Writing | Wang Yanwen Editor | Zhou Yebin
In recent years, the treatment of tumors has changed from broad-spectrum cytotoxic drugs such as chemotherapy to precise treatment suitable for individual patients, and the therapeutic effect has been greatly improved. Precision therapy relies on molecular and cellular analysis of a patient’s tumor, however, molecular approaches have only identified druggable tumor driver genes in a small subset of patients. For those patients who do not carry the target mutation or have a low tumor mutational burden, it is difficult to select appropriate drugs through genomic analysis.
This problem is common in childhood tumors such as rhabdomyosarcoma (RMS). RMS is the most common soft tissue sarcoma in children, and 30% of patients have poor clinical outcomes. So far, no targeted therapy for RMS is available. Patients with such tumors urgently need more effective drug selection methods.
September 15, 2020, Phenotypic profiling with a living by Gabriele Manzella et al in Nature Communications The article in the biobank of primary rhabdomyosarcoma unravels disease heterogeneity and AKT sensitivity describes a functional drug discovery platform constructed by it. The platform optimizes the growth and culture conditions of RMS cells and enables precise screening of patient-specific drugs.
First, the study optimizes RMS cell cultures. The authors selected 20 RMS human-derived tumor tissue xenograft (PDX)-derived cells (PDX-derived primary cells, PPCs) to compare the cell viability, proliferation, differentiation and tumor-to-tumor differences in various media. cytoplasmic contamination. Among them, Neurobasal (NB) medium combined with an adhesion matrix can support the growth of most PPCs, and shows low mouse mesenchymal cell contamination and low cell differentiation.
Long-term cell proliferation studies have also found that NB-cultured cells can maintain exponential proliferation. The optimized medium is also able to support 3D proliferation of cells. In vitro cultured PPCs cells and parental PDX cells were injected into NSG mice, respectively, and the engraftment rate reached 100% in both models, and the tumor growth and survival rate of tumor-bearing mice were also similar. This indicates that this platform can well support the growth of heterogeneous tumor cells.
a. The process of establishing a preclinical drug screening platform. b. Optimization of PPCs medium. c. Growth curves of two PPCs in DMEM and NB medium supplemented with growth factors.
To assess whether culture conditions lead to clonal selection, molecular analyses of PDX and corresponding PPCs were performed. Whole-genome copy number detection, exome sequencing, and methylation profiling all found that PDXs and PPCs were highly consistent, indicating that the epigenetic and genotypes of cultured cells were stable. To test whether the cultured PPC cells retained the histological features of the tumor, we generated xenografts (CDX) from PPC cells at passages 4 to 6 and found that the histological features of CDX and PDX were consistent with the original RMS tissue.
Then, the authors investigated whether PPCs cultured in vitro could be used for personalized drug screening. Drug screening uses a compound library of 204 drugs covering a range of targets. After treatment of 17 PPCs and 4 established cell lines with 500 nM of each drug class for 72 hours, approximately 30.9% of the drugs resulted in a 40% or greater reduction in activity in at least one sample. Most drug effect patterns appear patient-specific. Among them, both AKT and MEK inhibitors showed selective sensitivity. Further experiments demonstrated that drug toxicity was also not affected by the culture state (2D or 3D).
To make this platform suitable for clinical use, the researchers next step is to directly culture patient tumor tissue in vitro. The researchers selected lymph node biopsies from patients with RMS after the second relapse, which grew well in NB medium supplemented with B27 and bFGF. The drug screening results found effective drugs including proteasome and HDAC inhibitors, proving that the PPCs drug screening platform can also use cells isolated directly from patient tumors, so that the use of shorter time for personalized drug selection in the clinic has become a possible.
a. Clinical diagnosis, treatment, and drug screening process for RMS patients. b. Brightfield image of cells isolated from a patient’s lymph node biopsy. c. FOXO1 gene fluorescence in situ hybridization detection. d. Screening the activity of the drug. e. Left: Effects of Panobinostat and bortezomib alone and in combination on cell viability; Right: Synergistic effect of Panobinostat and bortezomib.
Among the drugs screened, AKTThe patient-specific sensitivity exhibited by and MEK inhibitors has attracted the attention of researchers. The investigators divided PPCs into 3 subgroups based on their response to the drug. Two groups responded to either AKT or EKR inhibitors, and the other group did not respond to either. To further understand the mechanism, the researchers treated eight cultures of PPCs with an AKT inhibitor (afuresertib) and a MEK inhibitor (trametinib). The two drugs exhibited synergy in seven PPCs, and this synergy was more pronounced in RAS-mutated PPCs. When PPCs were treated with auresertib and trametinib alone, respectively, IC50 showed significant individual variability, with the most drug-sensitive samples already reaching the concentration range used for treatment.
To further verify the reliability of the drug screening platform, the researchers conducted an in vivo study: auresertib was continuously treated with sensitive and resistant PDX for at least 3 cycles. The results showed that auresertib-sensitive tumor-bearing mice lived significantly longer than drug-resistant mice. The researchers further administered auresertib combined with trametinib to auresertib-sensitive mice, which was consistent with the in vitro results and also showed a synergistic effect.
In conclusion, this study constructs a preclinical drug screening platform for rhabdomyosarcoma (RMS). During the construction and validation of the platform, the heterogeneity of RMS sensitivity to different drugs was also revealed. Through this platform, it was also screened that the combination of AKT and MEK inhibitors may be effective for some RMS patients. This result provides an important theoretical basis for clinical drug screening, and it is believed that it can be used to provide patients with personalized treatment decisions in the future.
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