LACK OF TRANSFORMING GROWTH FACTOR- Β SIGNALING PROMOTES COLLECTIVE CANCER CELL INVASION THROUGH TUMOR- STROMAL CROSSTALK PRESENTED BY JONAKI BOSE

LEAD REFERENCES  LAUREN A MATISE, TRENIS D PALMER, WILLIAM J ASHBY, ABUDI NASHABI, ANNA CHYTIL, MARY AAKRE, MICHAEL W PICKUP, AGNIESZKA E GORSKA, ANDRIES ZIJLSTRA, HAROLD L MOSES.

INTRODUCTION  What is TGF-β ?  TGF-β is a Secreted protein that exists in atleast three isoforms called TGF-β1, TGF-β2 & TGF-β3. TGF-β is a Pleiotropic cytokine that is it affects the activity of multiple cell types. Functions:-  TGF-β Regulates growth arrest, cell motility, development, and differentiation.  TGF-β signaling is also instrumental in the tumor microenvironment by influencing both tumor development and metastasis.  Transforming growth factor beta (TGF-β) has a dual role during tumor progression, initially as a suppressor and then as a promoter.  TGF-β is a critical regulator of tumor-stromal interactions that promote mammary tumor cell migration and invasion. TGF-β SIGNALLING  This Pathway is involved in many cellular Processes in both the adult organism and the developing embryo including cell growth, cell differentiation, apoptosis, cellular homeostasis and other cellular functions.

METHODS MURINE MAMMARY CARCINOMA CELLS (Isolated from either mouse mammary tumor virus MMTV-PyVmT transforming growth factor – beta receptor II knockout T βRII KO or TβRII control nice. Combined with mammary fibroblast. Xenografted onto the chicken embryo chorioallantoic membrane. Combinatorial Xenografts were used as a model to study epithelial-stromal crosstalk. * Intravital imaging of migration was monitored ex ovo, and metastasis as investigated in ovo.

Ex Ovo CAM Assay On day 10 post incubation Mammary Epithellial Cells alone or in combination with fibroblasts were grafted on to the CAM Intravital imaging began on day 12 post incubation

In Ovo CAM Assay The CAM was dropped from the egg shell on day 10 post incubation Mammary Epithellial Cells alone or in combination with fibroblasts were grafted on to the CAM Tumor bearing animals were scarified and tumor tissue and distant CAM were collected 7 – 10 days post grafting and any piece of distant CAM is a metastatic site. To collect distant CAM, the egg shell was cut radially in to two equivalent halves. The resulting four pieces of CAM were then analyzed via Murine Alu PCR for the presence of disseminated cells.

Image of Intravital imaging Image of Metastatis

Utilization of Ex ovo & In ovo CAM Models Using both the ex ovo and in ovo CAM models, it was characterised how tumor cells migration and invasion utilises TGF-β mediated epithelial-stromal interactions.

Laser Capture Microdissection (LCM) LCM was performed on 5µm frozen in ovo tumor sections LCM-Captured RNA was isolated and validated for array quality Samples, 3 control tumors and 3 KO tumors were individually assayed on quantitative PCR arraysUtilization LCM was done to identify gen expression changes in response to TGF-β signalling loss.

Image of LCM of mouse tumor sections.

Expression Analysis Total cell RNA was collected Then it was further Purified  The Primer sequences used to amplify murine coding sequences of interest.

Results Intravital microscopy of xenografts revealed that mammary fibroblast promoted two migratory phenotypes dependent on epithelial TGF-β signalling; single cell/strand migration or collective migration. TβRII KO tumors also exhibited a two fold greater metastasis than TβRII control tumors.

Conclusion Fibroblast – Stimulated Carcinoma Cells utilize TGF-β signalling to drive single cell / strand migration but migrate collectively in the absence of TGF-β signalling. These migration patterns involve the signalling regulation of several epithelial – to – mesenchymal transition pathways.

AIM  These findings concerning TGF- β signalling in epithelial – stromal interactions are important in identifying migratory mechanisms that can be targeted as recourse for breast cancer treatment.