (T1DM) can be challenging over time given that T1DM may cause a negative emotional burden and, consequently, result in poor metabolic control of the disease. The objectives of this study are to translate the Problem Area in Diabetes Survey–Pediatric version (PAID-Peds) into Spanish, adapt it culturally and validate it. Methods A multicenter, observational, prospective and analytical study based on a sample of 636 patients aged 8–17 years, diagnosed with T1DM, under treatment with insulin and follow-up at the Miguel Servet University Hospital in Zaragoza (Aragón, Spain), the Ramón y Cajal University Clinical Hospital in Madrid (Spain) and at the Sant Joan de Déu Hospital in Barcelona (Catalonia, Spain) between 1 January 2023 and 31 December 2024. The study will consist of two phases: 1) Translation and cultural adaptation of the original PAID-Peds® survey into Spanish following eight steps; 2) Validation of the Spanish version of the PAIS-Peds® survey. The statistical analysis will be performed using Jamovi® 2.1.23, the reliability or internal consistency will be calculated using Cronbach’s alpha index (considering an index higher than 0.8 to be good) and the test-retest will be evaluated using the intraclass correlation coefficient. This study has been approved by the ethics and research committees at each center. Discussion Detecting the burden of having diabetes could favor early interventions to reduce anxiety and prevent the worsening of distress, exhaustion, and depressive symptoms, and subsequent poor diabetes control. Therapeutic education in diabetes—recommended by the WHO and the Diabetes Education Study Group—has shown encouraging results in glycemia and psychosocial and behavioral factors in T1DM.

Amyotrophy lateral sclerosis is a disease that causes disability, chronicity and physical handicap. Moreover, the dependence of the patient will increase. Objectives To assess the efficacy of specific interventions designed to train non-professional caregivers of patients diagnosed with amyotrophic lateral disease. Method Descriptive cross-sectional study of an intervention designed for non-professional caregivers of patients with ALS. Pilot study. The target audience of this study are non-professional caregivers of patients with ALS. The level of dependence of the patients is measured with the Barthel index, the Revised Scale of Functional Assessment of Amyotrophic Lateral Sclerosis and Cognitive Behavioural Screening in ALS. The needs of these patients’ caregivers were evaluated according to the Kreutzer questionnaire. A multidisciplinary intervention designed to modify this situation was designed and re-evaluated. Results Finally, seven caregivers were included in the pilot study. They were women, with an average age of 56 years, partners of the patients, with parallel economic activity and with an average level of education. The patients were men with a mean age of 63 years and 1-3 years of disease duration; they presented a moderate-mild Barthel, cognitive impairment, gross motility and respiratory function. The most demanded needs were for "medical / health information" and "involvement in treatment and care". Conclusion Specific interventions to meet the needs of non-professional caregivers are highly effective. Using tools that identify them contributes to improving caregivers' care.

Sickle cell disease (SCD) is a common hemoglobinopathy, secondary to alterations in the β globin chain, resulting in an abnormal hemoglobin variant named as hemoglobin S. These disorders show a wide phenotypical spectrum, and the prevalence of these disorders has significantly changed over the time because of multiple factors such as migration. We report a case of a 17-year-old black male, born in Gambia, diagnosed with sickle cell disease, who presented an associated mutation only described in a Japanese family (Oshima et al., 1996).

Objetivos. Comparar las diferentes capas retinianas y coroides en pacientes con diabetes mellitus tipo 1 (DM1) mediante tecnología swept-source, detectar presencia de retinopatía diabética (RD) y edema macular diabético (EMD) y analizar la relación entre sus espesores y marcadores analíticos de control metabólico. Material y métodos. Estudio transversal con muestreo consecutivo de pacientes ≥18 años diagnosticados DM1, al menos 5 años de evolución, ausencia de RD o RD leve o moderada. Se excluyeron a los pacientes con edema macular secundario a otras patologías. Se les realizó una retinografía no esteroscópica de 45º y una tomografía de coherencia óptica swept-source (SS-OCT) macular radial de 12mm con DRI Triton plus (Topcon, Japón). Se analizó el espesor de las diferentes capas de la retina y coroides y se correlacionó con los últimos parámetros metabólicos de control de la enfermedad. El análisis estadístico se realizó mediante Jamovi (versión 1.2). Resultados. Se examinaron 192 ojos (96 pacientes), el 49% hombres, edad media: 42±12.5 años; el 31.1% con RD; media de glucosa: 147±70.1mg/dl; urea: 34.6±10.6mg/dl; creatinina: 0.80±0.19mg/dl; triglicéridos: 78.8±33.4mg/dl; colesterol: 189±32.4mg/dl; colesterol HDL: 63.2±13.5mg/dl; LDL: 111±26.6mg/dl y hemoglobina glicosilada: 7.61±1.24%. Se hallaron correlaciones inversas leves entre retina, capa de células ganglionares (CCG+) y tiempo de evolución (p

