Drug-Resistant Epithelial Ovarian Cancer: Current and Future Perspectives.

Epithelial ovarian cancer (EOC) is a complex disease with diverse histological subtypes, which, based on the aggressiveness and course of disease progression, have recently been broadly grouped into type I (low-grade serous, endometrioid, clear cell, and mucinous) and type II (high-grade serous, high-grade endometrioid, and undifferentiated carcinomas) categories. Despite substantial differences in pathogenesis, genetics, prognosis, and treatment response, clinical diagnosis and management of EOC remain similar across the subtypes. Debulking surgery combined with platinum-taxol-based chemotherapy serves as the initial treatment for High Grade Serous Ovarian Carcinoma (HGSOC), the most prevalent one, and for other subtypes, but most patients exhibit intrinsic or acquired resistance and recur in short duration. Targeted therapies, such as anti-angiogenics (e.g., bevacizumab) and PARP inhibitors (for BRCA-mutated cancers), offer some success, but therapy resistance, through various mechanisms, poses a significant challenge. This comprehensive chapter delves into emerging strategies to address these challenges, highlighting factors like aberrant miRNAs, metabolism, apoptosis evasion, cancer stem cells, and autophagy, which play pivotal roles in mediating resistance and disease relapse in EOC. Beyond standard treatments, the focus of this study extends to alternate targeted agents, including immunotherapies like checkpoint inhibitors, CAR T cells, and vaccines, as well as inhibitors targeting key oncogenic pathways in EOC. Additionally, this chapter covers disease classification, diagnosis, resistance pathways, standard treatments, and clinical data on various emerging approaches, and advocates for a nuanced and personalized approach tailored to individual subtypes and resistance mechanisms, aiming to enhance therapeutic outcomes across the spectrum of EOC subtypes.

Correction: Mukherjee et al. Homo and Heterotypic Cellular Cross-Talk in Epithelial Ovarian Cancer Impart Pro-Tumorigenic Properties through Differential Activation of the Notch3 Pathway. Cancers 2022, 14, 3365.

In the original publication [...].

Triple Reporter Assay: A Non-Overlapping Luciferase Assay for the Measurement of Complex Macromolecular Regulation in Cancer Cells Using a New Mushroom Luciferase-Luciferin Pair.

This study demonstrates the development of a humanized luciferase imaging reporter based on a recently discovered mushroom luciferase (Luz) from Neonothopanus nambi. In vitro and in vivo assessments showed that human-codon-optimized Luz (hLuz) has significantly higher activity than native Luz in various cancer cell types. The potential of hLuz in non-invasive bioluminescence imaging was demonstrated by human tumor xenografts subcutaneously and by the orthotopic lungs xenograft in immunocompromised mice. Luz enzyme or its unique 3OH-hispidin substrate was found to be non-cross-reacting with commonly used luciferase reporters such as Firefly (FLuc2), Renilla (RLuc), or nano-luciferase (NLuc). Based on this feature, a non-overlapping, multiplex luciferase assay using hLuz was envisioned to surpass the limitation of dual reporter assay. Multiplex reporter functionality was demonstrated by designing a new sensor construct to measure the NF-κB transcriptional activity using hLuz and utilized in conjunction with two available constructs, p53-NLuc and PIK3CA promoter-FLuc2. By expressing these constructs in the A2780 cell line, we unveiled a complex macromolecular regulation of high relevance in ovarian cancer. The assays performed elucidated the direct regulatory action of p53 or NF-κB on the PIK3CA promoter. However, only the multiplexed assessment revealed further complexities as stabilized p53 expression attenuates NF-κB transcriptional activity and thereby indirectly influences its regulation on the PIK3CA gene. Thus, this study suggests the importance of live cell multiplexed measurement of gene regulatory function using more than two luciferases to address more realistic situations in disease biology.

The Power of Kinetic Inertness in Improving Platinum Anticancer Therapy by Circumventing Resistance and Ameliorating Nephrotoxicity.

