Final Report Summary - CELL THERAPY (Harnessing of hematopoietic stem cells for targeting of brain metastases) BackgroundBrain metastases develop in 20 to 35% of all cancer patients and mostly originate from lung cancer, breast cancer and melanoma. Approximately 15% of breast cancer patients develop brain metastases and the incidence is significantly higher in those with triple-negative and HER2-positive disease, reaching 30-55%. The median survival time of patients with brain metastases is only 4 to 19 months. Management of brain metastases is becoming increasingly important due to their rising incidence. Moreover, the brain is also increasingly the sole site of tumour relapse in patients with breast and lung cancer. Current treatment options for brain metastases are very limited. The methods of choice are surgical resection combined with whole-brain radiation or stereotactic radiosurgery. Systemic chemotherapies had little success so far, mostly due to the blood-brain barrier (BBB) that strongly limits the delivery of drugs into the brain. It has been shown in experimental models that although the blood vessel permeability in brain metastases is increased as compared to the normal brain, it reaches less than 15% of the permeability seen in other organs. Thus, novel approaches for the effective delivery of drugs into the brain are urgently required. Different stem cells have been explored as vehicles for the delivery of therapeutic molecules to brain tumours. Neural stem cells (NSCs) showed strong promise in animal models of glioma. However, primary autologous NSCs cannot be isolated in the quantities required for the therapy and oncogene-transduced NSCs remain a safety concern. Mesenchymal stem cells (MSCs) have also been used for delivery of therapeutic genes to experimental glioma. MSCs can be obtained from different sources and they are efficiently retained within tumours upon intra-tumoural injection. However, they show suboptimal homing to the brain upon systemic administration. In comparison to NSCs and MSCs, the hematopoietic stem cells (HSCs) received little attention as delivery vehicles so far.HSCs are precursors of blood cells and capable of giving rise to all different blood cell populations. Recent studies demonstrated that differentiated progeny of HSCs homes to diseased brain in animal models as well as in patients (e.g. autoimmune encephalitis, multiple sclerosis, Alzheimer’s disease, glioma), while their homing to brain metastases has not yet been investigated. Primary human HSCs can be isolated in large quantities from the cord blood or mobilized peripheral blood and procedures for the clinical use of autologous HSCs are well established, which is expected to accelerate clinical translation of therapies that are based on HSCs. Importantly, human CD34+ HSCs transduced with lentiviral vector have been successfully used in a phase I/II clinical trial to treat adrenoleukodistrophy, a severe demyelinating brain disease, thus demonstrating that the progeny of systemically administered HSCs can efficiently home to the diseased brain in patients. Based on these properties, we reasoned that the progeny of HSCs which have been engineered to express genes with anti-cancer properties could carry the latter across the BBB to metastases in the brain.ResultsIn the present study we demonstrated that myeloid cells (a subpopulation of white blood cells) derived from the exogeneously introduced HSCs infiltrate metastatic brain lesions in large quantities and can efficiently deliver green fluorescent protein (GFP) that has been introduced into HSCs by lentiviral transduction. This was observed for murine as well as human HSC progeny in our pre-clinical models. The homing of HSC progeny to brain metastases was specific for the tumour region, as infiltration into the healthy brain was not observed. Importantly, the myeloid progeny of HSCs homed to large established metastases, as well as to micrometastases consisting of only few cancer cells. Thus, the progeny of HSC has a potential to target small cancer lesions that are asymptomatic and undetectable with current diagnostic approaches, and therefore this strategy is suitable also for the development of preventive therapies.One drawback of our approach is the low specificity of the delivery due to the naturally occurring accumulation of the HSC progeny in organs like the spleen and bone marrow. To demonstrate that this can be circumvented, we identified gene promoters that can be used for specific expression of therapeutic genes within the brain metastases-infiltrating myeloid cells. This was achieved by comparative whole transcriptome microarray analysis of the HSC progeny infiltrating brain metastases, the bone marrow and the spleens. Further validation of promoter candidates by gene expression and immunofluorescence staining identified 3 promoter candidates with high specificity and activity within myeloid cells infiltrating brain metastases. Subsequently, promoter reporter constructs of different lengths were generated for the 3 candidates and their specificity was validated in vivo. This identified 2 different promoter constructs with significantly increased activity within brain metastases as compared to the spleen, bone marrow and the lungs. These promoters can be used for specific delivery of different therapeutic agents to brain metastases, as well as to primary tumours in pre-clinical models.Lastly, we demonstrated a potential for the clinical translation of the HSC therapy by confirming our findings in human HSCs and patient-derived brain metastases tissue. We have shown that the brain metastases-specific promoters identified in our pre-clinical models also display activity and specificity within the brain metastases-infiltrating myeloid cells in human brain tumours. In summary, we here established a proof-of-principle for the HSC therapy targeting brain metastases.ImpactTwenty to forty percent of all cancer patients develop metastases in the brain. The incidence of brain metastases in the clinic is increasing and the brain is increasingly the sole site of cancer relapse. Current treatment options for brain metastases are very limited and inefficient. Novel approaches for the delivery of drugs into the brain are expected to improve therapeutic efficacy. In line with this unmet need, our project developed a therapeutic approach for improved delivery of therapeutic agents to brain metastases. In long-term, the clinical translation of this strategy has a potential to improve therapeutic options for the underserved population of cancer patients with brain metastases. Importantly, HSC can be adapted for the delivery of different therapeutic agents like monoclonal antibodies or cytokines to multi-organ metastases including brain lesions, and thereby widen the therapeutic spectrum for brain metastases. Such improved therapies are expected to have an important impact on the overall management and disease outcome of cancer patients.
