Morphological Characterization of Human Lung Cancer Organoids Cultured in Type I Collagen Hydrogels: A Histological Approach.

The malignity of lung cancer is conditioned by the tumor microenvironment (TME), in which cancer-associated fibroblasts (CAFs) are relevant. In this work, we generated organoids by combining A549 cells with CAFs and normal fibroblasts (NF) isolated from adenocarcinoma tumors. We optimized the conditions for their manufacture in a short time. We evaluated the morphology of organoids using confocal microscopy analysis of F-actin, vimentin and pankeratin. We determined the ultrastructure of the cells in the organoids via transmission electron microscopy and the expression of CDH1, CDH2 and VIM via RT-PCR. The addition of stromal cells induces the self-organization of the organoids, which acquired a bowl morphology, as well as their growth and the generation of cell processes. They also influenced the expression of genes related to epithelial mesenchymal transition (EMT). CAFs potentiated these changes. All cells acquired a characteristic secretory phenotype, with cohesive cells appearing inside the organoids. In the periphery, many cells acquired a migratory phenotype, especially in organoids that incorporated CAFs. The deposit of abundant extracellular matrix could also be observed. The results presented here reinforce the role of CAFs in the progression of lung tumors and could lay the foundation for a useful in vitro pharmacological model.

SARS-CoV-2-encoded small RNAs are able to repress the host expression of SERINC5 to facilitate viral replication.

Serine incorporator protein 5 (SERINC5) is a key innate immunity factor that operates in the cell to restrict the infectivity of certain viruses. Different viruses have developed strategies to antagonize SERINC5 function but, how SERINC5 is controlled during viral infection is poorly understood. Here, we report that SERINC5 levels are reduced in COVID-19 patients during the infection by SARS-CoV-2 and, since no viral protein capable of repressing the expression of SERINC5 has been identified, we hypothesized that SARS-CoV-2 non-coding small viral RNAs (svRNAs) could be responsible for this repression. Two newly identified svRNAs with predicted binding sites in the 3'-untranslated region (3'-UTR) of the SERINC5 gene were characterized and we found that the expression of both svRNAs during the infection was not dependent on the miRNA pathway proteins Dicer and Argonaute-2. By using svRNAs mimic oligonucleotides, we demonstrated that both viral svRNAs can bind the 3'UTR of SERINC5 mRNA, reducing SERINC5 expression in vitro. Moreover, we found that an anti-svRNA treatment to Vero E6 cells before SARS-CoV-2 infection recovered the levels of SERINC5 and reduced the levels of N and S viral proteins. Finally, we showed that SERINC5 positively controls the levels of Mitochondrial Antiviral Signalling (MAVS) protein in Vero E6. These results highlight the therapeutic potential of targeting svRNAs based on their action on key proteins of the innate immune response during SARS-CoV-2 viral infection.

In Vitro Effect of Δ9-Tetrahydrocannabinol and Cannabidiol on Cancer-Associated Fibroblasts Isolated from Lung Cancer.

There is evidence that demonstrates the effect of cannabinoid agonists inhibiting relevant aspects in lung cancer, such as proliferation or epithelial-to-mesenchymal transition (EMT). Most of these studies are based on evidence observed in in vitro models developed on cancer cell lines. These studies do not consider the complexity of the tumor microenvironment (TME). One of the main components of the TME is cancer-associated fibroblasts (CAFs), cells that are relevant in the control of proliferation and metastasis in lung cancer. In this work, we evaluated the direct effects of two cannabinoid agonists, tetrahydrocannabinol (THC) and cannabidiol (CBD), used alone or in combination, on CAFs and non-tumor normal fibroblasts (NFs) isolated from adenocarcinoma or from healthy lung tissue from the same patients. We observed that these compounds decrease cell density in vitro and inhibit the increase in the relative expression of type 1 collagen (COL1A1) and fibroblast-specific protein 1 (FSP1) induced by transforming growth factor beta (TGFß). On the other hand, we studied whether THC and CBD could modulate the interactions between CAFs or NFs and cancer cells. We conditioned the culture medium with stromal cells treated or not with THC and/or CBD and cultured A549 cells with them. We found that culture media conditioned with CAFs or NFs increased cell density, induced morphological changes consistent with EMT, inhibited cadherin-1 (CDH1) gene expression, and induced an increase in the relative expression of cadherin-2 (CDH2) and vimentin (VIM) genes in A549 cells. These changes were inhibited or decreased by THC and CBD administered alone or in combination. In another series of experiments, we conditioned culture media with A549 cells treated or not with THC and/or CBD, in the presence or absence of TGFß. We observed that culture media conditioned with A549 in the presence of TGFß induced an increase in the expression of COL1A1 and VIM, both in CAFs and in non-tumor NFs. Both THC and CBD ameliorated these effects. In summary, the results presented here reinforce the usefulness of cannabinoid agonists for the treatment of some relevant aspects of lung cancer pathology, and demonstrate in a novel way their possible effects on CAFs as a result of their relationship with cancer cells. Likewise, the results reinforce the usefulness of the combined use of THC and CBD, which has important advantages in relation to the possibility of using lower doses, thus minimizing the psychoactive effects of THC.

