Building consensus on the application of organoid-based drug sensitivity testing in cancer precision medicine and drug development.

Patient-derived organoids (PDOs) have emerged as a promising platform for clinical and translational studies. A strong correlation exists between clinical outcomes and the use of PDOs to predict the efficacy of chemotherapy and/or radiotherapy. To standardize interpretation and enhance scientific communication in the field of cancer precision medicine, we revisit the concept of PDO-based drug sensitivity testing (DST). We present an expert consensus-driven approach for medication selection aimed at predicting patient responses. To further standardize PDO-based DST, we propose guidelines for clarification and characterization. Additionally, we identify several major challenges in clinical prediction when utilizing PDOs.

Exploring the impact of PDGFD in osteosarcoma metastasis through single-cell sequencing analysis.

PURPOSE: The overall survival rate for metastatic osteosarcoma hovers around 20%. Responses to second-line chemotherapy, targeted therapies, and immunotherapies have demonstrated limited efficacy in metastatic osteosarcoma. Our objective is to validate differentially expressed genes and signaling pathways between non-metastatic and metastatic osteosarcoma, employing single-cell RNA sequencing (scRNA-seq) and additional functional investigations. We aim to enhance comprehension of metastatic mechanisms and potentially unveil a therapeutic target. METHODS: scRNA-seq was performed on two primary osteosarcoma lesions (1 non-metastatic and 1 metastatic). Seurat package facilitated dimensionality reduction and cluster identification. Copy number variation (CNV) was predicted using InferCNV. CellChat characterized ligand-receptor-based intercellular communication networks. Differentially expressed genes underwent GO function enrichment analysis and GSEA. Validation was achieved through the GSE152048 dataset, which identified PDGFD-PDGFRB as a common ligand-receptor pair with significant contribution. Immunohistochemistry assessed PDGFD and PDGFRB expression, while multicolor immunofluorescence and flow cytometry provided insight into spatial relationships and the tumor immune microenvironment. Kaplan-Meier survival analysis compared metastasis-free survival and overall survival between high and low levels of PDGFD and PDGFRB. Manipulation of PDGFD expression in primary osteosarcoma cells examined invasion abilities and related markers. RESULTS: Ten clusters encompassing osteoblasts, osteoclasts, osteocytes, fibroblasts, pericytes, endothelial cells, myeloid cells, T cells, B cells, and proliferating cells were identified. Osteoblasts, osteoclasts, and osteocytes exhibited heightened CNV levels. Ligand-receptor-based communication networks exposed significant fibroblast crosstalk with other cell types, and the PDGF signaling pathway was activated in non-metastatic osteosarcoma primary lesion. These results were corroborated by the GSE152048 dataset, confirming the prominence of PDGFD-PDGFRB as a common ligand-receptor pair. Immunohistochemistry demonstrated considerably greater PDGFD expression in non-metastatic osteosarcoma tissues and organoids, correlating with extended metastasis-free and overall survival. PDGFRB expression showed no significant variation between non-metastatic and metastatic osteosarcoma, nor strong correlations with survival times. Multicolor immunofluorescence suggested co-localization of PDGFD with PDGFRB. Flow cytometry unveiled a highly immunosuppressive microenvironment in metastatic osteosarcoma. Manipulating PDGFD expression demonstrated altered invasive abilities and marker expressions in primary osteosarcoma cells from both non-metastatic and metastatic lesions. CONCLUSIONS: scRNA-seq illuminated the activation of the PDGF signaling pathway in primary lesion of non-metastatic osteosarcoma. PDGFD displayed an inhibitory effect on osteosarcoma metastasis, likely through the suppression of the EMT signaling pathway.

Targeted inhibition of Wnt signaling with a Clostridioides difficile toxin B fragment suppresses breast cancer tumor growth.