Even in the modern era of precision medicine and immunotherapy, chemotherapy with platinum (Pt) drugs remains among the most commonly prescribed medications against a variety of cancers. Unfortunately, the broad applicability of these blockbuster Pt drugs is severely limited by intrinsic and/or acquired resistance, and high systemic toxicity. Considering the strong interconnection between kinetic lability and undesired shortcomings of clinical Pt drugs, we rationally designed kinetically inert organometallic Pt based anticancer agents with a novel mechanism of action. Using a combination of in vitro and in vivo assays, we demonstrated that the development of a remarkably efficacious but kinetically inert Pt anticancer agent is feasible. Along with exerting promising antitumor efficacy in Pt-sensitive as well as Pt-resistant tumors in vivo, our best candidate has the ability to mitigate the nephrotoxicity issue associated with cisplatin. In addition to demonstrating, for the first time, the power of kinetic inertness in improving the therapeutic benefits of Pt based anticancer therapy, we describe the detailed mechanism of action of our best kinetically inert antitumor agent. This study will certainly pave the way for designing the next generation of anticancer drugs for effective treatment of various cancers.

Through the Looking Glass: Updated Insights on Ovarian Cancer Diagnostics.

Epithelial ovarian cancer (EOC) is the deadliest gynaecological malignancy and the eighth most prevalent cancer in women, with an abysmal mortality rate of two million worldwide. The existence of multiple overlapping symptoms with other gastrointestinal, genitourinary, and gynaecological maladies often leads to late-stage diagnosis and extensive extra-ovarian metastasis. Due to the absence of any clear early-stage symptoms, current tools only aid in the diagnosis of advanced-stage patients, wherein the 5-year survival plummets further to less than 30%. Therefore, there is a dire need for the identification of novel approaches that not only allow early diagnosis of the disease but also have a greater prognostic value. Toward this, biomarkers provide a gamut of powerful and dynamic tools to allow the identification of a spectrum of different malignancies. Both serum cancer antigen 125 (CA-125) and human epididymis 4 (HE4) are currently being used in clinics not only for EOC but also peritoneal and GI tract cancers. Screening of multiple biomarkers is gradually emerging as a beneficial strategy for early-stage diagnosis, proving instrumental in administration of first-line chemotherapy. These novel biomarkers seem to exhibit an enhanced potential as a diagnostic tool. This review summarizes existing knowledge of the ever-growing field of biomarker identification along with potential future ones, especially for ovarian cancer.

Photothermal therapy (PTT) is an effective treatment measure against solid tumors which fails to respond conventional chemo/radiation therapies in clinic.

Photothermal therapy (PTT) has emerged as a fast, precisive, and cost-effective anticancer therapy protocol. Here we applied our previously designed nanomaterial (Tocophotoxil) for prospective PTT application to manage radiation- and chemo-resistant cancers in a preclinical model. A PTT dose vs. efficacy relationship was established for radioresistant breast (ZR-75-1 50Gy, 4T1 20Gy) and chemo-resistant ovarian (A2780LR) cancer cells and tumors in mice models. Compared to the sensitive cases, resistant cells treated with PTT for a shorter duration show higher endurance. However, preclinical tumor xenografts treated with optimal PTT dose show 2-3 fold higher longevity (P ≤ 0.05) of treated mice monitored by non-invasive imaging methods. Elevated ERK and AKT activation in radioresistant or only AKT activation in chemo-resistant cells were contributory to higher cell survival in sub-optimal PTT dose. A comprehensive single-cell Raman map of PTT treated ZR-75-1 cell reveals broad-spectrum macromolecular deformities, including protein damage features. Marked induction of pJNK, unfolded protein response (UPR) pathway, increased reactive oxygen species (ROS), and lipid peroxidation in PTT-treated cells disrupted the intracellular homeostasis. Analyzing cellular ultrastructure, the coexistence of swollen endoplasmic reticulum, and autophagic bodies after PTT indicate possible coordination between UPR and autophagy pathways. Therefore, this comprehensive study provides new evidence on the potential impact of PTT as a standalone therapy for ablation of failed conventional therapy-resistant cancers in vivo, the success of which is intricately linked to the PTT dose optimization. The study, for the first time, also illustrates that under PTT treatment, concerted action of novel molecular switches such as JNK activation and UPR activation plays a vital role in triggering autophagy and cancer cell death.

