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Background There is overwhelming evidence that in vitro three-dimensional tumor cell cultures more accurately reflect the complex in vivo microenvironment than simple two-dimensional cell monolayers, not least with respect to gene expression profiles, signaling pathway activity and drug sensitivity. However, most currently available three-dimensional techniques are time consuming and/or lack reproducibility; thus standardized and rapid protocols are urgently needed. Results To address this requirement, we have developed a versatile toolkit of reproducible three-dimensional tumor spheroid models for dynamic, automated, quantitative imaging and analysis that are compatible with routine high-throughput preclinical studies. Not only do these microplate methods measure three-dimensional tumor growth, but they have also been significantly enhanced to facilitate a range of functional assays exemplifying additional key hallmarks of cancer, namely cell motility and matrix invasion. Moreover, mutual tissue invasion and angiogenesis is accommodated by coculturing tumor spheroids with murine embryoid bodies within which angiogenic differentiation occurs. Highly malignant human tumor cells were selected to exemplify therapeutic effects of three specific molecularly-targeted agents: PI-103 (phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitor), 17- N -allylamino-17-demethoxygeldanamycin (17-AAG) (heat shock protein 90 (HSP90) inhibitor) and CCT130234 (in-house phospholipase C (PLC)γ inhibitor). Fully automated analysis using a Celigo cytometer was validated for tumor spheroid growth and invasion against standard image analysis techniques, with excellent reproducibility and significantly increased throughput. In addition, we discovered key differential sensitivities to targeted agents between two-dimensional and three-dimensional cultures, and also demonstrated enhanced potency of some agents against cell migration/invasion compared with proliferation, suggesting their preferential utility in metastatic disease. Conclusions We have established and validated a suite of highly reproducible tumor microplate three-dimensional functional assays to enhance the biological relevance of early preclinical cancer studies. We believe these assays will increase the translational predictive value of in vitro drug evaluation studies and reduce the need for in vivo studies by more effective triaging of compounds.

Macrophage polarization is relevant for tumor biology. M2 polarized macrophages favor tumor growth and survival, while M1 macrophages support tumor destruction and antigen presentation. Markers identifying M1/M2 polarization are a subject of debate. We conducted a systematic review and meta-analysis to investigate the association of proposed macrophage markers with prognosis across epithelial tumors and melanoma. The Medline search engine was used and 195 articles were recovered for full review. Only articles which measured markers using immunohistochemistry or immunofluorescence and had overall survival (OS) as the primary endpoint were included. One hundred and thirteen articles were finally accepted for analysis. CD68 was associated with worse survival across tumors (hazard ratio (HR) = 1.24, 95% CI = 1.11–1.37). Tumor anatomical location influenced this association. Colorectal tumors showed an inverse association between CD68 and OS in contrast to the rest of cancer types (HR = 0.56 vs. 1.34). The approach taken to measure CD68 had an impact on prognosis; when macrophages were measured at the tumor invasion front prognosis was more favorable than when they were measured intratumorally (HR = 0.94 vs. 1.4). CD163, CD204, and CD206 showed a robust association with worse OS (HR = 1.63, 1.95, 1.65, respectively). Tumors arising in the lung and the liver showed a weaker association between CD163 and OS as compared with other locations ( β  = −0.5401 for the lung and −0.5940 for the liver compared with other anatomical locations). The counting strategy also had an impact on CD163 association with OS, with hot-spot counting having higher HRs compared with averaging macrophage counts across spots or absolute cell counting ( β  = −0.4678). In conclusion, proposed M2 markers are associated with worse survival across epithelial tumors and melanoma. The anatomical origin of tumors influences this association. The compartment where the macrophages were scored and counting strategy influenced the association with OS of CD68 and CD163, respectively.

Between 20% and 30% of endometrial cancer (EC) cases show mismatch repair deficiency (dMMR), and its characterisation is recommended in these tumours for molecular classification, screening of Lynch syndrome, and as a predictive biomarker for immunotherapy. The aim of this study was to explore two tests developed by Promega (OncoMate MSI Dx Analysis System and long mononucleotide repeat (LMR) microsatellite instability (MSI) analysis system). DNA from 126 EC tumours had been screened for MMR status by immunohistochemistry (IHC). Overall, 67 (53.2%) dMMRs and 59 (46.8%) proficient (pMMR) were included. The same cases were additionally explored for MMR genomic status, with 55 (43.7%) cases presenting an altered genomic pattern, and 69 (54.8%) with no genomic alterations. There were 71 (56.3%) microsatellite stability (MSS) cases for OncoMate and 69 (54.8%) for LMR, and 37 (29.4%) microsatellite instability (MSI) cases for OncoMate and 44 (34.9%) for LMR. Differences between the test assignments were significant ( p  < 0.001), with an increased proportion of correctly classified cases for the LMR assay, taking the IHC result as the reference. The respective sensitivity and specificity of the LMR assay was 95.5% and 68.1%, versus 86.5% and 53.5% for the OncoMate assay. In conclusion, the new LMR MSI Analysis System had a higher correlation with IHC, including cases that could be misdiagnosed due to minimal shifts, as well as higher sensitivity and specificity than the OncoMate panel. The best method regarding the use of dMMR/MSI as a response biomarker for immune checkpoint inhibitors requires further investigation.

