Contemporaneous Perioperative Inflammatory and Angiogenic Cytokine Profiles of Surgical Breast, Colorectal, and Prostate Cancer Patients: Clinical Implications.

Surgery-induced tumor growth acceleration and synchronous metastatic growth promotion have been observed for decades. Surgery-induced wound healing, orchestrated through growth factors, chemokines, and cytokines, can negatively impact patients harboring residual or metastatic disease. We provide detailed clinical evidence of this process in surgical breast, prostate, and colorectal cancer patients. Plasma samples were analyzed from 68 cancer patients who had not received treatment before surgery or adjuvant therapy until at least four weeks post-surgery. The levels of plasma cytokines, chemokines, and growth factors were simultaneously quantified and profiled using multiplexed immunoassays for eight time points sampled per patient. The immunologic processes are induced immediately after surgery in patients, characterized by a drastic short-term shift in the expression levels of pro-inflammatory and angiogenic molecules and cytokines. A rapid and significant spike in circulating plasma levels of hepatocyte growth factor (HGF), interleukin-6 (IL-6), placental growth factor (PLGF), and matrix metalloproteinase-9 (MMP-9) after surgery was noted. The rise in these molecules was concomitant with a significant drop in transforming growth factor-ß1 (TGF-ß1), platelet-derived growth factor (PDGF-AB/BB), insulin-like growth factor-1 (IGF-1), and monocyte chemoattractant protein-2 (MCP-2). If not earlier, each plasma analyte was normalized to baseline levels within 1-2 weeks after surgery, suggesting that surgical intervention alone was responsible for these effects. The effects of surgical tumor removal on disrupting the pro-inflammatory and angiogenic plasma profiles of cancer patients provide evidence for potentiating malignant progression. Our findings indicate a narrow therapeutic window of opportunity after surgery to prevent disease recurrence.

Repositioning of Old Drugs for Novel Cancer Therapies: Continuous Therapeutic Perfusion of Aspirin and Oseltamivir Phosphate with Gemcitabine Treatment Disables Tumor Progression, Chemoresistance, and Metastases.

Metastatic pancreatic cancer has an invariably fatal outcome, with an estimated median progression-free survival of approximately six months employing our best combination chemotherapeutic regimens. Once drug resistance develops, manifested by increased primary tumor size and new and growing metastases, patients often die rapidly from their disease. Emerging evidence indicates that chemotherapy may contribute to the development of drug resistance through the upregulation of epithelial-mesenchymal transition (EMT) pathways and subsequent cancer stem cell (CSC) enrichment. Neuraminidase-1 (Neu-1) regulates the activation of several receptor tyrosine kinases implicated in EMT induction, angiogenesis, and cellular proliferation. Here, continuous therapeutic targeting of Neu-1 using parenteral perfusion of oseltamivir phosphate (OP) and aspirin (ASA) with gemcitabine (GEM) treatment significantly disrupts tumor progression, critical compensatory signaling mechanisms, EMT program, CSC, and metastases in a preclinical mouse model of human pancreatic cancer. ASA- and OP-treated xenotumors significantly inhibited the metastatic potential when transferred into animals.

Next Generation of Cancer Drug Repurposing: Therapeutic Combination of Aspirin and Oseltamivir Phosphate Potentiates Gemcitabine to Disable Key Survival Pathways Critical for Pancreatic Cancer Progression.

Resistance to chemotherapeutics and high metastatic rates contribute to the abysmal survival rate in patients with pancreatic cancer. An alternate approach for treating human pancreatic cancer involves repurposing the anti-inflammatory drug, aspirin (ASA), with oseltamivir phosphate (OP) in combination with the standard chemotherapeutic agent, gemcitabine (GEM). The question is whether treatment with ASA and OP can sensitize cancer cells to the cytotoxicity induced by GEM and limit the development of chemoresistance. To assess the key survival pathways critical for pancreatic cancer progression, we used the AlamarBlue cytotoxicity assay to determine the cell viability and combination index for the drug combinations, flow cytometric analysis of annexin V apoptosis assay to detect apoptotic and necrotic cells, fluorometric QCM™ chemotaxis migration assay to assess cellular migration, fluorometric extracellular matrix (ECM) cell adhesion array kit to assess the expression of the ECM proteins, scratch wound assay using the 96-well WoundMaker™, and the methylcellulose clonogenic assay to assess clonogenic potential. The combination of ASA and OP with GEM significantly upended MiaPaCa-2 and PANC-1 pancreatic cancer cell viability, clonogenic potential, expression of critical extracellular matrix proteins, migration, and promoted apoptosis. ASA in combination with OP significantly improves the effectiveness of GEM in the treatment of pancreatic cancer and disables key survival pathways critical to disease progression.