A Multistep Tumor Growth Model of High Grade Serous Ovarian Carcinoma Identifies Hypoxia Associated Signatures.

High-grade serous ovarian carcinoma (HGSC) is associated with late-stage disease presentation and poor prognosis, with limited understanding of early transformation events. Our study presents a comprehensive analysis of tumor progression and organ-specific metastatic dissemination to identify hypoxia-associated molecular, cellular, and histological alterations during HGSC tumor growth. H&E staining and subsequent histological assessment of tumor volume-based categories revealed recapitulation of numerous clinical features, including the prevalence of >0.0625≤0.5cm3 volume tumors and metastatic spread by orthotopic xenografts. The constant evolution of the tissue architecture concerning increased hyaluronic acid deposition, tumor vasculature, necrosis, altered proliferative potential, and gland forming ability of the tumor cells was identified. Flow cytometry and label chase-based molecular profiling across the tumor regenerative hierarchy identified the hypoxia-vasculogenic niche and the hybrid epithelial-mesenchymal tumor-cell state as determinants of self-renewal capabilities of progenitors and cancer stem cells (CSCs). A regulatory network and mathematical model based on tumor histology and molecular signatures predicted hypoxia-inducible factor 1-alpha (HIF1A) as a central node connecting epithelial-mesenchymal transition, metabolic and necrotic pathways in HGSC tumors. Thus, our findings provide a temporal resolution of hypoxia-associated events that sculpt HGSC tumor growth, and an in-depth understanding of it may aid in the early detection and treatment of HGSC.

Homo and Heterotypic Cellular Cross-Talk in Epithelial Ovarian Cancer Impart Pro-Tumorigenic Properties through Differential Activation of the Notch3 Pathway.

An active fluidic microenvironment governs peritoneal metastasis in epithelial ovarian cancer (EOC), but its critical functional/molecular cues are not fully understood. Utilizing co-culture models of NIH3T3 cells (differentially overexpressing Jagged1) and SKOV3 cells expressing a Notch3 luciferase reporter-sensor (SNFT), we showed that incremental expression of Jagged1 led to proportional Notch3 activation in SNFT. With no basal luciferase activity, this system efficiently recorded dose-dependent Notch3 activation by rh-Jag1 peptide and the non-appearance of such induction in co-culture with NIH3T3Δjag1 cells indicates its sensitivity and specificity. Similar Notch3 modulation was shown for the first time in co-cultures with HGSOC patients' ascites-derived cancer-associated fibroblasts and Jagged1-expressing EOC cell lines. NIH3T3J1-A and OVCAR3 co-cultured SNFT cells showed maximum proliferation, invasion, and cisplatin resistance among all the heterotypic/homotypic cellular partners. VEGFA and CDKN1A are the two most upregulated genes identified across co-cultures by the gene profiler array. Co-culture induced VEGFA secretion from SNFT cells which also reduced cancer stem cell differentiation in platinum-resistant A2780 cells. rh-Jag1-peptide promoted enhanced nuclear-cytoplasmic p21 expression. Additionally, metastatic HGSOC tumors had higher VEGFA than corresponding primary tumors. This study thus demonstrates the tumoral and non-tumoral cell-mediated differential Notch3 activation imparting its tumorigenic effects through two critical molecular regulators, VEGFA and p21, during EOC progression.

Developing Clinically Relevant Acquired Chemoresistance Models in Epithelial Ovarian Cancer Cell Lines.