Correction: Pardo-Sánchez et al. Increased Tumor Growth Rate and Mesenchymal Properties of NSCLC-Patient-Derived Xenograft Models during Serial Transplantation. Cancers 2021, 13, 2980.

The authors would like to make a correction to their published paper [...].

Increased Tumor Growth Rate and Mesenchymal Properties of NSCLC-Patient-Derived Xenograft Models during Serial Transplantation.

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. The high mortality is very often a consequence of its late diagnosis when the cancer is already locally advanced or has disseminated. Advances in the study of NSCLC tumors have been achieved by using in vivo models, such as patient-derived xenografts. Apart from drug screening, this approach may also be useful for study of the biology of the tumors. In the present study, surgically resected primary lung cancer samples (n = 33) were implanted in immunodeficient mice, and nine were engrafted successfully, including seven adenocarcinomas, one squamous-cell carcinoma, and one large-cell carcinoma. ADC tumors bearing the KRAS-G12C mutation were the most frequently engrafted in our PDX collection. Protein expression of vimentin, ezrin, and Ki67 were evaluated in NSCLC primary tumors and during serial transplantation by immunohistochemistry, using H-score. Our data indicated a more suitable environment for solid adenocarcinoma, compared to other lung tumor subtypes, to grow and preserve its architecture in mice, and a correlation between higher vimentin and ezrin expression in solid adenocarcinomas. A correlation between high vimentin expression and lung adenocarcinoma tumors bearing KRAS-G12C mutation was also observed. In addition, tumor evolution towards more proliferative and mesenchymal phenotypes was already observed in early PDX tumor passages. These PDX models provide a valuable platform for biomarker discovery and drug screening against tumor growth and EMT for lung cancer translational research.

CXCR7 Reactivates ERK Signaling to Promote Resistance to EGFR Kinase Inhibitors in NSCLC.

Although EGFR mutant-selective tyrosine kinase inhibitors (TKI) are clinically effective, acquired resistance can occur by reactivating ERK. We show using in vitro models of acquired EGFR TKI resistance with a mesenchymal phenotype that CXCR7, an atypical G protein-coupled receptor, activates the MAPK-ERK pathway via ß-arrestin. Depletion of CXCR7 inhibited the MAPK pathway, significantly attenuated EGFR TKI resistance, and resulted in mesenchymal-to-epithelial transition. CXCR7 overexpression was essential in reactivation of ERK1/2 for the generation of EGFR TKI-resistant persister cells. Many patients with non-small cell lung cancer (NSCLC) harboring an EGFR kinase domain mutation, who progressed on EGFR inhibitors, demonstrated increased CXCR7 expression. These data suggest that CXCR7 inhibition could considerably delay and prevent the emergence of acquired EGFR TKI resistance in EGFR-mutant NSCLC. SIGNIFICANCE: Increased expression of the chemokine receptor CXCR7 constitutes a mechanism of resistance to EGFR TKI in patients with non-small cell lung cancer through reactivation of ERK signaling.

Clinical Significance of Molecular Micrometastasis in the Sentinel Lymph Node of Early-stage Non-Small Cell Lung Cancer Patients.

