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Cervical cancer (CC), the second most common in developing countries and the third most common in developed nations, is the fourth most common type of cancer in women overall. The HPV16 high-risk genotype of the virus, which is responsible for about 61% of cervical cancer incidences, was found to have higher LCN2 levels in advanced clinical CC stages. In this study, we assessed the impact of suppressing LCN2 activity after treatment with an anti-LCN2 monoclonal antibody (MAb) in bothin vitroandin vivosettings. Anti-LCN2 antibody was found to reduce proliferation and invasion of HeLa cells, the first immortal cells from a HPV positive aggressive adenocarcinoma of the cervix. LCN2 and its ligand MMP9 was found to be highly expressed in the cells and abrogated on treatment with anti-LCN2. The five receptors of LCN2 - SLC22A17, MC1R, MC2R, MC4R and LRP2 were barely detected with or without treatment. Anti-LCN2 Mab caused tumors to regress and softenin vivo, in a xenograft mouse model. Analysis of histology images of the treated and untreated tumor established the necrotic capability of the therapeutic molecule explaining the regression and softening of the tumor. Differential gene expression analysis between untreated and treated tumor proved that LCN2 inhibition abolished the migratory, invasive, and hypoxic pathways while significantly increasing the necrosis and cell death pathways in tumor after treatment with the monoclonal antibody. LCN2 inhibition was shown molecularly to lead to tumor regression via a negative feedback loop of LCN2 through the TNFα-IL17 axis exponentially increasing the effect of the anti-LCN2 monoclonal antibody. In conclusion, LCN2 appears to be a viable therapeutic target, and the monoclonal antibody used in this study can be further developed for clinical usage in cervical cancer.

Due to the high rates of tobacco chewers, smokers, and alcohol consumers in India, Head and Neck Squamous Cell Carcinoma (HNSCC) is one of the primary causes of mortality. Being profoundly varied in nature, treating patients diagnosed with HNSCC can be difficult. An in vitro cell line model is needed to better comprehend the heterogeneity especially via the interaction with components of the microenvironment like Cancer Associated Fibroblasts (CAFs). The effectiveness of creating cell lines from head and neck cancers is, however, poor. Furthermore, except for the two reported earlier by us, no other CAF cell lines are available to study the cross-talk of the tumor with its microenvironment. In this study, we report three novel CAF lines, spontaneously immortalized from HPV negative male patients with habits of tobacco and diagnosed with squamous cell carcinoma of the upper alveolus, larynx and buccal mucosa as opposed to HPV positive non-habitual female patients with cancer of the tongue as previously reported. The CAFs increased the tumorigenicity of epithelial cells in indirect co-culture experiments. Negative staining with EpCAM, CD31 and CD45, while positive staining with FSP-1 determined their fibroblast specific lineage. The developed CAF cultures are the first of their kind from the mentioned sites, and they will be an invaluable tool for learning how tumor-stroma and tumor interact with one another and discovering newer targets and pathways for treating HNSCC.

Oral tongue squamous cell carcinoma (OTSCC) is one of the major causes of fatality in India due to very high percentage of patients with habits of smoking and chewing tobacco and associated products. Being highly heterogeneous in nature, every patient poses a different challenge clinically. To understand disease progression in an improved way, knowledge of cross‑talk between tumor stroma and the tumor cells becomes indispensable. Patient‑derived

Oral tongue squamous cell carcinoma (OTSCC) is one of the major causes of fatality in India owing to very high percentages of patients with smoking and chewing habits. Being highly heterogeneous in nature, every patient poses a different challenge clinically. To better understand disease progression, knowledge of cross talk between tumor stroma and the tumor cells becomes indispensable. Patient-derived in vitro cell line models are helpful to understand the complexity of diseases. However, they have very low efficiency of establishment from the tumor samples, especially the cancer associated fibroblasts (CAFs). In the present study, two novel autologous pairs have been immortalized spontaneously from non-habitual, HPV-positive patients, presented with tongue squamous cell carcinoma. The epithelial and fibroblast primary lines had typical polygonal and spindle shaped morphology, respectively. Positive staining with Pan-cytokeratin (PanCK) and Fibroblast Specific Protein (FSP-1) further confirmed their epithelial and fibroblast origin. Unique Short Tandem Repeat (STR) profile of the cultures confirmed their novelty, while the similarity of the STR profiles between the epithelial and fibroblast cells from the same patient, confirmed their autologous nature. DNA analysis revealed aneuploidy of the established cultures. Increase in the tumorigenic potential of the established epithelial cultures upon treatment with CAF-conditioned medium proved the “CAF-ness” of the established fibroblast cells. The established cultures are the first of their kind which would serve as an useful platform in understanding the cross talk between tumor-stroma and tumor, along with studying tongue cancer progression.

Lipocalin 2 is a siderophore binding protein that regulates iron homeostasis. Lipocalin 2 expression is elevated in multiple tumor types; however, the mechanisms that drive tumor progression upon Lipocalin 2 expression remain unclear. When Lipocalin 2 is over-expressed it leads to resistance to 5-fluorouracil in colon cancer cell lines in vitro and in vivo by inhibiting ferroptosis. Lipocalin 2 inhibits ferroptosis by decreasing intracellular iron levels and stimulating the expression of glutathione peroxidase4 and a component of the cysteine glutamate antiporter, xCT. The increase in xCT levels is dependent on increased levels of ETS1 in Lipocalin 2 over-expressing cells. Inhibiting Lipocalin 2 function with a monoclonal antibody leads to a decrease in chemo-resistance and transformation in vitro, and a decrease in tumor progression and chemo-resistance in xenograft mouse models. Lipocalin 2 and xCT levels exhibit a positive correlation in human tumor samples suggesting that the pathway we have identified in cell lines is operative in human tumor samples. These results indicate that Lipocalin 2 is a potential therapeutic target and that the monoclonal antibody described in this study can serve as the basis for a potential therapeutic in patients that do not respond to chemotherapy.