Chemoresistance, the ability of cancer cells to overcome therapeutic interventions, is an area of active research. Studies on intrinsic and acquired chemoresistance have partly succeeded in elucidating some of the molecular mechanisms in this elusive phenomenon. Hence, drug-resistant cellular models are routinely developed and used to mimic the clinical scenario in-vitro. In an attempt to identify the underlying molecular mechanisms that allow ovarian cancer cells to gradually acquire chemoresistance, we have developed isogenic cellular models of cisplatin and paclitaxel resistance (singularly and in combination) over six months, using a clinically relevant modified pulse method. These models serve as important tools to investigate the underlying molecular players, modulation in genetics, epigenetics, and relevant signaling pathways, as well as to understand the role of drug detoxification and drug influx-efflux pathways in development of resistance. These models can also be used as screening tools for new therapeutic molecules. Additionally, repurposing therapeutic agents approved for diseases other than cancer have gained significant attention in improving cancer therapy. To investigate the effect of metformin on acquirement of chemoresistance, we have also developed a combinatorial model of metformin and platinum-taxol, using two different strategies. All these models were subsequently used to study modulation in receptor tyrosine kinase pathways, cancer stem cell functionalities, autophagy, metastasis, metabolic signatures, and various biological processes during development of chemoresistance. Herein, we outline the protocols used for developing these intricate resistant cellular models.

IGF1R-α6 integrin-S100A4 network governs the organ-specific metastasis of chemoresistant epithelial ovarian cancer cells.

Recurrent metastatic epithelial ovarian cancer (EOC) is challenging and associated with treatment limitations, as the mechanisms governing the metastatic behavior of chemoresistant EOC cells remain elusive. Using orthotopic xenograft mouse models of sensitive and acquired platinum-taxol-resistant A2780 EOC cells, we studied the mechanistic role of insulin like growth factor 1 receptor (IGF1R) signaling in the regulation of organ-specific metastasis of EOC cells undergoing acquirement of chemoresistance. Biochemical assays and organ-specific fibroblast-EOC cell co-culture were used to study the differential metastatic characteristics of sensitive vs. chemoresistant EOC cells, and the key molecule/s underlying the organ-specific homing of chemoresistant EOC cells were identified through subtractive LC/MS profiling of the co-culture secretome. The role of the identified molecule was validated through genetic/pharmacologic perturbation experiments. Acquired chemoresistance augmented organ-specific metastasis of EOC cells and enhanced lung homing, particularly for the late-stage chemoresistant cells, which was abrogated after IGF1R silencing. Escalation of chemoresistance (intrinsic and acquired) conferred EOC cells with higher adhesion toward primary lung fibroblasts, largely governed by the α6 integrin-IGF1R dual signaling axes. Subtractive analysis of the co-culture secretome revealed that interaction with lung fibroblasts induced the secretion of S100A4 from highly resistant EOC cells, which reciprocally activated lung fibroblasts. Genetic and pharmacologic inhibition of S100A4 significantly lowered distant metastases and completely abrogated lung-tropic nature of late-stage chemoresistant EOC cells. These results indicate that chemoresistance exacerbates organ-specific metastasis of EOC cells via the IGF1R-α6 integrin-S100A4 molecular network, of which S100A4 may serve as a potential target for the treatment of recurrent metastatic EOC.

Predicting response to platinum and non-platinum drugs through bioluminescence resonance energy transfer (BRET) based bio-molecular interactions in platinum resistant epithelial ovarian cancer.

Therapy induced rewiring of signalling networks often lead to acquirement of platinum-resistance, thereby necessitating the use of non-platinum agents as second-line treatment particularly for epithelial ovarian cancer (EOC). A prior subject-specific assessment can guide the choice of optimal non-platinum agent/s and possible targeted therapeutic/s. Assessment of protein-protein interactions are superior to simple cytotoxicity assays to determine therapeutic efficacy and associated molecular responses. Utilizing improved PIP3-AKT and ERK1/2 activation Bioluminescence Resonance Energy Transfer (BRET) sensors, we report chemotherapy-induced ERK1/2 activation predominantly in cisplatin-paclitaxel resistant EOC cells and increased activation of both ERK1/2 and AKT in malignant ascites derived cancer cells from platinum-resistant patients but not from treatment-naive or platinum-sensitive relapse patients. Further, majority of the non-platinum drugs except irinotecan increased ERK1/2 activation in platinum-taxol resistant cells as observed by live-cell BRET assessment which were associated with p90RSK1/2 and BAD activation along with upregulation of multidrug transporter gene ABCC1 and cell survival genes like cyclin D1 and Bcl2. Interestingly, only irinotecan was able to sensitize these resistant cells. Altogether, this first report of BRET based sensing of molecular pathway activations in platinum resistant cell lines and patient's derived cancer cells highlight the clinical potential of BRET sensors in management of therapy resistant cancer.