OBJECTIVES: Metastatic affectation of lymph node is the main prognostic factor in localized lung cancer. A pathologic study of the obtained samples, even after adequate lymphadenectomy, showed tumor relapses for 20% of stage I patients after oncological curative surgery. We evaluated the prognostic value of molecular micrometastasis in the sentinel lymph node of patients with early-stage lung cancer. PATIENTS AND METHODS: The sentinel node was marked immediately after performing thoracotomy by peritumorally injecting 0.25 mCi of nanocoloid of albumin (Nanocol1) labeled with Tc-99m in 0.3 mL. Guided by a Navigator1 gammagraphic sensor, we proceeded to its resection. The RNA of the tissue was extracted, and the presence of genes CEACAM5, BPIFA1, and CK7 in mRNA was studied. The significant association between the presence of micrometastasis, clinicopathologic characteristics, and patients' outcome was assessed. RESULTS: Eighty-nine stage I-II non-small cell lung cancer patients were included in the study. Of the 89 analyzed sentinel lymph nodes, 44 (49.4%) were positive for CK7, 24 (26.9%) for CEACAM5, and 17 (19.1%) for BPIFA1, whereas 10 (11.2%) were positive for the 3 analyzed genes. A survival analysis showed no significant relation between the presence of molecular micrometastasis in the sentinel node and patients' progression. CONCLUSIONS: The molecular analysis of the sentinel node in patients with early-stage lung cancer shows node affectation in cases staged as stage I/II by hematoxylin-eosin or an immunohistochemical analysis. However, this nodal affectation was not apparently related to patients' outcome.

[Genetic analysis of a family with Von Hippel-Lindau syndrome]. / Análisis genético en una familia con síndrome de Von Hippel-Lindau.

Von Hippel-Lindau syndrome (VHL) is an autosomal dominant inherited disease associated with mutations in the VHL tumour suppressor gene located on chromosome 3p25. VHL is characterized by the development of multiple malignant and benign tumours in the central nervous system and internal organs, including liver, pancreas and the adrenal gland. More than 823 different mutations of the VHL gene have currently been identified. In the present study we describe the case of a family affected by VHL treated at the University Hospital of La Ribera and the results of the genetic analysis of three relatives, identifying the mutation R167G in exon 3 of VHL gene as the cause of VHL syndrome in this family.

Association of PD-1, PD-L1, and CTLA-4 Gene Expression and Clinicopathologic Characteristics in Patients With Non-Small-Cell Lung Cancer.

INTRODUCTION: Recent studies show a potential benefit of therapies that target programmed death receptor 1 (PD-1)/programmed death-ligand 1 (PD-L1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) inhibitory checkpoints in a subgroup of patients with non-small-cell lung cancer (NSCLC), without the clinicopathologic characteristics related to positive responses to these treatments being well determined. The aim of this study was to determine PD-1, PD-L1, and CTLA-4 gene expression at the mRNA level in tumoral tissue from patients with NSCLC and analyze their possible relationship with the clinicopathological characteristics and their potential prognostic role. PATIENTS AND METHODS: PD-1, PD-L1, and CTLA-4 expression levels were analyzed using real-time quantitative reverse transcriptase polymerase chain reaction in fresh-frozen tumor and normal adjacent lung tissue samples from 78 patients with NSCLC. Later, a significant association between mRNA levels, clinicopathologic characteristics, and patient's survival was assessed. RESULTS: No significant correlation between gene expression levels and sex, age, histological type, smoking status, pathologic stage, or tumor differentiation was found. However, higher levels of PD-1 were significantly associated with worse prognosis in patients with NSCLC, and PD-L1 overexpression was associated with a worse prognosis in stage I patients and in Grade 1 to 2 tumors. CONCLUSION: Alterations in PD-1/PD-L1 and CTLA-4 expression in lung tumoral tissue seem not to be related to age, sex, smoking status, histological type, pathological stage, or tumor differentiation degree. However, PD-1 and PD-L1 overexpression might predict worse survival in patients with stage I NSCLC and in well differentiated tumors.

Fluorescence-guided surgery and biopsy in gliomas with an exoscope system.