Wnt signaling pathways are transmitted via 10 homologous frizzled receptors (FZD1-10) in humans. Reagents broadly inhibiting Wnt signaling pathways reduce growth and metastasis of many tumors, but their therapeutic development has been hampered by the side effect. Inhibitors targeting specific Wnt-FZD pair(s) enriched in cancer cells may reduce side effect, but the therapeutic effect of narrow-spectrum Wnt-FZD inhibitors remains to be established in vivo. Here, we developed a fragment of C. difficile toxin B (TcdBFBD), which recognizes and inhibits a subclass of FZDs, FZD1/2/7, and examined whether targeting this FZD subgroup may offer therapeutic benefits for treating breast cancer models in mice. Utilizing 2 basal-like and 1 luminal-like breast cancer models, we found that TcdBFBD reduces tumor-initiating cells and attenuates growth of basal-like mammary tumor organoids and xenografted tumors, without damaging Wnt-sensitive tissues such as bones in vivo. Furthermore, FZD1/2/7-positive cells are enriched in chemotherapy-resistant cells in both basal-like and luminal mammary tumors treated with cisplatin, and TcdBFBD synergizes strongly with cisplatin in inhibiting both tumor types. These data demonstrate the therapeutic value of narrow-spectrum Wnt signaling inhibitor in treating breast cancers.

A pilot study of multi-antigen stimulated cell therapy-I plus camrelizumab and apatinib in patients with advanced bone and soft-tissue sarcomas.

BACKGROUND: Cell-based  immunotherapy shows the therapeutic potential in sarcomas, in addition to angiogenesis-targeted tyrosine kinase inhibitor (TKI) and immune checkpoint inhibitor (ICI). Multi-antigen stimulated cell therapy-I (MASCT-I) technology is a sequential immune cell therapy for cancer, which composes of multiple antigen-loaded dendritic cell (DC) vaccines followed by the adoptive transfer of anti-tumor effector T-cells. METHODS: In this phase 1 study, we assessed MASCT-I plus camrelizumab (an ICI against PD-1) and apatinib (a highly selective TKI targeting VEGFR2) in patients with unresectable recurrent or metastatic bone and soft-tissue sarcoma after at least one line of prior systemic therapy. One MASCT-I course consisted of 3 DC subcutaneous injections, followed by 3 active T cell infusions administered 18-27 days after each DC injection. In schedule-I group, 3 DC injections were administered with a 28-day interval in all courses; in schedule-II group, 3 DC injections were administered with a 7-day interval in the first course and with a 28-day interval thereafter. All patients received intravenous camrelizumab 200 mg every 3 weeks and oral apatinib 250 mg daily. RESULTS: From October 30, 2019, to August 12, 2021, 19 patients were enrolled and randomly assigned to schedule-I group (n = 9) and schedule-II group (n = 10). Of the 19 patients, 11 (57.9%) experienced grade 3 or 4 treatment-related adverse events. No treatment-related deaths occurred. Patients in schedule-II group showed similar objective response rate (ORR) with those in schedule-I group (30.0% versus 33.3%) but had higher disease control rate (DCR; 90.0% versus 44.4%) and longer median progression-free survival (PFS; 7.7 versus 4.0 months). For the 13 patients with soft-tissue sarcomas, the ORR was 30.8%, DCR was 76.9%, and median PFS was 12.9 months; for the 6 patients with osteosarcomas, the ORR was 33.3%, the DCR was 50.0%, and median PFS was 5.7 months. CONCLUSIONS: Overall, MASCT-I plus camrelizumab and apatinib was safe and showed encouraging efficacy in advanced bone and soft-tissue sarcoma, and schedule-II administration method was recommended. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04074564.

Standardization of organoid culture in cancer research.

Establishing a valid in vitro model to represent tumor heterogeneity and biology is critical but challenging. Tumor organoids are self-assembled three-dimensional cell clusters which are of great significance for recapitulating the histopathological, genetic, and phenotypic characteristics of primary tissues. The organoid has emerged as an attractive in vitro platform for tumor biology research and high-throughput drug screening in cancer medicine. Organoids offer unique advantages over cell lines and patient-derived xenograft models, but there are no standardized methods to guide the culture of organoids, leading to confusion in organoid studies that may affect accurate judgments of tumor biology. This review summarizes the shortcomings of current organoid culture methods, presents the latest research findings on organoid standardization, and proposes an outlook for organoid modeling.

