Jerry T. Thornthwaite

Correlation of cell cycle analysis with Duke's staging in colon cancer patients

The biologic potential of a tumor for growth and metastasis is relatively inaccurately mirrored by the present staging system in colon cancers [29]. Except for Stage IV disease, traditional morphologic criteria approaches only 50-75% accuracy for predicting recurrent disease. Reliable staging, however, has become more critical in the light of the current thrust in adjuvant therapy research. First, the patients receive toxic and expensive therapy. Second, and of even greater importance, a central tenet of adjuvant therapy research is that treatment and control groups under study are comparable. Inadequate staging not only undermines analysis, especially when small improvements of lo-20% may be expected, but also demands very large study groups to offset this defect. A significant amount of research has attempted to quantitate the metastatic potential of a tumor. In the animal model some factors such as cell adhesiveness, tumor angiogenesis, detachability, thromboplasticity, and shedding of membrane antigen have been associated with an increased propensity of a tumor to spread [23]. In the clinical forum, analysis of factors such as tumor grade [4, 71, vascular invasion [7], host immune status [ 1, 8, 91 have not been established as having a clear-cut prognostic value. Hopefully prospective studies, objective criteria, and more sophisticated statistical analysis [6] may yield less disappointing results in the future. In this respect, accumulating evidence indicates that the analysis of tumor kinetics will be a major determinant of the metastatic potential of a tumor. It is well established that kinetic data of tumor doubling time and labeling index (LI) is associated with length of survival [ 11, 18, 21, 271, but the effect of tumor doubling time on ultimate survival was reported by Joseph et al. [12, 161. In his report, patients with resection of solitary pulmonary metastasis with doubling times less than 40 days subsequently died of metastasis whereas 60% of the patients with tumor doubling time over 40 days were cured. Other measures of tumor proliferative activity, i.e, mitotic index in sarcomas 1281 and labeling index in breast cancer patients 1151 have recently been implicated as important prognostic determinants, and in the former instance has been incorporated into the A.J.C. stating system [20] as a factor influencing tumor grading. Finally, the development of flow cytometry [24] has now made the rapid and objective analysis of cell cycle kinetics an exciting clinical reality. In this study, the predictive potential of cell cycle analysis by flow cytometry was examined by comparing the proliferative activity of colorectal adenocarcinoma with Duke’s staging and modified Broder’s grading.

A Review of DNA Flow Cytometric Preparatory and Analytical Methods

The detection of cell surface antigens is the most familiar application of flow cytometry in clinical immunology. Rapid advances in immunocytochemical applications of monoclonal antibodies, which may now be regarded as “true” reagents in the clinical laboratory, have made possible the immunological characterization of various tumors (1–3) and the classification of cells of the immune system (4–6). This extensive immunological characterization was virtually unattainable a few years earlier.

Characteristics of Monoclonal Antibody Measurements in Human Peripheral Blood

Three areas of monoclonal antibody measurements using flow cytometry have been presented. These include a description of a dual immunofluorescent method for measuring two antibodies simultaneously, the effects of blood storage on enumeration of helper (H) and suppressor (S) cells, and the relationship between absolute lymphocyte count and H/S ratio in both control and AIDS patients. These studies reveal that a dual immunofluorescent labeling method is useful for enumerating lymphocytes from peripheral blood which bear the helper, suppressor and/or thymus-derived (T) cell receptors. Fluorescein (FL)-conjugated Leu-3a + 3b antibodies were used to enumerate helper T-lymphocytes, while the B-phycoerythrin (B-PE)-conjugated Leu-2a antibodies were utilized for enumerating suppressor T-lymphocytes. Dual immunofluorescently stained lymphocytes, prepared from whole blood, were analyzed by flow cytometry. Two light scatter parameters, (forward and 90 degree scatter) were used to define the lysed erythrocyte, lymphocyte, monocyte, and granulocyte populations. Only the lymphocytes were analyzed for dual immunofluorescence activity. The helper and suppressor distributions from 167 control patients were as follows: The average percentage +/- SD of the helper and suppressor cells were 42.8 +/- 7.5 and 21.6 +/- 6.4, respectively. The H/S ratio was 2.17 +/- .75. These studies show that the H/S ratio can be determined in a single preparative sample and analyzed by dual immunofluorescence in a single flow cytometric analysis even though the H/S ratio may vary from normal during a disease condition. The dual immunofluorescent assay enables one to correlate the activities of two antibodies against cell surface receptors and allows the measurement of a large number of samples in a minimal time. This study also compared the effects of anticoagulant, storage time, and temperature on the phenotypic determination of the percentages of helper and suppressor T-lymphocytes in human peripheral blood. Blood was drawn in ACD, heparin, and EDTA and stored for up to 4 days at room temperature or 4 degrees C. Phenotypic determination of helper/suppressor lymphocytes was most stable for ACD or heparinized blood at room temperature. Marked changes were observed in the percentages of helper cells at 4 degrees C, whereas the percentages of suppressor cells did not change appreciably regardless of the anticoagulant storage time or temperature. Finally, the relationship between ALC and the H/S ratio in control and AIDS patients was determined. The ALC varied considerably in both control and patient populations as a function of time. Conversely, the H/S ratio remained constant.(ABSTRACT TRUNCATED AT 400 WORDS)

