Novel combinatorial autophagy inhibition therapy for triple negative breast cancers.

BACKGROUND: Triple negative breast cancer (TNBC) has the worst prognosis among breast cancer subtypes. It is characterized by lack of estrogen, progesterone and human epidermal growth factor 2 receptors, and thus, have limited therapeutic options. Autophagy has been found to be correlated with poor prognosis and aggressive behaviour in TNBC. This study aimed to target autophagy in TNBC via a novel approach to inhibit TNBC progression. METHODS: Immunoblotting and confocal microscopy were carried out to examine the effect of tumor microenvironmental stressors on autophagy. Cellular proliferation and migration assays were used to test the effect of different autophagy inhibitors and standard chemotherapy alone or in combination. In vivo xenograft mouse model was utilized to assess the effect of autophagy inhibitors alone or in combination with Paclitaxel. High resolution mass spectrometry based proteomic analysis was performed to explore the mechanisms behind chemoresistance in TNBC. Lastly, immunohistochemistry was done to assess the correlation between autophagy related proteins and clinical characteristics in TNBC tissue specimens. RESULTS: Metabolic stressors were found to induce autophagy in TNBC cell lines. Autophagy initiation inhibitors, SAR405 and MRT68921, showed marked synergy in their anti-proliferative activity in both chemosensitive and chemoresistant TNBC cell models. Paradoxically, positive expression of autophagosome marker LC3 was shown to be associated with better overall survival of TNBC patients. CONCLUSION: In this study, a novel combination between different autophagy inhibitors was identified which inhibited tumor cell proliferation in both chemosensitive and chemoresistant TNBC cells and could result in development of a novel treatment modality against TNBC.

Inhibition of autophagy initiation: A novel strategy for oral squamous cell carcinomas.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is one of the most common forms of oral cancer and is known to have poor prognostic outcomes. Autophagy is known to be associated with aggressive tumor biology of OSCC. Hence, this study aimed to develop a novel therapeutic strategy against OSCC by targeting the autophagic pathway. METHODS: Immunoblotting, and confocal microscopy were used to examine the effect of tumor microenvironmental stressors on the autophagy activity. Cellular proliferation and migration assays were performed to assess the anti-cancer activity of standard chemotherapy and autophagy initiation inhibitors, either alone or in combination. High resolution mass-spectrometry based proteomic analysis was utilized to understand the mechanisms behind chemoresistance in OSCC models. Finally, immunohistochemistry was performed to determine associations between autophagy markers and clinicopathological characteristics. RESULTS: Tumor microenvironmental stressors were shown to induce autophagy activity in OSCC cell lines. Novel combinations of chemotherapy and autophagy inhibitors as well as different classes of autophagy inhibitors were identified. Combination of MRT68921 and SAR405 demonstrated marked synergy in their anti-proliferative activity and also showed synergy with chemotherapy in chemoresistant OSCC cell models. Autophagy was identified as one of the key pathways involved in mediating chemoresistance in OSCC. Furthermore, TGM2 was identified as a key upstream regulator of chemoresistance in OSCC models. Finally, positive staining for autophagosome marker LC3 was shown to be associated with low histological grade OSCC. CONCLUSION: In conclusion, this study identified a combination of novel autophagy inhibitors which can potently inhibit proliferation of both chemosensitive as well as chemoresistant OSCC cells and could be developed as a novel therapy against advanced OSCC tumors.

Gene expression profiling of pancreatic ductal adenocarcinomas in response to neoadjuvant chemotherapy.

AIM: Pancreatic ductal adenocarcinoma (PDAC) has the lowest survival rate of all major cancers. Chemotherapy is the mainstay systemic therapy for PDAC, and chemoresistance is a major clinical problem leading to therapeutic failure. This study aimed to identify key differences in gene expression profile in tumors from chemoresponsive and chemoresistant patients. METHODS: Archived formalin-fixed paraffin-embedded tumor tissue samples from patients treated with neoadjuvant chemotherapy were obtained during surgical resection. Specimens were macrodissected and gene expression analysis was performed. Multi- and univariate statistical analysis was performed to identify differential gene expression profile of tumors from good (0%-30% residual viable tumor [RVT]) and poor (>30% RVT) chemotherapy-responders. RESULTS: Initially, unsupervised multivariate modeling was performed by principal component analysis, which demonstrated a distinct gene expression profile between good- and poor-chemotherapy responders. There were 396 genes that were significantly (p < 0.05) downregulated (200 genes) or upregulated (196 genes) in tumors from good responders compared to poor responders. Further supervised multivariate analysis of significant genes by partial least square (PLS) demonstrated a highly distinct gene expression profile between good- and poor responders. A gene biomarker of panel (IL18, SPA17, CD58, PTTG1, MTBP, ABL1, SFRP1, CHRDL1, IGF1, and CFD) was selected based on PLS model, and univariate regression analysis of individual genes was performed. The identified biomarker panel demonstrated a very high ability to diagnose good-responding PDAC patients (AUROC: 0.977, sensitivity: 82.4%; specificity: 87.0%). CONCLUSION: A distinct tumor biological profile between PDAC patients who either respond or not respond to chemotherapy was identified.