BACKGROUND: The introduction of fluorescence-guided resection allows a better identification of tumor tissue and its more radical resection. We describe our experience with a modified exoscope to detect 5 ALA-induced fluorescence in neuronavigation-guided brain surgery or biopsy of malignant brain tumors. METHODS: Thirty-eight patients with a suspected preoperative diagnosis of high-grade astrocytoma were included. We used a neuronavigation device and a high-definition exoscope system with a built-in filter to detect 5-ALA fluorescence in all cases. Thirty patients underwent craniotomy with tumor resection and 8 underwent frameless stereotactic brain biopsy. RESULTS: Histopathological diagnosis confirmed the presence of high-grade gliomas in 34 patients. Total resection was achieved in 23 cases and subtotal in 7. No relevant complications related to the administration of 5-ALA were detected. CONCLUSIONS: The use of the exoscope in 5-ALA fluorescence-guided tumor surgery has twofold implications: during brain tumor surgery it can be considered a valuable tool to achieve a more radical resection of the lesion, and when applied to a biopsy of a suspected brain high-grade glioma, it decreases the possibility of a negative biopsy.

Fluorescence-guided surgery in high grade gliomas using an exoscope system.

BACKGROUND: Fluorescence-guided microsurgical resections of high-grade gliomas using 5-aminolevulinic acid (5-ALA) is superior to conventional microsurgery. An optical device, usually a modified microscope, is needed for these procedures. However, an exoscope may be implemented for fluorescence techniques. We present the use of an exoscope to perform tumor resection guided by 5-ALA fluorescence in 21 consecutive patients with high-grade glioma and two neuronavigation-guided biopsies. METHODS: Twenty-three patients underwent operations. Tumor volume and localization were quantified with pre- and postoperative volumetric MRI in non-biopsy cases. RESULTS: In non-biopsy cases, the age range was 20 to 79 years, with a median of 56 (interquartile range = 45-66). Histological analysis indicated that 14 had glioblastoma multiforme, 2 grade-III oligodendrogliomas and 1 anaplastic astrocytoma, 3 metastases and 1 low-grade astrocytoma. Total resection was achieved in 15 cases; subtotal resection was performed in 5 patients. The result was partial resection in one case. There was no perioperative mortality. The median fluorescence intensity, on a scale of 1-5, was 4.5 in the GBM group (IQR = 4-5), 3 (IQR = 2.5-3.5) in anaplastic glioma, and 2.5 (IQR = 2.25-2.75) for oligodendrogliomas. Of the three metastases, one showed fluorescence level 4. As for the two biopsy cases, one was anaplastic astrocytoma and one glioblastoma multiforme. The samples obtained were fluorescent in both cases. CONCLUSIONS: An exoscope can be also used for fluorescence-guided surgery with 5-aminolevulinic acid (5-ALA) and neuronavigation-guided biopsy. With an important advantage of low cost, this allows the surgeon to perform collaborative surgeries and adds agility to the procedure.

[Analysis of the COL1A1-PDGFB fusion gene in a case of dermatofibrosarcoma protuberans with a fibrosarcoma component]. / Análisis del gen de fusión COL1A1-PDGFB en un caso de dermatofibrosarcoma protuberans con componente de fibrosarcoma.

Dermatofibrosarcoma protuberans (DFSP) is an infrequent tumor of intermediate malignancy, with little tendency to develop metastases but with a high rate of local recurrence. Cytogenetically, DFSP is characterized by a reciprocal translocation, t(17;22)(q22;q13), which is a conditioning factor in the fusion of the collagen type I alpha I gene (COL1A1) in chromosome 17q with the platelet-derived growth factor beta chain gene (PDGFB) in chromosome 22q. The fusion of these genes is variable, involving one of the 51 exons of the COL1A1 gene and exon 2 of the PDGFB gene. We present the case of a 37-year-old woman with a tumor on the arm whose histology showed a neoplastic infiltration of the subcutaneous cellular tissue made up of fusiform cells with an elongated nucleus in a storiform pattern and other more pleomorphic cells in a herringbone pattern, compatible with DFSP with a fibrosarcoma component. The molecular biology study with RT-PCR analysis of paraffin-embedded material and later sequencing showed a new fusion of exon 19 of the COL1A1 gene and exon 2 of PDGFB, supporting a diagnosis of DFSP. A study of the COL1A1-PDGFB fusion products is useful in cases where histology and immunohistochemistry are insufficient for the differential diagnosis of DFSP versus other sarcomas. It also justifies the use of new avenues of treatment with tyrosine kinase inhibitors.