An examination of the macrophage response after challenge with murine, syngeneic sarcoma cells

Abstract The role that macrophages play in the reticuloendothelial organs may be an important determinant in tumor progression. Enzymatic staining for lipase activity was used in this study to quantitate macrophage infiltrates of primary tumors, metastases, spleens, and thymuses during progressive tumor growth and metastasis utilizing an MCA-induced, syngeneic, antigenic, spontaneously metastasizing murine sarcoma. After a single im injection of sarcoma tumor cells, the red pulp, but not the white pulp, of the spleens showed a progressive increase of macrophages as the tumors grew. Correspondingly, colonies of macrophages appeared in the thymuses of mice with tumor growth. Prior to the appearance of macrophage colonies in the thymus, a significant depression in the number of spleen and thymus cells undergoing DNA synthesis was seen. Infiltration of macrophages was scarcely seen in either the original MCA-induced or transplanted sarcomas regardless of tumor size. The development of lung metastases was dependent on the primary tumor size before amputation of the tumor-bearing legs. The pulmonary metastases that developed from mice that had their primary tumors amputated at a metastasizing size were void of macrophages regardless of metastatic size (1–10 mm diameter). If the primary tumor-bearing legs were amputated at a premetastasizing size, the macrophage activity in the spleen and thymus decreased to control levels within 2 weeks. However, if the tumors were allowed to grow to metastasizing sizes, the macrophage activity in the spleens and thymuses did not decrease after amputation.

Abstract 3209: The anticancer effects of chemically derived natural products

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CAnnDespite the advances in the early detection of tumors and in the use of surgery, radiation and chemical therapies for disease management, cancer deaths are projected to more than double worldwide over the next two decades. New approaches to prevention and therapy for cancer include enhancement of Natural Killer Cells, antiangiogenic processes, antioxidant defense systems and direct cytotoxicity against cancer cells without harming normal cells are urgently needed. An assay system is described that addresses the ability of chemically defined natural products, such as Curcumin (derived from Curry), Genistein (derived from Soy), Resveratrol (derived from grape skins) and Artemisinin (derived from the Sweet Wormwood plant) to actually destroy cancer cells in vitro. These compounds are used as food supplements in many parts of the world and are important in the prevention of cancer, heart disease and immunological diseases such as arthritis. The purpose of this research is to develop an accurate, fast assay method to determine cell concentrations of viable cells in culture that are exposed to various compounds developed from natural sources. K562 Erythroleukemic cells were added to 48 well plates in 500ul portions [10(5) cells/ml], cultured 48 hr. and exposed to the various compounds for up to 72 hr. All compounds were dissolved in DMSO and diluted sufficiently to prevent solvent effects. A unique cell viability stain, which allowed the rapid staining of dead cells by membrane penetration using propidium iodide, was used to measure the cell viability of the surviving cells by gating on forward light scatter (size) and fluorescence flow cytometry. The control cultures were greater than 95% viable during and at the end of the experiments and were used as a baseline to measure the degree of killing. All samples were run as 3-6 replicates and SD determined. Both the Phytosome Curcumin derivative and pure Curcumin showed complete killing of as many as 10(6) cells/ml (zero detected cells) at a concentration of 25 µM as early as 20 hr. of culture. Genistein showed 94% killing at 65 hrs. but only 16% killing at 20 hrs. at a concentration of 100 µM. Artemisinin revealed 71% killing at 65 hrs. but only 2% killing at 20 hrs. for 100 µM. The delayed killing may reflect apoptotic events. Transferrin was used up to 800µg/ml without affecting K562 cell viability to potentially load the cells with iron. No enhancement in killing at 72 hr. was seen with Artemisinin, which requires iron for its cytotoxic peroxide activity, suggesting saturation with iron in the K562 cells had already occurred. Resveratrol at 50 µM completely destroyed the cancer cells at 67 hrs. N-acetylcysteine (NAC) showed minimal cytotoxicity of 13% for 200 µM at 65 hrs. This assay system will allow for the study of synergism of the compounds in a rapid, precise method to assess cancer cytotoxicity.nnCitation Format: Jerry T. Thornthwaite, Brandon England, Spencer England, Michelle Clarke, Lee Roland, Hare Shah. The anticancer effects of chemically derived natural products. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3209. doi:10.1158/1538-7445.AM2014-3209