PSMA Expression Correlates with Improved Overall Survival and VEGF Expression in Glioblastoma.

BACKGROUND: Glioblastomas are the most common and fatal primary brain malignancy in adults. There is a growing interest in identifying the molecular mechanisms of these tumors to develop novel treatments. Glioblastoma neo-angiogenesis is driven by VEGF, and another potential molecule linked to angiogenesis is PSMA. Our study suggests the potential for an association between PSMA and VEGF expression in glioblastoma neo-vasculature. METHODS: Archived IDH1/2 wild-type glioblastomas were accessed; demographic and clinical outcomes were recorded. PSMA and VEGF expression by IHC were examined. Patients were dichotomized into PSMA expression high (3+) and low (0-2+) groups. The association between PSMA and VEGF expression was evaluated using Chi2 analysis. OS in PSMA high and low expression groups were compared using multi-linear regression. RESULTS: In total, 247 patients with IDH1/2 wild-type glioblastoma with archival tumor samples (between 2009-2014) were examined. PSMA expression correlated positively with VEGF expression (p = 0.01). We detected a significant difference in median OS between PSMA vascular endothelial expression high and low groups-16.1 and 10.8 months, respectively (p = 0.02). CONCLUSION: We found a potential positive correlation between PSMA and VEGF expression. Secondly, we showed a potential positive correlation between PSMA expression and overall survival.

The Right Treatment Strategy for the Right Patient: A Biomarker-Driven Approach to Neoadjuvant vs. Surgery-First Management of Resectable and Borderline Resectable Pancreatic Cancer.

The genomic heterogeneity of pancreatic ductal adenocarcinoma (PDAC) is becoming increasingly appreciated. We aimed to evaluate the ability of a triple biomarker panel (S100A4, Ca-125, and mesothelin) to predict: (i) genetic PDAC subtypes; (ii) clinical phenotypes; and (iii) the optimal treatment strategy (neoadjuvant vs. surgery-first) in resectable and borderline resectable PDAC. Patients who underwent resection for resectable and borderline resectable PDAC were included from one single-institutional cohort and one multi-institutional cohort from the Australian Pancreatic Genome Initiative (APGI). Tumors were immunohistochemically evaluated for S100A4, Ca-125, and mesothelin, and a subset from the APGI cohort underwent RNA sequencing. This study included 252 and 226 patients from the single institution and the APGI cohorts, respectively. Triple-negative biomarker status correlated with non-squamous PDAC genotypes (p = 0.020), lower rates of distant recurrence (p = 0.002), and longer median overall survival (mOS) with the surgery-first approach compared with neoadjuvant treatment (33.3 vs. 22.2 mths, p = 0.038) in resectable PDAC. In contrast, the triple-positive disease was associated with longer mOS with neoadjuvant treatment compared with the surgery-first approach (29.5 vs. 13.7 mths, p = 0.021) in resectable and borderline resectable PDAC. In conclusion, the triple biomarker panel predicts genetic PDAC subtypes, clinical phenotypes, and optimal treatment strategies in resectable and borderline resectable PDAC.

Tissue biomarker panel as a surrogate marker for squamous subtype of pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has been recently classified into four subtypes based on the gene expression levels, with squamous subtype having worst prognostic outcomes. However, gene expression analysis for each individual patient is not clinically feasible due to very high associated cost. We previously reported that levels of three biomarkers (S100A4, Ca-125 and Mesothelin) can be used to classify PDAC patients based on their survival outcomes. This project aimed to determine if this novel biomarker panel can be used as a surrogate to identify squamous PDAC subtype. METHODS: Using the Nanostring gene expression platform, tumor tissue from 24 PDAC patients were analysed for our novel biomarkers and markers associated with four PDAC subtypes. RESULTS: Gene expression of our biomarker panel (S100A4, Ca-125 and Mesothelin) closely clustered together with markers for squamous PDAC subtype. CONCLUSION: These results highlight the potential of our biomarkers to be utilized for identification of squamous PDAC subtype.

Biomarker panel predicts survival after resection in pancreatic ductal adenocarcinoma: A multi-institutional cohort study.