First-in-Maintenance Therapy for Localized High-Grade Osteosarcoma: An Open-Label Phase I/II Trial of the Anti-PD-L1 Antibody ZKAB001.

PURPOSE: We investigated the safety and preliminary efficacy of anti-PD-L1 antibody (ZKAB001) as maintenance therapy for localized patients with high-grade osteosarcoma to reduce the risk of recurrence and metastasis. PATIENTS AND METHODS: This open-label Phase I/II study was divided into dose-escalation Phase I and expansion Phase II. Phase I used a 3+3 design with ZKAB001 at three escalating doses ranging: 5, 10, 15 mg/kg every 2 weeks in 9 patients with localized high-grade osteosarcoma and Phase II tested 10 mg/kg in 12 patients for up to 24 cycles. Primary endpoints were safety and tolerability assessed using CTCAE4.0.3. RESULTS: Between October 2018 and 2019, 21 eligible patients were enrolled and accepted ZKAB001 treatment: 9 in the dose-escalation phase, and 12 in expansion phase. Six patients with disease progression withdrew from this study and follow-up is ongoing. The MTD was not defined in Phase I. All doses had a manageable safety profile. The recommended dose in Phase II was set at 10 mg/kg. Most frequent immune-related adverse events were thyroiditis (76.2%) and dermatitis (42.9%). Only 1 (4.8%) of 21 patients had a Grade 3 skin rash. The median 3-year event-free survival (EFS) and overall survival (OS) were not established; however, 24-month EFS was 71.4% (95% confidence interval, 47.2-86.0) and 2-year OS was 100%. Preliminary efficacy data showed EFS benefits in patients with PD-L1 positive or an MSI-H sub-population. CONCLUSIONS: Switching to maintenance using ZKAB001 showed an acceptable safety profile and provided preliminary evidence of clinical activity in localized patients with osteosarcoma.

Colorectal cancer organoid models uncover oxaliplatin-resistant mechanisms at single cell resolution.

PURPOSE: Oxaliplatin-based chemotherapy is a standard treatment for advanced colorectal cancer (CRC) patients. However, chemoresistance-induced resistance is an essential cause for mortality. Therefore, it is necessary to study the mechanism of drug resistance in CRC. METHODS: Here, we established two strains of patient-derived organoids (PDOs) selected from oxaliplatin-resistant and treatment-naïve CRC patients. To dissect the drug-resistant mechanisms, these CRC-PDOs were subjected to single-cell RNA sequencing (scRNA-Seq). RESULTS: We found that the drug sensitivity test outcome from these organoids subjected to oxaliplatin and 5-FU exposure was consistent with the clinic readout. CRC-PDOs well recapitulated the morphology and histology of their parental biopsies based on HE and IHC staining of pathological biomarkers. The scRNA-Seq data filtered drug-resistant cell populations and related signaling pathways (e.g. oxidative phosphorylation and ATP metabolic process). The data also revealed several putative drug resistant-driven genes (STMN1, VEGFA and NDRG1) and transcription factors (E2F1, BRCA1, MYBL2, CDX2 and CDX1). CONCLUSION: We generated an oxaliplatin-resistant CRC organoid model that was employed to provide potential therapeutic targets for treating CRC patients exhibiting oxaliplatin-resistance.

Application of Organoids in Carcinogenesis Modeling and Tumor Vaccination.

Organoids well recapitulate organ-specific functions from their tissue of origin and remain fundamental aspects of organogenesis. Organoids are widely applied in biomedical research, drug discovery, and regenerative medicine. There are various cultivated organoid systems induced by adult stem cells and pluripotent stem cells, or directly derived from primary tissues. Researchers have drawn inspiration by combination of organoid technology and tissue engineering to produce organoids with more physiological relevance and suitable for translational medicine. This review describes the value of applying organoids for tumorigenesis modeling and tumor vaccination. We summarize the application of organoids in tumor precision medicine. Extant challenges that need to be conquered to make this technology be more feasible and precise are discussed.

Ailanthone Inhibits Proliferation, Migration and Invasion of Osteosarcoma Cells by Downregulating the Serine Biosynthetic Pathway.