Head and neck squamous cell carcinoma (HNSCC) represents 6% of all cancers. The overall 5-year survival rate for patients with this type of cancer is among the lowest of the major cancer types and has not improved dramatically during the last decade. The pathological staging, in particular the nodal stage, is the most important factor in HNSCC. The lack of progress in head and neck oncology emphasizes the importance of molecular genetic studies to define alterations that may correlate with tumor behavior. The molecular alterations observed in HNSCC are mainly due to oncogene activation and tumor suppressor gene inactivation, leading to deregulation of cell proliferation. These alterations include gene amplification and overexpression of oncogenes such as ras, myc, EGFR and cyclin D1, and mutations and deletions leading to p16 and TP53 tumor suppressor genes inactivation. This article reviews the molecular changes commonly observed in HNSCC. The biological function of these markers and the potential clinical application are discussed. Advances in the understanding of the molecular basis of HNSCC will help in the identification of new molecular markers that could be used for a more accurate diagnosis and assessment of prognosis and may open the way for novel approaches to treatment and prevention.

Glycosyltransferase enzyme GCNT3, has been proposed as a biomarker for prognosis in colorectal cancer (CRC). Our study goes in depth into the molecular basis of GCNT3 role in tumorigenesis and drug resistance, and it explores its potential role as biomarker in epithelial ovarian cancer (EOC). High levels of GCNT3 are associated with increased sensibility to 5-fluoracil in metastatic cells. Accordingly, GCNT3 re-expression leads to the gain of anti-carcinogenic cellular properties by reducing cell growth, invasion and by changing metabolic capacities. Integrated transcriptomic and proteomic analyses reveal that GCNT3 is linked to cellular cycle, mitosis and proliferation, response to drugs and metabolism pathways. The vascular epithelial growth factor A (VEGFA) arises as an attractive partner of GCNT3 functions in cell invasion and resistance. Finally, GCNT3 expression was analyzed in a cohort of 56 EOC patients followed by a meta-analysis of more than one thousand patients. This study reveals that GCNT3 might constitute a prognostic factor also in EOC, since its overexpression is associated with better clinical outcome and response to initial therapy. GCNT3 emerges as an essential glycosylation-related molecule in CRC and EOC progression, with potential interest as a predictive biomarker of response to chemotherapy.

Deregulation of Src kinases is associated with cancer. We previously showed that SrcDN conditional expression in MCF7 cells reduces tumorigenesis and causes tumor regression in mice. However, it remained unclear whether SrcDN affected breast cancer stem cell functionality or it reduced tumor mass. Here, we address this question by isolating an enriched population of Breast Cancer Stem Cells (BCSCs) from MCF7 cells with inducible expression of SrcDN. Induction of SrcDN inhibited self-renewal, and stem-cell marker expression (Nanog, Oct3-4, ALDH1, CD44). Quantitative proteomic analyses of mammospheres from MCF7-Tet-On-SrcDN cells (data are available via ProteomeXchange with identifier PXD017789, project DOI: 10.6019/PXD017789) and subsequent GSEA showed that SrcDN expression inhibited glycolysis. Indeed, induction of SrcDN inhibited expression and activity of hexokinase, pyruvate kinase and lactate dehydrogenase, resulting in diminished glucose consumption and lactate production, which restricted Warburg effect. Thus, c-Src functionality is important for breast cancer stem cell maintenance and renewal, and stem cell transcription factor expression, effects linked to glucose metabolism reduction.

Tumours arising in BRCA1 mutation carriers and sporadic basal-like breast carcinomas have similar phenotypic, immunohistochemical and clinical characteristics. SOX2 is an embryonic transcription factor located at chromosome 3q, a region frequently gained in sporadic basal-like and BRCA1 germline mutated tumours. The aim of the study was to establish whether sox2 expression was related to basal-like sporadic breast tumours. Two hundred and twenty-six sporadic node-negative invasive breast carcinomas were immunohistochemically analysed for oestrogen receptor (ER), progesterone receptor (PR), CK5/6, EGFR, vimentin, HER2, ki67, p53 and sox2 using tissue microarrays. Tumours were considered to have basal-like phenotype if they were ER/HER2-negative and CK5/6 and/or EGFR-positive. Thirty cases of this series (13.7%) displayed a basal-like phenotype. Sox2 expression was observed in 16.7% of cases and was significantly more frequently expressed in basal-like breast carcinomas (43.3% in basal-like, 10.6% in luminal and 13.3% in HER2+ tumours, P<0.001). Moreover, Sox2 showed a statistically significant inverse association with ER and PR (P=0.001 and 0.017, respectively) and direct association with CK5/6, EGFR and vimentin (P=0.022, 0.005 and <0.001, respectively). Sox2 is preferentially expressed in tumours with basal-like phenotype and may play a role in defining their less differentiated/‘stem cell' phenotypic characteristics.

