Introduction to current state of the art
Sergio Roman-roman (Institut Curie, Paris, France), Rob Clarke (University of Manchester, UK)
EACR sponsored speaker: Human specific models for studying ER+ and ER- breast cancer bone metastasis
Penny Ottewell (University of Sheffield, UK)
Bone metastasis is commonly modelled using xenograft transplantation in immunocompromised mice and are limited by the lack of a true metastatic pathway. It is also likely that there are species-specific differences involved in growing human cells in a murine environment. We have developed models where human breast cancer cell lines and PDX metastasise to human bone in mice.
Material and Methods
Bone discs from femoral heads of patients undergoing hip replacement surgery were implanted subcutaneously into NOD/SCID mice. For metastasis studies PDX (BB3RC32, ER+PR+HER2-; BB2RC08, ER+PR-ER2-; BB6RC37, ER-PR-HER2- and BB6RC39, ER+PR+HER2+), MDA-MB-231-luc2 or T47D-luc2 cells were injected directly into human bone implants or into mammary fat pads and metastases detected by luciferase imaging. Bone discs were harvested weekly for 4 weeks, osteoblast viability measured by calcein uptake and bone integrity assessed by uCT. Osteoclasts/osteoblasts and blood vessels were identified following TRAP and CD31 staining.
Results and Discussion
Following implantation of subchondral bone, MDA-MB-231 cells metastasised from mammary fat pads to the human bone discs in 70% of mice within 6-8 weeks and T47D cells in 60% of mice within 8-10 weeks. Interestingly, MDA-MB-231 cells specifically metastasised to the human bone implants whereas T47D cells BB3RC32, BB2RC08, and BB6RC37 metastasised to both human bone and mouse long bones, although metastasis to human bone implants was significantly more frequent especially from PDX (metastases from PDX occurred 80-100% of human bone grafts and 10-50% of mouse hind limbs).
Analysis of bone implants in the absence of tumours showed re-vascularisation by both human and mouse endothelial cells. No change in bone volume was detected, however, osteocyte viability was reduced to 40% of control by day 7 (P<0.0001) and then stabilised. Osteoblast activity was confirmed by active calcein uptake from day 21 and presence of osteoclasts on the bone surface increased with time.
In vivo implantation of human bone discs provides a species-specific metastatic site for breast cancer cells.
Cancer cell plasticity on skin carcinoma progression and therapy response
Purificación Muñoz (IDIBELL, Barcelona, Spain)
Laura Lorenzo-Sanz1, Adrià Bernat-Peguera1, Victoria da Silva-Diz12, Marta Lopez-Cerda1, Pilar Simón-Extremera1, Rosa M. Penin3, Diana Pérez Sidelnikova4, Oriol Bermejo4, Joan Maria Viñals4, Purificación Muñoz1
1 Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain
2 Present Address: Rutgers Cancer Institute of New Jersey, Rutgers University, New Jersey, USA
3 Pathology Service, 4 Plastic Surgery Unit, Hospital Universitario de Bellvtige/IDIBELL, Barcelona, Spain
Squamous cell carcinoma (SCC) is the second most frequent skin cancer in humans. Most of SCCs are treated by surgical excision, but 5-8% of patients develop aggressive recurrent tumors that show enhanced metastasis. Advanced SCCs are treated with chemotherapy and radiotherapy with poor clinical benefits. Therefore, it is important to design new targeted therapies. Cancer cells show a strong plasticity, which endow them with stemness and may also impact on therapy response. Cancer stem-like cells (CSC) play key roles in long-term tumor propagation and metastasis, but their dynamics during disease progression are not understood.
Material and Methods
To analyze CSC dynamics, we generated lineages of mouse skin SCC progression by orthotopically engrafting well-differentiated SCCs (WD-SCCs), exhibiting epithelial-differentiation traits in immunodeficient mice. After serial engraftments, these tumors evolved to poorly differentiated and spindle SCCs (PD/S-SCCs) with mesenchymal-like features. We also generated PDXs from patient WD/MD-SCC and PD-SCC samples, and characterized phenotypic features and regulatory mechanisms of cancer cells during SCC progression and therapy response.
Results and Discussion
Mouse advanced PD/S-SCCs showed an aggressive growth and enhanced metastasis, as compared to their WD-SCC precursors, which was associated with an expansion of CSC population. Molecular characterization of CSCs of WD-SCCs, intermediate MD/PD-SCCs and advanced PD/S-SCCs demonstrated that these plastic cells progressively and dynamically switch from CSCs that conserve epithelial differentiation capability toward full mesenchymal-like CSCs. Furthermore, regulatory mechanisms that control CSC proliferation and dissemination change together with this phenotypic progression.
In contrast to mouse skin SCCs, WD/MD-SCC PDXs conserved the epithelial traits and did not evolved to PD-SCCs after serial engraftments, suggesting that a crosstalk between tumor cells and microenvironment could promote SCC progression. Analysis of patient samples and PDXs demonstrated that, similarly to mouse advanced SCCs, PD-SCC PDXs exhibit an expansion of CSC population, a strong induction of EMT and upregulation of PDGFR signaling, which is responsible to promote tumor cell invasion and metastasis.
Furthermore, autocrine activation of EGFR signaling promotes WD/MD-SCC cell proliferation, and Gefitinib treatment blocked WD/MD-SCC PDX growth. However, resistance was generated after long-term Gefitinib treatment, which may be associated with a switch from EGFR to FGFR signaling.
Cancer (stem-like) cells show a strong plasticity that allows cell conversion from epithelial to mesenchymal state and the switch of regulatory mechanisms controlling SCC growth and metastasis during tumor progression. Plasticity also favors the acquisition of therapy resistance by switching signaling pathways that control proliferation and survival.