J.L. Jordan

Core needle biopsy of breast cancer tumors increases distant metastases in a mouse model.

INTRODUCTION: Incisional biopsies, including the diagnostic core needle biopsy (CNB), routinely performed before surgical excision of breast cancer tumors are hypothesized to increase the risk of metastatic disease. In this study, we experimentally determined whether CNB of breast cancer tumors results in increased distant metastases and examine important resultant changes in the primary tumor and tumor microenvironment associated with this outcome. METHOD: To evaluate the effect of CNB on metastasis development, we implanted murine mammary 4T1 tumor cells in BALB/c mice and performed CNB on palpable tumors in half the mice. Subsequently, emulating the human scenario, all mice underwent complete tumor excision and were allowed to recover, with attendant metastasis development. Tumor growth, lung metastasis, circulating tumor cell (CTC) levels, variation in gene expression, composition of the tumor microenvironment, and changes in immunologic markers were compared in biopsied and non-biopsied mice. RESULTS: Mice with biopsied tumors developed significantly more lung metastases compared to non-biopsied mice. Tumors from biopsied mice contained a higher frequency of myeloid-derived suppressor cells (MDSCs) accompanied by reduced CD4 + T cells, CD8 + T cells, and macrophages, suggesting biopsy-mediated development of an increasingly immunosuppressive tumor microenvironment. We also observed a CNB-dependent up-regulation in the expression of SOX4, Ezh2, and other key epithelial-mesenchymal transition (EMT) genes, as well as increased CTC levels among the biopsy group. CONCLUSION: CNB creates an immunosuppressive tumor microenvironment, increases EMT, and facilitates release of CTCs, all of which likely contribute to the observed increase in development of distant metastases.

IFN-γ and Fas/FasL pathways cooperate to induce medial cell loss and neointimal lesion formation in allograft vasculopathy

Using a clinically relevant, fully disparate, allogeneic aortic transplant mouse model of allograft vasculopathy, we have demonstrated that neointimal proliferation is dependent on CD8(+) T cell effector pathways in the presence of therapeutic doses of calcineurin inhibitor (CNI) immunosuppression. CD4(+) T cell pathways are ablated by CNI immunosuppression. In the current study, we examined the relationship between CD8(+) T cell activities, medial SMC loss and neointimal hyperplasia. We demonstrate that at 5-6wk post transplantation in a wild type/wild type transplant CD8(+) T cell infiltration, CD8(+) CTL effector cell mediator expression and medial SMC loss all occur within aortic interposition grafts in the face of CNI immunosuppression. Both IFN-gamma and CTL mediated effector function is required for SMC loss and lesion formation under these conditions. Using strain combinations and reconstitution models, we provide data that blockade of the perforin/granzyme pathway does not prevent lesion formation but that blockade of the Fas/FasL pathway of cytotoxicity dramatically reduces SMC loss and prevents neointimal lesion formation. Both of these blockade strategies are in the face of an active IFN-gamma pathway. These data suggest a cooperative role between Fas/FasL and IFN-gamma mediated effector functions in medial SMC loss and neointimal lesion formation.

C. sinensis ablates allograft vasculopathy when used as an adjuvant therapy with cyclosporin A

Immunosuppressive treatments are available to suppress acute cardiac rejection; however, no viable treatment exists for long-term cardiac graft failure. Moreover, extended use of calcineurin inhibitor immunosuppressants, the mainstay of the current therapeutic for cardiac transplantation, leads to significant associated pathologies such as nephrotoxicity and increased risk of cardiac disease. For the last ten years alternatives to calcineurin inhibitors, or adjuvant therapies designed to complement their activities, have been explored. In tandem with this development, there has been considerable interest in Traditional Chinese Medicines (TCM) as sources for novel therapeutics. Our study examines the ability of the TCM Cordyceps sinensis to reduce acute and chronic rejection associated with cardiac transplantation. The objectives of this study were to first determine if oral delivery of the extract could reduce acute rejection in a rat heterotopic heart model of transplantation. The second objective was to determine, in vitro, if a sterile, aqueous extract of C. sinensis could decrease CD8+ T cell activity. The third objective was to determine if oral delivery of the extract could ablate allograft vasculopathy in a mouse abdominal aortic transplant model. We found that oral delivery of the extract demonstrated a reduction in acute rejection when used in conjunction with a sub-therapeutic dose of Cyclosporine. Further, we found, using a mixed lymphocyte reaction, that the extract was able to significantly reduce CD8+ T cell activity. Finally, we demonstrate that oral delivery of the extract, used with a therapeutic dose of Cyclosporine to suppress acute rejection, ablates allograft vasculopathy.

