Tumor-associated macrophages (TAMs) play a pivotal role in facilitating tumor growth and metastasis. This tumor-promoting propensity of TAMs sets in as a result of their complex cross-talk with tumor cells mediated primarily by tumor cell-secreted proteins in the tumor microenvironment. To explore such interactions, we employed an immunoscreening approach involving the immunization of Balb-c mice with model human lung carcinoma cell line, A549. From serological examination combined with mass spectrometric analysis, EDA-containing fibronectin (EDA FN) was identified as a conspicuous immunogenic protein in A549 cell secretome. We showed that A549 secreted EDA FN engages TLR-4 on THP-1 monocytes to drive the proinflammatory response via NF-κB signaling cascade. Conversely, A549 derived EDA FN potentiates their metastatic capacity by inducing epithelial-mesenchymal transition through its autocrine activity. In conclusion, the study proposes a possible mechanism of cellular cross-talk between lung cancer cells and associated monocytes mediated by lung cancer-derived EDA FN and resulting in the establishment of proinflammatory and metastatic tumor microenvironment.

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Objective Isolating high-quality RNA is a basic requirement while performing high throughput sequencing, microarray, and various other molecular investigations. However, it has been quite challenging to isolate RNA with absolute purity from plants like Crocus sativus that are rich in secondary metabolites, polysaccharides, and other interfering compounds which often irreversibly co-precipitate with the RNA. While many methods have been proposed for RNA extraction including CTAB, TriZol, and SDS-based methods, which invariably yield less and poor quality RNA and hence it necessitated the isolation of high-quality RNA suitable for high throughput applications. Results In the present study we made certain adjustments to the available protocols including modifications in the extraction buffer itself and the procedure employed. Our method led to the isolation of clear and non-dispersive total RNA with an RNA Integrity Number (RIN) value greater than 7.5. The quality of the RNA was further assessed by qPCR-based amplification of mRNA and mature miRNAs such as Cs-MIR166c and Cs-MIR396a.

Isolating high quality RNA is a basic requirement while performing high throughput sequencing, microarray and various other molecular investigations. However, it has been quite challenging to isolate RNA with absolute purity from plants like Crocus sativus that are rich in secondary metabolites, polysaccharides and other interfering compounds which often irreversibly co-precipitate with the RNA. While many methods have been proposed for RNA extraction that include CTAB, TriZol, SDS based methods, they invariably yield less and poor quality RNA. In the present study we made certain changes in the available protocols including modifications in the extraction buffer and procedure viz-a-viz solutions used for precipitation of RNA. Our method led to the isolation of clear and non-dispersive total RNA with an RNA Integrity Number (RIN) greater than 7.5. The quality of the RNA was further assessed by qPCR based amplification of mature miRNAs such as Cs-MIR166c and Cs- MIR396a. In conclusion, the study describes an efficient method of RNA extraction that is highly ideal for high throughput sequencing of small RNAs.