Owing to their unique combination of chemical and physical properties, inorganic nanoparticles show a great deal of potential as suitable agents for early diagnostics and less invasive therapies. Yet, their translation to the clinic has been hindered, in part, by the lack of non-invasive methods to quantify their concentration in vivo while also assessing their effect on the tissue physiology. In this work, we demonstrate that diffuse optical techniques, employing near-infrared light, have the potential to address this need in the case of gold nanoparticles which support localized surface plasmons. An orthoxenograft mouse model of clear cell renal cell carcinoma was non-invasively assessed by diffuse reflectance and correlation spectroscopies before and over several days following a single intravenous tail vein injection of polyethylene glycol-coated gold nanorods (AuNRs-PEG). Our platform enables to resolve the kinetics of the AuNR-PEG uptake by the tumor in quantitative agreement with ex vivo inductively coupled plasma mass spectroscopy. Furthermore, it allows for the simultaneous monitoring of local tissue hemodynamics, enabling us to conclude that AuNRs-PEG do not significantly alter the animal physiology. We note that the penetration depth of this current probe was a few millimeters but can readily be extended to centimeters, hence gaining clinical relevance. This study and the methodology presented here complement the nanomedicine toolbox by providing a flexible platform, extendable to other absorbing agents that can potentially be translated to human trials.

Here, we used preclinical models and patient samples to identify predictive biomarkers to these drugs. We evaluated the antitumor activity of an FGFRi and an MTKI in a collection of 17 breast cancer patient-derived xenografts (PDXs) harboring amplification inHighTailored therapy with FGFRis in molecularly selected MBC based on high

Plasmonic photothermal therapy is an emerging complementary method for treating tumors usually studied pre-clinically in mice with small subcutaneous tumors. In this project, it is conducted on a more realistic orthotopic xenograft renal carcinoma mouse model, while tissue optical properties are monitored non-invasively by diffuse optics. Hybrid diffuse reflectance spectroscopy and diffuse correlation spectroscopy are used to quantify tissue hemodynamics, the accumulated plasmonic nanoparticle concentration and therapy induced physiological changes. The results show the need for personalization and the predictive capabilities of the monitoring, which, in turn, would allow for therapy optimization.

The aim of this study was to estimate the incidence of diabetes mellitus in the Basque Country and the risk factors involved in the disease by reassessing an adult population after 7 years of follow-up. In the previous prevalence study, 847 people older than 18 years were randomly selected from all over the Basque Country and were invited to answer a medical questionnaire, followed by a physical examination and an oral glucose tolerance test. In the reassessment, the same variables were collected and the resulting cohort comprised 517 individuals of whom 43 had diabetes at baseline. The cumulative incidence of diabetes was 4.64% in 7 years and the raw incidence rate was 6.56 cases/1000 person-years (95%CI: 4.11-9.93). Among the incident cases, 59% were undiagnosed. The most strongly associated markers by univariate analyses were age >60 years, dyslipidaemia, prediabetes and insulin resistance. We also found association with hypertension, obesity, family history of diabetes and low education level. Multivariate analysis adjusted for age and sex showed that a set of risk factors assessed together (dyslipidaemia, waist-to-hip-ratio and family history of diabetes) had great predictive value (AUC-ROC=0.899, 95%CI: 0.846-0.953, p=0.942), which suggests the need for early intervention before the onset of prediabetes This work was partially supported by grants from the Department of Health of the Basque Country Government (2015111020); ISCIII (PI14/01104), co-funded by ERDF/ESF, "A way to make Europe"/"Investing in your future"; UPV/EHU (IT1281-19); Menarini Group Spain (BCA16/029); Endocrine-European Reference Network (EndoERN 739527); and CIBERDEM (Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders). The study funders were not involved in the design of the study; the collection, analysis, and interpretation of data; writing the report; and did not impose any restrictions regarding the publication of the report.