Osteosarcoma (OS) is the most common primary bone sarcoma, chemoresistance becomes an obstacle to its treatment. Metabolic reprogramming is a hallmark of malignancy, targeting the metabolic pathways might provide a reasonable therapeutic strategy for OS. Here we demonstrated that Ailanthone (AIL), a major component of the Chinese medicine Ailanthus altissima, significantly suppressed OS cell growth in vitro and in vivo. Furthermore, AIL dose-dependently inhibited cell migration and invasion, induced cell cycle arrest and apoptosis in OS cells. Combined transcriptomics, proteomics and metabolomics analyses revealed that AIL induced widespread changes in metabolic programs in OS cells, while the serine biosynthetic pathway (SSP) was the most significantly altered pathway. qRT-PCR and Western blot assay confirmed that the transcript and protein levels of the SSP genes (PHGDH, PSAT1 and PSPH) were downregulated dose-dependently by AIL. In addition, we found out that many downstream pathways of the SSP including the one-carbon pool by folate, purine metabolism, pyrimidine metabolism, DNA replication and sphingolipid metabolism were downregulated after AIL treatment. In the revere test, PHGDH overexpression but not exogenous serine supplementation clearly attenuated the effects of AIL on OS cells. Taken together, AIL exerts antitumor effects on OS through mediating metabolic reprogramming, at least in part, by suppressing the SSP. Our findings suggest that AIL could emerge as a potential therapeutic strategy in OS.

The timing of targeted therapy initiation in metastatic sarcoma as an adjuvant to first-line chemotherapy or a second-line agent.

58 cases of metastatic sarcoma were reviewed retrospectively in order to compare the efficacy and safety of concurrent (n=24, group A) versus sequential (n=34, group B) use of chemotherapy and targeted therapy in metastatic sarcoma. Progression-free survival (PFS) 1 was defined as the duration between initiation of first-line treatment to disease progression or recurrence. PFS' was defined as the duration between initiation of first-line treatment to the failure of chemotherapy and targeted therapy, and overall survival (OS) was defined as the duration between initiation of first-line treatment to the date of last follow-up or death. The results revealed that patients in group A possessed a higher tumor burden compared to those in group B (P=0.049). Survival curves revealed that the median PFS1 (15.2 vs. 5.4 months, P=0.000), median PFS' (15.2 vs. 10.8 months, P=0.049), and median OS (42.3 vs. 25.3 months, P=0.041) of subjects in group A were remarkably longer than those of group B. Subgroup analysis showed that patients in group A experienced more favorable PFS1 (15.2 vs. 3 months, P=0.000), PFS' (15.2 vs. 5.8 months, P=0.003), and OS (35.2 vs. 15.7 months, P=0.011) than those in group B, with findings especially prominent in patients with tumor burden ≥ 10 cm in comparison to patients with tumor burden < 10 cm (P ≥ 0.05). All grades of leukopenia, thrombocytopenia, fatigue, and oral mucositis were more frequently diagnosed in patients of group A compared to those of group B. However, there were no significant differences between the rates of Grade 3-4 adverse events between the two groups. This investigation suggests that the concurrent use of targeted therapy and chemotherapy may be useful and safe as a first-line treatment in patients with metastatic sarcoma who possess a high tumor burden.

Enhanced Magnetic Properties of FeSiAl Soft Magnetic Composites Prepared by Utilizing PSA as Resin Insulating Layer.

Thermosetting organic resins are widely applied as insulating coatings for soft magnetic powder cores (SMPCs) because of their high electrical resistivity. However, their poor thermal stability and thermal decomposition lead to a decrease in electrical resistivity, thus limiting the annealing temperature of SMPCs. The large amount of internal stress generated by soft magnetic composites during pressing must be mitigated at high temperatures; therefore, it is especially important to find organic resins with excellent thermal stabilities. In this study, we prepared SMPCs using poly-silicon-containing arylacetylene resin, an organic resin resistant to high temperatures, as an insulating layer. With 2 wt % PSA as an insulating layer and annealed at 700 °C for 1 h, the FeSiAl SMPCs achieved the best magnetic properties, including the lowest core loss of 184 mW/cm3 (measured at 0.1 T and 50 kHz) and highest permeability of 96.