Dysregulation of BCL2 is a pathophysiology observed in haematological malignancies. For implementation of available treatment-options it is preferred to know the relative quantification of BCL2 mRNA with appropriate reference genes. For the choice of reference genes-(i) Reference Genes were selected by assessing variation of >60,000 genes from 4 RNA-seq datasets of haematological malignancies followed by filtering based on their GO biological process annotations and proximity of their chromosomal locations to known disease translocations. Selected genes were experimentally validated across various haematological malignancy samples followed by stability comparison using geNorm, NormFinder, BestKeeper and RefFinder. (ii) 43 commonly used Reference Genes were obtained from literature through extensive systematic review. Levels of BCL2 mRNA was assessed by qPCR normalized either by novel reference genes from this study or GAPDH, the most cited reference gene in literature and compared. The analysis showed PTCD2, PPP1R3B and FBXW9 to be the most unregulated genes across lymph-nodes, bone marrow and PBMC samples unlike the Reference Genes used in literature. BCL2 mRNA level shows a consistent higher expression in haematological malignancy patients when normalized by these novel Reference Genes as opposed to GAPDH, the most cited Reference Gene. These reference genes should also be applicable in qPCR platforms using Taqman probes and other model systems including cell lines and rodent models. Absence of sample from healthy-normal individual in diagnostic cases call for careful selection of Reference Genes for relative quantification of a biomarker by qPCR.BCL2 can be used as molecular diagnostics only if normalized with a set of reference genes with stable yet low levels of expression across different types of haematological malignancies.

Background Quantitative real time PCR (qPCR) remains by far the most cost-effective, fast yet sensitive technique to check the gene expression levels in various systems. The traditionally used reference genes over the years were found to be regulated heavily based on sample sources and/or experimental conditions. This paper therefore presents a data science driven -omic approach for selection of reference genes from ~60,000 candidates from The Cancer Genome Atlas (TCGA) and Broad Institute Cancer Cell Line Encyclopaedia (CCLE) for gene expression studies in head and neck squamous cell carcinoma (HNSCC). mRNA-sequencing data of 500 patient samples and 33 cell lines from publicly available databases were analysed to assess stability of genes in terms of multiple statistical measures. A final set of 12 candidate genes were studied in the Indian set of data in Gene Expression Omnibus (GEO) and validated experimentally using qPCR in 35 different types of samples from platforms like drug sensitive and resistant cell lines, normal and tumor samples, fibroblast and epithelial primary culture derived from HNSCC patients from India. Result The study lead to the choice of five most stable reference genes –TYW5, RIC8B, PLEKHA3, CEP57L1 and GPR89B across three experimental platforms. Conclusion In addition to providing a set of five most stable reference genes for future gene expression analysis experiments across different types of samples in HNSCC, the study also presents an objective framework for assessing reference genes for other disease areas as well.

e15608 Background: Lipocalin2 (LCN2, also known as neutrophil gelatinase-associated lipocalin) is a protein that in humans is encoded by the LCN2 gene. Its abnormal expression serves critical roles in EMT transition, angiogenesis, cell migration and invasion in many cancers. We aim to assess the in vitro and in vivo effects of LCN2 as a potential chemo and radiosensitizer. Methods: Normalized RNAseq RSEM values of LCN2 were compared between normal and tumour samples from TCGA. Differences between median expression levels were assessed using Wilcoxon rank sum test. Kaplan-Meier model was used for survival analysis. Immune cell population in publicly available Colon Adenocarcinoma dataset was estimated using MCP Counter tool. Cell systems used to experimentally study the role of LCN2 in therapy resistance and tumor progression were HCT116, HT29 and DLD1. PKP3 and/or LCN2 were knocked down by shRNA. Tumor regression and therapy (5FU and radiation) sensitivity upon Anti-LCN2 treatment were demonstrated in Xenograft mouse models. Results: Analysis of 23 TCGA datasets containing gene expression data for both tumour and adjacent normal samples indicated that LCN2 levels are elevated in colon tumors. Colon cancer cell line HCT116 derived PKP3 knock-down or LCN2 over-expressing cells showed therapy resistance. A comparison of the tumor cell lines HCT116, HT29 and DLD1 show that increased LCN2 expression correlates with therapy resistance. LCN2 levels correlated with resistance to 5FU (p = 0.006) and its ability to clear ROS (p < 0.05) in vitro. Inhibiting LCN2 led to a decrease in invasion in vitro (p = 0.0005), increased sensitivity to 5FU in vitro (p = 0.001) and inhibition in tumor growth and increased sensitivity to 5FU and radiation (p = 0.005) in xenograft mouse models. On MCP counter analysis of TCGA, in Colon adenoca the normal samples show a correlation between LCN2 expression and T-cells (Pearson r = 0.45, p = 0.0028) and with the T-cell chemoattractant CXCL10 (Pearson r = 0.5, p < 0.0001). Such correlations are broken in tumour samples. Conclusions: LCN2 expression leads to chemo and radio resistance in colon cancer cell lines and xenograft mouse models. Inhibiting LCN2 function can inhibit tumor progression and sensitizes tumors to radiation and 5FU. These results suggest that LCN2 expression could be a marker that can be used to determine the choice of therapy offered to patients and that LCN2 could serve as a therapeutic target that sensitizes cells to radio and chemotherapy. LCN2 affects tumor progression and therapy sensitivity probably through T cell mediated immune pathway.