BACKGROUND: Up to 60% of patients who undergo curative-intent pancreatic ductal adenocarcinoma (PDAC) resection experience disease recurrence within six months. We recently published a systematic review of prognostic immunohistochemical biomarkers in PDAC and shortlisted a panel of those reported with the highest level of evidence, including p53, p16, Ca-125, S100A4, FOXC1, EGFR, mesothelin, CD24 and UPAR. This study aims to discover and validate the prognostic significance of a combinatorial panel of tumor biomarkers in patients with resected PDAC. METHODS: Patients who underwent PDAC resection were included from a single institution discovery cohort and a multi-institutional validation cohort. Tumors in the discovery cohort were stained immunohistochemically for all nine shortlisted biomarkers. Biomarkers significantly associated with overall survival (OS) were reevaluated as a combinatorial panel in both discovery and validation cohorts for its prognostic significance. RESULTS: 224 and 191 patients were included in the discovery and validation cohorts, respectively. In both cohorts, S100A4, Ca-125 and mesothelin expression were associated with shorter OS. In both cohorts, the number of these biomarkers expressed was significantly associated with OS (discovery cohort 36.8 vs. 26.4 vs 16.3 vs 12.8 months, P < 0.001; validation cohort 25.2 vs 18.3 vs 13.6 vs 11.9 months, P = 0.008 for expression of zero, one, two and three biomarkers, respectively). On multivariable analysis, expression of at least one of three biomarkers was independently associated with shorter OS. CONCLUSION: Combinations of S100A4, Ca-125 and mesothelin expression stratify survival after resection of localized PDAC. Co-expression of all three biomarkers is associated with the poorest prognostic outcome.

Biodistribution and Clearance of Stable Superparamagnetic Maghemite Iron Oxide Nanoparticles in Mice Following Intraperitoneal Administration.

Nanomedicine is an emerging field with great potential in disease theranostics. We generated sterically stabilized superparamagnetic iron oxide nanoparticles (s-SPIONs) with average core diameters of 10 and 25 nm and determined the in vivo biodistribution and clearance profiles. Healthy nude mice underwent an intraperitoneal injection of these s-SPIONs at a dose of 90 mg Fe/kg body weight. Tissue iron biodistribution was monitored by atomic absorption spectroscopy and Prussian blue staining. Histopathological examination was performed to assess tissue toxicity. The 10 nm s-SPIONs resulted in higher tissue-iron levels, whereas the 25 nm s-SPIONs peaked earlier and cleared faster. Increased iron levels were detected in all organs and body fluids tested except for the brain, with notable increases in the liver, spleen, and the omentum. The tissue-iron returned to control or near control levels within 7 days post-injection, except in the omentum, which had the largest and most variable accumulation of s-SPIONs. No obvious tissue changes were noted although an influx of macrophages was observed in several tissues suggesting their involvement in s-SPION sequestration and clearance. These results demonstrate that the s-SPIONs do not degrade or aggregate in vivo and intraperitoneal administration is well tolerated, with a broad and transient biodistribution. In an ovarian tumor model, s-SPIONs were shown to accumulate in the tumors, highlighting their potential use as a chemotherapy delivery agent.

Establishing a panel of chemo-resistant mesothelioma models for investigating chemo-resistance and identifying new treatments for mesothelioma.

Mesothelioma is inherently chemo-resistant with only 50% of patients responding to the standard of care treatments, and consequently it has a very grim prognosis. The aim of this study was to establish a panel of chemo-resistant mesothelioma models with clinically relevant levels of resistance as tools for investigating chemo-resistance and identifying new treatments for mesothelioma. Chemo-resistant cell lines were established in vitro and characterized in vivo using syngeneic Fischer rats. Tumors derived from all chemo-resistant cell lines were immunohistochemically classified as mesothelioma. Homozygous deletion of p16(INK4A)/p14(ARF) and increased expression of several ATP-binding cassette transporters were demonstrated, consistent with findings in human mesothelioma. Further, the acquisition of chemo-resistance in vitro resulted in changes to tumor morphology and overall survival. In conclusion, these models display many features corresponding with the human disease, and provide the first series of matched parental and chemo-resistant models for in vitro and in vivo mesothelioma studies.

Streptavidin: a novel immunostimulant for the selection and delivery of autologous and syngeneic tumor vaccines.