Novel Molecular Mechanism of Aspirin and Celecoxib Targeting Mammalian Neuraminidase-1 Impedes Epidermal Growth Factor Receptor Signaling Axis and Induces Apoptosis in Pancreatic Cancer Cells.

BACKGROUND: Aspirin (acetylsalicylic acid) and celecoxib have been used as potential anti-cancer therapies. Aspirin exerts its therapeutic effect in both cyclooxygenase (COX)-dependent and -independent pathways to reduce tumor growth and disable tumorigenesis. Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, reduces factors that cause inflammation and pain. The question is whether aspirin and celecoxib have other molecular targets of equal or more therapeutic efficacy with significant anti-cancer preventive benefits. AIM: Here, we propose that aspirin and celecoxib exert their anti-cancer effects by targeting and inhibiting mammalian neuraminidase-1 (Neu-1). Neu-1 has been reported to regulate the activation of several receptor tyrosine kinases (RTKs) and TOLL-like receptors and their downstream signaling pathways. Neu-1 in complex with matrix metalloproteinase-9 (MMP-9) and G protein-coupled receptors (GPCRs) has been reported to be tethered to RTKs at the ectodomain. MATERIALS AND METHODS: The WST-1 cell viability assay, Caspase 3/7 assay, and Annexin V assay were used to evaluate the cell viability and detect apoptotic and necrotic cells following treatment in MiaPaCa-2, PANC-1 and the gemcitabine-resistant PANC-1 variant (PANC-1 GemR) cells. Microscopic imaging, lectin cytochemistry, and flow cytometry were used to detect levels of α-2,3 sialic acid. Epidermal growth factor (EGF)-stimulated live cell sialidase assays and neuraminidase assays were used to detect Neu-1 activity. Immunocytochemistry was used to detect levels of EGFR and phosphorylated EGFR (pEGFR) following treatment. RESULTS: For the first time, aspirin and celecoxib were shown to significantly inhibit Neu-1 sialidase activity in a dose- and time-dependent manner following stimulation with EGF. Aspirin blocked Neu-1 desialylation of α-2,3-sialic acid expression following 30 min stimulation with EGF. Aspirin and celecoxib significantly and dose-dependently inhibited isolated neuraminidase (Clostridium perfringens) activity on fluorogenic substrate 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MUNANA). Aspirin inhibited phosphorylation of the EGFR in EGF-stimulated cells. Aspirin dose- and time-dependently induced CellEvent caspase-3/7+ cells as well as apoptosis and necrosis on PANC-1 cells. CONCLUSION: These findings signify a novel multimodality mechanism(s) of action for aspirin and celecoxib, specifically targeting and inhibiting Neu-1 activity, regulating EGF-induced growth receptor activation and inducing apoptosis and necrosis in a dose- and time-dependent manner. Repurposing aspirin and celecoxib as anti-cancer agents may also upend other critical targets involved in multistage tumorigenesis regulated by mammalian neuraminidase-1. SIGNIFICANCE: These findings may be the missing link connecting the anti-cancer efficacy of NSAIDs to the role of glycosylation in inflammation and tumorigenesis.

A Triple Combination of Metformin, Acetylsalicylic Acid, and Oseltamivir Phosphate Impacts Tumour Spheroid Viability and Upends Chemoresistance in Triple-Negative Breast Cancer.