The Plasma Concentration of D-Dimer is Associated with Neoadjuvant-Chemotherapy Efficacy and the Prognosis in Osteosarcoma.

PURPOSE: This retrospective study explored the clinical value of the plasma D-dimer level in osteosarcoma. MATERIALS AND METHODS: We measured the plasma D-dimer level before neoadjuvant chemotherapy (D0) and the plasma D-dimer level after four courses of neoadjuvant chemotherapy (D1) in 103 patients with stage-IIB high-grade osteosarcoma of the limb. The change in the D-dimer level (ΔD) was defined as D1 minus D0. The chi-square test was used to compare categorical variables. Analyses of receiver operating characteristic (ROC) curves were undertaken to determine the optimal cutoff points for D0, D1, and ΔD. The area under the ROC (AUC) of D0, D1, and ΔD was calculated to evaluate their discriminatory abilities in monitoring the response to neoadjuvant chemotherapy (tumor necrosis). Survival curves were generated according to Kaplan-Meier analyses and compared using the Log rank test. Univariate analyses and multivariate analyses were carried out to determine independent prognostic factors. RESULTS: Kaplan-Meier curves showed that a high D-dimer level at D0 and tumor diameter ≥8 cm were associated significantly with worse overall survival (OS) (P<0.05). Multivariate Cox regression analyses revealed a high D-dimer level at D0 (hazard ratio, 3.92; 95% confidence interval, 1.756-5.804; P=0.000) was an independent unfavorable prognostic factor. The chi-square test showed ΔD to be associated significantly with tumor necrosis. Analyses of ROC curves showed the D-dimer level at D0 and ΔD had better ability compared to that at D1 to discriminate the response to neoadjuvant chemotherapy. CONCLUSION: The D-dimer level was correlated with the prognosis and response to chemotherapy in patients with stage-IIB high-grade osteosarcoma of the limb. The D-dimer level may serve as a risk factor of the response to chemotherapy and prognosis of localized osteosarcoma.

Single-cell RNA landscape of intratumoral heterogeneity and immunosuppressive microenvironment in advanced osteosarcoma.

Osteosarcoma is the most frequent primary bone tumor with poor prognosis. Through RNA-sequencing of 100,987 individual cells from 7 primary, 2 recurrent, and 2 lung metastatic osteosarcoma lesions, 11 major cell clusters are identified based on unbiased clustering of gene expression profiles and canonical markers. The transcriptomic properties, regulators and dynamics of osteosarcoma malignant cells together with their tumor microenvironment particularly stromal and immune cells are characterized. The transdifferentiation of malignant osteoblastic cells from malignant chondroblastic cells is revealed by analyses of inferred copy-number variation and trajectory. A proinflammatory FABP4+ macrophages infiltration is noticed in lung metastatic osteosarcoma lesions. Lower osteoclasts infiltration is observed in chondroblastic, recurrent and lung metastatic osteosarcoma lesions compared to primary osteoblastic osteosarcoma lesions. Importantly, TIGIT blockade enhances the cytotoxicity effects of the primary CD3+ T cells with high proportion of TIGIT+ cells against osteosarcoma. These results present a single-cell atlas, explore intratumor heterogeneity, and provide potential therapeutic targets for osteosarcoma.

Organoid culture system for patient-derived lung metastatic osteosarcoma.

Osteosarcoma (OS) is the most common primary bone malignancy with high rates of recurrence and metastasis. OS often spreads to lungs, an optimized model for studying lung metastatic OS cells may help develop potential therapies for patients with lung metastasis. Here we firstly report an organoid culture system for lung metastatic OS tissues. We provided a fully described formula that was required for establishing lung metastatic OS organoids (OSOs). Using this protocol, the lung OSOs were able to be maintained and serially propagated for at least six months; the OSOs can also be generated from cryopreserved patient samples without damaging the morphology. The patient-derived lung OSOs retained the cellular morphology and expression of OS markers (Vimentin and Sox9) that recapitulate the histological features of the human OS. The microenvironment of primary lung metastatic OSOs preserved a similar T cell distribution with the human lung OS lesions; this provided a possible condition to explore how OS cells may react to immunotherapy. OSOs established from this protocol can be further utilized for studying various aspects of OS biology (e.g., tumorigenesis and drug screen/discovery) for precision medicine.