Induction of antitumor immunity using autologous tumor proteins is an attractive approach to cancer therapy. However, better methods and stimulants to present these autologous proteins back to the immune system are needed. Here, we identify streptavidin as a novel carrier protein and stimulant, and test the efficacy of both syngeneic (rat) and autologous vaccines (dogs) using streptavidin in combination with reduced soluble tumor proteins. Initial syngeneic vaccine studies in the 9L rat glioma model were used to optimize vaccine dose and selectivity. Cytokine and blood analysis was used to monitor the response. Rats receiving two vaccinations of syngeneic tumor vaccine demonstrated a statistically significant (P < 0.05) survival advantage compared with controls (adjuvant only). Notably, vaccination also led to remission rates of between 30% and 60% in the aggressive 9L glioma model. Antibodies to streptavidin were detected in the serum of vaccinated rats; however, antibody levels did not correlate with the response. The cytokine TNF-α was upregulated in vaccine-treated rats, whereas ICAM1 was downregulated. After engraftment, vaccinated rats maintained CD4(+), CD8(+) T cells, and total lymphocyte levels closer to normal baseline than those in the controls. Twenty-five dogs treated with autologous vaccine preparations using streptavidin as a stimulant showed no adverse reactions, irrespective of additional chemotherapy and other medications. In this study, we developed a novel method for producing syngeneic and autologous vaccines using streptavidin selectivity and immunogenicity. These vaccines show efficacy in the 9L glioma rat model. Safety was also demonstrated in canine patients presenting with cancer treated with autologous vaccine.

Maps of cardiovascular and respiratory regions of rat ventral medulla: focus on the caudal medulla.

The ventral medulla oblongata is critical for cardiorespiratory regulation. Here we review previous literature relating to sites within the ventral medulla that have been identified as having a 'cardiovascular' or 'respiratory' function. Together with the maps generated here, of sites from which cardiovascular and respiratory responses were evoked by glutamate microinjection, specific 'cardiovascular' regions have been defined and delineated. Commonly investigated regions, including the vasopressor rostral ventrolateral medulla (RVLM) and vasodepressor caudal ventrolateral medulla (CVLM), or areas only described by others, such as the medullary cerebral vasodilator area, are included for completeness. Emphasis is given to the caudal medulla, where three pressor regions, the caudal pressor area (CPA), the intermediate pressor area (IPA) and the medullo-cervical pressor area (MCPA), caudal to the vasodepressor CVLM were defined in the original data provided. The IPA is most responsive under pentobarbitone rather than urethane anaesthesia clearly delineating it from both the rostrally located CPA and the caudally located MCPA. The description of these multiple pressor areas appears to clarify the confusion that surrounds the identification of the 'CPA'. Also noted is a vasopressor region adjacent to the vasodepressor CVLM. Apart from the well described ventral respiratory column, a region medial to the pre-Bötzinger is described, from which increases in both phrenic nerve frequency and amplitude were evoked. Limitations associated with the technique of glutamate microinjection to define functionally specific regions are discussed. Particular effort has been made to define and delineate the regions with respect to ventrally located anatomical landmarks rather than the commonly used ventral surface or dorsal landmarks such as the obex or calamus scriptorius that may vary with the brain orientation or histological processing. This should ensure that a region can easily be defined by all investigators. Study of defined regions will help expedite the identification of the role of the multiple cell groups with diverse neurotransmitter complements that exist even within each of the regions described, in coordinating the delivery of oxygenated blood to the tissues.

Lateralisation of projections from the rostral ventrolateral medulla to sympathetic preganglionic neurons in the rat.

Spinally projecting sympathoexcitatory neurons in the rostral ventrolateral medulla (RVLM), synapse with sympathetic preganglionic neurons (SPN) and regulate the activity of sympathetic nerves that control the heart, blood pressure and the adrenal medulla (AM). However, the degree of lateralization of the bulbospinal projections to SPN innervating specific targets is poorly understood. Three approaches were used in this study. Anterograde tracer was iontophoresed into a pressor site in the RVLM (left or right) and retrograde tracer injected into the superior cervical ganglion (SCG, right) and the AM (left). Close appositions between anterogradely labelled axons and retrogradely labelled SCG- or AM-SPN were counted. Projections to the SCG were bilateral. Projections to the AM were markedly ipsilateral. In the second part, retrograde tracers were injected unilaterally into the region of the intermediolateral cell column at spinal segment T2 or T8 on one side and the number of labelled neurons in the RVLM counted. The results from each level of injection were similar showing that approximately 63-64% of the neurons were ipsilateral. Responses to glutamate microinjection into the RVLM on adrenal nerve (left) and superior cervical nerve (left) activity were measured. The ratio of the nerve responses was the same even when different sides of the RVLM were injected. The anterograde data strongly suggest that the RVLM projections to AM-SPN are predominantly ipsilateral. Although other experimental approaches also attempted to investigate lateralization, the retrograde data target different and functionally heterogeneous pools of SPN that may mask the ipsilateral projection to the AM. Similarly, chemical stimulation of the RVLM will excite not only monosynaptic projections but also polysynaptic projections that may also mask the predominant ipsilateral monosynaptic projection to AM.