INTRODUCTION: Targeted multimodal approaches need to be strategically developed to control tumour growth and prevent metastatic burden successfully. Breast cancer presents a unique clinical problem because of the variety of cellular subtypes that arise. The tumour stage and cellular subtypes often dictate the appropriate clinical treatment regimen. Also, the development of chemoresistance is a common clinical challenge with breast cancer. Higher doses and additional drug agents can produce additional adverse effects leading to a more aggressive malignancy. Acetylsalicylic acid (ASA), metformin (Met), and oseltamivir phosphate (OP) were investigated for their efficacy to sensitize MDA-MB-231 triple-negative breast cancer and its tamoxifen (Tmx) resistant variant (MDA-MB-231-TmxR) together in combination with Tmx treatment. METHODS: Microscopic imaging, the formation of 3D multicellular tumour spheroids, immunocytochemistry, flow cytometry, Annexin V Assay, Caspase 3/7 Apoptosis Assay, tube formation assay and analysis, and WST-1 cell viability assay evaluated the formation of MCTS, morphologic changes, cell viability, apoptosis activity and the expression levels of ALDH1A1, CD44 and CD24 on the cell surface, MDA-MB231 triple-negative breast cancer, tamoxifen (Tmx) resistant variant (MDA-MB-231-TmxR). RESULTS: The results using a triple combination of ASA, Met and OP on MDA-MB-231 and MDA-MB-231-TmxR cells and their matrix-free 3D multicellular tumour spheroids (MCTS) formed by using the cyclic Arg-Gly-Asp-D-Phe-Lys peptide modified with 4-carboxybutyl-triphenylphosphonium bromide (cyclo-RGDfK(TPP)) peptide method demonstrate a consistent and significant decrease in cell and tumour spheroid viability and volume with increased apoptotic activity, and increased sensitivity to Tmx therapy. Tmx treatment of MDA-MB-231 cells in combination with ASA, Met and OP markedly reduced the CD44/CD24 ratio by 6.5-fold compared to the untreated control group. Tmx treatment of MDA-MB-231-TmxR cells in combination with ASA, Met and OP markedly reduced the ALDH1A1 by 134-fold compared to the same treatment for the parental cell line. Also, the triple combination treatment of ASA, Met, and OP inhibited vasculogenic endothelial cell tube formation and induced endothelial cell apoptosis. CONCLUSION: For the first time, the findings demonstrate that repurposing ASA, Met, and OP provides a novel and promising targeted multimodal approach in the treatment of triple-negative breast cancer and its chemoresistant variant.

Therapeutic Options for Metastatic Breast Cancer.

Metastatic breast cancer is the most common cancer in women after skin cancer, with a 5-year survival rate of 26%. Due to its high prevalence, it is important to develop therapies that go beyond those that just provide palliation of symptoms. Currently, there are several types of therapies available to help treat breast cancer including: hormone therapy, immunotherapy, and chemotherapy, with each one depending on both the location of metastases and morphological characteristics. Although technological and scientific advancements continue to pave the way for improved therapies that adopt a targeted and personalized approach, the fact remains that the outcomes of current first-line therapies have not significantly improved over the last decade. In this chapter, we review the current understanding of the pathology of metastatic breast cancer before thoroughly discussing local and systemic therapies that are administered to patients diagnosed with metastatic breast cancer. In addition, our review will also elaborate on the genetic profile that is characteristic of breast cancer as well as the local tumor microenvironment that shapes and promotes tumor growth and cancer progression. Lastly, we will present promising novel therapies being developed for the treatment of this disease.

Alternative therapies for metastatic breast cancer: multimodal approach targeting tumor cell heterogeneity.