CDKL3 promotes osteosarcoma progression by activating Akt/PKB.

Osteosarcoma (OS) is a primary malignant bone neoplasm with high frequencies of tumor metastasis and recurrence. Although the Akt/PKB signaling pathway is known to play key roles in tumorigenesis, the roles of cyclin-dependent kinase-like 3 (CDKL3) in OS progression remain largely elusive. We have demonstrated the high expression levels of CDKL3 in OS human specimens and comprehensively investigated the role of CDKL3 in promoting OS progression both in vitro and in vivo. We found that CDKL3 regulates Akt activation and its downstream effects, including cell growth and autophagy. The up-regulation of CDKL3 in OS specimens appeared to be associated with Akt activation and shorter overall patient survival (P = 0.003). Our findings identify CDKL3 as a critical regulator that stimulates OS progression by enhancing Akt activation. CDKL3 represents both a biomarker for OS prognosis, and a potential therapeutic target in precision medicine by targeting CDKL3 to treat Akt hyper-activated OS.

Impact of chemotherapy cycles and intervals on outcomes of nonspinal Ewing sarcoma in adults: a real-world experience.

BACKGROUND: Adult Ewing sarcoma (ES) is a rare disease, the optimal treatment model is unknown. This study aimed to retrospectively analyze treatment-related prognostic factors of nonspinal ES in Chinese adults. METHODS: Eighty-one patients treated between January 2005 and December 2017 were included in the present study. Thirty-three (40.7%) presented with metastatic disease at diagnosis. Eight patients were submitted to primary surgery followed by chemotherapy, while 73 patients received chemotherapy before and after surgery and/or local radiotherapy. The chemotherapy regimen included 8-17 cycles of vincristine, doxorubicin, and cyclophosphamide (VDC) alternating with ifosfamide and etoposide (IE) every 3 weeks. Clinical outcomes and safety were analyzed. RESULTS: VDC/IE chemotherapy was well tolerated in adult patients with ES. Multivariate Cox regression analyses revealed that chemotherapy of at least 12 cycles was a favorable independent prognostic factor of event-free survival (hazard ratio, 0.558; 95% confidence interval, 0.323-0.965; P = 0.037) and overall survival (hazard ratio, 0.424; 95% confidence interval, 0.240-0.748; P = 0.003). Similarly, a low frequency of chemotherapy delays was an independent prognostic factor of improved OS (hazard ratio, 0.438; 95% confidence interval, 0.217-0.887; P = 0.022). CONCLUSION: Our study suggests that adults with ES should be treated with an aggressive multidisciplinary approach, intensive chemotherapy with adequate cycles and appropriate intervals can be recommended in this group.

Inadequate DNA Damage Repair Promotes Mammary Transdifferentiation, Leading to BRCA1 Breast Cancer.

Loss of BRCA1 p220 function often results in basal-like breast cancer (BLBC), but the underlying disease mechanism is largely opaque. In mammary epithelial cells (MECs), BRCA1 interacts with multiple proteins, including NUMB and HES1, to form complexes that participate in interstrand crosslink (ICL) DNA repair and MEC differentiation control. Unrepaired ICL damage results in aberrant transdifferentiation to a mesenchymal state of cultured, human basal-like MECs and to a basal/mesenchymal state in primary mouse luminal MECs. Loss of BRCA1, NUMB, or HES1 or chemically induced ICL damage in primary murine luminal MECs results in persistent DNA damage that triggers luminal to basal/mesenchymal transdifferentiation. In vivo single-cell analysis revealed a time-dependent evolution from normal luminal MECs to luminal progenitor-like tumor cells with basal/mesenchymal transdifferentiation during murine BRCA1 BLBC development. Growing DNA damage accompanied this malignant transformation.