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Exclusion of brain metastases from clinical trials is a major cause of the limited therapeutic options for this growing population of cancer patients. Here, we report a medium-throughput drug-screening platform (METPlatform) based on organotypic cultures that allows to evaluate inhibitors against metastases growing in situ. By applying this approach to brain metastasis, we identified several hits from a library of FDA approved inhibitors and others being tested in clinical trials. A blood-brain barrier permeable HSP90 inhibitor showed high potency against mouse and human brain metastases at clinically relevant stages of the disease, including a novel model of local relapse after neurosurgery. Furthermore, in situ proteomic analysis applied to organotypic cultures with metastases treated with the chaperone inhibitor revealed novel biomarkers in human brain metastasis and actionable mechanisms of resistance. Our work validates METPlatform as a potent resource for metastasis research integrating drug-screening and unbiased omic approaches that is fully compatible with human samples. We envision that METPlatform could be established as a clinically relevant strategy to personalize the management of metastatic disease in the brain and elsewhere.SummarySystemic spread of cancer continues to be the key aspect associated with lethality. In this publication, Zhu et al. describes a drug-screening platform specifically designed to study vulnerabilities of metastasis when colonizing secondary organs and demonstrates its value in difficult-to-treat brain metastasis using new models and patient-derived samples.

BACKGROUND Finding effective treatment options for patients with brain metastasis remains an unmet need. Given the limitations imposed by the blood-brain-barrier for systemic approaches, radiotherapy offers a superior ability to access the brain. While clinical practice recently adapted the use of stereotactic radiosurgery (SRS), Whole-Brain-Radiotherapy (WBRT) continuous to be an important treatment option, since many patients present with multifocal lesions or bad performance scores, rendering them ineligible for SRS. Unfortunately, overall survival of patients remains unaffected by radiotherapy. Despite this clinical data, the molecular mechanisms that allow metastatic cells to resist radiotherapy in the brain is unknown. MATERIAL AND METHODS We have applied WBRT to experimental brain metastasis from lung and breast adenocarcinoma and validated their resistance in vivo. RESULTS An unbiased search to identify potential mediators of resistance identified the S100A9-RAGE-NFκB-JunB pathway. Targeting this pathway genetically reverts the resistance to radiotherapy and increases therapeutic benefits in vivo. In two independent cohorts of brain metastasis from lung and breast adenocarcinoma patients, levels of S100A9 correlate with the response to radiotherapy, offering a novel approach to stratify patients according to their expected benefit. In order to make this biomarker also available for brain metastasis patients receiving palliative WBRT without preceding surgery, we complemented our tumor-specimen based approach with the less invasive detection of S100A9 from liquid biopsies. Here, serum S100A9 also correlated with a worse response to WBRT in brain metastasis patients. Furthermore, we have validated the use of a blood-brain-barrier permeable RAGE inhibitor to restore radio-sensitivity in experimental brain metastasis models in vivo and in patient-derived organotypic cultures of radio-resistant brain metastasis ex vivo. CONCLUSION We identified S100A9 as a major mediator of radio-resistance in brain metastasis and offer the molecular framework to personalize radiotherapy by exploiting it as a biomarker and as a therapeutic target, thus maximizing the benefits for the patient.

Finding effective treatment options for patients with brain metastasis remains an unmet need. Given the limitations imposed by the blood-brain-barrier for systemic approaches, radiotherapy offers a superior ability to access the brain. While clinical practice recently adapted the use of stereotactic radiosurgery (SRS), Whole-Brain-Radiotherapy (WBRT) continuous to be an important treatment option, since many patients present with multifocal lesions or bad performance scores, rendering them ineligible for SRS. Unfortunately, overall survival of patients remains unaffected by radiotherapy. Despite this clinical data, the molecular mechanisms that allow metastatic cells to resist radiotherapy in the brain is unknown. We have applied WBRT to experimental brain metastasis from lung and breast adenocarcinoma and validated their resistance in vivo. An unbiased search to identify potential mediators of resistance identified the S100A9-RAGE-NFkB-JunB pathway. Targeting this pathway genetically reverts the resistance to radiotherapy and increases therapeutic benefits in vivo. In two independent cohorts of brain metastasis from lung and breast adenocarcinoma patients, levels of S100A9 correlate with the response to radiotherapy, offering a novel approach to stratify patients according to their expected benefit. In order to make this biomarker also available for brain metastasis patients receiving palliative WBRT without preceding surgery, we complemented our tumor-specimen based approach with the less invasive detection of S100A9 from liquid biopsies. Here, serum S100A9 also correlated with a worse response to WBRT in brain metastasis patients. Furthermore, we have validated the use of a blood-brain-barrier permeable RAGE inhibitor to restore radio-sensitivity in experimental brain metastasis models in vivo and in patient-derived organotypic cultures of radio-resistant brain metastasis ex vivo. In conclusion, we identified S100A9 as a major mediator of radio-resistance in brain metastasis and offer the molecular framework to personalize radiotherapy by exploiting it as a biomarker and as a therapeutic target, thus maximizing the benefits for the patient.