Molecular imaging of the kinetics of hyperactivated ERK1/2-mediated autophagy during acquirement of chemoresistance.

Alterations in key kinases and signaling pathways can fine-tune autophagic flux to promote the development of chemoresistance. Despite empirical evidences of strong association between enhanced autophagic flux with acquired chemoresistance, it is still not understood whether an ongoing autophagic flux is required for both initiation, as well as maintenance of chemoresistance, or is sufficient for one of the either steps. Utilizing indigenously developed cisplatin-paclitaxel-resistant models of ovarian cancer cells, we report an intriguing oscillation in chemotherapy-induced autophagic flux across stages of resistance, which was found to be specifically elevated at the early stages or onset of chemoresistance. Conversely, the sensitive cells and cells at late stages of resistance showed stalled and reduced autophagic flux. This increased flux at early stages of resistance was found to be dictated by a hyperactive ERK1/2 signaling, which when inhibited either pharmacologically (U0126/Trametinib) or genetically, reduced p62 degradation, number of LC3+veLAMP1+ve puncta, autophagolysosome formation, and led to chemo-sensitization and apoptosis. Inhibition of ERK1/2 activation also altered the level of UVRAG and Rab7, the two key proteins involved in autophagosome-lysosome fusion. Noninvasive imaging of autophagic flux using a novel autophagy sensor (mtFL-p62 fusion reporter) showed that combinatorial treatment of platinum-taxol along with Trametinib/chloroquine blocked autophagic flux in live cells and tumor xenografts. Interestingly, Trametinib was found to be equally effective in blocking autophagic flux as chloroquine both in live cells and tumor xenografts. Combinatorial treatment of Trametinib and platinum-taxol significantly reduced tumor growth. This is probably the first report of real-time monitoring of chemotherapy-induced autophagy kinetics through noninvasive bioluminescence imaging in preclinical mouse model. Altogether our data suggest that an activated ERK1/2 supports proper completion of autophagic flux at the onset of chemoresistance to endure initial chemotherapeutic insult and foster the development of a highly chemoresistant phenotype, where autophagy becomes dispensable.

Decoding molecular interplay between RUNX1 and FOXO3a underlying the pulsatile IGF1R expression during acquirement of chemoresistance.

Hyperactive Insulin like growth factor-1-receptor (IGF1R) signalling is associated with development of therapy resistance in many cancers. We recently reported a pulsatile nature of IGF1R during acquirement of platinum-taxol resistance in Epithelial Ovarian Cancer (EOC) cells and a therapy induced upregulation in IGF1R expression in tumors of a small cohort of high grade serous EOC patients. Here, we report Runt-related transcription factor 1 (RUNX1) as a novel transcriptional regulator which along with another known regulator Forkhead Box O3 (FOXO3a), drives the dynamic modulation of IGF1R expression during platinum-taxol resistance development in EOC cells. RUNX1-FOXO3a cooperatively bind to IGF1R promoter and produce a transcriptional surge during onset of resistance and such co-operativity falls apart when cells attain maximal resistance resulting in decreased IGF1R expression. The intriguing descending trend in IGF1R and FOXO3a expressions is caused by a Protein Kinase B (AKT)-FOXO3a negative feedback loop exclusively present in the highly resistant cells eliciting the pulsatile behaviour of IGF1R and FOXO3a. In vivo molecular imaging revealed that RUNX1 inhibition causes significant attenuation of the IGF1R promoter activity, decreased tumorigenicity and enhanced drug sensitivity of tumors of early resistant cells. Altogether our findings delineate a dynamic interplay between several molecular regulators driving pulsatile IGF1R expression and identify a new avenue for targeting EOC through RUNX1-IGF1R axis during acquirement of chemoresistance.