Based on the excellent results of the clinical trials with ALK-inhibitors, the importance of accurately identifying ALK positive lung cancer has never been greater. However, there are increasing number of recent publications addressing discordances between FISH and IHC. The controversy is further fuelled by the different regulatory approvals. This situation prompted us to investigate two ALK IHC antibodies (using a novel ultrasensitive detection-amplification kit) and an automated ALK FISH scanning system (FDA-cleared) in a series of non-small cell lung cancer tumor samples. Forty-seven ALK FISH-positive and 56 ALK FISH-negative NSCLC samples were studied. All specimens were screened for ALK expression by two IHC antibodies (clone 5A4 from Novocastra and clone D5F3 from Ventana) and for ALK rearrangement by FISH (Vysis ALK FISH break-apart kit), which was automatically captured and scored by using Bioview's automated scanning system. All positive cases with the IHC antibodies were FISH-positive. There was only one IHC-negative case with both antibodies which showed a FISH-positive result. The overall sensitivity and specificity of the IHC in comparison with FISH were 98% and 100%, respectively. The specificity of these ultrasensitive IHC assays may obviate the need for FISH confirmation in positive IHC cases. However, the likelihood of false negative IHC results strengthens the case for FISH testing, at least in some situations.

The purpose of this study was to evaluate the suitability of formalin-fixed and paraffin-embedded (FFPE) samples and fixed fresh (FF) samples for single-cell RNA sequencing (scRNAseq). To this end, we compared single-cell profiles from FFPE and matched FF tissue samples of one invasive carcinoma of no special type carcinoma (invasive ductal carcinoma–IDC) and one invasive lobular carcinoma (ILC) to assess consistency in cell type distribution and molecular profiles. The results were validated using immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and electron microscopy. Additionally, immune cell proportions identified by IHC were quantified using QuPath and compared to the scRNAseq results. FFPE- and FF-derived libraries demonstrated high-quality sequencing metrics, and cellular heterogeneity was similar. No exclusive cell populations were identified by either approach. The four samples analysis identified six types of epithelial cells, as well as tumoral microenvironment populations. The scRNAseq results from epithelial neoplastic cells were concordant with common IHC markers. The proportion of immune cells identified by IHC in FFPE sections were similar to those obtained by scRNAseq. We identified and validated a previously poorly recognized subpopulation of neoplastic multi-ciliated cells (MCCs) (FOXJ1, ROPN1L). Analysis of FOXJ1 in 214 ER-positive invasive carcinomas demonstrated protein expression in one third of tumors, suggesting frequent focal MCC differentiation. Our results support the suitability of scRNAseq analysis using FFPE tissue, and identified a subpopulation of neoplastic MCC in breast cancer.

Introduction: Following up on treated high-grade cervical intraepithelial neoplasia (HSIL/CIN) lesions poses a challenge. Cervical cytology often has a high false-negative rate, while high-risk human papillomavirus (HR-HPV) DNA testing, though sensitive, lacks specificity. The detection of messenger RNA of the HR-HPV E6 and E7 oncoproteins (E6/E7 mRNA) is proposed as an indicator of viral integration, which is crucial for identifying severe lesions. Additionally, HPV vaccination could reduce recurrence rates in patients treated for high-grade cervical intraepithelial neoplasia. Objective: Our study aimed to assess the clinical utility of E6/E7 mRNA determination in the follow-up of HPV-immunized patients who were treated for HSIL/CIN. Methods: We conducted a retrospective observational study including 407 patients treated for HSIL/CIN. The recurrence rate and the validity parameters of E6/E7 mRNA testing were analyzed. Results: The recurrence rate for high-grade lesions was 1.7%. This low percentage might be related to the vaccination of patients who were not immunized before treatment. The sensitivity of the E6/E7 mRNA test was 88% at the first clinical visit, reaching 100% in the second and third reviews. Specificity was 91% at the first visit, 92% at the second, and 85% at the third. Regarding predictive values, the positive predictive value was 18% at the first visit, 10% at the second, and 14% at the third, while the negative predictive value was 100% across all follow-up visits. Conclusions: The E6/E7 mRNA test appears to be an effective tool for ruling out recurrence after treatment for HSIL/CIN lesions in HPV-immunized patients.