Project description:Residual disease represents a major obstacle to the successful treatment of breast cancer, however little is known about the biology of residual tumor cells in vivo. To identify genes differentially expressed in vivo in residual tumor cells compared to primary tumor cells or recurrent tumor cells, we used flow cytometry to isolate GFP-labeled tumor cells from primary, residual, or recurrent tumors grown orthotopically in nu/nu mice. WTA was used to amplify RNA from all tumor cells.
Project description:Residual disease represents a major obstacle to the successful treatment of breast cancer, however little is known about the biology of residual tumor cells in vivo. To identify genes differentially expressed in vivo in residual tumor cells compared to primary tumor cells or recurrent tumor cells, we used flow cytometry to isolate GFP-labeled tumor cells from primary, residual, or recurrent tumors grown orthotopically in nu/nu mice. WTA was used to amplify RNA from all tumor cells.
Project description:This experiment investigates the genome wide DNA methylation landscape of residual breast cancer cells. Primary mammary epithelial cells taken from female mice with doxycycline-inducible hMyc- and Neu/Her2-oncogenes were grown in 3D organoid cultures. Upon the addition of doxycycline (200ng/mL) to the medium the expression of the oncogenes gets activated and the cells start uncontrolled proliferation resembling tumor growth. Tumor samples were taken after 5 days of oncogene induction. Subsequently, doxycycline was removed from the medium, which silences oncogene expression and results in rapid tumor regression. Residual samples reminiscent to the non-induced (normal) structures were taken after 7 days of de-induction (12 days overall). Non-induced structures were grown and sampled in parallel to the residual structures.
Project description:This experiment investigates the expression characteristics of residual breast cancer cells. Primary mammary epithelial cells taken from female mice with doxycycline-inducible hMyc- and Neu/Her2-oncogenes were grown in 3D organoid cultures. Upon the addition of doxycycline (200ng/mL) to the medium the expression of the oncogenes gets activated and the cells start uncontrolled proliferation resembling tumor growth. Tumor samples were taken after 5 days of oncogene induction. Subsequently, doxycycline was removed from the medium, which silences oncogene expression and results in rapid tumor regression. Residual samples reminiscent to the non-induced (normal) structures were taken after 7 days of de-induction (12 days overall). Non-induced structures were grown and sampled in parallel.
Project description:HER2 / Neu is amplified and overexpressed in a large proportion of human breast cancers, but the signaling pathways that contribute to tumor development and metastatic progression are not completely understood. Using gene expression data and pathway signatures we predicted a role for activator E2F transcription factors in Neu induced tumors. This was genetically tested by interbreeding Neu transgenics with knockouts of the three activator E2Fs. Loss of any E2F delayed Neu induced tumor onset. E2F1 loss accelerated tumor growth while E2F2 and E2F3 loss did not. Strikingly, it was observed that loss of E2F1 or E2F2 significantly reduced the metastatic capacity of the tumor and this was associated with a reduction in circulating tumor cells in the E2F2 knockout. Gene expression analysis between the tumors in the various E2F mutant backgrounds revealed that there was extensive compensation by other E2F family members in the individual knockouts, underscoring the importance of the E2Fs in HER2 / Neu induced tumors. Extension to HER2 positive human breast cancer revealed a number of HER2+ subtypes based on E2F activity with differences in relapse free survival times. Taken together these data demonstrate that the E2F transcription factors are integral to HER2+ tumor development and progression.
Project description:Trastuzumab (Herceptin™), a humanized monoclonal antibody targeting the extracellular domain of human epidermal growth factor receptor-2 (HER2), is one of the most successful examples of targeted therapies for HER2-positive breast cancer. However, drug resistance remains daunting challenges. New combinatorial regimen of CDK4/6 inhibitors plus trastuzumab is currently under active clinical investigations. In this study, we seek to prospectively model the in vivo response to CDK4/6 inhibitor Palbociclib (Pal) plus trastuzumab (Ab) using transgenic Her2/Neu mouse model in parallel with the current clinical trial scenario. We performed single cell RNA-seqencing (Drop-seq) to profile and compare tumor cells and infiltrated immune cells derived from control, Ab+Pal sensitive/residual (APS) and resistant/progressive (APR) tumors. We revealed that although Ab+Pal treatment enhanced antigen processing, presentation and interferon signaling on tumor cells, a distinct immunosuppressive immature myeloid cells (IMCs) infiltrated in the resistant tumor microenvironment to promote resistant phenotype. Based on single cell gene set enrichment analysis (profiling) guided drug screening, we identified and evaluated a combinatorial immunotherapy regimen. We found that combinatorial immunotherapy with receptor tyrosine kinase inhibitor Cabozantinib and immune checkpoint blockades overcome Ab+Pal resistance by inhibiting IMCs and enhancing anti-tumor immunity. Moreover, our rationally designed sequential combinatorial regimens enabled durable response and sustained controlling of the emergence of acquired resistance, thus significantly improved outcomes of rapidly evolving Her2/Neu positive breast cancers. Our results implicate that single-cell RNA sequencing profiling guided combinatorial immunotherapy as a strategy to mitigate the emergence of resistance and to achieve long-term therapeutic benefit merits clinical translation.
Project description:Transcriptional expression of a restricted set of cancer signal transduction-related gesen were comparatively quantified in HER2/neu transgenic mammary tumors, mesenchymal and epithelial tumor cell lineages, both estabilished from HER-2/neu transgenic tumors, and syngeneic mesenchymal stem cells.
Project description:We identified a 17-gene Her2-enriched tumor initiating cell (HTIC) signature in MMTV-Her2/Neu mouse mammary TICs. Here, we show that patients with HTICS+ HER2+:ERα− tumors are more likely to achieve a pathologic complete response to trastuzumab-based neoadjuvant chemotherapy compared with HER2+:ER+ tumors. Neoadjuvant study of 50 HER2-positive breast cancer cases treated with trastuzumab-based chemotherapy pre-operatively. Pre-treatment FNA from primary tumors were obtained and RNA extracted and hybridized to Affymetrix microarrays according to manufacturer protocol. Pathologic response was assessed at the end of neoadjuvant treatment.