Subtype specific biomarkers associated with chemoresistance in epithelial ovarian cancer.

In spite of the advent of many high throughput technologies, tumor tissue biomarkers are still the gold standard for diagnosis and prognosis of different malignancies including epithelial ovarian cancer (EOC). EOC is a heterogeneous disease comprised of five major subtypes which show distinct clinicopathological features and therapy response. Acquirement of chemoresistance toward therapy is a major challenge for successful treatment outcome in EOC patients. Several markers have been tested by immunohistochemical method to evaluate their prognostic merit to predict clinical outcome. However, a vast majority of such markers have been assessed for high-grade serous and clear cell ovarian cancer, among all subtypes of EOC. The current review elaborates upon those biomarkers that can potentially predict chemoresistance with subtype specificity.

EpCAM-Mediated Cellular Plasticity Promotes Radiation Resistance and Metastasis in Breast Cancer.

Substantial number of breast cancer (BC) patients undergoing radiation therapy (RT) develop local recurrence over time. During RT therapy, cells can gradually acquire resistance implying adaptive radioresistance. Here we probe the mechanisms underlying this acquired resistance by first establishing radioresistant lines using ZR-75-1 and MCF-7 BC cells through repeated exposure to sub-lethal fractionated dose of 2Gy up to 15 fractions. Radioresistance was found to be associated with increased cancer stem cells (CSCs), and elevated EpCAM expression in the cell population. A retrospective analysis of TCGA dataset indicated positive correlation of high EpCAM expression with poor response to RT. Intriguingly, elevated EpCAM expression in the radioresistant CSCs raise the bigger question of how this biomarker expression contributes during radiation treatment in BC. Thereafter, we establish EpCAM overexpressing ZR-75-1 cells (ZR-75-1EpCAM), which conferred radioresistance, increased stemness through enhanced AKT activation and induced a hybrid epithelial/mesenchymal phenotype with enhanced contractility and invasiveness. In line with these observations, orthotopic implantation of ZR-75-1EpCAM cells exhibited faster growth, lesser sensitivity to radiation therapy and increased lung metastasis than baseline ZR-75-1 cells in mice. In summary, this study shows that similar to radioresistant BC cells, EpCAM overexpressing cells show high degree of plasticity and heterogeneity which ultimately induces radioresistant and metastatic behavior of cancer cells, thus aggravating the disease condition.

Selectivity Enhancement of Paclitaxel Liposome Towards Folate Receptor-Positive Tumor Cells by Ligand Number Optimization Approach.

The present work aims to develop folate-targeted paclitaxel liposome (F-PTX-LIP), which will selectively target tumor cells overexpressing folate receptor (FR) and leave normal cells. Liposomes were prepared by thin-film hydration method followed by post-insertion of synthesized ligand 1,2-distearoyl-sn-glycero-phosphoethanolamine-polyethyleneglycol 2000-folic acid (DSPE-PEG2000-FA) on the outer surface of the liposome. The synthesized ligand was evaluated for in vivo acute toxicity in Balb/c mice. Developed liposomal formulations were characterized using transmission electron microscopy (TEM) and small-angle neutron scattering (SANS). We have investigated the effect of ligand number on cell uptake and cytotoxicity by confocal laser scanning microscopy (CLSM), competitive inhibition and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay. Compared to lung adenocarcinoma cells (A549), uptake in human ovarian carcinoma cells (SKOV3) was 2.2- and 1.2-fold higher for liposome with 480 and 240 ligand number respectively. Competitive inhibition experiment shows that prior incubation of SKOV3 cells with free folic acid significantly reduced the cell uptake of F-PTX-LIP with 480 ligand number (480 F-PTX-LIP) by 2.6-fold. 480 F-PTX-LIP displays higher cytotoxicity than free drug and PTX liposome. Moreover, it specifically targets the cells with higher folate receptor expression. Optimized 480 F-PTX-LIP formulation can be potentially useful for the treatment of folate receptor-positive tumors.

