Jennifer A. Chan

Glioblastoma is a lethal brain tumour for which current multimodal treatment rarely prevents recurrence ¹ . Therapeutic failure is driven by extensive intratumoural cellular heterogeneity ² with a microenvironment dominated by tumour-associated macrophages that sustain tumour growth and immunosuppression ³ . Although chimeric antigen receptor (CAR)-T cell therapies are being developed for glioblastoma, sustained response has been undermined by non-uniform antigen expression, antigen loss and microenvironmental barriers that are not directly engaged by tumour-targeting designs ⁴ . These limitations motivate new strategies that address the disease as a coupled tumour–immune system rather than a single malignant compartment. Here we use a multi-omic target discovery platform to identify GPNMB as a dual-compartment antigen in glioblastoma. Anti-GPNMB CAR-T cells showed potent anti-tumour activity, with long-term disease control in orthotopic patient-derived xenografts and syngeneic glioma models through concomitant depletion of GPNMB ⁺ tumour and immunosuppressive myeloid populations. By collapsing tumour control and microenvironmental reprogramming, these findings provide a new strategy for antigen selection and targeting in heterogenous, myeloid-rich solid cancers.

Chimeric antigen receptor (CAR) T cell therapy for solid tumors is constrained by the scarcity of safe, uniformly expressed cell-surface targets. Here we identify glycoprotein NMB (GPNMB)—an MiT/TFE-family fusion-driven protein—as being highly, homogeneously and stably expressed in primary and relapsed alveolar soft-part sarcoma (ASPS) and translocation renal cell carcinoma. We develop a GPNMB-directed CAR T cell product, GCAR1, which demonstrates potent activity against patient-matched cells, organoids and xenograft models. Post hoc interim analysis of a first-in-human open-label, individual-participant trial (NCT07104682) for a participant with relapsed/refractory, metastatic ASPS showed that GCAR1 induces stable disease for up to 3 months, accompanied by resolution of many nontarget lesions (primary endpoint), and is well tolerated. GCAR1 T cells expand in peripheral blood as a polyclonal population and remain detectable for 1 month. Spatial transcriptomics identified immunosuppressive niches in a treatment-resistant lesion and immune checkpoint blockade synergized with GCAR1 in a xenograft model. Altogether, our data provide a proof of concept for treating GPNMB-expressing solid tumors with GCAR1 and more broadly targeting surface antigens driven by oncogenic gene fusions with CAR T cell therapies.

Oligodendrogliomas (ODGs) are slow-growing tumors with poorly defined biology. Here, we identify SMPD3, encoding neutral sphingomyelinase 2 (nSMase2), as a negative regulator of ODG progression via extracellular vesicle (EV) biogenesis and autocrine-paracrine signaling. High SMPD3 expression correlates with longer survival in ODG and low-grade glioma patients. SMPD3 knockdown in patient-derived BT54 and BT88 cells increases proliferation, enhances BT88 invasion in cerebral organoids, and accelerates BT88-tumor growth in mouse xenografts and chick CAM assays. Patient tumors display abnormal non-neoplastic glial proliferation and a ribosome-rich transcriptional signature in tumor-associated oligodendrocytes. Concordantly, ODG-derived EVs, enriched in ribosomal proteins, suppress astrocyte proliferation. Pharmacological inhibition of nSMase2 (GW4869) or ribosome biogenesis (Rbin1, diazaborine) increases ODG cell proliferation while reducing protein synthesis. Blocking EV synthesis amplifies ribosome inhibition effects, whereas exogenous EVs attenuate them. Thus, SMPD3-mediated EV signaling and ribosomal stress converge to regulate ODG proliferation, establishing SMPD3 as a key regulator and potential therapeutic target.

Despite advances in precision medicine, 30% of high-risk pediatric cancers lack an actionable molecular target, hindering effective treatment and affecting survival outcomes. Although mouse patient-derived xenograft (PDX) models offer additional insights into clinical drug responses, delivering findings from these models within a clinically actionable time frame remains challenging. This international collaboration between two national precision medicine programs demonstrates proof-of-principle that individualized larval zebrafish PDXs can robustly and rapidly assess clinical responses in high-risk pediatric cancers. Retrospective zebrafish PDX testing was performed on tumor samples from 10 pediatric patients with high-risk cancers. Drug responses in zebrafish models were correlated with clinical responses for each patient and directly compared with responses in cognate mouse PDX models. Responses to conventional and targeted therapies, administered as single agents or in combinations, were assessed. Zebrafish PDXs were successfully established from all 10 patients and provided robust drug response data in every case, including from three patients whose tumor samples could not be engrafted in mice. Remarkably, zebrafish models accurately recapitulated patient responses for 11 of 12 treatment regimens. These findings highlight the potential of larval zebrafish PDX models to provide real-time, clinically relevant drug response data, supporting their potential use in prospective precision medicine studies.Significance:This proof-of-principle study is the first to compare drug responses in larval zebrafish and mouse PDX models with patient outcomes in pediatric precision oncology, showing high concordance. Results highlight the potential of zebrafish PDX models to predict drug responses in high-risk cancers more accurately, rapidly, and cost-effectively in prospective studies.

Mouse and larval zebrafish PDX drug efficacy for nonresponsive patients. A to C, Patient information, alongside mouse PDX waterfall plot of maximum tumor regression, and larval zebrafish tumor cell numbers for each therapy for neuroblastoma zccs373 (A), sarcoma zccs262 (B), and osteosarcoma zccs43 (C). For mouse PDX data, colored columns represent individual mice. For larval zebrafish data, error bars represent mean ± SEM, each colored dot represents an individual larva, and a threshold is based on SEM of 1 dpi. Statistical analysis was conducted using a one-way ANOVA with the Dunnett multiple comparisons test, comparing the mean of each treatment group with control (4 dpi), P values: *, < 0.05; **, < 0.01; ***, < 0.001; ****, < 0.0001). mut, mutant; dpi, days post-injection.

Features of mouse PDX, larval zebrafish PDX and in vitro models

CONSORT flow diagram for patients included in the study and the criteria not met for those excluded

Patient cohort details, including molecular targets, clinical response to therapy, and mouse PDX development

Larval zebrafish drug efficacy studies for patients with no available mouse PDX. A to C, Patient information, alongside larval zebrafish tumor cell numbers for each therapy for rhabdomyosarcoma zccs170 (A), gastrointestinal stromal tumor zccs15 (B), and Ewing sarcoma zccs276 (C). For larval zebrafish data, error bars represent mean ± SEM, each colored dot represents an individual larva, and a threshold is based on SEM of 1 dpi. Statistical analysis was conducted using a one-way ANOVA with the Dunnett multiple comparisons test, comparing the mean of each treatment group with control (4 dpi), P values: *, < 0.05; **, < 0.01; ***, < 0.001; ****, < 0.0001. D, Representative image of a five microns sectioned control larval zebrafish xenograft for patient zccs170. Deep Red–labeled patient cells present in the YS 4 dpi and labeled with DAPI to confirm viability. White circle in the images point to zccs170 tumor cells. Images were taken with 10× and 20× magnification. The scale bar is 100 µm. dpi, days post-injection.

Single agent and combination doses established in the larval zebrafish PDX at 35°C for 3 to 7 days post fertilization zebrafish for 72-hour treatment.