One of the primary challenges in developing effective therapies for malignant tumors is the specific targeting of a heterogeneous cancer cell population within the tumor. The cancerous tumor is made up of a variety of distinct cells with specialized receptors and proteins that could potentially be viable targets for drugs. In addition, the diverse signals from the local microenvironment may also contribute to the induction of tumor growth and metastasis. Collectively, these factors must be strategically studied and targeted in order to develop an effective treatment protocol. Targeted multimodal approaches need to be strategically studied in order to develop a treatment protocol that is successful in controlling tumor growth and preventing metastatic burden. Breast cancer, in particular, presents a unique problem because of the variety of subtypes of cancer that can arise and the multiple drug targets that could be exploited. For example, the tumor stage and subtypes often dictate the appropriate treatment regimen. Alternate multimodal therapies should consider the importance of time-dependent drug administration, as well as targeting the local and systemic tumor environment. Many reviews and papers have briefly touched on the clinical implications of this cellular heterogeneity; however, there has been very little discussion on the development of study models that reflect this diversity and on multimodal therapies that could target these subpopulations. Here, we summarize the current understanding of the origins of intratumoral heterogeneity in breast cancer subtypes, and its implications for tumor progression, metastatic potential, and treatment regimens. We also discuss the advantages and disadvantages of utilizing specific breast cancer models for research, including in vitro monolayer systems and three-dimensional mammospheres, as well as in vivo murine models that may have the capacity to encompass this heterogeneity. Lastly, we summarize some of the current advancements in the development of multitarget therapeutics that have shown promising results in clinical and preclinical studies when used alone or in combination with traditional regimens of surgery, chemotherapy, and/or radiation.

Cancer treatments transform residual cancer cell phenotype.

BACKGROUND: Physiologic wound repair and tissue regeneration are associated with distinct cellular behaviors triggered by tissue damage. Normally quiescent stem cells proliferate to regenerate damaged tissue, while relatively immobile epithelial cells can transform into a motile, tissue invasive phenotype through a partial epithelial-mesenchymal transition. These distinct cellular behaviors may have particular relevance to how cancer cells can be predicted to behave after treatments damaging a tumor. PRESENTATION OF THE HYPOTHESIS: Surgery, chemotherapy, and radiation therapy trigger highly conserved wound healing pathways that: (1) facilitate the phenotypic transformation of surviving cancer cells into a highly mobile, metastatic phenotype through an EMT or epithelial-mesenchymal transition and (2) induce residual cancer stem cell proliferation. TESTING THE HYPOTHESIS: Tissue damage caused by cancer treatments will trigger the release of distinct cytokines with established roles in physiologic wound healing, EMT induction, and stem cell activation. They will be released rapidly after treatment and detectable in the patient's blood. Careful histologic evaluation of cancerous tissue before and after treatment will reveal cellular changes suggestive of EMT induction (down regulation of cytokeratin expression) and cancer stem cell enrichment (stem cell markers upregulated). IMPLICATIONS OF THE HYPOTHESIS: Cancer cells surviving treatment will be more capable of metastasis and resistant to conventional therapies than the pre-treatment population of cancer cells. These changes will develop rapidly after treatment and, in distinct contrast to selection pressures fostering such changes, be triggered by highly conserved wound repair signals released after tissue damage. This pattern of tissue (tumor) repair may be amenable to treatment intervention at the time it is upregulated.

Defining the next generation journal: The NLM-Elsevier interactive publications experiment.