Finding effective treatment options for patients with brain metastasis remains an unmet need. Given the limitations imposed by the blood-brain-barrier for systemic approaches, radiotherapy offers a superior ability to access the brain. While whole-brain-radiation-therapy (WBRT) historically has been the gold standard in the management of brain metastasis patients, clinical practice rapidly adapted the frequent use of stereotactic radiosurgery (SRS), given the high incidence of neurotoxicity when giving WBRT. However, WBRT continuous to be an important treatment option, since many patients present with numerous multifocal lesions, high risk of SRS-associated radionecrosis, bad performance scores or probable distant intracranial recurrence, rendering them ineligible for SRS. Unfortunately, overall survival of patients remains unaffected by radiotherapy. In spite of this clinical data, the underlying molecular mechanisms that allow metastatic cells to resist radiotherapy in the brain is unknown. We have applied WBRT to experimental brain metastasis from lung and breast adenocarcinoma and validated their resistance in vivo. An unbiased search to identify potential mediators of resistance identified the S100A9-RAGE-NFkB-JunB pathway. Targeting this pathway genetically reverts the resistance to radiotherapy and increases therapeutic benefits in vivo. In two independent cohorts of brain metastasis from lung and breast adenocarcinoma patients, levels of S100A9 correlate with the response to radiotherapy, offering a novel approach to stratify patients according to their expected benefit. As a proof of concept, we also detected S100A9 in serum and CSF samples from brain metastasis patients, complementing our biomarker strategy with a less invasive approach. Furthermore, we have validated the use of a blood-brain-barrier permeable RAGE inhibitor to restore radio-sensitivity in experimental brain metastasis models in vivo and in patient-derived organotypic cultures of radio-resistant brain metastasis ex vivo. In conclusion, we identified S100A9 as a major mediator of radio-resistance in brain metastasis and offer the molecular framework to personalize radiotherapy by exploiting it as a biomarker and as a therapeutic target, thus maximizing the benefits for the patient. Citation Format: Lauritz Miarka, Catia Monteiro, Celine Dalmasso, Natalia Yebra, Coral Fustero-Torre, Aisling Hegarty, Stephen Keelan, Eduardo Caleiras, Vareslija Damir, Leonie Young, Riccardo Soffietti, Juan M. Sepúlveda, Angel Pérez, Aurelio Lain, Aurore Siegfied, Elizabeth Cohen-Jonathan Moyal, Manuel Valiente. S100A9 mediates radiation resistance of brain metastasis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-068.

The diagnosis of brain metastasis involves high morbidity and mortality and remains an unmet clinical need in spite of being the most common tumor in the brain. Exclusion of these cancer patients from clinical trials is a major cause of their limited therapeutic options. In this study, we report a novel drug-screening platform (METPlatform) based on organotypic cultures which allows identifying effective anti-metastasis agents in the presence of the organ microenvironment. We have applied this approach to clinically relevant stages of brain metastasis using both experimental models and human tumor tissue (by performing patient-derived organotypic cultures). We identified heat shock protein 90 (HSP90) as a promising therapeutic target for brain metastasis. Debio-0932, a blood-brain barrier permeable HSP90 inhibitor, shows high potency against mouse and human brain metastases from melanoma, lung and breast adenocarcinoma with distinct oncogenomic profiles at clinically relevant stages of the disease, including a novel model of local relapse after neurosurgery. Furthermore, we have also used METPlatform to perform unbiased proteomics of brain metastases in situ. By applying this analysis to brain metastases treated with the chaperone inhibitor, we uncovered non-canonical clients of HSP90 as potential novel mediators of brain metastasis and actionable mechanisms of resistance driven by autophagy. Combined therapy using HSP90 and autophagy inhibitors showed synergistic effects compared to sublethal concentrations of each monotherapy, demonstrating the potential of METPlatform to design and test rationale combination therapies to target metastasis more effectively. In conclusion, our work validates METPlatform as a potent resource for metastasis research integrating drug-screening and unbiased omic approaches that is fully compatible with human samples and questions the rationale of excluding patients with brain metastasis from clinical trials. We envision that METPlatform will be established as a clinically relevant strategy to personalize the management of metastatic disease in the brain and elsewhere.