Abstract 4797: The antioxidation microsphere flow cytometer assay.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnIncreased production of reactive oxygen species is a feature of most, if not all, human disease, including cardiovascular disease and cancer. Dietary antioxidants may be especially important in protecting against human diseases associated with free radical damage to cellular DNA, lipids, and proteins. Yet, present data are not sufficient to quantify micronutrient requirements needed to protect against oxidative damage. The antioxidant roles of many food constituents, including herbs and spices, have not been clarified. The AMFC assay measures the oxidation of R-Phycoerythrin attached to microspheres via a protein coupling linker. A 25mM stock solution of ABTS.+ [2,2’-Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) free radical], prepared during a 12 hour, 4 °C incubation with potassium persulfate to cause removal of one electron generating a metastable ABTS. This stock solution was diluted to 100μM in 150μl, which resulted in 80% quenching of 15μl of 104 PE microspheres. The antioxidant effects were measured by first incubating 50μl the 80 % quenching ABTS.+ with 100μl portions of serial diluted antioxidants for 30 min. at 22 °C to determine the antioxidants neutralize the ABTS.+. Subsequently, 15μl of PE-tagged microspheres were added to each sample and incubated one hour at 22 °C. The peak fluorescent channel number was determined for 5,000 microspheres. The typical percentage protection by the antioxidant standard at 500 ng/ml for Trolox (water soluble Vitamin E derivative) was 8, while 500 ng/ml of Vitamin C restored 78% The range of 500-5,000 ng/ml showed the protective effects of Trolox and Vitamin C remained constant. On the average, equivalent percentage protection for both the hydrophilic and lipid soluble fractions of 14 spices (courtesy of McCormick Science Institute) was not achieved until the 20-50 μg/ml concentration of the spices with Turmeric, Cinnamon, Oregano and Cloves, showing the highest antioxidant protection. Interestingly, Rosemary, Sage, Thyme and Turmeric revealed a significant reduction in antioxidant activity at 200 μg/ml. These are still significant antioxidant levels that show a secondary reason besides flavoring to use spices for achieving clinically important antioxidant activity. The AMFC assay is very sensitive to the effects of oxidants, functions under physiological conditions, requires little sample and allows application to the study of serum samples. Thus, one may monitor antioxidant activity in healthy volunteers taking compounds to boost the antioxidant activity to offset the long-term effects of oxidative radiation and chemotherapy.nnCitation Format: Jerry T. Thornthwaite, Hare R. Shah. The antioxidation microsphere flow cytometer assay. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4797. doi:10.1158/1538-7445.AM2013-4797nnNote: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Abstract 621: The importance of antioxidant, antiangiogenesis and immune stimulation in cancer

In our quest over the last nine years to find comprehensive over-the- counter supplements, designed to facilitate current classical treatments in fighting diseases, include antiangiogenic, immune and antioxidant defense systems. These supplements include, in part, Acetyl L-Carnitine (ALC), Alpha Lipoic Acid (ALA), Coenzyme Q10 (CoQ10), Curcumin with Piperine, Genistein, Lentinan, N-AcetylCysteine (NAC), Resveratrol, selenium, Vitamin B Complex, Vitamin C, Vitamin E and zinc. These ingredients have been demonstrated, either individually or collectively, to have antioxidant, anti-angiogenesis, and immune stimulation and antioxidant properties. Furthermore, the scientific literature supports direct cancer cell cytotoxicity for Curcumin, Geinstein and NAC. The dietary supplement activities are supported by over 50,000 references in the scientific literature (PubMed.com) and over 150 clinical trials (clinicaltrials.gov), not including the human-cancer publications (23,000) and clinical trials (2,100) with zinc and the vitamins. From the literature there is evidence that some of the antiangiogenesis components that affect the majority if not all pathways of angiogenesis, such as Curcumin, Genistein and NAC, actually stimulate the in vivo production of natural antiangiogenic compounds that include Angiostatin, Endostatin and Thrombospotin 1. All of the components play a role in serving as either water or lipid soluble (able to cross the blood-brain barrier) antioxidants. Curcumin, Genistein, Resveratrol, Lentinen, NAC, zinc, selenium and the B and C vitamins all stimulate the immune system. Except for ALC and CoQ10, the other components show anti-inflammatory activity. ALC, Resveratrol along with the B and C vitamins are helpful in treating fatigue. The components that help protect the brain and promote nerve regeneration include ALA, CoQ10, Resveratrol, NAC, selenium, zinc and the B/C vitamins. In conclusion, effective prevention and treatment from and for diseases such as cancer, heart disease and immune deficiency will require multiple compounds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 621. doi:1538-7445.AM2012-621