OBJECTIVE: A unique collaborative project to identify interactive enhancements to conventional-print journal articles, and to evaluate their contribution to readers' learning and satisfaction. HYPOTHESIS: It was hypothesized that (a) the enhanced article would yield more knowledge acquisition than the original article; (b) the interactivity aspects of the enhanced article would measurably contribute to the acquisition of knowledge; and (c) the enhancements to the original article would increase reader acceptance. METHODS: Fifteen SNMA medical students, assumed to have a greater generational familiarity and comfort level with interactive electronic media, reviewed 12 articles published in three Elsevier clinical and basic science journals. They used the Student National Medical Association's asynchronous online discussion forum over a four month period to suggest desired enhancements to improve learning. "Prognostic Factors in Stage T1 Bladder Cancer", published in the journal Urology was selected by the investigators as presenting the best opportunity to incorporate many of the students' suggested interactive and presentational enhancements in the limited timeframe available prior to the established test date. Educational, statistical, and medical consultants assisted in designing a test protocol in which 51 second to fourth year medical students were randomly assigned to experimental and control conditions, and were administered either the original or enhanced interactive version of the article on individual computer workstations. Test subjects consisted of 23 participants in the control group (8 males, 15 females) and 28 participants in the experimental group (9 males, 19 females). All subjects completed pre- and post-test instruments which measured their knowledge gain on 30 true-false and multiple-choice questions, along with 7 Likert-type questions measuring acceptance of the articles' format. Time to completion was recorded with the experimental group taking 22 min on average compared to 18 min for the controls; pre- and post-test times were 6 and 7 min, respectively. Statistical comparisons were based on change scores using either the Student t-test or the Two Way Analysis of Variance or Covariance. Significance was set at α = 0.05 or better. RESULTS: on the dependent measure of knowledge acquisition showed no difference overall on the 30 questions, but learning gain was statistically significant for the subset of 10 questions that measured gain on content that was accessible by the user-invoked interactive features of the enhanced article. Further analyses revealed significant interactions by student year and gender. Second year students (11 in the control group, 8 in the experimental group) were the best performers in terms of knowledge acquisition from both articles. The female medical students received a larger learning gain from journal enhancements and interactivity components than their male counterparts. Acceptance overall was greater for the experimental group who rated the experience more favorably than the controls. CONCLUSIONS: Failure to consider human factors such as gender and learning style may obscure underlying differences and their impact on the interactive aspects of scientific publications. Preliminary findings suggest the need for further study to include a heavier focus on interactivity apart from presentational enhancements; a more rigorous treatment of time as a specific variable; and an expanded experimental design that evaluates acquisition, understanding, integration and acceptance as dependent measures.

Revisiting perioperative chemotherapy: the critical importance of targeting residual cancer prior to wound healing.

BACKGROUND: Scientists and physicians have long noted similarities between the general behavior of a cancerous tumor and the physiological process of wound healing. But it may be during metastasis that the parallels between cancer and wound healing are most pronounced. And more particularly and for the reasons detailed in this paper, any cancer remaining after the removal of a solid tumor, whether found in micrometastatic deposits in the stroma or within the circulation, may be heavily dependent on wound healing pathways for its further survival and proliferation. DISCUSSION: If cancer cells can hijack the wound healing process to facilitate their metastatic spread and survival, then the period immediately after surgery may be a particularly vulnerable period of time for the host, as wound healing pathways are activated and amplified after the primary tumor is removed. Given that we often wait 30 days or more after surgical removal of the primary tumor before initiating adjuvant chemotherapy to allow time for the wound to heal, this paper challenges the wisdom of that clinical paradigm, providing a theoretical rationale for administering therapy during the perioperative period. SUMMARY: Waiting for wound healing to occur before initiating adjuvant therapies may be seriously compromising their effectiveness, and patients subsequently rendered incurable as a result of this wait. Clinical trials to establish the safety and effectiveness of administering adjuvant therapies perioperatively are needed. These therapies should target not only the residual cancer cells, but also the wound healing pathway utilized by these cells to proliferate and metastasize.

Evaluation of a virtual dialogue method for breast cancer patient education.

OBJECTIVE: Virtual dialogue--a voice-activated, interactive computer model that enables a user to have an individual virtual conversation with a real person--was evaluated as a method to educate women about breast cancer. Virtual dialogues with breast cancer experts were developed and used in a field study by women with and without the disease to assess the method's effectiveness. METHODS: In a clinical setting, subjects engaged in one-on-one virtual dialogues with an oncologist and a breast surgeon, and completed automated pre-post instruments developed to measure the feasibility of the method, and subjects' knowledge gain and acceptance of the method. RESULTS: Seventy women, 39 with breast cancer and 31 without the disease, participated. The data demonstrated the feasibility and acceptance of the method, and significant learning gain across all subjects. CONCLUSION: Virtual dialogues with knowledgeable health professionals can be a new and effective method for educating women about breast cancer. PRACTICE IMPLICATIONS: Virtual dialogues may systematically comfort patients while providing them with comprehensive and reliable basic information about breast cancer. The method may also give health care providers more time to address their patients' specific concerns.

A fatal outcome in a patient with glioblastoma multiforme after receiving high-dose methotrexate.