IGF1R predicts better survival in high-grade serous epithelial ovarian cancer patients and correlates with hCtr1 levels.

Aim: To evaluate the potential of IGF1R as a prognostic marker for high-grade serous ovarian cancer (HGSOC) patients. Patients & methods: The expression levels of IGF1R and drug transporters (ABCB1, hCtr1) were measured longitudinally in chemo-naive and chemo-treated tumor samples from 19 HGSOC patients, and their correlation with the clinical outcome was examined. Results:IGF1R expression was significantly upregulated in treated tumor samples, which positively correlated with hCtr1 levels. Patients with metastatic tumors with IGF1R expression higher than median showed better overall survival (median not reached) and disease-free survival (26.7 months) than those with less than median expression (overall survival: 27.5 months [p = 0.029]; disease-free survival: 11.9 months [p = 0.014]). Conclusion: IGF1R prognosticates prolonged survival in HGSOC patients, possibly due to its positive correlation with hCtr1.

Long term treatment of metformin impedes development of chemoresistance by regulating cancer stem cell differentiation through taurine generation in ovarian cancer cells.

Development of resistance poses a significant challenge to effective first-line platinum based therapy for epithelial ovarian cancer patients. Cancer Stem Cells are envisaged as a critical underlying factor for therapy resistance. Thus, there is a critical need for developing approaches to diminish the enrichment of cancer stem cells and acquirement of resistance. Administration of metformin, a commonly prescribed drug against Type II diabetes exhibited promising effect in the management of ovarian cancer. However, the effect of long term administration of low dose of metformin as an adjuvant to cisplatin and paclitaxel during acquirement of chemoresistant phenotype has not been investigated so far. Using two isogenic cellular chemoresistant models (A2780 and OAW42) developed in the presence or absence of metformin, we demonstrated the ability of metformin to impede the development of resistance through increased drug sensitivity, increased proliferation, and reduced migratory abilities of the resistant cells. Metformin introduction also decreased the cancer stem cell population, expression of specific biomarkers and pluripotent genes. Further metabolic profiling of these cells using 1H-Nuclear Magnetic Resonance spectroscopy revealed significant modulation in taurine and histidine levels in resistant cells developed in the presence of metformin. Intriguingly, taurine treatment considerably reduced the cancer stem cell population and chemoresistance in resistant cells, indicating a novel role of taurine in differentiation of ovarian cancer stem cells. Altogether this is the first report on the potential role of metformin for targeting the cancer stem cell population via up regulation of taurine, leading to impediment in the acquirement of chemoresistance.

Dual Modality Imaging of Promoter Activity as a Surrogate for Gene Expression and Function.

Molecular functional imaging with optical reporter genes (both bioluminescence and fluorescence) is a rapidly evolving method that allows noninvasive, sensitive, real-time monitoring of many cellular events in live cells and whole organisms. These reporter genes with optical signatures when expressed from gene-specific promoters or Cis/Trans elements mimic the endogenous expression pattern without perturbing cellular physiology. With advanced recombinant molecular biology techniques, several strategies for optimal expression from constitutive or inducible, tissue-specific and weak promoters have been developed and used for dynamic and functional imaging. In this chapter, we provide an overview of the applications of this powerful technology for imaging gene expression in living cells and rodent models.

Construction of Dual Modality Optical Reporter Gene Constructs for Bioluminescent and Fluorescent Imaging.

Dual modality reporter genes are powerful means of tracking cellular processes in cell culture systems and whole animals. In this chapter, we describe the methods for construction of a plasmid reporter gene vector expressing a fluorescent and a bioluminescent gene and its validation by in vitro assays in mammalian cells as well as by noninvasive imaging methods in small animal models.