Transcending the biomarker mindset: deciphering disease mechanisms at the single cell level.

The application of proteomics to disease research promises to enhance the understanding and treatment of many human maladies through the identification of molecular profiles associated with each disease. However, although much is made of the utility of molecular signatures as markers of disease state, insufficient emphasis is often placed on the simultaneous need for biological mechanism inquiry. Focused and detailed analyses of disease-associated signaling networks have the potential to be more mechanistically informative than large-scale proteomic profiling approaches, providing insight into the cellular processes involved in pathogenesis, disease progression and therapeutic resistance; while still providing diagnostic or clinical management direction. Phospho-specific flow cytometry provides a method for the analysis of pathological signaling networks, enabling the investigation of disease mechanisms at the single-cell level.

Surgical removal of primary tumor reverses tumor-induced immunosuppression despite the presence of metastatic disease.

Immunotherapy is a promising approach for the management of malignancies. It may be particularly useful for tumors that do not respond to conventional therapies, such as many metastatic cancers. The efficacy of immunotherapy will depend on many factors, one of which is the immunocompetence of the host. Patients with large primary tumors frequently are immunosuppressed, making them poor candidates for immunotherapy. Although a few studies have reported that surgical removal of primary tumor reverses immunosuppression, it is not known whether metastatic disease in postsurgery patients inhibits this recovery. To determine the role of metastatic disease, we examined tumor-free mice versus mice with primary tumor and metastatic disease versus mice whose primary tumors were removed surgically but who had metastatic disease. We have used the mouse 4T1 mammary carcinoma, a BALB/c-derived transplantable tumor that shares many characteristics with human breast cancer and is an established model for spontaneous, metastatic cancer. Cell-mediated and humoral adaptive immunity, as measured by rejection of allogeneic tumor, antigen-specific T-cell proliferation, and antigen-specific antibody responses, was suppressed in 4T1-bearing nonsurgery mice relative to tumor-free mice. Surgical removal of primary tumor resulted in rebounding of antibody and cell-mediated responses, even in mice with metastatic disease. Macrophage activity, as measured by lipopolysaccharide responsiveness, and dendritic cell function, as measured by nominal and alloantigen presentation, were not suppressed in tumor-bearing mice. Therefore, the presence of primary tumor suppresses T-cell and antibody responses; however, surgical removal of primary tumor restores immunocompetence even when disseminated metastatic disease is present.

Antagonists of tumor-specific immunity: tumor-induced immune suppression and host genes that co-opt the anti-tumor immune response.

Metastatic disease is the principle cause of death for most patients with breast cancer. Conventional therapies including radiation therapy and chemotherapy are largely uneffective against metastatic disease. It is now generally appreciated that the immune system can destroy tumor cells, and numerous novel immunotherapies are currently under development. Many of these immunotherapies are dependent on activation of the host's immune system so the success of a cancer vaccine will depend on the immune status of the patient. Tolerance to tumor antigens, tumor-induced immune suppression, and the presence of immunomodulatory genes that block the development of tumor-specific immunity can potentially interfere with the therapeutic efficacy of immune-based therapies. Studies from the authors' laboratory demonstrate that although mice with bulky primary mammary tumors are immunosuppressed for T cell and antibody-mediated immunity, surgical removal of the primary tumor reverses the suppression, even when disseminated metastatic disease is present. The post-surgical reversal is associated with a large decrease in myeloid suppressor cells. In addition to tumor-induced suppression, two genes, the Stat6 and CD1 genes, are also associated with inhibiting tumor-specific immunity, since mice deficient for these genes have dramatically enhanced resistance to metastatic mammary carcinoma. Therefore, optimal delivery of immunotherapy should be coordinated with methodology that decreases immune suppression and eliminates or blocks inhibitory factors.

Single-cell phospho-protein analysis by flow cytometry.

This protocol describes methods for monitoring intracellular phosphorylation-dependent signaling events on a single-cell basis. This approach measures cell signaling by treating cells with exogenous stimuli, fixing cells with formaldehyde, permeabilizing with methanol, and then staining with phospho-specific antibodies. Thus, cell signaling states can be determined as a measure of how cells interact with their environment. This method has applications in clinical research as well as mechanistic studies of basic biology. In clinical research, diagnostic or drug efficacy information can be retrieved by discovering how a disease affects the ability of cells to respond to growth factors. Basic scientists can use this technique to analyze signaling events in cell lines and human or murine primary cells, including rare populations, like B1 cells or stem cells. This technique has broad applications bringing standard biochemical analysis into primary cells in order to garner valuable information about signaling events in physiologic settings.

Single-cell phospho-protein analysis by flow cytometry.

This protocol describes methods for monitoring intracellular phosphorylation-dependent signaling events on a single-cell basis. This approach measures cell signaling by treating cells with exogenous stimuli, fixing cells with formaldehyde, permeabilizing with methanol, and then staining with phospho-specific antibodies. Thus, cell signaling states can be determined as a measure of how cells interact with their environment. This method has applications in clinical research as well as mechanistic studies of basic biology. In clinical research, diagnostic or drug efficacy information can be retrieved by discovering how a disease affects the ability of cells to respond to growth factors. Basic scientists can use this technique to analyze signaling events in cell lines and human or murine primary cells, including rare populations, like B1 cells or stem cells. This technique has broad applications to take standard biochemical analysis into primary cells to garner valuable information about signaling events in physiologic settings.