The most common adult primary brain tumor is glioblastoma multiforme (GBM). Current treatment is surgical resection, adjuvant radiation and chemotherapy, which can extend the median survival 20-36 weeks (Mansky et al. Central nervous system tumors. In Abraham J, Allegra CJ, Gulley J, eds. Bethesda Handbook of clinical oncology, 2nd edn. Philadelphia, Pennsylvania: Lippincott Williams and Wilkins, 2000: 440-2; Knox S. Intracranial tumors. In Pillot G, Chantler M, Magiera H, Peles S, et al., eds. The Washington Manual Hematology and Oncology Subspecialty Consult. Philadelphia, Pennsylvania: Lippincott Williams and Wilkins, 2004: 204-6.). But treatment efficacy is limited, mandating the exploration of more effective treatments. We report on a patient with GBM treated as per a clinical protocol with high-dose methotrexate (12 g/m(2)), who expired within hours after the initiation of treatment secondary to transtentorial herniation. Although it is not completely clear what caused the patient's herniation, we think that high-dose methotrexate therapy may have played a crucial role. We suggest that high-dose methotrexate should be used cautiously in patients with GBM.

An assessment of the virtual conversations method for prostate cancer patient education.

INTRODUCTION: Virtual dialogue is a specific educational methodology that employs speech recognition, digital video, and computer technologies in a symbiotic relationship to allow users to have the illusion of a face-to-face conversation with a person in cyberspace. A voice-activated, interactive, virtual dialogue with a prostate cancer specialist was developed and tested in a clinical setting at the Walter Reed Army Medical Center (WRAMC). OBJECTIVES: The purpose of this study was two-fold: (a) to create a prototype virtual dialogue program on the subject of prostate cancer and (b) to evaluate the effectiveness of this method to educate men about their disease and treatment. METHOD: Participants were recruited using a convenience sample of patients attending the Center for Prostate Disease Research multidisciplinary clinic at WRAMC. An automated pretest and post-test instrument was developed by the investigators to assess patients' knowledge before and after the virtual dialogue session. RESULTS: A total of 33 patients volunteered for the study. Results from this convenience sample showed an increase in patients' knowledge and positive acceptance of this innovative method of patient education. CONCLUSIONS: The data resulting from this study provide persuasive evidence that a virtual dialogue with a knowledgeable health professional can be a useful and highly effective method for educating men about prostate cancer. This method may also offer an effective way for health professionals to systematically provide their patients with comprehensive and reliable information.

Cancer: A medical emergency.

Over the last decade clinical trials have established the effectiveness of adjuvant chemotherapy in eradicating micrometastases in many different cancers, including breast, colon, and lung. This success stands in sharp contrast to our failure to cure clinically evident metastatic cancer. These dramatic polarities illustrate the critical importance of treatment timing if residual cancer is to be eradicated. Adjuvant chemotherapy is started only after recovery from surgery, a period of time that can exceed 30 days. During this time any cancer that remains after surgery will continue to divide. Although adjuvant chemotherapy has proven effective despite this time delay, there are reasons, both conceptual and quantitative, to think that its effectiveness could be magnified by a more prompt administration. The extent of this magnification is mathematically modeled in this paper. Surgery and the process of wound healing after surgery create a very favorable environment for the growth of the metastatic clone. Surgery can increase the number of circulating tumor cells and induce an immunosuppressive effect that might facilitate metastatic spread. And the process of wound healing can stimulate growth factors that have been shown to accelerate tumor cell growth. This situation is a double-edged sword. Although the metastatic clone should proliferate rapidly during this time, it should also, at least theoretically, be more sensitive to the effects of chemotherapy as more cells are pushed into a cycling phase. We derive a mathematical model based upon empirical data predicting that the effectiveness of a given chemotherapeutic regimen is inversely proportional to the tumor burden that has to be eradicated, which, in turn, is a function of when chemotherapy is started after surgery. Although the critical importance of timing in the treatment of cancer is intuitive, this knowledge has not yet been fully translated into the clinical practice of medical oncology. If the model presented is accurate, many people are dying unnecessarily of their cancer today because we are waiting too long after surgery to use highly effective chemotherapies, following well-trod clinical paths and established paradigms for how cancer should be treated.