Recipient cells form the proliferative lesion in the rat heterotopic tracheal allograft model of obliterative airway disease

To determine the nature of the proliferative lesion in obliterative airway disease, heterotopic tracheal allograft transplantation was performed between fully disparate Brown Norway and Lewis rat strains. Four weeks after transplantation, the resulting lumenal occlusive lesion was stripped from the underlying tissue. The lesion was probed using immunohistochemical analysis with monoclonal antibodies and for DNA using strain-specific primers for Brown Norway or Lewis major histocompatibility complex Class I alleles. The lesions were alpha-actin positive, and polymerase chain reaction probing revealed only recipient DNA in the lesion tissue, regardless of the direction of transplantation, with no amplification of donor DNA. From this, we conclude that the proliferative lesion in the rat heterotopic tracheal model is of recipient origin a finding with important implications for the pathobiology of obliterative airway disease.

Abstract 1665: Biopsy induced metastasis: Role of SOX4/TGF-β driven EMT and immunosuppressive microenvironment

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA As a consequence of advances in digital diagnostic imaging, breast cancers are being diagnosed earlier in their development. These smaller tumors are less likely to be metastatic at time of discovery. The majority of newly diagnosed breast cancers in North America are confirmed using an incisional core needle biopsy (CNB). However an increasing number of studies highlight the pro-metastatic inflammatory and microenvironment changes occurring after incisional procedures on breast cancer tumors. This may account for reported higher incidences of lymph node metastases with increased risk of local recurrence and distant metastasis among patients undergoing incisional procedures. Evidence from our studies using a metastatic mouse model shows that incisional core needle biopsies lead to increased pulmonary metastasis. To study the mechanism(s) involved we monitored changes in circulating tumor cells (CTC), cytokine levels, variations in gene expression, microenvironment alteration and pulmonary metastatic burden, following CNB. Gene expression analysis by real-time qPCR showed increased TGF-β expression accompanied by SOX4 upregulation at 3 hours post biopsy (SOX4 is a Master regulator of EMT). This was followed, at 24 hours post biopsy, by significantly increased levels of the SOX4-associated Ezh2 gene and other EMT genes (ZEB2, SNAI2, SNAI3, and CDH2). Monitoring of CTCs over the same time frame showed them to be significantly increased in biopsied mice from 6 to 24 hours post biopsy, indicating continual CTC egress from biopsied tumors. Flow cytometry analysis of tumor microenvironment showed an immunosuppressive microenvironment with increased MDSC levels, reduced CD4+ and CD8+ T cells, macrophage and NKT cell. Interestingly, similar analysis of the Lung microenvironment 4 days later (day 7 post biopsy) revealed an inversion of the tumor situation, in the form of a significant uptick in Th1 type changes described by increased CD4+ and CD8+ type T cells, macrophages and NK cells, as well as increased TNF-β gene expression indicators corresponding to an early response to growth of metastases. We also compared H&E stained histological sections of lungs from biopsied and un-biopsied mice and recorded significant increase in pulmonary metastasis for the biopsied group. The findings of our study indicate that performance of CNB results in increased metastasis with immunomodulation of the tumor microenvironment and unregulated SOX4/TGF- β. Additional studies are needed to further elucidate the mechanism, and explore mitigating strategies to counter CNB related metastasis. Citation Format: Edward G. Mathenge, Cheryl Dean, Derek Clements, Ahmad Vagharkashani, Steffany Photopoulos, Krysta Coyle, Benjamin A. Malueth, Mike Giacomantonio, Anna Nunokawa, Julie Jordan, Shashi Gujar, Paola Marcato, Partick Lee, Carman A. Giacomantonio. Biopsy induced metastasis: Role of SOX4/TGF-β driven EMT and immunosuppressive microenvironment. [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 1665. doi:10.1158/1538-7445.AM2014-1665